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Phenotype-driven approach to understanding the function of craniofacial regulators using IMPC-generated mouse strains

使用 IMPC 产生的小鼠品系的表型驱动方法来了解颅面调节器的功能

基本信息

批准号：
9899973
负责人：
Jeffrey Ohmann Bush
金额：
$ 74.16万
依托单位：
UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
依托单位国家：
美国
项目类别：
财政年份：
2019
资助国家：
美国
起止时间：
2019-04-01 至 2024-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9899973
关键词：
3-Dimensional Ablation Adult Affect Algorithms Alleles Animal Model Applications Grants Area Binding Biological Process Biomedical Research Birth Branchial arch structure Candidate Disease Gene Catalogs Cell Adhesion Cell physiology Cells ChIP-seq Classification Cleft Palate Code Congenital Abnormality Craniofacial Abnormalities Data Data Set Databases Defect Development E1A-associated p300 protein Ectoderm Effectiveness Embryo Embryonic Development Enhancers Exhibits Experimental Models Face FaceBase Fetus Genes Genetic Genome Goals Health Homeostasis House mice Human Informatics International Knock-out Knockout Mice Knowledge Laboratory mice Life Light Manuals Maxilla Mesenchyme Modeling Molecular Morphogenesis Morphology Mouse Strains Mus Mutant Strains Mice Mutate Mutation Neoplastic Cell Transformation Organism Pattern Perinatal mortality demographics Phenotype Physiologic Ossification Pregnancy Premaxillary palate Process Proteins Reporting Research Role Secondary Palate Shapes Signaling Molecule Structural Congenital Anomalies Syndrome Time Tissues Transgenic Organisms United States National Institutes of Health Validation base conditional mutant craniofacial craniofacial complex craniofacial development cranium design experience genome-wide malformation mammalian genome microCT mouse genome mutant phenome phenotypic data response suture fusion transcription factor transcriptome transcriptome sequencing

项目摘要

ABSTRACT Many of the datasets resulting from genome-wide approaches lack functional validation in living organisms. While the laboratory mouse is often used as an experimental model, a large number of mouse genes have unknown functions. The International Mouse Phenotyping Consortium (IMPC) is building the first catalogue of mammalian genome function by generating knockout (KO) mouse strains for every protein-coding gene in the genome. Taking advantage of this opportunity, the two co-PIs have designed this proposal in response to NIH PAR-17-005 for phenotyping IMPC embryonic and perinatal lethal KO mouse lines. Our focus is on mutations that affect the craniofacial complex, based on our expertise in modeling craniofacial malformations in the mouse. While craniofacial defects represent one third of all human birth defects, our knowledge of the underlying cellular and molecular mechanisms remains poor. To select mutant mouse lines for in-depth phenotyping of craniofacial abnormalities, we generated algorithms to intersect the current list of IMPC lethal /subviable KO strains exhibiting craniofacial defects with: 1) all genes present in transcriptomes of mouse embryonic craniofacial domains that were either generated in our labs or available in the FaceBase database; and 2) all significant ChIP-Seq peaks for binding of the enhancer-associated protein p300 in mouse embryonic whole faces present in the FaceBase database. By searching the Mouse Genome Informatics (MGI) database for phenotypic data and by prioritizing genes that have unknown or poorly defined roles in craniofacial develop- ment, we restricted the number of chosen genes to N=30. These comprise regulators of various cellular functions that cause embryonic lethality and craniofacial defects when disrupted in the mouse. Preliminary phenotyping of the KO mouse line for Zfhx4, a gene that exhibited highest maxillary enrichment in our RNA- Seq dataset, revealed that all Zfhx4 KO embryos present cleft palate and maxillary hypoplasia, providing proof of concept for the effectiveness of the proposed strategy. Accordingly, we will characterize 30 mutant lines (15 per lab over 5 years) via the following specific aims: AIM 1: Examination and classification of 30 IMPC mutant mouse lines for craniofacial phenotypes. Employing a two-stage phenotyping pipeline, we will categorize the craniofacial defects for each line and narrow the time of onset. AIM 2. Deep characterization of craniofacial phenotypes. Based on three phenotyping platforms, we will uncover the function(s) of the 30 chosen genes in craniofacial development through in-depth analyses. Platform A will characterize early craniofacial anomalies including defects in branchial arch patterning, as well as primary palate morphogenesis and fusion (E8.5-11.5); Platform B will characterize later abnormalities of secondary palate fusion (E11.5-15.5); and Platform C will dissect perturbations of craniofacial shape/morphology and skull ossification. Shedding light on the cellular and molecular processes controlled by genes that are essential for embryonic development and cause human birth defects when disrupted will be vital to the health of the fetus before and long after birth.

摘要从全基因组方法得到的许多数据集缺乏在活生物体中的功能验证。虽然实验室小鼠经常被用作实验模型，但大量的小鼠基因具有未知的功能国际小鼠表型鉴定联盟（IMPC）正在建立第一个哺乳动物基因组功能，通过产生敲除（KO）小鼠品系中的每个蛋白质编码基因，基因组利用这一机会，两位共同主持人设计了这一提案，以回应 NIH PAR-17-005用于IMPC胚胎和围产期致死KO小鼠系的表型分型。我们的重点是基于我们在颅面畸形建模方面的专业知识，影响颅面复合体的突变鼠标虽然颅面缺陷占所有人类出生缺陷的三分之一，但我们对颅面缺陷的了解潜在的细胞和分子机制仍然很差。为了选择突变小鼠品系进行深入研究，颅面异常的表型，我们生成了算法，以交叉目前的IMPC致死性 /亚存活KO菌株，其表现出具有以下缺陷的颅面缺陷：1）小鼠转录组中存在的所有基因在我们的实验室中生成或在FaceBase数据库中可用的胚胎颅面域; 和2）小鼠胚胎中增强子相关蛋白p300结合的所有显著ChIP-Seq峰 FaceBase数据库中的所有面孔。通过搜索小鼠基因组信息学（MGI）数据库对于表型数据，并优先考虑在颅面发育中具有未知或不明确作用的基因，此外，我们将选择的基因的数量限制为N=30。这些包括各种细胞的调节剂在小鼠中被破坏时导致胚胎死亡和颅面缺陷的功能。初步 Zfhx 4基因的KO小鼠品系的表型分析，Zfhx 4基因在我们的RNA中表现出最高的上颌富集， Seq数据集显示，所有Zfhx 4 KO胚胎均存在腭裂和上颌骨发育不全，提供了证据对拟议战略的有效性进行概念评估。因此，我们将表征30个突变株系（15 每个实验室超过5年）通过以下具体目标：目的1：检查和分类30 IMPC 颅面表型的突变小鼠品系。采用两阶段表型分析管道，我们将对每一行颅面缺损进行分类，缩小发病时间。AIM 2.深层表征颅面表型基于三个表型分析平台，我们将揭示30个基因的功能，通过深入的分析选择颅面发育的基因。平台A将在早期颅面异常，包括鳃弓图案缺陷以及原发性腭形态发生和融合（E8.5-11.5）;平台B将表征继发性腭融合的后期异常（E11.5-15.5）; 平台C将解剖颅面形状/形态和颅骨骨化的扰动。脱落了解胚胎发育所必需的基因控制的细胞和分子过程并导致人类出生缺陷时，中断将是至关重要的健康胎儿出生前和出生后很长一段时间。