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：
10400255
负责人：
Jeffrey Ohmann Bush
金额：
$ 7.76万
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：
2021
资助国家：
美国
起止时间：
2021-07-01 至 2024-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10400255
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.

抽象的由全基因组方法产生的许多数据集缺乏活生物体的功能验证。虽然实验室小鼠通常被用作实验模型，但大量小鼠基因具有未知功能。国际老鼠表型联盟（IMPC）正在建立第一个目录哺乳动物基因组功能通过为每个蛋白质编码基因产生基因敲除（KO）小鼠菌株基因组。利用这一机会，这两个共同提出了这一建议，以响应 NIH PAR-17-005用于表型IMPC胚胎和围产期致死性KO小鼠系。我们的重点是基于我们在建模颅面畸形方面的专业知识，影响颅面复合物的突变鼠标。尽管颅面缺陷占所有人类出生缺陷的三分之一，但我们对潜在的细胞和分子机制仍然很差。为深度选择突变鼠标线颅面异常的表型，我们生成算法以与当前的IMPC致命列表相交 /可亚科菌株表现出颅面缺陷，有：1）所有基因存在于小鼠的转录组中在我们的实验室中生成的胚胎颅面结构域或在面库数据库中可用； 2）所有显着的CHIP-SEQ峰，用于在小鼠胚胎中结合增强子相关蛋白p300 面孔数据库中存在的整个面孔。通过搜索鼠标基因组信息学（MGI）数据库用于表型数据以及通过优先考虑在颅面发育中未知或定义不明的基因我们将所选基因的数量限制为n = 30。这些包括各种细胞的调节剂在小鼠中破坏时会导致胚胎致死性和颅面缺陷的功能。初步的 ZFHX4的KO小鼠系的表型，该基因在我们的RNA中表现出最高的上颌富集 SEQ数据集，揭示了所有ZFHX4 KO胚胎呈现裂口和上颌发育不全，提供证明提出策略的有效性的概念。因此，我们将表征30条突变线（15 每个实验室超过5年）通过以下特定目的：目标1：检查和分类30 IMPC 颅面表型的突变小鼠系。使用两阶段的表型管道，我们将对每条线的颅面缺陷进行分类，并缩小发作时间。目标2。深刻的特征颅面表型。基于三个表型平台，我们将发现30个功能通过深入分析中选择的颅面发育中选择的基因。平台A将提早特征颅面异常，包括分支拱形图案中的缺陷，以及原发性的形态发生和融合（E8.5-11.5）;平台B将表征后来的次级味融合异常（E11.5-15.5）；平台C将剖析颅面形状/形态和颅骨骨化的扰动。脱落对胚胎发育至关重要的基因控制的细胞和分子过程的光并在出生前后很久以前和胎儿的健康至关重要。