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

• 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
• 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

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 由全基因组方法产生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 未知函数。国际老鼠表型联盟(IMPC)正在建立第一个老鼠表型鉴定目录 哺乳动物基因组的功能是为每个编码蛋白质的基因产生敲除(KO)小鼠品系 基因组。利用这个机会，两个共同的私营部门督导制定了这项建议，以回应 NIH PAR-17-005用于IMPC胚胎和围产期致死的KO小鼠品系的表型鉴定。我们的重点是 影响颅面复合体的突变，基于我们在建模颅面畸形方面的专业知识 那只老鼠。虽然头面部缺陷占人类所有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。选择突变小鼠品系进行深入研究 颅面异常的表型，我们生成了与当前IMPC致死列表相交的算法 /亚活的KO菌株表现出头面部缺陷：1)小鼠转录本中存在的所有基因 胚胎头面部区域，要么是在我们的实验室生成的，要么是在FaceBase数据库中找到的； 2)小鼠胚胎中与增强子相关蛋白p300结合的所有重要的芯片序列峰 在FaceBase数据库中显示完整的人脸。通过搜索小鼠基因组信息学(MGI)数据库 对于表型数据，并通过优先处理在颅面部发育中具有未知或不明确角色的基因- 我们将所选基因的数量限制在N=30。它们由不同的细胞调节因子组成 当小鼠受到干扰时，会导致胚胎死亡和头面部缺陷的功能。初步 KO小鼠品系Zfhx4的表型，该基因在我们的RNA中表现出最高的上颌丰度。 SEQ数据集，显示所有Zfhx4 KO胚胎都存在腭裂和上颌发育不良，提供了证据 关于拟议战略的有效性的概念。因此，我们将鉴定30个突变系(15个 每个实验室5年以上)，具体目标如下：目标1：对30个IMPC进行检查和分类 颅面部表型突变小鼠系。采用两阶段表型流水线，我们将 对每条线的颅面缺损进行分类，并缩短发病时间。目标2.深入刻画 头面部的表型。基于三个表型平台，我们将揭示30个表型平台的功能(S) 通过深入分析筛选出影响颅面发育的基因。平台A将成为早期的特征 颅面畸形，包括颧弓图案缺陷以及原发的腭部形态发生 和融合(E8.5-11.5)，B平台表现为晚期继发性腭裂融合异常(E11.5-15.5)； 平台C将解剖颅面形状/形态和头骨骨化的扰动。脱毛 阐明胚胎发育所必需的基因控制的细胞和分子过程 而造成人类出生缺陷时，中断将对胎儿的健康至关重要，出生前和出生后很长一段时间。