Dissecting the Genetic Basis of Congenital Diaphragmatic Hernias in Mice

剖析小鼠先天性膈疝的遗传基础

• 批准号: 10569504
• 负责人: Eric Bogenschutz
• 金额: $ 6.95万
• 依托单位: JACKSON LABORATORY
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-02-03 至 2024-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10569504
• 关键词: Abdomen; Alleles; Animal Model; Animals; Appearance; Automobile Driving; Biological Assay; Breeding; Bypass; Candidate Disease Gene; Cell Lineage; Cells; Cessation of life; Child; Clustered Regularly Interspaced Short Palindromic Repeats; Complement; Complex; Congenital Abnormality; Congenital diaphragmatic hernia; Consumption; Data Set; Defect; Development; Disease; Dorsal; Embryo; Embryonic Development; Engineering; Etiology; Fibroblasts; Gene Expression; Gene Expression Profiling; Generations; Genes; Genetic; Genetic Diseases; Genetic Transcription; Goals; Hernia; High-Throughput Nucleotide Sequencing; Human; In Vitro; Invaded; Knockout Mice; Lead; Live Birth; Liver; Location; Lung; Mediating; Mesothelium; Methods; Modeling; Molecular; Morbidity - disease rate; Morphogenesis; Morphology; Mus; Muscle; Muscular Atrophy; Mutation; Nature; Organogenesis; Pathway interactions; Patients; Penetrance; Phenotype; Population; Process; Protein-Serine-Threonine Kinases; Rapid screening; Reproductive Biology; Respiratory Diaphragm; Role; Severities; Skeletal Muscle; Speed; Structural Congenital Anomalies; Structure; Techniques; Technology; Tendon structure; Testing; Thoracic cavity structure; Time; Tissues; Translating; Validation; Variant; Ventral Hernia; Work; cell motility; cell type; cohort; congenital heart disorder; cost; genetic pedigree; genetic variant; genome sequencing; improved; in vivo; insertion/deletion mutation; insight; lung development; migration; model organism; mortality; mouse model; muscular structure; mutation screening; novel; pharmacologic; programs; renal agenesis; repaired; screening; single-cell RNA sequencing; validation studies

PROJECT SUMMARY Congenital diaphragm hernia (CDH) is a common and severe structural birth defect arising in 1 of every 3,000 live births and leading to mortality in an estimated 30-50% of patients. CDH is caused by either a weakening or partial loss of diaphragm skeletal muscle, allowing abdominal contents to herniate into the thoracic cavity, often leading to severe lung hypoplasia. The severity of CDH is directly related to the location in the diaphragm muscle sheet where the defect arises, with more dorsal hernias correlating with higher severity and morbidity. Though it is clear from human studies that CDH is largely a genetic disease, the genetics are complex and much remains unknown about the number and nature of the genes and pathways that underlie the defect. This is in part due to limitations in the number of functional validation studies as the diaphragm is a mammalian- specific structure, and thus only murine model organisms are available to study CDH. The goal of this proposal is to study both the genetics and cellular mechanisms underlying CDH using new gene-editing and transcriptional profiling techniques in the mouse model organism. From high-throughput sequencing of CDH patients, the number of novel variants predicted to underly CDH has grown exponentially. In the first aim of this proposal, I will optimize a method to rapidly create precise gene edits in the mouse embryo and screen embryos at the appropriate developmental stage without the need of further breeding. Using this discovery platform, I will then screen CDH-associated variants discovered from patient genome sequencing cohorts. To dissect the mechanism by which hernias arise in discrete regions of diaphragm, in aim 2 I will study the cellular dynamics leading to CDH within in mice homozygous null for the serine kinase encoding gene Cdc42bpb, a novel mouse model of CDH which develop unique ventral hernias. These studies will reveal key differences in the development of the symptomatic and asymptomatic forms of CDH. Finally, in aim 3 I will use single cell RNA sequencing to understand the cell types and lineages driving diaphragm development both temporally and spatially within the mouse. Using novel spatial gene expression techniques, I will specifically assay for two hypothesized cell populations in the developing diaphragm, a mesothelial layer surrounding the tissue and a fibroblast population driving the migration of the tissue across the liver through embryonic development. Overall, the work proposed will not only further our understanding of the mechanism by which CDH arises, it will create a novel transcriptional dataset to assist in prioritization of CDH-associated genes and provide a powerful method to screen disease-associated alleles rapidly in mouse, advancing our understanding of the genetic basis for CDH.

项目摘要 先天性膈疝（CDH）是一种常见且严重的结构性出生缺陷，每3000人中就有1人发生 活产并导致估计30-50%的患者死亡。CDH是由减弱或 膈肌骨骼肌部分缺失，使腹腔内容物疝入胸腔，通常 导致严重的肺发育不全CDH的严重程度与其在膈肌中的位置直接相关 缺损处的肌肉片，更多的背疝与更高的严重性和发病率相关。 虽然从人类研究中可以清楚地看出，CDH在很大程度上是一种遗传性疾病，但遗传学是复杂的， 关于造成这种缺陷的基因和途径的数量和性质，仍有许多未知之处。这 部分原因是功能验证研究的数量有限，因为横膈膜是哺乳动物- CDH是一种特异性结构，因此只有鼠模型生物体可用于研究CDH。这项提案的目的是 是使用新的基因编辑研究CDH的遗传学和细胞机制， 在小鼠模型生物体中的转录谱分析技术。从CDH的高通量测序 在患者中，预测为CDH基础的新变体的数量呈指数级增长。第一个目标是 根据这项提议，我将优化一种方法，在小鼠胚胎中快速创建精确的基因编辑， 胚胎在适当的发育阶段，而不需要进一步育种。利用这一发现 平台，然后我将筛选从患者基因组测序队列中发现的CDH相关变异。到 在目的2中，我将研究隔膜的细胞结构， 在丝氨酸激酶编码基因Cdc 42 bpb纯合子缺失的小鼠中， CDH的新小鼠模型，其产生独特的腹疝。这些研究将揭示关键的差异， 有症状和无症状形式的CDH的发展。最后，在目标3中，我将使用单细胞 RNA测序，以了解驱动横膈膜发育的细胞类型和谱系 以及在空间上在小鼠内。使用新的空间基因表达技术，我将专门分析两个 假设的细胞群在发展中的隔膜，间皮层周围的组织和 成纤维细胞群体通过胚胎发育驱动组织迁移穿过肝脏。 总的来说，建议的工作不仅将进一步加深我们对CDH产生机制的理解， 将创建一个新的转录数据集，以帮助优先考虑CDH相关基因，并提供一个新的转录数据集。 一种在小鼠中快速筛选疾病相关等位基因的强有力的方法， CDH的遗传基础