Dissecting the Genetic Basis of Congenital Diaphragmatic Hernias in Mice

项目总结 先天性隔膜疝气(CDH)是一种常见且严重的结构性出生缺陷，每3,000人中就有1人发生 活产并导致约30-50%的患者死亡。慢性萎缩性脑病的原因有两种： 部分横隔肌丢失，使腹部内容物突出进入胸腔，通常 导致严重的肺发育不全。CDH的严重程度与其在横隔膜中的位置直接相关 肌片出现的缺陷，与更多的背部疝气相关的严重程度和发病率更高。 尽管人类研究清楚CDH在很大程度上是一种遗传性疾病，但遗传学是复杂的和 关于这种缺陷背后的基因和途径的数量和性质，仍有许多未知之处。这 部分是由于功能验证研究的数量有限，因为横隔膜是一种哺乳动物- 特定的结构，因此只有小鼠模型生物可用于研究CDH。这项提案的目标是 是研究CDH的遗传学和细胞机制，使用新的基因编辑和 小鼠模型生物体的转录图谱技术。来自CDH的高通量测序 患者中，预测CDH不足的新变种的数量呈指数级增长。在这个项目的第一个目标中 建议，我将优化一种方法，在小鼠胚胎和屏幕上快速创建精确的基因编辑 胚胎处于适当的发育阶段，不需要进一步培育。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相关变体。至 在目标2中，我将研究腹股沟的细胞 导致小鼠体内CDH的动力学因素丝氨酸激酶编码基因CDC42bpb，a 新的CDH小鼠模型，形成独特的腹股沟。这些研究将揭示 有症状和无症状的CDH的发展。最后，在目标3中，我将使用单细胞 RNA测序以了解驱动横隔膜发育的细胞类型和谱系 并在空间上位于鼠标内。使用新的空间基因表达技术，我将专门检测两个 假设发育中的横隔膜中的细胞群，组织周围的间皮层和 成纤维细胞群体通过胚胎发育推动组织跨越肝脏的迁移。 总体而言，拟议的工作不仅将加深我们对CDH产生机制的了解，它 将创建一个新的转录数据集，以帮助确定CDH相关基因的优先顺序，并提供 快速筛选小鼠疾病相关等位基因的有效方法，促进了我们对 先天性巨结肠的遗传学基础。

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.