Dissecting the Genetic Basis of Congenital Diaphragmatic Hernias in Mice

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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.