PROJECT SUMMARY/ABSTRACT Congenital diaphragmatic hernia (CDH) is polygenic condition in which the fetal intestines and liver herniate into the thoracic cavity, resulting in lung compression and impaired pulmonary development. Despite being one of the most common and expensive surgical birth defects managed in neonatal intensive care units worldwide, the underlying biomolecular mechanisms of CDH lung hypoplasia remain unknown. Advances in state-of-the-art surgical critical care, including novel pharmacologic agents, extracorporeal membrane oxygenation, and fetal surgery have failed to make a substantial impact in improving clinical outcomes in severely affected children, with overall mortality remaining at 30%, largely due to the devastating degree of lung pathology. There remains a critical need to better understand the mechanisms underlying CDH lung hypoplasia to offer hope for affected patients and their families. YAP/TAZ is the core kinase of the Hippo signaling pathway that has been shown to respond to mechanosensory stimuli during fetal lung patterning and differentiation. The proximal- distal fetal lung abnormalities observed with the human CDH phenotype are consistent with those of YAP/TAZ dysregulation in the setting of mechanical compression (reduced intrapulmonary pressures). Although transpulmonary pressures have also been shown to regulate FGF10, an essential growth factor and major downstream regulator of branching morphogenesis and epithelial differentiation, the interplay between the YAP/TAZ translocation and FGF10-mediated lung growth in CDH is not well understood. The central hypothesis of this proposal is that reduced nuclear YAP/TAZ activation during the canalicular stage of lung development results in profound lung hypoplasia in CDH. Four research teams, led by Shaun Kunisaki (surgery), Jason Spence (cell biology), Celeste Nelson (engineering), and Enid Neptune (pulmonary), will bring together complementary backgrounds and technologies to address this hypothesis. Using the established nitrofen mouse model, human lung organoids derived from induced pluripotent stem cells, and high throughput micro- mechanical compression devices, three distinct aims are proposed. In Specific Aim 1, they will investigate the impact of nuclear YAP/TAZ regulation during CDH canalicular lung development. In Specific Aim 2, they will determine whether mechanical forces modulate CDH fetal lung morphogenesis ex vivo through YAP/TAZ signaling. In Specific Aim 3, they will evaluate how nuclear YAP/TAZ activation affects in vivo lung growth in a large animal fetal model of CDH. Completion of these Aims will have facilitated a better understanding of the role of an important mechanosensing pathway during CDH lung development and will have potentially uncovered new therapeutic targets for affected children at the more severe end of the CDH disease spectrum.