Supplementary Material for: Nuclear YAP Activation in an Ex Vivo Murine Lung Model of Fetal Tracheal Occlusion

Introduction: In this study, we aimed to investigate the expression of nuclear Yes-associated protein (nYAP), a molecule involved in mechanotransduction, during fetal lung development in a novel ex vivo mouse model of congenital diaphragmatic hernia (CDH) lung hypoplasia.
Methods: Pregnant CD1 mice were gavaged nitrofen at E8.5 to induce CDH lung hypoplasia. At E16.5, fetal lungs were harvested and cultured ex-vivo for 72hrs with or without tracheal occlusion (hypo-TO and hypo, respectively). Age-matched normal fetal lung controls (norm-TO and norm) were evaluated in parallel. Whole lungs at E16.5+3 were analyzed for nYAP and markers of distal epithelial differentiation by immunohistochemistry and quantitative gene expression.
Results: There was robust nYAP expression in norm-TO lungs. Analysis of the distal lung parenchyma in normal and hypoplastic lungs showed enhanced epithelial nYAP expression in the distal airways of both norm-TO and hypo-TO lungs relative to their respective controls. Hypo lungs had the lowest expression of nYAP among the groups. There was significantly increased expression of both Ctgf and Cyr61 in hypo-TO lungs compared to hypo lungs without tracheal occlusion (Ctgf: 1.57±0.43 and 1.02±0.23, p=0.016; Cyr61: 1.60±0.34 and 1.01±0.17, p=0.003).
Conclusion: In this ex vivo model, tracheal occlusion upregulates nYAP in both control and hypoplastic lungs during the canalicular stage of development, suggesting a critical role of mechanosensory response molecule in CDH lung hypoplasia.