PROJECT SUMMARY Congenital diaphragmatic hernia (CDH) is characterized by incomplete closure of the diaphragm and lung hypoplasia in newborns. The diaphragm defect can be surgically repaired; however, lung hypoplasia remains the main cause of mortality and morbidity in CDH. The etiology of lung hypoplasia in CDH is multifactorial and involves compression by herniated viscera, genetic and environmental factors. To date, over 100 CDH- associated genes were identified in patient cohorts. However, the function of most CDH-associated genes, including Zinc Finger Homeobox 4 (ZFHX4), in the fetal lung is unknown. In Preliminary Studies, we show that ZFHX4 is selectively expressed in mesenchymal cells in fetal lungs of humans and mice. Functional disruption of Zfhx4 in mice causes thickened mesenchyme, defective alveolar fibroblast differentiation, and impaired alveologenesis, which resemble the clinical hallmarks of lung hypoplasia in CDH. Interestingly, Zfhx4-/- mice have no diaphragmatic henia, which suggests that additional factors may cause diaphragm defects in CDH patients with ZFHX4 variants. A lack of confounding diaphragm defects also makes Zfhx4-/- mice an excellent model to investigate the direct role of Zfhx4 in lung mesenchyme development. We show that Zfhx4 expression peaks at the pseudoglandular stage of lung development; however, Zfhx4-/- mice have no lung mesenchymal phenotypes until E16.5 when Zfhx4 is no longer detectable. Supporting the impact of early Zfhx4 expression on late mesenchyme development, transcriptome profiling of wildtype and Zfhx4-/- lung mesenchymal cells at E16.5 identified differentiatlly expressed genes enriched in matrix and mesenchyme developmental pathways. The observed temporal disparity between Zfhx4 expression and the phenotype in Zfhx4-/- mice are reminiscent of two defining features of a pioneer factor -- by binding to silent gene loci early on and by recruiting transcriptional factors and epigenetic regulators, drives later lineage specification. Based on these preliminary findings, we hypothesize that the pioneer factor ZFHX4 accesses silent gene sites and recruits transcriptional regulators in embryonic mesenchymal progenitors thereby orchestrating alveolar fibroblast differentiation. Utilizing the R03 funding mechanism and leveraging available Zfhx4-/- and Zfhx4v5 mouse lines, we propose two pilot assays to interrogate Zfhx4 regulation of lung mesenchyme development during embryogenesis. The first assay is to investigate dynamics of Zfhx4 binding, chromatin opening, and gene regulation in lung mesenchymal progenitors (Aim 1) and the second assay is to identify Zfhx4-binding transcriptional regulators in lung mesenchymal progenitors (Aim 2). The results of these two proposed assays will identify canidate mediators of Zfhx4 for future functional investigation to inform lung mesenchyme development and pathogenesis of lung hypoplasia in CDH.