The diaphragm is an essential mammalian skeletal muscle, as it is vital for respiration and serves as a barrier between the thoracic and abdominal cavities. Defects in diaphragm development are the cause of congenital diaphragmatic hernias (CDHs), a common birth defect (1:3000 births) that results in severe morbidity and 50% mortality. Given the diaphragm's functional importance and the frequency and severity of CDH, an understanding of diaphragm development normally and during herniation is critical. Using mouse genetics, we definitively established that the pleuroperitoneal folds, transient embryonic structures, and the muscle connective tissue fibroblasts derived from them critically regulate development of the diaphragm muscle (Merrell et al. 2015). Furthermore, we showed that mutations in Gata4 in these fibroblasts cause CDH. Although conditional Gata4 mouse mutants have been critical for dissecting the role of PPFs and Gata4 in CDH, they are not representative of the genetic and phenotypic characteristics of CDH. We have identified and created new hypomorphic alleles of Gata4 that faithfully recapitulate both the genotypic and phenotypic characteristics of human CDH. Based on preliminary data with these new Gata4 alleles, we will test the hypothesis that Gata4 regulates in a dose-dependent manner development of development of the diaphragm and CDH. Genetic and environmental disruptions in retinoic acid (RA) signaling have also been proposed to be an important source of CDH. Using a series of mouse genetic alleles and protocols for modulating maternal vitamin A, we will explicitly test a role for RA and as well as RA and Gata4 interactions in diaphragm development and CDH. Our research will provide insights into the multifaceted genetic, molecular, cellular, and environmental mechanisms underlying the etiology of CDH and its phenotypic and clinical variability.