Whole or hole? Development of the diaphragm and congenital diaphragmatic hernias

The mammalian diaphragm is the most critical skeletal muscle and defects in the development of the diaphragm give rise to congenital diaphragmatic hernias (CDH), which are common and frequently lethal birth defects. The diaphragm has been proposed to develop from multiple embryonic sources, but how these sources are integrated to form the diaphragm and how defects in diaphragm development induce CDH is poorly understood. Using mouse genetics we demonstrate that the pleuroperitoneal folds (PPF), transient developmental structures, will give rise to connective tissue fibroblasts throughout the diaphragm. Furthermore, we show that the PPFs non-cell autonomously regulate the somitically-derived muscle of the diaphragm and control the morphogenesis of the diaphragm. Deletion of candidate CDH genes, Gata4 and Porcn, specifically within the PPF generates CDH with 100% penetrance, demonstrating that the PPFs are the cellular source of CDH defects. Deletion of Gata4 in the PPF causes early defects in muscle progenitor number and localization, creating amuscularized regions that will form CDHs. Interestingly, PPF-specific deletion of Porcn, which is required for secretion of Wnt ligand, produces late defects in diaphragm development and prevents the diaphragm muscle from spreading completely to the most ventral region of the diaphragm. This demonstrates that Wnt signals from the PPF are critical for the formation of the ventral muscle of the diaphragm. Furthermore, we propose that signals from the PPF may be critical for inducing muscle precursors to migrate from the somites to the diaphragm and acquisition of these signals may have allowed evolution of the muscularized mammalian diaphragm. This work demonstrates the essential roles of the PPF-derived connective tissue fibroblasts in all steps of diaphragm development.