The role of epicardial GATA factors in coronary vascular development

The early embryonic heart forms as an avascular structure that requires a vascular network to complete development. The formation of the coronary vasculature is dependent on the epicardium, a thin layer of cells covering the developing heart. The epicardium provides progenitor cells for the coronary vasculature and signals to direct the assembly of the early coronary vascular plexus. While this system was initially described almost 20 years ago, the extent to which the epicardium provides progenitor cells and mechanisms by which it directs coronary vascular formation are still not fully understood. The goal of the work in this dissertation was to advance the understanding of the epicardium's role in coronary vascular development by focusing on the contribution of epicardial derived cells to the coronary vasculature and the transcriptional control of epicardial function in coronary vascular formation.;To investigate the contribution of epicardial derived cells to the coronary vasculature, the ability to label epicardial cells and follow them through early coronary vascular development was necessary. To accomplish this I utilized a Cre-loxP system to direct expression of fluorescent reporter proteins specifically in the epicardium and epicardial derived cells. To target the epicardium, I used a previously unpublished WT1-Cre mouse line. Characterization of WT1-Cre expression revealed it was expressed in the epicardium and epicardial derived cells. Within the cells of the coronary vasculature, the WT1-Cre was expressed primarily in smooth muscle cells, consistent with previous fate mapping studies of cells from the epicardium. A small percentage of coronary endothelial cells also expressed the WT1-Cre reporter suggesting the epicardium may contribute to a small extent to the coronary endothelium.;GATA transcription factors have been shown to be required for the formation of the epicardium; however, their role within the epicardium is unclear. To investigate whether epicardial GATA factors may be playing a role in epicardial mediated coronary vascular development, I utilized the WT1-Cre to ablate expression of the floxed GATA4 and GATA6 in the epicardium. The WT1-Cre driven loss of GATA4 and GATA6 was embryonic lethal at approximately E15.5, a time of developmental arrest associated with cardiac and coronary vascular defects. Visualization of the coronary plexus in double knockout hearts revealed a disruption in its formation. The loss of endothelial plexus formation was determined to be the result of a decrease in the appearance of subepicardial endothelial cells. Based on these results, I have proposed a model in which loss of GATA4/GATA6 impairs the earliest steps of coronary vascular development due to a defect in epicardial signaling. Sonic hedgehog is an epicardial-signaling factor previously shown to be required for the formation of the coronary plexus. By immunofluorescence I observed a decrease in Sonic hedgehog expression in the epicardium upon loss of epicardial GATA4 and GATA6, correlating with a loss of subepicardial endothelial cells. Supplementation with Sonic hedgehog in a whole heart culture with GATA4/GATA6 null epicardium rescued the loss of these sub-epicardial endothelial cells. Together these findings support a model in which epicardial GATA factors regulate coronary plexus formation via Sonic hedgehog signaling pathway.;These results provide new insight concerning the role of the epicardium in coronary vascular formation. They support the current model of limited epicardial contribution to endothelium of the coronary vasculature. Additionally, I determined epicardial GATA factors are crucial not only for the formation of the coronary vasculature, but also the function of the epicardium in coronary vascular development. Future research should further investigate the mechanism by which GATA factors regulate epicardial signaling particularly the relationship with Sonic hedgehog. Increased understanding of this developmental process could direct the investigation of new therapies to promote coronary vascular regeneration.