The coronary arterial system is essential for cardiac function. We previously identified that a unique extracardiac cell population, the proepicardium (PE), is a major source of coronary vascular progenitors. We have fuither shown that PE-heatI fusion depends on atrioventricular junction (AVJ) myocardium-derived bone morphogenetic protein 2 (BMP2) which promotes directional PE extension toward the heart. However, nothing is known about how BMP2 signaling directs PE extension or how the extended PE fuses to the heail specifically at the AVJ. Furthermore, when and how the fate of PE-derived individual coronary vascular cell types is specified remains unknown. We have recently found that (i) PE cells that are exposed to BMP2 undergo an epithelial cell shape extension and decreased mitotic activity. In contrast, those distant from BMP2 remain mitotically active without cell shape change; (ii) In our chick-quail chimera model both in vivo and in vitro, host PE mesothelial villi successfully attach and open up on the surface of donor quail AVJ implants within 2 hours with high frequency. In contrast, they display no or little attachment or opening to the surface of atrium implants. Our in situ hybridization analysis of candidate factors has already identified that ephrinSA is expressed in the PE and only EphB3 is expressed in the heart, predominantly at the AVJ; and (iii) Live imaging of intracellular Ca2-i- has detected three PE subpopulations that display distinct Ca2+ handling patterns. Further, a subpopulation of PE cells begin to express differentiation markers for either endothelial or smooth muscle cells but not both. These surprising findings lead to the following three new hypotheses which will each be test experimentally in individual but related specific aims. These preliminary data lead to the hypothesis that induction and recruitment of coronary progenitor cells to the heaiT are mediated by three distinct mechanisms: 1) PE extension towards the heart is mediated by localization ofa BMP-dependent cellular extension zone at the distal PE and a growth zone at the proxiiTial PE. 2) Selective PE fusion to the AVJ is defined by the specific cell adhesion properties that different regions ofthe heart exert. 3) Fate specification of individual PE cells, into coronary smooth muscle, endothelial cells, or fibroblasts, takes place within the extending PE prior to fusion with the heart. The studies proposed here will identify the mechanisms that regulate the three earliest steps in coronary arterial development and build a foundation for rational therapeutics of coronary disorders in adults. RELEVANCE (See instructions): The coronary arterial system is essential for cardiac function and survival. Limited options for effective treatments contribute to the continued prevalence of coronary disease. The proposed study will explore the molecular mechanisms that regulate entry of coronary progenitor cells to the heart and fate specification of individual coronary vascular cell types. A clearer understanding of these earliest developmental steps may provide the basis for future therapeutic approaches in this essential cardiac tissue.