Project Summary Humans, like all mammals, possess limited natural ability to efficiently replace lost myocardium with new contractile tissue. This deficiency contributes to heart failure, the leading cause of morbidity and mortality in the United States. By contrast, teleost fish efficiently regenerate new cardiac tissues after heart injury, representing a good model for studying heart repair and regeneration. We are interested to understand how regenerative responses to injury have been optimized in adult zebrafish, to discover new targets that underlie the regenerative deficiencies in mammals. In previous work, we have investigated the function of the epicardium on zebrafish heart regeneration and found that epicardial depletion decreases CM proliferation, causes defective myocardial regeneration, and reduces coronary revascularization. However, the epicardium itself is a heterogeneous tissue and it is not known which epicardial subpopulations offer benefits to heart regeneration. Studies of subpopulations of epicardial cells have been limited by the lack of genetic tools to specifically label and manipulate distinct epicardial cell types. In preliminary studies, we employed deep sequencing, in situ hybridization and BAC transgenic technology in search for novel genetic markers specific for these elusive cell types. We have identified a new transgenic strain hapln1a:EGFP to specifically mark a subpopulation of epicardial cells. We also identified a novel transgenic strain deltaC:EGFP specifically labeling coronary endothelial cells to help define the role of hapln1a+ cells on coronary revascularization. With a panel of new tools, we found that: 1) hapln1a+ cells surround growing cardiomyocytes and could pioneer coronary growth during development;; 2) hapln1a+ cells rapidly accumulate in the sites of cardiac wound prior to myocardial regeneration and coronary revascularization;; and 3) depleting hapln1a+ cells blocks myocardial and coronary growth. In this proposal, we will address central questions about functions of hapln1a+ cells for the two key cardiac regenerative events: myocardial regeneration and coronary revascularization. Our overall hypothesis is that hapln1a+ cells are a specialized epicardial subpopulation controlling myocardial and coronary growth during development and regeneration. To test this hypothesis, we will: 1) define requirements of hapln1a+ cells for myocardial growth during development and regeneration with new methods of depleting hapln1a+ cells;; 2) define requirements of hapln1a+ cells for coronary vascularization and revascularization;; and 3) define molecular nature of hapln1a+ cells during heart regeneration, by utilizing deep sequencing and in situ screen to ide...