Abstract The human heart shows little regenerative capacity following an injury such as myocardial infarction (MI). Instead, the heart scars, decreasing cardiac function, and leading to heart failure. There is no clinically meaningful regenerative therapy available for MI patients. By contrast, adult zebrafish regenerate heart muscle after severe cardiac damage without significant scarring. This is achieved through proliferation of existing cardiomyocytes (CMs), aided by the environment provided by non-muscle cells, such as the epicardium, a mesothelial cell sheet covering the surface of the heart. An analogous regenerative machinery of CM proliferation and epicardium contributions also exists in the adult mammalian heart; however, it is not sufficiently activated for significant regeneration. Recent studies demonstrated that restoring epicardial factors through the application of epicardial patches or co-transplantation of human stem cell-derived epicardial cells together with stem cell- derived CMs after an MI benefit heart regeneration. Thus, enhancing the pro-regenerative activation of the epicardium may benefit mammalian heart regeneration after MI. We and others previously found that the zebrafish epicardium is activated by injury and aids muscle regeneration through paracrine effects and as a source of multipotent cells. However, little is known about the cellular and molecular mechanisms controlling epicardial activation that lead to successful heart regeneration. To this end, understanding how regenerative responses of the epicardium are regulated in adult zebrafish will lead to new therapeutic targets that underlie the regenerative deficiencies in mammals. To address this, using single-cell RNA-sequencing, we have identified a transient adult epicardial progenitor cell (aEPC) subpopulation within the epicardium after heart injury. Transplantation assays implicate a capacity of aEPCs to give rise to perivascular cells, which are critical for coronary revascularization. Genetic ablation of these aEPCs blocks heart regeneration, suggesting an indispensable role. Pharmacological manipulations and transcriptome analyses yielded candidate genes that underlie the activation of aEPCs. Further, unbiased genome-wide profiling of chromatin accessibility using ATAC-seq revealed putative regulatory elements that exert transcriptional regulation of these genes. We hypothesize that activation of a progenitor cell state in the epicardium underlies successful heart regeneration. To test this hypothesis, we propose to 1) define the cell fates and functions of the aEPCs in adult zebrafish heart regeneration using genetic fate mapping, genetic ablation, and single-cell transplantation approaches; and 2) define the molecular mechanisms underlying aEPC activation through genetic manipulations and analyzing dozens of transgenic lines and mutants. The outcome of this proposal may ultimately inform approaches for activating the epicardial progenitors to enhance...