Recent studies indicate that mammals, including humans, maintain some capacity to renew cardiomyocytes throughout postnatal life1 ,2 . Yet, there is little or no significant cardiac muscle regeneration after an injury like acute myocardial infarction (MI) 3 . By contrast, zebrafish efficiently regenerate lost cardiac muscle, providing a model for understanding how natural heart regeneration may be blocked or enhanced4 , 5. In the absence of lineage-tracing technology applicable to adult zebrafish, the cellular origins of newly regenerated cardiac muscle have remained unclear. Here, we used new genetic fate-mapping approaches to identify a population of cardiomyocytes that become activated after resection of the ventricular apex and contribute prominently to cardiac muscle regeneration. Through use of a transgenic reporter strain, we found that cardiomyocytes throughout the subepicardial ventricular layer trigger expression of the embryonic cardiogenesis gene gata4 within a week of trauma, before expression localizes to proliferating cardiomyocytes surrounding and within the injury site. Cre recombinase-based lineage-tracing of cells expressing gata4 before evident regeneration, or of cells expressing the contractile gene cmlc2 before injury, each labeled a majority of cardiac muscle in the ensuing regenerate. By optical voltage mapping of surface myocardium in whole ventricles, we found that electrical conduction is re-established between existing and regenerated cardiomyocytes between 2 and 4 weeks post-injury. After injury and prolonged Fgf receptor inhibition to arrest cardiac

Zebrafish possess a unique yet poorly understood capacity for cardiac regeneration. Here, we show that regeneration proceeds through two coordinated stages following resection of the ventricular apex. First a blastema is formed, comprised of progenitor cells that express precardiac markers, undergo differentiation, and proliferate. Second, epicardial tissue surrounding both cardiac chambers induces developmental markers and rapidly expands, creating a new epithelial cover for the exposed myocardium. A subpopulation of these epicardial cells undergoes epithelial-to-mesenchymal transition (EMT), invades the wound, and provides new vasculature to regenerating muscle. During regeneration, the ligand fgf17b is induced in myocardium, while receptors fgfr2 and fgfr4 are induced in adjacent epicardial-derived cells. When fibroblast growth factors (Fgf) signaling is experimentally blocked by expression of a dominant-negative Fgf receptor, epicardial EMT and coronary neovascularization fail, prematurely arresting regeneration. Our findings reveal injury responses by myocardial and epicardial tissues that collaborate in an Fgf-dependent manner to achieve cardiac regeneration.