Myocardial infarction (MI) is a common injury that causes permanent loss of hundreds of millions of cardiac muscle cells, increasing susceptibility to heart failure and sudden death. Major goals of regenerative medicine are methodologies to enhance cardiomyocyte recovery after MI and to restore cardiac function to heart failure patients. Heart regeneration is limited in adult mammals, but occurs naturally in adult zebrafish and neonatal mice through the activation of cardiomyocyte division. Whereas the research community has identified several factors important for heart regeneration over the past decade, we still know little of the regulatory mechanisms needed to activate regeneration programs in injured cardiac tissue. In particular, questions of whether regulatory enhancer elements are employed, and how specific they are to regeneration, are virtually unexplored. This is a critical deficiency, as the identification and manipulation of such elements could both expand our understanding of regeneration and have applications in regenerative medicine. In a recent collaboration between our groups, we found evidence for tissue regeneration enhancer elements (TREEs) that trigger gene expression in injury sites and can be engineered to modulate the regenerative potential of vertebrate organs including the heart. Here, we propose a multi-PI project exploiting the strengths of the zebrafish and mouse model systems to delineate regulatory sequences that control regeneration programs, and to create TREE-based factor delivery constructs to optimize heart regeneration in higher and lower vertebrates. 1) We will define the cis-regulatory motifs and binding factors necessary for activity of a TREE that is linked to the zebrafish leptin b gene. 2) We will use this TREE to define effects of enhancer-delivered pro-regenerative factors after cardiac injury in zebrafish and mice, focusing initially on provision of mitogenic and angiogenic molecules. 3) We will use open chromatin profiling approaches to identify new TREEs that activate expression in endothelial and/or endocardial cell types during zebrafish heart regeneration. We will perform sequence comparisons in mice, and we will initiate a program to generate transgenic reporter and TREE deletion animals to test the sufficiency and requirements for these sequences in directing regeneration programs. With these approaches, we will test the hypothesis that cardiac injury activates regeneration enhancer elements to facilitate heart regeneration.