Project Summary Human hearts fail to regenerate after injury, instead replacing injured regions of muscular myocardium with rigid scar tissue that impairs cardiac output. Promoting the proliferation of cardiomyocytes (CMs) to replenish injured heart muscle represents a promising strategy for improving the quality of life and preventing premature death of patients recovering from myocardial infarction. Attempts at regenerative therapies have thus far failed to promote meaningful restoration of lost myocardium. A promising source of therapeutic strategies involves the study of vertebrates with the inherent ability to regenerate cardiac tissue, including the zebrafish model organism. Studies comparing the cardiac injury response in non-regenerating mammals to zebrafish are made difficult, however, by vast evolutionary divergence. More evolutionarily relevant comparisons are therefore needed to determine the cellular behaviors that differentiate regenerating and non-regenerating species. This proposal describes experiments aimed at identifying the unique genetic and cellular features that facilitate cardiac regeneration in zebrafish through comparisons with medaka, a non-regenerating teleost fish species. In the first aim of this fellowship proposal, I will generate organ-wide cell atlases of cardiac injury response in zebrafish and medaka over several time points through single-cell RNA sequencing. Analyses of these datasets will reveal how key cell types differ in their gene expression patterns in regenerating and non-regenerating species. In the second aim, I utilize a recently developed single-cell lineage tracing technique to reconstruct the lineage relationships of cells participating in cardiac regeneration in zebrafish. These experiments will provide a comprehensive view of which CMs and immune cells are activated to restore injured tissue. In the final aim, I will test the hypothesis that the cell-autonomous behaviors of responding immune cells facilitate cardiac regeneration in zebrafish. I will assay heart regeneration following reciprocal transplantations of hematopoietic stem cells between zebrafish and medaka, revealing whether immune cell functions are sufficient to impart regenerative potential. Together, these experiments will provide important steps toward identifying the exact cell populations and gene expression patterns that mediate cardiac regeneration and have the potential to aid in the development of targeted therapies that promote cardiac regeneration in humans.