Summary Adult zebrafish have a remarkable capacity to regenerate the heart with minimal scarring. Understanding the underlying cellular and molecular mechanisms will help addressing the regenerative deficiency in the adult mammalian heart. We recently found that the zebrafish epicardium (the outermost layer of vertebrate hearts) regenerates after injury by the creation of a leader region of polyploid cells (having two or more copies of the genome). Polyploidy has been observed in many mammalian organs following injury and recently has been invoked in mechanisms of tissue repair. However, the functional significance of polyploidy, as well as its underlying mechanisms in tissue repair, remains elusive, representing a major knowledge gap in harnessing the advantages of polyploidy in tissue repair. We found that, through collective cell migration, these leader epicardial cells guide a trailing population of much smaller, dividing follower cells to repopulate the wound. The leader cell population is established and maintained by endoreplication and is eliminated through apoptosis upon completion of regeneration, indicating a transient role. The elevated cellular tension in the leader cells drives endoreplication. This coordinated behavior of leader and follower cells facilitates robust regeneration of the epicardium. Also, we found that the polyploid epicardial cells are a major source of paracrine secretion for heart regeneration. The overall objective of our proposal is to understand the mechanisms that regulate spatiotemporal cell behavior of the epicardium and how defects in this behavior impact heart regeneration. Through single-cell RNA sequencing, reporter assays, and pharmacological treatments, we have discovered a novel signaling pathway together with Yap signaling that participate in the spatiotemporal polyploidization in the epicardium. We will 1) characterize the signaling cascade that involves mechanical cues, Yap, and the new pathway in regulating spatiotemporal polyploidization during epicardial regeneration, 2) define the leader signals that drive leader- follower coordination in epicardial regeneration, and 3) investigate the functional significance of epicardial polyploidy in heart regeneration. The proposed research will define a new signaling paradigm in guiding cell cycle decisions for efficient heart regeneration. Moreover, polyploid cells are present in normal tissues such as the mammalian cardiomyocytes, as well as in pathological processes such as lung injury, acute kidney injury, and cancer. Results from our study will unearth conceptual innovations concerning the regulation of cell cycle decisions to mediate physiological and pathological polyploidization and robust tissue regeneration.