Junsu Kang

Background Zebrafish possess a remarkable regenerative capacity, which is mediated by the induction of various genes upon injury. Injury‐dependent transcription is governed by the tissue regeneration enhancer elements (TREEs). Here, we utilized leptin b ( lepb ), an injury‐specific factor, and its TREE to dissect heterogeneity of noncardiomyocytes (CMs) in regenerating hearts. Results Our single‐cell RNA sequencing (scRNA‐seq) analysis demonstrated that the endothelium/endocardium(EC) is activated to induce distinct subpopulations upon injury. We demonstrated that lepb can be utilized as a regeneration‐specific marker to subset injury‐activated ECs. lepb + ECs robustly induce pro‐regenerative factors, implicating lepb + ECs as a signaling center to interact with other cardiac cells. Our scRNA‐seq analysis identified that lepb is also produced by subpopulation of epicardium (Epi) and epicardium‐derived cells (EPDCs). To determine whether lepb labels injury‐emerging non‐CM cells, we tested the activity of lepb ‐linked regeneration enhancer ( LEN ) with chromatin accessibility profiles and transgenic lines. While nondetectable in uninjured hearts, LEN directs EC and Epi/EPDC expression upon injury. The endogenous LEN activity was assessed using LEN deletion lines, demonstrating that LEN deletion abolished injury‐dependent expression of lepb , but not other nearby genes. Conclusions Our integrative analyses identify regeneration‐emerging cell‐types and factors, leading to the discovery of regenerative features of hearts.