leptin b and its regeneration enhancer illustrate the regenerative features of zebrafish hearts

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 non-cardiomyocytes (CMs) in regenerating zebrafish hearts. Our single-cell RNA sequencing (scRNA-seq) analysis demonstrated that the endothelium/endocardium(EC) is activated to induce distinct subpopulations upon cardiac 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 specific subpopulation of epicardium (Epi) and epicardium-derived cells (EPDCs). To determine 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 non-detectable 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. Our integrative analyses identify regeneration-emerging cell types and factors, leading to the discovery of regenerative features of hearts.