Cloche is a pro-regenerative platelet factor during zebrafish heart regeneration [10x scRNA-seq]

Zebrafish heart regeneration is a complex process consisting of tempo-spatial coordination of cardiomyocyte (CM) and endothelial cell (EC) regeneration, fibrosis and inflammation. While myocardial, endocardial, and epicardial signaling have been reported to modulate this process, little is known about how leukocyte especially platelet signaling is involved in this regenerative process. Here we report that cloche/npas4l (neuronal PAS domain protein 4 like) is a pro-regenerative platelet factor for adult zebrafish heart regeneration. We found that injury triggered npas4l expression as early as 1 h post ventricular amputation, and haploinsufficiency of npas4l disrupted CM and EC proliferation and heart regeneration in clofv087b/+ and clom39/+ mutants after ventricular resection or nitroreductase (NTR)-mediated CM ablation. By constructing a single-cell transcriptomic atlas, we discovered that npas4l was dynamically expressed in platelets in response to heart injury with robust platelet-CM or -EC interactions via ligand-receptor activity analysis. Decreasing platelets in NTR-mediated depletion or mpl mutants impaired CM and EC proliferation, and over-expression of npas4l in platelets sufficiently made uninjured and injured CM reentry into the cell-cycle and rescued CM and EC proliferation in clofv087b/+ mutants. Furthermore, Npas4l positively regulated Bmp6 expression in platelets and either BMP6 inhibitors or siRNAs decreased CM proliferation and heart regeneration. This work demonstrates, for the first time, that injury-induced platelets are essential for zebrafish heart regeneration and Npas4l is a core platelet transcription factor for fine-tuning heart regeneration partially via Bmp6 signaling. Wild type and clofv087b/+ adult zebrafish hearts (injured or uninjured) were dissected and cardiac cells were disassociation at 4℃. Fluorescence-activated cell sorting (FACS) was used to remove cell debris and sort for single cell according to hochest signal intensity. Sorted cell concentration and vaibility were determined on a Countstar Rigel system. 12,000 cells with more than 85% cell vaibility were loaded into 10x Genomics Chromium chip and proceeded to single-cell RNA library construction and sequencing.