Cloche is a pro-regenerative platelet factor during zebrafish heart regeneration [CUT&Tag]

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. The ventricles (30 adult ventricles per sample) were gently chopped, transferred, and digested in digestion buffer for 15 min at 4℃ with constant gentle agitation using a rotator. The digestion was then neutralized with 10% FBS (fetal bovine serum) and the disassociated cells were filtered through a 100 μm-strainer to remove large clumps and pelleted at 500 × g for 10 min at 4℃. The pellet which mainly contained platelets and erythrocytes was re-suspended in Hank’s balance salt solution (HBSS). Then, around 2000,000 cells per sample were fixed in 0.2% formaldehyde for 2 min and neutralized by adding glycine to a final concentration 25 mM. The fixed cells were pelleted at 800 × g for 5 min at 4℃, washed twice with PBS and loaded for CUT&TAG reaction using Hyperactive Universal CUT&TAG Assay Kit for Illumina Pro (Vazyme, TD904) and Mouse anti-HA Tag antibody (Santa Cruz, sc-7392) according to the manufacturer’s instructions. The concentrations of library were measured by a Qubit dsDNA high sensitivity assay. The sequencing was performed on a BGI DNBSEQ-T7 PE150 platform operated by Peking GenePlus Co.