Functional heterogeneity in the developing zebrafish epicardium

The epicardium is a sheet of cells enveloping the heart muscle and is essential during cardiac development. Yet fundamental insights into epicardium formation, lineage heterogeneity and functional cross-talk with other cell types in the heart are currently lacking. Here, we investigated epicardial heterogeneity and the functional diversity of discrete epicardial subpopulations in the developing zebrafish heart. Smart-seq2 based single cell RNA-sequencing uncovered three epicardial subpopulations (Epi1-3) with specific genetic programmes and distinctive spatial distribution in the developing heart. Functional perturbation identified tgm2b, a transglutaminase gene highly enriched in Epi1, as necessary for the proper development of the epicardial cell sheet. Epi2 was spatially localised in the cardiac outflow tract and expressed the chemokine sema3fb. Loss of sema3fb increased the number of tbx18+ cells in the outflow tract, suggesting it controls the spatiotemporal access of epicardial cells to this tissue. Epi3 was enriched for cell guidance cues such as cxcl12a, loss of which decreased the number of ptprc/CD45+ leukocytes on the epicardial surface. Understanding which mechanisms cells employ to establish a functional epicardium and to communicate with other cardiovascular cell types during development will bring us closer to repairing cellular relationships that are disrupted during cardiovascular disease.