AbstractThe epicardium is essential during cardiac development, homeostasis and repair and yet fundamental insights into its underlying cell biology, notably epicardium formation, lineage heterogeneity and functional cross-talk with other cell types in the heart, are currently lacking. In this study, we investigated epicardial heterogeneity and the functional diversity of discrete epicardial subpopulations in the developing zebrafish heart. Single-cell RNA-sequencing uncovered three epicardial subpopulations with specific genetic programmes and distinctive spatial distribution within the developing heart. Perturbation of unique gene signatures uncovered distinct functions associated with each subpopulation and established novel epicardial roles in cell adhesion, migration, and chemotaxis as a mechanism for recruitment of leukocytes into the heart. This work elucidates the mutual spatiotemporal relationships between different epicardial subpopulations and assigns unique function to each during cardiac development. 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.