Distinct populations of vascular smooth muscle and sinoatrial progenitors are localized adjacent to pro-epicardium

The developmental potential of cells adjacent to pro-epicardial (PE) cells (pro-EpiCs) that also express pro-EpiC markers is unknown. We apply single-cell mRNA deep sequencing paired with RNAScope validation, bioinformatic analysis, and lineage tracing to determine the heterogeneity and specification of cells in the PE region. We find that the PE region contains three adjacent cell clusters that have distinct gene expression profiles and distinct spatial locations. Cluster 1 is pro-EpiCs, cluster 2 is vascular smooth muscle cell (VSMC)-like cells, and cluster 3 is pacemaker progenitor cells (PMPCs). Generating a PE-region-specific Myrf DreERT2/+ -knockin line and using Cre-Lox and/or Dre-Rox reporters, we determine that VSMC-like cells localized posterior to the PE region give rise to VSMCs, and PMPCs localized to the PE region proximal to the atrial wall give rise to sinoatrial node cells. Thus, cells in the PE region adjacent to pro-EpiCs are specified to give rise to coronary VSMCs or pacemaker cells.