Quantitation analysis by flow cytometry shows that Wt1 is required for development of the proepicardium and epicardium

The epicardium is a cell layer found on the external surface of the heart. During de-velopment it has an epithelial identity and contains progenitor cells for coronary smooth muscle and cardiac fibroblasts. The epicardium has been suggested to have therapeutic potential in cardiac repair. Study of epicardial development has been diffi-cult because it is dynamic and morphologically complex. We developed a flow cytom-etry-based method to quantify cardiac development including the epicardial lineage. This provided accurate and sensitive analysis of (1) the emergence of epicardial pro-genitors within the proepicardium (2) their transfer to the heart to form the epicardi-um, and (3) their epithelial-to-mesenchymal transition (EMT) to create the subepicar-dium. Platelet-derived growth factor alpha (Pdgfra) and Wilms tumor protein (Wt1) have both been reported to be pro-mesenchymal during epicardial EMT. Quantitative analysis with flow cytometry confirmed a pro-mesenchymal role for Pdgfra but not for Wt1. Analysis of Wt1 null embryos showed that they had (1) poor formation of proepicardial villi, (2) reduced transfer of proepicardial cells to the heart, (3) a discon-tinuous epicardium with poor epithelial identity, and (4) a proportionally excessive number of mesenchymal-like cells. This data shows that Wt1 is essential for epicardial formation and maintenance rather than being pro-mesenchymal.