Enhancer Activity Defines Unipotent Progenitors for Left Ventricular Cardiomyocytes in Juxta-cardiac Field of Early Mouse Embryo

The cardiac crescent is the first structure of the heart and contains progenitor cells of the first heart field, which primarily differentiate into left ventricular cardiomyocytes. The interface between the forming cardiac crescent and extraembryonic tissue is known as the juxta-cardiac field (JCF), and progenitor cells in this heart field contribute to the myocardium of the left ventricle and atrioventricular canal as well as the epicardium. However, it is unclear whether there are progenitor cells that differentiate specifically into left ventricular cardiomyocytes. We have previously demonstrated that an enhancer of the gene encoding the Hey2 bHLH transcriptional repressor is activated in the ventricular myocardium during mouse embryonic development. In this study, we aimed to investigate the characteristics of cardiomyocyte progenitor cells and their cell lineages by analyzing enhancer activity at the earliest stages of heart formation. We found that the enhancer initiated its activity prior to cardiomyocyte differentiation within the JCF. enhancer-active cells were present rostrally to the -expressing region at the early phase of cardiac crescent formation and differentiated exclusively into left ventricular cardiomyocytes in a lineage distinct from the -positive lineage. By the late phase of cardiac crescent formation, enhancer activity became significantly overlapped with expression in cells that contribute to the left ventricular myocardium. Our study reveals that a population of unipotent progenitor cells for left ventricular cardiomyocytes emerge in the JCF, providing further insight into the mode of cell type diversification during early cardiac development.