Coronary endothelial cells receive Notch1 signal during heart development

The Notch1 signaling pathway, via the Notch1 receptor and its endogenous ligands, has been suggested to play a role in heart development, as mutations in Notch1 and associated genes are associated with abnormal development of the myocardium, aortic valve, and outflow tract. The cell lineages receiving this signaling are, however, still unclear, partly owing to technical difficulties in labeling Notch1 signaling rather than Notch1 receptor expression. To identify the cell lineages that receive Notch1 signal during heart development, we re-analyzed publicly available single-cell RNA-sequencing data from human embryos (post-conception weeks 6, 8, 12, and 19) and mouse embryos (embryonic days 12.5, 14.5, and 16.5). We found that endothelial cells expressed Notch1 in both species. To confirm Notch1 signaling rather than Notch1 receptor expression in heart development, we carried out transgenic mouse experiments that enabled tracing of both past and ongoing Notch1 signaling by combining the Notch1 receptor protein with the Gal4/UAS and Cre/loxP systems. Past Notch1 signal analysis confirmed that coronary endothelial cells, in addition to a small proportion of endocardial cells, received Notch1 signals during development, consistent with our findings in single-cell RNA-seq re-analysis. Ongoing Notch1 signaling was observed in the surface-covering cells of the vascular lumens, although its colocalization with an endothelial marker (Pecam1/Cd31) was unclear. Collectively, our findings suggested that coronary endothelial cells are Notch1 signal receivers, which should contribute to understanding the pathogenesis of congenital heart diseases.