Abstract 857: Notch Regulates Cardiac Progenitor Cell Commitment and Myocyte Formation in the Embryonic Mouse Heart
The identification of c-kit-positive cardiac progenitor cells (c-kit-CPCs) in the adult heart raises the question whether similar cells are present in the embryonic-fetal myocardium and whether this primitive cell pool is responsible for cardiomyogenesis. We report that cells expressing c-kit are located within the primitive heart and their number progressively increases with gestational age. The colocalization of c-kit with transcription factors and contractile proteins specific of myocytes pointed to a lineage relationship between c-kit-CPCs and myocytes. To document unequivocally this possibility, we defined the molecular mechanisms regulating the myocyte commitment of embryonic c-kit-CPCs. The Notch pathway is a major determinant of stem cell fate but whether Notch exerts an inhibitory or an inductive role in the growth and differentiation of c-kit-CPCs in embryonic life remains to be established. A perfect consensus site for the target gene of Notch, RBP-Jk, was recognized on the promoter of Nkx2.5 indicating that Nkx2.5 may be a novel target gene of Notch-1. The formation of a complex between the Nkx2.5 promoter and RBP-Jk was determined by electrophoretic mobility assay. The DNA-protein complex was super-shifted by incubation with specific RBP-Jk antibodies. This result was strengthened by chromatin immunoprecipitation that documented that the endogenous Nkx2.5 promoter was occupied by the RBP-Jk protein. The active form of Notch, i.e., Notch intracellular domain (NICD), consistently co-localized with Nkx2.5 in nuclei of forming myocytes in the ventricular trabeculae and compact myocardium at E9.5 and throughout the ventricular myocardium at E19.5. Developing myocytes were surrounded by the Notch ligand Jagged 1. Most NICD-Nkx2.5-positive cells were negative for c-kit. However, a small proportion of these cells expressed c-kit providing evidence in favor of a lineage relationship between c-kit-CPCs and cardiomyogenesis. In conclusion, resident c-kit-CPCs are critically involved in the growth of the embryonic and fetal heart and the activation of the Notch pathway is involved in cardiomyogenesis through the upregulation of Nkx2.5.