Murine cardiac progenitor cells require visceral embryonic endoderm and primitive streak for terminal differentiation

Cardiac progenitor cells in avian and amphibian embryos are known to commit to cardiac lineage during gastrulation or early neurulation. These cells require cell interaction with anterior endoderm for their differentiation into cardiomyocytes. However, little is known about cell interaction in mammalian cardiogenesis. We investigated the staging of murine cardiomyocyte commitment and the role of cell interaction in differentiation of cardiac progenitor cells into cardiomyocytes, using cultures of various embryonic regions at 7.25 and 7.5 days post coitum (p.c.), respectively. To evaluate the terminal differentiation of cardiac progenitor cells, we employed three parameters; expression of spontaneous beating, myosin heavy chain (MHC) protein, and cardiac-specific genes (alpha myosin heavy chain, Csx/Nkx2.5 and myosin light chain 2V genes). mRNAs of cardiac-specific genes were detected in 7.25-day p.c. mesoderm by RT-PCR, suggesting that the genetic specification to cardiac lineage initiated in the mesoderm by 7.25 days p.c. The 7.25-day p.c. isolated mesoderm in 48 hr culture, however, failed to differentiate into spontaneous beating cardiomyocytes and exhibited non-organized MHC protein in 19% of these culture. In contrast, all of the 7.5-day p.c. isolated mesoderm differentiated into beating cardiomyocytes even in 24 hr culture. The 7.25-day p.c. mesoderm associated with primitive streak increased MHC protein expression in 93% of these cultures, although they formed beating foci in 3%. The 7.25-day p.c. explants containing both visceral embryonic endoderm and primitive streak succeeded in terminal differentiation into spontaneous beating cardiomyocytes. Our study suggests that cardiac progenitor cells obtain the potency to complete terminal differentiation autonomously at 7.5 days p.c., as a consequence of the multistep induction by cell interactions with both the primitive streak and visceral embryonic endoderm, following the genetic specification to cardiac lineage in the early gastrula stage.