Hedgehog signaling induces cardiomyogenesis in P19 cells

Sonic Hedgehog (Shh) is a critical signaling factor for a variety of developmental pathways during embryogenesis, including the specification of left-right asymmetry in the heart. Mice that lack Hedgehog signaling show a delay in the induction of cardiomyogenesis, as indicated by a delayed expression of Nkx2-5. To further examine a role for Shh in cardiomyogenesis, clonal populations of P19 cells that stably express Shh, termed P19(Shh) cells, were isolated. In monolayer P19(Shh) cultures the Shh pathway was functional as shown by the up-regulation of Ptc1 and Gli1 expression, but no cardiac muscle markers were activated. However, Shh expression induced cardiomyogenesis following cellular aggregation, resulting in the expression of factors expressed in cardiac muscle including GATA-4, MEF2C, and Nkx2-5. Furthermore, aggregated P19 cell lines expressing Gli2 or Meox1 also up-regulated the expression of cardiac muscle factors, leading to cardiomyogenesis. Meox1 up-regulated the expression of Gli1 and Gli2 and, thus, can modify the Shh signaling pathway. Finally, Shh, Gli2, and Meox1 all up-regulated BMP-4 expression, implying that activation of the Hedgehog pathway can regulate bone morphogenetic protein signals. Taken together, we propose a model in which Shh, functioning via Gli1/2, can specify mesodermal cells into the cardiac muscle lineage.     

Cross talk between hedgehog and bone morphogenetic proteins occurs during cardiomyogenesis in P19 cells

Hedgehog (Hh) signaling plays a role in heart morphogenesis and can initiate cardiomyogenesis in P19 cells. To determine if Hh signaling is essential for P19 cell cardiomyogenesis, we determined which Hh factors are expressed and the effect of Hh signal transduction inhibitors. Here, we find that the Hh gene family and their downstream mediators are expressed during cardiomyogenesis but an active Hh signaling pathway is not essential. However, loss of Hh signaling resulted in a delay of BMP-4, GATA-4, Gli2, and Meox1 expression during cardiomyogenesis. By using Noggin-overexpressing P19 cells, we determined that Hh signaling was not active during Noggin-mediated inhibition of cardiomyogenesis. Thus, there is cross talk between the Hh and BMP signaling pathways and the Hh pathway appears important for timely cardiomyogenesis.