Abstract 311: Genome-wide Dissection of Wnt- and BMP-dependent Pathways: Identification of Sox17 as Essential for Cardiac Myocyte Formation by Embryonic Stem Cells
Early steps for cardiac specification are problematic to study in mammalian embryos, which has favored using pluripotent cells that recapitulate cardiac myogenesis. Furthermore, circuits governing cardiac specification have relevance to the use of embryonic stem (ES) cells and other cells for cardiac repair. In pluripotent mouse teratocarcinoma cells, canonical Wnts that are inhibitors of heart formation in avian or amphibian explants activate cardiogenesis, paradoxically. Here, we address the following inter-related questions: Do canonical Wnts activate cardiogenesis in mouse ES cells, a more natural and translationally applicable model? What molecular targets in differentiating mouse ES cells are dependent on canonical Wnts and BMPs? And, which, if any, of such targets is essential for cardiogenesis? We have used embryoid body culture as an ES cell differentiation model and complementary Wnt antagonists, soluble Frizzled protein and Dickkopf-1 (Dkk-1), to evaluate the requirement of Wnts in cardiogenesis. Genome-wide expression profiling was used to dissect Wnt- and BMP-dependent pathways, and small hairpin RNA (shRNA)-mediated RNA interference was used to determine the function of potential candidates. We determined that inhibiting Wnt signaling with soluble Frizzled protein or Dkk-1 blocked the induction of cardiogenic transcription factors, cardiac structural genes, and spontaneous beating in mouse ES cells. Early targets shared by the Wnt and BMP pathways were associated with gastrulation, mesoderm formation, and mesoderm patterning. Sox17, the endodermal HMG-box transcription factor, was also contingent on both pathways. Two independent lentiviral Sox17 shRNA vectors specifically suppressed endogenous Sox17, as well as the associated induction of cardiogenic transcription factors, cardiac structural genes and spontaneous beating. In conclusion, the Wnt/β-catenin pathway is essential for cardiac myogenesis in ES cells, acting at a gastrulation-like stage. Sox17 is essential for cardiomyocyte formation from undifferentiated mouse ES cells.