Introduction: Recruiting multi-potent embryonic cells to a cardiac myocyte lineage requires extracellular signals by several protein families, including Wnts, TGF-bs, BMPs, and FGFs. Understanding this circuitry eventually may enable us to induce embryonic stem (ES) cells to become cardiac myocytes more selectively for cell therapies. Recent evidence suggests that Sox17, an Sry-box-containing transcription factor that interacts with the Wnt pathway and is essential for gut endoderm development, might be involved in cardiac myogenesis, perhaps even directly: Sox17 is expressed at an elevated level in cardiac crescent, adult heart side population (SP) cells, and differentiating adult heart Sca-1þ cells. Hypotheses: (1) Sox17 expression is dependent on activation of a Wnt pathway, and (2) the induction of Sox17 is important for cardiac myogenesis. Methods: Lentivirus-mediated RNAinterference: High-titer lentiviral particles (O10 IU/ml) encoding shRNAs that knock down Sox17 with O90% efficacy (pLL3.7-sh172 and -sh176) were transduced into AB2.2 cells. Transduced cells, distinguished by an EGFP reporter encoded by the viral vector, were flow-sorted and subjected to embryonic body (EB) culture. Analysis of phenotypes: Total RNA was extracted from EBs at 8 time points. Real-time QRTPCR was carried out for representative genes related to cardiac myogenesis. Immunostaining for sarcomeric a-actin was performed and spontaneously beating EBs were scored. Results: (1) Endogenous Sox17 first appeared at day 4, one day after the peak of Wnt3A and one day before the appearance of Nkx2.5. (2) The Wnt antagonist Frizzled-8/Fc markedly suppressed Sox17 induction. (3) Both Sox17 shRNAs blocked Sox17 induction (left, shown for one shRNA vs. control). (4) Both Sox17 shRNAs successfully suppressed the known Sox17 targets Foxa1 and Foxa2, as well as cardiac transcription factors Nkx2.5 (center), Tbx5, Mef2c and Myocardin. (5) Both Sox17 shRNAs suppressed the expression of cardiac structural genes a-MHC (right) and Ryr2, sarcomeric a-actin staining, and the number of beating EBs. Conclusion: Sox17 induction is regulated by a Wnt pathway, and plays an essential role in cardiac myogenesis by mouse ES cells.