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A bstract   Cardiac muscle differentiation in vivo is guided by sequential growth factor signals, including endoderm‐derived diffusible factors, impinging on cardiogenic genes in the developing mesoderm. Previously, by RNA interference in AB2.2 mouse embryonic stem cells (mESCs), we identified the endodermal transcription factor  Sox17  as essential for Mesp1 induction in primitive mesoderm and subsequent cardiac muscle differentiation. However, downstream effectors of  Sox17  remained to be proven functionally. In this study, we used genome‐wide profiling of  Sox17 ‐dependent genes in AB2.2 cells, RNA interference, chromatin immunoprecipitation, and luciferase reporter genes to dissect this pathway.  Sox17  was required not only for  Hhex  (a second endodermal transcription factor) but also for  Cer1 , a growth factor inhibitor from endoderm that, like  Hhex , controls mesoderm patterning in  Xenopus  toward a cardiac fate. Suppressing  Hhex  or  Cer1  blocked cardiac myogenesis, although at a later stage than induction of  Mesp1/2 .  Hhex  was required but not sufficient for  Cer1  expression. Over‐expression of  Sox17  induced endogenous  Cer1  and sequence‐specific transcription of a  Cer1  reporter gene. Forced expression of Cer1 was sufficient to rescue cardiac differentiation in  Hhex ‐deficient cells. Thus,  Hhex  and  Cer1  are indispensable components of the  Sox17  pathway for cardiopoiesis in mESCs, acting at a stage downstream from  Mesp1/2 . S tem  C ells   2014;32:1515–1526

Hhex and Cer1 Mediate the Sox17 Pathway for Cardiac Mesoderm Formation in Embryonic Stem Cells