SRA

In hESCs, Wnt3/ß-catenin activity is low and Activin/SMAD signaling ensures NANOG expression to sustain pluripotency. In response to exogenous Wnt3 effectors, Activin/SMADs switch to cooperate with ß-catenin and induce mesendodermal differentiation genes. We show here that the HIPPO effector YAP binds to the WNT3 gene enhancer and prevents the gene from being induced by Activin in proliferating hESCs. In the absence of YAP, Activin signaling is sufficient to induce expression of the endogenous Wnt3 cytokine, which stabilizes ß-catenin and selectively activates genes required for cardiac mesoderm (ME) formation. Interestingly, Activin-stimulated YAP-knockout hESCs strongly express ß-catenin-dependent cardiac mesoderm markers (BAF60c and HAND1), but unlike WT hESCs, fail to express cardiac inhibitor genes (CDX2, MSX1). Accordingly, YAP-/- cells treated with Activin alone can differentiate efficiently to beating cardiomyocytes in culture, bypassing the need for sequential treatment with exogenous Wnt ligand and Wnt inhibitors. Similarly, Activin in combination with small-molecule YAP inhibitors generates beating cardiomyocytes from wild-type hESCs following a one- step protocol. Our findings highlight an unanticipated role of YAP as an upstream regulator of WNT3 to maintain hESC pluripotency in the presence of Activin, and uncover a direct route for the development of human embryonic cardiac mesoderm. Overall design: We performed a set of sequencing experiments to identify the role of YAP in hESCs and whether YAP influences the activity of the Activin and Wnt signaling pathways during the mesoderm differentiation. We performed transcriptional profile of WT and YAP-KO cells under different Wnt or Activin treatments and performed ChIP-seq analysis of the Wnt and Activin effectors beta-catenin and Smad2,3. In parallel we also mapped the binding of the YAP protein together with TEAD4 DNA-binding protein in hESCs. As a read-out of transcriptional activity under the different treatments we performed ChIP-seq of the active elongation mark CTD-Ser7P RNAPII.