Activin/SMAD signaling in human embryonic stem cells (hESCs) ensures expression and stem cell pluripotency. In the presence of Wnt ligand, the Activin/SMAD transcription network switches to cooperate with Wnt/β-catenin and induce mesendodermal (ME) differentiation genes. We show here that the Hippo effector YAP binds to the gene enhancer and prevents the gene from being induced by Activin in proliferating hESCs. ChIP-seq (chromatin immunoprecipitation [ChIP] combined with high-throughput sequencing) data show that YAP impairs SMAD recruitment and the accumulation of P-TEFb-associated RNA polymerase II (RNAPII) C-terminal domain (CTD)-Ser7 phosphorylation at the gene. CRISPR/ knockout of YAP in hESCs enables Activin to induce Wnt3 expression and stabilize β-catenin, which then synergizes with Activin-induced SMADs to activate a subset of ME genes that is required to form cardiac mesoderm. Interestingly, exposure of YAP hESCs to Activin induces cardiac mesoderm markers ( and ) without activating Wnt-dependent cardiac inhibitor genes ( and ). Moreover, canonical Wnt target genes are up-regulated only modestly, if at all, under these conditions. Consequently, YAP-null hESCs exposed to Activin differentiate precisely into beating cardiomyocytes without further treatment. We conclude that YAP maintains hESC pluripotency by preventing expression in response to Activin, thereby blocking a direct route to embryonic cardiac mesoderm formation.

Pioneer transcription factors initiate cell-fate changes by binding to silent target genes. They are among the first factors to bind key regulatory sites and facilitate chromatin opening. Here, we identify an additional role for pioneer factors. In early Caenorhabditis elegans foregut development, the pioneer factor PHA-4/FoxA binds promoters and recruits RNA polymerase II (Pol II), often in a poised configuration in which Pol II accumulates near transcription start sites. At a later developmental stage, PHA-4 promotes chromatin opening. We found many more genes with poised RNA polymerase than had been observed previously in unstaged embryos, revealing that early embryos accumulate poised Pol II and that poising is dynamic. Our results suggest that Pol II recruitment, in addition to chromatin opening, is an important feature of PHA-4 pioneer factor activity.

SummarySpecifying the primitive streak (PS) guides stem cell differentiation in vitro; however, much remains to be learned about the transcription networks that direct anterior and posterior PS cells (APS and PPS, respectively) to differentiate to distinct mesendodermal subpopulations. Here, we show that APS genes are predominantly induced in YAP1−/− human embryonic stem cells (hESCs) in response to ACTIVIN. This finding establishes the Hippo effector YAP1 as a master regulator of PS specification, functioning to repress ACTIVIN-regulated APS genes in hESCs. Moreover, transient exposure of wild-type hESCs to dasatinib, a potent C-SRC/YAP1 inhibitor, enables differentiation to APS-derived endoderm and cardiac mesoderm in response to ACTIVIN. Importantly, these cells can differentiate efficiently to normal beating cardiomyocytes without the cytoskeletal defect seen in YAP1−/− hESC-derived cardiomyocytes. Overall, we uncovered an induction mechanism to generate APS cells using a cocktail of ACTIVIN and YAP1i molecules that holds practical implications for hESC and induced pluripotent stem cell differentiation into distinct mesendodermal lineages.

The gastrulation process relies on complex interactions between developmental signaling pathways that are not completely understood. Here, we interrogated the contribution of the Hippo signaling effector YAP1 to the formation of the three germ layers by analyzing human embryonic stem cell (hESC)-derived 2D-micropatterned gastruloids. YAP1 knockout gastruloids display a reduced ectoderm layer and enlarged mesoderm and endoderm layers compared with wild type. Furthermore, our epigenome and transcriptome analysis revealed that YAP1 attenuates Nodal signaling by directly repressing the chromatin accessibility and transcription of key genes in the Nodal pathway, including the NODAL and FOXH1 genes. Hence, in the absence of YAP1, hyperactive Nodal signaling retains SMAD2/3 in the nuclei, impeding ectoderm differentiation of hESCs. Thus, our work revealed that YAP1 is a master regulator of Nodal signaling, essential for instructing germ layer fate patterning in human gastruloids.

During gastrulation, the coordinated activity of BMP, WNT and NODAL signaling pathways guide the differentiation of the pluripotent epiblast into the three germinal layers. Recent studies underline the role of the Hippo-effector YAP1 regulating WNT and NODAL signaling pathways and repressing mesoendodermal differentiation in human embryonic stem cells (hESCs). However, the contribution of YAP to the cell-fate patterning decisions that transform the epiblast in a three-germ layer gastrula remains unknown. We address this question by analyzing micropatterned 2D-gastruloids derived from hESCs, in the presence and absence of YAP1. Our findings show that YAP1 is necessary for gastrulation. YAP KO-gastruloids display reduced ectoderm layer and enlarged mesoderm and endoderm layers, compared to WT. Furthermore, YAP1 regulates the self-organized patterning of the hESCs, as the discrete position of the three germ layers is altered in the YAP1 KO-gastruloids. Our epigenome (single-nuclei ATACseq) and transcriptome (RNA-seq) analysis revealed that YAP1 directly represses the chromatin accessibility and transcription of key genes in the NODAL pathway, including the NODAL and FOXH1 genes. In WT gastruloids, a gradient of NODAL: SMAD2.3 signaling from the periphery to the center of the colony regulates the exit of pluripotency toward endoderm, mesoderm and ectoderm, respectively. Hence, in the absence of YAP1, a hyperactive NODAL signaling retains SMAD2.3 in the nuclei impeding the self-organized differentiation of hESCs. Accordingly, the partial inhibition of NODAL signaling is sufficient to rescue the differentiation and pattern -defective phenotypes of the YAP1 KO gastruloids. Our work revealed that YAP1 is a master regulator of NODAL signaling, essential to instruct germ layer fate patterning in human gastruloids.