Hippo-YAP signaling pathway functions in early lineage differentiation of pluripotent stem cells, but the detailed mechanisms remain elusive. We found that knockout (KO) of Mst1 and Mst2, two key components of the Hippo signaling in mouse embryonic stem cells (ESCs), resulted in a disruption of differentiation into mesendoderm lineage. To further uncover the underlying regulatory mechanisms, we performed a series of ChIP-seq experiments with antibodies against YAP, ESC master transcription factors and some characterized histone modification markers as well as RNA-seq assays using wild type and Mst KO samples at ES and day 4 embryoid body stage respectively. We demonstrate that YAP is preferentially co-localized with super-enhancer (SE) markers such as Nanog, Sox2, Oct4 and H3K27ac in ESCs. The hyper-activation of nuclear YAP in Mst KO ESCs facilitates the binding of Nanog, Sox2 and Oct4 as well as H3K27ac modification at the loci where YAP binds. Moreover, Mst depletion results in novel SE formation and enhanced liquid-liquid phase-separated Med1 condensates on lineage associated genes, leading to the upregulation of these genes and the distortion of ESC differentiation. Our study reveals a novel mechanism on how Hippo-YAP signaling pathway dictates ESC lineage differentiation.

Wnt signaling, a highly conserved signaling pathway, plays important roles in endochondral ossification which is a key process for skeletal development and bone repair. Wnt16, as one of the nineteen Wnt ligands, is reported to repress osteoclastogenesis, prevent cortical bone fragility fractures and to be upregulated in osteoarthritis. But how Wnt16 mediates chondrocyte differentiation during endochondral ossification is still unclear. Here, we investigate the roles of Wnt16 specifically in chondrocytes during endochondral ossification. First, we generated Col2a1‐Wnt16 transgenic mice in which Wnt16 was overexpressed in chondrocytes under the control of Col2a1 promoter and enhancer. The transgenic mice showed a great reduction of tissue mineralization during embryonic development. We also genetically knocked out Wnt16 by generating Wnt16Loxp/Loxp;Col2a1‐Cre mutant mice to examine whether Wnt16 is required for skeletal development. The mutant mice showed no severe phenotype in early skeletal development. However, after 2‐month‐old, the mutant mice displayed a smaller body size and lower bone mass as compared to that of control littermates. In vitro, our studies showed that Wnt16 delays chondrocyte hypertrophy and subsequent maturation. Mechanistically, we found that Wnt16 mainly activates the planar cell polarity (PCP) pathway through activation of JNK in primary chondrocyte. After treated chondroprogenitor cell line ATDC5 with SP600125, a JNK specific inhibitor, Wnt16‐induced delay of chondrocyte hypertrophy is eliminated. In addition, our data suggest that Wnt16 mainly interacts with Ror2 or CD146, co‐receptors of PCP pathway, but not Vangl2 or Ryk. Collectively, our current study provides evidence that Wnt16 delays chondrocyte hypertrophy through PCP pathway partially by binding to Ror2 and CD146. Our findings deepen the understanding of chondrocyte differentiation during endochondral ossification.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.