ABSTRACT: The trophectoderm (TE), the outer layer of the blastocyst, is the first lineage specified during mammalian development. It initiates attachment and implantation into the maternal endometrium and gives rise to trophoblasts, the epithelial cells of the placenta that support development in utero. Animal models have identified key signaling pathways, including BMP, WNT, and HIPPO, that regulate extra-embryonic ectoderm development, but species-specific differences in regulatory networks underlying TE specification and placental development emphasize the need for human-specific models. We were the first to previously identify Vestigial-like Family Member 1 (VGLL1), a coactivator of the TEAD family of transcription factors, as a human-specific placental marker highly expressed in first trimester trophoblast progenitors. The HIPPO signaling pathway effector TEAD4 in complex with YAP1 regulates initial TE specification by acting on key TE transcription factors (TEtra factors): GATA2, GATA3, TFAP2A, and TFAP2C. Pluripotent stem cells (PSCs) represent a unique resource to study species-specific genetic and epigenetic mechanisms of TE and trophoblast development. Here, we use a PSC model in which BMP4 treatment and WNT inhibition (via IWP2) induce TE-like cells. We examined BMP4-dependent chromatin accessibility during the transition from PSC to TE-like cells in relation to gene expression and WNT activation. We found that BMP4 drives chromatin remodeling toward a trophoblast identity and that mesoderm genes are only transiently expressed with canonical WNT activation. Using this model, we determined that, while VGLL1 expression activation is temporally downstream of the TEtra factors, it is necessary for complete trophoblast differentiation by regulating EGFR expression and reinforcing GATA3 activation through positive feedback. We also found that VGLL1 enhanced canonical WNT signaling by directly regulating WNT receptors and effector genes. Furthermore, we identified KDM6B as a direct target of VGLL1. As a chromatin remodeler, KDM6B removes the repressive H3K27me3 at bivalent promoters, enabling the transcriptional activation of TE markers. These findings establish a mechanistic framework that determine the lineage outcomes at the interplay of three developmentally important signaling pathways, HIPPO, BMP, and WNT, positioning VGLL1 at the center of the genetic and epigenetic mechanisms supporting the maintenance and differentiation of human TE and trophoblast cells, key aspects of early placental development.
