Project Summary The amniotic membrane forms a tough fluid-filled sac that protects the developing embryo and is essential for a successful pregnancy. Amniogenesis initiates early in human development as the embryo implants into the uterine wall: the epiblast cells first polarize to form a pluripotent lumenal cyst and, subsequently, one half of this polarized cyst loses pluripotency and becomes squamous amniotic ectoderm while the other side remains pluripotent. In published work using an hPSC-based Gel-3D system, we showed that a mechanical cue initiates amnion specification by triggering a BMP signaling cascade in individual cells of pluripotent cysts. Recently, we established a new hPSC-based amniogenic model called Glass-3D+BMP, in which the mechanical cue is bypassed and uniform addition of BMP ligand to pluripotent hPSC cysts leads to simultaneous activation of BMP signaling in all cells within 10 minutes. Over the next 48 hours, all cells give rise to squamous amnion cells, forming fully squamous amnion cysts. This highly robust amniogenic system, which enables reproducible mechanistic analyses specifically into amniogenesis, will be used throughout this study to investigate transcriptional, signaling and epigenetic machineries driving amnion fate progression. Interestingly, although all cells are equally exposed to BMP in the Glass-3D+BMP model, amniogenesis initiates focally then spreads laterally to form fully squamous amnion cysts. Importantly, similar focal amniogenesis followed by spreading was also seen in the Gel-3D amniogenic system that we previously developed. Moreover, in early cynomolgus macaque embryos, molecular signatures associated with focal initiation followed by spreading are readily seen at transcript and protein levels, indicating that this two-step process is an important aspect of successful amnion fate determination both in vivo and in vitro. The goal of this proposal is to understand the mechanisms that initiate (Aim 1) and spread (Aim 2) amnion fate specification. In vitro findings will be grounded using early cynomolgus macaque embryo samples. Our preliminary loss-of-function data lead us to hypothesize that GATA2 and GATA3 are critical for focal initiation of amniogenesis, while spreading is controlled by TFAP2A and canonical WNT signaling. Proposed transcriptomic and epigenetic studies will further expose these transcriptional and signaling machineries. Functional genetic deletion studies of candidate genes will be used to test for potential master regulators of amnion fate. Overall, the work proposed here will greatly accelerate the pace of discovery regarding critical but previously inaccessible post-implantation events and thus will have enormous implications for understanding early processes that impact embryonic development and human fertility.