Mechanically sensitive regulators of human amnion and primordial germ cell formation from a common progenitor

About This Grant

Project Summary Infertility is a complex disease that impacts approximately 1 in 10 couples of reproductive age in the United States. However, the pathogenesis underlying many of these cases is unknown. Clinically, this proposal aims to gain molecular insights into infertilities associated with defective formation of amnion, a component of the fetal membrane, and primordial germ cells (PGC, precursors of gametes). Formation of human amnion is first seen in the pluripotent epiblast cells as implantation is initiated (within two weeks after fertilization). Thus, couples with pregnancies repeatedly accompanied by defective amnion formation would be diagnosed with infertility. Recent data indicate that, in implanting primate embryos, PGC are specified in the nascent amnion and a growing body of evidence supports the notion that a subset of the pluripotent epiblast cells form a bi- potential amnion/PGC progenitor cell population at the amnion-epiblast boundary of developing embryos. In published work using a human pluripotent stem cell (hPSC)-based Gel-3D amniogenic system, we showed that a mechanical cue initiates amnion specification by triggering a Bone Morphogenetic Protein (BMP) signaling cascade in individual cells of pluripotent cysts. Moreover, our recent single cell transcriptomic analyses of developing Gel-3D showed that, in fact, PGC-like cells (PGC-LC) are also present in Gel-3D, and that cells that are progressing to amnion and PGC-LC lineages appear to share a common progenitor labeled by Claudin 10 (CLDN10). Indeed, our preliminary results revealed the presence of CLDN10/TFAP2C/SOX17-triple positive cells at the posterior amnion-epiblast boundary of a cynomolgus macaque peri-gastrula, suggesting that this progenitor formation as well as the amnion/PGC lineage diversification from the progenitor is an important aspect of successful amniogenesis and PGC formation both in vivo and in vitro. These findings present Gel-3D as a robust, transcriptomically well-characterized and mechanosensitive model to investigate these previously unexplored aspects of amniogenesis and PGC-LC formation. Together, using the Gel-3D model, the goal of this proposal is to understand the mechanisms that triggers amniogenic BMP signaling to initiate CLDN10+ progenitor cell specification (Aim 1), as well as the mechanisms that control amnion-PGC lineage decisions in the progenitor (Aim 2). In vitro findings will be grounded using early cynomolgus macaque embryo samples. Our preliminary data lead us to hypothesize that mechanically triggered WNT/?-catenin signaling activates amniogenic BMP signaling, and, in the progenitor population, Yes-associated protein 1 (YAP1) activity determines amnion or PGC fate. Proposed studies will further expose these signaling and transcriptional machineries. Overall, the work proposed here will greatly accelerate the pace of discovery regarding critical but previously inaccessible peri-implantation events and thus will have enormous implications for understanding early processes that impact embryonic development and human fertility.