Abstract: Nearly 60% of human conceptions are miscarried surrounding the window of implantation. During this stage within a week after fertilization, the pluripotent epiblast tissue of the embryo transforms into a polarized epithelium with a central lumen. The causes of significant pregnancy loss at this period are still not well understood owing to the substantial challenges associated with human embryo research. Critical gaps in knowledge include mechanistic understanding of the cellular morphogens driving epiblast tissue development at the implantation stages. Further, animal models such as rodents, while highly valuable, have been found to demonstrate distinct processes from humans at the implantation stages. Thus, an in-depth understanding of the levels of conservation or divergence between key mammalian species at this stage remains incomplete, making it difficult to extrapolate findings around the implantation period from model species to human health. The central goal of this proposal is to enhance our comprehension of the human-specific mechanisms that govern embryonic development by examining the regulatory mechanisms through which WNT/β-catenin signaling guides epiblast tissue remodeling during implantation across different species. Based on my preliminary results, the overall hypothesis that WNT/β-catenin signaling will show species-specific differences in its role in epiblast tissue remodeling. I hypothesize that these differences are mechanistically tied to regulation of Ezrin-Radixin-Moesin (ERM) proteins which control cell surface tension and actin architecture. Aim 1 will identify the biomechanical effects of WNT/β-catenin signaling on human epiblast development at implantation using novel human 3D stem cell-based models. Using these highly reproducible models, I will confirm that WNT/β-catenin acts through pERM via Western Blot and loss-of-function experiments. I will then utilize live-cell imaging to analyze changes in actin architecture over development, and then analyze changes in cell surface tension through use a fluorescent membrane tension probe that will be quantified using fluorescence lifetime imaging microscopy (FLIM) to validate previous preliminary data using a secondary 5-dimensional state-of-the-art imaging software-based readout of cell surface tension. Aim 2 will define species-specificity of the mechanism underlying WNT/β-catenin epiblast remodeling control across humans, non-human primates and mice, through performing similar perturbation techniques and characterizations using Western Blot and IF, live-cell imaging with actin reporters, 5D imaging analyses, and FLIM. This proposal directly addresses the NICHDD’s research theme 1: “Understanding the Molecular, Cellular, and Structural Basis of Development” through use of novel models to understand correct processes of early human embryonic development, as well as how abnormal processes lead to undesirable outcomes. A detailed understanding of the morphogen-driv...