Shining light on the mechanisms of primate gastrulation: development of high-throughput, optogenetic tools for understanding embryonic development - ABSTRACT Gastrulation is a pivotal process in mammalian embryogenesis that is essential for the establishment of definitive germ layers and the formation of the body plan. Despite decades of research, the precise mechanisms that regulate cell differentiation, migration, and patterning during mammalian gastrulation remain poorly understood, particularly in primates. The study of primate gastrulation is especially challenging because this process occurs after the embryo has implanted into the uterus, making it difficult to directly observe. Recently, several three-dimensional (3D), stem cell-based models of primate embryos, termed “stembryos”, have been developed for in vitro studies of embryonic development. However, most approaches for generating stembryos exploit the inherent self-organizing capacity of stem cells to form 3D constructs and are unable to precisely control the number of cells and cell types per construct. A lack of tools for engineering stembryos has resulted in low yields and has limited the ability of stembryos to faithfully recapitulate primate gastrulation. We have recently developed a stembryo model using induced pluripotent stem cells (iPSCs) from chimpanzees along with an optofluidic (i.e., combination of optogenetic and microfluidic) device for performing Chip-based, High-throughput Investigations into Morphogenesis in Primates, called the OptoCHIMP platform. Our OptoCHIMP platform utilizes microfluidic encapsulation to place cells into hydrogel droplets. A downstream droplet sorter unit allows us to select droplets of interest with prescribed cell numbers of cell ratios. This enables us to fabricate stembryos in a precise and high-throughput manner. In this proposal, we will utilize the OptoCHIMP platform to define the roles that 3D structure, local tissue mechanics, and spatiotemporal signaling dynamics play in symmetry breaking during primate gastrulation. By utilizing optogenetic tools to stimulate signaling pathways in chimpanzee stembryos, we can recreate the complex interplay of signaling pathways between embryonic and extraembryonic tissues, induce symmetry breaking in our stembryo, and develop a model for understanding how morphogen signaling dynamics impact tissue patterning. This proposal will be the first time that microfluidic droplet encapsulation of stem cells, which offers precise control over the initial conditions, has been combined with optogenetic, providing a promising approach to uncovering the mechanisms that drive primate gastrulation.
