PROJECT SUMMARY Although the “master regulators” that maintain the self-renewal of pluripotent embryonic stem cells and extraembryonic trophoblast stem cells are relatively well understood, little is known about how these transcription factors are regulated in vivo at early stages of embryogenesis. Molecular genetics and embryology studies have identified signaling pathways required to direct cells to become trophoblast or embryonic epiblast, but the transcription factors that activate expression of pluripotency master regulators (PMRs) or trophoblast master regulators (TMRs) in each cell remain largely unknown. This major gap in our understanding of early development is due to at least two factors. First, technical barriers prevent the use of many molecular and genomics approaches in embryos consisting of low cell numbers. Second, there are significant discrepancies between the totipotent blastomeres of early embryos (where most PMRs and TMRs begin to be expressed) and cell culture models of totipotent cells. Therefore, a new approach that combines comprehensive methods for identification of regulators of PMR and TMR expression with sensitive new methods of testing their functions in vivo is necessary to fill this major gap in our understanding of the early embryonic gene regulatory network (GRN). We propose three aims to address this problem. Aim 1 is focused on comprehensive identification of transcription factors regulating PMRs and TMRs and uncovering their regulatory functions. Aim 2 will generate a lineage-resolved atlas of epigenetic changes that occur as cells select either the epiblast or trophoblast fate, as well as identify the direct targets of transcription factors that participate in cell fate specification. In Aim 3, we propose to dissect the mechanisms by which developmental transcription factors help elicit these epigenetic changes and generate a model of the totipotent GRN that mediates this decision. Successful completion of these studies will reshape our understanding of early embryonic gene regulation, as well as specification of epiblast and trophoblast cell fate.

PROJECT NARRATIVE A thorough understanding of the gene regulatory network (GRN) functioning in early mammalian embryos will aid in the diagnosis and treatment of human developmental disorders, along with facilitating more effective strategies for regenerative medicine. Until recently, tools for comprehensive characterization of the early embryonic GRN were unavailable. We will combine powerful cell-based methods with new in vivo approaches to fill this void, providing insight into the first cell fate decision during embryogenesis.