Abstract (taken from the parent grant) We aim to understand the normal biology of neuromesodermal progenitors (NMPs), as well as to use NMPs to model cellular and molecular mechanisms in vivo. NMPs are basal progenitor cells located in the tailbud of vertebrate embryos that continue to make a germ-layer decision after gastrulation to generate ectoderm and mesoderm. As a primary source of cells generating the spinal cord, skeletal muscle, and other mesodermal derivatives, NMPs are a key cell type contributing to the formation of the vertebrate body plan. Studying NMPs has advanced our understanding of how body plans are generated, improved techniques for in vitro tissue generation, and provided critical insights into signaling pathway mechanisms. I discovered zebrafish NMPs as a postdoctoral fellow and my lab continues to focus on them. Our recent work has uncovered important roles for the transcription factor Sox2 in maintaining NMPs in an undifferentiated state through interactions with the canonical Wnt signaling pathway. This, combined with the past research showing the Wnt signaling effector bcat physically interacts with Sox2, indicates Wnt signaling can modulate gene expression and biological activity directly via Sox2. Little is known about this exciting branch of the Wnt/ bcat pathway. We will interrogate this pathway of Sox2 and Wnt/ bcat interactions at a number of biological levels, focusing on direct physical interactions between Sox2 and bcat, as well as genome wide analysis of Sox2/ bcat mediated transcriptional regulation. We hypothesize that Sox2/ bcat signaling represents a new arm of the Wnt/ bcat pathway distinct from the canonical TCF/LEF transcription factor family mediated signaling. We also recently developed a new Cyclin Dependent Kinase biosensor transgenic zebrafish and observed that NMPs and some of their derivatives exist in restricted cell cycle phases. The NMPs are held primarily in the G2 phase, while mesodermal notochord progenitors are restricted to the G1 phase. Cell cycle phase is broadly implicated in various aspects of stem cell maintenance, cell differentiation, and cell migration and invasion. We will manipulate the cell cycle in NMPs and their derivatives to understand how the cell cycle phase impacts normal NMP development. We hypothesize that the G2 phase restriction of NMPs is essential for maintenance of the undifferentiated state and for receipt of Wnt signaling based on G2 dependent receptor activation. We also hypothesize that the G1 phase of notochord progenitors is essential for their morphogenetic behavior of convergence and extension. Together, our work will shed important light on not only NMP development and vertebrate body plan formation, but also basic principles of cell biology and signaling. Wnt/ bcat signaling and Sox2 are found together and play important roles in numerous normal and diseased cellular contexts, including stem cells and cancer. The cell cycle is a fundamental aspect of nor...