PROJECT SUMMARY / ABSTRACT Complex morphogenetic processes are required for proper organismal development. These morphogenetic processes require various combinations of cell migration, proliferation, invasion, and fate acquisition. The coordination of these behaviors must be tightly regulated, as dysregulation of these processes can lead to developmental disorders and disease states such as cancer. To study the regulation of complex morphogenetic process we turn to zebrafish development. In zebrafish, morphogenesis of midline tissue structures such as the notochord, floor plate, and hypochord drive axis elongation of the developing embryo. These tissues are derived from a population of progenitors residing in the tailbud known as midline progenitor cells (MPCs). MPCs undergo a morphogenetic process called convergent extension (CE) to give rise to the notochord. During CE, adjacent MPCs migrate and intercalate between one another to form the notochord. The decision of MPCs to adopt a notochord, floor plate, or hypochord fate is based on local signaling cues such as Wnt and Notch. While these signaling pathways have been shown to regulate morphogenetic cell behaviors, there is growing evidence to suggest that the cell cycle can also modulate cell behaviors. However, the mechanisms by which cell cycle state dictates cell behavior and cell fate during tailbud morphogenesis remain unclear. I will address this gap in knowledge and elucidate the relationship between cell cycle state and cell fate/morphogenesis during development using CE of the zebrafish notochord during tailbud morphogenesis as a model. Published data from my lab and others show notochord progenitor cells are in G1, while floor plate and hypochord progenitors can be found in all phases of the cell cycle. Furthermore, these notochord progenitors undergo CE in G1 and reenter the cell cycle after joining the notochord, suggesting that G1 arrest facilitates this morphogenetic process and notochord fate acquisition. In Aim 1 of this project, I will perturb G1 arrest specifically in the MPCs and determine the effects on CE. Using transgenic zebrafish lines, including a CDK activity biosensor to establish cell cycle state, and spinning disk confocal microscopy, I will time-lapse image these cell cycle perturbed embryos and quantify CE and fate acquisition. In Aim 2, I will explore the gene regulatory network (GRN) responsible for inducing G1 arrest in the MPCs, with a focus on the T-box transcription factor Brachyury (tbxta). Published data show tbxta to be indispensable to notochord formation and moreover, preliminary data from our lab show knockdown of tbxta drives notochord progenitors to cycle and be excluded from the notochord, adopting a floor plate or hypochord fate. The combination of a CDK activity biosensor, cell cycle perturbation constructs, midline directed cell transplantation, and spinning disk confocal microscopy will allow me to test my hypotheses thoroughly and rigorously.