Project Summary Collective cell migration is a highly varied process involved in development and disease. Depending on context, the appropriate degree of order in cell movements can range from a nearly completely ordered or laminar flow to a very disordered or turbulent flow. How order is controlled is not well understood. The zebrafish tailbud provides a unique opportunity to investigate this question. Cells entering the tailbud move through the dorsal medial (DM) zone in a processive, orderly fashion until they reach the tip of the tailbud. There mesoderm fated cells undergo an EMT to enter the progenitor zone (PZ). Cell movements in the PZ are non-persistent and characterized by a high degree of disorder. Nevertheless, cell movement in this domain is still collective as indicated by the presence of frequently reversing, transient, anisotropic left-right fluxes generated by transient, local order. Ultimately PZ cells move into the presomitic mesoderm (PSM) which has very little cell movement. In vivo and in silico experiments indicate that both orderly and disorderly migration are necessary to produce a properly formed trunk and tail. Excessively disordered motions in the DM decrease the flux of cells through the tailbud leading to the formation of a short body axis while excessively ordered cell motions in the PZ induce inappropriately prolonged anisotropic fluxes, the formation of stable cell vortices, asymmetric allotment of cells to the left or right side of the embryo and a bent body axis. While Wnt, FGF, and BMP are known to regulate the degree of order in the tailbud, mechanisms for tuning local order remain poorly understood. This project will take a cross-scale approach from the transcriptional to cytoskeletal polarity to tissue organization to investigate this process. Preliminary analyses of single cell RNA sequencing (scRNAseq) of wildtype embryos and embryos subject to treatments that decrease order (FGF or BMP inhibition) have identified genes subject to temporal and signaling regulation. In Aim 1, the scRNAseq results will be validated by fluorescent in situ hybridization. Additionally, scRNAseq of embryos with excessive order induced by Wnt inhibition will also be performed. In Aim 2, changes in cytoskeletal polarity between the DM and PZ in wildtype and Wnt, FGF, and BMP inhibited embryos will be assayed using live fluorescent reporters imaged by confocal. In Aim 3, two hypotheses as to the cause of local order in the PZ, as a cell crowding induced emergent property or by cells aligning their polarity with an external signal, will be tested. First, a pipeline will be constructed to identify local order in lightsheet, timelapse microscopy data. Then correlation between local order and cell crowding will be measured using a secreted GFP to detect extracellular space. To investigate the role of directed migration, order will be quantified in the tbx16/mesogenin1 double mutant which lacks directional bias. We will also perform pert...