Project Summary The overall goal of the proposal is to determine how the extracellular matrix (ECM) contributes to the morphogenesis of a three-dimensional tissue in vivo. The ECM is well studied in the field of cell migration; however, limited examples exist analyzing the role of the ECM in other developmental contexts. To understand the role of the ECM during development, a three-dimensional system in which the ECM can be tracked, quantified, and manipulated in vivo during development is needed. The elongating neural tube in zebrafish is well-suited to examine the ECM during three-dimensional tissue morphogenesis, due to the ease of imaging, the ability to quantify Fibronectin matrix remodeling, and the numerous mutants and tools that are available in zebrafish to manipulate the Fibronectin matrix and tissue mechanics. In this system, Fibronectin is present between two tissues, the presomitic mesoderm (PSM) and neural tube. Fibronectin acts as a glue to hold the two tissues together and is required for proper neural tube convergence along the medio-lateral axis. However, the neural tube also moves posteriorly in relation to the PSM and Fibronectin matrix. When the main integrin receptor that binds cells to the Fibronectin matrix is removed, no significant changes in cell motion are observed in the neural tube. Furthermore, when Fibronectin is removed, the neural tube elongates fully. Together these data indicate that cell migration along the Fibronectin matrix is not required for posterior movement of the neural tube. Overall, this indicates that the neural tube moves past the PSM through a mechanism other than cell migration along the Fibronectin matrix while also maintaining adhesion to the Fibronectin matrix. One model for how this might occur is through changes in integrin dynamics in the neural tube, in which decreases in integrin activation and stability could allow for tissue level motion while still maintaining adhesion. In aim 1, I will investigate integrin binding dynamics in the context of posterior motion of the neural tube. Using techniques, including FRET/FLIM, antibody staining, and live imaging, a cross-scale view of integrin dynamics will be generated. These data will generate new hypotheses for how integrins contribute to neural tube motion which will be tested using mutations that affect posterior motion of the neural tube and mutations that strengthen or weaken integrin binding activity. In aim 2, I will determine how adhesion of neural tube cells to the ECM influences their cell motion. This will be completed using live tracking of Fibronectin matrix remodeling and cell motion in relation to their position to the Fibronectin matrix. This data will generate hypotheses for how cell-cell and cell-ECM contacts influence cell behavior, which will be further tested using established mutations that affect neural tube convergence and by altering cell adhesion and cell contractility.