The tailbud is the posterior growth zone of the post-gastrulation vertebrate embryo containing the neural and mesodermal progenitors of the spinal column. The posterior neural tube undergoes convergence along the medial-lateral axis while extending posteriorly, and failure of neural tube convergence leads to birth defects such as spina bifida. The left and right paraxial mesoderm are assembled from motile mesodermal progenitors and subsequently segmented into somites. Failure to segment the paraxial mesoderm or maintain bilateral symmetry leads to birth defects such as congenital scoliosis. This project uses zebrafish as a model to study the molecular biophysics and systems morphogenesis in early spinal column development. The lab recently found that inter- tissue adhesion mediated by the extracellular matrix (ECM) protein Fibronectin mechanically couples the neural tube and paraxial mesoderm. This inter-tissue adhesion resists convergence of the neural tube which predisposes the embryo to spina bifida. However, inter-tissue adhesion ensures bilaterally symmetric morphogenesis of the paraxial mesoderm and thus prevents scoliosis. The interfaces between the neural tube and the left and right paraxial mesoderm resemble adhesive lap joints which are used in engineering to efficiently bond two objects. This project uses transgenic zebrafish to examine how the medial-lateral lap joint mechanics are maintained (Fibronectin acts as a glue) while the neural tube simultaneously slides posteriorly relative to the mesoderm (Fibronectin acts as a grease). Integrin heterodimers are the primary cell surface receptors for the ECM and are central to cell and tissue mechanics because they link the ECM to the actomyosin cytoskeleton. The lab has studied integrin activity in early spinal column development using proteomics, an in vivo FRET-FLIM assay for Integrin conformational changes, and FCS/FCCS to measure integrin intra-heterodimer affinities in vivo. The lab found that Integrin 51 and V1 are the two main Fibronectin receptors at this stage of zebrafish development and that intra-heterodimer affinity sets the threshold for integrin activation. Here, the project will quantify the molecular dynamics of single heterodimers in the neural tube and paraxial mesoderm using a new single molecule spectroscopy and machine learning protocol that we have developed. For the first time, we can measure the movement and conformational dynamics of a single protein in a living embryo.