A key feature of embryonic development is the assembly of cells into tissues and organs. How cells re-arrange and resolve over time into complex three-dimensional arrays remains a central question. It is clear, however, that morphogenesis requires the orchestration of interactions of cells with one another and with the extracellular matrix (ECM). The central hypothesis to be evaluated is that the ECM defines spatial boundaries in the embryo within which cell movements and rearrangements are coordinated and confined. The first aim of this proposal is to establish the relationship between the non-canonical wnt signaling pathway and fibronectin (FN) in regulating morphogenetic movements at gastrulation in Xenopus laevis. Integrin adhesion and signaling will be disrupted in embryos and explants and affects on downstream targets of wnt signaling analyzed to explore possible points of convergence between these pathways. Building on findings from the previous project period, the goal of aim 2 is to investigate the relationships between integrin binding to FN and cell-cell adhesion mediated by cadherins. Cadherin adhesion and bead binding assays will be used to identify and characterize molecules likely to be involved in the putative integrin-cadherin cross talk. Changes in gene expression in explants and embryos under different adherent conditions will also be investigated using available X. laevis gene chip microarrays. The third aim is to evaluate the role of FN in establishing boundaries important for patterning tissues and/or confining morphogenetic movements within whole embryos and in novel explant preparations. Matrix deposition will be inhibited or inappropriately increased by manipulating integrin receptors and regions of FN normally involved in matrix assembly. Cell behaviors will then be imaged by high-resolution time-lapse microscopy. The final aim is to develop methods for analyzing tension and force generation in the embryo. Biosensors of cell and ECM-generated tension will be developed and analyzed by FRET microscopy. Traction force microscopy and computational automata simulations will also be applied to the Xenopus system in order to address the general question of how force is generated in the embryo. This problem area has general significance for our understanding of birth defects, cell adhesion and motility, and the basic science of morphogenesis with potential practical applications to organogenesis and tissue engineering.