| The extracellular matrix (ECM) is a rich microenvironment that provides biochemical and mechanical cues to regulate cell survival, differentiation, and morphogenesis. Cells perceive and respond to ECM stimuli by engaging cell surface receptors, predominantly integrins. The purpose of this dissertation is to evaluate the importance of bidirectional signaling through integrins during Xenopus laevis development.;First, I examined the progressive assembly and 3-D fibrillar organization of the ECM molecule fibronectin (FN) and its role in regulating cell and tissue movements during gastrulation. A 70 kD-N terminal fragment was used to block FN fibril assembly at gastrulation but not initial FN binding to integrins at the cell surface. I found that fibrillar FN is necessary to maintain cell polarity and to promote epiboly, possibly through maintenance of tissue-surface tension. In contrast, FN fibrils were dispensable for the convergence and extension movements required for axis elongation. Closure of the migratory mesendodermal mantle was accelerated in the absence of a fibrillar matrix. Thus, the macromolecular assembly of FN matrices constitutes a regulatory mechanism for coordination of distinct morphogenetic movements.;Second, I examined the roles of kindlins, which regulate talin-dependent inside-out integrin activation, during embryonic morphogenesis. In mammals, loss of kindlin2 is embryonic lethal whereas mutations in kindlin1 and 3 result in the skin pathology Kindler Syndrome and the bleeding disorder Leukocyte Adhesion Deficiency III (LAD-III), respectively. Using antisense morpholino knockdowns in Xenopus laevis, I observed kindlin2-dependent defects in angiogenesis, fluid homeostasis and vascular maintenance that required kindlin2 function as an integrin activator. Maternal depletion of kindlin2 resulted in early cleavage defects attributable to failures in cytokinesis, and ultimately death by mid-blastula stage. Kindlin3 knockdown resulted in reversible inhibition of epidermal ciliary beating and embryo paralysis. Knockdown of kindlin1 also inhibited tadpole development potentially through disruptions of skin and gut epithelia. These data provide evidence for the importance of tissue and stage specific regulation of integrin activation through kindlin isoforms. These studies also illustrate the utility of using developmental models like Xenopus laevis to uncover novel in vivo functions of proteins and signaling pathways not readily apparent in cell culture. |
