It is well established that cell-cell matrix adhesion effect changes in cellular morphology, differentiation and migration throughout embryogenesis and during physiological processes such as hemostasis, wound healing, immune surveillance and malignant transformation. In the next three years, we will investigate molecular aspects of cell-extracellular matrix (ECM) interactions that occur during vertebrate development. The major focus of this work will involve detailed analyses of the structure, function and expression of two groups of molecules; the ECM glycoprotein fibronectin (FN), and the integrin family of ECM receptors. The eggs and embryos of the amphibian Xenopus laevis will be used as the primary experimental system in these studies because they are ideally suited to biochemical and molecular analyses of very early development. In order to elucidate the functional significance of FNs and integrins it is necessary to analyze their structures, and this will involve the isolation and characterization of Xenopus cDNAs that encode these proteins. Structural information derived from these cDNAs will result in the preparation of synthetic peptide and fusion protein antibodies, which will be used to determine the spatial and temporal expression of FN isoforms and integrin heterodimers in embryos. Studies will concentrate on gastrulation because it is at this stage of development when cellular differentiation commences in part, as a consequence of mesodermal induction and morphogenesis. Evidence suggests that integrins and FNs may participate in these events by providing positional information involved in directing cell migration. After establishing when and where these molecules are distributed in the embryo, their expression will be manipulated in order to determine their precise roles in development. Reverse genetic approaches techniques will complement a second major approach, which will be to express full length and engineered (mutagenized) forms of FNs and integrins by injecting transcripts prepared from cloned cDNAs into fertilized eggs. The knockout and expression experiments will enable us to analyze the functions of FNs integrins and identity structurally important features of these molecules. The objective of this research is to enhance current knowledge concerning the biology of cell-ECM interactions (which is based primarily on in vitro evidence) and provide insights into the cellular and molecular basis of vertebrate morphogenesis.
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