INVIVO ANALYSES OF INTEGRIN BETA-1 SUBUNIT FUNCTION IN FIBRONECTIN MATRIX ASSEMBLY

Early development of the urodele amphibian Pleurodeles waltl is accompanied by a process of progressive fibronectin (FN) fibrillogenesis. FN begins to assemble into fibrils on the inner surface of the blastocele roof at the early blastula stage and progressively forms a complex extracellular matrix. We have analyzed the mechanisms of FN-fibril formation under normal and experimental conditions in vivo with the following probes: iodinated FN, fluorescein-labeled FN, synthetic peptides containing the Arg-Gly-Asp (RGD) cell surface recognition sequence of FN, and polyclonal antibodies against both β1 subunit of the amphibian FN receptor and the cytoplasmic domain of β1 subunit. We report that in living embryos, exogenous labeled mammalian FN injected into the amphibian blastocele undergoes FN-fibril formation in spatiotemporal patterns similar to those of endogenous FN. This indicates regulation of fibrillogenesis by the cell surface rather than by changes in the type of FN. Fibrillogenesis is inhibited in a dose-dependent manner both by the GRGDS peptide and monospecific antibodies to amphibian integrin β1 subunit. Furthermore, when injected intracellularly into uncleaved embryos or into selected blastomeres, antibodies to the cytoplasmic domain of integrin β1 subunit produce a reversible inhibition of FN-fibril formation that follows early cell lineages and causes delays in development. Together, these data indicate that in vivo, the integrin β1 subunit and the RGD recognition signal are essential for the proper assembly of FN fibrils in early amphibian development.