The epithelial-mesenchymal interactions required for kidney organogenesis are disrupted in mice lacking the integrin alpha8beta1. None of this integrin's known ligands, however, appears to account for this phenotype. To identify a more relevant ligand, a soluble integrin alpha8beta1 heterodimer fused to alkaline phosphatase (AP) has been used to probe blots and cDNA libraries. In newborn mouse kidney extracts, alpha8beta1-AP detects a novel ligand of 70-90 kD. This protein, named nephronectin, is an extracellular matrix protein with five EGF-like repeats, a mucin region containing a RGD sequence, and a COOH-terminal MAM domain. Integrin alpha8beta1 and several additional RGD-binding integrins bind nephronectin. Nephronectin mRNA is expressed in the ureteric bud epithelium, whereas alpha8beta1 is expressed in the metanephric mesenchyme. Nephronectin is localized in the extracellular matrix in the same distribution as the ligand detected by alpha8beta1-AP and forms a complex with alpha8beta1 in vivo. Thus, these results strongly suggest that nephronectin is a relevant ligand mediating alpha8beta1 function in the kidney. Nephronectin is expressed at numerous sites outside the kidney, so it may also have wider roles in development. The approaches used here should be generally useful for characterizing the interactions of novel extracellular matrix proteins identified through genomic sequencing projects.

Two different monoclonal antibodies, characterized initially as binding synaptic terminal regions of rat brain, bind a 65,000-dalton protein, which is exposed on the outer surface of brain synaptic vesicles. Immunocytochemical experiments at the electron microscope level demonstrate that these antibodies bind the vesicles in many different types of nerve terminals. The antibodies have been used successfully to purify synaptic vesicles from crude brain homogenates by immunoprecipitation onto the surface of polyacrylamide beads. The profiles of the structures precipitated by these beads are almost exclusively vesicular, confirming the vesicle-specificity of the antibodies. In SDS gels, the antibodies bind a single protein of 65,000 daltons. The two antibodies are not identical, but compete for binding sites on this protein. Immune competition experiments also demonstrate that the antigenic components on the 65,000-dalton protein are widely distributed in neuronal and neural secretory tissues. Detectable antigen is not found in uninnervated tissue--blood cells and extrajunctional muscle. Low levels are found in nonneural secretory tissues; it is not certain whether this reflects the presence of low amounts of the antigen on all the exocytotic vesicles in these tissues or whether the antigen is found only in neuronal fibers within these tissues. The molecular weight and at least two antigenic determinants of the 65,000-dalton protein are highly conserved throughout vertebrate phylogeny. The two antibodies recognize a 65,000-dalton protein present in shark, amphibia, birds, and mammals. The highly conserved nature of the determinants on this protein and their specific localization on secretory vesicles of many different types suggest that this protein may be essential for the normal function of neuronal secretory vesicles.

In Xenopus laevis development, beta-catenin plays an important role in the Wnt-signaling pathway by establishing the Nieuwkoop center, which in turn leads to specification of the dorsoventral axis. Cadherins are essential for embryonic morphogenesis since they mediate calcium-dependent cell-cell adhesion and can modulate beta-catenin signaling. alpha-catenin links beta-catenin to the actin-based cytoskeleton. To study the role of endogenous alpha-catenin in early development, we have made deletion mutants of alphaN-catenin. The binding domain of beta-catenin has been mapped to the NH2-terminal 210 amino acids of alphaN-catenin. Overexpression of mutants lacking the COOH-terminal 230 amino acids causes severe developmental defects that reflect impaired calcium-dependent blastomere adhesion. Lack of normal adhesive interactions results in a loss of the blastocoel in early embryos and ripping of the ectodermal layer during gastrulation. The phenotypes of the dominant-negative mutants can be rescued by coexpressing full-length alphaN-catenin or a mutant of beta-catenin that lacks the internal armadillo repeats. We next show that coexpression of alphaN-catenin antagonizes the dorsalizing effects of beta-catenin and Xwnt-8. This can be seen phenotypically, or by studying the effects of expression on the downstream homeobox gene Siamois. Thus, alpha-catenin is essential for proper morphogenesis of the embryo and may act as a regulator of the intracellular beta-catenin signaling pathway in vivo.