This proposal investigates the role of a new signaling molecule (X-Fringe) in Xenopus development. Fringe-type molecules were originally identified in Drosophila because of their boundary specific properties in the wing disc and their apparent role in modulating Notch/Serrate/Delta interactions. Rao has cloned two Xenopus homologues. Based on their sequence and preliminary biochemical assays, Rao argues that they function as secreted growth factors, that they exist as dimers and that their processing and expression may be developmentally regulated. The central result in the current proposal, however, is the demonstration that supernatant from COS cells expressing His tagged Fringe induces mesoderm differentiation in animal caps. The proposal describes several experiments designed to investigate the role of Fringe in Xenopus development. Antibodies will be generated to X-Fringe and used to compare protein expression to the distribution of Fringe RNA at various embryonic stages. The mesodermalizing activity in COS supernatants will be characterized and attempts to deplete it using nickel agarose beads will test whether the effects are a direct consequence of Fringe protein in the supernatant. Dominant negative mutations in FGF receptor, ras, BMP- and activin receptor will be used to position fringe inducing activity relative to these well characterized mesodermalizing pathways. Rao will also attempt to generate dominant negative alleles of fringe for use in these epistasis experiments. A second set of experiments investigate fringe role in the patterning of primary neurons in the neural tube. Rao will determine the expression of Fringe relative to Delta and other primary neuron specific markers and will use existing dominant alleles of Notch and Delta , as well as his newly generate Fringe mutations to analyze function in the nervous system.
