The present application includes the further development of the genetic system and the generation of site-directed mutants in each of the hemagglutinin (HA) genes. In addition, the application includes approaches to determine if either HA also has protease activity (as circumstantial evidence indicates), to identify functional domains of the HA molecules, and to determine the biological/adhesive function of the HAs using the isogenic mutants in comparison with the wild type parent in in vitro adherence assays. The ultimate goal is to test the virulence of the mutants in animal models. In addition, the erythrocyte receptor molecule will be identified for each HA, employing a variety of biochemical approaches. GRANT=R29DE10150 The long term objectives of these studies are to understand the molecular basis for interactions between epithelial- and mesenchymal-derived tissues that direct tissue morphogenesis, and cell differentiation of mammalian submandibular glands. The hypothesis for this proposal is that epidermal growth factor (EGF) gene products are synthesized by mesenchymal-derived cells in a time- and space-restricted manner and participate in regulating submandibular gland epithelial morphogenesis and differentiation. These EGF gene products interact with receptors on adjacent epithelial cell surfaces and thereby activate signal transduction pathways to convey instructive information from mesenchyme to salivary epithelium. The information is imparted to the epithelial cell nucleus and genome ultimately inducing the expression of a finite family of genes. The epithelial gene products include proteins that i) regulate cell cycle progression; ii) participate in the assembly and degradation of the basement membrane; and iii) induce the expression of cell-specific gene products. To test this hypothesis, the following studies will be performed: i) the temporal and spatial expression of EGF gene products by mesenchymal-derived cells in developing mouse submandibular glands will be studied by high resolution in situ hybridization; ii) simultaneous in situ hybridization and immunochemistry will be used to correlate localized synthesis of EGF gene products with EGF-receptors in the developing mouse submandibular gland; iii) the localized loss or accumulation of specific basement membrane components will be examined in relationship to localized EGF transcription by combined in situ hybridization and immunohistochemistry, and this relationship will be perturbed by the addition of either tyrphostins or antisense EGF oligonucleotides to organ cultures; iv) EGF signal transduction will be perturbed in cultures of developing submandibular glands and the alteration of localized expression of a putative cell cycle control gene, p58, will be determined by in situ hybridization; and v) modification of mitotic indices, tissue morphogenesis and cell differentiation will be determined by mitotic labeling and morphometric analysis in cultured submandibular glands in which EGF signal transduction has been perturbed. The data derived from these studies will contribute to the understanding of molecular mechanisms operant during epithelial-mesenchymal interactions that direct the morphogenesis and differentiation of salivary glands. A knowledge of the molecular mechanisms that direct salivary gland development is critical to understanding the function and behavior of salivary cells as well as the pathogenesis of salivary gland diseases.
