The long-term objective is to increase the usefulness and """"""""connectivity"""""""" of easily cultured Haliotis (molluscan) larvae as a model system for biomedical research. Development in these larvae is synchronously induced by chemosensory recognition of an exogenous morphogen; the receptors, signal transducers and pathways involved in this control have been found to be closely homologous to counterparts in mammalian systems. We propose to increase the connectivity of this model system by characterizing the link between the chemosensory signal recognition/transduction mechanisms already characterized at the molecular level, and the developmental and genetic mechanisms that regulate specific target gene expression, cellular differentiation and proliferation.
The specific aims of this proposal are: (1) to identify the """"""""primary response"""""""" genes and transcription regulators they may encode, that control larval metamorphosis and early post-larval growth in Haliotis in response to chemosensory recognition of exogenous morphogens; (2) to identify the molecular mechanisms by which these genes and transcription activators interact with gene- and tissue-specific cis-acting DNA (and mRNA) regulatory sequences to control the expression of essential """"""""secondary response"""""""" target genes, leading to metamorphosis; (3) to identify, through the analysis of G protein and phosphoprotein cDNAs, the centrally important mechanisms by which receptor-regulated G proteins and protein phosphorylations transduce the inductive signals generated by larval recognition of exogenous chemical morphogens; and (4) to establish the relatedness of the mechanisms of chemosensory signal-dependent gene expression found in this research to corresponding mechanisms in mammals. Morphogen-induced primary response gene transcripts and their cDNAs will be purified by (a) PCR-amplification and hybridization using highly conserved oligonucleotides, corresponding to mammalian transcription factors (c-myc, c-fos, c-jun, and homeoprotein gene families), and by (b) direct isolation by subtractive hybridization. These will be cloned and sequenced, and the mechanisms and locations of action of their protein products determined by in situ hybridization, and blockade of target gene induction with anti- sense RNA. The mechanisms of these morphogen-induced transcription factors will be further characterized by footprinting analyses of the regulatory sites with which they interact on the genomic DNA sequences of their target (secondary response) genes. Results of the proposed studies should be significant in providing the most detailed understanding yet available of the molecular mechanisms by which chemosensory recognition of exogenous morphogens and regulatory chemical signals controls gene expression, differentiation and cellular proliferation in multicellular organisms. This link is of fundamental biomedical importance in the processes of normal development, hormonal regulation, wound healing, antibody synthesis, long-term memory, and cancer. The proposed research thus should increase the usefulness of the Haliotis larvae as a model system with high connectivity to research in these areas.
