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Show Judy Vu Biochemistry Faculty Sponsor Anthea Letsou UNCOVERING PATTERNS OF GENETIC EXPRESSION IN DROSOPHILA MELANOGASTER EMBRYOS Judy Vu, (Anthea Letsou, Ph.D.) Department of Human Genetics, University of Utah Research on Drosophila melanogaster has provided much valuable in- sight into biological processes such as gene expression and morpho-genesis. Dorsal closure in the Drosophila embryo is a morphogenetic process that refers to the dorsalward migration of the lateral and ventral epidermis to enclose the embryo. It has become an ideal model sys-tem for studying coordinated cell migration and fusion processes during development. Genetic studies show that a network of interacting signal-ing molecules, which includes the Jun N-terminal kinase (JNK) cascade, acts at the leading edge of the migrating epidermis to regulate dorsal closure. The JNK cascade triggers signaling by decapentaplegic (Dpp), a secreted protein member of the transforming growth factor- (TGF-) superfamily. Genes that are regulated by and function downstream of dpp, may affect the cytoskeletal changes that occur during dorsal clo-sure. In order to isolate new genes that function downstream of dpp, we will perform a molecular screen to determine the expression pat-tern for all Drosophila genes, and then subsequently identify spatially restricted transcripts. The Drosophila genome has been sequenced and over 15,000 genes have been identified. We have obtained 16 384-well plates contain-ing cDNA bacterial stocks of these genes representing the Drosophila unigene collection (Research Genetics). With the cDNAs in hand, we are now performing a large-scale hybridization screen in situ to determine the expression pattern of each gene. We are using robotic automation in order to quickly assay all expression patterns. We first perform PCRs on all the cDNA bacterial stocks to create templates for the preparation of riboprobes. Given that the DNA templates are amplified from bacte-rial stocks, they must be purified with ethanol washes before they are used in the riboprobe reactions. By means of robotic animation, we will make antisense riboprobes labeled with digoxigenin-UTP. We will then use a well-established automated protocol to perform our whole mount embryo RNA hybridizations in situ. In our hybridization screens, we will use wild-type Canton-S embryos 8-12 hours after egg lay, which corre-spond to dorsal closure stage embryos. After the whole mount embryo hybridizations in situ are complete, the embryos will be mounted onto glass slides and scored using standard light microscopes. We will doc-ument the genetic expression patterns of the embryos. Using this re-search strategy, we expect to identify genes with novel patterns, as well as genes with patterns consistent with their known functions in dorsal closure and morphogenesis. We expect to find relationships between expression pattern and gene function; thus, information obtained from our screens will be used to facilitate prediction of novel gene functions in Drosophila, particularly those involved in dorsal closure. |