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Show posters on the hill Using Robots to Assay for Gene Expression in Morphogenesis of Drosophila melanogaster Embryos Judy Vu, Anthea Letsou Department of Human Genetics A new approach to studying and understanding gene function is the use of reverse genetics. In the conventional forward genetics approach, an animal's genes are mutated one at a time, and the effects of the mutations are observed in order to uncover each gene function. The method of reverse genetics instead identifies genes first; mutation analysis occurs afterward. Genetic studies show that a network of interacting signaling molecules, including the Jun N-terminal kinase (JNK) cascade, act at the first row of cells in the migrating epidermis of Drosophila embryos to regulate dorsal closure, a morphogenetic process. This process is analogous to several vertebrate closure processes, including neurulation, eyelid fusion, and wound healing. In order to identify targets of the JNK signaling cascades, which are involved in these morphogenetic processes, we have undertaken a highthroughput screen to identify genes that have shared patterns of expression. We will use reverse genetic methods subsequently to reveal gene functions. We expect that genes with shared patterns of expression will function together in spatially-restricted differentiation programs, and thus that this strategy will enable us to Using Robots to Assay for Gene Expression in Morphogenesis of Drosophila melanogaster Embryos Judy Vu, Anthea Letsou Department of Human Genetics u Classical Forward Genetics vs. Reverse Genetics Embryogenesis DJNK Signaling Cascade s"""' Leading mut edge cell A Robotics Screen for Transcriptionally Regulated Targets of Signaling Robotics Workstation ¦j identify novel genes that are transcriptionally regulated targets of known signaling cascades. The Drosophila melanogaster genome has been sequenced and over 15,000 genes have been identified. We have obtained 16 384-well plates containing cDNA bacterial stocks of these genes representing the Drosophila unigene collection (Research Genetics). With these cDNAs in hand, we are 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. In our screen we use wild- type embryos 0-4 and 8-12 hours after egg lay, which correspond to patterning and morphogenetic stages of embryogenesis. After the whole mount embryo hybridizations in situ are complete, we will document the genetic expression patterns and perform further screens that include sequence analysis of the genes, molecular epistasis experiments, and reverse genetic functional studies. |
