The goal of the experiments proposed in this application is to understand how complex patterns of cell differentiation are established in epithelial cells during Drosophila embryonic development. The studies will concentrate on bridging the gap between the expression pattern of segmentation genes at the blastoderm stage and the final pattern of cell differentiation in the epidermis. Much of the work will concentrate on armadillo, the Drosophila homolog of vertebrate beta- catenin, which provides a direct link between cell signaling and cell adhesion. The initial phase of the proposal is a structure function analysis of the arm protein. Most alleles of arm previously identified were selected based on its segment polarity phenotype. The various functions of arm in cell signalling and adhesion uncovered by Dr. Wieschaus during the past funding period form the basis for a new screening based on criteria other than the cuticle phenotype, that should produce new classes of mutations. One of the screens will be based on isolating lethals that fail to complement a null arm allele; the second strategy is based on the ability of some arm mutations to act as suppressors of the sterility of zw3 germline clones. These alleles will be tested for their effects on lethality, cuticular phenotype, and imaginal disc development. Molecular analysis of the lessions associated with these alleles will indicate whether defined domains of the protein have specific functions. The arm protein is localized in the zonula adherens (ZA) of adhesive junctions in epithelial cells. Dr. Wieschaus' lab has demonstrated that genes located in the X chromosome are important in the formation of a proper ZA during early gastrula stages. He proposes to continue this work and identify genes involved in the establishment and maintenance of the ZA by carrying out a screen of embryos deficient for defined regions of the autosomes. These embryos will be examined at the confocal and EM levels using arm and antiphosphotyrosine antibodies to determine the presence and morphology of the ZA in epithelial cells. The temporal and spatial sequence of arm incorporation into the ZA will be examined at the EM level for each mutant. The third part of the proposal will examine the role of arm in wg signaling, both in the maintenance of en expression and induction of wg expression. A comparison between the distribution of wg-containing vesicles and the width of arm stripes will be carried out. Using EM, Dr. Wieschaus will examine whether the increase in arm staining in wg- responding cells is restricted to the cytoplasm or levels also increase at the cell surface. Wg seems to induce its own expression in cells that are not expressing wg due to pair-rule activation, and the role of arm in this process will also be examined. The role of the ZA in wg signaling will be studied in nullo-X embryos. The mechanism of signaling in the wg/zw3/arm pathway will be dissected by identifying new members of the pathway through a genetic screen for suppressors of the sterility of zw3 germline clones. Interesting genes will be cloned and analyzed. The last part of the proposal represents a morphological analysis at the cellular level of the processes of germ band extension and cephalic furrow formation. Scanning EM of embryos broken in cross section will be used to examine changes in cell morphology during intercalation. Changes in Za position and morphology will be followed during extension using arm antibodies. Mutations I the eve gene interfere with the formation of the cephalic furrow, and the role of eve in cell behavior at the cephalic furrow will be examined using a temperature sensitive allele. These effects of eve must take place through its control of transcription of other genes. Dr. Wieschaus will identify these genes by testing defined deficiencies encompassing the whole genome for their effects on early gastrulation that mimic those of eve. Genes of interest will be cloned. These genetic analyses will be complemented with molecular approaches, using differential display to identify RNAs differentially expressed in embryos that fail to undergo germ band extension and cephalic furrow formation.
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