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A high throughput screen to identify secreted and transmembrane proteins involved in Drosophila embryogenesis.

ABSTRACT: Secreted and transmembrane proteins play an essential role in intercellular communication during the development of multicellular organisms. Because only a small number of these genes have been characterized, we developed a screen for genes encoding extracellular proteins that are differentially expressed during Drosophila embryogenesis. Our approach utilizes a new method for screening large numbers of cDNAs by whole-embryo in situ hybridization. The cDNA library for the screen was prepared from rough endoplasmic reticulum-bound mRNA and is therefore enriched in clones encoding membrane and secreted proteins. To increase the prevalence of rare cDNAs in the library, the library was normalized using a method based on cDNA hybridization to genomic DNA-coated beads. In total, 2,518 individual cDNAs from the normalized library were screened by in situ hybridization, and 917 of these cDNAs represent genes differentially expressed during embryonic development. Sequence analysis of 1,001 cDNAs indicated that 811 represent genes not previously described in Drosophila. Expression pattern photographs and partial DNA sequences have been assembled in a database publicly available at the Berkeley Drosophila Genome Project website (). The identification of a large number of genes encoding proteins involved in cell-cell contact and signaling will advance our knowledge of the mechanisms by which multicellular organisms and their specialized organs develop.

SUBMITTER: Kopczynski CC

PROVIDER: S-EPMC21446 | biostudies-literature | 1998 Aug

REPOSITORIES: biostudies-literature

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A high throughput screen to identify secreted and transmembrane proteins involved in Drosophila embryogenesis.

Kopczynski C C CC Noordermeer J N JN Serano T L TL Chen W Y WY Pendleton J D JD Lewis S S Goodman C S CS Rubin G M GM

Proceedings of the National Academy of Sciences of the United States of America 19980801 17

Secreted and transmembrane proteins play an essential role in intercellular communication during the development of multicellular organisms. Because only a small number of these genes have been characterized, we developed a screen for genes encoding extracellular proteins that are differentially expressed during Drosophila embryogenesis. Our approach utilizes a new method for screening large numbers of cDNAs by whole-embryo in situ hybridization. The cDNA library for the screen was prepared from ...[more]

PMID: 9707585

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Project description:BackgroundMorphogenetic events that shape the Drosophila melanogaster embryo are tightly controlled by a genetic program in which specific sets of genes are up-regulated. We used a suppressive subtractive hybridization procedure to identify a group of developmentally regulated genes during early stages of D. melanogaster embryogenesis. We studied the spatiotemporal activity of these genes in five different intervals covering 12 stages of embryogenesis.ResultsMicroarrays were constructed to confirm induction of expression and to determine the temporal profile of isolated subtracted cDNAs during embryo development. We identified a set of 118 genes whose expression levels increased significantly in at least one developmental interval compared with a reference interval. Of these genes, 53% had a phenotype and/or molecular function reported in the literature, whereas 47% were essentially uncharacterized. Clustering analysis revealed demarcated transcript groups with maximum gene activity at distinct developmental intervals. In situ hybridization assays were carried out on 23 uncharacterized genes, 15 of which proved to have spatiotemporally restricted expression patterns. Among these 15 uncharacterized genes, 13 were found to encode putative secreted and transmembrane proteins. For three of them we validated our protein sequence predictions by expressing their cDNAs in Drosophila S2R+ cells and analyzed the subcellular distribution of recombinant proteins. We then focused on the functional characterization of the gene CG6234. Inhibition of CG6234 by RNA interference resulted in morphological defects in embryos, suggesting the involvement of this gene in germ band retraction.ConclusionOur data have yielded a list of developmentally regulated D. melanogaster genes and their expression profiles during embryogenesis and provide new information on the spatiotemporal expression patterns of several uncharacterized genes. In particular, we recovered a substantial number of unknown genes encoding putative secreted and transmembrane proteins, suggesting new components of signaling pathways that might be incorporated within the existing regulatory networks controlling D. melanogaster embryogenesis. These genes are also good candidates for additional targeted functional analyses similar to those we conducted for CG6234.See related minireview by Vichas and Zallen: http://www.jbiol.com/content/8/8/76.

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A high throughput screen to identify secreted and transmembrane proteins involved in Drosophila embryogenesis.

Publications

A high throughput screen to identify secreted and transmembrane proteins involved in Drosophila embryogenesis.

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