Genome-wide RNA interference (RNAi) screens have identified near-complete sets of genes involved in cellular processes. However, this methodology has not yet been used to study complex developmental processes in a tissue-specific manner. Here we report the use of a library of Drosophila strains expressing inducible hairpin RNAi constructs to study the Notch signalling pathway during external sensory organ development. We assigned putative loss-of-function phenotypes to 21.2% of the protein-coding Drosophila genes. Using secondary assays, we identified 6 new genes involved in asymmetric cell division and 23 novel genes regulating the Notch signalling pathway. By integrating our phenotypic results with protein interaction data, we constructed a genome-wide, functionally validated interaction network governing Notch signalling and asymmetric cell division. We used clustering algorithms to identify nuclear import pathways and the COP9 signallosome as Notch regulators. Our results show that complex developmental processes can be analysed on a genome-wide level and provide a unique resource for functional annotation of the Drosophila genome.Genome-wide RNAi screens have been performed in cultured cells 1 -4 or by ubiquitous gene silencing in worms 5 , 6 or planarians 7 . To study complicated developmental processes, however, genes need to be inactivated in a tissue-specific manner in intact animals. This has become possible through the creation of a transgenic RNAi library targeting 88% of the Drosophila 8 protein-coding genes. To test the feasibility of this new approach, we focused on the Notch pathway, one of the most important regulators of development 9 , 10 . Notch is activated by binding to its ligands Delta or Serrate. After ligand binding, Notch is cleaved by Presenilin and the intracellular domain acts in the nucleus as a transcriptional co-activator. Europe PMC Funders Group Genome-wide RNAi screenHairpin constructs in the RNAi library are expressed under UAS/GAL4 control 18 . We tested scabrous-GAL4 18 , pannier (pnr)-GAL4 19 and fzIII-GAL4 (also known as MS248-GAL4 or P{GawB}MS248) 20 using a set of 40 RNAi lines targeting 21 genes involved in external sensory organ development (Supplementary Table 1). Consistently, phenotypes were stronger and lethality lower with pnr-GAL4, and this line was selected for large-scale analysis.A total of 20,262 transgenic RNAi lines were screened; these are predicted to target 11,619 of the 14,139 protein-coding genes (82.2%) in release 5.7 of the Drosophila genome 21 . Ten flies each were analysed and phenotypic abnormalities were recorded in a database (http:// bristlescreen.imba.oeaw.ac.at). Because pnr-GAL4 is only expressed in a central region of the notum (Fig. 1b), lateral areas were unaffected and served as internal controls. Phenotypes were described using controlled vocabulary (Fig. 1b, Supplementary Table 2). Phenotypic strength (P x ) was expressed on a scale of 0 (not affected) to 10 (completely affected) as the fraction of the pnr-GAL4 expression a...
In Drosophila, planar cell polarity (PCP) molecules such as Dachsous (Ds) may function as global directional cues directing the asymmetrical localization of PCP core proteins such as Frizzled (Fz). However, the relationship between Ds asymmetry and Fz localization in the eye is opposite to that in the wing, thereby causing controversy regarding how these two systems are connected. Here, we show that this relationship is determined by the ratio of two Prickle (Pk) isoforms, Pk and Spiny-legs (Sple). Pk and Sple form different complexes with distinct subcellular localizations. When the amount of Sple is increased in the wing, Sple induces a reversal of PCP using the Ds-Ft system. A mathematical model demonstrates that Sple is the key regulator connecting Ds and the core proteins. Our model explains the previously noted discrepancies in terms of the differing relative amounts of Sple in the eye and wing.
talia. In the female disc, the A9 segment is much smaller than the A8, and it has been shown that the gene double sex (dsx) represses growth in the former, probably by repressing decapentaplegic (dpp) expression. We show here that the absence of Abd-B or homothorax (hth) in this segment also increases
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