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Genome-wide analysis of Notch signalling in Drosophila by transgenic RNAi

Abstract

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.

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Figure 1: Phenotypic categories and screen results.
Figure 2: Secondary assays for general components of Notch signalling.
Figure 3: Secondary assays for asymmetric cell division genes.
Figure 4: An interaction network for Notch signalling.
Figure 5: Nuclear import and the COP9 signallosome regulate Notch signalling and asymmetric cell division.

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Acknowledgements

Antibodies, plasmids and fly stocks were provided by the Vienna Drosophila RNAi Center, the Bloomington Drosophila Stock Center, the National Institute of Genetics Fly Stock Center (NIG-FLY), the Developmental Studies Hybridoma Bank, C.-T. Chien, R. J. Fleming, T. Klein, R. Lehmann, A. Nose, G. S. Suh and F. Wirtz-Peitz. We thank C. Cowan for comments on the manuscript, V. Rolland for help with secondary analysis, E. Kleiner, G. Haas, S. Reiter, T. Pritz, Z. Topalovic, S. Farina Lopez, S. Wculek and Ö. Copur for technical assistance in constructing the custom second RNAi line collection, C. Gallagher for generating the Su(H) antibody, P. Pasierbek for bio-optics support, F. Stocker for graphics assistance and P. Serrano Drozdowskyj for creating the online database. M.Y. was supported by a European Union Marie Curie Mobility Fellowship. Work in J.A.K.’s laboratory is supported by the Austrian Academy of Sciences, the Wiener Wissenschafts-, Forschungs- und Technologiefonds (WWTF), the Austrian Science Fund (FWF) and the EU network ONCASYM.

Author Contributions J.L.M.-W., M.Y. and J.A.K. designed the experiments. J.L.M.-W. and M.Y. carried out the genetic screen and secondary analysis. T.S. contributed to the secondary analysis. M.N. performed Bioinformatics data analysis. D.C. and S.B. contributed to generation of the second RNAi lines. G.D. and B.J.D. generated and provided the RNAi library. J.L.M.-W. and J.A.K. wrote the paper.

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Correspondence to Juergen A. Knoblich.

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Supplementary Figures

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Supplementary Tables

This file contains Supplementary Tables 1, and 3-10. (PDF 660 kb)

Supplementary Table 2 - Genome-wide bristle screen results

This file contains information such as chromosome insertion site and viability, and target gene mapping for release 5.7 of the Drosophila melanogaster genome is presented together with phenotpic strength in each of 11 categories, stage of lethality (if any) and classification in the "lateral inhibition" category for each transgenic line in the genome-wide screen. (XLS 4442 kb)

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Mummery-Widmer, J., Yamazaki, M., Stoeger, T. et al. Genome-wide analysis of Notch signalling in Drosophila by transgenic RNAi. Nature 458, 987–992 (2009). https://doi.org/10.1038/nature07936

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