Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

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

Nature volume 458, pages 987–992 (2009)Cite this article

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.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

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.

Similar content being viewed by others

References

  1. Kiger, A. A. et al. A functional genomic analysis of cell morphology using RNA interference. J. Biol. 2, 27 (2003)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Boutros, M. et al. Genome-wide RNAi analysis of growth and viability in Drosophila cells. Science 303, 832–835 (2004)

    Article  ADS  CAS  PubMed  Google Scholar 

  3. Bettencourt-Dias, M. et al. Genome-wide survey of protein kinases required for cell cycle progression. Nature 432, 980–987 (2004)

    Article  ADS  CAS  PubMed  Google Scholar 

  4. Goshima, G. et al. Genes required for mitotic spindle assembly in Drosophila S2 cells. Science 316, 417–421 (2007)

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  5. Kamath, R. S. et al. Systematic functional analysis of the Caenorhabditis elegans genome using RNAi. Nature 421, 231–237 (2003)

    Article  ADS  CAS  PubMed  Google Scholar 

  6. Gonczy, P. et al. Functional genomic analysis of cell division in C. elegans using RNAi of genes on chromosome III. Nature 408, 331–336 (2000)

    Article  ADS  CAS  PubMed  Google Scholar 

  7. Reddien, P. W., Bermange, A. L., Murfitt, K. J., Jennings, J. R. & Sanchez Alvarado, A. Identification of genes needed for regeneration, stem cell function, and tissue homeostasis by systematic gene perturbation in planaria. Dev. Cell 8, 635–649 (2005)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Dietzl, G. et al. A genome-wide transgenic RNAi library for conditional gene inactivation in Drosophila . Nature 448, 151–156 (2007)

    Article  ADS  CAS  PubMed  Google Scholar 

  9. Artavanis-Tsakonas, S., Rand, M. D. & Lake, R. J. Notch signaling: cell fate control and signal integration in development. Science 284, 770–776 (1999)

    Article  ADS  CAS  PubMed  Google Scholar 

  10. Schweisguth, F. Notch signaling activity. Curr. Biol. 14, R129–R138 (2004)

    Article  CAS  PubMed  Google Scholar 

  11. Bang, A. G., Hartenstein, V. & Posakony, J. W. Hairless is required for the development of adult sensory organ precursor cells in Drosophila . Development 111, 89–104 (1991)

    CAS  PubMed  Google Scholar 

  12. Schweisguth, F. & Posakony, J. W. Antagonistic activities of Suppressor of Hairless and Hairless control alternative cell fates in the Drosophila adult epidermis. Development 120, 1433–1441 (1994)

    CAS  PubMed  Google Scholar 

  13. Le Borgne, R., Bardin, A. & Schweisguth, F. The roles of receptor and ligand endocytosis in regulating Notch signaling. Development 132, 1751–1762 (2005)

    Article  CAS  PubMed  Google Scholar 

  14. Nagel, A. C., Maier, D. & Preiss, A. Su(H)-independent activity of hairless during mechano-sensory organ formation in Drosophila . Mech. Dev. 94, 3–12 (2000)

    Article  CAS  PubMed  Google Scholar 

  15. Rhyu, M. S., Jan, L. Y. & Jan, Y. N. Asymmetric distribution of numb protein during division of the sensory organ precursor cell confers distinct fates to daughter cells. Cell 76, 477–491 (1994)

    Article  CAS  PubMed  Google Scholar 

  16. O’Connor-Giles, K. M. & Skeath, J. B. Numb inhibits membrane localization of sanpodo, a four-pass transmembrane protein, to promote asymmetric divisions in Drosophila . Dev. Cell 5, 231–243 (2003)

    Article  PubMed  Google Scholar 

  17. Hutterer, A. & Knoblich, J. A. Numb and α-Adaptin regulate Sanpodo endocytosis to specify cell fate in Drosophila external sensory organs. EMBO Rep. 6, 836–842 (2005)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Brand, A. H. & Perrimon, N. Targeted gene expression as a means of altering cell fates and generating dominant phenotypes. Development 118, 401–415 (1993)

    CAS  PubMed  Google Scholar 

  19. Calleja, M. et al. Generation of medial and lateral dorsal body domains by the pannier gene of Drosophila . Development 127, 3971–3980 (2000)

    CAS  PubMed  Google Scholar 

  20. Cavodeassi, F., Rodriguez, I. & Modolell, J. Dpp signalling is a key effector of the wing-body wall subdivision of the Drosophila mesothorax. Development 129, 3815–3823 (2002)

    CAS  PubMed  Google Scholar 

  21. Wilson, R. J., Goodman, J. L. & Strelets, V. B. FlyBase: integration and improvements to query tools. Nucleic Acids Res. 36, D588–D593 (2008)

    Article  CAS  PubMed  Google Scholar 

  22. Kulkarni, M. M. et al. Evidence of off-target effects associated with long dsRNAs in Drosophila melanogaster cell-based assays. Nature Methods 3, 833–838 (2006)

    Article  CAS  PubMed  Google Scholar 

  23. Hartenstein, V. & Posakony, J. W. A dual function of the Notch gene in Drosophila sensillum development. Dev. Biol. 142, 13–30 (1990)

    Article  CAS  PubMed  Google Scholar 

  24. Blair, S. S. Wing vein patterning in Drosophila and the analysis of intercellular signaling. Annu. Rev. Cell Dev. Biol. 23, 293–319 (2007)

    Article  CAS  PubMed  Google Scholar 

  25. Furriols, M. & Bray, S. A model Notch response element detects Suppressor of Hairless-dependent molecular switch. Curr. Biol. 11, 60–64 (2001)

    Article  CAS  PubMed  Google Scholar 

  26. Dove, S. K. et al. Vac14 controls PtdIns(3,5)P(2) synthesis and Fab1-dependent protein trafficking to the multivesicular body. Curr. Biol. 12, 885–893 (2002)

    Article  CAS  PubMed  Google Scholar 

  27. Rusten, T. E. et al. Fab1 phosphatidylinositol 3-phosphate 5-kinase controls trafficking but not silencing of endocytosed receptors. Mol. Biol. Cell 17, 3989–4001 (2006)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Zhang, Y. et al. Loss of Vac14, a regulator of the signaling lipid phosphatidylinositol 3,5-bisphosphate, results in neurodegeneration in mice. Proc. Natl Acad. Sci. USA 104, 17518–17523 (2007)

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  29. Koo, E. H. & Kopan, R. Potential role of presenilin-regulated signaling pathways in sporadic neurodegeneration. Nature Med. 10 (Suppl). S26–S33 (2004)

    Article  PubMed  Google Scholar 

  30. Pi, H., Huang, S. K., Tang, C. Y., Sun, Y. H. & Chien, C. T. phyllopod is a target gene of proneural proteins in Drosophila external sensory organ development. Proc. Natl Acad. Sci. USA 101, 8378–8383 (2004)

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  31. Milan, M., Perez, L. & Cohen, S. M. Boundary formation in the Drosophila wing: functional dissection of Capricious and Tartan. Dev. Dyn. 233, 804–810 (2005)

    Article  CAS  PubMed  Google Scholar 

  32. Milan, M., Weihe, U., Perez, L. & Cohen, S. M. The LRR proteins capricious and Tartan mediate cell interactions during DV boundary formation in the Drosophila wing. Cell 106, 785–794 (2001)

    Article  CAS  PubMed  Google Scholar 

  33. Shinza-Kameda, M., Takasu, E., Sakurai, K., Hayashi, S. & Nose, A. Regulation of layer-specific targeting by reciprocal expression of a cell adhesion molecule, capricious. Neuron 49, 205–213 (2006)

    Article  CAS  PubMed  Google Scholar 

  34. Giot, L. et al. A protein interaction map of Drosophila melanogaster . Science 302, 1727–1736 (2003)

    Article  ADS  CAS  PubMed  Google Scholar 

  35. Gavin, A. C. et al. Functional organization of the yeast proteome by systematic analysis of protein complexes. Nature 415, 141–147 (2002)

    Article  ADS  CAS  PubMed  Google Scholar 

  36. Gavin, A. C. et al. Proteome survey reveals modularity of the yeast cell machinery. Nature 440, 631–636 (2006)

    Article  ADS  CAS  PubMed  Google Scholar 

  37. Crowner, D., Le Gall, M., Gates, M. A. & Giniger, E. Notch steers Drosophila ISNb motor axons by regulating the Abl signaling pathway. Curr. Biol. 13, 967–972 (2003)

    Article  CAS  PubMed  Google Scholar 

  38. Tekotte, H. et al. Dcas is required for importin-α3 nuclear export and mechano-sensory organ cell fate specification in Drosophila . Dev. Biol. 244, 396–406 (2002)

    Article  CAS  PubMed  Google Scholar 

  39. Meshorer, E. & Gruenbaum, Y. Gone with the Wnt/Notch: stem cells in laminopathies, progeria, and aging. J. Cell Biol. 181, 9–13 (2008)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Bosu, D. R. & Kipreos, E. T. Cullin-RING ubiquitin ligases: global regulation and activation cycles. Cell Div. 3, 7 (2008)

    Article  PubMed  PubMed Central  Google Scholar 

  41. Mistry, H., Wilson, B. A., Roberts, I. J., O’Kane, C. J. & Skeath, J. B. Cullin-3 regulates pattern formation, external sensory organ development and cell survival during Drosophila development. Mech. Dev. 121, 1495–1507 (2004)

    Article  CAS  PubMed  Google Scholar 

  42. Wu, J. T., Lin, H. C., Hu, Y. C. & Chien, C. T. Neddylation and deneddylation regulate Cul1 and Cul3 protein accumulation. Nature Cell Biol. 7, 1014–1020 (2005)

    Article  CAS  PubMed  Google Scholar 

  43. Bellaiche, Y., Gho, M., Kaltschmidt, J. A., Brand, A. H. & Schweisguth, F. Frizzled regulates localization of cell-fate determinants and mitotic spindle rotation during asymmetric cell division. Nature Cell Biol. 3, 50–57 (2001)

    Article  CAS  PubMed  Google Scholar 

  44. von Mering, C. et al. STRING 7—recent developments in the integration and prediction of protein interactions. Nucleic Acids Res. 35, D358–D362 (2007)

    Article  CAS  PubMed  Google Scholar 

  45. Breitkreutz, B. J. et al. The BioGRID Interaction Database: 2008 update. Nucleic Acids Res. 36, D637–D640 (2008)

    Article  CAS  PubMed  Google Scholar 

  46. Shannon, P. et al. Cytoscape: a software environment for integrated models of biomolecular interaction networks. Genome Res. 13, 2498–2504 (2003)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  47. Bader, G. D. & Hogue, C. W. An automated method for finding molecular complexes in large protein interaction networks. BMC Bioinformatics 4, 2 (2003)

    Article  PubMed  PubMed Central  Google Scholar 

  48. Gallagher, C. M. & Knoblich, J. A. The conserved c2 domain protein lethal (2) giant discs regulates protein trafficking in Drosophila . Dev. Cell 11, 641–653 (2006)

    Article  CAS  PubMed  Google Scholar 

  49. Lu, B., Ackerman, L., Jan, L. Y. & Jan, Y. N. Modes of protein movement that lead to the asymmetric localization of partner of Numb during Drosophila neuroblast division. Mol. Cell 4, 883–891 (1999)

    Article  CAS  PubMed  Google Scholar 

  50. Rozen, S. & Skaletsky, H. Primer3 on the WWW for general users and for biologist programmers. Methods Mol. Biol. 132, 365–386 (2000)

    CAS  PubMed  Google Scholar 

  51. Groth, A. C., Fish, M., Nusse, R. & Calos, M. P. Construction of transgenic Drosophila by using the site-specific integrase from phage phiC31. Genetics 166, 1775–1782 (2004)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  52. Gho, M., Lecourtois, M., Geraud, G., Posakony, J. W. & Schweisguth, F. Subcellular localization of Suppressor of Hairless in Drosophila sense organ cells during Notch signalling. Development 122, 1673–1682 (1996)

    CAS  PubMed  Google Scholar 

  53. Vaessin, H. et al. prospero is expressed in neuronal precursors and encodes a nuclear protein that is involved in the control of axonal outgrowth in Drosophila . Cell 67, 941–953 (1991)

    Article  CAS  PubMed  Google Scholar 

  54. Remm, M., Storm, C. E. & Sonnhammer, E. L. Automatic clustering of orthologs and in-paralogs from pairwise species comparisons. J. Mol. Biol. 314, 1041–1052 (2001)

    Article  CAS  PubMed  Google Scholar 

Download references

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.

Author information

Author notes

  1. Masakazu Yamazaki

    Present address: Present address: The Global COE program, Akita University School of Medicine, 1-1-1 Hondo, Akita 010-8543, Japan.,

  2. Jennifer L. Mummery-Widmer and Masakazu Yamazaki: These authors contributed equally to this work.

Authors and Affiliations

  1. Institute of Molecular Biotechnology of the Austrian Academy of Sciences (IMBA), Dr Bohr-Gasse 3,,

    Jennifer L. Mummery-Widmer, Masakazu Yamazaki, Thomas Stoeger, Maria Novatchkova, Sheetal Bhalerao & Juergen A. Knoblich

  2. Research Institute of Molecular Pathology (IMP), Dr Bohr-Gasse 7, and,,

    Maria Novatchkova, Sheetal Bhalerao, Georg Dietzl & Barry J. Dickson

  3. Department of Biochemistry, Max F. Perutz Laboratories (MFPL), Dr Bohr-Gasse 9, A-1030 Vienna, Austria,

    Doris Chen

Authors
  1. Jennifer L. Mummery-Widmer
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Masakazu Yamazaki
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Thomas Stoeger
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Maria Novatchkova
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Sheetal Bhalerao
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Doris Chen
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Georg Dietzl
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Barry J. Dickson
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Juergen A. Knoblich
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Juergen A. Knoblich.

Supplementary information

Supplementary Figures

This file contains Supplementary Figures 1-6 with Legends. (PDF 5312 kb)

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)

PowerPoint slides

Rights and permissions

Reprints and permissions

About this article

Cite this article

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

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nature07936

This article is cited by

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing