Advertisement

The Notch repressor complex in Drosophila: in vivo analysis of Hairless mutants using overexpression experiments

  • Thomas K. Smylla
  • Markus Meier
  • Anette Preiss
  • Dieter Maier
Original Article

Abstract

During development of higher animals, the Notch signalling pathway governs cell type specification by mediating appropriate gene expression responses. In the absence of signalling, Notch target genes are silenced by repressor complexes. In the model organism Drosophila melanogaster, the repressor complex includes the transcription factor Suppressor of Hairless [Su(H)] and Hairless (H) plus general co-repressors. Recent crystal structure analysis of the Drosophila Notch repressor revealed details of the Su(H)-H complex. They were confirmed by mutational analyses of either protein; however, only Su(H) mutants have been further studied in vivo. Here, we analyse three H variants predicted to affect Su(H) binding. To this end, amino acid replacements Phenylalanine 237, Leucines 245 and 247, as well as Tryptophan 258 to Alanine were introduced into the H protein. A cell-based reporter assay indicates substantial loss of Su(H) binding to the respective mutant proteins HFA, HLLAA and HWA. For in vivo analysis, UAS-lines HFA, HLLAA and HWA were generated to allow spatially restricted overexpression. In these assays, all three mutants resembled the HLD control, shown before to lack Su(H) binding, indicating a strong reduction of H activity. For example, the H variants were impaired in wing margin formation, but unexpectedly induced ectopic wing venation. Concurrent overexpression with Su(H), however, suggests that all mutant H protein isoforms are still able to bind Su(H) in vivo. We conclude that a weakening of the cohesion in the H-Su(H) repressor complex is sufficient for disrupting its in vivo functionality.

Keywords

Drosophila melanogaster Notch antagonist Hairless Notch signalling Notch repression Repressor complex Suppressor of Hairless Overexpression 

Notes

Acknowledgements

We are indebted to Rafael Saup for establishing the NTCTLL245/247AA replacement mutation, to Adriana Schulz, Helena Mastel and Thomas Stößer for technical assistance and to Anja C. Nagel for critical reading of the manuscript, manifold input and discussions.

Author’s contributions

D.M. and A.P. conceived and designed the experiments; T.K.S., M.M., and D.M. conducted the experiments, T.K.S., M.M., A.P. and D.M. collected and analysed the data and performed the statistical analysis; A.P. wrote the manuscript; and all authors have approved the final manuscript.

Funding

This work was supported by grants from the German Science Foundation to DM (MA 1328/10-1 and MA 1328/11-1). The funders had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript.

Compliance with ethical standards

Conflict of interests

The authors declare that they have no conflict of interest.

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

References

  1. Artavanis-Tsakonas S, Matsuno K, Fortini ME (1995) Notch signaling. Science 268:225–232CrossRefGoogle Scholar
  2. Barolo S, Stone T, Bang AG, Posakony JW (2002) Default repression and Notch signaling: Hairless acts as an adaptor to recruit the corepressors Groucho and dCTBP to Suppressor of Hairless. Genes Dev 16:1964–1976CrossRefGoogle Scholar
  3. Bischof J, Maeda RK, Hediger M, Karch F, Basler K (2007) An optimized transgenesis system for Drosophila using germ-line-specific PhiC31 integrases. Proc Natl Acad Sci U S A 104:3312–3317CrossRefGoogle Scholar
  4. Blair SS (2007) Wing vein patterning in Drosophila and the analysis of intercellular signaling. Annu Rev Cell Dev Biol 23:293–319CrossRefGoogle Scholar
  5. Borggrefe T, Oswald F (2009) The Notch signaling pathway: transcriptional regulation at Notch target genes. Cell Mol Life Sci 66:1631–1646CrossRefGoogle Scholar
  6. Borggrefe T, Oswald F (2016) Setting the stage for Notch: the Drosophila Su(H)-Hairless repressor complex. PLoS Biol 14(7):e1002524CrossRefGoogle Scholar
  7. Brand AH, Perrimon N (1993) Targeted gene expression as a means of altering cell fates and generating dominant phenotypes. Development 118:401–415Google Scholar
  8. Bray SJ (2006) Notch signalling: a simple pathway becomes complex. Nat Rev Mol Biol 7:678–689CrossRefGoogle Scholar
  9. Bray SJ, Gomez-Lamarca M (2016) Notch after cleavage. Curr Opin Cell Biol 51:103–109CrossRefGoogle Scholar
  10. Bray S, Musisi H, Bienz M (2005) Bre1 is required for Notch signaling and histone modification. Dev Cell 8:279–286CrossRefGoogle Scholar
  11. Bunch TA, Grinblat Y, Goldstein LS (1988) Characterization and use of the Drosophila metallothionein promoter in cultured Drosophila melanogaster cells. Nucleic Acids Res 16:1043–1061CrossRefGoogle Scholar
  12. Campbell S, Inamdar M, Rodrigues V, Raghavan V, Palazzolo M, Chovnick A (1992) The scalloped gene encodes a novel, evolutionarily conserved transcription factor required for sensory organ differentiation in Drosophila. Genes Dev 6:367–379CrossRefGoogle Scholar
  13. Collins KJ, Yuan Z, Kovall RA (2014) Structure and function of the CSL-KyoT2 corepressor complex: a negative regulator of Notch signaling. Structure 22:70–81CrossRefGoogle Scholar
  14. de Celis JF (1998) Positioning and differentiation of veins in the Drosophila wing. Int J Dev Biol 42:335–344Google Scholar
  15. de Celis JF, Bray SJ (1997) Feed-back mechanisms affecting Notch activation at the dorsoventral boundary in the Drosophila wing. Development 124:3241–3251Google Scholar
  16. de Celis JF, Garcia-Bellido A (1994) Roles of the Notch gene in Drosophila wing morphogenesis. Mech Dev 46:109–122CrossRefGoogle Scholar
  17. Djiane A, Krejci A, Bernard F, Fexova S, Millen K, Bray SJ (2013) Dissecting the mechanisms of Notch induced hyperplasia. EMBO J 32:60–71CrossRefGoogle Scholar
  18. Furriols M, Bray S (2000) Dissecting the mechanisms of Suppressor of Hairless function. Dev Biol 227:520–532CrossRefGoogle Scholar
  19. Ghysen A, Dambly-Chaudière C, Jan LY, Jan YN (1993) Cell interactions and gene interactions in peripheral neurogenesis. Genes Dev 7:723–733CrossRefGoogle Scholar
  20. Go MJ, Eastman DS, Artavanis-Tsakonas S (1998) Cell proliferation control by Notch signaling in Drosophila development. Development 125:2031–2040Google Scholar
  21. Gyuris J, Golemis E, Chertkov H, Brent R (1993) Cdi1, a human G1 and S phase protein phosphatase that associates with cdk2. Cell 75:791–803CrossRefGoogle Scholar
  22. Hartenstein V, Posakony JA (1990) A dual function of the Notch gene in Drosophila sensillum development. Dev Biol 142:13–30CrossRefGoogle Scholar
  23. Hasson P, Paroush Z (2006) Crosstalk between the EGFR and other signalling pathways at the level of the global transcriptional corepressor Groucho/TLE. Br J Cancer 94:771–775CrossRefGoogle Scholar
  24. Hasson P, Egoz N, Winkler C, Volohonsky G, Jia S, Dinur T, Volk T, Courey AJ, Paroush Z (2005) EGFR signaling attenuates Groucho-dependent repression to antagonize Notch transcriptional output. Nat Genet 37:101–105CrossRefGoogle Scholar
  25. Hay BA, Wolff T, Rubin GM (1994) Expression of baculovirus P35 prevents cell death in Drosophila. Development 120:2121–2129Google Scholar
  26. Jiménez G, Paroush Z, Ish-Horowicz D (1997) Groucho acts as a corepressor for a subset of negative regulators, including Hairy and Engrailed. Genes Dev 11:3072–3082CrossRefGoogle Scholar
  27. Johannes B, Preiss A (2002) Wing vein formation in Drosophila melanogaster Hairless is involved in the cross-talk between Notch and EGF signaling pathways. Mech Dev 115:3–14CrossRefGoogle Scholar
  28. Kim J, Irvine KD, Caroll SB (1995) Cell recognition, signal induction, and symmetrical gene activation at the dorsal-ventral boundary of the developing Drosophila wing. Cell 82:795–802CrossRefGoogle Scholar
  29. Klein T, Martinez Arias A (1998) Differential spatial and temporal interaction between Notch, wingless, and vestigial specify proximal and distal pattern elements of the wing in Drosophila. Dev Biol 194:196–212CrossRefGoogle Scholar
  30. Koelzer S, Klein T (2003) A Notch-independent function of Suppressor of Hairless during the development of the bristle sensory organ precursor cell of Drosophila. Development 130:1973–1988CrossRefGoogle Scholar
  31. Kopan R, Ilagan MXG (2009) The canonical Notch signaling pathway: unfolding the activation mechanism. Cell 137:216–233CrossRefGoogle Scholar
  32. Kovall RA, Blacklow SC (2010) Mechanistic insights into Notch receptor signaling from structural and biochemical studies. Curr Top Dev Biol 92:31–71CrossRefGoogle Scholar
  33. Kurth P, Preiss A, Kovall RA, Maier D (2011) Molecular analysis of the Notch repressor-complex in Drosophila: characterization of potential Hairless binding sites on Suppressor of Hairless. PLoS One 6(11):e277986CrossRefGoogle Scholar
  34. Lecuit T, Brook WJ, Ng M, Calleja M, Sun H, Cohen SM (1996) Two distinct mechanisms for long-range patterning by Decapentaplegic in the Drosophila wing. Nature 381:387–393CrossRefGoogle Scholar
  35. Lindsley D, Zimm GG (1992) The genome of Drosophila melanogaster. Academic Press, San DiegoGoogle Scholar
  36. Maier D (2006) Hairless, the ignored antagonist of the Notch signalling pathway. Hereditas 143:212–221CrossRefGoogle Scholar
  37. Maier D, Marquart J, Thompson-Fontaine A, Beck I, Wurmbach E, Preiss A (1997) In vivo structure-function analysis of Drosophila Hairless. Mech Dev 67:97–106CrossRefGoogle Scholar
  38. Maier D, Nagel AC, Preiss A (2002) Two isoforms of the Notch antagonist Hairless are produced by differential translation initiation. Proc Natl Acad Sci U S A 99:15480–15485CrossRefGoogle Scholar
  39. Maier D, Chen AX, Preiss A, Ketelhut M (2008) The tiny Hairless protein from Apis mellifera: a potent antagonist of Notch signaling in Drosophila melanogaster. BMC Evol Biol 8:175CrossRefGoogle Scholar
  40. Maier D, Kurth P, Schulz A, Russel A, Yuan Z, Gruber K et al (2011) Structural and functional analysis of the repressor complex in the Notch signaling pathway of Drosophila melanogaster. Mol Cell Biol 22:3242–3252CrossRefGoogle Scholar
  41. Möckli N, Auerbach D (2004) Quantitative beta-galactosidase assay suitable for high-throughput applications in the yeast two-hybrid system. Biotechniques 36(5):872–876CrossRefGoogle Scholar
  42. Morel V, Lecourtois M, Massiani O, Maier D, Preiss A, Schweisguth F (2001) Transcriptional repression by Suppressor of Hairless involves the binding of a Hairless-dCtBP complex in Drosophila. Curr Biol 11(10):789–792CrossRefGoogle Scholar
  43. Nagel AC, Maier D, Preiss A (2000) Su(H)-independent activity of Hairless during mechano-sensory organ formation in Drosophila. Mech Dev 94:3–12CrossRefGoogle Scholar
  44. Nagel AC, Krejci A, Tenin G, Bravo-Patino A, Bray S, Maier D, Preiss A (2005) Hairless-mediated repression of Notch target genes requires the combined activity of Groucho and CtBP co-repressors. Mol Cell Biol 25:10433–10441CrossRefGoogle Scholar
  45. Praxenthaler H, Smylla TK, Nagel AC, Preiss A, Maier D (2015) Generation of new Hairless alleles by genomic engineering at the Hairless locus in Drosophila melanogaster. PLoS ONE 10(10):e0140007Google Scholar
  46. Praxenthaler H, Nagel AC, Schulz A, Zimmermann M, Meier M, Schmid H, Preiss A, Maier D (2017) Hairless-binding deficient Suppressor of Hairless alleles reveal Su(H) protein levels are dependent on complex formation with Hairless. PLoS Genet 13(5):e1006774CrossRefGoogle Scholar
  47. Protzer CE, Wech I, Nagel AC (2008) Hairless induces cell death by downregulation of EGFR signalling activity. J Cell Sci 121:3167–3176CrossRefGoogle Scholar
  48. Rooke JE, Xu T (1998) Positive and negative signals between interacting cells for establishing neural fate. BioEssays 20:209–214CrossRefGoogle Scholar
  49. Schweisguth F, Gho M, Lecourtois M (1996) Control of cell fate choices by lateral signaling in the adult peripheral nervous system of Drosophila melanogaster. Dev Genet 18:28–39CrossRefGoogle Scholar
  50. Tabaja N, Yuan Z, Oswald F, Kovall RA (2017) Structure-function analysis of RBP-J-interacting and tubulin-associated (RITA) reveals regions critical for repression of Notch target genes. J Biol Chem 292:10549–10563CrossRefGoogle Scholar
  51. VanderWielen BD, Yuan Z, Friedmann DR, Kovall RA (2011) Transcriptional repression in the Notch pathway: thermodynamic characterization of CSL-MINT (Msx2-interacting nuclear target protein) complexes. J Biol Chem 286:14892–14902CrossRefGoogle Scholar
  52. Yuan Z, Praxenthaler H, Tabaj N, Torella R, Preiss A, Maier D et al (2016) Structure and function of the Su(H)-Hairless repressor complex, the major antagonist of Notch signalling in Drosophila melanogaster. PLoS Biol 14(7):e1002509CrossRefGoogle Scholar
  53. Zehender A, Bayer M, Bauer M, Zeis B, Preiss A, Maier D (2017) Conservation of the Notch antagonist Hairless in arthropods: functional analysis of the crustacean Daphnia pulex Hairless gene. Dev Genes Evol 227(5):339–353CrossRefGoogle Scholar
  54. Zimmermann M, Kugler SJ, Schulz A, Nagel AC (2015) Loss of putzig activity results in apoptosis during wing imaginal development in Drosophila. PLoS One 10(4):e0124652CrossRefGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Institut für GenetikUniversität HohenheimStuttgartGermany

Personalised recommendations