Skip to main content
SpringerLink
Log in

Visualizing Notch Signaling In Vivo in Drosophila Tissues

  • Protocol
  • First Online:
Notch Signaling

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1187))

Abstract

The ability to visualize Notch pathway activity in vivo is invaluable for studying the functions and mechanisms of Notch signaling. A variety of tools have been developed to enable monitoring of pathway activity in Drosophila, including endogenous Notch-responsive genes and synthetic transcriptional reporter constructs. Here we summarize some of the different Notch signaling reporters that are available, discuss their relative merits, and describe two methods for visualizing their expression (immunostaining and X-gal staining). These approaches are widely applicable to a range of tissues and stages in Drosophila development.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution
Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 89.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 119.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 169.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  1. Bray SJ (2006) Notch signalling: a simple pathway becomes complex. Nat Rev Mol Cell Biol 7:678–689

    Article  CAS  PubMed  Google Scholar 

  2. Fryer CJ, Lamar E, Turbachova I et al (2002) Mastermind mediates chromatin-specific transcription and turnover of the Notch enhancer complex. Genes Dev 16:1397–1411

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  3. Fryer CJ, White JB, Jones KA (2004) Mastermind recruits CycC:CDK8 to phosphorylate the Notch ICD and coordinate activation with turnover. Mol Cell 16:509–520

    Article  CAS  PubMed  Google Scholar 

  4. Schroeter EH, Kisslinger JA, Kopan R (1998) Notch-1 signalling requires ligand-induced proteolytic release of intracellular domain. Nature 393:382–386

    Article  CAS  PubMed  Google Scholar 

  5. Micchelli CA, Rulifson EJ, Blair SS (1997) The function and regulation of cut expression on the wing margin of Drosophila: Notch, wingless and a dominant negative role for Delta and Serrate. Development 124:1485–1495

    CAS  PubMed  Google Scholar 

  6. Bailey AM, Posakony JW (1995) Suppressor of hairless directly activates transcription of enhancer of split complex genes in response to Notch receptor activity. Genes Dev 9:2609–2622

    Article  CAS  PubMed  Google Scholar 

  7. Jennings B, Preiss A, Delidakis C et al (1994) The Notch signalling pathway is required for Enhancer of split bHLH protein expression during neurogenesis in the Drosophila embryo. Development 120:3537–3548

    CAS  PubMed  Google Scholar 

  8. Lai EC, Bodner R, Posakony JW (2000) The enhancer of split complex of Drosophila includes four Notch-regulated members of the bearded gene family. Development 127:3441–3455

    CAS  PubMed  Google Scholar 

  9. Lecourtois M, Schweisguth F (1995) The neurogenic suppressor of hairless DNA-binding protein mediates the transcriptional activation of the enhancer of split complex genes triggered by Notch signaling. Genes Dev 9:2598–2608

    Article  CAS  PubMed  Google Scholar 

  10. Wech I, Bray S, Delidakis C et al (1999) Distinct expression patterns of different enhancer of split bHLH genes during embryogenesis of Drosophila melanogaster. Dev Genes Evol 209:370–375

    Article  CAS  PubMed  Google Scholar 

  11. de Celis JF, de Celis J, Ligoxygakis P et al (1996) Functional relationships between Notch, Su(H) and the bHLH genes of the E(spl) complex: the E(spl) genes mediate only a subset of Notch activities during imaginal development. Development 122:2719–2728

    PubMed  Google Scholar 

  12. Nellesen DT, Lai EC, Posakony JW (1999) Discrete enhancer elements mediate selective responsiveness of enhancer of split complex genes to common transcriptional activators. Dev Biol 213:33–53

    Article  CAS  PubMed  Google Scholar 

  13. Furriols M, Bray S (2001) A model Notch response element detects suppressor of hairless-dependent molecular switch. Curr Biol 11:60–64

    Article  CAS  PubMed  Google Scholar 

  14. Housden BE, Millen K, Bray SJ (2012) Drosophila reporter vectors compatible with phiC31 integrase transgenesis techniques and their use to generate new Notch reporter fly lines. G3 Bethesda 2:79–82

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  15. Saj A, Arziman Z, Stempfle D et al (2010) A combined ex vivo and in vivo RNAi screen for notch regulators in Drosophila reveals an extensive notch interaction network. Dev Cell 18:862–876

    Article  CAS  PubMed  Google Scholar 

  16. Go MJ, Eastman DS, Artavanis-Tsakonas S (1998) Cell proliferation control by Notch signaling in Drosophila development. Development 125:2031–2040

    CAS  PubMed  Google Scholar 

  17. Li X, Zhao X, Fang Y et al (1998) Generation of destabilized green fluorescent protein as a transcription reporter. J Biol Chem 273:34970–34975

    Article  CAS  PubMed  Google Scholar 

  18. Presente A, Shaw S, Nye JS et al (2002) Transgene-mediated RNA interference defines a novel role for notch in chemosensory startle behavior. Genesis 34:165–169

    Article  CAS  PubMed  Google Scholar 

  19. Helms W, Lee H, Ammerman M et al (1999) Engineered truncations in the Drosophila mastermind protein disrupt Notch pathway function. Dev Biol 215:358–374

    Article  CAS  PubMed  Google Scholar 

  20. McGuire SE, Le PT, Osborn AJ et al (2003) Spatiotemporal rescue of memory dysfunction in Drosophila. Science 302:1765–1768

    Article  CAS  PubMed  Google Scholar 

  21. Brand AH, Perrimon N (1993) Targeted gene expression as a means of altering cell fates and generating dominant phenotypes. Development 118:401–415

    CAS  PubMed  Google Scholar 

  22. Xu T, Rubin GM (1993) Analysis of genetic mosaics in developing and adult Drosophila tissues. Development 117:1223–1237

    CAS  PubMed  Google Scholar 

  23. Lee T, Luo L (1999) Mosaic analysis with a repressible cell marker for studies of gene function in neuronal morphogenesis. Neuron 22:451–461

    Article  CAS  PubMed  Google Scholar 

  24. Classen AK, Aigouy B, Giangrande A et al (2008) Imaging Drosophila pupal wing morphogenesis. Methods Mol Biol 420:265–275

    Article  CAS  PubMed  Google Scholar 

  25. Muller HA (2008) Immunolabeling of embryos. Methods Mol Biol 420:207–218

    Article  PubMed Central  PubMed  Google Scholar 

  26. Stocker H, Gallant P (2008) Getting started : an overview on raising and handling Drosophila. Methods Mol Biol 420:27–44

    Article  PubMed  Google Scholar 

  27. Cooper MT, Tyler DM, Furriols M et al (2000) Spatially restricted factors cooperate with notch in the regulation of Enhancer of split genes. Dev Biol 221:390–403

    Article  CAS  PubMed  Google Scholar 

  28. de Celis JF, Tyler DM, de Celis J et al (1998) Notch signalling mediates segmentation of the Drosophila leg. Development 125:4617–4626

    PubMed  Google Scholar 

  29. Pines MK, Housden BE, Bernard F et al (2010) The cytolinker Pigs is a direct target and a negative regulator of Notch signalling. Development 137:913–922

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  30. Assa-Kunik E, Torres IL, Schejter ED et al (2007) Drosophila follicle cells are patterned by multiple levels of Notch signaling and antagonism between the Notch and JAK/STAT pathways. Development 134:1161–1169

    Article  CAS  PubMed  Google Scholar 

  31. Almeida MS, Bray SJ (2005) Regulation of post-embryonic neuroblasts by Drosophila Grainyhead. Mech Dev 122:1282–1293

    Article  CAS  PubMed  Google Scholar 

  32. Cooper MT, Bray SJ (1999) Frizzled regulation of Notch signalling polarizes cell fate in the Drosophila eye. Nature 397:526–530

    Article  CAS  PubMed  Google Scholar 

  33. Kramatschek B, Campos-Ortega JA (1994) Neuroectodermal transcription of the Drosophila neurogenic genes E(spl) and HLH-m5 is regulated by proneural genes. Development 120:815–826

    CAS  PubMed  Google Scholar 

  34. Castro B, Barolo S, Bailey AM et al (2005) Lateral inhibition in proneural clusters: cis-regulatory logic and default repression by suppressor of hairless. Development 132:3333–3344

    Article  CAS  PubMed  Google Scholar 

  35. Barolo S, Castro B, Posakony JW (2004) New Drosophila transgenic reporters: insulated P-element vectors expressing fast-maturing RFP. Biotechniques 36:436–440, 442

    CAS  PubMed  Google Scholar 

  36. Afek Y, Alon N, Barad O et al (2011) A biological solution to a fundamental distributed computing problem. Science 331:183–185

    Article  CAS  PubMed  Google Scholar 

  37. Williams JA, Paddock SW, Vorwerk K et al (1994) Organization of wing formation and induction of a wing-patterning gene at the dorsal/ventral compartment boundary. Nature 368:299–305

    Article  CAS  PubMed  Google Scholar 

  38. Djiane A, Krejci A, Bernard F et al (2013) Dissecting the mechanisms of Notch induced hyperplasia. EMBO J 32:60–71

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  39. Blochlinger K, Bodmer R, Jack J et al (1988) Primary structure and expression of a product from cut, a locus involved in specifying sensory organ identity in Drosophila. Nature 333:629–635

    Article  CAS  PubMed  Google Scholar 

  40. Brook WJ, Cohen SM (1996) Antagonistic interactions between wingless and decapentaplegic responsible for dorsal-ventral pattern in the Drosophila Leg. Science 273:1373–1377

    Article  CAS  PubMed  Google Scholar 

  41. Yip ML, Lamka ML, Lipshitz HD (1997) Control of germ-band retraction in Drosophila by the zinc-finger protein HINDSIGHT. Development 124:2129–2141

    CAS  PubMed  Google Scholar 

  42. Giráldez AJ, Pérez L, Cohen SM (2002) A naturally occurring alternative product of the mastermind locus that represses notch signalling. Mech Dev 115:101–105

    Article  PubMed  Google Scholar 

  43. Lieber T, Kidd S, Alcamo E et al (1993) Antineurogenic phenotypes induced by truncated Notch proteins indicate a role in signal transduction and may point to a novel function for Notch in nuclei. Genes Dev 7:1949–1965

    Article  CAS  PubMed  Google Scholar 

  44. Seugnet L, Simpson P, Haenlin M (1997) Requirement for dynamin during Notch signaling in Drosophila neurogenesis. Dev Biol 192:585–598

    Article  CAS  PubMed  Google Scholar 

  45. Rebay I, Fehon RG, Artavanis-Tsakonas S (1993) Specific truncations of Drosophila Notch define dominant activated and dominant negative forms of the receptor. Cell 74:319–329

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

We would like to thank Hamid Moosavi for contributing data used in Fig. 1.

Author information

Authors and Affiliations

  1. Department of Genetics, Harvard Medical School, 77 Avenue Louis Pasteur, NRB 339, 02115, Boston, MA, USA

    Benjamin E. Housden

  2. Department of Physiology, Development and Neuroscience, University of Cambridge, Downing Street, CB2 3DY, Cambridge, UK

    Benjamin E. Housden, Jinghua Li & Sarah J. Bray

Authors
  1. Benjamin E. Housden
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Jinghua Li
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Sarah J. Bray
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Sarah J. Bray .

Editor information

Editors and Affiliations

  1. Dept.of Molecular and Human Genetics, Program in Developmental Biology, Dept. of Neuroscience, Jan and Dan Duncan Neurological Research Institute at Texas Children's Hospital, Howard Hughes Medical Institute, Baylor College of Medicine, Houston, Texas, USA

    Hugo J. Bellen

  2. Dept. of Molecular and Human Genetics, Program in Developmental Biology, Jan and Dan Duncan Neurological Research Institute at Texas Children's Hospital, Baylor College of Medicine, Houston, Texas, USA

    Shinya Yamamoto

Rights and permissions

Reprints and permissions

Copyright information

© 2014 Springer Science+Business Media New York

About this protocol

Cite this protocol

Housden, B.E., Li, J., Bray, S.J. (2014). Visualizing Notch Signaling In Vivo in Drosophila Tissues. In: Bellen, H., Yamamoto, S. (eds) Notch Signaling. Methods in Molecular Biology, vol 1187. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-1139-4_8

Download citation

  • DOI: https://doi.org/10.1007/978-1-4939-1139-4_8

  • Published:

  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-1138-7

  • Online ISBN: 978-1-4939-1139-4

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation