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.

  • Protocol
  • Published:

Determination of gene expression patterns using in situ hybridization to Drosophila testes

Nature Protocols volume 4, pages 1807–1819 (2009)Cite this article

Abstract

We describe a whole-mount RNA in situ hybridization (ISH) method optimized for detection of the cellular and subcellular distributions of specific mRNA within Drosophila testes and male genital tract. Digoxygenin (dig)-labeled antisense RNA probes are in vitro transcribed from a template synthesized by (RT)-PCR; the probe length is reduced by hydrolysis. Testes and male genital tracts are dissected from adult flies, fixed and processed for hybridization. Both probe and fixed testes can be stored before use. Extensive post-hybridization washing reduces the background. Detection is through alkaline phosphatase-conjugated anti-dig antibodies followed by a color reaction. This protocol is suitable for low-medium throughput applications with parallel processing of 2–48 samples, and takes 4–5 d to complete. We have used this protocol, which is similar to other RNA ISH protocols, but optimized for whole-mount Drosophila testes, to document the expression of about 1,000 genes in Drosophila melanogaster male genital tract.

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: Optimization of the protocol.
Figure 2: Flow diagram showing an overview of the ISH procedure.
Figure 3: Example of a positive and negative control.
Figure 4: Anticipated results for control genes.
Figure 5: Preparation of hydrolyzed and unhydrolyzed probes.
Figure 6: Comparison of the performance of hydrolyzed and unhydrolyzed probes.
Figure 7: Anticipated results.

Similar content being viewed by others

References

  1. Fuller, M.T. Spermatogenesis. In The Development of Drosophila. (eds. Bate, M. & Martinez-Arias, A.) 71–147 (Cold Spring Harbor Press, Cold Spring Harbor, New York, 1993).

    Google Scholar 

  2. Barreau, C., Benson, E., Gudmannsdottir, E., Newton, F. & White-Cooper, H. Post-meiotic transcription in Drosophila testes. Development 135, 1897–1902 (2008).

    Article  CAS  PubMed  Google Scholar 

  3. Olivieri, G. & Olivieri, A. Autoradiographic study of nucleic acid synthesis during spermatogenesis in Drosophila melanogaster . Mutat. Res. 2, 366–380 (1965).

    Article  CAS  PubMed  Google Scholar 

  4. Schäfer, M., Nayernia, K., Engel, W. & Schäfer, U. Translational control in spermatogenesis. Dev. Biol. 172, 344–352 (1995).

    Article  PubMed  Google Scholar 

  5. Hales, K.G. & Fuller, M.T. Developmentally regulated mitochondrial fusion mediated by a conserved novel predicted GTPase. Cell 90, 121–129 (1997).

    Article  CAS  PubMed  Google Scholar 

  6. Vardanyan, A. et al. Dumpy-30 family members as determinants of male fertility and interaction partners of metal-responsive transcription factor 1 (MTF-1) in Drosophila . BMC Dev. Biol. 8, 68 (2008).

    Article  PubMed  PubMed Central  Google Scholar 

  7. Kuhn, R., Schafer, U. & Schafer, M. Cis-acting regions sufficient for spermatocyte-specific transcriptional and spermatid-specific translational control of the Drosophila melanogaster gene mst(3)gl-9 . EMBO J. 7, 447–454 (1988).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. White-Cooper, H., Schafer, M.A., Alphey, L.S. & Fuller, M.T. Transcriptional and post-transcriptional control mechanisms coordinate the onset of spermatid differentiation with meiosis I in Drosophila . Development 125, 125–134 (1998).

    CAS  PubMed  Google Scholar 

  9. Chintapalli, V., Wang, J. & Dow, J. Using FlyAtlas to identify better Drosophila models of human disease. Nat. Genet. 39, 715–720 (2007).

    Article  CAS  PubMed  Google Scholar 

  10. Tautz, D. & Pfeifle, C. A non-radioactive in situ hybridization method for the localization of specific RNAs in Drosophila embryos reveals translational control of the segmentation gene hunchback . Chromosoma 98, 81–85 (1989).

    Article  CAS  PubMed  Google Scholar 

  11. Lehmann, R. & Tautz, D. In situ hybridization to RNA. Methods Cell Biol. 44, 575–598 (1994).

    Article  CAS  PubMed  Google Scholar 

  12. Roche. Nonradioactive In Situ Hybridization Application Manual (Roche Diagnostics GmbH, Penzberg, Germany, 2002).

  13. Alphey, L. et al. twine, a cdc25 homologue that functions in the male and female germlines of Drosophila . Cell 69, 977–988 (1992).

    Article  CAS  PubMed  Google Scholar 

  14. Wang, F. et al. Laminin in hte male germ cells of Drosophila . J. Cell Biol. 119, 977–988 (1992).

    Article  CAS  PubMed  Google Scholar 

  15. Lecuyer, E. et al. Global analysis of mRNA localisation reveals a prominent role in organizing cellular architecture and function. Cell 131, 174–187 (2007).

    Article  CAS  PubMed  Google Scholar 

  16. Robida, M.D. & Singh, R. Drosophila polypyrimidine-tract binding protein (PTB) functions specifically in the male germline. EMBO J. 22, 2924–2933 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Harhangi, H.R. et al. RADHA—a new male germ line-specific chromosomal protein of Drosophila . Chromosoma 108, 235–242 (1999).

    Article  CAS  PubMed  Google Scholar 

  18. Rothwell, W.F. & Sullivan, W. Fluorescent analysis of Drosophila embryos. In Drosophila Protocols (eds. Sullivan, W., Ashburner, M. & Hawley, R.S.) 141–157 (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 2000).

  19. White-Cooper, H. Spermatogenesis: Analysis of meiosis and morphogenesis. In Drosophila Cytogenetics Protocols. (ed. Henderson, D.) 45–75 (Humana Press, Totowa, New Jersey, 2004).

    Google Scholar 

  20. Weiszmann, R., Hammonds, A.S. & Celniker, S.E. Determination of gene expression patterns using high-throughput RNA in situ hybridisation to whole-mount Drosophila embryos. Nat. Protoc. 4, 605–618 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

We thank the succession of project students, technicians, PhD students and post-docs who have over the years carried out RNA in situ hybridization in the lab. Between all of us we have probably made every mistake possible in this protocol, thus identifying the important steps. We are very grateful for support from the UK Biotechnology and Biological Sciences Research Council for our testis RNA in situ hybridization projects (Grants BB/C503903/1 and BB_D009324_1). H.W.-C. is a Royal Society University Research Fellow.

Author information

Author notes

  1. Elizabeth Benson

    Present address: Present address: Journal of Biology, London, UK.,

Authors and Affiliations

  1. Department of Biosciences, University of Cardiff, Cardiff, UK

    Ceri A Morris & Helen White-Cooper

  2. Department of Zoology, University of Oxford, Oxford, UK

    Elizabeth Benson

Authors
  1. Ceri A Morris
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Elizabeth Benson
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Helen White-Cooper
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

All authors contributed extensively to the work presented in this paper. H.W.-C. adapted and optimized the non-radioactive ISH methods from Drosophila ovaries and testes to work reliably and with minimal background in Drosophila testes and contributed extensively to the paper writing. E.B. previously performed many experiments with the method and contributed to making it a robust assay, particularly by teaching others. She acted as the 'experienced operator' and gave feedback/technical tips on the protocol/paper. C.M. acted as the 'inexperienced operator,' contributed extensively to the writing of the paper and ran many of the experiments used in the figures, all of which are novel and have not been previously published.

Corresponding author

Correspondence to Helen White-Cooper.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Morris, C., Benson, E. & White-Cooper, H. Determination of gene expression patterns using in situ hybridization to Drosophila testes. Nat Protoc 4, 1807–1819 (2009). https://doi.org/10.1038/nprot.2009.192

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nprot.2009.192

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