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:

MAZe: a tool for mosaic analysis of gene function in zebrafish

Nature Methods volume 7, pages 219–223 (2010)Cite this article

Subjects

Abstract

To trace cell lineages in a developing vertebrate and to observe, in vivo, how behaviors of individual cells are affected by the genes they express, we created a zebrafish line containing a transgene called mosaic analysis in zebrafish (MAZe), built around a self-excising hsp70:Cre cassette. Heat shock triggers Cre recombinase–mediated recombination in a random subset of cells, bringing the transcriptional activator Gal4:VP16 under control of the EF1α promoter. Gal4-VP16 then activates expression of a fluorescent protein from an upstream activating sequence (UAS) promoter. Marked clones of cells expressing any desired gene product can be generated by crossing MAZe fish with other lines containing UAS-driven transgenes. The number of clones induced, and their time of origin, could be varied by adjusting heat-shock timing and duration. As an alternative to heat shock, we introduced Cre under a tissue-specific promoter in MAZe fish to generate clones in a designated tissue.

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: The MAZe transgene.
Figure 2: Persistence of MAZe-labeled clones.
Figure 3: Coexpression of nlsRFP with the product of another UAS-driven transgene in MAZe clones.
Figure 4: Activity of proteins expressed from a UAS-driven transgene in MAZe clones.
Figure 5: Tissue-specific induction of MAZe clones.
Figure 6: Live imaging of myoblast fusion using MAZe labeling.

Similar content being viewed by others

References

  1. Stern, C. Somatic crossing over and segregation in Drosophila melanogaster . Genetics 21, 625–730 (1936).

    CAS  PubMed  PubMed Central  Google Scholar 

  2. Stern, C. Genetic mosaics, and other essays (Harvard University Press, Cambridge, Massachusetts, USA, 1968).

  3. Le Douarin, N.M. & Kalcheim, C. The Neural Crest (Cambridge University Press, New York, 1999).

  4. McLaren, A. Mammalian Chimaeras (Cambridge University Press, Cambridge, 1976).

  5. Xu, T. & Rubin, G.M. Analysis of genetic mosaics in developing and adult Drosophila tissues. Development 117, 1223–1237 (1993).

    CAS  PubMed  Google Scholar 

  6. Lee, T. & Luo, L. Mosaic analysis with a repressible cell marker (MARCM) for Drosophila neural development. Trends Neurosci. 24, 251–254 (2001).

    Article  CAS  Google Scholar 

  7. Duffy, J.B. GAL4 system in Drosophila: a fly geneticist's Swiss army knife. Genesis 34, 1–15 (2002).

    Article  CAS  Google Scholar 

  8. Branda, C.S. & Dymecki, S.M. Talking about a revolution: the impact of site-specific recombinases on genetic analyses in mice. Dev. Cell 6, 7–28 (2004).

    Article  CAS  Google Scholar 

  9. Westerfield, M. The zebrafish book. A guide for the laboratory use of zebrafish (Danio rerio) (University of Oregon Press, Eugene, Oregon, USA, 2000).

  10. Halloran, M.C. et al. Laser-induced gene expression in specific cells of transgenic zebrafish. Development 127, 1953–1960 (2000).

    CAS  PubMed  Google Scholar 

  11. Hans, S. et al. Temporally-controlled site-specific recombination in zebrafish. PLoS One 4, e4640 (2009).

    Article  Google Scholar 

  12. Halpern, M.E. et al. Gal4/UAS transgenic tools and their application to zebrafish. Zebrafish 5, 97–110 (2008).

    Article  CAS  Google Scholar 

  13. Scheer, N. & Campos-Ortega, J.A. Use of the Gal4-UAS technique for targeted gene expression in the zebrafish. Mech. Dev. 80, 153–158 (1999).

    Article  CAS  Google Scholar 

  14. Lewis, J. Notch signalling and the control of cell fate choices in vertebrates. Semin. Cell Dev. Biol. 9, 583–589 (1998).

    Article  CAS  Google Scholar 

  15. Kim, C.H. et al. Zebrafish elav/HuC homologue as a very early neuronal marker. Neurosci. Lett. 216, 109–112 (1996).

    Article  CAS  Google Scholar 

  16. Offield, M.F., Hirsch, N. & Grainger, R.M. The development of Xenopus tropicalis transgenic lines and their use in studying lens developmental timing in living embryos. Development 127, 1789–1797 (2000).

    CAS  PubMed  Google Scholar 

  17. Dutton, J.R. et al. An evolutionarily conserved intronic region controls the spatiotemporal expression of the transcription factor Sox10. BMC Dev. Biol. 8, 105 (2008).

    Article  Google Scholar 

  18. Hardy, M.E., Ross, L.V. & Chien, C.B. Focal gene misexpression in zebrafish embryos induced by local heat shock using a modified soldering iron. Dev. Dyn. 236, 3071–3076 (2007).

    Article  Google Scholar 

  19. Urasaki, A., Morvan, G. & Kawakami, K. Functional dissection of the Tol2 transposable element identified the minimal cis-sequence and a highly repetitive sequence in the subterminal region essential for transposition. Genetics 174, 639–649 (2006).

    Article  CAS  Google Scholar 

  20. Bunting, M. et al. Targeting genes for self-excision in the germ line. Genes Dev. 13, 1524–1528 (1999).

    Article  CAS  Google Scholar 

  21. Grabher, C. & Wittbrodt, J. Efficient activation of gene expression using a heat-shock inducible Gal4/Vp16-UAS system in medaka. BMC Biotechnol. 4, 26 (2004).

    Article  Google Scholar 

  22. Shaner, N.C. et al. Improved monomeric red, orange and yellow fluorescent proteins derived from Discosoma sp. red fluorescent protein. Nat. Biotechnol. 22, 1567–1572 (2004).

    Article  CAS  Google Scholar 

  23. Okada, A. et al. Imaging cells in the developing nervous system with retrovirus expressing modified green fluorescent protein. Exp. Neurol. 156, 394–406 (1999).

    Article  CAS  Google Scholar 

  24. Tawk, M. et al. A mirror-symmetric cell division that orch-estrates neuroepithelial morphogenesis. Nature 446, 797–800 (2007).

    Article  CAS  Google Scholar 

  25. Kajita, M. et al. Interaction with surrounding normal epithelial cells influences signalling pathways and behaviour of Srctransformed cells. J. Cell Sci. 123, 171–180 (2010).

    Article  CAS  Google Scholar 

  26. Grabher, C., Joly, J.S. & Wittbrodt, J. Highly efficient zebrafish transgenesis mediated by the meganuclease I-SceI. Methods Cell Biol. 77, 381–401 (2004).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank M. Tada (University College London) for the UAS:gfp transgenic line; R. Kelsh (University of Bath) for the Sox10:gfp transgenic line and the Sox10 promoter sequence; J. Clarke (Kings College London) for the Gap42-GFP plasmid; M. Capecchi (University of Utah School of Medicine) for the pACN plasmid; H. Gerhardt (London Research Institute, Cancer Research UK) for the Cre antibody; L. Zimmerman (National Institute for Medical Research, London) for the γ-crystallin promoter; P. Taylor and D. Martin for fish husbandry; J. Clarke, S. Wilson and D. Ish-Horowicz for comments on the manuscript; and Cancer Research UK for funding.

Author information

Author notes

  1. Claudia Linker

    Present address: Present address: Randall Division of Cell & Molecular Biophysics, King's College London, Guy's Campus, London, UK.,

Authors and Affiliations

  1. Vertebrate Development Laboratory, Cancer Research UK London Research Institute, London, UK

    Russell T Collins, Claudia Linker & Julian Lewis

Authors
  1. Russell T Collins
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Claudia Linker
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Julian Lewis
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

R.T.C. conceived, designed and generated the MAZe transgenic fish; R.T.C. and C.L. characterized the MAZe line; and R.T.C., C.L. and J.L. wrote the paper.

Corresponding authors

Correspondence to Russell T Collins or Julian Lewis.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–2 (PDF 7981 kb)

Supplementary Video 1

Myoblast fusion (MAZe + membrane GFP). A MAZe-labeled myoblast (left red arrow) fuses with neighboring unlabeled myoblast in the dorsal part of a trunk somite. The unlabeled myoblast nucleus becomes fluorescent after cytoplasm continuity is observed (yellow arrow). Thereafter, both nuclei are fluorescent (red arrows) and the intensity of their fluorescence increases over time. MAZe embryos were injected with 100 pg of mRNA encoding membrane-targeted GFP at the 1-2 cell stage and heat-shocked at 39°C for 45 min at 8 h.p.f.; confocal images were obtained from ~18 h.p.f. every 14 min. Each time point corresponds to a single optical section of 3 μm depth. Anterior to the left, dorsal up. (MOV 1667 kb)

Supplementary Video 2

Myoblast fusion (MAZe + UAS:gfp). A myoblast labeled with MAZe and GFP (red arrow) in the medial part of a trunk somite fuses with a neighboring unlabeled myoblast. The myoblast cytoplasm (observed with GFP fluorescence) elongates to span the entire length of the somite, and subsequently a second fluorescent nucleus (second red arrow) is observed in the same cell. Embryos obtained from crossing MAZe to UAS:gfp fish were heat shocked at 39°C for 45 min at 8 h.p.f. and confocal images were obtained from ~18 h.p.f. every 14 min. Each time point corresponds to a single optical section of 3 μm depth. (MOV 1031 kb)

Supplementary Video 3

Dividing MAZe-labeled cell. A MAZe-labeled cell situated just dorsal to the notochord (white arrow) divides. As the nuclear membrane breaks down the fluorescent protein diffuses to fill the cytoplasm of the cell. After cytokinesis the nlsRFP becomes re-localized to the nuclei of the daughter cells, labeling each nucleus with equal intensity. MAZe embryos were heat shocked at 39°C for 45 min at 8 h.p.f. and confocal and bright-field images were obtained from ~30 h.p.f. every 8 min. Each time point corresponds to a projection of confocal sections merged with the corresponding bright field image. Anterior to the left, dorsal up. (MOV 909 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Collins, R., Linker, C. & Lewis, J. MAZe: a tool for mosaic analysis of gene function in zebrafish. Nat Methods 7, 219–223 (2010). https://doi.org/10.1038/nmeth.1423

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nmeth.1423

This article is cited by

Search

Advanced search

Quick links

Nature Briefing: Translational Research

Sign up for the Nature Briefing: Translational Research newsletter — top stories in biotechnology, drug discovery and pharma.

Get what matters in translational research, free to your inbox weekly. Sign up for Nature Briefing: Translational Research