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:

Studying cell behavior in whole zebrafish embryos by confocal live imaging: application to hematopoietic stem cells

Nature Protocols volume 6, pages 1897–1904 (2011)Cite this article

Subjects

Abstract

Confocal live imaging is a key tool for studying cell behavior in the whole zebrafish embryo. Here we provide a detailed protocol that is adaptable for imaging any progenitor cell behavior in live zebrafish embryos. As an example, we imaged the emergence of the first hematopoietic stem cells from the aorta. We discuss the importance of selecting the appropriate zebrafish transgenic line as well as methods for immobilization of embryos to be imaged. In addition, we highlight the confocal microscopy acquisition parameters required for stem cell imaging and the software tools we used to analyze 4D movies. The whole protocol takes 2 h 15 min and allows confocal live imaging from a few hours to several days.

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: Time-lapse confocal live imaging: three methods for immobilization of a zebrafish embryo.
Figure 2: Proper embryo orientation in lateral position determines the quality of EHT imaging.
Figure 3: Imaging of the kdrl:GFP+ definitive blood progenitors (HSPCs) emerging in the zebrafish AGM.
Figure 4: 3D analyses of confocal time-lapse sequence of endothelial cells exiting the ventral wall of the aorta to become free HSPCs.

Similar content being viewed by others

References

  1. Godin, I. & Cumano, A. The hare and the tortoise: an embryonic haematopoietic race. Nat. Rev. Immunol. 2, 593–604 (2002).

    Article  CAS  Google Scholar 

  2. Murayama, E. et al. Tracing hematopoietic precursor migration to successive hematopoietic organs during zebrafish development. Immunity 25, 963–975 (2006).

    Article  CAS  Google Scholar 

  3. Jin, H., Xu, J. & Wen, Z. Migratory path of definitive hematopoietic stem/progenitor cells during zebrafish development. Blood 109, 5208–5214 (2007).

    Article  CAS  Google Scholar 

  4. Gering, M. & Patient, R. Hedgehog signaling is required for adult blood stem cell formation in zebrafish embryos. Dev. Cell 8, 389–400 (2005).

    Article  CAS  Google Scholar 

  5. Yoshimoto, M. & Yoder, M.C. Developmental biology: birth of the blood cell. Nature 457, 801–803 (2009).

    Article  CAS  Google Scholar 

  6. Dieterlen-Lievre, F., Pouget, C., Bollerot, K. & Jaffredo, T. Are intra-aortic hemopoietic cells derived from endothelial cells during ontogeny? Trends Cardiovasc. Med. 16, 128–139 (2006).

    Article  CAS  Google Scholar 

  7. Bertrand, J.Y. et al. Characterization of purified intraembryonic hematopoietic stem cells as a tool to define their site of origin. Proc. Natl. Acad. Sci. USA 102, 134–139 (2005).

    Article  CAS  Google Scholar 

  8. Kissa, K. & Herbomel, P. Blood stem cells emerge from aortic endothelium by a novel type of cell transition. Nature 464, 112–115 (2010).

    Article  CAS  Google Scholar 

  9. Kimmel, C.B., Ballard, W.W., Kimmel, S.R., Ullmann, B. & Schilling, T.F. Stages of embryonic development of the zebrafish. Dev. Dyn. 203, 253–310 (1995).

    Article  CAS  Google Scholar 

  10. Hutson, L.D. & Chien, C.B. Pathfinding and error correction by retinal axons: the role of astray/robo2. Neuron 33, 205–217 (2002).

    Article  CAS  Google Scholar 

  11. Langenberg, T., Brand, M. & Cooper, M.S. Imaging brain development and organogenesis in zebrafish using immobilized embryonic explants. Dev. Dyn. 228, 464–474 (2003).

    Article  CAS  Google Scholar 

  12. Kamei, M. & Weinstein, B.M. Long-term time-lapse fluorescence imaging of developing zebrafish. Zebrafish 2, 113–123 (2005).

    Article  Google Scholar 

  13. Yaniv, K. et al. Live imaging of lymphatic development in the zebrafish. Nat. Med. 12, 711–716 (2006).

    Article  CAS  Google Scholar 

  14. Lecaudey, V., Cakan-Akdogan, G., Norton, W.H. & Gilmour, D. Dynamic Fgf signaling couples morphogenesis and migration in the zebrafish lateral line primordium. Development 135, 2695–2705 (2008).

    Article  CAS  Google Scholar 

  15. Kardash, E. et al. A role for Rho GTPases and cell-cell adhesion in single-cell motility in vivo. Nat. Cell Biol. 12, 47–53 (2009).

    Article  Google Scholar 

  16. Boisset, J.C. et al. In vivo imaging of haematopoietic cells emerging from the mouse aortic endothelium. Nature 464, 116–120 (2010).

    Article  CAS  Google Scholar 

  17. Boisset, J.-C., Andrieu-Soler, C., van Cappellen, W.A., Clapes, T. & Robin, C. Ex vivo time lapse confocal imaging of the mouse embryo aorta. Nat. Protoc. 6, 1792–1805 (2011).

    Article  CAS  Google Scholar 

  18. Jin, S.W., Beis, D., Mitchell, T., Chen, J.N. & Stainier, D.Y. Cellular and molecular analyses of vascular tube and lumen formation in zebrafish. Development 132, 5199–5209 (2005).

    Article  CAS  Google Scholar 

  19. Bussmann, J., Lawson, N., Zon, L. & Schulte-Merker, S. Zebrafish VEGF receptors: a guideline to nomenclature. PLoS Genet. 4, e1000064 (2008).

    Article  Google Scholar 

  20. Bertrand, J.Y. et al. Haematopoietic stem cells derive directly from aortic endothelium during development. Nature 464, 108–111 (2010).

    Article  CAS  Google Scholar 

  21. Kissa, K. et al. Live imaging of emerging hematopoietic stem cells and early thymus colonization. Blood 111, 1147–1156 (2008).

    Article  CAS  Google Scholar 

  22. Zhu, H. et al. Regulation of the lmo2 promoter during hematopoietic and vascular development in zebrafish. Dev. Biol. 281, 256–269 (2005).

    Article  CAS  Google Scholar 

  23. Huisken, J., Swoger, J., Del Bene, F., Wittbrodt, J. & Stelzer, E.H. Optical sectioning deep inside live embryos by selective plane illumination microscopy. Science (New York, NY) 305, 1007–1009 (2004).

    Article  CAS  Google Scholar 

  24. Huisken, J. & Stainier, D.Y. Selective plane illumination microscopy techniques in developmental biology. Development (Cambridge, England) 136, 1963–1975 (2009).

    Article  CAS  Google Scholar 

  25. Swoger, J., Muzzopappa, M., Lopez-Schier, H. & Sharpe, J. 4D retrospective lineage tracing using SPIM for zebrafish organogenesis studies. J. Biophotonics 4, 122–134 (2011).

    Article  Google Scholar 

  26. Rasband, W.S. ImageJ (US National Institutes of Health) http://imagej.nih.gov/ij/ (1997–2011).

  27. Westerfield, M. The Zebrafish Book: A Guide for the Laboratory Use of Zebrafish (Danio rerio) (University of Oregon, 2000).

Download references

Acknowledgements

This work was supported by the Caisse Nationale Autonome de la Sécurité Sociale dans les Mines.

Author information

Authors and Affiliations

  1. Institut Curie, Centre de Recherche, PICT-IBiSA, Paris, France

    Olivier Renaud

  2. Centre National de la Recherche Scientifique, UMR3215, Génétique et Biologie du Développement, Paris, France

    Olivier Renaud

  3. Institut National de la Santé et de la Recherche Médicale, Génétique et Biologie du Développement, Paris, France

    Olivier Renaud

  4. Institut Pasteur, Unité Macrophages et Développement de l'Immunité, Paris, France

    Philippe Herbomel & Karima Kissa

  5. Centre National de la Recherche Scientifique, URA2578, Paris, France

    Philippe Herbomel & Karima Kissa

Authors
  1. Olivier Renaud
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Philippe Herbomel
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Karima Kissa
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

K.K. designed and performed the experimental procedures. O.R. and K.K. wrote the protocol with input from P.H.

Corresponding author

Correspondence to Karima Kissa.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Renaud, O., Herbomel, P. & Kissa, K. Studying cell behavior in whole zebrafish embryos by confocal live imaging: application to hematopoietic stem cells. Nat Protoc 6, 1897–1904 (2011). https://doi.org/10.1038/nprot.2011.408

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nprot.2011.408

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