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.

  • Brief Communication
  • Published:

In vitro whole-organ imaging: 4D quantification of growing mouse limb buds

Abstract

Quantitative mapping of the normal tissue dynamics of an entire developing mammalian organ has not been achieved so far but is essential to understand developmental processes and to provide quantitative data for computational modeling. We developed a four-dimensional (4D) imaging technique that can be used to quantitatively image tissue movements and dynamic GFP expression domains in a growing transgenic mouse limb by time-lapse optical projection tomography (OPT).

This is a preview of subscription content, access via your institution

Access options

Rent or buy this article

Prices vary by article type

from$1.95

to$39.95

Prices may be subject to local taxes which are calculated during checkout

Figure 1: The 4D time-lapse OPT.
Figure 2: Tracking growth for 6 h (E11.0–E11.25).
Figure 3: The 4D monitoring of a dynamic gene expression pattern (Scx-GFP) in limb buds.
Figure 4: Comparison of physical sections with OPT sections.

Similar content being viewed by others

References

  1. Megason, S.G. & Fraser, S.E. Cell 130, 784–795 (2007).

    Article  CAS  Google Scholar 

  2. Damle, S., Hanser, B., Davidson, E.H. & Fraser, S.E. Dev. Biol. 299, 543–550 (2006).

    Article  CAS  Google Scholar 

  3. Svetic, V. et al. Development 134, 1011–1022 (2007).

    Article  CAS  Google Scholar 

  4. Clarke, J.D.W. & Tickle, C. Nat. Cell Biol. 1, E103–E109 (1999).

    Article  CAS  Google Scholar 

  5. Muneoka, K., Wanek, N. & Bryant, S.V. J. Exp. Zool. 249, 50–54 (1989).

    Article  CAS  Google Scholar 

  6. Vargesson, N. et al. Development 124, 1909–1918 (1997).

    CAS  PubMed  Google Scholar 

  7. Welten, M.C.M., Verbeek, F.J., Meijer, A.H. & Richardson, M.K. Evol. Dev. 7, 18–28 (2005).

    Article  CAS  Google Scholar 

  8. Sharpe, J. et al. Science 296, 541–545 (2002).

    Article  CAS  Google Scholar 

  9. Sharpe, J. Annu. Rev. Biomed. Eng. 6, 209–228 (2004).

    Article  CAS  Google Scholar 

  10. Niswander, L. & Martin, G.R. Nature 361, 68–71 (1993).

    Article  CAS  Google Scholar 

  11. Zuniga, A., Haramis, A.P., McMahon, A.P. & Zeller, R. Nature 401, 598–602 (1999).

    Article  CAS  Google Scholar 

  12. Ochiya, T., Sakamoto, H., Tsukamoto, M., Sugimura, T. & Terada, M. J. Cell Biol. 130, 997–1003 (1995).

    Article  CAS  Google Scholar 

  13. Tickle, C. Dev. Cell 4, 449–458 (2003).

    Article  CAS  Google Scholar 

  14. Fernandez-Teran, M.A., Hinchliffe, J.R. & Ros, M.A. Dev. Dyn. 235, 2521–2537 (2006).

    Article  CAS  Google Scholar 

  15. Pryce, B.A., Brent, A.E., Murchison, N.D., Tabin, C.J. & Schweitzer, R. Dev. Dyn. 236, 1677–1682 (2007).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank M. Logan (MRC National Institute for Medical Research) for providing the Scleraxis-GFP line, the Zeller lab for their time and helpful suggestions on the in vitro mouse limb bud culturing technique, L. Hay for technical support in the design and building of the 4D time-lapse OPT scanner, D.A. Kleinjan and V. van Heyningen for Pax6-GFP embryos, C. DeAngelis for performing the Fgf-8 in-situ hybridization, B. Pryce for sectioning the Scx-GFP limbs, J. Swoger for help with 3D reconstructions, and the Edinburgh Mouse Atlas Project (EMAP) for software to support the computational analysis. This project was supported as part of the EU Integrated Project grant 'Molecular Imaging' (to M.J.B.), by a research grant from the Human Frontier Science Program (to C.H.W.), by a grant from the Spanish Ministry of Science and Education (BFU2006-10978/BMC; to J.S.-E. and M.T.), by MRC and Centre for Genomic Regulation (to J.C. and J.S.) and by ICREA (to J.S.).

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to James Sharpe.

Supplementary information

Supplementary Text and Figures

Supplementary Note, Supplementary Methods (PDF 7122 kb)

Supplementary Movie 1

Raw data of microsphere landmarks at 0 hours. Movie showing the raw data obtained from multiple angles during an OPT scan at the first time-point for one of the fluorescent landmark-tracking experiments. (MOV 576 kb)

Supplementary Movie 2

Timelapse of microsphere landmarks over 6 hours. Timelapse movie from one angle of the same limb shown in Supplementary Movie 1. (MOV 225 kb)

Supplementary Movie 3

Annotated timelapse of microsphere landmarks over 6 hours. Annotated timelapse movie from one angle of the same limb shown in Supplementary Movie 1. (MOV 180 kb)

Supplementary Movie 4

Raw data of Scx-GFP expression pattern at 0 hours. Movie showing the raw data obtained from multiple angles during an OPT scan at the first time-point for one of the Scx-GFP imaging experiments. (MOV 725 kb)

Supplementary Movie 5

Raw data of Scx-GFP expression pattern after 19 hours. Movie showing the raw data obtained after 19 hours in culture for the same Scx-GFP imaging experiment shown in Supplementary Movie 4. (MOV 786 kb)

Supplementary Movie 6

4D timelapse of dynamic gene expression. Timelapse movie showing how the 3D domain of Scx-GFP expression changes over. (Same limb as shown in Supplementary Movie 4). All expression above a certain threshold level has been highlighted by the green isosurface. (MOV 578 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Boot, M., Westerberg, C., Sanz-Ezquerro, J. et al. In vitro whole-organ imaging: 4D quantification of growing mouse limb buds. Nat Methods 5, 609–612 (2008). https://doi.org/10.1038/nmeth.1219

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nmeth.1219

This article is cited by

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