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:

Cells keep a memory of their tissue origin during axolotl limb regeneration

Nature volume 460, pages 60–65 (2009)Cite this article

Abstract

During limb regeneration adult tissue is converted into a zone of undifferentiated progenitors called the blastema that reforms the diverse tissues of the limb. Previous experiments have led to wide acceptance that limb tissues dedifferentiate to form pluripotent cells. Here we have reexamined this question using an integrated GFP transgene to track the major limb tissues during limb regeneration in the salamander Ambystoma mexicanum (the axolotl). Surprisingly, we find that each tissue produces progenitor cells with restricted potential. Therefore, the blastema is a heterogeneous collection of restricted progenitor cells. On the basis of these findings, we further demonstrate that positional identity is a cell-type-specific property of blastema cells, in which cartilage-derived blastema cells harbour positional identity but Schwann-derived cells do not. Our results show that the complex phenomenon of limb regeneration can be achieved without complete dedifferentiation to a pluripotent state, a conclusion with important implications for regenerative medicine.

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: Dermis does not make muscle but makes cartilage and tendons.
Figure 2: Cartilage cells do not make muscle.
Figure 3: Muscle does not make cartilage or epidermis.
Figure 4: Schwann cells give rise to Schwann cells and do not form cartilage even during nerve-rescue of irradiated limbs.
Figure 5: Schwann cell-derived blastema cells do not possess proximo-distal positional identity but cartilage-derived cells do.

Similar content being viewed by others

References

  1. Umanski, E. E. The regeneration potencies of axolotl skin studied by means of exclusion of the regeneration capacity of tissues through exposure to x-rays. Bull. Biol. Med. Exp. USSR 6, 141–145 (1938)

    Google Scholar 

  2. Weiss, P. Unabhängigkeit der Extremitätenregeneration vom Skelett (bei Triton cristatus) ‘Wilhelm Roux’ . Arch. Entwicklungsmech. Organ. 104, 359–394 (1925)

    Google Scholar 

  3. Thornton, C. S. The histogenesis of the regenerating fore limb of larval Amblystoma after exarticulation of the humerus. J. Morphol. 62, 219–235 (1938)

    Article  Google Scholar 

  4. Namenwirth, M. The inheritance of cell differentiation during limb regeneration in the axolotl. Dev. Biol. 41, 42–56 (1974)

    Article  CAS  Google Scholar 

  5. Dunis, D. A. & Namenwirth, M. The role of grafted skin in the regeneration of x–irradiated axolotl limbs. Dev. Biol. 56, 97–109 (1977)

    Article  CAS  Google Scholar 

  6. Lheureux, E. The origin of tissues in the X-irradiated regenerating limb of the newt Pleurodeles waltlii . Prog. Clin. Biol. Res. 110, 455–465 (1983)

    PubMed  Google Scholar 

  7. Wallace, B. M. & Wallace, H. Participation of grafted nerves in amphibian limb regeneration. J. Embryol. Exp. Morphol. 29, 559–570 (1973)

    CAS  PubMed  Google Scholar 

  8. Echeverri, K. & Tanaka, E. M. Mechanisms of muscle dedifferentiation during regeneration. Semin. Cell Dev. Biol. 13, 353–360 (2002)

    Article  CAS  Google Scholar 

  9. Lo, D. C., Allen, F. & Brockes, J. P. Reversal of muscle differentiation during urodele limb regeneration. Proc. Natl Acad. Sci. USA 90, 7230–7234 (1993)

    Article  ADS  CAS  Google Scholar 

  10. Morrison, J. I., Loof, S., He, P. & Simon, A. Salamander limb regeneration involves the activation of a multipotent skeletal muscle satellite cell population. J. Cell Biol. 172, 433–440 (2006)

    Article  CAS  Google Scholar 

  11. Burns, T. C. et al. Thymidine analogs are transferred from prelabeled donor to host cells in the central nervous system after transplantation: a word of caution. Stem Cells 24, 1121–1127 (2006)

    Article  CAS  Google Scholar 

  12. Sobkow, L., Epperlein, H. H., Herklotz, S., Straube, W. L. & Tanaka, E. M. A germline GFP transgenic axolotl and its use to track cell fate: dual origin of the fin mesenchyme during development and the fate of blood cells during regeneration. Dev. Biol. 290, 386–397 (2006)

    Article  CAS  Google Scholar 

  13. Gargioli, C. & Slack, J. M. Cell lineage tracing during Xenopus tail regeneration. Development 131, 2669–2679 (2004)

    Article  CAS  Google Scholar 

  14. Hay, E. D. & Fischman, D. A. Origin of the blastema in regenerating limbs of the newt Triturus viridescens. An autoradiographic study using tritiated thymidine to follow cell proliferation and migration. Dev. Biol. 3, 26–59 (1961)

    Article  CAS  Google Scholar 

  15. Carlson, B. M. Morphogenetic interactions between rotated skin cuffs and underlying stump tissues in regenerating axolotl forelimbs. Dev. Biol. 39, 263–285 (1974)

    Article  CAS  Google Scholar 

  16. Maden, M. & Mustafa, K. The structure of 180 degrees supernumerary limbs and a hypothesis of their formation. Dev. Biol. 93, 257–265 (1982)

    Article  CAS  Google Scholar 

  17. Slack, M. J. Morphogenetic properties of the skin in axolotl limb regeneration. J. Embryol. Exp. Morphol. 58, 265–288 (1980)

    CAS  PubMed  Google Scholar 

  18. Muneoka, K., Fox, W. F. & Bryant, S. V. Cellular contribution from dermis and cartilage to the regenerating limb blastema in axolotls. Dev. Biol. 116, 256–260 (1986)

    Article  CAS  Google Scholar 

  19. Rollman-Dinsmore, C. & Bryant, S. V. The distribution of marked dermal cells from small localized implants in limb regenerates. Dev. Biol. 106, 275–281 (1984)

    Article  CAS  Google Scholar 

  20. Asakura, A., Komaki, M. & Rudnicki, M. Muscle satellite cells are multipotential stem cells that exhibit myogenic, osteogenic, and adipogenic differentiation. Differentiation 68, 245–253 (2001)

    Article  CAS  Google Scholar 

  21. Wada, M. R., Inagawa-Ogashiwa, M., Shimizu, S., Yasumoto, S. & Hashimoto, N. Generation of different fates from multipotent muscle stem cells. Development 129, 2987–2995 (2002)

    CAS  PubMed  Google Scholar 

  22. Wallace, H. The components of regrowing nerves which support the regeneration of irradiated salamander limbs. J. Embryol. Exp. Morphol. 28, 419–435 (1972)

    CAS  PubMed  Google Scholar 

  23. Horstadius, S. & Sellmann, S. Experimentelle Untersuchungen uber die Determination des knorpeligen Kopfskelettesbei Urodelen. Nova Acta Regiae Soc. Sci. Ups. Ser. 13, 1–170 (1946)

    Google Scholar 

  24. Epperlein, H., Meulemans, D., Bronner-Fraser, M., Steinbeisser, H. & Selleck, M. A. Analysis of cranial neural crest migratory pathways in axolotl using cell markers and transplantation. Development 127, 2751–2761 (2000)

    CAS  PubMed  Google Scholar 

  25. Crawford, K. & Stocum, D. L. Retinoic acid coordinately proximalizes regenerate pattern and blastema differential affinity in axolotl limbs. Development 102, 687–698 (1988)

    CAS  PubMed  Google Scholar 

  26. Iten, L. E. & Bryant, S. V. The interaction between the blastema and stump in the establishment of the anterior–posterior and proximal–distal organization of the limb regenerate. Dev. Biol. 44, 119–147 (1975)

    Article  CAS  Google Scholar 

  27. Nardi, J. B. & Stocum, D. L. Surface properties of regenerating limb cells: evidence for gradation along the proximodistal axis. Differentiation 25, 27–31 (1983)

    Article  Google Scholar 

  28. Echeverri, K. & Tanaka, E. M. Proximodistal patterning during limb regeneration. Dev. Biol. 279, 391–401 (2005)

    Article  CAS  Google Scholar 

  29. Mercader, N. et al. Conserved regulation of proximodistal limb axis development by Meis1/Hth. Nature 402, 425–429 (1999)

    Article  ADS  CAS  Google Scholar 

  30. Mercader, N., Tanaka, E. M. & Torres, M. Proximodistal identity during vertebrate limb regeneration is regulated by Meis homeodomain proteins. Development 132, 4131–4142 (2005)

    Article  CAS  Google Scholar 

  31. Gardiner, D. M., Blumberg, B., Komine, Y. & Bryant, S. V. Regulation of HoxA expression in developing and regenerating axolotl limbs. Development 121, 1731–1741 (1995)

    CAS  PubMed  Google Scholar 

  32. Fromental-Ramain, C. et al. Hoxa-13 and Hoxd-13 play a crucial role in the patterning of the limb autopod. Development 122, 2997–3011 (1996)

    CAS  PubMed  Google Scholar 

  33. Kumar, A., Velloso, C. P., Imokawa, Y. & Brockes, J. P. Plasticity of retrovirus-labelled myotubes in the newt limb regeneration blastema. Dev. Biol. 218, 125–136 (2000)

    Article  CAS  Google Scholar 

  34. Kumar, A., Velloso, C. P., Imokawa, Y. & Brockes, J. P. The regenerative plasticity of isolated urodele myofibers and its dependence on MSX1. PLoS Biol. 2, E218 (2004)

    Article  Google Scholar 

  35. Echeverri, K., Clarke, J. D. & Tanaka, E. M. In vivo imaging indicates muscle fiber dedifferentiation is a major contributor to the regenerating tail blastema. Dev. Biol. 236, 151–164 (2001)

    Article  CAS  Google Scholar 

  36. Tanaka, E. M. Regeneration: if they can do it, why can't we? Cell 113, 559–562 (2003)

    Article  CAS  Google Scholar 

  37. Echeverri, K. & Tanaka, E. M. Ectoderm to mesoderm lineage switching during axolotl tail regeneration. Science 298, 1993–1996 (2002)

    Article  ADS  CAS  Google Scholar 

  38. Mchedlishvili, L., Epperlein, H. H., Telzerow, A. & Tanaka, E. M. A clonal analysis of neural progenitors during axolotl spinal cord regeneration reveals evidence for both spatially restricted and multipotent progenitors. Development 134, 2083–2093 (2007)

    Article  CAS  Google Scholar 

  39. Sobkow, L., Epperlein, H. H., Herklotz, S., Straube, W. L. & Tanaka, E. M. A germline GFP transgenic axolotl and its use to track cell fate: dual origin of the fin mesenchyme during development and the fate of blood cells during regeneration. Dev. Biol. 290, 386–397 (2006)

    Article  CAS  Google Scholar 

  40. Gargioli, C. & Slack, J. M. Cell lineage tracing during Xenopus tail regeneration. Development 131, 2669–2679 (2004)

    Article  CAS  Google Scholar 

  41. Okabe, M., Ikawa, M., Kominami, K., Nakanishi, T. & Nishimune, Y. 'Green mice' as a source of ubiquitous green cells. FEBS Lett. 407, 313–319 (1997)

    Article  CAS  Google Scholar 

  42. Brady, G. & Iscove, N. N. Construction of cDNA libraries from single cells. Methods Enzymol. 225, 611–623 (1993)

    Article  CAS  Google Scholar 

  43. Habermann, B. et al. An Ambystoma mexicanum EST sequencing project: analysis of 17,352 expressed sequence tags from embryonic and regenerating blastema cDNA libraries. Genome Biol. 5, R67,–1–19 (2004)

    Article  Google Scholar 

Download references

Acknowledgements

This work was supported by grants from the Volkswagen Foundation I/78 766; DFG SFB655, SPP1109, SPP 1356, the BMBF Biofutures program, funds from the Max Planck Institute, and the Center of Regenerative Therapies, Dresden. D.K. was a fellow of the Alexander von Humboldt Foundation. We are grateful to K. Agata, H. Tarui, T. Hayashi and M. Saitou for advice on the single-cell PCR technique. We thank I. Nüsslein for assistance on FACS analysis and A. Merseburg for assistance on the nerve rescue experiments. We thank H. Andreas, T. Richter and M. Schuez for technical assistance. We are grateful to L. Rohde, C. Antos, G. Weidinger and A. Tóth for comments on the manuscript.

Author Contributions M.K., D.K. and E.M.T. designed the experiments. M.K., D.K., E.N. and H.H.E. performed embryonic grafting and specificity assessment. M.K. imaged and performed the histological analysis of regenerating limbs, cell counting and single-cell PCR on all experiments. D.K. performed all work related to Schwann cells, with M.M.’s advice. S.K. generated the nucCherry transgenic animals. E.M.T. advised on experiments, examined samples and evaluated data. M.K., D.K. and E.M.T. wrote the manuscript.

Author information

Author notes

  1. Martin Kragl

    Present address: Present address: Institute of Metabolic Physiology, Heinrich-Heine-Universität Düsseldorf, Universitätsstrasse 1, 40225 Düsseldorf, Germany.,

  2. Martin Kragl and Dunja Knapp: These authors contributed equally to this work.

Authors and Affiliations

  1. Max Planck Institute of Molecular Cell Biology and Genetics, Pfotenhauerstrasse 108,,

    Martin Kragl, Dunja Knapp, Eugen Nacu, Shahryar Khattak & Elly M. Tanaka

  2. Institute of Anatomy, Medical Faculty, University of Technology Dresden, Fetscherstrasse 74,,

    Hans Henning Epperlein

  3. Center for Regenerative Therapies, University of Technology Dresden, Tatzberg 47/49, 01307 Dresden, Germany ,

    Martin Kragl, Dunja Knapp, Eugen Nacu, Shahryar Khattak & Elly M. Tanaka

  4. The Regeneration Project, McKnight Brian Institute, University of Florida, Rm 326 Bartram Hall, Gainesville, Florida 32611, USA ,

    Malcolm Maden

Authors
  1. Martin Kragl
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Dunja Knapp
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Eugen Nacu
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Shahryar Khattak
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Malcolm Maden
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Hans Henning Epperlein
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Elly M. Tanaka
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Elly M. Tanaka.

Supplementary information

Supplementary Information

This file contains Supplementary Figures 1-10 with Legends and Supplementary Tables 1-6 with Supplementary Methods and Notes. (PDF 4462 kb)

PowerPoint slides

Rights and permissions

Reprints and permissions

About this article

Cite this article

Kragl, M., Knapp, D., Nacu, E. et al. Cells keep a memory of their tissue origin during axolotl limb regeneration. Nature 460, 60–65 (2009). https://doi.org/10.1038/nature08152

Download citation

  • Received:

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nature08152

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