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Amputation-induced reactive oxygen species are required for successful Xenopus tadpole tail regeneration

Nature Cell Biology volume 15, pages 222–228 (2013)Cite this article

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Abstract

Understanding the molecular mechanisms that promote successful tissue regeneration is critical for continued advancements in regenerative medicine. Vertebrate amphibian tadpoles of the species Xenopus laevis and Xenopus tropicalis have remarkable abilities to regenerate their tails following amputation1,2, through the coordinated activity of numerous growth factor signalling pathways, including the Wnt, Fgf, Bmp, Notch and TGF-β pathways3,4,5,6. Little is known, however, about the events that act upstream of these signalling pathways following injury. Here, we show that Xenopus tadpole tail amputation induces a sustained production of reactive oxygen species (ROS) during tail regeneration. Lowering ROS levels, using pharmacological or genetic approaches, reduces the level of cell proliferation and impairs tail regeneration. Genetic rescue experiments restored both ROS production and the initiation of the regenerative response. Sustained increased ROS levels are required for Wnt/β-catenin signalling and the activation of one of its main downstream targets, fgf20 (ref. 7), which, in turn, is essential for proper tail regeneration. These findings demonstrate that injury-induced ROS production is an important regulator of tissue regeneration.

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Figure 1: Production of ROS during Xenopus tadpole tail regeneration.
Figure 2: Amputation-induced ROS production does not depend on inflammatory cells.
Figure 3: Pharmacologically lowering ROS levels impairs tail regeneration.
Figure 4: Morpholino-mediated knockdown of cyba results in lowered amputation-induced ROS production and decreased regenerative tissue formation.
Figure 5: Amputation-induced ROS are important for proper growth factor signalling during tail regeneration.

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References

  1. Slack, J. M., Lin, G. & Chen, Y. The Xenopus tadpole: a new model for regeneration research. Cell Mol. Life Sci. 65, 54–63 (2008).

    Article  CAS  Google Scholar 

  2. Love, N. R. et al. Genome-wide analysis of gene expression during Xenopus tropicalis tadpole tail regeneration. BMC Dev. Biol. 11, 70 (2011).

    Article  CAS  Google Scholar 

  3. Lin, G. & Slack, J. M. Requirement for Wnt and FGF signaling in Xenopus tadpole tail regeneration. Dev. Biol. 316, 323–335 (2008).

    Article  CAS  Google Scholar 

  4. Sugiura, T., Tazaki, A., Ueno, N., Watanabe, K. & Mochii, M. Xenopus Wnt-5a induces an ectopic larval tail at injured site, suggesting a crucial role for noncanonical Wnt signal in tail regeneration. Mech. Dev. 126, 56–67 (2009).

    Article  CAS  Google Scholar 

  5. Beck, C. W., Christen, B. & Slack, J. M. Molecular pathways needed forregeneration of spinal cord and muscle in a vertebrate. Dev. Cell 5, 429–439 (2003).

    Article  CAS  Google Scholar 

  6. Ho, D. M. & Whitman, M. TGF-β signaling is required for multiple processes during Xenopus tail regeneration. Dev. Biol. 315, 203–216 (2008).

    Article  CAS  Google Scholar 

  7. Chamorro, M. N. et al. FGF-20 and DKK1 are transcriptional targets of β-catenin and FGF-20 is implicated in cancer and development. EMBO J. 24, 73–84 (2005).

    Article  CAS  Google Scholar 

  8. Finkel, T. & Holbrook, N. J. Oxidants, oxidative stress and the biology of ageing. Nature 408, 239–247 (2000).

    Article  CAS  Google Scholar 

  9. Lambeth, J. D. NOX enzymes and the biology of reactive oxygen. Nature Rev. Immunol. 4, 181–189 (2004).

    Article  CAS  Google Scholar 

  10. Belousov, V. V. et al. Genetically encoded fluorescent indicator for intracellular hydrogen peroxide. Nat. Methods 3, 281–286 (2006).

    Article  CAS  Google Scholar 

  11. Niethammer, P., Grabher, C., Look, A. T. & Mitchison, T. J. A tissue-scale gradient of hydrogen peroxide mediates rapid wound detection in zebrafish. Nature 459, 996–999 (2009).

    Article  CAS  Google Scholar 

  12. Yoo, S. K., Starnes, T. W., Deng, Q. & Huttenlocher, A. Lyn is a redox sensor that mediates leukocyte wound attraction in vivo. Nature 480, 109–112 (2011).

    Article  CAS  Google Scholar 

  13. Love, N. R. et al. pTransgenesis: a cross-species, modular transgenesis resource. Development 138, 5451–5458 (2011).

    Article  CAS  Google Scholar 

  14. Owusu-Ansah, E., Yavari, A., Mandal, S. & Banerjee, U. Distinct mitochondrial retrograde signals control the G1-S cell cycle checkpoint. Nat. Genet. 40, 356–361 (2008).

    Article  CAS  Google Scholar 

  15. Miesenbock, G., De Angelis, D. A. & Rothman, J. E. Visualizing secretion and synaptic transmission with pH-sensitive green fluorescent proteins. Nature 394, 192–195 (1998).

    Article  CAS  Google Scholar 

  16. West, A. P. et al. TLR signalling augments macrophage bactericidal activity through mitochondrial ROS. Nature 472, 476–480 (2011).

    Article  CAS  Google Scholar 

  17. Costa, R. M., Soto, X., Chen, Y., Zorn, A. M. & Amaya, E. Spib is required for primitive myeloid development in Xenopus. Blood 112, 2287–2296 (2008).

    Article  CAS  Google Scholar 

  18. O’Donnell, B. V., Tew, D. G., Jones, O. T. & England, P. J. Studies on the inhibitory mechanism of iodonium compounds with special reference to neutrophil NADPH oxidase. Biochem. J. 290, 41–49 (1993).

    Article  Google Scholar 

  19. Kahles, T. & Brandes, R. P. NADPH oxidases as therapeutic targets in ischemic stroke. Cell Mol. Life Sci. 69, 2345–2363 (2012).

    Article  CAS  Google Scholar 

  20. Stefanska, J. & Pawliczak, R. Apocynin: molecular aptitudes. Mediators Inflamm. 2008, 106507 (2008).

    Article  CAS  Google Scholar 

  21. Wind, S. et al. Comparative pharmacology of chemically distinct NADPH oxidase inhibitors. Br. J. Pharmacol. 161, 885–898 (2010).

    Article  CAS  Google Scholar 

  22. Otomo, E. Effect of a novel free radical scavenger, edaravone (MCI-186), on acute brain infarction. Randomized, placebo-controlled, double-blind study at multicenters. Cerebrovasc. Dis. 15, 222–229 (2003).

    Article  Google Scholar 

  23. Yoneyama, M., Kawada, K., Gotoh, Y., Shiba, T. & Ogita, K. Endogenous reactive oxygen species are essential for proliferation of neural stem/progenitor cells. Neurochem. Int. 56, 740–746 (2010).

    Article  CAS  Google Scholar 

  24. Ambasta, R. K. et al. Direct interaction of the novel Nox proteins with p22phox is required for the formation of a functionally active NADPH oxidase. J. Biol. Chem. 279, 45935–45941 (2004).

    Article  CAS  Google Scholar 

  25. Le Belle, J. E. et al. Proliferative neural stem cells have high endogenous ROS levels that regulate self-renewal and neurogenesis in a PI3K/Akt-dependant manner. Cell Stem. Cell 8, 59–71 (2011).

    Article  CAS  Google Scholar 

  26. Sundaresan, M., Yu, Z. X., Ferrans, V. J., Irani, K. & Finkel, T. Requirement for generation of H2O2 for platelet-derived growth factor signal transduction. Science 270, 296–299 (1995).

    Article  CAS  Google Scholar 

  27. Yanes, O. et al. Metabolic oxidation regulates embryonic stem cell differentiation. Nat. Chem. Biol. 6, 411–417 (2010).

    Article  CAS  Google Scholar 

  28. Dickinson, B. C., Peltier, J., Stone, D., Schaffer, D. V. & Chang, C. J. Nox2 redox signaling maintains essential cell populations in the brain. Nat. Chem. Biol. 7, 106–112 (2011).

    Article  CAS  Google Scholar 

  29. Hendzel, M. J. et al. Mitosis-specific phosphorylation of histone H3 initiates primarily within pericentromeric heterochromatin during G2 and spreads in an ordered fashion coincident with mitotic chromosome condensation. Chromosoma 106, 348–360 (1997).

    Article  CAS  Google Scholar 

  30. Nutt, S. L., Bronchain, O. J., Hartley, K. O. & Amaya, E. Comparison of morpholino based translational inhibition during the development of Xenopus laevis and Xenopus tropicalis. Genesis 30, 110–113 (2001).

    Article  CAS  Google Scholar 

  31. Whitehead, G. G., Makino, S., Lien, C. L. & Keating, M. T. fgf20 is essential for initiating zebrafish fin regeneration. Science 310, 1957–1960 (2005).

    Article  CAS  Google Scholar 

  32. Lee, Y., Grill, S., Sanchez, A., Murphy-Ryan, M. & Poss, K. D. Fgf signaling instructs position-dependent growth rate during zebrafish fin regeneration. Development 132, 5173–5183 (2005).

    Article  CAS  Google Scholar 

  33. Stoick-Cooper, C. L., Moon, R. T. & Weidinger, G. Advances in signaling in vertebrate regeneration as a prelude to regenerative medicine. Genes Dev. 21, 1292–1315 (2007).

    Article  CAS  Google Scholar 

  34. Denayer, T., Tran, H. T. & Vleminckx, K. Transgenic reporter tools tracing endogenous canonical Wnt signaling in Xenopus. Methods Mol. Biol. 469, 381–400 (2008).

    Article  CAS  Google Scholar 

  35. Funato, Y., Michiue, T., Asashima, M. & Miki, H. The thioredoxin-related redox-regulating protein nucleoredoxin inhibits Wnt-β-catenin signalling through dishevelled. Nat. Cell Biol. 8, 501–508 (2006).

    Article  CAS  Google Scholar 

  36. Galliot, B. & Chera, S. The Hydra model: disclosing an apoptosis-driven generator of Wnt-based regeneration. Trends Cell Biol. 20, 514–523 (2010).

    Article  CAS  Google Scholar 

  37. Whyte, J. L., Smith, A. A. & Helms, J. A. Wnt signaling and injury repair. Cold Spring Harb. Perspect. Biol. 4, a008078 (2012).

    Article  Google Scholar 

  38. Nieuwkoop, P. D. & Faber, J. Normal Table of Xenopus laevis (Daudin): A Systematical and Chronological Survey of the Development from the Fertilized Egg Till the End of Metamorphosis (Garland Publishing, 1994).

    Google Scholar 

  39. Kroll, K. L. & Amaya, E. Transgenic Xenopus embryos from sperm nuclear transplantations reveal FGF signaling requirements during gastrulation. Development 122, 3173–3183 (1996).

    CAS  PubMed  Google Scholar 

  40. Turner, D. L. & Weintraub, H. Expression of achaete-scute homolog 3 in Xenopus embryos converts ectodermal cells to a neural fate. Genes Dev. 8, 1434–1447 (1994).

    Article  CAS  Google Scholar 

  41. Chen, Y. et al. C/EBPα initiates primitive myelopoiesis in pluripotent embryonic cells. Blood 114, 40–48 (2009).

    Article  CAS  Google Scholar 

  42. Lea, R., Papalopulu, N., Amaya, E. & Dorey, K. Temporal and spatial expression of FGF ligands and receptors during Xenopus development. Dev. Dyn. 238, 1467–1479 (2009).

    Article  CAS  Google Scholar 

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Acknowledgements

We thank P. Niethammer (Sloan-Kettering Institute, USA) for the pCS2+ HyperYFP construct, the University of Manchester Bioimaging Facility for guidance with imaging, and R. Paredes and Y. Matsubayashi for advice on statistical analyses. We also thank N. Papalopulu and C. Thompson for comments on the manuscript. This work was supported by a Wellcome Trust Program Grant (E.A.), a Wellcome Trust Career Development Fellowship (J.L.G.), a Wellcome Trust PhD Studentship (P.K.), and grants from the BBSRC (K.D.), The Healing Foundation (N.R.L., Y.C., E.A.) and The National Science Foundation (N.R.L.).

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  1. Yaoyao Chen

    Present address: Present address: Wellcome Trust Centre for Stem Cell Research, University of Cambridge, Tennis Court Road, Cambridge CB2 1QR, UK,

Authors and Affiliations

  1. Faculty of Life Sciences, University of Manchester, Oxford Road, Manchester M13 9PT, UK

    Nick R. Love, Yaoyao Chen, Shoko Ishibashi, Paraskevi Kritsiligkou, Robert Lea, Yvette Koh, Karel Dorey & Enrique Amaya

  2. The Healing Foundation Centre, Michael Smith Building, University of Manchester, Oxford Road, Manchester M13 9PT, UK

    Nick R. Love, Yaoyao Chen, Shoko Ishibashi, Robert Lea, Yvette Koh, Karel Dorey & Enrique Amaya

  3. The Wellcome Trust/Cancer Research UK Gurdon Institute, University of Cambridge, Tennis Court Road, Cambridge CB2 1QN, UK

    Jennifer L. Gallop

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Contributions

N.R.L. designed and carried out most of the experiments in this study and co-wrote the manuscript. Y.C. established the HyPerYFP assay in Xenopus, generated spib morphants, and assisted in many of the other experiments in the study. S.I. generated the cyba constructs, and P.K. generated the pHlourin constructs. K.D. performed western blot analyses on the tagged cyba constructs and helped prepare the manuscript. Y.K. performed the C1-blastomere injections and cell-tracking analysis of the inflammatory cells. R.L. performed the whole-mount in situ hybridizations and whole-mount immunohistochemistry experiments. J.L.G. generated the initial finding that the antioxidant MCI-186 could be used to lower ROS in Xenopus. E.A. supervised the project, aided with embryo experiments and co-wrote the manuscript.

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Correspondence to Yaoyao Chen or Enrique Amaya.

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Supplementary information

Supplementary Information

Supplementary Information (PDF 1198 kb)

Simultaneous imaging of HyPerYFP and RFP labeled inflammatory cells for the first 6 h following tail amputation.

Upper left panel shows transillumination, lower left panel shows HyPerYFP ratio, upper right panel shows RFP labelled inflammatory cells, lower right panel shows RFP labelled inflammatory cells and their tracked migratory behaviour. Images were captured at one-minute intervals. (MOV 33848 kb)

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Love, N., Chen, Y., Ishibashi, S. et al. Amputation-induced reactive oxygen species are required for successful Xenopus tadpole tail regeneration. Nat Cell Biol 15, 222–228 (2013). https://doi.org/10.1038/ncb2659

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