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

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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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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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.

Corresponding authors

Correspondence to Yaoyao Chen or Enrique Amaya.

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The authors declare no competing financial interests.

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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