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 amputation, through the coordinated activity of numerous growth factor signalling pathways, including the Wnt, Fgf, Bmp, Notch and TGF-β pathways. 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.

Figure 1

Production of ROS during Xenopus tadpole tail regeneration. (a) Panels show brightfield and fluorescence images of a tadpole derived from the F1 generation of a transgenic Xenopus laevis line that expresses the H₂O₂ sensor HyPerYFP ubiquitously . (b) Panels show HyPerYFP imaging of a representative regenerating tadpole tail. [H₂O₂] is derived from the excitation ratio of HyPerYFP490nm/HyPerYFP402nm. mpa, minutes post-amputation; hpa, hours post-amputation; dpa, days post-amputation. Due to the size of the regenerated tail, the 6-day after amputation time point panels are derived from the merging of three images.

Figure 2

Amputation-induced ROS production does not depend on inflammatory cells. (a) Frames from the Supplementary Video showing transillumination (Trans), H₂O₂ production (HyPerYFP), and inflammatory cells (labeled with RFP) recruitment during the first 6 hrs following amputation. Closed red arrow points to blood clot that quickly forms in the distal site of the dorsal aorta following tail amputation . Open red arrow points to first inflammatory cell recruited into the area under examination. The colored lines show the migratory paths of the recruited inflammatory cells. (b) Quantification showing the change in average HyPerYFP ratio in relation to the number or recruited inflammatory cells into the area examined in the Supplementary Movie. (c) Sudan Black B staining of inflammatory cells in regenerative bud tissue at 24hpa, showing the decreased number of inflammatory cells in spib versus control morphant tadpoles. (d) Representative HyPerYFP imaging of control and spib morphants at 24 hours after tail amputation. (e) Quantification of H₂O₂ production using the HyPerYFP probe in control and spib morphants. Error bars indicate standard deviation (s.d.) of the mean. n tadpole tails analyzed indicated by brackets. n.s.; P > 0.05.

Figure 3

Pharmacologically lowering ROS impairs tail regeneration. (a) Panels show representative HyPerYFP imaging and quantification of tadpole tails treated with DMSO, 2μM DPI, 200μM apocynin (APO), or 200μM MCI-186 (MCI) treatments at 12 hours after amputation. (b) Representative tails and quantification of regenerated tail length at 72 hours after amputation following the indicated inhibitor treatments. (c) Images and quantification of tadpoles that were exposed to DMSO, DPI, APO, or MCI (same doses as above) from 0-72hpa, and then cultured in normal media until 7dpa. Error bars indicate standard deviation of the mean (s.d.) of (n) specimens. Red lines indicate initial point of amputation. Significance was determined using one-way ANOVA versus DMSO control. **, P < 0.01; ***, P < 0.001; no significance (n.s.), P > 0.05.

Figure 4

Morpholino mediated knockdown of cyba results in lowered amputation-induced ROS production and decreased regenerative tissue formation. (a) Western-blot against the FLAG epitope in cyba-flag mRNA injected cyba morphant or control embryos. (b) Western-blot against the myc epitope in myc-cyba mRNA injected cyba morphant or control embryos. (c) Representative transillumination and HyPerYFP imaging at amputation (T0) and 24 hpa in control, cyba morphants, and myc-cyba rescue cyba morphants, showing inhibition of ROS production and regenerative tissue formation by the cyba MO and rescue by co-injection of the MO-resistant myc-cyba mRNA. Black closed arrow shows regenerative bud. The quantification of HyPerYFP ratio increases following amputation in control, cyba morphants, and myc-cyba rescue construct injected cyba morphants is shown to the right of the panels. (d) Quantification of regenerative bud formation in control, cyba morphants, and myc-cyba rescue construct injected cyba morphants. Error bars indicate standard deviation (s.d.) of (n) specimens. Significance was determined using one-way ANOVA versus control. **, P < 0.01; ***, P < 0.001.

Figure 5

Amputation induced ROS are important for proper growth factor signaling during tail regeneration. (a) Representative images and quantification of X. laevis tadpole tail mitotic cells at 36hpa when cultured in control (DMSO) or ROS inhibitors DPI (2μM), APO (200μM), and MCI (200μM). (b) Representative images and relative Wnt/β-catenin signaling reporter dsGFP fluorescence at 36hpa in control (DMSO) or ROS inhibitors DPI (2μM), APO (50μM), and MCI (200μM) treated X. tropicalis tadpoles. (c) RT-PCR reactions amplifying fgf20 or reference gene rpl8 in control (DMSO) or ROS inhibitors DPI (2μM), APO (50μM), and MCI (200μM) at 36hpa in X. tropicalis tadpoles. (d) In situ hybridization of fgf20 in DMSO controls versus DPI treated X. tropicalis tadpole tails during the tail regrowth phase of tail regeneration. (e) RT-PCR reactions detecting fgf20 in X. tropicalis control or fgf20 morphants. (f) Representative regenerated control or fgf20 morphant tadpole tails at 48hpa. (g) Quantification of regenerated tail length in control or fgf20 morphants. Error bars indicate standard error of the mean (s.e.m.) of (n) specimens. Significance was determined using one-way ANOVA or unpaired t-tests. *, P < 0.05; **, P < 0.01; ***, P < 0.001.