Chemical genetics of regeneration: Contrasting temporal effects of CoCl₂ on axolotl tail regeneration

Background: Histone deacetylases (HDACs) regulate transcriptional responses to injury stimuli that are critical for successful tissue regeneration. Previously we showed that HDAC inhibitor romidepsin potently inhibits axolotl tail regeneration when applied for only 1-minute postamputation (MPA). Results: Here we tested CoCl_2, a chemical that induces hypoxia and cellular stress, for potential to reverse romidepsin inhibition of tail regeneration. Partial rescue of regeneration was observed among embryos co-treated with romidepsin and CoCl₂ for 1 MPA, however, extending the CoCl₂ dosage window either inhibited regeneration (CoCl₂:0 to 30 MPA) or was lethal (CoCl₂:0 to 24 hours postamputation; HPA). CoCl₂:0 to 30 MPA caused tissue damage, tissue loss, and cell death at the distal tail tip, while CoCl₂ treatment of non-amputated embryos or CoCl₂:60 to 90 MPA treatment after re-epithelialization did not inhibit tail regeneration. CoCl₂-romidepsin:1 MPA treatment partially restored expression of transcription factors that are typical of appendage regeneration, while CoCl₂:0 to 30 MPA significantly increased expression of genes associated with cell stress and inflammation. Additional experiments showed that CoCl₂:0 to 1 MPA and CoCl₂:0 to 30 MPA significantly increased levels of glutathione and reactive oxygen species, respectively. Conclusion: Our study identifies a temporal window from tail amputation to re-epithelialization, within which injury activated cells are highly sensitive to CoCl₂ perturbation of redox homeostasis.