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

Abstract

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.

Keywords: CoCl2; axolotl; chemical genetics; hypoxia; tail regeneration.

Figure 1.. CoCl₂ partially rescues axolotl tail regeneration.

A) 7 DPA Embryos treated continuously with 0.01% DMS0. B) 7 DPA embryos treated continuously with 10 μM romidepsin. C) 7 DPA embryos treated with 10 μM romidepsin and 10 mM CoCl₂ for 1-minute post amputation (MPA). D) 7 DPA embryos treated with 10 μM romidepsin for 1 MPA. E) Higher magnification image of embryo treated with 10 μM romidepsin for 1 MPA. F) Higher magnification image of embryo treated with 10 mM romidepsin and 10 mM CoCl₂ for 1 MPA. G) 7 DPA embryos treated with 10 μM romidepsin and 10 mM CoCl₂ for 1 MPA. H) Quantification of regenerated tail tissue among embryos treated with romidepsin and or CoCl₂. Embryos that were treated with 10 μM romidepsin and CoCl₂ (5, 7.5, and 10 mM) regenerated significantly (* ANOVA, p < 0.05) more tissue than embryos that were only treated with 10 μM romidepsin.

Figure 2.. Genes identified as significantly differentially expressed between romidepsin and CoCl2/romidepsin treated embryos at 3 hours post amputation.

Fold change is relative to expression level measured at the time of amputation. Control refers to non-chemically treated axolotls that were previously administered tail and limb amputations.

Figure 3.. Contrasting effects of CoCl₂ on axolotl tail regeneration.

A) 7 DPA embryos treated with 7.5 mM CoCl₂ for 0-30 MPA presented blunt tail tips, consistent with inhibition of tail regeneration. B) 7 DPA embryos treated with 7.5 mM CoCl₂ for 60–90 MPA regenerated their tail tips. C) Methylene blue staining of damaged tissue in a 3 HPA embryo treated with 7.5 mM CoCl₂ for 0-30 MPA. Arrows indicate areas of tail tissue loss. D) Methylene blue staining of a 3 HPA embryo treated with 7.5 mM CoCl₂ for 60-90 MPA. E) 16 HPA embryos treated with 7.5 mM CoCl₂ for 0-30 MPA presented abnormal tail tips. F) Comparison of 0-30 MPA vs 60-90 MPA CoCl₂ treated embryos at 16 HPA. G) Higher magnification image of 7.5 mM CoCl₂ 0-30 MPA treated embryo at 16 HPA. H) Propidium iodide staining reveals dead cells in 0-30 MPA CoCl₂ treated embryo tail tips at 16 HPA.

Figure 4.. Histological evaluation of the effect of CoCl2 treatment on regenerating tails at 3 hpa.

Compared to control embryos (A), CoCl2 treatment (B) disrupted wound epidermis formation. Clumps of blood cells (yellow arrow heads) indicate hemorrhage in CoCl2-treated embryo tails. SC: spinal cord, NC: notochord, WE: wound epidermis.

Figure 5.. CoCl₂ treatment regime differentially affected gene expression.

CoCl₂ treatment for 0-30 MPA inhibited regeneration and was associated with increased transcription of genes that encode protein interactants of HIF1α, and genes that are typically expressed by red blood cells and platelets. CoCl₂ treatment for 60-90 MPA did not inhibit regeneration and was associated with increased transcription of matrix metalloproteinases, cell signaling pathway components, and genes that are typically upregulated during axolotl tail and limb regeneration. Rep = replicate.

Figure 6.. Effect of Romidepsin-CoCl2 co-treatment on ROS and GSH at 3 HPA.

(A) Representative images of ROS (top row) and GSH production (bottom row) at 3 HPA for control, Romidepsin:0-1 MPA, CoCl₂:0-1 MPA, Romidepsin and CoCl2:0-1 MPA, CoCl₂:0-30 MPA, CoCl₂:0-60 MPA, and CoCl₂:60-90 MPA. (B) Quantification of ROS and GSH production. Error bars represent standard deviations of the mean (N=7-8 embryos/group). *: indicates statistical significance (Student’s T-test P value < 0.01) compared to control.

Figure 7.. Models for transcriptional regulation and windows of CoCl₂ sensitivity after tail amputation.

(A) HDAC activity at the time of amputation is informed by injury stimuli, including hypoxia and oxidative stress, to regulate a regeneration specific, transcriptional network. (B) CoCl₂ inhibits tail regeneration if administered continuously before wound closure (re-epithelialization), but not after.