Project name: Chemical Genetics of Regeneration: Contrasting Temporal Effects of CoCl2 on Axolotl Tail Regeneration

Description: The Mexican axolotl provides a powerful model to investigate mechanisms of tissue regeneration. A recent chemical screen found that HDAC inhibitor romidepsin, administered for only 1-minute post amputation (1 MPA), blocks axolotl tail regeneration. Here, we tested the potential for cobalt chloride (CoCl2), a chemical stabilizer of HIF1a and inducer of hypoxia, to rescue romidepsin-inhibition of tail regeneration. Tail regeneration was partially rescued when embryos with amputated tails were co-treated with romidepsin and CoCl2. However, extending the CoCl2 dosage window either inhibited regeneration (CoCl2:0-30 MPA) or was lethal (CoCl2:0-24 hours post amputation; HPA). CoCl2:0-30 MPA caused tissue damage, tissue loss, and cell death at the distal tail tip, and blocked regeneration. In contrast, CoCl2 treatment of non-amputated embryos or CoCl2:60-90 MPA treatment of amputated embryos did not affect wound healing or inhibit tail regeneration. To further investigate the contrasting effects of CoCl2, microarray analysis was performed to identify differentially expressed genes at 3 HPA. CoCl2-romidepsin:1 MPA treatment significantly increased the expression of transcription factors associated with appendage regeneration, while CoCl2:0-30 MPA significantly increased expression of hemoglobin and platelet-specific transcripts, consistent with hemorrhage and an impaired hemostatic response. Also, CoCl2:0-30 MPA significantly increased expression of hypoxia inducible genes, including genes that encode Hif1a interacting proteins and heat shock proteins (HSP); in contrast, genes encoding TGFB signaling components were significantly downregulated. Using additional chemical inhibitors of tail regeneration, we identified transcriptional responses associated with HSP90 activity and TGFB signaling. Notably, geldanamcin decreased transcription of matrix metalloproteinases and sustained muscle-specific gene expression, suggesting a role for HSP90 in regulating extracellular matrix remodeling and muscle dedifferentiation. Our study shows the power of using chemical tools to precisely identify temporal windows within which critical biological processes are enacted during tissue regeneration.Overall design: A microarray experiment was performed to determine the effect of 7.5 mM CoCl2 treatment on axolotl embryo gene expression at 3 hours post amputation. For each treatment tested (0-30 MPA CoCl2, 60-90 MPACoCl2, 10 uM romidepsin and CoCl2, Control), tail amputations were performed on 36 embryos (2 mm removed from distal tip with a razor blade) and then embryos were placed into microtiter plates containing rearing water (Control) or rearing water with chemicals. Exactly 1 mm of the distal tail tip was removed from embryos at 3 HPA. Tissues from 12 embryos were pooled into a 1.5 ml tubes with 0.5 ml of RNA-later (Qiagen) to obtain three replicate pools per treatment. In addition, a microarray experiment was performed to determine the effect of 1 um geldanamycin and 10 uM SB505124 on axolotl embryo gene expression at 24 hours post amputation. For each treatment tested (geldanamycin vs control, SB505124 vs control), tail amputations were performed on 48 embryos (2 mm removed from distal tip with a razor blade) and then embryos were placed into microtiter plates containing rearing water or rearing water (Control) with chemical. Exactly 1 mm of the distal tail tip was removed from embryos at 3 HPA. Tissues from 12 embryos were pooled into a 1.5 ml tubes with 0.5 ml of RNA-later (Qiagen) to obtain four replicate pools per treatment.

Data type: Transcriptome or Gene expression

Sample scope: Multiisolate

Relevance: Other

Last updated: 2020-05-20