Tissue regeneration is associated with complex changes in gene expression and post-translational modifications of proteins, including transcription factors and histones that comprise chromatin. We tested 172 compounds designed to target epigenetic mechanisms in an axolotl ( Ambystoma mexicanum ) embryo tail regeneration assay. A relatively large number of compounds (N = 55) inhibited tail regeneration, including 18 histone deacetylase inhibitors (HDACi). In particular, romidepsin, an FDA-approved anticancer drug, potently inhibited tail regeneration when embryos were treated continuously for 7 days. Additional experiments revealed that romidepsin acted within a very narrow, post-injury window. Romidepsin treatment for only 1-minute post amputation inhibited regeneration through the first 7 days, however after this time, regeneration commenced with variable outgrowth of tailfin tissue and abnormal patterning. Microarray analysis showed that romidepsin altered early, transcriptional responses at 3 and 6-hour post-amputation, especially targeting genes that are implicated in tumor cell death, as well as genes that function in the regulation of transcription, cell differentiation, cell proliferation, pattern specification, and tissue morphogenesis. Our results show that HDAC activity is required at the time of tail amputation to regulate the initial transcriptional response to injury and regeneration.

Background Among vertebrates, salamanders are unparalleled in their ability to regenerate appendages throughput life. However, little is known about early signals that initiate regeneration in salamanders. Results Ambystoma mexicanum embryos were administered tail amputations to investigate the timing of reactive oxygen species (ROS) production and the requirement of ROS for regeneration. ROS detected by dihydroethidium increased within minutes of axolotl tail amputation and levels remained high for 24 hr. Pharmacological inhibition of ROS producing enzymes with diphenyleneiodonium chloride (DPI) and VAS2870 reduced ROS levels. Furthermore, DPI treatment reduced cellular proliferation and inhibited tail outgrowth. Conclusions The results show that ROS levels increase in response to injury and are required for tail regeneration. These findings suggest that ROS provide instructive, if not initiating cues, for salamander tail regeneration. Developmental Dynamics 248:189‐196, 2019. © 2018 Wiley Periodicals, Inc.

Sal-Site serves axolotl research efforts by providing Web access to genomic data and information, and living stocks that are reared and made available by the Ambystoma Genetic Stock Center (AGSC). In this chapter, we detail how investigators can search for genes of interest among Sal-Site resources to identify orthologous nucleotide and protein-coding sequences, determine genome positions within the Ambystoma meiotic map, and obtain estimates of gene expression. In the near future, additional genomic resources will be made available for the axolotl, including a listing of genes that are partially or wholly contained within Bacterial Artificial Chromosome (BAC) vectors, a prioritized collection of deeply sequenced BAC clones, chromosome-specific assemblies of genomic DNA, and transgenic axolotls that are engineered using TALENs and CRISPRs. Also, services provided by the AGSC will be expanded to include microinjection of user constructs into single cell embryos and distribution of axolotl tissues, DNA, and RNA. In conclusion, Sal-Site is a useful resource that generates, shares, and evolves Ambystoma associated information and databases to serve research and education.

Highlights d Gli3 repressor (Gli3R) activity level governs tetrapod limb axis formation polarity d 5 0 Hoxd-Gli3 balance modulates cell-cycle exit to determine mouse limb axis polarity d Alternating A-P digit appearance in mammals is linked to primary limb axis polarity d Axolotl Gli3 knockdown shifts the ancestral preaxial dominance to postaxial

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 CoCl 2 for 1 MPA, however, extending the CoCl 2 dosage window either inhibited regeneration (CoCl 2 :0 to 30 MPA) or was lethal (CoCl 2 :0 to 24 hours postamputation; HPA). CoCl 2 :0 to 30 MPA caused tissue damage, tissue loss, and cell death at the distal tail tip, while CoCl 2 treatment of nonamputated embryos or CoCl 2 :60 to 90 MPA treatment after re-epithelialization did not inhibit tail regeneration. CoCl 2 -romidepsin:1 MPA treatment partially restored expression of transcription factors that are typical of appendage regeneration, while CoCl 2 :0 to 30 MPA significantly increased expression of genes associated with cell stress and inflammation. Additional experiments showed that CoCl 2 :0 to 1 MPA and CoCl 2 :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 CoCl 2 perturbation of redox homeostasis.Amphibians and fish are well-suited for studies of tissue regeneration, not only because they regenerate damaged tissues, but also because they are amenable to chemical screening. Chemicals can be readily delivered to aquatic fish, frogs, and salamanders for the purpose of altering developmental, cellular, and molecular mechanisms that regulate tissue regeneration. [1][2][3][4][5][6] A variety of chemicals with relatively well-characterized targets and mechanisms of action have been shown to block regeneration. These include chemicals that inhibit primary developmental signaling pathways, including retinoic acid, BMP, TGFα, FGF, Wnt, and HSP90. 4,[7][8][9][10][11][12] When a chemical with a known biological activity is shown to block regeneration, it provides strong evidence that the biological