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Amphibians such as Xenopus laevis and Ambystoma mexicanum are capable of whole structure regeneration. However, transcriptional control over these events is not well understood. Here, we investigate the role of histone deacetylase (HDAC) enzymes in regeneration using HDAC inhibitors. The class I/II HDAC inhibitor valproic acid (VPA) inhibits tail regeneration in embryos of the anuran amphibian Xenopus laevis, confirming a recent report by others (Tseng et al., 2011). This inhibition correlates with a sixfold reduction in endogenous HDAC activity. VPA also inhibited tail regeneration in post-refractory stage Xenopus larvae and larvae of the urodele A. mexicanum (axolotl). Furthermore, Xenopus limb regeneration was also significantly impaired by post-amputation treatment with VPA, suggesting a general requirement for HDAC activity in the process of appendage regeneration in amphibians. The most potent inhibition of tail regeneration was observed following treatment with VPA during the wound healing, pre-blastema phase. A second HDAC inhibitor, sodium butyrate, was also shown to inhibit tail regeneration. While both VPA and sodium butyrate are reported to block sodium channel function as well as HDACs, regeneration was not inhibited by valpromide, an analogue of VPA that lacks HDAC inhibition but retains sodium channel blocking activity. Finally, although VPA is a known teratogen, we show that neither tailbud nor limb bud development are affected by exposure to this compound. We conclude that histone deacetylation is specifically required for the earliest events in appendage regeneration in amphibians, and suggest that this may act as a switch to trigger re-expression of developmental genes.
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22947425
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Fig. 1. Valproic acid (VPA) inhibits tail regeneration in both larval and embryonic stage Xenopus laevis tadpoles and VPA inhibits HDACs in regenerating X. laevis. (A–C) Examples of tadpole larval tails five days after amputation at stage 48, white arrowheads indicate the approximate cut site. (A) Untreated tadpole showing full regeneration. (B) Tadpole treated with 0.1 mg/ml VPA showing partial regeneration (white arrow), (C) Example of tadpole treated with 0.1 mg/ml VPA that failed to undergo any regeneration. (D) Bar graph showing mean tail regeneration score out of 10 (mean RS) in stage 48 tadpoles treated with differing concentrations of VPA. All concentrations shown significantly inhibited regeneration (∗∗Indicates t-test p value of <0.01), full data and analysis is shown in Table 1. (E)–(G) Examples of embryo tails five days after amputation at stage 40, white arrowheads indicate the approximate cut site. (E) Example of untreated embryo showing full regeneration. (F) Example of embryo treated with 0.2 mg/ml VPA showing partial regeneration and characteristic “poodle” phenotype, consisting of fintissue bubble formed at the end of the notochord (white arrow). (G) Example of stage Stage 40 embryo treated with 0.2 mg/ml VPA showing failed regeneration. (H) Graph showing mean tail regeneration score out of 10 (mean RS) in stage 40 tadpoles treated with 0.2 mg/ml VPA. This concentration significantly inhibited regeneration (∗∗Indicates t-test p value of <0.01). (I) Colourimetric HDAC activity assay in stage 40 embryos treated with VPA (0.2 mg/ml) for 24 h post tail amputation, where HDAC activity is indicated by the amount deacetylated lysine standard per ng of total nuclear protein.
Fig. 2. HDAC inhibitors further inhibit tail regeneration in refractory stage Xenopus laevis. Stage 46/47 tadpoles are normally unable to regenerate tails following partial amputation. (A and B) Tadpole tails shown five days after amputation of the posterior 30–50% during the refractory period. White arrowheads indicate approximate level of amputation. (A) Example of failed regeneration in control, untreated tadpole. (B) Representative tadpole treated with 0.2 mg/ml VPA. (C) Bar graph showing mean tail regeneration score out of ten (mean RS) in refractory stage tadpoles. Despite the low background regeneration in controls at this stage, treatment with VPA or NaBu still significantly inhibited regeneration (∗∗Indicates t-test p value of <0.01). Full data and analysis is shown in Table 3.
Fig. 3. Sodium butyrate (NaBu) inhibits tail regeneration in Xenopus laevis. (A) Example 0.5 mg/ml NaBu treated embryotail shown five days after amputation of 30–50% of the tail and allowed to regenerate showing partial regeneration (compare to control regeneration in Fig. 1E). Arrowheads indicate the approximate cut site. NaBu treated stage 40 tadpoles do not show a “poodle” phenotype. (B) Bar graph showing mean tail regeneration score out of ten (mean RS) in stage 40 tadpoles treated with 0.5 mg/ml NaBu compared to untreated controls. (∗∗Indicates t-test p value of <0.01). Full data and analysis is shown in Table 2.
Fig. 4. VPA inhibits tail regeneration in stage 46 Ambystoma mexicanum. (A–C) stage 46 axolotl larvae tails shown 7 days after amputation of the posterior third of the tail. Arrowheads indicate the approximate cut site (A) untreated tail showing full regeneration. (B) Tail of axolotl larva treated with 0.2 mg/ml VPA post amputation showing example of partial regeneration. (C) Tail of axolotl larva treated with 0.2 mg/ml VPA post amputation showing example of failed regeneration. (D) Bar graph showing mean tail regeneration score out of ten (mean RS) in stage 46 axolotl larvae treated with VPA (0.2 mg/ml) compared to untreated controls. (∗∗Indicates t-test p value of <0.01). Full data and analysis is shown in Table 4.
Fig. 5. VPA reduces hind limb regeneration in stage 52 Xenopus tadpoles. In (B–D) digit numbers are indicated (I–V), p: phalanges, mt: metatarsal, t: tarsus. (A) Stacked column graph showing the percentage of left hind limbs that had failed to regenerate (stump), or that had regenerated 1–5 toes by stage 58. The left hind limbs of stage 52 tadpoles were amputated at the approximate level of the future knee. The group treated with 0.2 mg/ml VPA regenerated significantly fewer toes than the control, untreated group (∗∗Indicates p value of <0.01, Chi squared test). Full data and analysis is shown in Table 5. (B) VPA treated limb that was not amputated showing perfect development. (C) VPA treated limb after regeneration, in this example only digit II is present and the single tarsus is deformed. (D) Control limb after regeneration, in this example one phalange is missing from digit IV showing that control regeneration is often imperfect. (E) Graph showing the percentage of each type of bone present in the total limbs of control and VPA treated tadpoles comparing amputated and non-amputated limbs. Blue indicates normal bone formation and red indicates some deformation in the bone including bending and malformation (Control n = 10, VPA n = 17).
Fig. 7. VPA does not inhibit tail development in Xenopus laevis: Stage 32 tail buds were extirpated and cultured in the absence (A) or presence (B) of 0.2 mg/ml VPA for 24 h until stage 40 (stage of photographs) Both cohorts exhibited normal automomous development of the somites, spinal cord, notochord and fin.
