January 1, 2011;
HDAC activity is required during Xenopus tail regeneration.
The ability to fully restore damaged or lost organs is present in only a subset of animals. The Xenopus tadpole tail
is a complex appendage
, containing epidermis
, nerves, spinal cord
, and vasculature
, which regenerates after amputation. Understanding the mechanisms of tail
regeneration may lead to new insights to promote biomedical regeneration in non-regenerative tissues. Although chromatin remodeling is known to be critical for stem cell
pluripotency, its role in complex organ regeneration in vivo remains largely uncharacterized. Here we show that histone deacetylase (HDAC
) activity is required for the early stages of tail
is expressed during the 1(st
) two days of regeneration. Pharmacological blockade of HDACs using Trichostatin A (TSA) increased histone acetylation levels in the amputated tail
. Furthermore, treatment with TSA or another HDAC
inhibitor, valproic acid, specifically inhibited regeneration. Over-expression of wild-type Mad3
, a transcriptional repressor known to associate in a complex with HDACs via Sin3
, inhibited regeneration. Similarly, expression of a Mad3
mutant lacking the Sin3
-interacting domain that is required for HDAC
binding also blocks regeneration, suggesting that HDAC
may act together to regulate regeneration. Inhibition of HDAC
function resulted in aberrant expression of Notch1
, two genes known to be required for tail
regeneration. Our results identify a novel early role for HDAC
regeneration and suggest that modulation of histone acetylation is important in regenerative repair of complex appendages
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References [+] :
Figure 1. HDAC1 and Mad3 are Expressed During Xenopus Tail Regeneration.
(A) RNA in situ hybridization to detect gene expression in tail regenerates at 24, 48 and 72 hpa for HDAC1, (D) Mad3, and (G and H) HDAC6. Probe targets are shown at the left. Black arrowheads indicate presence of RNA whereas open arrowheads indicate absence of expression. Anterior is to the left.
Figure 2. Pharmacological HDAC Blockade using TSA or VPA Inhibits Tail Regeneration.
(A) After st. 40 tail amputation, tadpoles were assayed for tail regeneration at 7 days post amputation (dpa). Yellow arrowheads demarcate amputation site. (B) Control tadpoles (RI = 290, n = 69). 25 nM TSA treatment (RI = 109, n = 69), * denotes p<0.001. (C) Control tadpoles (RI = 283, n = 72). 500 Valproic Acid treatment (RI = 126, n = 53), * denotes p<0.001. (D) Temporal requirement for HDAC activity during regeneration. Percentage of regeneration shows total number of tail regenerates scored as full or good. TSA treatment as follows: Control/untreated (98.6%, RI = 290, n = 69), 0 dpa (10.8%, RI = 105, n = 65), 0 dpa (22.1%, RI = 118, n = 77), and 2 dpa (1.5%, RI = 272, n = 65). * denotes p<0.01 as compared to either Control or 2 dpa treatment.
Figure 3. Over-Expression of Mad3 Inhibits Tail Regeneration.
(A) Schematic showing the structure of Mad3 protein. Green indicates the Sin3-interacting domain (SID) important for the Mad3 interaction with HDACs. The DNA binding domain is represented in blue. Mad3 WT shows the full-length sequence and Mad3-ΔSID showing the construct that lacks the SID domain. (B) Mad3 WT, Mad3-ΔSID and lacZ mRNA were injected into early embryos at the 4 cell stage. Embryos were allowed to develop until st. 40, when tail amputation was performed. Graph showing effects of ectopic expression of Mad3 WT and Mad3-ΔSID on tail regeneration at 7 dpa. Control regenerates (RI = 250, n = 95). Wild-type Mad3 expression (RI = 188, n = 68, p<0.01). Mad3 with SID deletion expression (RI = 159, n = 88, p<0.01). p value denotes comparison to control. Comparison of the 2 ectopic expression experiments yielded p>0.05.
Figure 4. HDAC Inhibition Increases Histone Acetylation During Regeneration.
The acetylation state of tail regenerates were examined at 24 hpa using an anti-acetylated Histone H4 antibody. Yellow arrowheads denote regeneration bud. Top row shows untreated controls. Bottom row shows tadpoles treated with 25 nM TSA after tail amputation. (A, D) Acetylated Histone H4. (B, E) Hoechst DNA stain. (C) merge of A and B. (F) merge of D and E.
Figure 5. HDAC Inhibition Induces Mis-Expression of BMP2 and Notch1 During Regeneration.
Wholemount in situ hybridization of st. 40 24 hpa regeneration buds. DIG-labeled probes specfic to Xenopus BMP2 and Notch1 were used to detect expression during regeneration without (A, A', C, and C') or with (B, B', D, and D') TSA treatment immediately after tail amputation. Wholemount views are shown in (A, B, C, and D) and sagittal sections through the regenerate tail are shown in (A', B', C', and D'). Anterior is to the left. Full arrowheads indicate gene expression. Open arrowheads show lack of expression. (A, A', B, and B') BMP2. (C, C', D, and D') Notch1. Scale bar = 100 .
Tail Regeneration Assay. Individual animals for each specific treatment were scored as follows: Full: complete regeneration. Good: robust regeneration with minor defects (missing fin, curved axis). Weak: poor regeneration (hypomorphic/defective regenerates). None: no regeneration. Shown are representative examples of each regenerate class.
Expression of HDAC1 and Mad3 in Xenopus Tail. RNA in situ hybridization to detect gene expression in st. 40 uncut and amputated tail for (A) HDAC1, (C) Mad3, and (E) β-gal. Probe targets are shown to the left of the panels.
H+ pump-dependent changes in membrane voltage are an early mechanism necessary and sufficient to induce Xenopus tail regeneration.