Yokoyama H et al. (2011), Different requirement for Wnt/β-catenin signali...

XB-ART-43635

PLoS One 2011 Jan 01;67:e21721. doi: 10.1371/journal.pone.0021721.

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Different requirement for Wnt/β-catenin signaling in limb regeneration of larval and adult Xenopus.

Yokoyama H , Maruoka T , Ochi H , Aruga A , Ohgo S , Ogino H , Tamura K .

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BACKGROUND: In limb regeneration of amphibians, the early steps leading to blastema formation are critical for the success of regeneration, and the initiation of regeneration in an adult limb requires the presence of nerves. Xenopus laevis tadpoles can completely regenerate an amputated limb at the early limb bud stage, and the metamorphosed young adult also regenerates a limb by a nerve-dependent process that results in a spike-like structure. Blockage of Wnt/β-catenin signaling inhibits the initiation of tadpole limb regeneration, but it remains unclear whether limb regeneration in young adults also requires Wnt/β-catenin signaling. METHODOLOGY/PRINCIPAL FINDINGS: We expressed heat-shock-inducible (hs) Dkk1, a Wnt antagonist, in transgenic Xenopus to block Wnt/β-catenin signaling during forelimb regeneration in young adults. hsDkk1 did not inhibit limb regeneration in any of the young adult frogs, though it suppressed Wnt-dependent expression of genes (fgf-8 and cyclin D1). When nerve supply to the limbs was partially removed, however, hsDkk1 expression blocked limb regeneration in young adult frogs. Conversely, activation of Wnt/β-catenin signaling by a GSK-3 inhibitor rescued failure of limb-spike regeneration in young adult frogs after total removal of nerve supply. CONCLUSIONS/SIGNIFICANCE: In contrast to its essential role in tadpole limb regeneration, our results suggest that Wnt/β-catenin signaling is not absolutely essential for limb regeneration in young adults. The different requirement for Wnt/β-catenin signaling in tadpoles and young adults appears to be due to the projection of nerve axons into the limb field. Our observations suggest that nerve-derived signals and Wnt/β-catenin signaling have redundant roles in the initiation of limb regeneration. Our results demonstrate for the first time the different mechanisms of limb regeneration initiation in limb buds (tadpoles) and developed limbs (young adults) with reference to nerve-derived signals and Wnt/β-catenin signaling.

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Species referenced: Xenopus laevis
Genes referenced: ctnnb1 dkk1 fgf10 fgf8 gsk3a gsk3b hoxa13 hsp70 hspa1l lmx1a msx2 prrx1 prx tbx5 wnt3a
GO keywords: Wnt signaling pathway [+]

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	Figure 1. Wnt-3a is expressed in the blastema of both froglets and tadpoles. (A) RT-PCR of the total RNA from tadpole blastemas at 5 dpa or froglet blastemas at 9 dpa showed that wnt-3a was expressed in both the tadpole and froglet blastemas. (B) In situ hybridization of froglet blastemas at 9 dpa with a wnt-3a antisense probe revealed expression of wnt-3a in the epidermal layer of the blastema, while no specific signal was detected with the wnt-3a sense probe. Each section was hybridized with the antisense or sense probe, at the same time and by the same procedure, and the development of the staining reaction was stopped at exactly the same time. The lines indicate the estimated amputation plane. Scale bar = 100 mm. doi:10.1371/journal.pone.0021721.g001
	Figure 2. Refined procedure for heat-shock-inducible inhibition of Wnt/b-catenin signaling in Xenopus laevis. (A) Map of the heat- shock-inducible Dkk1GFP transgene with lens labeling. Details are described in Experimental Procedures. The bright RFP variant, tdTomato under control of the 2.2-kb crystallin promoter, was introduced downstream of hsp70-Dkk1GFP. (B) Prior to heatshock, a tadpole containing the transgene could be recognized by the tdTomato fluorescence in its lenses. After heatshock, ubiquitous Dkk1GFP expression was induced in the lens-labeled tadpoles. Note that the tdTomato protein in the lens was detected through a GFP filter as well as through an RFP filter. (C) Dorsal view of the left hindlimb bud at stage 52. (D) Induction of hsDkk1 expression at stage 52 completely blocked regeneration of the hindlimb bud. Nothing was regenerated from the amputation level. Lines indicate the estimated amputation planes (knee level for the hindlimb bud). Arrows indicate the lens region of tadpoles. Scale bar = 1 mm for (B) and (D) and 250 m for (C). See also Figure S1. doi:10.1371/journal.pone.0021721.g002
	Figure 3. hsDkk1 does not block spike regeneration in the froglet. (A) Experimental scheme for froglets. One heatshock (hs: represented as a red circle) was applied to the froglets 3 to 4 h before amputation. The forelimb was amputated through the distal zeugopodium (amp: represented as a blue square) followed by a heatshock every other day until 8 dpa. (B and C) The hsDkk1 froglets (C) regenerated a spike like the wild-type froglets (B). (D-F) hsDkk1 had a different effect on regenerative capacity of the tadpole and froglet within the same individual. hsDkk1 expression induced at the tadpole stage (st. 52) blocked regeneration of the left hindlimb bud amputated at the presumptive knee level (D). After this hsDkk1 tadpole had become a froglet, hsDkk1GFP expression could still be induced effectively by heatshock (E). However, repeated heatshocks as shown in (A) could not block forelimb regeneration of the same hsDkk1 froglet (F). Lines indicate the estimated amputation plane (knee level for the hindlimb bud, distal zeugopod level for the forelimb). Scale bar = 500 mm for (B) and (C) and 1 mm for (D), (E), and (F). doi:10.1371/journal.pone.0021721.g003
	Figure 4. Histological examination of limb blastemas/stumps with or without blockage of Wnt/b-catenin signaling. (A) Experimental scheme for tadpoles. One heatshock (hs: represented as a red circle) was applied to stage 52 tadpoles 3 to 4 h before amputation. The hindlimb buds were amputated at the presumptive knee level (amp: represented as a blue square), and fixation was done at 5 dpa. (B) Longitudinal sections of the limb blastema/stump of wild-type (B, D) and Dkk1GFP (C, E) tadpoles. Panels D and E are high-power views of the boxed regions in B and C, respectively. The distinct basement membrane is indicated by arrowheads. No distinct basement membrane was seen at the tip of the blastema, indicated by arrowheads in (D), while the basement membrane covered the entire amputation plane of the limb stump in (E). (F) Experimental scheme for froglets. One heatshock was applied 3 to 4 h before amputation. The forelimbs of the froglets were amputated through the distal zeugopodium followed by a heatshock every other day, and fixation was done at 10 dpa. (G) Longitudinal sections of the limb blastema/stump of wild-type (G, J) and Dkk1GFP (H, K) froglets. Panels J and K are high-power views of the boxed regions in G and H, respectively. Cone-shaped blastemas were formed in both wild-type (G) and Dkk1GFP (H) froglets. These blastemas were covered with dermis-free epidermis, and no skin gland was seen in the blastemal region of wild type (J) or Dkk1GFP froglets (K). In the denervated forelimb, no cone-shaped blastema was formed (I) and a differentiated dermis with skin glands covered the amputation plane at 10 dpa (L). ep, epidermis; dm, dermis. Arrows indicate skin glands. Arrowheads indicate the edge of a distinct basement membrane. Lines indicate the estimated amputation planes. Scale bar = 100 mm for (B), (C), (G), (H), and (I) and 50 mm for (D), (E), (J), (K), and (L). doi:10.1371/journal.pone.0021721.g004
	Figure 5. Wnt/b-catenin signaling is blocked in hsDkk1 froglets. (A) Experimental scheme for in situ hybridization. Upper: Froglet forelimbs were amputated (amp: blue square) and heat-shocked (hs: red circle) at 8 dpa, and their blastemas were excised and fixed (fix: black triangle) 16 h after the heatshock for in situ hybridization. Lower: Froglet forelimbs were amputated (amp: blue square) repeatedly heat-shocked (hs: red circles) every other day until 8 dpa, and their blastemas were excised and fixed (fix: black triangle) 16 h after the last heatshock for in situ hybridization. (B and C) In situ hybridization on sectioned samples of froglet blastemas that had been heat-shocked as shown in the upper scheme in (A). The sectioned samples were hybridized with the fgf-8 antisense probe. (D and E) In situ hybridization of sectioned samples of froglet blastemas that were repeatedly heat-shocked as shown in the lower scheme in (A). Sectioned samples were hybridized with the fgf-8 antisense probe. To guarantee correct comparisons of gene expression levels, wild-type (B or D) and hsDkk1GFP froglet (C or E) sections were treated in exactly the same way. Scale bar = 100 mm. doi:10.1371/journal.pone.0021721.g005
	Figure 6. hsDkk1 expression blocked spike regeneration of froglets after partial denervation. (A and B) The dorsal (A) and ventral (B) sides of a froglet forelimb each contain a thick bundle of nerve trunks. The forelimb was amputated through the distal zeugopod level. For observation, an incision was made in the skin on the dorsal side of the shoulder and the nerve trunks were pinched out (A), while nerve trunks can be seen through the intact skin on the ventral side (B). (C and D) After denervation on only the ventral side of a forelimb, the forelimbs of hsDkk1 froglets were amputated, and the animals were repeatedly heat-shocked until 8 dpa. Only one-third of the forelimbs regenerated a spike (C), and most of the forelimbs did not show any regeneration (D). Arrowheads indicate the bundle of nerve trunks. Lines indicate the estimated amputation planes. Scale bar = 500 mm. doi:10.1371/journal.pone.0021721.g006
	Figure 7. GSK3 inhibitor BIO can rescue spike regeneration of a denervated limb. (A) A DMSO-treated (100 ml/l) control froglet showed no regeneration. (B and C) BIO-treated (1 mM) froglets regenerated a short spike (B) or a long spike (C). Lines indicate the estimated amputation planes. Scale bar = 500 mm. doi:10.1371/journal.pone.0021721.g007
	Figure 8. Model for Wnt/β-catenin and nerve signals in early limb regeneration in Xenopus froglets.(A) Successful blastema formation in the intact forelimb of the hsDkk1 froglet. Wnt/β-catenin signaling (presumably mediated by wnt-3a in the epidermis) induces fgf-8 expression in the epidermis. Induction of Dkk1GFP expression by heatshock diminishes epidermal fgf-8 expression. However, nerve signals released from the nerves around the limb stump have some redundant function with FGF-8 and/or the product(s) of another Wnt downstream gene(s), whereas Dkk1GFP does not markedly affect the function of the nerve signals. The nerve signals can substitute for the role of the Wnt downstream gene, so that the limb stump can continue the blastema formation process and form a spike, even after blockage of Wnt/β-catenin signaling. (B) Interrupted blastema formation in the half-denervated forelimb of an hsDkk1 froglet. When the limb stump is partially denervated, the amount of nerve signals is thought to be around half of that in an intact limb stump. This level of nerve signals is insufficient to substitute for the role of the Wnt downstream gene. Thus, most of the limb stumps cannot continue the blastema formation process, resulting in no regeneration, after blockage of Wnt/β-catenin signaling. Considering the redundant function of the nerve signals and a Wnt downstream gene, the nerve signals may be mediated by another member of the FGFs. Scissors indicate the amputation plane. nv, nerves; ct, cartilage.

References [+] :

BODEMER, Observations on the mechanism of induction of supernumerary limbs in adult Triturus viridescens. 1959, Pubmed