Mitogawa K et al. (2014), Ectopic blastema induction by nerve deviation a...

XB-ART-53358

Regeneration (Oxf) 2014 May 28;12:26-36. doi: 10.1002/reg2.11.

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Ectopic blastema induction by nerve deviation and skin wounding: a new regeneration model in Xenopus laevis.

Mitogawa K , Hirata A , Moriyasu M , Makanae A , Miura S , Endo T , Satoh A .

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Recently, the accessory limb model (ALM) has become an alternative study system for limb regeneration studies in axolotls instead of using an amputated limb. ALM progresses limb regeneration study in axolotls because of its advantages. To apply and/or to compare knowledge in axolotl ALM studies to other vertebrates is a conceivable next step. First, Xenopus laevis, an anuran amphibian, was investigated. A Xenopus frog has hypomorphic regeneration ability. Its regeneration ability has been considered intermediate between that of non-regenerative higher vertebrates and regenerative urodele amphibians. Here, we successfully induced an accessory blastema in Xenopus by skin wounding and rerouting of brachial nerve bundles to the wound site, which is the regular ALM surgery. The induced Xenopus ALM blastemas have limited regenerative potential compared with axolotl ALM blastemas. Comparison of ALM blastemas from species with different regenerative potentials may facilitate the identification of the novel expression programs necessary for the formation of cartilage and other tissues during limb regeneration.

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Species referenced: Xenopus laevis
Genes referenced: bmp2 fgf10 fgf8 fn1 hoxa13 myh6 prrx1 sox9

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	Figure 1. ALM blastemaâlike formation (bump) in Xenopus laevis. (A, B) Nerve deviation to the skin wound resulted in induction of the bump. Arrow in A indicates the induced bump. Arrowheads in B indicate the border of the wounded skin. (C, D) Histological observations. (C) In the bump region, many mesenchymal cells can be observed. Arrows indicate the border of the dermal collagen layer. (D) Higher magnification of (C). The distal epithelium, which is expected to be an AEC, was thickened. Scale bars in (A) and (C) are 1 mm.
	Figure 2. Gene expression pattern in the bump. (A) Neural tubulin (acetylated alphaâtubulin) was visualized by immunofluorescence. The white arrow indicates the thick nerve bundle observed in the center of the bump. The deviated nerves abundantly penetrate into the overlying epithelium (inset). (B) One of the classical blastema marker genes, fibronectin, was investigated. The signal of fibronectin can be seen in the blastemal region and the epitheliumâmesenchyme boundary shows intense fibronectin signal (inset). (C) Type II collagen expression. Type II collagen is a cartilage marker gene and negative in the bump. The inset shows the signal in the limb cartilage located in the proximal region as the positive control. (D, E) Prrx1 expression. Prrx1 can be seen in the blastemal mesenchyme in the ALM blastema (D) and the regular blastema in the amputated limb (E). The dotted line in (E) indicates the presumptive amputation plane. All conditions of the in situ hybridization were the same in (D) and (E). Scale bars in (A) and (D) are 0.5 mm.
	Figure 3. RTâPCR analysis of the induced bump. Gene expression pattern was investigated by RTâPCR. Samples were prepared from the induced bumps (2 weeks after the surgery), regular blastemas (MB; 2 weeks after amputation), and intact skin. Msx1b, Fgf10, Fgf8, and Prrx1 were positive in both the bump and the regular blastema, while they were negative in intact skin. Hoxa13 could not be detected in the bump but could be detected in the regular blastema. Bmp genes were also investigated by RTâPCR. Bmp2, 4, and 7 were positive in both the bump and the regular blastema. Type II collagen and Sox9 were unexpectedly detectable in the bump, but the regular blastema showed much higher expression level. Ef1Î± was an internal control.
	Figure 4. The induced bump did not contain cartilaginous cells. (A, B) Dorsal views of the day 20 bump. The induced bump stopped growing at day 20. (C) Histology of the bump. There are no Alcianâblueâpositive cartilage cells in the mesenchymal region. (D, E) Higher magnification of the boxed regions in (C). Arrows in (D) and (E) indicate developing basal laminae. (F) Axons were visualized by immunofluorescence using antiâacetylated alphaâtubulin. In the distal region, highly innervated axon fibers in the epithelium were not observed in the day 20 bump (inset). (G) Myosin heavy chain (MHC) expression. There were no MHCâpositive myogenic cells in the bump. Scale bars in (A) and (B) are 1 mm. The scale bar in (C) is 500 Î¼m. (C), (F), and (G) are the same magnification. The scale bar in (D) is 20 Î¼m. (D) and (E) are the same magnification.
	Figure 5. Cartilaginous spike induction by deep wounding. (A) Time course of the induced bump and spike induction. Skin wounding plus nerve deviation result in bump formation. In this case, a small bump can be seen within 12 days, but it starts regressing by day 19. On the other hand, when a deep wound is created enough to damage a stylopod bone, a cartilaginous spike can be observed. In this case, a relatively smaller bump can be observed at day 12. The induced bump kept growing up to spike formation. (B, C) Induced cartilaginous spike at day 45. (D) Wholeâmount Alcian blue staining. Scale bars in (A), (B), (C), and (D) are 2 mm.
	Figure 6. Section of the cartilaginous spike induction by deep wounding. (A) Histological observation. Alcianâblueâpositive cartilage extends from the stylopod. (B, C) Acetylated alphaâtubulin expression. (D, E) Type II collagen (T2 Col.) expression. (F, G) MHC expression. (B)â(G) are the boxed region of (A). The scale bar in (A) is 1 mm.

References [+] :

Cannata, Nerve-independence of limb regeneration in larval Xenopus laevis is correlated to the level of fgf-2 mRNA expression in limb tissues. 2001, Pubmed, Xenbase