XB-ART-35803Dev Biol June 1, 2007; 306 (1): 170-8.
Wnt/beta-catenin signaling has an essential role in the initiation of limb regeneration.
Anuran (frog) tadpoles and urodeles (newts and salamanders) are the only vertebrates capable of fully regenerating amputated limbs. During the early stages of regeneration these amphibians form a "blastema", a group of mesenchymal progenitor cells that specifically directs the regrowth of the limb. We report that wnt-3a is expressed in the apical epithelium of regenerating Xenopus laevis limb buds, at the appropriate time and place to play a role during blastema formation. To test whether Wnt/beta-catenin signaling is required for limb regeneration, we created transgenic X. laevis tadpoles that express Dickkopf-1 (Dkk1), a specific inhibitor of Wnt/beta-catenin signaling, under the control of a heat-shock promoter. Heat-shock immediately before limb amputation or during early blastema formation blocked limb regeneration but did not affect the development of contralateral, un-amputated limb buds. When the transgenic tadpoles were heat-shocked following the formation of a blastema, however, they retained the ability to regenerate partial hindlimb structures. Furthermore, heat-shock induced Dkk1 blocked fgf-8 but not fgf-10 expression in the blastema. We conclude that Wnt/beta-catenin signaling has an essential role during the early stages of limb regeneration, but is not absolutely required after blastema formation.
PubMed ID: 17442299
PMC ID: PMC2703180
Article link: Dev Biol
Species referenced: Xenopus
Genes referenced: dkk1 fgf10 fgf8 hoxa13 lmx1a msx2 wnt3a
Article Images: [+] show captions
|Fig. 2. Wnt/β-catenin signaling is required for Xenopus limb regeneration. (A) Experimental scheme. Hindlimb buds of F0 tadpoles were amputated at the presumptive knee level (amp: represented as blue square). One heat-shock (hs: represented as red circle) was applied to tadpoles at 3 to 4 h prior to amputation (yellow line), 3 dpa (days post amputation; green line) or 5 dpa (blue line). (B) Map of the heat-shock inducible Dkk1GFP transgene. Details are described in Materials and methods. Expression of Dkk1GFP was induced in a transgenic tadpole carrying this transgene within 3 to 4 h after heat-shock (left panel, bright field; right panel, GFP). No GFP expression was detected in the same tadpole before heat-shock (inset). (C) Live limb buds were photographed when tadpoles were heat-shocked (st. 52–53; a–d). The same amputated limb buds were photographed again when regenerated limbs became obvious in controls (st. 57; e–h). A wild-type tadpole heat-shocked prior to amputation regenerated the amputated limb bud completely (a and e). While the hsDkk1GFP tadpoles heat-shocked prior to amputation (b and f) or at 3 dpa (c and g) failed to regenerate, hsDkk1GFP tadpoles heat-shocked at 5 dpa regenerated incomplete hindlimbs (d and h). Note that un-amputated right limb buds developed normally (black arrows). Arrowheads show the presumptive knee level (amputation level). Scale bars: 500 μm.|
|Fig. 4. Expression of wnt-3a and fgf-8 in regenerating limb buds. (A and D) Stage 52 limb buds. (B, C, E and F) Regenerating blastemas at 3 dpa. Right panels (C and F) show in situ hybridization on sectioned samples. Wnt-3a and fgf-8 are expressed in the inner layer of thickened apical epithelium of the blastema at 3 dpa. No specific hybridization signal was detected with an wnt-3a sense probe (C, inset). Arrowheads show amputation level (knee level). a, anterior; p, posterior; d, dorsal; v, ventral. Scale bars: 100 μm.|
|Fig. 5. Dkk1GFP represses fgf-8 expression in the regenerating blastema, but not expression of fgf-10, Lmx-1, Hoxa-13 or msx-2. (A) Experimental scheme. One heat-shock was applied to tadpoles at 5 dpa (blue line). Wild-type and hsDkk1GFP tadpoles were fixed 8 h after the heat-shock. (B) In situ hybridization on sectioned samples of blastemas. Sectioned samples were hybridized with fgf-8 (a and b), fgf-10 (c and d), Lmx-1 (e and f), Hoxa-13 (g and h) or msx-2 (i and j). To guarantee the correct comparisons of the gene expression level, wild-type (a, c, e, g and i) and hsDkk1GFP (b, d, f, h and j) tadpole sections treated exactly the same way. Arrowheads show amputation level (knee level). D, dorsal; V, ventral. Scale bar: 100 μm.|
|wnt3a (wingless-type MMTV integration site family, member 3A ) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 52 hindlimb bud, lateral view, posterior up, anterior down.|
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