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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.
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 rightlimb 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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