Abstract
BackgroundIn 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 FindingsWe 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/SignificanceIn 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.
Highlights
Limb regeneration in amphibians is one of the most fascinating examples of organ or appendage regeneration among tetrapods
Wnt-3a is expressed in the froglet blastema In most experiments, we used forelimbs to analyze froglet limb regeneration because hindlimbs are essential for swimming, and hindlimb amputation can result in drowning or exsanguination of the animal
Wnt-3a is expressed in ectodermal cell layers during formation of the apical ectodermal ridge (AER), a specialized ectodermal structure essential for the outgrowth of amniote limb buds, and induces fgf-8 expression in a b–catenindependent manner [35]
Summary
Limb regeneration in amphibians is one of the most fascinating examples of organ or appendage regeneration among tetrapods. In the case of anuran amphibians, such as Xenopus laevis, the tadpole can completely regenerate its developing hindlimb buds prior to the onset of metamorphosis, but the regenerative ability declines gradually as metamorphosis proceeds [3,4]. It is possible that elucidation of the critical factor(s) for blastema formation in the early stage of amphibian limb regeneration will enable us to control limb regenerative ability and will contribute to organ replacement therapy [12,14]. 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 regenerates a limb by a nerve-dependent process that results in a spike-like structure. Blockage of Wnt/b-catenin signaling inhibits the initiation of tadpole limb regeneration, but it remains unclear whether limb regeneration in young adults requires Wnt/b-catenin signaling
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