Acquisition of nerve dependence for the formation of a regeneration blastema in amputated hindlimbs of larval Xenopus laevis: the role of limb innervation and that of limb differentiation.

Abstract

In larval and adult urodeles and late-stage larval anurans, blastema formation after limb amputation requires an adequate nerve supply. Experimental evidence obtained from aneurogenic limbs indicates that, in urodeles, the acquisition of nerve dependence during embryonic development is due to the "addiction" of limb tissues to factors released by the ingrowing nerves rather than to limb differentiation. The aim of this work was to establish whether, in the toad Xenopus laevis, nerve-dependence for blastema formation after hindlimb amputation, which is acquired gradually during larval development and becomes complete at stage 57 is due to limb innervation or to limb differentiation. Two series of experiments were carried out. In the first series, limb differentiation was inhibited by treating the larvae with an anti-thyroid drug, and innervation was maintained for an interval much longer than that normally required for development from nerve-independent stages to stage 57. In the second series, the limb was caused to differentiate in the absence of nerves by maintaining the limbs denervated. Limb differentiation was often accelerated by treating early-stage larvae with thyroxine or by grafting early-stage limbs onto denervated limbs of late larvae, which, being near metamorphic climax, possessed high levels of circulating thyroid hormones. Results showed that in the first series of experiments the denervated limbs formed regeneration blastemas after amputation, but in the second series they did not. It was therefore concluded that the acquisition of nerve dependence for blastema formation in larval Xenopus laevis is not directly imposed by factors released by the nerve fibers, but is strongly related to differentiation of limb tissues.