Morphogenesis and differentiation of grafted blastemas formed in vitro from amputated hindlimbs of larval Xenopus laevis.

The present study was designed to test the morphogenetic potency of limb blastemas formed in vitro from amputated limbs of larval Xenopus laevis. Hindlimbs of larvae at stage 55 (according to Nieuwkoop and Faber [1956] Normal Table of Xenopus laevis (Daudin)) were amputated through the tarsalia, excised at the base of the thigh and cultured in Leibovitz's L-15 supplemented with 2% FCS. After 8-10 days, 50% of the cultured limbs formed a conic blastema on the amputation surface. However, on the excision surface no blastema was present. Three different parts (blastema, blastema with the shank region and proximal part of the limb) of the cultured limbs were then grafted to the axial musculature or to the hindlimb of stage 57 host larvae. Results showed that the blastema formed in vitro were true autodifferentiating regeneration blastemas, since they were able to form well-differentiated autopodia not only when grafted with the shank region to a neutral territory (axial musculature) or to the limb territory, but also when transplanted alone to the two environments. The morphological complexity (no. of toes) of the autopodia differentiated from the grafted blastemas was superimposable to that observed in vivo. Moreover, as in vivo, the entire regeneration process was nerve-independent. In fact, the regeneration blastemas, formed in vitro in the complete absence of nerves, could grow and differentiate also when grafted to denervated host limbs. The grafted proximal parts of the cultured limbs never formed a regenerate.