REGENERATIVE RESPONSES IN CULTURED HINDLIMB STUMPS OF LARVAL XENOPUS-LAEVIS

The regenerative capacity of larval Xenopus laevis hindlimbs amputated through the tarsalia at different stages of development and explanted in vitro was tested. In the first experimental series hindlimb stumps from stage 53, 54, 55, and 57 larvae (according to Nieuwkoop and Faber, '56) were cultured in Leibovitz's L-15 medium supplemented with 10% FCS, and 0.04 U of insulin and 10(-8)mg of L-thyroxine per ml of medium. Results showed that the distal part of the limb stumps from stages 53, 54, and 55 formed a regeneration blastema composed of proliferating mesenchymal cells beneath a typical apical cap. No blastema was formed in the proximal part of the stump. In limb stumps from stage 57, a regeneration blastema did not form either in the proximal or in the distal part of the stump. In a second experimental series, hindlimb stumps from stage 55 larvae, denervated 5 days prior to amputation in order to eliminate any residual neurotrophic factor, were cultured in a simplified L-15 medium containing 2% FCS and lacking insulin and thyroxine. Results showed that also in these experimental conditions the stumps from stage 55 formed a conical regeneration blastema at the distal tip. The blastema cells duplicated their own DNA and divided. At the proximal extremity no regeneration blastema was formed. In the same culture medium, the stumps of larvae at stage 57 did not form a regeneration blastema. Overall these results confirm those obtained in vivo on denervated and amputated limbs of larval Xenopus (Filoni and Paglialunga: Differentiation 43:10-19, 1990), namely, that the early stage limbs are able to form a regeneration blastema in the absence of nerves while the late stage limbs are nerve-dependent for regeneration. The capacity of the early stage limbs for forming a regeneration blastema on the distal tip seems related to the presence, in the amputation area, of a population of undifferentiated mesenchymal cells capable of proliferating in the absence of mitogenous nerve factors.