Autoradiographic analyses of35S-sulfate uptake in regenerating limbs of larvalAmbystoma.

Abstract

Autoradiographic and histochemical techniques were used to determine whether chondrocytes continue to synthesize chondroitin sulfate or closely related compounds during morphological dedifferentiation of these cells in regenerating limbs of larvalAmbystoma. Forelimbs were amputated either through the mid-diaphysis or the distal epiphysis of the humerus and each animal was subsequently injected with Na235SO4 at an appropriate stage of regeneration. Incorporation of the isotope and metachromatic staining responses were used as indices of cell specialization.In autoradiographs of unamputated limbs, epiphyseal chondrocytes exhibited moderate sulfate incorporation, whereas isotope uptake was slight in diaphyseal regions. Accordingly, in early stages of regeneration, limbs amputated through the diaphysis showed a low level of sulfate incorporation by cartilage-derived cells; since these cells dispersed during blastema formation, they were not identifiable in later stages. When limbs were amputated through the epiphysis, the matrix here underwent slow dissolution and epiphyseal-derived chondrocytes and their progeny consequently remained identifiable as they contributed to the blastema. These cells continued to exhibit isotope uptake, even during early and middle stages of regeneration -results which support the idea of tissue-specific regeneration of cartilage.Further inspection of the stained autoradiographs revealed that in addition to chondrocytes and blastema cells derived from chondrocytes, fibroblast-like cells located lateral to the limb skeleton and seemingly derived from muscle or muscle-associated cells also exhibited a moderate label during certain stages in the restoration of the limb. In several respects isotope incorporation and related metachromatic responses by these two types of cells during blastemal and early redifferentiating stages of regeneration were seen to parallel results reported in the literature of histochemical and autoradiographic studies of differentiating chick fimb buds. These observations, which may be added to previous analogies concerning developing and regenerating limbs, suggest a similar mechanism of cytodifferentiation in the two systems. The possibility is also considered that the observed isotope uptake by cells of non-cartilaginous origin may indicate the synthesis of sulfated glycosaminoglycans which alfect cell interactions during the regenerative processes.