Hyaluronate accumulation and nerve-dependent growth during regeneration of larval Ambystoma limbs

Hyaluronate-mediated expansion of the extracellular matrix has been suggested as an important element of growth and morphogenesis in several developing systems. In vitro, various growth factors have been shown to stimulate hyaluronate synthesis as well as cell proliferation. A similar link between proliferation and hyaluronate production during in vivo growth is difficult to demonstrate, because in most systems the source of growth-promoting factors is either not known or not amenable to experimental manipulation. During amphibian limb regeneration, cell proliferation depends upon paracrine release of factors from axons in the limb stump, and the nerve supply can be eliminated or augmented experimentally for study of growth in this system. Denervated and amputated limbs of larval salamanders do not begin to regenerate until distal areas of the limb stumps are reinnervated. We have used such limbs to examine the effect exerted by the reappearance of nerves on the amount of hyaluronate in the tissue undergoing the growth response. Hyaluronate was demonstrated by the metachromatic dye Ethyl Stains-all, which stains hyaluronate blue while sulfated glycosaminoglycans (GAGs) and proteins in the extracellular matrix stain various shades of violet, and by microspectrophotometry of alcian-blue-stained GAGs in serial sections pretreated with buffer or with Streptomyces hyaluronidase (SH) to remove hyaluronate specifically. Both methods showed little hyaluronate in the distal region of limb stumps prior to reinnervation, while reinnervated stumps had amounts of hyaluronate similar to those of control blastemas. Autoradiography of 3H-glucosamine-labeled limbs indicated that hyaluronate in the blastemas of reinnervated limb stumps included material newly synthesized by cells throughout the growing tissue.(ABSTRACT TRUNCATED AT 250 WORDS)