A comparative study of gland cells implicated in the nerve dependence of salamander limb regeneration

Abstract

Limb regeneration in salamanders proceeds by formation of the blastema, a mound of proliferating mesenchymal cells surrounded by a wound epithelium. Regeneration by the blastema depends on the presence of regenerating nerves and in earlier work it was shown that axons upregulate the expression of newt anterior gradient (nAG) protein first in Schwann cells of the nerve sheath and second in dermal glands underlying the wound epidermis. The expression of nAG protein after plasmid electroporation was shown to rescue a denervated newt blastema and allow regeneration to the digit stage. We have examined the dermal glands by scanning and transmission electron microscopy combined with immunogold labelling of the nAG protein. It is expressed in secretory granules of ductless glands, which apparently discharge by a holocrine mechanism. No external ducts were observed in the wound epithelium of the newt and axolotl. The larval skin of the axolotl has dermal glands but these are absent under the wound epithelium. The nerve sheath was stained post-amputation in innervated but not denervated blastemas with an antibody to axolotl anterior gradient protein. This antibody reacted with axolotl Leydig cells in the wound epithelium and normal epidermis. Staining was markedly decreased in the wound epithelium after denervation but not in the epidermis. Therefore, in both newt and axolotl the regenerating axons induce nAG protein in the nerve sheath and subsequently the protein is expressed by gland cells, under (newt) or within (axolotl) the wound epithelium, which discharge by a holocrine mechanism. These findings serve to unify the nerve dependence of limb regeneration. © 2010 The Authors. Journal compilation © 2010 Anatomical Society of Great Britain and Ireland.