Fine structure of the surfaces of the epithelium and mesenchyme of the newt limb regenerate, separated in salt solution: 14 to 25 days.

Abstract

Forelimbs of the newt Notophthalmus (Triturus) viridescens were amputated at the wrist, and at 14 to 25 days, the new limb regenerates were removed by a razor-blade cut. After incubation of the isolated regenerates in a medium containing only inorganic salts, the epithelium was removed easily from the underlying mesenchyme. The surfaces separated in the salt solution were examined using scanning and transmission electron microscopy to evaluate the completeness of the separation and the extent of tissue damage near the plane of separation. Rarely was an epithelial cell found clinging to the mesenchymal surface, supporting previous light microscopic observations. The most apparent morphological damage was sustained by the basal epithelial cell surfaces, which exhibited blebbing; the formation of vacant vesicles within the cytoplasm near the newly exposed plasma membranes; the structural disruption of some organelles; and the tearing away of fragments of the basal epithelial cell plasma membranes, which remained adherent to the mesenchymal surface during separation procedure. Examination of the structure of the epithelial-mesenchymal interface of intact regenerates showed that the plane of epithelial-mesenchymal separation was, for the most part, between the basal epithelial cell membrane and its substrate, an extracellular material which remained at the mesenchymal surface after separation. This substrate was in the form of an extracellular material closely adherent to the basal epithelial cell membrane at the distal tips of the regenerates; a developing basal lamina at the periphery of regenerates undergoing blastema accumulation; or a mature basal lamina at the base of digit stage regenerates. The usefulness of these separated tissues for future experimentation and the evaluation of the nature of the epithelial substratum throughout the course of limb regeneration are discussed.