Histological analysis of limb regeneration in postmetamorphic adult Ambystoma

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Abstract
Previous investigation into the regenerative ability of postmetamorphic adult land phase Ambystoma has revealed that (1) these species have the capacity to completely regenerate a limb, given optimal environmental conditions, and (2) the gross morphological characteristics of limb regeneration in these species compared favorably with the external regeneration morphology of aquatic phase forms. The present study concerns a histological and histochemical examination of the regenerating limb tissues and their respective extracellular and intracellular tissue matrices. Postmetamorphic adult Ambystoma were amputated through the forearm, placed within optimal environmental conditions, and allowed to regenerate. The tissues were harvested at designated intervals after amputation and prepared for light microscopic examination. The limb tissues were assayed histologically for similarities to and differences from previously established regeneration morphologies. It was noted that specific correlations (i.e., apical epidermal cap formation, bud outgrowth and elongation, palette formation, and digit formation) existed between regeneration histologies in these species and those previously reported for the aquatic urodeles, newt, axolotl, and larval salamander. By utilizing the histological and histochemical characteristics of the tissue, the regenerate limb was divided into five tissue units: epidermal, blastemal, soft, hard, and neuro/vascular. Based on the unique morphology of their extracellular matrices and respective histochemical staining patterns, four distinct blastemal regions were delineated within the blastemal units: subregenerate epidermal blastema, soft-tissue blastema, hard-tissue blastema, and core blastema. Histochemically, changing patterns of highly sulfated, weakly sulfated, and carboxylated polysaccharides and glycosylated compounds were located within both the extra- and intracelluler stump and regenerate tissue matrices during regeneration. In addition, these patterns of intra- and extracellular macromolecular material correlated to previous reports of similar-type compounds assayed during regeneration in aquatic urodeles. With this in mind, the adult land phase Ambystoma can be considered an appropriate model system for studies concerning normal limb regeneration.