Maculatin 1.1.1

Previous investigation into the regenerative ability of postmetamorphic adult land phase Ambystoma has revealed that these species have the capacity to completely regenerate a limb, given optimal environmental conditions, and 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, but 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 intracellular 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.

Historically, postmetamorphic adult land-phase salamanders have been shown to exhibit minimal to nonexistent limb regeneration. Hence, it has been generally accepted that these forms have lost the intrinsic capacity to regenerate a limb. Due to the experimental protocols used, an alternate explanation is also possible: that this intrinsic capacity cannot be expressed when the salamanders are maintained under adverse laboratory environmental conditions. Therefore, this study addresses two questions: 1) What are the optimal environmental conditions for long-term survival of adult land-phase salamanders; and 2) will complete limb regeneration occur in these salamanders if they are maintained under survival conditions. A mixed population of adult Ambystoma were tested under varying conditions of habitat, temperature, humidity, photoperiod, and food source. Complete limb regeneration was possible in 100% of four species of adult postmetamorphic land-phase Ambystoma salamanders given the proper environmental laboratory conditions of a peat moss and potting soil habitat with a controlled temperature of 25 degrees C +/- 5 degrees C, 70% or greater humidity, a 12/12 light/dark photoperiod, a diet including nightcrawlers released into their respective terraria, and an extended observation time of up to 370 days postamputation (dpa). Regeneration was completed during the following range periods for the adult salamanders: A. annulatum, 324 to 370 dpa; A. maculatum, 255 to 300 dpa; A. texanum, 215 to 250 dpa; and A. tigranum, 155 to 180 dpa.

A series of normal stages describing the regeneration of larval A. maculatum limbs after amputation through the upper arm or wrist is described. Nine discrete stages were recognized, based on external morphological and associated histological features. These stages are Initial Dedifferentiation (ID), Early Bud (EB), Medium Bud (MB), Late Bud (LB), Early Redifferentiation (ER), Notch (N), 2-Fingerbud (2-FB), 3-Fingerbud (3-FB) and 4-Fingerbud (4-FB). Similarities and differences between this and other staging systems for urodele limb regeneration are discussed. The absence of osteoclasts was a striking feature during dedifferentiation of the wrist, in contrast to their presence in large numbers during dedifferentiation of the upper arm.