Due to the great disparity between regeneration times for the larval salamander (40 days), axolotl (30+ days), newt (44 days), and adult salamander (155 to 370 days), a staging system was devised so correlative comparisons could be made between regenerative model systems. The sequence was based on two criteria: 1) the stages should be similar to previously reported sequences for the newt, axolotl, and larval salamander, and 2) the stages must be readily recognizable by examination of the external morphology in the living state. Postmetamorphic adult land-phase Ambystoma were amputated through the forearm, placed within survival conditions, and observed until regeneration was completed. Of the initial 160 salamanders, 157 (98%) progressed through 11 clearly defined stages of regeneration. A side-by-side comparison of the staging sequence for land and aquatic phase urodeles is given along with a summary of key external morphological characteristics for the adult salamander. It was noted that as the length of time for regeneration decreased, the relative ratio of the nerves innervating the limb (spinal nerves 4, 5, and 6) increased for the four species of Ambystoma examined: A. annulatum, 324 to 370 days postamputation (dpa) with a 1:1:1 neural tissue ratio; A. maculatum, 255 to 300 dpa with a 2:2:1 ratio; A. texanum, 215 to 250 dpa with a 2:2:2 ratio; and A. tigranum, 155 to 180 dpa with a 2:3:3 ratio.
The present study identifies, localizes, and reports the relative composition of specific glycosaminoglycans within tissue matrices during the initiation phase of limb regeneration. The regenerate tissues were harvested and assayed morphologically, histochemically, and chemically. We observed 1) a population of cells interspersed among the cells of the dermis, epimysium, perimysium, perichondrium, and periosteum. 2) This population was distinguishable by a unique pattern of glycoconjugate staining, i.e., intracellular and pericellular heparan sulfate and glycoproteins and extracellularly associated hyaluronate and glycoproteins. 3) Cells with these staining characteristics aggregated to a position directly beneath the apical epidermal cap. 4) Extracellular hyaluronate and glycoproteins colocalized with undifferentiated tissues. And 5) extracellular chondroitin sulfate, dermatan sulfate, and keratan sulfate glycosaminoglycans colocalized with differentiated tissues. The correlations of distinct glycoconjugate compositions with specific regeneration morphologies suggest the possibility that these components may be related to the phenotypic expression of tissues during regeneration.
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