A staging system has been devised for normal regeneration from the upper arm in the mature axolotl. It consists of seven externally definable stages: (1) Wound healing (WH): (2) Dedifferentiation (DD); (3) Early bud (EB); (4) Medium bud (MB); (5) Late bud (LB); (6) Palette (Pal), and (7) Digital outgrowth (DO). Serial histological sections of 38 regenerating limbs were used to correlate gross stages with microscopic events in the regenerative process.

A comparison between the surface features of embryonic limb development and limb regeneration was made in the axolotl. Scanning electron microscopy revealed an overall similarity between embryonic and regenerating limbs. A notable feature was the lack of a morphologically discrete apical epidermal specialization on the surface of any of the limbs. Histological preparations revealed no thickening of the apical epidermis in embryonic limbs. There is a definite thickening of the apical epidermis in regenerating limbs during the stages of dedifferentiation and the early blastemal phases, but the thickening projects into the underlying tissues rather than altering the smooth surface contours of the blastema. Minor differences in epidermal specializations (ciliated cells scattered over the entire body and very irregularly shaped cells with knob-like projections at the tips of digits in embryos) may be attributable to differences in overall activity of the animals at various stages in their life history.

Cellular behavior along the anteroposterior axis of the regenerating axolotl forelimb was studied by use of triploid (3N) tissue grafted into diploid (2N) hosts and three-dimensional computer reconstructions. Asymmetrical upper forelimbs were surgically constructed with one half (anterior or posterior) 3N and the other half 2N. Limbs were amputated immediately after grafting or were permitted to heal for 5 or 30 days prior to amputation. When regenerates had attained the stage of digital outgrowth, the limbs were harvested and sectioned in the transverse axis for histological analysis. When all limbs bearing anterior grafts were considered as a group, 77% of the 3N mesodermal cells were observed in the anterior side of the regenerates and 23% were located in the posterior side of the regenerates. When all limbs bearing posterior grafts were considered as a group, 76% of the 3N mesodermal cells were found in the posterior side of the regenerate and 24% had crossed into the anterior side. Healing times of 0, 5, or 30 days prior to amputation had no effect on the experimental outcome. Three-dimensional computer reconstructions revealed that most 3N cells of mesodermal origin underwent short-distance migration from anterior to posterior or from posterior to anterior and intermixed with diploid mesodermal cells near the midpoint of the regenerated anteroposterior axis. Some 3N cells were observed at greater distances from the graft-host interface. By contrast, labeled epidermal cells from both anterior and posterior grafts exhibited long-distance migration across all surfaces of regenerated limbs. Details of a computer-assisted reconstructive method for studying the three-dimensional distribution of labeled cells in tissues are presented.

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Almost perfect fits of the Gompertz equation to the growth in length of tail regenerates in the lizard, Lacerta lepida, and the newt, Notophthalmus viridescens, were obtained. Comparison of certain parameters of the equation with published mitotic index data suggests that the Gompertz equation characterizes each system at least from the time that significant mitotic activity is first observed histologically. An objective method for comparing the regeneration periods of the two species is described and applied. A unified hypothesis derived from consideration of properties of the Gompertz equation successfully accounts for the following phenomena reported, but previously unexplained, in the literature: (1) proximal amputations result in longer regenerates than do distal amputations; (2) proximal amputations elicit greater absolute rates of elongation (in mm/day) than do distal amputations; (3) the percent replaced of the length removed is rather constant, regardless of the absolute length regenerated; and (4) one of the parameters of the Gompertz equation appears to be lognormally distributed in a regenerating population. (See text for references.) A computerized interactive graphical system for normalizing growth equations of individual regenerates and integrating the mathematical model with potential candidates for biological control factors is briefly described.