Stages and Major Features of Limb Regeneration

Figure 1 shows a series of drawings following amputation either through the midradius and ulna (the zeugopodium level) or the midhumerus (stylopo-dium level) of an adult newt limb. This emphasizes several principles—whatever the level of amputation, the regenerate is a perfect copy of what was removed; the stages are the same whatever the level of amputation; regeneration from the lower arm is completed more quickly than from the upper arm. The latter occurs because there is less tissue to be replaced from lower arm amputations and the rate of cell division of blastemal cells is the same at lower arm levels as at upper arm levels (9).

Staging systems for various species have been published: adult N. viridescens (10), large axolotls (11), and larval A. maculatum, also valid for small larval axolotls (12). In the following description, the major events of regeneration are described rather than a precise staging system, which can be obtained from the above references. Also, times are not included, since this depends on the ambient temperature, age of the animal, and species. As mentioned above, in small larval axolotls, this is a very rapid process, being completed in 2-3 wk.

1. Wound healing (Fig. 1, stage 1; Fig. 2A): This is achieved in a matter of hours after amputation by migration of the epidermis from the cut edges of the stump. It is slower if the protruding bones are not trimmed away. There are large numbers of damaged mesodermal cells and blood clots that the wound epidermis has covered, and during the first few days after amputation, phagocytes accumulate to remove this cell debris. This debris can also be seen between the cells of the wound epithelium, so some must be ejected via this route. Fluid often accumulates at the tip, but a circulating blood supply is rapidly re-established. Mitosis in the wound epithelium is inhibited for several days after amputation.

2. Dedifferentiation (Fig. 2B,C): Once the local damage has been repaired, the net effect of wound healing can be appreciated—the juxtaposition of a naked epidermis and mesodermal tissues. Normally the dermis and basal lamina are present to prevent interactions. Indeed, if full thickness skin is sewn over an amputated stump, then limb regeneration is inhibited, as described above (2,13). The wound epithelium seems to be active in inducing histolysis of the mesodermal tissues it is now in contact with and the process of dedifferentiation begins. The cytoplasm of the myotubes fragments and surrounds individual nuclei, and osteocytes appear along the cut ends of the bones breaking down the matrix and releasing individual chondrocytes (Fig. 2B,C). This process is clearly an organized one unrelated to simple phagocytic breakdown, because the result of dedifferentiaion is the appearance of embryonic cells with large pale nuclei and active cytoplasms rich in rough endoplasmic reticulum. These dedifferentiated cells now begin cell division and accumulate under the apical cap.

3. Apical cap (Fig. 2D): After closure of the wound, the epidermis continues to migrate and piles up at the tip forming a very thick apical epidermis or apical cap. This structure has been likened to the apical ectodermal ridge (AER) of the chick limb bud, since it is responsible for the accumulation of dedifferentiated cells beneath it, for the direction of outgrowth of the blastema, and it behaves like the AER in inducing an accessory outgrowth when transplanted (14). Perhaps it also generates fibroblast growth factor (15), as the AER does.

4. Blastema: The accumulation of dedifferentiated cells beneath the apical cap results in the appearance of a small, conical structure known as the early bud blastema (Fig. 2D). The blastemal cells now begin to divide rapidly and increase in number by proliferation rather than by continued dedifferentiation, the latter now ceasing. As more and more cells are generated, the blastema increases in size, forming a larger and larger cone at the limb apex (stage 2 in Fig. 1) and is known as the medium bud blastema (Fig. 2E). Nerve fibers are present throughout the blastema, but the vasculature appears sparse.

Fig. 2. Stages of limb regeneration. (A) Wound healing. Within a few hours after amputation a wound epithelium (we) migrates over the stump to heal the wound completely. Solid line marks the level of amputation through the radius and ulna. (B,C) Dedifferentiate. In B, muscle dedifferentiation is shown. The loss of myofiber structure can clearly be seen stretching back a considerable distance from the amputation plane, between the two arrowheads. In C, cartilage dedifferentiation is shown.

Fig. 2. Stages of limb regeneration. (A) Wound healing. Within a few hours after amputation a wound epithelium (we) migrates over the stump to heal the wound completely. Solid line marks the level of amputation through the radius and ulna. (B,C) Dedifferentiate. In B, muscle dedifferentiation is shown. The loss of myofiber structure can clearly be seen stretching back a considerable distance from the amputation plane, between the two arrowheads. In C, cartilage dedifferentiation is shown.

5. Redifferentiation (Fig. 1, stage 3; Fig. 2F): As the blastema enlarges and becomes an elongated cone, the cells in the proximal region adjacent to the stump begin to segregate into precartilage and premuscle masses. This stage is known as the late bud or palette stage. As redifferentiation commences, the first new cartilage element (e.g., the distal half of the humerus if amputation was through the midhu-merus level) appears as a continuation from the stump, as if the remaining piece of humerus serves as a model. The same applies to the newly differentiating muscles. Redifferentiation is occurring proximally, while distally, proliferation of the blastemal cells continues. Clearly then, redifferentiation progresses in a generally proximal-to-distal fashion (except for a blip in the wrist), since the appearance of the humerus is soon followed by the appearance of a completely new radius and ulna, followed by the digits, followed by the wrist elements. This sequence is a repeat of the sequence of appearance of elements during development, even down to the wrist elements and digits, which develop and regenerate in an anterior to posterior sequence. When the digits first appear (Fig. 1, stage 4 on the left and stage 5 on the right), the regenerate is at the early digit stage, and when all the digits have appeared (Fig. 1, stage 5 on the left and stage 6 on the right), the regenerate is at the late digit stage.

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