The influence of spinal cord on differentiation of skeletal muscle in regenerating limb blastema of Amblystoma larvae*

SUMMARY

In the regenerating limb of the salamander larva, do myogenically competent cells exist at all stages of the blastema? Spinal cord is known to promote muscle differentiation. To test the question, regenerating limb blastemata of varying ages were transplanted to the dorsal fin in company with segments of anterior spinal cord. Histological examination, 30 days later, revealed mature skeletal muscle fibers in experiments with blastemata of all ages. This was true in 5 of 16 cases using the 6-day blastema, 14 of 29 experiments with the 7-day blastema, and in every case using a blastema of more advanced stages (8, 11 and 15 day). Patterns of muscle fibers did not suggest limb-type morphology. While myogenically competent cells appear to contribute to the blastema, doubt exists that these are the very same cells that would have produce muscle in the normal limb regenerate.

Regeneration of the salamander limb is a phenomenon attended by a large, conjectural and speculative literature. Some text book writers have abstracted from the technical writings a belief that the regenerated limb is the product of differentiation within a primitive, indifferent mesenchymal aggregation (viz. the so-called blastema). Decisive evidence in support of the latter opinion is lacking. However, one need only examine the bulk of more recent articles to see that the belief in question constitutes a major premise upon which are built the main lines of thinking about regeneration.

Pietsch ('60a, b, ) performed a number of experiments whose results suggest that the blastema is not an aggregation of indifferent mesenchyme. Blastemata of various ages were cultivated in chambers in the dorsal fins of Amblystoma larvae. In some cases a piece of stump was left attached at the base of the blastema; in others the stump was excluded. With stump present both the limb skeleton and the limb musculature differentiated in the fin chamber. With stump absent typical limb cartilages developed but the musculature did not. The blastema, from the outset of its history as a discrete entity, failed to behave as though ontologically neutral or developmentally indifferent. At the same time, there was no evidence that the musculature arose from blastema cells. In some of the transplants an occasional myogenic cell was identified by the presence within it of one or two myofibrils. Might these and perhaps other myogenic cells in the blastema produce multinucleated fibers? In other words, are the cells in the blastemal aggregation capable of forming muscle?

An experimental approach was indicated by the independent findings of several other investigators who, using a variety of materials and methods have convincingly demonstrated that spinal cord enhances myogenesis in systems known a priori to contain myoblasts (Holtzer and Detwiler, '53; Holtzer, Holtzer and Avery, '55; Avery, Chow and Holtzer, '56; Holtzer, '56; Muchmore, '58). There was good reason to suspect that if myogenically competent cells enter into the formation of the blastema, these cells would respond to the presence of spinal cord by producing skeletal muscle tissue. If the latter expectation were vindicated, would the new muscle exhibit the organization found in the normally regenerated limb? Accordingly, blastema and spinal cord were transplanted together to the dorsal fin.

MATERIALS AND METHODS

Specimens were preserved in Bouin's fluid 30 days after transplantation. Processed routinely for paraffin sectioning at 10 u, specimens were stained with iron hematoxylin and examined histologically for muscle. Some tissue were silver stained, but this approach did not prove of significant value to the study. Cases in which either the blastema or the spinal had degenerated were excluded from analysis.

Experiments also were performed in which either spinal cord or blastema was transplanted alone. In the former, the only foreign tissue in the fin was the transplanted piece of spinal cord. Blastema by itself responded as previously reported (see Pietsch, '61b), namely by producing limb cartilages but no mature skeletal muscle fibers. These experiments shall not be considered further.

The term blastema is used here to denote the cone of new tissue, observed under the dissecting microscope, growing distal to the plane of amputation between the fifth and sixteen days following limb amputation. Under the conditions of this laboratory regeneration is usually complete within a month, but definitive tissues begin to supplant the blastema cells by about day 16.

RESULTS

Transplanting the 6-day blastema along with spinal cord resulted in the differentiation of mature skeletal muscle fibers in approximately one-third of the cases (see the table). In experiments with spinal cord and 7-day blastemata, 12 of 29 cases exhibited newly differentiated skeletal muscle tissue. Blastemata of more advanced age at the time of transfer to the dorsal fin chamber invariably produced muscle fibers in response to spinal cord (see the table and fig. 1).

The 6-day and 7-day cases lacking in muscle were re-examined at high magnification. Three specimens in each series showed an occasional myofibril-containing cell in the immediate vicinity of newly differentiated limb cartilages. Several re-examinations failed to reveal even this sign of muscle in the remaining cases (note last column in the table).

Multinucleated fibers in the transplants were narrow, cross-striated bundles of myofibrils similar in appearance and size to the muscle fibers in newly regenerated larval limbs. All regions of the sarcomere were identified.

Rough estimates were made of the amounts of muscle relative to that found in normal limb regenerates. Lacking other criteria for comparing transplant and normal regenerates, it was postulated that the amount of cartilage would bear an approximated relationship to overall limb size. Specimens were chosen in which antibrachial cartilages appeared in transverse section. Images were projected onto standard index cards at 70 X and the areas representing muscle and cartilage were traced. Traced areas were cut out, washed in acetone, dried and weighed. Muscle:cartilage ratios were computed from these weights. In a case such as that photographed in figure 2, the relative amount of muscle was about 75 % of that found in a normal regenerate of the same age. More rigorous quantification of the data concerning muscle volumes was not possible, owing to the absence of a suitable frame of reference. However, the crude estimates employed indicated that the amounts of muscle ranged from about 40-100 % of normal.

Muscle fibers that differentiated in the transplants assumed any of three relationships to the newly developed cartilages:

• uniformly distributed around the cartilages (figs. 2 and 4);
• concentrated in from one to several clumps at one side of the cartilage (figs. 3 and 5);
• in clusters of varying sizes located 50 to 300 u from the cartilages.

The distribution of the fibers could not be predicted on basis of the age of the blastema at transplantation. It is also noted that combinations of the three mentioned configurations were sometimes encountered in the same specimen (as is the case in figures 1 and 2).

While appreciable in amount, and although sometimes parceled into two or more clumps, in not one case was there an arrangement of muscle tissue according to the patterns that characterize the normally regenerated forelimb musculature (compare especially fig. 2 of this article with those in Piatt, '57; or with fig. 1 in Pietsch, '61b).

DISCUSSION

Although there is good evidence that muscle plays an important role in regenerative myogenesis (see Holtzer, '56 for tail regeneration) and, despite the atypical morphogenesis observed in this study, any proffered explanation of limb regeneration must take into account the presence of myogenically competent cells among the blastemal aggregation.

Using different mesenchymal systems, several other workers have observed marked increases in myogenesis as a response to spinal cord (see references in the introduction). These workers concur that the effect is enhancement of proliferation, in contradistinction to de novo production of myoblasts. The experimental conditions necessary to decide this point would have to satisfy the following requisites:

• the test system must be free of cells with myofibrils or contractile protein molecules;
• the system must contain myogenically competent cells; i. e., cells able to form muscle given appropriate circumstances;
• the system must not give rise to muscle spontaneously under condition of isolation.

Removing the 6-day blastema to the exclusion of all subjacent stump tissue is problematical. An attempt was always made to leave some blastema cells on the stump as a means of checking against contamination. If this objective was not always achieved -- as might well have been the case owing to the small size of the blastema at 6 days -- then it is quite possible that muscle formed from 6-day blastemata came from myoblasts of stump, not blastemal origin. Contamination, however, does not explain the rise between 6 and 7 in the number of cases exhibiting skeletal muscle.

Because spinal cord does seem to stimulate mitotic activity (see Overton, '55) and because mitosis is an important concomitant of muscle regeneration elsewhere (see Pietsch, '61a) it seems worth considering the possibility that the molecular events leading to myofibril formation are related to cell division, perhaps to alterations in division rates. Pietsch ('61a), working with mammalian skeletal muscle regeneration, obtained evidence indicating altered mitotic rates in would coagulum cells prior to myotube formation. When these more rapidly dividing cells were arrested in division (with colchicine) regenerating myotubes did not develop.

LITERATURE CITED

De Haan, R. L. 1956 The serological determination of developing muscle protein in the regenerating limb of Amblystoma mexicanium. Ibid., 133: 73-86.

Hay, E. D. 1959 Electron microscopic observations of muscle dedifferentiation in regenerating Amblystoma limbs. Develop. Biol., 1: 555-585

Holtzer, H. and S. R. Detwiler 1953 An experimental analysis of the development of the spinal column. III. induction of skeletogenous cells. J. Exp. Zool., 123: 335-370.

Holtzer, H., S. Holtzer and G. Avery 1955 An experimental analysis of the development of the spinal column. IV Morphogenesis of tail vertebrae during regeneration. Ibid., 96: 145-172.

Holtzer, S. 1956 The inductive activity of the spinal cord in urodele tail regeneration. J. Morph., 99: 1-39.

Laufer, H. 1959 Immunochemical studies of muscle proteins in mature and regenerating limbs of the adult newt, Triturus viridescens. J. Embryol. Exp. Morph., 7:431-458.

Muchmore,. W. B. 1958 The influence of embryonic neural tissues on differentiation of striated muscle in Amblystoma. J. Exp. Zool., 139:181-188.

Overton, J. 1955 Mitotic responses in amphibian epidermis to feeding and grafting. Ibid., 130: 433-484.

Piatt, J. 1957 Studies on the problem of nerve pattern. III. Innervation of the regenerated forelimb in Amblystoma. Ibid., 136: 229-247.

Pietsch, P. 1960a The development of muscle and cartilage in deplanted regenerating limb blastemas of Amblystoma larvae. Diss. Abstr. 31: 723.

Pietsch, P. 1960b The differentiation of limb blastemas deplanted into the dorsal fin of Amblystoma. Anat. Rec. 136: 258.

Pietsch, P. 1961a The effects of colchicine on regeneration of mouse skeletal muscle. Ibid., 139: 167-172.

Pietsch, P. 1961b Differentiation in regeneration. I. The development of muscle and cartilage following deplantation of regenerating limb blastema of Amblystoma larvae. Develop. Biol., 3: 255-264.

Weiss, P. 1950 The deplantation of fragments of nervous system in amphibians. I. Central reorganization and the formation of nerves. J. Exp. Zool., 113: 397-462.

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