THE DEVELOPMENT OF MUSCLE AND CARTILAGE IN DEPLANTED REGENERATING LIMB BLASTEMAS OF AMBLYSTOMA LARVAE

Paul Pietsch

A dissertation in Anatomy

Doctor of Philosophy.

1960

________________________
Howard Holtzer, Ph.D.,
Department of Anatomy
Supervisor of Dissertation

________________________
Jean Piatt, Ph.D.,
Department of Anatomy,
Advisor

________________________
Louis B. Flexner, M.D.,
Chairman, Department of Anatomy,
Chairman of Group Committee

Introduction

The pluripotent blastema concept replaced the view that regeneration was a consequence of direct budding from preexisting tissues (Barfurth '91, Morgan '01, Wendelstadt '04). Weiss pointed out that the skeleton of the stump could be extirpated and yet the regenerate would form a skeleton (see also von Fritsch '11, Kurz '12, Morrill '18, Thornton '38b, Goss '56, S. Holtzer '56). Weiss, and many others later on, inferred that the cells contributing to the mesenchymal derivatives of the regenerating part must be both undetermined and capable of responding to a variety of inductive influences. In other words, the regenerative blastema was essentially an embryonal system which acquired, progressively, the characteristics of a definitive part. There is almost universal agreement that individual and alleged pluripotent blastema cells are capable of differentiating cartilage or muscle or other mesenchymal derivatives. However, Polezhaiev ('34) and especially Needham ('50) have rejected claims that the blastema is organogenetically plastic.

If limb regeneration in salamanders is based upon the differentiation of a pluripotent group of cells, then isolation of the blastema in a neutral environment should yield: 1) an undifferentiated mass if the blastemas are young or if the environment is unsuitable; 2) development of a limb replete with muscles and cartilages if the blastema is older.

In 1954, Holtzer et al reported that nine day limb blastemas placed into the dorsal fins of Amblystoma larvae would form limb cartilages, but not muscle. The results of Holtzer et al (op. cit.) suggested that the pluripotency concept, as applied to the limb blastema, might be inadequate. If still younger isolated blastemas will form cartilage, then it is unlikely that the cells in question are undetermined. Further, if blastemas will form cartilage but not muscle, then the possible fates of blastema cells are restricted and therefore not pluripotent.

The purpose of this work was to reevaluate the fate of regenerating limb blastemas. The results do no support the pluripotent blastema concept, but suggest that limb regeneration might be interpreted according to another hypothesis. The experiments to be described involved isolation of regenerating limb blastemas and evaluation of: 1) conditions under which mature skeletal muscle differentiated; 2) the extent to which cartilage formed.

Materials and Methods

A blastema began to appear on the larval limb stump at about the fifth day after amputation. The earlier the phase of regeneration, the more difficult it was to be sure of a sharp separation between blastema and subjacent stump tissues. Preliminary investigations suggested it would be impossible to exclude stump tissues from the base of the blastema before the sixth day of regeneration. It was quite probable that some six day blastemas were contaminated with bits of stump tissue. The experiments reported were with six, seven, eight, ten, eleven, twelve and fifteen day blastemas. Sixteen days after amputation the normal regenerates had acquired mature muscle and limb-like cartilages. Thus a blastema was present from the fifth through the fifteenth day of regeneration. The limbs were completely regenerated thirty days after initial amputation.

The method of deplanting to the dorsal fin of Amblystoma larvae is discussed by Weiss ('50). A tunnel was made in the jelly-like connective tissue of a dorsal fin of an Amblystoma larva and experiment tissues place therein. Deplanting is much like chorionic grafting, the rabbit ear chamber, the anterior eye chamber or the cheek pouch of the hamster, in that growth and differentiation of tissue may be studied in isolation. The deplantation method is particularly valuable in the study of limb regeneration since the blastema does not differentiate under standard in vitro conditions (Lecamp '47, Fimian '59, Pietsch, unpublished).

Blastemas were deplanted singly or in combinations of two, four or eight. In some cases skins were stripped mechanically from blastemas before deplantation. Number of blastemas per deplant and presence or absence of skin will be discussed only when they relate significantly to the results.

All tissues were grown in the dorsal fins for thirty days. Thus deplant regenerates were fixed thirty-six to forty-five days after the initial limb amputation.

The following experiments were performed:

1. DP-bud or experiments in which limb rudiments of Harrison stage 37 Amblystoma embryos were deplanted to establish the fact that myogenesis is not interfered with by the environment encountered in the dorsal fin.
2. DP or experiments in which blastemas were deplanted. A number in front of the letters refers to the age of the blastema when it was deplanted. Thus an '11-DP" denotes an experiment in which an eleven day blastema was deplanted.
3. DP-stump or experiments in which the subjacent stump was left attached to the deplanted blastema.
4. DP-detached stump or experiments in which a seven or eleven day blastema and subjacent stump were detached from each other. The blastema was deplanted in proximity with the distal end of its own stump. An interval of about 100-300 u separated the proximal end of the deplanted blastema and the distal end of the stump from which it was taken.
5. DP-arm or experiments in which the proximal end of a freshly cut piece of upper arm was placed in proximity with a deplanted seven or eleven day blastema.
6. TP-orbit or transplants of eleven day blastemas to the orbit in place of the eye. The base of the transplanted came into contact with cut extraocular muscles.
7. DP-cord or experiments in which a 2 mm segment of brachial level spinal cord was deplanted along with a six, seven, eleven or fifteen day blastema.

The terms 'mature' and 'immature' are used below in reference to muscle. 'Mature' refers to multinucleated fibers which have achieved diameters encountered in muscles of the normally regenerated limb. 'Immature' refers to muscle which is in the form of mononucleated myoblasts. These myoblasts are distinguished from other cells by the presence of cross striated myofibrils (see Fig. 14). Examination of representative six to fifteen day blastema failed to reveal the presence of muscle in any form except in the region of the stump immediately adjacent to the blastema. Thornton ('38a), however, has noted the presence of skeletal muscle fibers in fifteen day blastemas of Amblystoma larval limbs. Electron micrographs (Peterson, unpublished) revealed that there are occasionally myoblasts in the proximal parts of eleven day blastemas. Some myoblasts might have escaped detection during ordinary histological examination. Holtzer, Marshall and Finck ('57), for example, have demonstrated the presence of muscle with fluorescent antimyosin which would have escaped detection with iron hematoxylin. Moreover, since the presence of cross striated myofibrils was the only criterion for identification of myoblasts, the angle of sectioning may have caused muscle to go undetected. Thus in the present investigation, two things had to be kept in mind regarding muscle: 1) The blastemas may have possessed myoblasts at the time of their deplantation. 2) The failure to detect immature muscle might be a consequence of the limitations imposed by the techniques employed.

Selected deplant cases were counted for the total number of identifiable myoblasts in each. Again, it is emphasized that immature muscle may have escaped identification. In the cases counted it was known from previous examinations that immature muscle was present. Each section was inspected at high magnification and every identifiable myoblast was counted. The values are expressed in absolute numbers per specimen.

Large amounts of mature skeletal muscle were encountered in DP-stump, DP-cord and in TP-orbit experiments. To illustrate large amounts of mature muscle, cases were selected in which the forearm regions appeared in cross section. Images, 70 times the size of the specimen, were projected onto standard index cards. The areas of muscle and cartilage were traced onto the cards. These traced areas were weighed. Amounts of muscle were expressed in relation to the amounts of cartilage. The reasons for using cartilage (versus the entire limb) as a basis for comparing large amounts of mature muscle were: 1) deplants and transplants were smaller than normal regenerates; 2) the epithelium of the deplants usually degenerates making it impossible to compare the absolute sizes of normal and deplaned regenerates.

Results

A. Myogenesis

1. Will the dorsal fin support myogenesis?

Development of mature skeletal muscle thus can take place under the conditions presented by the dorsal fin.

2. Can isolated deplanted blastemas form muscle? (DP experiments)

The results for 153 cases are summarized in Table I. In 151 of these cases muscle was either completely undetected or present as sparsely distributed myoblasts (see Figs. 13, 14). In the two cases with mature muscle, the blastema was deplanted fifteen days after initial limb amputation and therefore might have had muscle fibers at the time of isolation. Attention is called to the observation of mature muscle in normal sixteen day limb regenerates.

It can be seen from the Graph that when blastemas were deplanted at ten days (10-DP) no detectable muscle developed, either mature or immature. Muscle, always immature, was found in about 10 percent of 7-DP and in about 50 percent of 6-DP cases. It is believed that the incidence of muscle in the experiments with younger blastemas represents contamination with muscle from the stump. The presence of immature muscle in the deplants of blastemas older than ten days (see Graph and Table I) is believed to be a consequence of the invasion by myoblasts from the stump before isolation of the blastema. Myoblasts have been detected in the basilar portions of intact blastemas as early as eleven days after initial limb amputation. The possibility cannot be denied that the myoblasts in the eleven day and older blastemas developed under some unknown influences of the stump.

The absence of detectable muscle in 10-DP and its paucity in 7-DP cases cannot be explained simply by assuming widespread cell death of blastema cells, for the cartilage is just as well developed in the 10-DP as in any other experiments in the entire investigation.

The amount of immature muscle in even the advanced aged blastemas is exceedingly small. To illustrate the paucity of muscle in the DP experiments six cases, known from previous examinations to contain myoblasts, were inspected and each fiber in each specimen was counted at high magnification. An average of sixteen fibers per specimen was encountered. At best, therefore, the muscle is only a minute fraction of what is encountered in even a single section through a normally regenerated limb (see Figs. 1 and 2).

It should be noted that when muscle was detected cartilage was always present in large amounts.

3. Will normal limb muscles develop if stump is left attached to the base of the deplanted blastema? (DP-stump experiments)

When stump was left attached to the deplanted blastema mature muscle developed. The muscle was not only large in amount but was organized into the typical pattern of the normally regenerated limb. On the other hand, when the deplanted blastema was deprived of stump, the quality of myogenesis was exceedingly poor even though the blastema had developed to an advanced stage before isolation.

4. Will the detached stump enhance myogenesis in the deplanted blastema? (DP-detached stump experiments)

5. Can newly injured limb tissue enhance myogenesis in the deplanted blastema? (DP-arm experiments)

6. Can injured, non-limb muscles enhance myogenesis in the blastema? (TP-orbit experiments)

After thirty days, normal-appearing hands projected from both the orbits and the tail fins. Those in the orbit were observed to execute movements in harmony with the intact eye on the contralateral side. The hands which projected from the fins never exhibited motion of their own, even upon mechanical stimulation.

Histologically, the deplants showed the same trends as already described. The orbital transplants possessed sizable masses of mature skeletal muscle extending without interruption between the limb cartilages and the orbital wall. The amount of muscle in the five cases examined was estimated to be about 40 percent of that encountered in the normal regenerate (see Table II). Whether the muscle in these orbital transplant experiments is arranged in a limb-like pattern is the subject of investigations now in progress.

The orbital transplant experiments as well as those with stumps and injured upper arm, demonstrate that when muscle was in the neighborhood of the blastema myogenesis was enhanced. In each case, the muscle of the regenerate was continuous with the preexisting muscle. The implication was that muscle made a direct contribution to myogenesis.

7. Can spinal cord enhance myogenesis in the deplanted blastema? (DP-cord experiments)

The results of the DP-cord experiments are summarized in Table III. Large amounts of mature skeletal muscle were encountered in each case when spinal cord was deplanted with an eight day or older blastema. With seven day blastemas and spinal cord, twelve of twenty exhibited large amounts of muscle. Eight of thirteen 6-DP-cord experiments led to the production of mature skeletal muscle. The amounts of muscle in each of the cases mentioned was sizable (see Table II; i.e., about seventy-five percent normal. The reason for an absence of mature muscle in a large percentage of young blastema experiments cannot be given. The simplest explanation seems to be that not all younger blastemas were equally endowed with myoblasts. Just as in experiments with the blastema (6-DP) alone, myoblasts of the six day blastemas were probably carried over from muscle of the stump. When acted upon by the spinal cord the myoblasts, whatever their true source, proliferated profusely. Unless myoblasts were present the cord was unable to exert beneficial effects on myogenesis. It is difficult to explain the absence of muscle in the large percentage of 6-DP-cord and 7-DP-cord by assuming widespread cell death for, even though devoid of detectable muscle, these particular cases has well organized cartilages.

It is interesting that the percentage of cases showing any muscle at all is about the same in experiments using six day blastemas with or without the presence of cord. On the basis of these and the observations of other workers (Holtzer and Detwiler '55, Avery et al '56, Muchmore '58) the simplest inference seems to the that mononucleated myoblasts are stimulated to divide under yet unknown influences of spinal cord.

There is a significant detail about the muscle in the DP-cord experiments. Though present in large amounts, the muscle in the spinal cord experiments was never arranged into the specific patterns encountered in either the normally regenerated limb (Figs. 1 and 2) or in experiments in which stump was attached to the deplanted blastemas (Figs. 3, 4, 5). Figs. 6 through 12 are presented to illustrate that the large masses of muscle encountered in the DP-cord experiments fail to assume typical limb order. Small foci of myoblasts seem to have proliferated into large, but unorganized, muscle masses. This observation was consistent for DP-cord cases, irrespective of age of the blastema at deplantation.

Another indication from the results with cord and blastema experiments is that a muscle pattern is not fixed within the blastema before mature muscle is already present. The 15-DP-cord experiments resulted in large amounts of muscle which were no better organized than in spinal cord experiments with younger blastemas. After sixteen days muscle was already present in the normal regenerate. It seems, therefore, that the blastema passes through almost all of its history as such without acquiring a presumptive organization of limb-like muscles.

As mentioned in connection with the simple blastema deplant experiments (DP), when muscle was present large amounts of organized cartilage were also to be found.

B. Chondrogenesis

1. Is the isolated deplanted blastema able to form cartilage?

2. What is the importance of stump to chondrogenesis in the deplanted blastema?

1. by comparing the frequency of identifiable limb cartilages in experiments with older and younger blastema;
2. by comparing the results of experiments with and without attached stumps. These two issues were approached simultaneously .

The results are summarized in Table V. Details were as follows:

1. 7-DP experiments. Nineteen cases were examined. In eight cases it was possible to make a specific identification of some portion of the limb skeleton. One case showed no cartilage at all. In the remaining ten cartilages resembled elements of the limb skeleton but could not be accurately identified. Digits, carpals and metacarpals were the most frequently identified cartilages. One of the 7-DP cases exhibited the distal ends of radius and ulna. A wrist was also present in the latter case with what seemed to be hand elements bent back over the carpals. Paired rods suggesting radius and ulna were observed in seven of eight cases with other identifiable elements of the skeleton.
2. 7-DP-stump experiments. Of eight cases, three exhibited identifiable components of the forelimb skeleton. One case showed no cartilages. Four cases possessed cartilages with rod or cuboid shapes. A radius and ulna were identified in two of the three mentioned positive cases.
3. 11-DP experiments. Twenty cases were examined. Specific limb cartilages were identified in eight cases. Radius, ulna and humerus were identified in one of the eight cases.
4. 11 DP-stump experiments. Six of sixteen cases possessed specific limb skeletal parts. Radius and ulna were present in three of these six cases.
5. Experiments with fifteen day blastemas. With or without stump, when cartilages were present in these cases, specific identifications were always possible. It should be noted that chondrogenesis is already manifested in the fifteen day blastema.

3. Will several deplanted blastemas regulate to form a single large limb?

Similar experiments were conducted in which the skins were stripped mechanically from the blastemas before deplantation. The results were the same as the experiments in which skins were not disturbed. In one particular case where two skinless eleven day blastemas were brought together into apposition, it seemed that there had been a coalescing of the distal elements of the two skeletons. Staining revealed that while the soft tissues had fused, the cartilages were arranged into two separate groups.

The extent to which blastemas retain morphological integrity is an object of investigations still progress. As far as the data go, it would seem that chondrogenic order in the blastema is not readily disrupted by influences from nearby cells of another blastema.

Discussion

Chondrogenesis takes places under a variety of conditions; i. e., ectopically (Nageotte '18, Heinen, Dabbs and Mason '49), in extirpation and chorion graft experiments (Holtzer and Detwiler '55, Avery et al '56), in regenerating limbs from which the cartilages have been removed (Thornton '38b), in explants of subcutaneous connective tissue (Nassonov '34), in situ cultures of dissociated limb bud cells (Moscona and Moscona '52). Muscle, on the other hand, has never been critically demonstrated to form in the post embryonic animals unless other muscle preexists.

Muscle formation thus seems to be predicated upon fairly specific circumstances while the requirements for chondrogenesis are much more generalized. Chondrogenesis, during regeneration, is not dependent upon preexisting cartilage (Thornton '38b, S. Holtzer '56). There is even the suggestion that chondrogenesis may proceed at the expense of myogenesis when there is a curtailment of available cells (see Holtzer and Detwiler '55, Moscona and Moscona '52).

The present investigation does not support the concept of pluripotency as applied to the regenerating limb blastemas of Amblystoma larvae. The main point of disagreement is in the fact that the early blastemas of the present investigation were not 'In a condition of have a large number of possible fates...'(see Needham's glossary '50). Rather it appeared that the majority of cells of the deplanted blastema were chondroblasts already set fairly rigidly along a specific course of differentiation. It is, therefore,. suggest that the view of Fraisse (1885) is to be taken seriously in regards to limb regeneration, namely that the blastema is essentially procartilage and the muscles are derived from myoblasts which are supplied directly from the stump (see also S. Holtzer '56). The basis for larval Ambylstoma limb regeneration would seem to lie in the readiness for wounded tissues of the stump to provide chondroblasts. The readiness to form cartilage is not the exclusive property of the regenerate.

Summary

1. The forelimb blastemas of regenerating Amblystoma larvae limbs were studied after isolation in dorsal fin deplant chambers. Attention was directed towards chondrogenesis and myogenesis.
2. Chondrogenesis was found to take place almost invariably in blastemas isolated at the earliest testable age (six days after initial limb amputation). The cartilages formed from blastemas of all ages were organized into discrete components of the limb skeleton. Age at time of deplantation and presence or absence of stump were variables which had no discernible relationship to chondrogenic activity in the deplanted blastemas.
3. The deplanting of two or more blastemas into the same fin chamber tended to yield the same number of individual skeletons. Thus the orderly pattern as regards the skeleton of the limb regenerate was not established early, but was able to resist possible influences of nearby cells of another blastema.
4. Myogenesis was not well represented in blastemas deplanted without other tissues. In experiments in which the blastema was deplanted without other tissues, muscle was either totally lacking or present in the form of sparsely distributed myoblasts. When deplanted in the presence of attached or detached arm tissues, the regenerate developed appreciable amounts of mature skeletal muscle fibers. When the blastema was transplanted to the orbit such as to come into contact with extraocular muscles sizable amounts mature skeletal muscle developed.
5. Spinal cord was found to enhance the quality of myogenesis particular among older deplanted blastemas. The muscle formed in these instances was large in amount but fail to assume the topographical distribution of normal limb regenerate muscles. Since the muscle occurred unorganized masses even in experiments with the latest staged blastemas (fifteen day), the conclusion was reached that there is no fixed pattern for muscle at any time in the history of the blastema as such. The spinal cord did not enhance myogenesis in all younger blastemas; the implication was that some of the younger blastemas did not posses myoblasts at all.
6. The data obtained in the present studies do not support the generally held belief that limb regeneration in salamanders is based upon a 'pluirpotent' blastema. The alternative hypothesis is proposed that the salamander limb regenerates because of the tendency of injured stump tissues to yield highly ordered chondroblasts. Muscle would then form secondarily from myoblasts supplied directly from the injured muscles of the stump.

Bibliography

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Emerson, H. S. 1940 Embryonic induction in regenerating tissue of Rana pipiens and Rana clamitans larvae. J. Exp. Zool. 83:191-224.

Fimian, W. J., jr. 1959 The in vitro cultivation of amphibian blastema tissue. J. Exp. Zool. 140: 125-144.

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Goss, R. J. 1956 The relation of bone to the histogenesis of cartilage in regenerating forelimbs and tails of adult Triturus viridescens.. J. Morph. 98: 89-124.

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Holtzer, H., Avery, G. and Holtzer, S. 1954 Some properties of the regenerating limb blastema cells of salamanders. Biol. Bull. 107: 313.

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FIGURES

Fig. 2 Cross section through normally regenerated thirty-seven day regenerate at lower level than in previous figure. 100 X.

Fig. 3 An 11-DP-stump case. Muscles are arranged in the typical pattern of the forearm. Amputation was just above the elbow joining. 100X.

Fig. 4 Another 11-DP-stump showing essentially the same thing as in Fig. 3. Amputation just above elbow joint. 100 X.

Fig. 5 A 7-DP-stump. Note orderly arrangement of muscles into forearm pattern. Amputation through the neck of humerus. 100 X.

Fig. 6 An-11 DP-cord case. Note that muscle is in one large continuous mass around the cartilages. 125 X.

Fig. 7 Low power micrograph of same 11-DP cord specimen in Fig. 6. Different region. Note muscle mass at top of photograph and spinal cord towards the bottom. 50 X.
Fig. 8 A 6-DP-cord case. Note large clump of muscle in lower half and cartilage above that mass. 100 X.
Fig. 9 A second 11-DP-cord case. Muscle swarms around cartilage in a single continuous mass. 100 X.

Fig. 10 A 7-DP-cord case. Muscle is in one uninterrupted mass around small cartilages. 100 X.
Fig. 11 A third 11-DP-cord case with one continuous muscle mass around centrally located cartilage. 470 X.
Fig. 12 A second 7-DP-cord case. Cartilage seems to form a wedge into otherwise uninterrupted muscle. 100 X.

Fig. 13 An 11-DP. No muscle is detectable at low magnification. Cartilages are the most prominent features of this picture. 100 X.

Fig. 14 'Immature muscle,' identifiable because of the myofibril (arrow). From the same section as in Fig. 13. 1455 X.

Fig. 15. A TP-orbit case. Note orbital wall on left of photograph. Extraocular muscle passes without interruption from the orbital wall and into limb cartilages on right side. 100 X
[Available through University Microfilms International, 330 N. Zeeb Road, Ann Arbor, MI (L.C. Card No. Mic 60-3604) or see similar figures in a later publication.

Fig. 16. 'Mature muscle' taken from the same TP-orbit as in Fig. 15. Muscle fibers lie in the interval between radius and ulna. 970 X.
[Available through University Microfilms International, 330 N. Zeeb Road, Ann Arbor, MI (L.C. Card No. Mic 60-3604) ]

Fig. 17. Seven day blastema. Black line indicates approximate interface between stump and blastema. 100 X.
[Available through University Microfilms International, 330 N. Zeeb Road, Ann Arbor, MI (L.C. Card No. Mic 60-3604) ]

Fig. 18 In toto photograph of two limb-like elements after deplantation of two seven day blastemas (7-DP). Note that the elements have not fused into a single structure. 50 X.

The Y axis represents the percentage of cases exhibiting immature muscle (myoblasts with myofibrils as seen in Fig. 14); the X axis represents the post-amputation day of deplantation.

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pietsch@indiana.edu