Differentiation in Regeneration

I. The development of muscle and cartilage following deplantation of regenerating limb blastemata of Amblystoma larvae*

Department of Anatomy,
School of Medicine,
The University of Pennsylvania, Philadelphia**

Originally published in Developmental Biology 3, 255-264 (1961)
(Accepted December 20, 1960)

SUMMARY
Six- to 15-day blastemata of regenerating forelimbs of Amblystoma larvae were deplanted to the dorsal fin region both with and without their stumps attached. With stump present the deplant regenerates developed skeletal and muscular elements of the limb. In the absence of stump tissues no blastema developed a musculature whereas blastemata of all ages gave rise to elements of the forelimb skeleton.

INTRODUCTION

Amputation of a salamander limb usually leads to the accumulation of sub-epithelial cells at the wound surface of the remaining stump. Morphologically similar to granulation tissue, the aggregation is generally considered a blastema of the regenerating limb. The term blastema has been in widespread usage in reference to regeneration since about the 1920's.*** Since that time it has played a key role in much experimentation and speculation. Many texts and reviews and a few technical papers contain ideas about regeneration that are based upon the assumption that the blastema has been firmly established as an embryonal and multipotential system of cells. However, the available evidence is not so incontrovertible as is sometimes believed ( see critical discussion of Needham, 1950), and during the 1930's the developmental fate of the blastema was the object of some debate (cf. Schotté, 1940, and Polezhaiev, 1934).

Holtzer et al. ( 1954) observed that, when deplanted to the dorsal fin, blastemata of regenerating Amblystoma larval limbs yielded cartilage, but not muscle. The work of Holtzer and colleagues indicated a convenient means for studying differentiation in the regenerate. More important, the work of the latter investigators emphasizes the need for the information necessary to appreciate regeneration in more specific terms.

This investigation is an attempt to extend the studies of Holtzer et al. with a view to elucidating some of the factors in the differentiation of muscle and cartilage in the limb regenerate. At what stage in its development call the blastema produce cartilage in the absence of stumps. Without benefit of the stump can the blastema of more advanced age produce a limb-like musculature?

MATERIALS AND METHODS

Amblystoma opacum and A. punctatum larvae (30-40 mm) were used as hosts and donors. The aquarium was kept at about 20deg. C. Limbs were amputated through the upper or lower third of the humerus. Blastemata were visible under the dissecting microscope 4- 5 days after initial limb amputation but could not be transplanted before day 6. By the sixteenth day definitive tissues were supplanting blastema cells of the intact regenerate (see also Thornton, 1938). Therefore, experiments were performed with 6- to 15- day blastemata.

Deplantation ( see Weiss, 1950) involved piercing the dorsal fin epithelium and reaming out some of the jelly like connective tissue. A blastema, with or without its stump attached, was placed into such a tunnel. Epithelial caps were not removed. In some experiments several blastemata of similar age, size, and level were deplanted into a common chamber. In experiments without the stump, a narrow band of blastema cells was left behind to limit this possibility of contamination. Tissues remained in deplant for 30 days.

Specimens to he studied for muscle were fixed in Bouin's fluid, processed for paraffin sectioning at 10 u, and stained in iron hematoxylin. Some of the deplants to he studied for cartilage were fixed in ethanol- formalin and were processed for histological examination or were stained in toto with Van Wijhe's methylene blue. Cartilage was also studied in the Bouin- fixed deplant tissues.

RESULTS

Deplantation of the Blastema with Stump Attached

With stump attached, most deplants developed cartilages of a limb type, as revealed by in toto examination. No consistent differences could be related to age of regenerate at transfer to the fin. Complete, four- fingered hands developed only rarely (3 of 51 cases), even in experiments involving deplantations of 15- day blastemata with their stumps attached. Cartilages could sometimes be identified with precision as humerus, radius, ulna, and elements of the wrist and hand.

Histological examination revealed the presence of mature skeletal muscle, and when specimens had been sectioned transversely, miniature, though typical, limb musculatures were revealed (Fig. 1) (note figures in Piatt, 1957). The patterns of the musculatures often made it possible to tell which cartilage was radius and which ulna. Deplant musculatures always came into immediate contact with host connective tissue or epithelium.

Myogenesis following Deplantation of the Blastema Alone

In 142 deplant experiments representing stumpless blastemata of all ages, none resulted in the formation of a typical musculature (Fig. 2; Table 1).

Mature skeletal muscle fibers were present in two cases, both of which were the outcome of deplanting 15- day blastemata. Experiments with 10- day blastemata showed no signs of muscle. Cells of myogenic origin were observed following deplant experiments with 6- , 7- , 11- , 12- , and 15- day blastemata. The myogenic cells were mononucleated and contained myofibrils (Fig. 3). The cells were few in number, scattered in distribution and were difficult to detect. The percentage of cases with myogenic cells exhibited first a drop and then a rise with age of the blastema at deplantation.

Counts were made of the myogenic cells in specimens selected from the 11- day stumpless blastema series. Fourteen cases were examined; each was known in advance to possess some of the cells in question. All sections were inspected at high magnification, and an average was found of 16 ( +/- 3.5 SD) myogenic cells per case. Considering all 11- day blastema deplant experiments and assuming random distribution, the calculated chance of finding a myogenic cell in any given section was approximately one in fifty.

Chondrogenesis following Deplantation of the Blastema Alone

Of 434 deplantations with stumpless blastemata of various ages, 423 exhibited cartilage (see Table 2). The cartilages bore the shapes and interrelationships of elements of the forelimb skeleton (Figs. 3 and 4). Cartilages articulated with each other by means of the rudimentary joints typical of Amblystoma larval limbs. Complete four- fingered hands were observed in only four instances. Each was a deplantation experiment with a stumpless 11-day blastema. Younger and older blastemata produced from one to three digits per deplant- hand. In experiments with more than one blastema in a common chamber, more than one partially formed limb always developed ( Fig. 5) .

One consistent difference was found in the experiments with stumpless blastemata of differing ages: radius, ulna, and humerus were not precisely identified in 6- and 7- day deplantations (Fig 6). Radius and ulna, but not humerus, were identified in about 10% of the cases with stumpless 11- day deplants and in approximately 50% of the 15- day stumpless deplants.

DlSCUSSION

Deplanting the blastema with its stump attached resulted in miniature regenerates With skeletons and musculatures characteristic of the limb. This indicates that the methods employed did not rule out chondrogenesis or myogenesis. Experiments with stumpless blastemata revealed that chondrogenesis is set into motion quite early in regeneration, perhaps before the blastema cells have amassed distal to the plane of amputation. On the other hand, stumpless regenerates failed, consistently, to develop limb musculatures even when deplanted at the close of the blastemal phase of regeneration. Cells of myogenic origin were encountered in certain stumpless blastema deplant experiments, and there were probably more of these cells than were observed. Whether or not the myogenic cells would have contributed to the regenerate musculatures or to other tissue still remains to be determined.

The information suggests that the continued presence of stump is indispensable for the development of a typical musculature in the limb regenerate. The specific contribution of the stump to regenerative muscle was not ascertained, but probably relates somehow to the injured stump musculature. Muscle in the tail regenerate is dependent upon the presence of muscle in the stump (see literature and evidence in Holtzer, 1956). Likewise, when transplanted to the cut ends of extraocular muscles, limb regenerates developed with appreciable amounts of skeletal muscle tissue (Pietsch, in progress).

Chondrogenesis had achieved considerable independence of the stump even among cells of the earliest testable blastema. Experiments with the 6- and 7- day blastemata were undertaken pessimistically. The blastemal aggregations were quite small, and yet some of the cells had to be left on the stump if these were to be isolation experiments. Furthermore, it is quite probable that the full complement of cells had not been added from the stump in a 6- or 7- day blastema (see data of Chalkley, 1954). In addition, interference with the nerve supply of the early blastema often results in poor differentiation (Butler and Schotté, 1949). Despite the many reasons for developmental failure, 6- and 7- day blastemata consistently produced cartilages with recognizable limb-like morphology. From this it is inferred that the blastema of regenerating Amblystoma larval limbs possesses at least some limb organization even at the outset of its history as a discrete entity. The failure of regenerates to develop in isolation experiments of other investigators (see literature and discussion in Goss, 1956; see also Skowron and Walknowska, 1959) reflects not upon the developmental capabilities of blastema cells, but upon the methods employed.

REFERENCES

BUTTLER, E. C., and SCHOTTÉ, O. E. ( 1949 ) . Effects of delayed denervation on regenerative activity in limbs of urodele larvae. J. Exptl. Zool. 112, 361- 392.

CHALKLEY, D. T. ( 1954 ) . A quantitative analysis of forelimb regeneration in Triturus viridescens. J. Morphol. 94, 21-70

GOSS, R. J. (1956). The relation of bone to the histogenesis of cartilage in regenerating forelimbs and tails of adult Triturus viridescens. J. Morphol. 98, 89-123.

HOLTZER, H., AVERY, G., and HOLTZER, S. ( 1954) . Some properties of regenerating limb blastema cells of salamanders. Biol. Bull. 107, 31:3.

HOLTZER, S. (1956). The inductive activity the spinal cord in urodele tail regeneration. J. Morphol. 99, 1- 39.

LOTZE, R. (1851). "Allegemeine Physiologie des körperlichen Lebens." Leipzig.

NEEDHAM, J. (1950). "Biochemistry and Morphogenesis." Cambridge Univ. Press, London and New York.

PIATT, J. (1957). Studies of the problem of nerve pattern. 111. Innervation of the regenerated forelimb in Amblystoma larvae. J. Exptl. Zool. 136, 229- 248.

POLEZHAIEV, L. ( 1934). Über die Determination des Regenerats. Compt. rend. acac. sci. U.R.S.S. 4, 171- 175

SCHOTTÉ, O. E. (1939). The origin and morphogenic potencies of regenerates. Growth Suppl., 59- 76.

SKOWRON, S., and WALKNOWSKA, J. (1959) . The fate of regenerating limb blastemas implanted into the body cavity. Folia Biol. (Warsaw) 7, 113- 119.

THORNTON, C. S. (1938). The histogenesis of muscle in the regenerating forelimb of Amblystoma punctatum J. Morphol. 62, 17- 47.

WEISS, P. (1950). The deplantation of fragments of nervous system in amphibians. l. Central reorganization and the formation of nerves. J. Exptl. Zool. 113, 397-461.

[figures and tables]:

Fig.1. Cross section through the forearm region of a limb regenerate which developed following deputation of a single 11- day blastema with the stump attached. Original limb amputation was through the upper portion of the humerus. The muscular pattern is typical of the limb (cf. figures in Piatt, 1957 ). Radius ( R ) and ulna (U) were identified by the position of the various forearm (antibrachial) muscles. C. T., host connective tissue. This is one of a series of five in which results were almost identical and were in harmony with previous and subsequent observations. Magnification: X 100.

Fig. 2. (Right) Deplant of a single stumpless 11-day blastema which resulted in the presence of cartilage and the absence of mature skeletal muscle. Magnification: X 100.

Fig. 3. A myofibril in a case resulting from the deplanting of a single stumpless 11-day blastema. Magnification: X 970.

Fig. 4. Cartilages that developed from a deplanted, single stumpless 6-day blastema. Magnification: X 100.

Fig. 5. Partially formed limbs developed from deplanted, stumpless 7-day blastemata. Eight blastemata were deplanted together in a common chamber. Eight entities developed, three of which are in the photographic plane. Magnification: X 51.

Fig. 6. Percentage of cases exhibiting the various components of the forelimb skeleton The above data were compiled from one series of stumpless deplant experiments. Sibling A. punctatum larvae, 30 mm in length, had their limbs amputated through the humeral neck. Blastemata were deplanted singly, 7 or 11 days after initial limb amputation. Examinations were made in toto after methylene blue staining. Each individual piece of cartilage was examined. When a given piece of cartilage could not be named precisely it was cataloged as "unidentified." The information gained from this series was in harmony with the trend in previous and subsequent deplantation studies.

TABLE 1. **MYOGENESIS AFTER DEPLANTING THE BLASTEMA WITHOUT ITS STUMP**
Postamputative age of blastemata at deplantation (days)	Number of cases	Cases with no muscle	Cases with muscle --Mature	Cases with muscle --Immature
6	13	7	0	6
7	36	33	0	3
10	14	14	0	0
11	69	40	0	29
12	6	0	0	6
15	17	0	2	15

TABLE 2. **CHONDROGENESIS AFTER DEPLANTING THE BLASTEMA WITHOUT ITS STUMP**
Postamputative Age of Blastema at deplantation (days)	Number of cases	Cases with cartilage present
6	13	11
7	77	74
10	118	116
11	201	197
12	6	6
15	19	19

This is to express my appreciation for having had the opportunity to work in the Department of Anatomy, The University of Pennsylvania, Louis B. Flexner, Chairman. The men and women of that department in the routine course of their everyday activities set a living example of humanitarianism, of scholarliness, and of intellectual integrity and courage.
My advisors were Howard Holtzer and Jean Piatt. Their wisdom and knowledge added meaningfulness to much of my activity. More important, they allowed me freedom not only to develop this problem, but to arrive at my own conclusions. This they could do because of the rare kind of man each is. Further acknowledgment of contributions to my endeavors would require a list of all members of the Department referred to above. [And 37 years after first having written those words, I still feel lucky to have known such persons and to have had the good sense to have said so then.]

*Submitted to the Faculty of the Graduate School, The University of Pennsylvania, in partial fulfillment of requirements for the degree of Doctor of Philosophy and supported by funds from the United States Public Health Service.

** Present address: Indiana University, Bloomiongton, Indiana, USA

***Blastema came into usage in reference to regeneration at least as early as 1851 (Lotze, p. 555).

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pietsch@indiana.edu