the effects of x-radiation on the regeneration of the fore limb of amblystoma larvae

THE EFFECTS OF X-RADIATION ON THE REGENERATION O F THE FORE LIMB

O F AMBLYSTOMA LARVAE

ELMER G. BUTLER Luboratory of Comparative Anatomy, Princeton University

FIFTEEN TEXT FIQURES AND SIX PLATES (~VENTY-FOUR FIQURES)

I. INTRODUOTION

Several investigators have demonstrated that x-rays and radium exert a pronounced effect on the process of regeneration. Nearly 30 years ago, Bardeen and Baetjer ('04) reported that exposure to x-rays prevented regeneration in Planaria and, in the same year, Schaper ('04) reported briefly a few experiments showing that radium emanation prevented the regeneration of the limb in Triton larvae. Within recent years, other species of animals, chiefly invertebrates, have been studied. Zawarzin ('29) and Strelin ('29) have prevented regeneration in Pelmatohydra by x-rays, Weigand ( '30) has prevented regeneration in Clavelina, as well as i n Planaria, by the use of radium emanation and, recently, Stone ('32) has reported his work on inhibiting regeneration in Tubifex by x-rays. Curtis and Hickman ('26) and Curtis ('28), in a histological study of the tissue of radiated planarians, have found that the so-called formative cells are de- stroyed by the x-rays. Since these formative cells, as Curtis and his students have shown, normally are the source of new tissue formed during regeneration, their destruction renders the planarian incapable to regenerate. Histological observations of a similar nature are reported by Stone ('32) in his work on Tubifex in which, during regeneration, the so-called neoblasts give rise to the new mesodermal tissue. X-radiation of Tubifex results in the destruction of the neoblasts.

271 THE JOURNAL OF KXI'ERIMENTAL zoOr.oau. VOL 65 , NO. 3

JULY, 1933

272 ELMER G. BUTLER

Recorded observations of the effects, either of x-rays or of radium, on regeneration in vertebrates are limited to the report of the few experiments on Triton larvae by Schaper ('04) and a brief reference to the same experiments in a paper by Levy ( '06). I have undertaken the present study, therefore, for the purpose of examining the effects of x-rays on regeneration in Amblystoma and particularly for the purpose of studying the histological changes associated with the loss of the regenerative ability. A brief preliminary report of some of the general results of this investigation has already been published (Butler, '31). The present report will deal with the results of a group of experiments conducted during the past 3 years which are concerned with the effects of x-radiation on the regeneration of the fore limb in Amblys- toma larvae. The fore limb of this animal is well adapted to experimental studies on regeneration, for the reason that under ordinary conditions it is readily regenerated and in larval stages of Amblystoma the regeneration takes place rather rapidly.

In the following pages I wish to deal in particular with two aspects of the problem; first, the effect of x-rays on fore limb regeneration at different stages of limb development and, secondly, with the histological changes which I have found associated with the loss of the regenerative capacity in the radiated larvae. For the histological study to be pre- sented at this time larvae have been used which possessed fore limbs in the 3-digit stage at the time of amputation and in each case the level of amputation has been through the proximal third of the humerus. A study of histological changes in non-regenerating limbs in which amputation was performed in the early limb bud stage, before the appearance of digits and before the formation of cartilage within the limb, is now being made by one of my students, Mr. W. 0. Puckett, and will be the subject of a later paper.

Most of the histological work included in this paper was done while I was a guest at the Zoologisches Institut, Frei-

X-RAYS AND REGENERATION I N AMBLYSTOMA 273

burg, i. Br., Germany. I wish to thank Prof. H. Spemann for the facilities of his laboratory which he so generously placed at my disposal. The photomicrographs are the work of Miss E. Blum, of the Zoologisches Institut, Freiburg, i. Br.

11. MATERIAL AND METHODS

The larvae of Amblystoma punctatum which were used for all experiments were reared in the laboratory from eggs collected in the vicinity of Princeton. After the feeding stage was reached the larvae were isolated in individual dishes to prevent the biting off of limbs and were fed chiefly on small crustaceans collected from ponds near the laboratory. The limbs were amputated with iridectomy scissors with the larva under chloretone anesthesia. Each amputation was made as nearly as possible in a plane at right angles to the long axis of the limb.

The source of radiation was a Coolidge medium focus tube. The factors governing the radiation, which have been kept constant for all experiments are as follows: 60 kv., 6 ma., distance from target to Amblystoma, 25 om. I n the different experiments the dosage has been somewhat altered by chang- ing the length of exposure, so in connection with each experiment the length of exposure will be stated. The rays were unfiltered.

The outline drawings used as text illustrations were made with a camera lucida from larvae under chloretone anesthesia. Bouin's fhid was used for a11 fixation. Sections were cut at 5 p and stained with Delafield 's haematoxylin.

111. EXPERIMENTS DEMONSTRATING T H E GENERAL EFFECTS O F X-RAYS ON REGENERATION O F T H E FORE LIMB

It can be easily demonstrated that exposure to x-rays in repeated doses will prevent limb regeneration. I shall describe here briefly three typical experiments to show this reaction, and especially to demonstrate that the loss of regenerative capacity occurs whether the limb be in a rather early or a rather late stage of limb development at the time

274 ELMER G . BUTLER

the amputation is made. The three experiments to be described have been selected from a group of twenty-five experiments of a similar nature which were done during the seasons of 1930,1931, and 1932.

2 . Amputatiorz of the limb in the 4-digit stage (figs. 1 t o 4)

One fore limb was amputated from each of thirty-two larvae. The larvae used were about 15 mm. in length and possessed, at the time of amputation, fore limbs in the early 4-digit stage. After amputation of the limb twelve of the thirty-two larvae were radiated for 2 minutes daily; twelve were radiated for 3 minutes daily; eight larvae were never radiated and served as controls. The amputations were performed on June 12th. Daily radiation was started on June 13th and continued through July 4th, when the experiment was ended.

I n none of the twenty-four radiated animals was there any sign of regeneration of the amputated fore limb. I n the eight controls normal regeneration was rapid, and by July 4th all fore limbs had regenerated as far as the 3-digit stage. Figures 1 to 4 demonstrate the results as shown in two typical animals of this experiment; one (D-6) is a larva radiated 2 minutes daily, which shows the entire absence of limb regeneration, and the other (D-28) is an unradiated control larva in which regeneration pro- gressed normally. Figures 1 and 2 represent the two larvae at the time of amputation; the broken line in each case shows the level at which the right fore limb was removed. Figure 3 shows the radiated animal (D-6) 22 days after the amputation. The wound has healed and the free end of the limb stump has rounded off, but there is no evidence of any regeneration. Figure 4, the unradiated control (D-28) 22 days after the amputation, demonstrates the amount of regeneration which took place normally during the 22-day period. A few days after this drawing was made, the fourth digit appeared in the control and the limb soon regained its original condition as before amputation.

The results were clear cut.

X-RAYS AND REGENERATION IN AMBLYSTOMA 275

Not only has there been a complete absence of regeneration in radiated larvae of this experiment, as figure 3 shows, but, in addition, it has been observed that the non-regenerating

Figs. l a n d 2 Two larvae, D-6 and D-28, on the day of limb amputation. The broken line in each case indicates the level a t which the right limb was amputated.

D-6 has received daily doses of x-rays.

Figs. 3 and 4 The same larvae 22 days after amputation. D-28 is an unradiated control.

276 ELMER 0. BUTLER

limb stump after several days of radiation is invariably shorter than the stump which remained attached to the body at the time of amputation. In association with the failure to regenerate, there has been evidently a resorption of material in the non-regenerating limb. This matter will be discussed in later pages in connection with the histological studies of non-regenerating limbs.

In this experiment and in other experiments of a similar nature no significant difference has been found between the larvae which were radiated 3 minutes and those which were given a 2-minute dosage.

2. Amputation. of the limb in. the .%digit stage (figs. 5 to 10) One fore limb was amputated from each of fifteen larvae in

which, at the time of amputation, the fore limbs were in the early 2-digit condition. I n each individual the amputation was made close to the body. After the amputation ten of the fifteen larvae were radiated for 5 minutes daily from May loth, the day of amputation, to June 4th, inclusive. Five larvae were never radiated and served as controls.

The results of this experiment are best understood by reference to figures 5 to 10. In figures 5 and 6 broken lines indicate the level of amputation. Figures 6, 8, and 10 show the normal course of regeneration of the right limb and the normal growth of the left limb over a period of 26 days in an unradiated control larva (H-21). In this unradiated individual the regenerating limb, 17 days after the operation (fig. 8), was again in the 2-digit condition as at the time of amputation. On June 5th, 26 days after amputation (fig. l o ) , the regenerating right limb was in the 3-digit condition and was only slightly less developed than the unamputated left limb on which the fourth digit had just appeared.

Figs.5and6 Two larvae, H-5 and H-21, on the day of limb amputation. The broken line in each case indicates the level a t which the right limb was amputated.

H-5 has received daily doses of x rays.

H-5 has continued to receive daily doses of x-rays.

Figs. 7 and 8

Figs. 9 and 10

The same larvae 1 7 days after amputation. H 2 1 is an unradiated control.

The same larvae 26 days after amputation. H-21 is the unradiated control.

7 I 1 May27

9 June 5

8 1 1 May27


Figures 5 t o 10

278 ELMER G. BUTLER

In the ten larvae exposed daily to x-rays the changes during the 26 days of the experiment were strikingly different than those in the control individuals. There was no sign of regeneration of the amputated limb. Figures 5, 7, and 9 represent one of the radiated animals (H-5). After the amputation the cut end of the limb healed and rounded off, but showed no evidence of regeneration. It remained as a non-regenerating stump on the side of the body. It is noteworthy, however, that the unamputated left limb of the same radiated larva continued development and that the third digit on this limb formed (fig. 7) as soon as did the third digit on the corresponding unamputated left limb of the control individual (fig. 8). Thus, it appears that the daily exposure to x-rays had no externally visible effect on the formation of the third digit. One should observe, however, that at the time the fourth digit appeared on the unamputated left limb of the control larva (fig. lo ) , no fourth digit was evident on the corresponding unamputated left limb of the radiated individual (fig. 9). Indeed, no fourth digit ever developed on this left limb of the radiated larva. This effect of x-radiation in preventing digit formation on otherwise normally developing limbs was evident in all radiated larvae of this experiment and has been observed repeatedly in other experiments. It appears to be one of the regular results of exposure to x-rays. I n every case studied, I have found that when the limb was in the 2-digit stage at the time daily radiation was begun the third digit developed in a manner apparently normal, but no fourth digit ever made an appearance. I have been able to prevent the development of the fourth digit, also, by beginning the radiation at the time when the limb was in the early 3-digit condition.

3. Amputa t ion of t he limb in the early limb bud stage before the a,ppeara.nce of digits (figs. 11 to 15)

The experiments briefly described in the two preceding sections demonstrate that in relatively advanced stages of limb development x-rays are effective in preventing limb regenera-

X-RAYS A N D REGENERATION IN AMBLYSTOMA 279

tion. I shall now describe an experiment, which is typical of several experiments of a similar nature, to show the effect of exposure to x-rays on limb regeneration when the amputation was made at a relatively early stage, soon after the limb bud became evident as a protrusion on the lateral body wall and before the appearance of digits. In this experiment to be described the right limb was amputated as close to the body wall as possible in twenty-four Amblystoma in the stage of development shown in figure 11. Amputation in this experiment consisted simply in the clipping off of the free part of the fore limb bud. Of the twenty-four Amblystoma from which the limb was so removed twelve were radiated for 5 minutes daily from the day of amputation, May 14th to June 6th, inclusive; the other twelve animals were never radiated and served as controls.

In general, the results of this experiment, which is understood best by reference to figures 11 to 15, were similar to the results of the experiments described in foregoing pages. Radiation prevented regeneration as effectively when the limb was amputated in this early limb bud stage (fig. ll), before the appearance of digits, as when the amputation was made in the more advanced stages of limb development here- tofore described. Figures 12 and 14 show the absence of the regeneration of the right fore limb in one of the radiated animals (L-3) and figures 13 and 15 demonstrate the normal course of regeneration in one of the unradiated controls (L-13). Thirty-four days after the amputation, when the right limb of the control had regenerated to the 3-digit condition (fig. 15), the right limb of the radiated individual (fig. 14) consisted of a short non-regenerating stump such as has been observed in the experiments previously described.

The effect of radiation in preventing normal digit formation, which has been previously referred to, was especially evident in this experiment. It should be noted, however, that the radiation did not affect the formation of the first two digits on the unamputated left limb (fig. 12). These first two digits were apparently as well formed on the radiated

280 ELMER G. BUTLER

12 May27 I

I I L-3 1 1 June17 14

I3 May 27 I L-13

Fig. 11

Figs. 12 and 13

A larvae showing the stage in which the right limb bud was amputated

Two larvae 13 days after amputation. L-3 has received daily I n each case amputation of the

The broken lines indicate the level of

The same larvae 34 days after the amputation of the right

in experiment L.

doses of x-rays. left limb was performed on May 27th. amputation.

limb and 21 days after the amput,ation of the left limb.

L-13 is an unradiated control.

Figs. 14 and 15


larva 7 days after the beginning of the daily exposure to x-rays as the first two digits on the unradiated control. How- ever, in the radiated individual no third digit ever appeared on the left limb. Thirteen days after the beginning of the experiment, when the control individual (L-13) possessed a newly formed third digit (fig. 13), the radiated larva (L-3) still possessed only the first two digits (fig 12). This failure to form a third digit occurred in all of the radiated Amblys- toma of this experiment. I n none of the radiated animals did a third digit ever form on the left limb, although some of the larvae were kept till the thirty-fourth day after the beginning of the experiment.

In twelve of the animals used in this experiment (six radiated larvae and six controls), a second amputation was performed on May 27th, 13 days after the first amputation. On this day the left limbs of the twelve larvae were amputated through the middle of the humerus as indicated by the broken lines in figures 12 and 13. The results were as anticipated. Twenty-one days after this amputation, the regenerating left limbs of the control animals had reached the 2-digit stage (fig. 15). The left limbs of the radiated larvae had not only failed to regenerate (fig. 14), but there was evidence of considerable resorption. The non-regenerating limb stumps of both right and left sides of the radiated larvae at the end of the experiment were of about equal size.

This experiment clearly demonstrates that limb regeneration can be as effectively prevented when amputation is made in the early limb bud stage as when more advanced stages of limb development are used. Recently, Mr. W. 0. Puckett, working in this laboratory, has removed the limb bud in earlier stages of development than shown in figure 11. For example, if the limb bud is removed in ‘stage 33’ of Harrison (%), regeneration of the limb can be completely prevented by radiation. An unamputated limb on the opposite side of the body will continue development under the influence of radiation only as far as the 2-digit stage. No third digit ever develops on these unamputated limbs as was the case in the experiment described above.


4. Discussion

The experiments described in foregoing pages are three selected from twenty-five experiments of a similar nature which have been made during the seasons of 1930, 1931, and 1932. In all experiments the results in essential respects have been the same. These experiments demonstrate that repeated exposure to x-rays in sufficient dosage will prevent the regeneration of the fore limb of Amblystoma larvae regardless of the age of the larvae, of the stage of limb development, or of the level at which the limb is amputated. Not only does the amputated limb fail to regenerate, but ordinarily there is considerable resorption, so that the non- regenerating limb stump is smaller several weeks after the amputation than the portion of the limb left attached to the body at the time of amputation.

The effect of x-rays on the formation of the digits on the unamputated limb of a radiated larva as demonstrated in the experiments described is, I believe, of particular signifi- cance. The general rules in regard to this effect of radiation on digit formation appear to be as follows. If the limb is about to enter the 2-digit condition at the time radiation is begun, then the first two digits will be formed in a manner apparently normal, but no third digit will ever develop. If the limb is in the 2-digit condition with the third digit about to appear at the time radiation is begun, then the third digit will be formed in a manner apparently normal, but no fourth digit will ever develop. Likewise, if the third digit is just appearing at the time radiation is begun, the third digit will be completely formed, but no fourth digit will ever develop.

The effect of x-radiation on digit formation suggests that the x-rays act on differentiating tissue at a certain critical period in the process of differentiation during which there is a heightened sensitivity to x-radiation. If the differentiation has passed this critical period before radiation is begun, then differentiation proceeds even though the animal be radiated. For example, when radiation is begun in the 2-digit stage the third digit will always develop, presumably because

X-RAYS AND REQENERATION I N AMBLYSTOMA 283

the critical period for the differentiation of the third digit was passed before the radiation became effective. However, in the same case the fourth digit will be ‘caught’ at the critical period and its differentiation prevented. In the process of regeneration there is, possibly, a similar critical period when the differentiating cells are especially susceptible to the influence of x-rays. That this may be the case is indicated by some experiments on regenerating limbs recently made in this laboratory which are still unpublished. These experiments show that if radiation is begun after a certain period in regeneration is passed, then regeneration will ensue regardless of radiation. However, if radiation is begun before the critical period. is passed, then the regeneration will be prevented as demonstrated by the experiments described in preceding pages.

The question of the dosage of x-rays necessary to prevent regeneration has not been thoroughly investigated. It is quite certain, however, that the dosages used in some of the early experiments were much larger than actually necessary. In some of the last experiments which have been conducted it has been found that daily exposures of 1 minute each have been as effective as daily exposures of longer durati0n.l The questions of the dosage and of the critical period are problems now under investigation.

IV. HISTOLOGY O F NON-REGENERATING LIMBS COMPARED WITH THE HISTOLOGY O F NORMALLY REGENERATING LIMBS

Having established the fact that exposure to x-rays will prevent regeneration of the fore limb of Amblystoma larvae and that x-rays produce this effect regardless of the age of the larvae or of the stage of limb development at the time of amputation, the next aspect of the problem undertaken was a study of the histological differences between normally regenerating limbs and non-regenerating limbs of radiated ani-

‘ I n all experiments the same voltage and the same milliamperage have been used and the distance from the target to the larvae has been kept constant. Only the length of exposure has been altered.


mals. A preliminary survey indicated that profound histological alterations occur within the non-regenerating limbs. One would expect these histological alterations to assume somewhat different aspects, depending on the stage of limb development at the time of amputation. It seemed best, therefore, first to choose one particular stage of limb development for a critical study of histological changes in non-regenerating limbs and later to extend the histological study to include other stages. For initial histological study the stage of limb development chosen was that in which the limb was in the 3-digit stage at the time of amputation. This stage possesses certain advantages, particularly the advantage that the component structures of the limb are well differentiated so that they provide an opportunity to observe the reaction of each of the components to the radiation.

The histology of normal regeneration of the amphibian limb has been the subject of numerous investigations, and its general aspects are rather well known. I shall deal with it here, therefore, very briefly. The first differentiated structure to appear as regeneration takes place is the limb skeleton. That the new skeleton of the regenerating limb develops not from the old preexisting skeleton in the stump, but from a new blastema which develops at the point of amputation and covers over the old stump is amply demonstrated by the work of many investigators, among others Wendelstadt ( '04), Fritsch ( 'll), Schaxel ( '21), Weiss ( '25), Guyhot ( '27) and his students, and Bohmel ( '29). Moreover, the blastema appears to be the source of all new tissue of the regenerating limb with the exception of epidermis and nerves. The source of the cells which make up the blastema is still a matter of question. Hellmich ( '29) has stated that some of the cells of the blastema which enter into the regenerative process are of histogenetic origin and others of hematogenetic origin. Unfortunately, the difficulties attendant on a histological problem so involved as that of regeneration makes it difficult to recognize on the basis of their morphological characteristics the actual origin of cells.


To obtain material for the histological study to be discussed in the following pages, the fore limbs of seventy-six Amblys- toma larvae (experiment M) were amputated through the proximal third of the humerus. Thirty-eight of the larvae were exposed to x-rays for 5 minutes daily beginning with the day of amputation, while the other thirty-eight were never radiated and served as controls. In all cases the limbs were in the 3-digit stage at the time of amputation and all amputations were bilateral. During the first 2 days after amputation radiated and control larvae were fixed at intervals of from 2 to 7 hours. After the first 2 days larvae were fixed at daily intervals up to the twenty-first day after amputation. In this manner a graded series of larvae was obtained for the study of the normal limb regeneration in the controls and the histological changes in non-regenerating limbs of radiated larvae over a period of 21 days. Serial sections were cut at 5 p, each larva being so oriented that the plane of section was as nearly parallel to the long axis of the limb as possible.

1. Normally regenerating limbs (figs. 16 to 27)

In order more completely to understand the changes which take place within the non-regenerating limbs of radiated Amblystoma, it is necessary first to be acquainted with the general histology of normal regeneration. I present here- with, therefore, a series of photomicrographs of sections through normally regenerating fore limbs (figs. 16 to 27). This series of photomicrographs shows the process of normal regeneration over a period of 15 days, beginning with the day of amputation and ending with the limb in the 2-digit stage on the fifteenth day after amputation. The larvae used for these sections were those which served as controls in the experiment (experiment M) now under discussion.

Figure 16 shows a section through the fore limb fixed immediately after amputation. This section demonstrates the stage of limb development at which the amputation was per-

THE JOURKAL OF EXPERIMENTAL 208LOaY, VOL. 65, NO. 3


formed as well as the level of amputation. The chief feature to be noted is that immediately after amputation the tissue surrounding the humerus contracts and pulls away leaving the cartilage stump bare for a short distance near the cut end. The extent to which the tissue pulls away is rather vari- able among different larvae and appears to depend to a COIL-

siderable extent on the level of amputation. If the amputation be through the distal third of the humerus, there is usually more retraction of surrounding tissue than when the amputation is close to the body. I n the case shown in figure 16 the amputation was through the proximal third of the humerus and there has been relatively little retraction of tissue.

Healing of the wound of amputation is ordinarily rather rapid. I ts rapidity depends, however, in a large measure on the extent to which the tissues pulled away from the cut end of the humerus at the time of amputation and conse- quently on how large an area must be covered with tissue to bring about complete healing. I n most cases healing is complete within 12 hours after amputation. In cases where only a slight amount of retraction of tissue takes place, the wound heals in much less than 12 hours.

Initial healing of the wound appears to be purely epidermal in character and takes place without any evidence of cell division. The epidermis gradually spreads over the limb stump until the operated area is entirely covered (fig. 17). This reaction of the epidermis in wound healing has been noted by many observers, not only in amphibians, but in other animals as well. Recently, Arey ('32) and also Herrick ('32) have described the process in considerable detail. These authors show that the initial epidermal healing of wounds takes place by a migration of epidermal cells from adjacent regions and that cellular proliferation is not involved. My own observations support this view. Sections through limb stumps of larvae fixed during the first 12 hours after amputation show no mitotic activity or other evidences of cell division in the epidermal layer, although during this period the epidermis is gradually spreading over the wound area.


I have noticed, also, that as the epidermis spreads over the cut end of the limb there is usually a temporary epidermal thickening which forms over the end of the humerus. This is shown in figure 17, but more clearly in figure 28. This transitory epidermal thickening is very probably the same phenomenon which Herrick ( ’32) has observed in Rana and has referred to as a ‘piling up’ of epidermal cells after the advancing edges of the wound have come together.

It is noteworthy, also, that as epidermal cells spread over the cut end of the limb the pigment layer does not follow (figs. 17, 18, and 19). This pigment layer, which is ordinarily sharply defined, does not begin to spread out under the epidermal cells of the wound area till several days after amputation. The edge of the pigment layer serves for some time, therefore, as a landmark which indicates approximately the level of amputation. However, in using this as a landmark one must take into account the retraction of tissue from the humerus which occurred at the time of amputation. Because of this retraction, the edge of the pigment layer never indicates exactly the level of amputation, but it is trustworthy as an approximate indication.

After epidermal healing has taken place, the next observable change in the limb stump is a vacuolation of the cut end of the cartilage. This change in the cartilage is shown in figures 18 and 19, which are limbs 48 and 72 hours, respec- tively, after amputation. I n the distal third of the cartilage stump the cells shrink away from their cartilage capsules, the capsules appear to swell to a slight extent and the matrix stains more lightly than in the normal healthy cartilage. This initial cartilage degeneration is due primarily, I believe, to the severe mechanical injury which the humerus suffered at the time of amputation. It marks the beginning of a rather extensive cartilage degeneration or, more properly, cartilage alteration which takes place within the first 10 days after amputation.

During the first 3 days after amputation (figs. 17, 18, and 19), therefore, the principal changes in the limb have been


the epidermal healing and the onset of cartilage alteration at the distal end of the humerus. On the third day a new activity becomes evident. Mesenchyme cells appear in the region between the cut end of the humerus and the overlying epidermis. Soon these mesenchyme cells are joined by numbers of blood cells. One observes a rather early stage in this accumulation of mesenchyme cells and blood cells in figure 20 which is a limb stump on the fourth day after amputation. It may be noted by a comparison of figures 19 and 20 that as the mesenchyme and blood cells appear the vacuolated region of the cartilage disappears. This coincidence suggests that the vacuolated cartilage is being removed through the phagocytic activity of some of the invading blood cells. Possibly this is the case.

Beginning with about the fourth day after amputation (fig. 20) an extensive cartilage alteration takes place. The exact nature of this change and the agencies which bring it about are very incompletely understood. I t has been referred to by some of the previous investigators as a period of cartilage degeneration and has been considered by some to be a result of phagocytosis. Very possibly phagocytosis does occur to some extent, as is suggested by the large numbers of blood cells which are clustered about the tip of the humerus 5 or 6 days after amputation (fig. 21). Helmich ( '29) has described these cells in considerable detail. However, the phagocytosis which may occur should be regarded as an accessory process and the change in the cartilage which takes place should be regarded, I am convinced, not as a cartilage degeneration in the strict sense, but rather as a cartilage alteration. This cartilage alteration which takes place during this particular period is a necessary forerunner of the regenerative processes which are to take place later. One of the fundamental aspects of the cartilage alteration appears to be a dedifferentiation of cartilage cells. I n my preparations I have observed that, beginning with about the fourth day after amputation, cartilage cells at the end of the humerus are freed from their surrounding capsules and the cartilage matrix in this region

X-RAYS A N D REGENERATION IN AMBLYSTOMA 289

gradually disappears. As these changes occur mitoses are not infrequent in the released cartilage cells and the cells appear to assume the general characteristics of mesenchyme cells. Indeed, one may note by reference to figures 22 and 23 that it is possible to trace, as one progresses from the head of the humerus distad, an even gradation of dedifferentiation in the cartilage cells. In figure 23, for example, if one starts near the humerus head and proceeds distad one goes from a region of typical cartilage cells into a region where the limits of the cartilage capsules are disappearing, thence into a region where there is no longer any visible cartilage matrix, and finally out into the blastema. In the preparation from which figure 23 was photographed it is impossible to deter- mine exactly where the freed cartilage cells leave off and where the cells of the new blastema begin. In other words, this process of dedifferentiation appears to result in a return of some of the cartilage cells to their previous mesenchymal state. And in general, therefore, this period of cartilage alteration which occurs through the medium of dedifferentiation should be regarded as a period during which the distal portion of the cartilaginous humerus is prepared to unite with cells of the blastema in the building up of the new limb skeleton.

It is interesting that as the cartilage alteration takes place the perichondrium becomes free from the distal end of the humerus and prominent vacuolated spaces appear in this region (figs. 21, 22, and 23). The region where the perichondrium is so separated from the humerus delimits almost exactly the region where the cartilage cells become entirely free from their capsules. This relationship is shown especially well in figures 22 and 23. The separation of cartilage and perichondrium is due undoubtedly to the dissolution of the cartilage matrix which occurs in connection with the dedifferentiation of the cartilage cells.

Extensive cartilage alteration takes place over a period of about 6 days, namely, from about the fourth day after amputation to about the tenth. During this same period the new


blastema, a group of cells between the cut end of the humerus and the tip of the limb, appears and grows rapidly in extent. The growth of the blastema is best understood by a comparison of figures 20, 21, 22, and 23. It is from the cells of this new blastema that all of the new components of the regenerated limb will be formed with the exception of epidermis and nerves.

Between the tenth and eleventh days after amputation the progress of cartilage alteration ceases. The loose envelope of perichondrium around the region of dedifferentiated cartilage disappears. On the eleventh day after amputation (fig. 24) one observed many closely packed cells in the region occupied originally by the cut end of the humerus. Some of these cells I regard as dedifferentiated cartilage cells and some as cells of the new blastema. These two sorts of cells are, I believe, indistinguishable.

The cartilage alteration being completed and the blastema being well formed, regeneration after the eleventh day progresses rapidly. I n the experiment under discussion the first two digits appeared on the twelfth day after amputation (fig. 25). The exact time, however, when the first two digits appear depends to some extent on the level at which the limb was amputated as well as on the stage of limb development at the time of amputation. The first indication of digits is the appearance of a shallow groove in the blunt end of the regenerating limb. It is interesting that the external appearance of digits antedates slightly any visual differentiation of cells in the blastema in the region of the digits. How- ever, very shortly after the shallow groove at the blunt end of the limb indicates externally the appearance of digits there is evident within the limb the formation of new cartilage at the distal tip of the humerus (fig. 25). Blastema cells and dedifferentiated cartilage cells line up in rows, their nuclei become somewhat ovoid and the region of the new humerus is rather clearly mapped out.

Once initiated, cartilage formation proceeds rather rapidly. Fourteen days after amputation (fig. 26), the first two digits


are prominent (both digits do not show in figure 26 because of the plane of the section) and there is an indication of the formation of radius and ulna. Moreover, aggregations of cells in precartilage stages can be followed nearly to the tips of the digits. This situation is noticeable in figure 27, which is a limb 15 days after amputation.

After the fifteenth day of regeneration (fig. 27) the changes within the limb closely resemble those of normal limb development. The elbow joint forms, radius and ulna become distinct, and the more distal regions of the limb skeleton are mapped out. These later aspects of regeneration do not con- cern us here.

2. No%-regeNerating limbs of s r a y e d Amblystorna (figs. 28 to 39)

By repeated experiments, three of which were described in the early part of this paper, it has been demonstrated that daily exposure to x-rays prevents limb regeneration in larval Amblystoma. The question arises, therefore, as to what histological changes within the amputated limb of radiated larvae are associated with the total loss of the capacity to regenerate. I n other words, how does the histology of non-regenerating limbs differ from the histology of normal regeneration which has been surveyed in preceding pages?

The radiated Amblystoma to be discussed in following pages are those individuals of experiment M, mentioned above, in which limb regeneration was prevented by daily exposure for 5 minutes to x-rays. During the first 2 days after limb amputation radiated larvae were fixed at intervals of from 2 to 7 hours. After the first 2 days larvae were fixed at daily intervals up to the twenty-first day after amputation. I n this manner a graded series of larvae was obtained showing the changes which take place within the non-regenerating limb stumps of radiated larvae over a period of 21 days.

I n x-rayed Amblystoma one finds that initial epidermal healing which follows immediately after amputation takes place as rapidly and as completely as in unradiated indi-

292 ELMER Q. BUTLER

viduals. In the larva shown in figure 28, for example, the healing was complete 11 hours after amputation. The absence of an effect on epidermal healing is quite in harmony with expectations, for we know from previous work that x-rays affect particularly cells which are in mitotic division or cells which are undergoing differentiation (Butler, '32). The epidermal healing of wounds appears to occur not through the agency of cell division, but rather through a migration over the wounded area of cells from regions adjacent to it. Cellu- lar differentiation is probably not involved in this process of wound healing. Thus, as one would expect, epidermal healing is a process which is apparently uninfluenced by x-radiation. In figure 28 one sees very clearly the little cap of thickened epidermis over the end of the humerus stump which has previously been mentioned as a regular aspect of the epidermal healing.

During the first 2 to 3 days after amputation (figs. 29 and 30) the changes which take place within the limb appear to be identical with those which one observes in the amputated limbs of unradiated individuals. These changes, which I have previously described as being a result of the mechanical injury to the humerus at the time of amputation consist in general of a breaking down of the cartilage tip of the humerus stump. This breakdown is accompanied by a disappearance of the cartilage cells, a vacuolation of the distal end of the cartilage stump and a loss of the staining capacity of the matrix. That these changes in radiated larvae are in no observable fashion different from the same changes in unradiated larvae one may see by comparing figures 29 and 30 of the radiated series with figures 18 and 19 of the unradiated controls.

On about the fourth day after amputation (fig. 31) there begins an extensive alteration in the cartilage of the humerus. At the outset this cartilage alteration resembles in some respects the alteration which begins in unradiated larvae at about this same time and which I have referred to in preceding pages as being primarily a cartilage dedifferentiation.


In its final results, however, this alteration which takes place in the limb stumps of radiated larvae is entirely different than that which occurs in normally regenerating limbs. On about the fourth day (fig. 31) it is evident that some of the cartilage cells are being released from their capsules and that the cartilage matrix at the distal end of the humerus stump is approaching a period of dissolution. The perichondrium becomes free, forming a loose envelope about the distal end of the humerus in a manner which closely resembles that previously described in unradiated embryos and shown in figure 21.

Figure 32 is the limb of a radiated larva on the fifth day after amputation. In this case the distal third of the humerus stump has broken off and has been pulled ventrad together with the loose envelope of perichondrium surrounding it. Obviously, there must have been a considerable loss of strength and eIasticity of the cartilage in the distal third of the humerus to permit this break. This alteration in the cartilage may be directly attributed, I believe, to the dedifferentiation of cartilage cells which is taking place and the attendant dissolution of the cartilage matrix.

One may observe, also, in figure 32 an aggregation of cells in the distal portion of the limb indicating the beginning of the formation of a blastema. These cells appear to assemble in this region much as do the cells of the blastema in normal regeneration. However, since they never enter into the regenerative process in radiated larvae they are more properly considered, I believe, as constituting a pseudoblastema. I shall use this term in following pages to designate the group of cells in a non-regenerating limb stump which occupy the region normally taken up by the blastema.

Up to the sixth day after amputation a similarity in histological changes has been evident between the limbs of radiated and control individuals. On the sixth day, however, this similarity disappears and changes of an extraordinary and far-reaching nature begin within the limb stumps of radiated larvae. These changes affect chiefly the cartilage of the humerus and the region of the limb occupied by the pseudo-


blastema. The changes which will first be dealt with are those which overtake the humerus and which are best understood by an inspection of figures 33 to 39, which show limbs of radiated larvae from the sixth day to the eighteenth day after amputation. I n general, this change in the humerus may be described as a gradual ‘melting’ of the cartilage. The ‘melting’ process begins at the distal end of the humerus stump and progresses proximad toward the head of the humerus. Coincident with the dissolution of the cartilage, there is a disappearance of the perichondriuni which up to this time has been prominent.

The process by which the humerus undergoes dissolution and fades away does not appear to be a degeneration process in the ordinary sense. There are very few evidences of de- generating cartilage cells. Now and then I have found a cartilage cell in which the nucleus is in advanced pycnosis, but this condition is rather rare. Also, there is no accumulation of blood cells about the end of the humerus as one would expect to find were phagocytosis taking place. What I have observed is as follows: First, there is a gradual loss of the staining capacity of the cartilage matrix. Next, there occurs a dissemination and disappearance of the matrix substance (figs. 34 and 35). Simultaneously with the disappearance of the matrix the cartilage cells undergo profound changes. As the matrix substance ‘melts away’ the cartilage cells are released from their capsules and their nuclei appear to swell slightly and to assume a more granular appearance than they possessed while they were still within normal cartilage. Cell boundaries are difficult to observe with certainty in these released cells. As the dissolution of the cartilage progresses further and further toward the head of the humerus the released cartilage cells mix indistinguishably with the cells of the pseudoblastema in the distal region of the limb stump.

The extent to which the cartilage ‘melting’ or cartilage dissolution progresses is rather startling. Between 10 to 13 days after amputation (figs. 36 and 37) all of the humerus has disappeared, except the head. During the next few days


the head disappears. I n figure 38, for example, which is a limb stump 16 days after amputation, only a slight condensation of cells indicates the former location of the head of the humerus. After the humerus has totally disappeared the scapula shows evidence of being affected and soon it also suffers dissolution. About 18 or 20 days after amputation (fig. 39) the scapula, except for a few remnants, has undergone dissolution and disappeared. Thus, about 20 days after limb amputation, one finds that a radiated larva possesses a non-regenerating limb stump totally devoid of a skeleton of any sort.

The dissolution of the cartilage skeleton of the amputated limb of an x-rayed larvae is all the more striking when one considers that at the same time this phenomenon is taking place cartilage may be forming on the opposite side of the body in an unamputated limb. This is the case, for example, in an experiment such as that described in section 111, part 2, of this paper and illustrated by figures 5 to 10. In this experiment (experiment H) the right limb in the 2-digit stage was amputated. Daily radiation prevented the regeneration of the amputated right limb, but did not prevent the unamputated left limb from developing from the 2-digit stage to the 3-digit stage (figs. 5,7, and 9). Sections of the radiated larvae of experiment H show a complete absence of cartilage in the non-regenerating right limb stump similar to the condition shown in figure 39 and the presence of an apparently normally developed cartilage skeleton within the 3-digit limb on the left side of the body. Here we have a situation, therefore, in which cartilage has undergone dissolution on one side of the body, while it has been developing on the other side.

It is extremely noteworthy, however, that no fourth digit ever appeared on the uiiamputated limbs of the radiated larvae of experiment H (fig. 9) . This effect of x-rays in suppressing digit formation has already heen discussed. On the basis of our present histological knowledge, one may say, therefore, that although cartilage may form on one side of the body while on the other side of the body cartilage is

296 ELMER G. BUTLER

undergoing dissolution, nevertheless, radiation will prevent the initiation of new cartilage structures such as, for example, the cartilage of the fourth digit. The effect of radiation, in other words, seems to be not on cartilage tissue as such but on the initiation and differentiation of new cartilage. Hence, it seems very possible that the critical period, referred to in preceding pages as the period when regenerating or normally developing limbs are most susceptible to radiation, may be identical with the period of cartilage initiation and differentiation. The critical period may simply be the expression of the sensitivity of cartilage differentiation to x-radiation.

Let us now turn our attention to other histological changes which take place within the non-regenerating limb stump. On about the fifth to sixth day (figs. 32 and 33) after amputation, a group of cells appears in the distal portion of the limb. As these cells assemble they resemble very closely in general characteristics the cells which in normal regeneration make up the blastema (figs. 22 and 23). The cytoplasm is finely granular. Cell boundaries are not sharply defined. The nuclei possess deeply staining nuclear membranes which enclose nucleoplasm containing many deeply stained granules. Within each nucleus there is always at least one deeply stained nucleolus ; often there are several prominent nucleoli. Because of their location, the time of their appearance and the cytological characteristics which they exhibit, there can be little doubt but that these cells of the non-regenerating limb stump of radiated larvae are homologous with those cells which compose the blastema of the normally regenerating limb of unradiated individuals. However, in the radiated larvae I consider these cells in the distal end of the limb stump as constituting a pseudoblastema, for the reason that they never exhibit any indication of differentiating into new limb structures.

Although the similarities between the cells of the blastema and the cells of the pseudoblastema are sufficient to convince one of their fundamental homology, nevertheless, many alterations which the x-radiation has induced in the cells of the

X-RAYS AND REGENERATION IN AMBLYSTONA 297

pseudoblastema are evident. I n the pseudoblastema the cells are never so closely packed together as in the true blastema. This scarcity of cells becomes more noticeable in the non- regenerating limb stump as the time subsequent to amputation increases. This may be observed by comparing figure 34, which is a limb stump of a radiated larva 7 days after amputation, with figure 38 which is a limb stump 16 days after amputation. Among the rather widely scattered nuclei of the limb stump one finds vacuolated spaces and a considerable amount of intercellular material which is apparently non- living. In part this non-living material is probably dis- seminated debris of broken-down cartilage matrix and in part it is probably the remnants of dead and disintegrating cells.

I n studying the non-regenerating limb stump as a whole one finds it difficult to distinguish between the cells which make up the pseudoblastema proper and the cells which have been released as the cartilage has undergone dissolution. These released cartilage cells and the cells of the pseudoblastema appear to be identical in general characteristics and they mix indistinguishably at the distal end of the cartilage humerus as the humerus undergoes dissolution. This condition, however, is not totally unlike a situation which occurs during normal regeneration, for, as already pointed out, there is a period in normal regeneration during which it is impossible to distinguish between dedifferentiated cartilage cells and the cells of the new blastema. In the non-regenerating limb stumps of radiated larvae the difficulty in distinguishing between these two types of cells is simply increased by the extreme dedifferentiation or dissolution which the cartilage of the humerus undergoes.

Another obvious difference between the cells of the non- regenerating limb stump and the cells of a true blastema con- cerns cell division. I n the blastema of a normally regenerating limb mitotic figures are rather frequent. In a non-regenerating limb stump one never finds normal mitoses. How- ever, many abnormal mitoses are evident which consist of tangled masses of chromosomes in no very definite orienta-

298 ELMER G. BUTLER

tion. This pronounced effect of x-rays on mitotic cell division has long been known and is one of the effects of radiation most readily observed.

One finds also other cellular abnormalities produced by the radiation. In general, the nuclei of the cells in non-regenerating limb stumps have increased in size and are con- siderably larger than the nuclei of the cells of a true blastema. After many days of radiation one finds evidences of disin- tegration of some of these swollen nuclei. Deeply stained granules have appeared within many of the nuclei, nuclear outlines have become irregular and finally, in those larvae which have suffered the most radiation, remnants of disin- tegrated nuclei make up a part of the intercellular debris already mentioned.

V. GENERAL CONSIDERATIONS

That properly controlled x-radiation will prevent regeneration of the fore limb of Amblystoma larvae has been demonstrated by many experiments, a few of which have been described in the preceding pages. Associated with loss of the ability to regenerate, one finds that profound histological alterations take place within the non-regenerating limb stump. These histological alterations are of two sorts. First, there are the changes which are apparent in the region of the regeneration blastema. Secondly, there are the changes which overtake the cartilaginous skeleton of the limb and result finally in the disappearance of the limb skeleton altogether.

I n normal regeneration all structures of the new limb, with the exception of the epidermis and the nerves, develop from the regeneration blastema which forms at the distal end of the limb stump. I n radiated larvae a condensation of cells occurs which resembles the formation of the blastema in a normally regenerating limb. These cells, however, show no indication of differentiating into the components of a new limb. X-radiation in some manner completely prevents differentiation of the cells within the blastema. Whether this effect on differentiation is due to a direct action of the x-rays

X-RAYS AND RE.GENERATION IN AMELYSTOMA 299

on the cells of the blastema, or whether it is an indirect effect which is the result of the action of the x-rays on the organism as a whole is open to question. In any case, it is certain that the cells of the blastema are particularly susceptible to x-radiation. This is indicated by the many abnormalities which appear among the cells of the blastema after continued radiation. Moreover, it has long been known that x-rays affect most severely cells which are in mitosis, or which are approaching mitosis, and also cells which are undergoing differentiation. The normal regeneration blastema is a region composed of actively dividing cells and of cells which, particularly in the later phases of regeneration, are undergoing extensive differentiation. One would expect, therefore, that the regeneration blastema would be a region which is especially sensitive to x-rays. This is very clearly the case. Examina- tion of the cells of the blastema, or, as it may more properly be termed, the pseudoblastema of a radiated larva reveals only undifferentiated cells and among these cells there are no normal mitoses. The prevention of regeneration in the limb of Amblystoma larvae is directly attributable, I am convinced, to the activity of the x-rays in preventing cell division and more particularly in preventing differentiation of the cells of the blastema which normally give rise to the components of the regenerating limb.

The second effect of x-radiation referred to above, namely, the dissolution and final disappearance of the cartilaginous skeleton of the limb stump of radiated larvae is one of the most surprising results of this investigation. In its general aspects it appears like cartilage dedifferentiation ‘run wild.’ The dissolution and disappearance of cartilaginous structures cannot be attributed to a direct action of x-rays on cartilage as such, for the reason that other cartilage structures of the body are not visibly affected. Moreover, a search of the literature reveals no record of a direct effect of x-rays on cartilage. It seems possible, therefore, that the ‘melting away’ or the dissolution which the cartilage undergoes may in some manner be associated with the effect of x-radiation on


differentiating cells. I n other words, prevention by x-rays of the differentiation of new cartilage within the blastema may bring about secondarily the dedifferentiation of cartilage which has already been formed. It has been noted that in normal regeneration there is a considerable amount of cartilage dedifferentiation which precedes the differentiation of new cartilage. Also, it has been noted that cartilage dedifferentiation always ceases as cartilage differentiation begins. This coincidence suggests that in normal regeneration the initiation of differentiation may, in some manner, serve as a check on the process of dedifferentiation. In radiated larvae differentiation of new cartilage is completely prevented. As a consequence cartilage dediff erentiation, which has already begun, is unchecked and continues without re- striction till all of the cartilage of the limb has dedifferentiated and disappeared.

The hypothesis, that the effect of x-rays on cartilage is primarily an effect on cartilage differentiation and that cartilage dedifferentiation occurs as a secondary result, is further supported by our knowledge of the influence of x-rays on digit formation. It has been clearly demonstrated that x-radiation will prevent normal digit formation in Amblystoma larvae. When this occurs, the differentiation of the cartilages which normally are present within the suppressed digit or digits is prevented. However, there is no evidence that the x-rays affect in any way the cartilages of the limb which were completely formed before radiation was begun. Here one has a situation, therefore, in which x-radiation definitely prevents cartilage differentiation, but does not bring about cartilage dedifferentiation such as occurs in the amputated limbs of radiated larvae. According to the present hypothesis, cartilage dedifferentiation does not occur in limbs in which x-radiation prevents digit formation for the reason that in the process of normal digit formation dedifferentiation is not involved. X-radiation, in other words, does not initiate cartilage dediff erentiation directly.

X-R.AYS A N D REGENERATION I N AMBLYSTOMA 301

All evidence, therefore, points to the fact that the primary effect of x-radiation on the regeneration of the fore limb of Amblystoma larvae is an effect on differentiation. Regenera- tion of limbs in radiated larvae is prevented because x-radiation prohibits cellular differentiation and thereby blocks all processes fundamentally dependent on it. Extreme cartilage dedifferentiation in radiated larvae appears to be a secondary effect dependent on the influence of x-radiation on differentiation.

SUMMARY

1. X-radiation in proper dosage prevents the regeneration of the fore limb of Amblystoma larvae. This effect occurs regardless of the age of a larva or the stage of limb development at the time of amputation.

2. The cells of the regeneration blastema are especially sensitive to x-rays. In a non-regenerating limb stump of a radiated larva a condensation of cells occurs which resembles the formation of the blastema of a normally regenerating limb. These cells in a non-regenerating limb stump, however, are incapable of differentiating into the components of a new limb.

3. In a normally regenerating limb the first component of the new limb to differentiate from the cells of the blastema is cartilage. In a radiated larva not only is there no indication of cartilage differentiation, but also the old cartilage within the limb stump is severely affected by the radiation and as a result gradually disappears. 4. The disappearance of the cartilaginous skeleton of the

limb is due to a progressive dedifferentiation of cartilage which is induced by the x-radiation. Dedifferentiation of cartilage continues till the entire limb skeleton has disappeared.

5. The primary effect of x-radiation on the regeneration of the fore limb of Amblystoma appears to be an effect on differentiation. Cellular differentiation is prevented through the action of the x-rays and regeneration, therefore, cannot

TIIE JOURNAL OR EXPERIMENTAL eoor,oaY, VOL. 65, NO. 3


take place. Extensive cartilage dedifferentiation is a secondary effect induced by the radiation and is dependent oil the influelice of x-radiation on diff ereiitiation.

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HARRISOX, R. G. 1918 Experiments on the development of the fore limb of Amblystoma, a self -diff crentiating equipotential system. J. Exp. Zool., vol. 25.

IIELLMICH, W. 1929 Untersuehungen uber Herkunft und Detcrmination des re- generativen Materials bei Amphibien. Arch. f . Entmmech., Bd. 121.

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(0

PL

AT

E 4

EX

PL

AN

AT

ION

OF

FIG

UR

ES

All

fig

ures

are

unr

ctou

chcd

pho

tom

icro

grap

hs

28

29

30

31

Lon

gitu

dina

l se

ctio

n th

roug

h th

e li

mb

stum

p of

a

radi

ated

lar

va (

M-1

3)

Lon

gitu

dina

l se

ctio

n th

roug

h th

e li

mb

stum

p of

a

radi

ated

lar

va

(&I-

8)

Lon

gitu

dina

l se

ctio

n th

roug

h th

e li

mb

stum

p of

a

radi

ated

lar

va

(M-3

7)

Lon

gitu

dina

l sc

ctio

ii t

hrou

gh t

he l

imb

stum

p of

a

radi

ated

lar

va

(M-2

3)

11 h

ours

aft

er a

mpu

tati

on.

24 h

ours

aft

er a

mpu

tati

on.

41

hou

rs

afte

r am

puta

tion

. X

120

.

4 da

ys a

fter

am

puta

tion

. x

120.

X 1

20.

x 12

0.

X-R

AY

S A

ND

RE

GE

NE

RA

TIO

N I

N A

AIE

LY

STO

MA

E

LM

ER

G

B

UT

LE

R

PL

AT

E 4

PL

AT

E 6

EX

PL

AN

AT

ION

OF

FIG

UR

ES

All

fig

ures

are

unr

etou

ched

pho

tom

icro

grap

hs

32

33

Lon

gitu

dina

l se

ctio

n th

roug

h th

e no

n-re

gene

rati

ng l

imb

stun

ip o

f a

radi

. at

ed l

arva

(M

-12)

5 d

ays

afte

r am

puta

tion

. L

ongi

tudi

nal

sect

ion

thro

ugh

the

non-

rege

nera

ting

lim

b st

ump

of

a ra

di

ated

lar

va

(M-3

6) 6

da

ys a

fter

am

puta

tion

. X

120

. C

ompa

re

with

fi

gure

21

of

the

unra

diat

ed c

ontr

ol s

erie

s.

Lon

gitu

dina

l se

ctio

n th

roug

h th

e no

n-re

gene

rati

ng l

imb

stum

p of

a

radi

at

ed l

arva

(M

-17)

7 d

ays

afte

r am

puta

tion

. X

120

. L

ongi

tudi

nal

sect

ion

thro

ugh

the

non-

rege

nera

ting

lim

b st

ump

of

a ra

di-

ated

lar

va

(M-2

5) 8

day

s af

ter

ampu

tati

on.

X 1

20.

Com

pare

wit

h fi

gure

22

of t

he u

nrad

iate

d co

ntro

l se

ries

.

X 1

20.

34

35

w

ci

M

X-R

AY

S A

ND

RE

GE

NE

RA

TIO

N I

N A

MB

LY

STO

MA

E

LX

ER

G

. B

UT

LE

R

PLATE 5

W

F

W

PL

AT

E 6

EX

PL

AN

AT

ION

O

F F

IGU

RE

S

All

fig

ures

are

unr

etou

ched

pho

toni

icro

grap

hs

Lon

gitu

dina

l se

ctio

n th

roug

h th

e no

n-re

gene

rati

ng l

imb

stum

p of

a

radi

- at

ed l

arva

(1

1.1-

34)

10 d

ays

afte

r am

puta

tion

. X

120

. C

ompa

re w

ith

figu

res

23

and

24 o

f th

e un

radi

ated

con

trol

ser

ies.

L

ongi

tudi

nal

sect

ion

thro

ugh

the

non-

rege

nera

ting

lim

b st

ump

of

a ra

di-

ated

lar

va (

hI-

6)

13 d

ays

afte

r am

puta

tion

. x

120.

C

ompa

re w

ith

figu

res

23

and

26 o

f th

e un

radi

ated

con

trol

ser

ies.

L

ongi

tudi

nal

sect

ion

thro

ugh

the

non-

rege

nera

ting

lim

b st

ump

of

a ra

di

ated

lar

va

(M-1

9) 1

6 da

ys a

fter

am

puta

tion

. X

120

. C

ompa

re w

ith

figu

re 2

7 of

th

e un

radi

ated

co

ntro

l se

ries

. L

ongi

tudi

nal

sect

ion

thro

ugh

the

non-

rege

nera

ting

lim

b st

ump

of

a ra

di-

ated

lar

va (

M-2

0)

18 d

ays

afte

r am

puta

tion

.

36

37

38

39

X 1

20.

X-R

AY

S A

ND

RE

GE

KE

RA

TIO

N I

N A

MB

LY

STO

MA

E

LM

ER

Q

. BUTLER

PLAT

E 6

the effects of x-radiation on the regeneration of the fore limb of amblystoma larvae

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