Influence of an eccentric epidermal cap on limb regeneration in Amblystoma larvae

DEVELOPMENTAL

BIOLOGY,

2, 551-569

(1960)

Influence of an Eccentric Epidermal Regeneration in Amblystoma

Cap on Limb Larvae1

CHARLES STEAD THORNTON Kenyon

College,

Gambier,

Ohio,

and the Jackson Moran, Wyoming

Accepted

August

17,

Hole

BioEogical

Research

Station,

1960

INTRODUCTION

TWO tissues are known to be essential for the activation of regeneration of the amphibian limb-nerves (Singer, 1952) and the skin (Thornton, 1958). Although the mechanism of the nerve influence on regeneration remains obscure (Singer, 1959), it has become firmly established that limb regeneration is dependent on a threshold quantity of nerve fibers. Less is known of the nature of the dependence of limb regeneration on the skin. Particular attention, however, has been devoted to the wound epithelium that covers the amputation surface of the limb stump. This becomes thickened to establish the apical epidermal cap beneath which mesenchyme cells aggregate to form the regeneration blastema. It has been proposed that the function of the epidermal cap in regenerating limbs of amphibian larvae is to direct, in a manner still unknown, the aggregation and outgrowth of the blastemal cells (Thornton, 1954). It has also been proposed (Polejaiev, 1945; Scheuing and Singer, 1957; Bodemer, 1958) that the wound epithelium and epidermal cap stimulate the limited histolysis of the injured stump tissues, a process which has been found to provide many of the cells of regeneration (Hay, 1959). Recently it has been found that the position of the epidermal cap can be shifted from its typical location at the apex of the limb stump tip to a new position at the posterior border of the limb tip. Beneath 1 This investigation was supported at Kenyon by a grant (B-578) National Institute of Neurological Diseases and Blindness of the National of Health, United States Public Health Service, and at Moran by a grant National Science Foundation. 551

from the Institutes from the

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such eccentric epidermal caps mesenchyme cells aggregate to establish correspondingly asymmetrical blastemata; these develop into regenerates that project from the limb stumps at a sharp angle. By dissociating, in a variety of ways, the processes of histolysis of stump tissues from the activity of the eccentric epidermal cap it has become possible to demonstrate an influence of the epidermal cap on the aggregation and outgrowth of the blastemal cells. MATERIALS

AND

METHODS

Premetamorphic larvae of Amblytioma tigrinum, Amblystoma opncum, and Amblystoma punctatum have been used in the experiments to be described below. The larvae ranged in length from 30 mm to 60 mm. Conditions of maintenance of the larvae were standard and were similar for all larvae. The basic experimental manipulation common to all experiments reported in succeeding pages was a shifting of the established epidermal cap from its normal position at the apex of the limb stump to the posterior (postaxial) border of the limb tip-an eccentric position at the edge of the healed amputation surface. The shifting of the epidermal cap was a consequence of the removal of a narrow strip of skin (dermis and epidermis), !,s mm wide and running parallel and adjacent to th e posterior border of the amputation surface of the limb for $5 mm. As will be described in subsequent pages, healing movements of the epidermis of the amputation surface over this narrow wound in the skin carried the epidermal cap to its eccentric position at the posterior border of the amputation surface. Variations of the operation and additional manipulations will be described in the appropriate sections below. All amputations were performed at the level of the mid-section of the humerus. Regenerated limbs were fixed in Bouin’s fluid and stained in hematoxylin and eosin for histological study. RESULTS

Migration

of the Epiderma! Cap

In experiments reported in the present investigation, the epidermal cap assumed an eccentric position at the limb tip after a narrow strip of skin had been excised from the posterior (postaxial) surface of the limb stump adjacent to the edge of the healed amputation surface. A

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series of experiments was undertaken to study the manner in which the shift in position of the epidermal cap occurred. Thus in 30 larvae, 5 days after amputation of the forelimb when an epidermal cap is well established, a strip of skin ( 45 mm by $6 mm) was removed adjacent to the posterior edge of the amputation surface. Limbs so treated were fixed immediately and at 1, 2, 3, 4, 6, 8, 12, and 18 hours after skin strip excision. Figure 1 is a photomicrograph of a longitudinal section through a limb fixed immediately after skin excision. Although the limb stump is sectioned longitudinally, the skin excision is cut in cross section and thus illustrates the width of the wound that initiates the epidermal movement that carries the epidermal cap to the posterior edge of the amputation surface. An early epidermal cap can be seen in its typical position at the apex of the limb tip. Blood cells are escaping from the wound. By 2 hours after skin excision the wound is healed; this is seen in Fig. 2, where it may be noted that a much greater epidermal migration has occurred from the epidermis covering the amputation surface than from that proximal to the new wound. Lash (1955)) in a detailed study of wound closure in salamander larvae, found that epidermal sheets of cells move more rapidly toward a wound after their attachments to the underlying dermis are broken. Quite possibly the differential mobility of the epidermal cells in the present experiments is correlated with the degree of dermal attachment, As is shown in Fig. 2, the epidermis proximal to the new wound is underlaid by thick dermal fibers while beneath the epidermis covering the amputation surface a new dermis has not yet regenerated. Thus this distal epidermis might well have had more freedom of movement than the proximal epidermis. The epidermal cells of the amputation surface move as a sheet, the cells bordering the wound undergoing extension and stretching as though actively engaged in ameboid activity. Nevertheless, the apical epidermal thickening persists during the migratory movements of the epidermis although it becomes somewhat flattened and extended, as may also be seen in Fig. 2. By 3 hours after skin excision there are no further indications of migratory movements of the epidermis, and the epidermal cap, in its new eccentric location, is regaining a more conical shape, as may be seen in Fig. 3. Typically by 12 hours after skin excision (Fig. 4) the epidermal cap is 7-8 cells thick and is well established at the posterior border of the limb tip and in close proximity to

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the area of skin excision. At this time it should be noted (Fig. 4) that there is no indication that the mesenchyme cells of the limb tip are in any way associated with the epidermal cap. The asymmetrical disposition of the epidermal cap persists, in the great majority of cases, during succeeding days of limb regeneration. The Effect of un Eccentric Epidcrmal Cap on Limb Regeneration The question now to be raised is: Will the asymmetry of the epidermal cap induce a corresponding asymmetry of regenerative outgrowth of the blastema? As a first approach to this problem 20 Amblystoma tigrinum larvae and 30 Amblystoma opacum larvae underwent amputation of the left forelimb, and 5 days later had a narrow strip of skin excised from the limb stump adjacent to the posterior border of the amputation surface. At 5 days after amputation the epidermal caps of larvae of Amblystoma are well established. Furthermore, the processesof stump tissue dedifferentiation are nearly completed. Of the total of 50 larvae which underwent skin excision, 42 developed correspondingly asymmetrical regenerates. The asymmetry of the regenerates in these as well as in subsequent experiments was expressed as a deviation in the direction of outgrowth of the blastema so that the regenerate formed an angle with the limb stump which varied from approximately 50° to 90°. In every case of asymmetry the regenerate grew out from the posterior edge of the amputation surface. The negative casesin these and subsequent experiments are apparently a result of a gradual return of the epidermal cap and blastema to a more apical position. In the negative cases, in other words, regeneration began

FIG.

excision. to the FIG. surface FIG.

surface FIG.

surface FIG. of limb FIG.

surface

PLATE I Amblystoma punctatum larva. Limb fixed immediately after skin Note epidermal cap at apex of limb stump. Postaxial surface of limb is right. Magnification: x 67. 2. A. punctutum larva. Limb fixed 2 hours after skin excision. Postaxial of limb is to the left. x 67. 3. A. ;ounctatum larva. Limb fixed 3 hours after skin excision. Postaxial of limb is to the right. x 67. 4. A. punctatum larva. Limb fixed I2 hours after skin excision. Postaxial is to the right. x 67. 5. A. opacum larva. Limb fixed 1 day after skin excision. Postaxial surface is to the left. Note eccentric accumulation of mesenchyme cells. x 72. 6. A. opacum larva. Limb fixed 3 days after skin excision. Postaxial of the limb is to the right. x 67. 1.

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THORNTON

eccentrically but gradually reduced the angle of asymmetry with the limb stump until the regenerate occupied the apical position characteristic for normally regenerating limbs. A similar return of eccentrically regenerating blastemata, induced by nerve deviation, to an apical position at the distal tip of the limb stump has been described by Guy¬ and associates (1948) in adult Triton. One day after skin excision (Fig. 5)) the epidermal cap (which in this case has not become fully concentrated again after epidermal migration) is situated at the posterior border of the amputation surface. A small accumulation of mesenchyme cells already appears to be somewhat more dense beneath the epidermal cap than elsewhere in the limb tip. Figure 6 illustrates a bulb blastema stage of regeneration in which the prominent epidermal cap is directed posteriorly from the limb tip. The aggregating blastemal cells are clustered beneath the epidermal cap in an obvious asymmetry of distribution. Many more blastemal cells have aggregated in association with the epidermal cap than in other sectors of the limb tip. In Fig. 7 is shown a blastema 5 days after skin excision. The acute eccentricity of outgrowth of this blastema is clearly correlated with the eccentric position of the epidermal cap. Both in the epidermal cap and in the underlying blastemal cells mitoses are frequent. Finally, in Fig. 8 is shown an Amblystomu tigrinum regenerate 2 weeks after skin PLATE II FIG. 7. Amblystomu opacum larva. Limb fixed 5 days after skin excision. Postaxial surface is to the right. Note prominent eccentric epidermal cap and blastema. Magnification: X 60. FIG. 8. A. tigrinum larva. Limb fixed 2 weeks after skin excision. Postaxial surface is to the right. Section illustrates the right-angled fusion of regenerated section of humerus with old humerus stump. Radius and ulna not shown. x 21. FIG. 9. A. opacum larva. Regressed limb fixed 1 day after skin break. Postaxial surface is to the right. Note eccentric cap and associated blastemal cells; also head of humerus. x 78. FIG. 10. A. opacum larva. Regressed limb fixed 8 days after skin break. Postaxial surface is to the right. Note asymmetrical blastema; also head of humerus at base of limb. x 78. FIG. 11. A. punctatum larva. Regressed limb bud fixed 1 day after skin break. Postaxial surface is to the left. Note eccentric epidermal cap at the skin break. No formed limb tissues present. x 67. FIG. 12. A. punctatum larva. Regressed limb bud fixed 3 days after skin break. Postaxial sur’ace is to the right. Note eccentric epidermal cap and associated blastemal cells. x 67.

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THORNTON

excision. The regenerate projects from the posterior surface of the limb stump at an angle of 90 O. This is not a result of the formation of an elbow joint since the differentiating cartilage of the regenerate has fused with the postaxial surface of the remnant of the humerus in the limb stump rather than with the distal end of the humerus, as occurs in normal regeneration. Thus a completed humerus with a right-angled curvature is produced and the regenerated segment of the limb is thus structurally asymmetrical. Several series of experiments were undertaken in which skin excisions were made at different levels of the limb stump and at various stages of regeneration. In all these experiments skin excision alone had no effect on the direction of regenerative outgrowth when the epidermal cap was not carried from its typical location at the apex of the limb stump. Thus, for example, a skin excision made 1 mm proximal to the posterior border of the amputation surface healed by migration of epidermis over the wound surface from all sides equally. The epidermal cap remained in its normal position at the apex of the limb tip and regenerative outgrowth proceeded in a quite normal manner without any indication of asymmetry. It is therefore possib!e to correlate the asymmetry of the epidermal cap with a like asymmetry of regenerative outgrowth of the blastema.

Regeneration

of Regressed Limbs with Eccentric Epiderm,al

Caps

Although the experiments described in the preceding section demonstrated a striking correlation between epidermal cap asymmetry and direction of outgrowth of the blastema, the possibility must be considered that the epidermal cap possesses histolytic activity and that tissues nearest to it would therefore be subjected to a more intensive and prolonged histolytic action than would tissues farther away. In such a situation an asymmetrical accumulation of blastemal cells could be the result primarily of a greater amount of tissue dedifferentiation in the areas of the limb stump nearest to the eccentric epidermal cap. One means of eliminating all possibility that the epidermal cap might influence asymmetrical regeneration by inducing differential histolysis of stump tissues would be to produce complete dedifferentiation of these tissues before allowing an epidermal cap to become established. Butler and Schottk (1941) have shown that the limb of the larval salamander can be induced to undergo excessive regression when amputation is combined with denervation of the limb. Typical re-

ASYMMETBICAL

LIMB

REGENERATION

559

generation subsequently osccurs when reinnervation of the regressed limbs is permitted. Consequently, this technique was used in an attempt to stimulate complete tissue dedifferentiation. The left forelimbs of 20 Amblystom~u opacu~n larvae were amputated and denervated according to the procedure of Schotte and Butler (1941). Redenervation at intervals of 10 days continued the nerveless condition of the limb stumps for as long as 30 days, when redenervation procedures became of questionable success. At the end of 30 days the denervated limb stumps had been reduced by regression to small buds on the shoulder. Nerves were allowed to regenerate into the regressed limb remnants and at the first indication of sensitivity (avoiding reactions of the larva induced by pricking the limb remnant with a needle), a break was made in the skin at the posterior border of the tip of the limb remnant, actual excision of a strip of skin being difficult owing to the small size of the limb bud. In all but two cases, asymmetrical regenerates were produced. Histological analysis, however, disclosed the fact that although limb muscle tissues had completely regressed, the shoulder girdle and head of the humerus still remained. Thus in Fig. 9, which shows a longitudinal section through a regressed limb 1 day after a break had been made in the skin of the posterior edge of the limb tip, an epidermal cap is forming eccentrically and beneath it may be seen an assemblage of mesenchyme cells probably derived from the excessive dedifferentiation of the stump tissues. As in all cases of early blastemata, mesenthyme cell accumulation is more concentrated directly beneath the epidermal cap than elsewhere in the limb. Although no histologically integrated muscle tissue can be found, the head of the humerus still persists. At 8 days, an early cone blastema has developed asymmetrically (see Fig. 10). Again, although there is no evidence of formed muscle tissues in the limb stump, the head and most proximal segment of the humerus still remain, as is just visible at the base of Fig. 10. It seems unlikely that a differential histolysis of the remaining cartilaginous skeleton could account for the asymmetrical outgrowth of the blastemata of these larvae. Nevertheless, these experiments have not met the rigorous requirements of a complete dedifferentiation of all stump tissues. Excessive tissue dedifferentiation can be induced, however, in another way. Denervation combined with fracture of the limb skeleton has been demonstrated to produce excessive limb regression in Ambly-

560

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STEAD

THORNTON

stoma larvae (Thornton and Kraemer, 1951). It was decided, therefore, to denervate the left forelimbs of 50 AmbZystMna punctatum larvae, to fracture the skeleton of the limb by pressure applied with watchmaker’s forceps, and, in order to assure complete skeletal dedifferentiation, to remove the shoulder girdle and head of the humerus. This was accomplished at the time of denervation. The incision in the skin of the shoulder which allowed access to the brachial nerves also allowed access to the shoulder girdle and head of the humerus. These cartilages were cut free and withdrawn through the incision. No open wound, other than the incision at the shoulder, was made. Regression of limbs treated in this manner was rapid, and no more than one redenervation was necessary before tissue dedifferentiation was complete. Two weeks after the original operation, the left forelimbs of the larvae had regressed either to short, stout spikes or to budlike mounds at the shoulder. In either type of regressed limb, tissue dedifferentiation was complete and the limb remnant consisted of an unorganized mass of mesenchyme cells. Reinnervation then proceeded, and when sensitivity had returned to the limb remnants, a break in the skin at the posterior edge of the tip of the limb was made. As has been described previously (Thornton, 1954)) regressed, unamputated limbs will form an epidermal cap at the point where a dermal break occurs. In the present experiments, the skin break interrupted the dermis. In 41 of the 47 surviving cases regeneration was asymmetrical. Furthermore, no histologically integrated tissues, muscle or cartilage, remained in the limb area to be histolyzed. The course of events may best be followed by means of the following cases. Figure 11 is a photomicrograph of a longitudinal section through a bud type of limb remnant 1 day after a skin break had been made. This limb bud is typical in showing the tangled mass of general connective tissue and undifferentiated mesenchyme cells that remain in the limb area after complete dedifferentiation of the limb tissues. Such limbs resemble embryonic limb buds yet are composed of larval cells. The thickened epidermal cap is located asymmetrically near the posterior edge of the tip of the limb remnant and occupies the area of the skin break. Thick dermal fibers underlay the epidermis in all but this one region. Particularly worthy of note is the close physical contact between the lowest layer of cells of the epidermal cap and the mesenchyme cells at the tip of the limb remnant. The nature of the

ASYMMETRICAL

LIMB

REGENERATION

561

epidermal cap-mesenchyme cell contact is being investigated with histochemical techniques (Schmidt, 1960). At 3 days (Fig. 12) the connective tissue and mesenchyme cells have become morphologically similar to blastemal cells. This is particularly noticeable in the dense accumulation of cells in close contact with the asymmetrical epidermal cap which is located at the posterior border of the limb tip-the region where the break in the skin of the limb remnant was originally made. The developing blastema is becoming asymmetrical in correspondence with the asymmetry of the epidermal cap. Figure 13 is a photomicrograph of a longitudinal section through a spike type of limb remnant fixed at 3 days after skin break and illustrates the acute angle of outgrowth of the epidermal cap and immediately subjacent blastemal cells in contact with the cap. The large accumulation of mesenchyme cells in the limb spike is derived from the dedifferentiation of the limb tissues. Figure 14 is a photomicrograph of a longitudinal section through a limb spike at the cone blastema stage of regeneration (8 days). The epidermal cap is no longer as prominent morphologically as it was at earlier stages of regeneration, but the asymmetry of regenerative outgrowth is expressed clearly by the approximately 65” angle which the blastema makes with the stump remnant. Finally, in Fig. 15 is shown a 20-day regenerate. In this case the upper arm regressed very rapidly and was resorbed before the more distal forearm components had regressed. The remaining limb spike consequently seemed to be largely composed of the regressed forearm components, It is of particular interest, therefore, to note that no signs of a regenerated humerus can be found in the limb that subsequently regenerated from the reinnervated limb remnant. Rather, the skeleton of the regenerate is composed of atypical radius, ulna, and the more distal cartilaginous elements. In this respect this regenerate is reminiscent of those that Weiss (1925) and Bischler (1926) obtained after removal of the skeleton and subsequent amputation of the limb of the adult newt. In both these investigations the nature of skeletal regeneration was determined by the level of the limb represented by the amputation surface and not by the amount of skeleton that was missing. The asymmetry of the regenerate shown in Fig. 15 is striking. As in the limb shown in Fig. 8, the sharp bend which the regenerate makes

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THORNTON

with the limb stump is not an indication of a joint articulation. Rather, as is shown in Fig. 16, a more highly magnified view of the regenerate shown in Fig. 15, the distal sections of the radius and ulna were completed by cartilage regeneration within the more distal and asymmetrical part of the regenerate. In these cases of limbs regenerating asymmetrically after complete regression, the eccentric epidermal cap could not have induced an eccentric aggregation of blastemal cells by means of a differential histolytic action on the stump tissues. The stump tissues had already undergone complete dedifferentiation before the epidermal cap was allowed to become established. DISCUSSION

Although the role of the skin in regeneration has received intensive analysis in recent years, the problem is as old, at least, as the work of Tornier (1906)) who inhibited limb regeneration by covering the fresh amputation surface with fully differentiated skin. His hypothesis that the inelasticity of the whole skin mechanically suppressed regenerative outgrowth was subsequently disproved by Godlewski (1928), who, by an ingenious amputation of the axolotl tail which provided a free flap of whole skin at the dorsal border of the tail stump, demonstrated that several days’ delay in sealing the amputation wound with the skin flap did not prevent the regenerate from rupturing this skin barrier. Godlewski proposed that the importance of the epidermis in regeneration resided in its contributing cells to the mesenchymal blastema. Rose (1948) supported this hypothesis of an epidermal contribution to the PLATE III FIG. 13. AnbZgrsEomu punctatum larva. Regressed limb spike fixed 3 days after skin break. Postaxial surface is to the left. Note acutely eccentric epidermal cap and associated mesenchyme cells. No formed limb tissues present. Magnification: x 87. FIG. 14. A. punctatum larva. Regressed limb spike fixed 8 days after skin break. Postaxial surface is to the left. Note acute eccentricity of cone blastema. x 67. FIG. 15. A. ~~unctatum larva. Regressed limb spike fixed 20 days after skiu break. Postaxial surface is to the right. Section illustrates the acute bend in the regenerated radius and ulna, but does not show the digits. x 39. FIG. 16. Same larva as in Fig. 15. View of the eccentric regeneration of the distal ends of the radius and ulna. This limb is rotated 90” to the right from the position shown in Fig. 15. X 160.

564

CHARLES

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THORNTON

blastema in an investigation in which a sharp decrease in the number of cells of the epidermal cap was correlated with a rise in the number of blastemal cells. Other investigators, however, have failed to find any evidence for an epidermal contribution to the blastema (see Chalkley, 1959). Indeed Hay and Fischman (1960), working with limb tissues labeled with tritiated thymidine, have recently reported direct evidence that apical epidermal cap cells do not contribute to the regeneration blastema in Triturus. That it is the epidermis, rather than the dermis, which is important for regeneration was also concluded by Effimov (1831; 1933) and Polejaiev and Favorina (1935). Thus, after skin taken from the head or back of an axolotl is transplanted to a limb which has been denuded of its own skin, and a subsequent amputation performed in the region of the skin graft, the wound surface is covered by an epithelium derived from the grafted head (or back) skin. But no blastema arises, and regeneration fails. A wound epithelium contributed by skin from the tail, abdomen, or limb, on the other hand, supports regeneration quite normally in the axolotl. Parenthetically, it should be noted that in some other urodeles [and in Xenopus, according to Tschumi ( 1957) ] skin from any region of the body may support limb regeneration. Thus, for example, in AmbEystoma tigrinum (Thornton, unpublished) a strip of head or back skin transplanted as a cuff to the forelimb will provide a wound epithelium, after amputation through the graft, which allows regeneration to occur. Polejaiev (1945) has proposed that the wound epithelium derived from normal limb skin possessesproteolytic activity which is important for the initiation of the limited dedifferentiation of the limb tissues injured at amputation, a process generally considered to give rise to the cells of regeneration. Adova and Feldt (1939) have indeed demonstrated an increased histolytic activity of wound epidermis as compared with normal epidermis. Scheuing and Singer (1957) and Bodemer (1958) have lately re-emphasized the possible histolytic function of the wound epidermis, particularly the epidermal cap, in limb regeneration of adult newts. The possibility, however, that a histolytic action of the epidermal cap is more commonly found in the regenerating limbs of adult salamanders than of larvae is well worth consideration. There is evidence that in the postmetamorphic limb of amphibians, tissue dedifferentiation is both more difficult to initiate and more limited in extent than in the larva (Goodwin, 1946; Polejaiev, 1936). Furthermore, a variety of treatments which stimulate excessive

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tissue regression in the larval limb have little effect on the dedifferentiation of limb tissues in the adult. Thus, for example, amputation and denervation produce extreme regression of the larval limb (Butler and Schotte, 1941) yet similar treatment in the adult results in a stabilized limb stump (Singer, 1952). Indeed, not even a wound epithelium is a prerequisite for excessive regression in denervated larval limbs (Thornton and Kraemer, 1951; Thornton, 1953). An injury that does not involve breaking the skin will produce fully as extensive a type of tissue dedifferentiation in denervated limbs as occurs after amputation. In these cases no break in the dermis occurs to bring the epidermis into intimate contact with internal mesodermal tissues, which nevertheless dedifferentiate. Although highly speculative, it is possible that a histolytic mechanism to stimulate tissue dedifferentiation has become a necessary feature of the epidermal cap of the regenerating adult limb whereas the stimulus of trauma alone is all that is needed in the larva to initiate dedifferentiation. The experiments presented in the foregoing pages support the view that, in larval Amblystoma at least, the epidermal cap of the regenerating limb functions to control the aggregation and outgrowth of the blastemal cells. Thus an eccentrically located epidermal cap is intimately associated with a correspondingly asymmetrical aggregation of blastemal cells, which subsequently grow into an eccentric regenerate. That the asymmetry of the blastema is not a result of an asymmetrically directed histolytic activity of the epidermal cap is made clear by inducing complete limb regression and tissue dedifferentiation before the epidermal cap is allowed to become established. The regressed limb remnant is composed of mesenchyme cells derived from dedifferentiation of the limb tissues. The aggregation of these mesenchyme cells beneath the eccentric cap must therefore be a consequence of a directing influence by the cap on their accumulation and outgrowth rather than of any kind of histolytic action by the epidermal cap. The activity of the apical epidermal cap in directing the accumulation and outgrowth of mesenchyme cells in the regenerating limbs of Amblystoma larvae recalls the essential role which the apical ectoderm plays in directing the outgrowth of the embryonic limb bud. Thus Saunders (1948) suppressed distal differentiation of the chick wing bud by surgical excision of the apical ectodermal ridge. Ectoderm other than that of the apical ridge failed to support distal wing bud

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outgrowth. Zwilling (1956, for review) has shown, by interchanging wing- and limb-bud apical ridges, that although continued distal outgrowth of the appendage bud is dependent on the ectodermal ridge, the direction of morphogenesis of the bud is controlled by its mesoderm. Furthermore, the continued existence of the apical ectodermal ridge is dependent on a “maintenance factor” resident in the mesodermal core. Recently, however, Bell and associates (1959a), using focused ultrasound to strip the ectoderm from chick limb buds, were able to demonstrate that a significant number (19%) of limb buds lacking apical ectoderm developed normally. In a joint reanalysis of chick limb bud morphogenesis, Bell et al. ( 1959b) conclude that the presence of a membranous layer subjacent to the ectodermal ridge may have a particularly important effect on limb bud development. On the other hand, in the urodele embryo Balinsky (1956) reports that a prerequisite for accessory limb induction is the localized breakdown of the basement membrane so that ectoderm and mesoderm are brought into intimate contact. Amphibian embryos do not possess prominent apical ectodermal caps, although Taylor (1943) illustrates a slightly thickened apical ectoderm in Rana pipiens limb buds and Tschumi (1957) has described an apical ectodermal ridge in hindlimb buds of Xenopus. Steiner (1928) destroyed the apical ectoderm of both anuran and urodele limb buds with the result that the development of the distal parts of the limb were inhibited. Distal differentiation of these limb buds resumed, however, after a new ectodermal covering had regenerated. In essential agreement with these results is the recent work of Tschumi (1957) in which epidermis-free limb buds of Xenopu,s were implanted into the body wall; there they developed only those limb parts that had already been laid down before implantation. He found, however, that undifferentiated nonlimb epidermis can also support distal differentiation of the limb. The results of the investigations of Steiner and of Tschumi allow some interesting comparisons to be made between amphibian limb development and regeneration. Thus, as in the limb bud, (1) the distal outgrowth of the regeneration blastema is dependent on the presence of an apical epidermal thickening, or cap; (2) nonlimb skin can also support apical outgrowth of regenerating limbs of Am.blysknna larvae (see page 564) ; and (3) limb regeneration is only temporarily halted by excision of the apical epidermal cap, since a new epidermal cap is quickly regenerated with a consequent resumption of

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REGENERATION

blastemal cell aggregation and outgrowth (Thornton, 1W’). It is to be hoped that future work will provide further evidence of similarities between amphibian limb development and limb regeneration. The nature of the influence of the epidermal cap on the blastemal cells of the regenerating limb is unknown. Recently, in this laboratory, successful transplantation of epidermal caps to blastemata has resulted in the production of reduplicated limb regenerates, but similar grafts of whole skin were negative. Further experiments are in progress designed to test the possibility that the epidermal cap may either stimulate blastemal cell mitosis or effect blastemal cell migration. SUMMARY

1. The epidermal cap of the regenerating forelimb of larval Anzblywas induced to become eccentrically located at the posterior border of the amputation surface by means of a skin excision (‘/ mm long by $5 mm wide) at the posterior edge of the amputation surface. Healing of the skin excision wound by epidermal migration, particularly from the epidermis covering the amputation surface, carried the epidermal cap to the postaxial border of the limb tip. 2. Blastemal cells aggregated beneath the eccentric epidermal cap to form a correspondingly eccentric blastema which developed into an asymmetrical regenerate. 3. Eccentric epidermal caps were induced in regressed limbs after all limb tissues had completely dedifferentiated. Blastemal cells aggregated beneath the eccentric epidermal caps to produce eccentric blastemata and regenerates. Thus the eccentric epidermal cap induced a corresponding eccentricity of regenerative outgrowth not by a differential histolysis of limb tissues, but by controlling the aggregation of blastemal cells.

S~NTUZ

I am technical

indebted to Mrs. Mary Thornton and Mr. Kenneth Jewel1 assistance they have provided throughout this investigation.

for

the

expert

REFERENCES Anov~, A. N., and FELDT, A. M. (1939). Biochemical regions of the axolotl’s body connected with form-building organizers. Compt. rend. acad. sci. U. R. S. S. 25, 43-45. BALINSKY, B. I. (1956). A new theory of limb induction.

peculiarities under Proc.

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M. E., and components

FESSENDEN,

in

the

L. M. (1959a). The role of mesodermal development of the chick limb. Deuelop.

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mol. 33,431-560. C. W. ( 1958). The development of nerve-induced supernumerary limbs in the adult newt, Triturus uiridescens. J. Morphol. 102, 555-582. BUTLER, E. G., and SCHOT&, 0. E. (1941). Histological alterations in denervated non-regenerating limbs of urodele larvae. .I. Exptl. Zool. 88, 307342. CHALKLEY, D. T. ( 1959). The cellular basis of limb regeneration. In “Regeneration in Vertebrates” (C. S. Thornton, ed.), pp. 34-58. Univ. of Chicago Press, Chicago, Illinois. EFFIMOV, M. L. ( 1931). Die hlnterialen zur Erlernung der Gesetzmaszigkeit in den Erscheinungen der Regeneration. Z. exptl. Biol. (Russ.) 7. [Summarized by Polejaiev and Favorina ( 1935) .I EFFIMOV, M. L. ( 1933). Die Rolle der Haut in prozess der Regeneration eines Organs beim Axolotl. Zhur. Bid. (Russ.) 2. [Summarized by Polejaiev and Favorina ( 1935 ) .] GOULEWSKI, E. ( 1928). Untersuchungen iiber Au&sung und Hemmung der Regeneration beim Axolotl. Wilhelm Roux’ Arch. Entt&ckEungsmech. Organ. BWEMER,

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anoures.

2 I am grateful chapter.

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LIMB

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REGENERATION

of the adult

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Triturus.

J. Exptl.

Zool.

136, 301-328. SCHMI~~~, A. J. (1960). A histochemical analysis of forelimb regeneration in the hypothyroid adult newt. Anat. Record 136,274 (Abstract ). SCHOLL, 0. E., and BUTLER, E. G. (1941). Morphological effects of denervation and amputation of limbs in urodele larvae. J. Erptl. Zool. 87, 279-322. SINGER, M. ( 1952). The influence of the nerve in regeneration of the amphibian extremity. Qwlrt. Reu. Bid. 27, 169-200. SINGER, M. (1959). The acetylcholine content of the normal forelimb regenerate of the adult newt, Triturus. Develop. Bid. 1, 603-620. STEINER, K. ( 1928). Entwicklungsmechaniche Untersuchungen iiber die Bedeutung des ektodermalen Epithels der Extremitatenknospe von Amphibien Larven. Wilhelm Roux’ Arch Entwicklungsmech. Organ. 113, l-11. TAYLOR, A. C. ( 1943). Development of the innervation pattern in the limb bud of the frog. Amt. Record 87, 379414. THORNTON, C. S. ( 1953). Histological modifications in denervated injured fore limbs of Amblystomu larvae. J. Exptl. 2002. 122, 119-150. THORNTON, C. S. (1954). The relation of epidermal innervation to limb regeneration in Amblystoma larvae. J. Exptl. Zool. 127, 577-602. THORNTON, C. S. (1957). The effect of apical cap removal on limb regeneration in Amblystomu larvae. J. Exptl. Zool. 134, 357382. THORNTON, C. S. (1958). The inhibition of limb regeneration in urodele larvae by localized irradiation with ultra-violet light. J. Exptl. Zool. 137, 153-180. THORNTON, C. S., and KRAEMER, D. W. (1951). The effect of injury on denervated unamputated fore limbs of Amblystoma larvae. J. Exptl. Zool. 117, 415-440. TORNIER, G. (1906). Kampf der Gewebe im Regenerat bei Begiinstigung der Hautregeneration. Wilhelm Roux’ Arch. Entwicklungsmech. Organ. 22, 346-369. TSCHUMI, P. A. (1957). The growth of the hind limb bud of Xenopus laevis and its dependence upon the epidermis. J. Anut. 91,149-173. WEISS, P. (1925). Unabhiingigkeit der Extremitatenregeneration vom Skelett (bei T&on &status). Wilhelm Roux’ Arch. Entwicklungsmech. Organ. 104,359-394. ZWILLING, E. (1956). Genetic mechanisms in limb development. Cold Spring Harbor Symposia Quunt. Biol. 21, 349-354.