The origin of skeletal structures during intercalary regeneration of larval Ambystoma limbs1, 2

DEVELOPMENTAL BIOLOGY 79, 255-275 (1980)

The Origin of Skeletal Structures during lntercalary Regeneration of Larval Ambystoma Limbs’

MAURICE J. PESCITELLI, JR.' AND DAVID L. STOCUM” Department of Genetics and Development, Universitv of Illinois, Urbana, Illinois 61801

Received December 7, 1979; accepted in revised form February II, 1980

Urodele limbs are able to regulate for intercalary deletions created when distal regeneration blastemas are grafted to a more proximal level. Using several morphological markers, pigmentation differences between white and dark axolotls, and the difference in nucleolar number between diploid and triploid animals, we show that the entire intercalary regenerate is derived from the stump when wrist or tarsus blastemas are grafted to the midstylopodium of the fore- or hindlimb. The transplanted prospective autopodium forms no more than would be expected in situ. Thus, the rule of distal transformation is not violated during intercalary regeneration in salamanders. The advantages and disadvantages of the several marking techniques are discussed.

INTRODUCTION

Amputation of a urodele limb at any level results in the faithful regeneration of the missing parts. One of the major problems in the study of this phenomenon is to un- derstand how cells from a given level of the limb are able to reconstitute the exact pattern of more distal levels. Little is under- stood about the rules which govern this process. However, it is a well-documented fact that terminal regeneration from both a normally oriented stump, as well as from a stump of reversed proximal-distal polarity, always forms structures that are normally distal to the amputation plane (Kurz, 1922; Milojevic and Grbic, 1925; Dent, 1954;

’ Part of a dissertation submitted by M. J. Pescitelli. Jr., to the Graduate College of the University of Illinois in partial fulfillment of the requirements for the degree of Doctor of Philosophy.

’ Present address: University of Dominica School of Medicine, P. 0. Box 266, Roseau, Commonwealth of Dominica, West Indies.

.I To whom correspondence and requests for re- prints should be addressed.

Butler, 1955; Deck and Riley, 1958; Ober- heim and Luther, 1958). These observations have been formalized by Rose (1962) as the “rule of distal transformation,” which states that regeneration produces only more distal parts regardless of the proximal-distal orientation of the amputation surface.

As a basis for the rule of distal transformation, Wolpert (1971) has suggested that the proximal boundary of the regenerate is determined by an intrinsic level-specific property of the cells at the amputation surface. Cells cannot have positional values more proximal than the level from which they were derived, though they can acquire more distal positional values. Whether this rule reflects an intrinsic property of cellular behavior is not known. The distal transformation of blastema cells during terminal regeneration might be due to the fact that the wound epidermis, which is an essential factor in regeneration (Thornton, 1968), acts in a distalizing capacity (Maden, 1977; Stocum, 1978). Interaction between blastema cells and the overlying wound epider-

255 0012-1606/80/120255-21$02.00/O Copyright 0 1980 by Academic Press, Inc. All rights of reproduction in any form reserved.

256 DEVELOPMENTAL BIOLOGY VOLUME 79,198O

mis during terminal regeneration would thus inevitably lead strictly to distal transformation. This constraint, however, does not obtain in the case of intercalary regeneration. When a distal regenerate is transplanted to a more proximal level, the graft and stump mesodermal tissues interact to intercalate the deleted limb structures (Iten and Bryant, 1975; Stocum, 1975). It is possible that interaction with the proximal stump cells could elicit proximal transformation of graft cells to form either all or part of the intercalary regenerate.

Proximal transformation is known to occur in several other developmental systems which have the capacity to intercalate missing parts. Using morphological (Bohn, 1976) and color (Bohn, 1971; French, 1976) markers it has been shown that proximal transformation is the rule during intercalary regeneration of cockroach legs. Avian limb buds are also capable of intercalary regulation when presumptive autopodial tissue is grafted to the presumptive stylopodium at stage 22 or earlier. Kieny and Patou (1976, 1977) have shown, using het- erospecific quail/chick recombinants, that both proximal and distal transformation occur during intercalary regulation. However, in larval Ambystoma maculatum limbs, Stocum (1975) concluded that the entire inter&u-y regenerate produced after proximal grafting of distal blastemas comes from the stump, and thus obeys the rule of distal transformation. This conclusion was based on histological observations and pre- liminary autoradiographic evidence. After grafting [3H]thymidine-labeled distal blastemas, Stocum observed a large number of heavily labeled cells in the wrist and hand, with a few lightly labeled cells scattered in the intercalary regenerate. The suggestive- ness of the histological data led Stocum to attribute the presence of labeled cells in the intercalary regenerate to a transfer of label that is lost from donor cells which die during the grafting process. Bohn (1976) has criticized this interpretation, and argues that one would expect to observe a re-

stricted number of lightly labeled cells in the intercalary regenerate as a result of dilution during mitosis if proximal transformation of graft cells had occurred. He has stressed the importance of resolving this issue in view of the many similarities between the regeneration of vertebrate and insect appendages.

There are instances where the rule of distal transformation appears to be violated under certain experimental conditions in regenerating urodele limbs. Carlson (1974) has reported several cases of A. mexicanum regenerates which duplicated the zeugopodial bones in series after rotating the arm skin 180”. DeBoth (1970) has also reported proximal transformation of blastema cells in A. mexicanum limbs. He massed three epidermis-free blastemal mesenchymes derived from the wrist in the enucleated orbit of the eye and capped them with an intact wrist blastema. Two of 28 cases which did not resorb were reported to have a single forearm element in addition to carpals and digits. He also reported 7 cases of 12 in which hand plate mesenchymes of palette- stage upper arm regenerates combined in the flank and capped with an intact hand plate regenerated a zeugopodial element in addition to carpals and digits. Since single prospective hand blastemas transplanted in the same fashion never formed anything but carpals and digits, DeBoth concluded that the occurrence and extent of proximal transformation is determined by the mass of mesenchyme present. Stocum (unpub- lished results) has repeated DeBoth’s experiment, but observed only autopodial elements in the regenerate.

In this paper, we report the results of experiments designed to determine more accurately whether or not the cells of salamander limb regeneration blastemas can undergo proximal transformationduring intercalary regeneration. Morphological, color, and cellular markers were used to trace the origin of the regenerated skeletal elements. The results indicate that, by all these criteria, a distal blastema grafted to

PESCITELLI AND STOCUM Origin of Zntercalary Regenerates 257

a more proximal limb level does not partic- ipate in the formation of the intercalary regenerate. Rather, the intercalated structures are derived from the host stump.

MATERIALS AND METHODS

Experiments were done on Ambystoma mexicanum and Ambystoma maculatum larvae. White and dark A. mexicanum larvae were obtained from laboratory spawn- ings or from the Indiana University axolotl colony. A. maculatum larvae were collected in the vicinity of Princeton, New Jersey (courtesy of Dr. Thomas Poole, Princeton University). All animals were raised in sep- arate containers in 1% Holtfreter solution and maintained on a diet of freshly hatched brine shrimp.

The axolotls used for the autografting and homografting experiments were 4-5 months old at the time of surgery, and were 60-70 mm in snout-tail length. In the heterografting experiments, the axolotls and A. maculatum larvae were 2 months old and 30-40 mm in snout-tail length at the time of transplantation.

The following groups of distal to proximal transplants were performed.

(1) Diploid Autografts and Homografts

Using dark and white axolotls, late bud- notch stage wrist blastemas were autografted or homografted with normal axial orientation to the ipsilateral thigh stumps (staging according to Stocum, 1979). Like- wise, late bud-notch stage tarsus blastemas were autografted or homografted in the same manner to the ipsilateral upper arm stumps. Thus, in these combinations the limbs were a composite of forelimb and hindlimb mesodermal tissue, and the homografted cases were, in addition, composites of white and dark epidermis. The derivation of the regenerated skeletal elements was determined from skeletal morphology and pattern, and from color pattern. The oper- ations are diagramed in Fig. 1A. Controls consisted of autografted or homografted

upper arm blastemas exchanged with ipsilateral thigh blastemas.

(2) Diploid Heterografts

Using A. maculatum and A. mexicanum larvae, wrist or tarsus blastemas of one species were heterografted to the ipsilateral thigh stumps of the other species (Fig. 1B). The grafts were made between larvae of approximately the same size. However, A. mexicanum limbs subsequently grow much more rapidly and to a larger size than those of A. maculatum. The origin of the regenerated skeletal elements was determined on the basis of size differences in the skeletal elements of the two species. Controls consisted of autografts of contralateral wrist or tarsus blastemas to the thigh of that side in both host and donor species.

(3) Triploid-diploid Homografis

Triploid A. mexicanum larvae were produced by heat treatment of freshly spawned eggs using the method of Fankhauser and Godwin (1948). Eggs were collected at 30- min intervals and transferred immediately to Holtfreter solution at 36.0 f 0.5”C for 10 min. The eggs were then allowed to develop at room temperature. Triploid larvae were

-

FIG. 1. (A) Method of autografting or homografting A. mexicanurn wrist or tarsus blastemas to the ipsilateral thigh or upper arm. (B) Method of heterografting A. mexicanum wrist blastemas to A. maculatum thighs, and A. maculatum tarsus blastemas to A. mexicanum thighs.

258 DEVELOPMENTAL BIOLOGY VOLUME 79.1980

identified by counting nucleoli under phase contrast in live cell preparations of tail-tip epidermis which had been removed and teased apart with watchmakers forceps. Approximately 70% of the animals surviving the heat treatment were triploid. Late bud wrist blastemas of triploid animals were homografted to the ipsilateral upper arm stumps of diploid animals, or late bud wrist blastemas of diploid animals were homografted to the ipsilateral upper arm stumps of triploid animals. Controls consisted of regenerates formed by the ampu- tated contralateral upper arms of the donor and host animals used in each combination. The origin of the regenerated skeletal elements was analyzed by counting, in longitudinal sections, the number of one nucleolar, two nucleolar, and three nucleolar cells in each proximal-distal segment of the experimental limbs and comparing the frequency distribution of these numbers with that obtained from the controls.

Histological Preparations

In experiments 1 and 2, limbs were allowed to regenerate for 30-79 days. They

were then removed, fixed in Gregg’s solution, and stained in toto with methylene blue for cartilage.

In the diploid-triploid combinations of experiment 3, the limbs were allowed to regenerate until they reached the three- to four-fingerbud stage. They were then removed, fixed in Bouin’s solution, and sec- tioned longitudinally at 10 pm. Deparaffln- ized and hydrated sections were treated for 10 min with 0.001% crystalline DNase (Sigma), in a 0.1 M Tris buffer, pH 7.3, containing 0.2 M MgCl* and 0.2 M CaClz at 37°C (Lillie, 1965). The DNase digests the heterochromatin blobs which interfere with the identification of nucleoli (Namenwirth, 1974). The sections were then stained with Heidenhain’s iron hematoxylin and light green. This procedure renders the nucleoli black, and they stand out clearly against the green background.

RESULTS

DESCRIPTION OF THE NORMAL FORELIMB AND HINDLIMB OF A. mexicanum

Figures 2 and 3 compare the normal morphology and skeletal pattern of the regen-

FIG. 2. Skeleton of normal forelimb regenerate of A. mexicanum after amputation through the mid-upper arm, dorsal view. The stylopodium consists of the humerus (H) and the zeugopodium consists of the radius (R) and the ulna (U). The basipodium consists of eight carpals arranged in three rows. The proximal row consists of the radiale (r), intermedium and ulnare (u); the middle row consists of cent&e 1 (cl), centrale 2, and carpal d, (d.,). The distal row consists of carpal dl-2 and carpal da. The metapodium has four metacarpals (mc), and the acropodium has a phalangeal formula of 2:2:3:2. Digits are numbered from anterior to posterior. Note the olecranon process of the ulna (arrow) and the bend at the elbow joint. Nomenclature according to Romer (1949). x14.

FIG. 3. Skeleton of normal hindlimb regenerate of A. mexicanum after amputation through the midthigh, dorsal view. The stylopodium consists of the femur (F) and the zeugopodium consists of the tibia (T) and the fibula (Fb). Note the flared proximal end of the tibia (compare with the radius) and the lack of an olecranon process on the fibula (compare with the ulna). The shapes of the distal ends of the tibia and fibula are also different from those of the radius and ulna. The basipodial skeleton is best viewed as three columns of elements, the posterior column being curved. The anterior column consists of cent&e 1 (cl) and the tibiale (t); the middle column consists of tarsal l-2, centrale 2, and the intermedium. The posterior column consists of tarsal 3, tarsal 4, tarsal 5 (ts), and the fibulare (f). This specimen has 10 tar&s instead of the usual 9, due to splitting of cent&e 1. The metapodium consists of five metatarsals (mt), and the acropodium has a phalangeal formula of 2:2:3:3 or 4:2. Digits numbered from anterior to posterior. ~14.

FIG. 4. Control regenerate produced after homografting the left mid-upper arm blastema of a white axolotl to the left midthigh of a dark axolotl, dorsal view. The graft developed as a donor forelimb both in morphology and color. The arrow indicates the graft-host junction. For abbreviations, see Figs. 1 and 2. x14.

FIG. 5. Control regenerate produced after homografting the left midthigh blastema of a white axolotl to the left mid-upper arm of a dark axolotl, dorsal view. The graft developed as a donor hindlimb, both in morphology and color. The arrow indicates the graft-host junction. For abbreviations, see Figs. 1 and 2. x 14.

PESCITELLIANDSTOCUM Origin of Zntercalary Regenerates

2

ts ,,,B

0 4

3 2

5 .

259


erated A. mexicanum forelimb and hindlimb.

The organization of the forelimb and hindlimb skeleton is very similar, with a remarkable parallelism of structure. Each possesses a proximal component (stylopodium) of a single element (humerus in the forelimb, femur in the hindlimb); an inter- mediate component (zeugopodium) of two elements (an anterior radius and posterior ulna, in the forelimb; an anterior tibia, posterior fibula in the hindlimb); and a distal component (autopodium) forming the hand in the forelimb and the foot in the hindlimb. The autopodium consists of three divisions. The basipodium (carpus or wrist in the forelimb, tarsus or ankle in the hindlimb) articulates with the zeugopodium. The basipodium is followed by the metapodium (metacarpals in the forelimb, metatarsals in the hindlimb). The terminal division of the autopod is the acropodium, consisting of several rows of phalanges in both hand and foot.

There are, however, certain differences in the morphology and pattern of the skeletal elements of forelimb and hindlimb within this basic structure that can be used to distinguish between the two. These differences will be described for each segment. The axial definitions used are those of Romer (1949), where dorsal and ventral correspond to the position of the extensor and flexor muscles of the limb, respectively.

(I) Stylopodium. The humerus of the forelimb has a broad distal end flattened in the anterior-posterior plane of the limb and possessing two prominent condyles. The distal end of the femur is also flattened in the anterior-posterior plane of the limb, but is broader than that of the humerus, and lacks prominent condyles.

(2) Zeugopodium. The proximal end of the radius is round or flat and articulates with the anterior condyle of the humerus, while its distal end is broadly triangular where it articulates with the radiale and intermedium of the carpus. The proximal end of the ulna lies posterior to the proxi-

mal end of the radius, and has a long, beveled olecranon process which articulates with the notch between the two humeral condyles. The olecranon process extends over the dorsal surface of the humerus. The distal end of the ulna is rounded or angled where it articulates with the ulnare and intermedium of the carpus.

The proximal end of the tibia is flattened and flared in the anterior-posterior plane of the limb, and articulates with the anterior two-thirds of the distal surface of the femur. Its distal end is much more slender than its proximal end and is flat or rounded where it articulates with the intermedium and tibiale of the tarsus. The proximal end of the fibula has a short bevel which faces directly toward the posterior edge of the tibia. Unlike the olecranon process of the ulna, however, the beveled surface does not project over the dorsal surface of the femur. The distal end of the fibula is broader than its proximal end, and is stouter than the distal end of the ulna.

Because of the morphological differences in the adjoining ends of their zeugopodial and stylopodial bones, the elbow joint of the forelimb and the knee joint of the hindlimb are anatomically distinct. In addition, flexion at the elbow joint is more pro- nounced than at the knee joint, giving the forelimb a forward bend which is not observed in the hindlimb.

(3) Autopodium. There are usually eight, but sometimes nine carpal elements in the basipodium of the forelimb (centrale 1 sometimes splits into two elements). The anterior two carpals (radiale and centrale 2) are distinctly different in shape from the posterior two carpals (ulnare and &). The hand possesses four digits (fingers) numbered from anterior (radial side) to posterior (ulnar side). Each finger has a single metacarpal followed by two or three phalanges. The phalangeal formula of the fingers from anterior to posterior is 2:2:3:2. The first metacarpal articulates with centrale 2 and carpal dl-2 of the basipodium; the second metacarpal with carpal dl-2; the

PESCITELLIANDSTOCUM Origin of Intercalaty Regenerates 261

third metacarpal with carpal d3; and the fourth metacarpal with carpal ch.

There are usually 9, but sometimes 10 tarsal elements in the basipodium of the hindlimb (again, cent&e 1 sometimes splits into two elements). Eight of the tarsals (tibiale, cent&e 1, intermedium, cent&e 2, tarsaL, tars&, and tarsah) are virtually indistinguishable in their morphology and pattern from the corresponding carpals in the wrist. The features that distinguish the tarsus from the carpus are that the fibulare of the tarsus is larger than the corresponding ulnare of the carpus and the element corresponding to carpal & of the carpus is split into two elements in the tarsus, tarsah and tan&,. TarsaL, lies at the posterior edge of the tarsus between tarsah and the fibulare and is much smaller than the latter two elements.

The foot possesses five digits (toes) numbered from the tibial (anterior) to fibular (posterior) side. Each toe consists of a single metatarsal followed by two to four phalanges. The phalangeal formula of the toes from anterior to posterior is 2:2:3:3 or 412. The fust metatarsal articulates with centrale 2 and tars&~; the second metatarsal with tarsal-~; the third metatarsal with tar- sala; the fourth metatarsal with tar&; and the fifth metatarsal with tar&. TarsaL sometimes does not form and both metatarsals 4 and 5 then articulate with tarsah. From this description, it can be seen that the features which distinguish the toes of the hindlimb from the fingers of the forelimb are the different numbers of digits and that the fourth toe has one or two more phalanges than the fourth finger.

The descriptions given above also apply to the skeletal elements of larval A. macu- l&urn limbs, However, the skeletal elements of A. mexicanum limbs are consid- erably larger than those of A. maculatum.

DISTAL TO PROXIMAL TRANSPLANTS

(1) Diploid Autografts and Homografts (A. mexicanum)

(a) Controls. The controls consisted of

11 cases (7 autograft, 4 homograft) of upper arm blastemas grafted to thigh stumps and 13 cases (9 autograft, 4 homograft) of thigh blastemas grafted to upper arm stumps. One hundred percent of the upper arm blastema-thigh stump combinations regenerated according to origin, forming limbs consisting of a femur-humerus, radius-ulna, and four-fingered hand (Fig. 4). Ninety-two percent of the reciprocal combinations also regenerated according to origin, forming limbs consisting of a humerus-femur, tibia- fibula, and five-toed foot (Fig. 5). The one case of this group which did not regenerate according to origin formed a skeleton that was completely hindlimb in morphology and pattern, suggesting that the graft resorbed, followed by host stump regeneration.

The pigmentation of the homografted cases was in accord with the morphology of the limb segments. Host-color epidermis was present over the stylopodium of the host stump, while the regenerated portion of the stylopodium and more distal segments were covered by epidermis of donor color (Figs. 4 and 5). These results demonstrate that neither the surgical procedure itself, nor the morphological character of the host stump, influence the development of a grafted blastema.

(b) Wrist blastemas to thigh stumps. This series consisted of 33 cases (25 autografts, 8 homografts). The results are summarized in Table 1. Two of the autografts and four of the homografts regenerated too poorly for analysis. Eighteen of the autografts and 2 of the homografts formed limbs possessing a four-digit hand with phalangeal formula 212~3~2 and 8 basipodial elements arranged in the normal wrist pattern (Figs. 6-8). The morphology and pattern of the zeugopodial and stylopodial elements of these cases were clearly hindlimb (tibia- fib& and femur). This composite forelimb-hindlimb morphology indicates that the grafts developed according to their wrist origin, while zeugopodial and stylopodial structures were intercalated from


TABLE 1

TYPE AND FREQUENCY OF REGENERATION AFTER RECIPROCAL AUTOGRAFTING OR HOMOGRAFTING OF WRIST AND TARSUS BLASTEMAS TO THIGH AND UPPER ARM STUMPS~

Transplant Poor development Intercalary regenera- Host-type regenera- Total tion tion

WBtoT Autograft 2 18 5 25 Homograft 4 2 2 8

Total 6 (18%) 20 (61%) 7 (21%) 33

TrB to UA Autograft Homograft

5 8 40 53 0 6 21 2,

Total 5 (6%) 14 (18%) 61 (76%) 80

(1 UA, upper arm stump; T, thigh stump; WB, wrist blastema; TrB, tarsus blastema.

cells derived from the host stump. The pigmentation pattern of the two homograft cases with composite forelimb and hindlimb morphology supports this interpretation. Figure 7 shows a case in which a wrist blastema from a white animal was homografted to the thigh of a dark animal. The epidermis of the regenerated limb is of host color up to the base of the digits, and of donor color more distally.

The fact that host-colored epidermis in this case extends further distally than would be expecaed if the graft had formed everything it normally would have in situ suggests that much or all of the prospective carpal material of the graft may have resorbed and been replaced by host cells.

Partial resorption of graft prospective digit material may also take place, as indicated by the fact that digit 4 of the hand in Fig. 7 contains considerable pigment. Thus, after a distal to proximal transplantation, an autopodium with hand and wrist morphology cot&l ‘be produced by the cooperation ‘of donor forelimb and host hindlimb &Us. Specific digital combinations might be digits 1, 2, and 3, digits 2, 3, and 4, or digits 2 and 3 surviving from the graft with digits 1 and/or 4 regenerating from the host. Figure 8 shows another homografted case (dark wrist blastema to white thigh) in which such cooperative development occurred. Digits 2 and 3 exhibit donor pig-

FIG. 6. Regenerate produced after autografting the right wrist blastema of a white axolotl to the ipsilateral midthigh, dorsal view. The autopodium of the regenerate is clearly graft derived (carpals and fingers), while the stylopodium and zeugopodium are clearly host derived (femur, tibia, and fibula). For abbreviations, see Figs. 1 and 2. ~14.

FIG. 7. Regenerate formed after homografting the right wrist blastema from a white axolotl to the right midthigh of a dark axolotl, dorsal view. Digits l-3 are graft derived, whereas the remainder of the regenerate is host derived. Arrow indicates graft-host junction. For abbreviations, see Figs. 1 and 2. X14.

FIG. 8. Regenerate formed after homografting the left wrist blastema from a dark axolotl to the left midthigh of a white axolotl, dorsal view. Digits 2 and 3 are derived from the graft, while the remainder of the regenerate is derived from the host. For abbreviations, see Figs. 1 and 2. ~14.

FIG. 9. Regenerate produced after homografting the right wrist blastema from a dark axolotl to the right midthigh of a white axolotl, dorsal view. The skeletal morphology and pattern is entirely host (hindlimb), as is the pigmentation (white). The third digit articulates with tare& 3 and 4, suggesting the fusion of digits 3 and 4. For abbreviations, see Figs. 1 and 2. ~14.

PESCITELLIANDSTOCUM Origin of Intercalary Regenerates

0 6

263


mentation, while digits 1 and 4 are of host color.

The remaining five autografts and two homografts regenerated limbs that exhibited hindlimb skeletal morphology and pattern in every segment, suggesting a partial or total resorption of the graft, followed by regeneration from the host thigh. Figure 9 illustrates one of the two homograft cases in this category (dark wrist blastema to white thigh). The zeugopodial bones are clearly tibia and fibula, and tarsal 5 is present on the postaxial side of the basipodium. The acropodium superficially appears to consist of only four digits, but the third digit articulates with both tarsal 3 and 4, indicating that the pattern for toes 3 and 4 was present, but fused to produce a single toe between toes 2 and 5. The second homograft case (white wrist blastema to dark thigh) was one in which a foot pattern was formed by cooperation between a partially resorbed graft and regeneration from the host thigh (Fig. 10). The phalanges of digits 1 and 2 were of graft origin (donor color). Digits 3 and 4 of the graft resorbed, and were replaced by digits 3,4, and 5 regenerating from the host (host color).

(c) Tarsus blastemas to upper arm stumps. This series consisted of 80 cases (53

autografts, 27 homografts). The results are summaried in Table 1. Five cases, all autografts, regenerated too poorly for analysis. Eight of the autografts and 6 of the homografts regenerated stylopodial and zeugopodial elements with host (forelimb) morphology and pattern, and autopodial elements of donor (hindlimb) morphology (Figs. 11,12). In 3 of these homograft cases, the epidermis over the stylopodium and zeugopodium was entirely of host color, and that over the autopodium of donor color. Thus, by both morphological and color criteria, the origin of the stylopodium and zeugopodium of these cases was by intercalary regeneration from the host stump. In the other 3 homograft cases, however, donor epidermis extended somewhat into the distal half of the zeugopodium (Fig. 12). These cases might represent the proximal transformation of graft cells to form the distal ends of the zeugopodial bones. How- ever, the zeugopodial elements definitely have host (radius-ulna) morphology and another interpretation of the color pattern is that the gap between stump and grafted blastema might be healed by the migration of both host and donor epidermis toward one another. Such a healing pattern would result in intercalated structures of host

FIG. 10. Regenerate produced after homografting the right wrist hlastema from a white axolotl to the right midthigh of a dark axolotl, anterodorsal view. The white color of digits 1 and 2 indicates their origin from the graft, but the remainder of the regenerate is host derived, by both morphology and pigmentation. The digits contributed by the host are toes 3-5. A foot acropodium has thus been created by the integrated development of hand and foot material. For abbreviations, see Figs. 1 and 2. X13.58.

Fro 11. Regenerate formed after autografting the right tarsus blastema of a white axolotl to the ipsilateral mid-upper arm, dorsal view. The autopodium is derived from the graft, and the stylopodium and zeugopodium are derived from the host upper arm. For abbreviations, see Figs. 1 and 2. ~13.58.

FIG. 12. Regenerate formed after homografting the left tarsus blastema of a dark axolotl to the left mid- upper arm of a white axolotl, dorsal view. The autopodium is derived from the graft according to morphology and color. The remainder of the regenerate is derived from the host upper arm according to the skeletal morphology, but donor pigmentation extends proximally over half the length of the radius and ulna. For abbreviations, see Figs. 1 and 2. X13.58.

FIG. 13. Regenerate formed after homografting the right tarsus blastema of a white axolotl to the left mid- upper arm of a dark azolotl, dorsal view. The autopodium has host (forelimb) skeletal morphology, but its epidermis has mainly donor color. For abbreviations, see Figs. 1 and 2. x13.58.

FIG. 14. Regenerate formed after homografting the left tarsus blastema from a dark axolotl to the left mid- upper arm of a white axolotl, dorsal view. The skeletal morphology and pattern of the whole regenerate is of host character (forelimb), but the epidermis of the autopodium has donor color. For abbreviations, see Figs. 1 and 2. ~13.58.

PESCITELLIANDSTOCUM Origin of Intercalary Regenerates 265

13 2

0 11

9


morphology being covered proximally by host epidermis and distally by donor epidermis.

The remaining 40 autograft and 21 homograft cases regenerated what appeared to be a normal forelimb with five to nine carpals and four digits with the hand phalangeal formula, suggesting complete or partial resorption of the grafts followed by regeneration from the host upper arm. Five of these 21 homograft cases possessed auto- podia with epidermis of entirely host color, and these cases are undoubtedly ones in which the graft completely resorbed. The autopodial epidermis of the remaining 16 homograft cases, however, exhibited donor pigmentation to varying degrees. Figure 13 illustrates a case (white tarsus blastema to dark upper arm) in which the digits have host morphological pattern but mainly do- nor color. Figure 14 shows a case (dark tarsus blastema to white upper arm) in which the digits have host morphological pattern but are covered with epidermis of entirely donor color. It is possible that these two regenerates are cases in which only the fourth toe and tarsal 5 of the graft were resorbed, which would create hand morphology. However, the selective resorption of tarsal 5 without resorption of its associ- ated fifth toe seems unlikely. A more prob- able explanation is that in these cases, the majority or all of the graft mesenchyme completely resorbed, but its epidermis sur-

vived, to be filled by mesenchymal cells contributed by the host. It should be noted that in all of the 21 homograft cases dis- playing forelimb morphology, the epidermis overlying the stylopodium and zeugopodium was of host color.

(2) Diploid Heterografts (A. mexicanum, A. maculatum)

This series consisted of three cases of A. maculatum tarsus blastemas grafted to A. mexicanum thighs, and five cases of A. mexicanum wrist blastemas grafted to A. maculatum thighs. These cases represent the survivors of a total of 20 heterografts. The development of heterografted blastemas was in general poorer than that of homografts, and there was a higher inci- dence of immunorejection. However, the results clearly show that intercalary regeneration of the stylopodium and zeugopodium takes place from the host stump, while the major portion of the autopodium develops from the graft. Skeletal structures could be easily identified as originating from graft or host on the basis of their size. Figure 15 illustrates the regenerate produced after heterografting an A. macula- turn tarsus blastema to a white A. mexicanum thigh, and Fig. 16 shows the contralateral control regenerate of the A. mexicanum host. The autopodium is derived from the graft (cf. with Fig. 16). The tibia is slightly shorter than the control tibia, but

FIG. 15. Regenerate produced after heterografting the left tarsus blastema of an A. maculatum larva to the midthigh of a white A. mexicanurn larva, dorsal view. The autopodium is derived from the maculatum graft (cf. with Fig. 16). The stylopodium is derived from the mexicanurn host. The zeugopodial elements are somewhat smaller than expected. For abbreviations, see Figs. 1 and 2. x14.

FIG. 16. Contralateral control regenerate for the case of Fig. 15, dorsal view. The right tarsus blastema of the white A. mexicanum host was autografted to the right midthigh. Note the large size of the toes compared to those of the regenerates in Figs. 15 and 18. For abbreviations, see Figs. 1 and 2. x14.

FIG. 17. Regenerate produced after heterografting the left tarsus blastema of a white A. mexicanurn larva to the midthigh of an A. maculatum larva, dorsal view. The basipodium and toes 1-4 are composed of donor-size elements. Toe 5 and the zeugopodium and stylopodium are composed of host-size elements (cf. with Fig. 18). For abbreviations, see Figs. 1 and 2. ~14.

FIG. 18. Contralateral control regenerate for the case of Fig. 17, dorsal view. The right tarsus blastema of the A. maculatum host was autografted to the right midthigh. Note the similarity in size of the stylopodial and zeugopodial skeletal elements to those of the experimental limb. The basipodium and toes, however, are much smaller. For abbreviations, see Figs. 1 and 2. ~14.

PESCITELLIANDSTOCUM Origin of Intercalary Regenerates 267

4

0 16

5

1

268 DEVELOPMENTAL BIOL~CY VOLUME 79,198O

the fibula is substantially smaller in all dimensions than the control fibula. In addition, pigment extends into the distal half of the zeugopodium. These facts suggest a possible graft-host interaction which might shift the size of host and/or graft elements toward each other, or a mixing of graft-host cells in the distal zeugopodium. It is not possible to judge whether either of these phenomena have actually occurred without analysis of a cell marker. However, the majority of the cases were not ambiguous. Figure 17 illustrates the 5-digit regenerate produced after heterografting a white A. mexicanurn tarsus blastema to an A. mu- culutum thigh, and Fig. 18 shows the contralateral control thigh regenerate of the A. maculatum host. Digits l-4 and the basipodial elements are clearly of graft origin, whereas the fifth digit is of host origin. The zeugopodial and stylopodial bones of the regenerate are of host origin. The pigment patterns conform precisely to the size differences in this case.

(3) Triploid-Diploid Homografts (A. mexicanum)

This series consisted of 28 cases. Of these, 14 were grafts of triploid wrist blastemas to diploid upper arm stumps and the remaining 14 were diploid wrist blastemas to triploid upper arm stumps. One case failed to regenerate. In 4 cases the grafts failed to heal in place properly and were discarded, and 4 other cases were excessively digested with DNase and were not analyzable. The remaining 19 cases were analyzed for nucleolar number. The digestion and staining procedure used was optimal for determining the nucleolar number in cartilage and procartilage condensations. The nucleoli in the other tissues of the regenerate were for the most part obscured by heterochromatin clumps which were only partially digested by the DNase. Hence, in most cases only the skeletal cartilage was examined for nucleolar number. In cases in which the nucleoli of the noncartilage tissues could be identified unambiguously, however, the nu-

cleolar number was identical to that of the adjacent cartilage.

Nucleolar counts were made in the control and experimental limbs of 7 of the 19 analyzable cases (4 cases were triploid grafts to diploid stumps; 3 cases were diploid grafts to triploid stumps). On alternate longitudinal sections, selected areas of the regenerated cartilage elements in each proximal-distal segment were counted with the aid of a grid divided into 100 squares mounted in the eyepiece of a Zeiss photo- microscope. All counts were made at 1000 X. All of the nuclei within the grid possessing one or more nucleoli were counted and the location of the grid within the limb was recorded. The whole proximal-distal extent of each limb segment was sampled.

The results are summarized in Fig. 19. In diploid control limbs about 50% of the cartilage nuclei contained 1 nucleolus and 50% contained 2 nucleoli. This distribution was constant throughout the five skeletal segments of the limb (Fig. 19A). In A. mexi- cunum there is one nucleolus per haploid set of chromosomes so that in diploid tissues a maximum of 2 nucleoli is expected per nucleus (Fankhauser and Humphrey, 1943). In all four diploid controls, however, a few nuclei were observed with 3 nucleoli. Whether these cells represent true triploid cells or are only mistaken as such due to the fortuitous staining of an undigested clump of heterochromatin is unclear. How- ever, their low frequency (less than 0.3% of 8181 nuclei counted in the diploid controls) does not interfere with the use of nucleolar number as a cellular label. In the triploid control limbs, 22% of the nuclei had 3 nucleoli, 44% had 2 nucleoli, and 34% had one nucleolus. This frequency was constant throughout the five skeletal segments (Fig. 19B). One of the three triploid controls had 6 nuclei, concentrated in the autopodium, each with 4 nucleoli, this was the only observed occurrence of nuclei with more than 3 nucleoli, and is possibly due to a sponta- neous polyploidization.

Figure 19C shows the results of grafting

PESCITELLI AND STOCUM Origin of Intercalary Regenerates 269

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100 q WITH , NUCLEOLUS 100

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FIG. 19. Mean percentage of nuclei in each of five skeletal regions (four proximal to distal segments) with 1, 2, or 3 nucleoli. The bars above each histogram represent the standard deviation. (A) Diploid controls. A total of 8181 nuclei were counted in four specimens. (B) Triploid controls. A total of 5079 nuclei were counted in three specimens. (C) Triploid wrist blastemas homografted to the ipsilateral mid-upper arms of diploid animals. A total of 7929 nuclei were counted in four specimens. (D) Diploid wrist blastemas homografted to the ipsilateral mid-upper arms of triploid antimals. A total of 5024 nuclei were counted in three specimens. C, carpals; D, digits.

triploid blastemas to diploid stumps. The nucleolar frequency in the stylopodial and zeugopodial elements is identical to that of the contralateral (diploid) controls, indicating their origin from the host stump. The nuclei in these regions have either one or two nucleoli in a 1:l ratio. In two cases, no nuclei with three or more nucleoli were observed within the stylopodium or zeugopodium. In one case six nuclei (0.36%) were counted with three nucleoli; however, this is less than the frequency of three nucleolar cells in the contralateral control (0.53%) and it is therefore unlikely that they represent a contribution from the triploid graft. The fourth case had one nucleus (0.08%) with three nucleoli located in the zeugopodium and none in the stylopodium. Again, this percentage is below the percentage of three-nucleolar cells present in the contralateral control (0.23%). Seven- teen percent of the nuclei within the carpus possessed three nucleoli. This is less than

the 22% present in triploid controls, but is significantly greater than the 0.1% occurring in the stylopodium and zeugopodium. The percentage of three-nucleolar cells in the proximal carpals is generally one-half that present in the distal carpals, suggesting some donor-host cell mixing at the blastema-stump junction. In one case the number of nuclei with three nucleoli was very low throughout the entire carpus, suggesting that prospective carpal material from the graft resorbed and was replaced by diploid cells from the stump. The frequency of nucleolar number in the digits was, overall, identical to that of control triploid tissue. However, in one case the percentage of three nucleolar cells in digit 1 was very low, suggesting resorption of the prospective graft material of that digit and replacement with host cells.

The results of grafting diploid blastemas to triploid stumps are summarized in Fig. 19D. The nucleolar frequency in the hu-

270 DEVELOPMENTAL BIOLOGY VOLUME X3,1980

merus, radius, and ulna is the same as that of the contralateral triploid controls, indicating that these parts are intercalated from the host stump. The autopodium was composed mainly of diploid graft cells except for some donor-host cell mixing in the proximal carpus where many nuclei with three nucleoli were observed. In one of the three cases, triploid cells were scattered throughout the carpus. In this case 18.4% of the nuclei in digit 1 had three nucleoli, while no three-nucleolar cells were observed in digits 2 and 3, indicating that the graft material destined to form the carpus and digit 1 resorbed to be replaced by triploid cells from the stump.

Figure 20 is a low magnification photo- graph of a longitudinal section through a diploid limb to which a triploid wrist blastema was transplanted. The region between the arrows, including the distal half of the stylopodium, the entire zeugopodium, and the proximal row of carpals, represents the presumptive intercalary regenerate. Fig- ures 21, 22, and 23 are high-magnification photographs of the distal humerus, radius, and ulna, showing the absence of nuclei with three nucleoli in these regions. Figures 24 and 25 are high-magnification photographs of the distal carpus and digit 2, showing the presence of many nuclei with three nucleoli.

The remaining 12 experimental cases were examined without making extensive cell counts in order to qualitatively determine the derivation of the regenerated structures. In all 12 cases the entire stylopodium and zeugopodium consisted of cells of host ploidy. There was no indication that any of the intercalary regenerate was de-

rived from graft cells. In 3 of these cases the entire autopodium except for the proximal carpus was of graft type ploidy. One case was predominantly of stump ploidy throughout the entire carpus, but the digits were of donor type (triploid) indicating resorption of the prospective graft carpals. In 3 cases host cells were observed throughout the carpus and digit 1, indicating that these structures resorbed in the graft to be replaced by stump cells, while the remaining graft cells survived. Finally, in 5 cases the entire limb, including the autopodium, was of host-type nucleolar number, indicating that the grafted regenerate totally resorbed to be replaced by cells from the host stump. These cases were especially interesting, since frequent inspection did not indicate any obvious reduction in volume of the grafts after transplantation. These results confirm, on a cellular level, the results obtained by the use of morphological and pigmentation markers.

DISCUSSION

The limbs of both adult Notophthalmus viridescens (Iten and Bryant, 1975) and Ambystoma maculatum (Stocum, 1975) are able to regulate to replace intercalary deletions created by grafting a distal blastema to a more proximal limb level. On the basis of histological evidence, Stocum (1975) concluded that the host stump is the sole source of the intercalary regenerate in A. maculatum. This evidence, however, is not conclusive, especially in view of Bohn’s (1976) demonstration, using morphological markers, that the cells of cockroach leg segments do contribute to intercalated structures when they are grafted to more

FIG. 20. Longitudinal section through the regenerate produced after homografting a triploid wrist blastema to a diploid mid-upper arm. The region between the arrows is the presumptive intercalary regenerate. x32.

FIG. 21. Higher magnification of a portion of the distal humerus of the specimen shown in Fig. 20. None of the chondrocyte nuclei have three nucleoli. x800.

FIG. 22. Higher magnification of a portion of the distal radius of the specimen shown in Fig. 20. None of the chondrocyte nuclei have three nucleoli. x512.

FIG. 23. Higher magnification of a portion of the distal ulna of the specimen shown in Fig. 20. None of the chondrocyte nuclei have three nucleoli. x640.

PESCITELLIANDSTOCUM Origin of Znterccdary Regenerates 271

272 DEVELOPMENTAL BIOLOGY VOLUME 79.1980

FIG. 24. Higher magnification of a portion of the distal carpus of the specimen shown in Fig. 20. Triploid nuclei (arrows) are now visible. X640.

FIG. 25. Higher magnification of a portion of digit 2 of the specimen shown in Fig. 20. Triploid nuclei (arrows) are visible. X512.

proximal or distal levels. In addition, De- Both (1970) and Carlson (1974) have reported the occurrence of proximal transformation in axolotl blastemas under other experimental conditions.

In the present study, we have endeavored to more accurately test the capacity of distal regeneration blastemas of A. mexicanum and A. maculatum limbs to undergo proximal transformation during the intercalary regeneration exhibited after grafting them to a more proximal limb level. The origin of the regenerated skeletal elements was determined by making recombinant limbs in which graft and host-derived tissues were distinguished by hindlimb-forelimb morphological differences, skin color differences, species-specific size differences, and differences in cell ploidy. By the collec-

tive use of these criteria, we have been able to demonstrate that when a blastema derived by amputation through the proximal basipodium is grafted to the midstylopodium, the intercalated stylopodial and zeugopodial skeletal elements originate from

I blastema cells supplied by the stump, while the autopodial elements originate from graft cells. The only uncertainty in the results stemmed from several cases in which dark or white axolotl wrist blastemas were homografted to the thighs of animals of the opposite color. In these cases, graft-type pigment extended over the distal portion of the zeugopodium. Looking at color pattern alone, this result might be interpreted as graft participation in the formation of the zeugopodium. Internally, however, the zeugopodial elements in these cases possessed

PESCITELLIANDSTOCIJM Origin of Intercalary Regenerates 273

host-type morphology. It is therefore more likely that, during the process of wound- healing, graft epidermis migrated proximally to fuse with distally migrating host epidermis to result in the covering of distal host-derived mesodermal structures with epidermis of graft origin. We therefore con- clude that proximal transformation of blastema cells does not occur during the intercalary regeneration of amphibian limbs, and that they possess intrinsic properties rendering them capable of changing positional value in a distal direction only, as postulated by Wolpert (1971). Similar results and conclusions have been obtained by Maden (1980), who used color markers and X-irradiated and unirradiated stumps and regenerates to determine tissue origins during intercalary regeneration. We disa- gree with Bohn (1976) that amphibian and insect appendages will eventually be found to possess similar pattern regulative capacities at the level of the individual cell. In- stead, we suggest that natural selection has endowed the limb cells of these organisms with different capacities for change in positional value. We have also obtained evidence that positional information is not organized the same way in insect and amphibian appendages (Pescitelli and Stocum, in preparation).

Our results with reciprocal forelimb- hindlimb homografts of different color also show that in a substantial number of cases, prospective basipodial and sometimes digit material of the graft resorbed to varying degrees, and was replaced by host-derived cells. This phenomenon was also clearly shown in diploid-triploid combinations, where cells of host ploidy were found in the autopodium at a higher than expected frequency. The degree of resorption ranged from total, in which case the regenerate was entirely of host character, to resorption of basipodial material plus anterior and/or posterior digits. In the latter case, the au- topodia of the regenerates possessed the overall morphology and skeletal pattern of either the donor or host limb, but were

actually composites of host and donor regions. It is thus clear that blastema cells derived from forelimb and hindlimb can develop in an integrated fashion to produce an autopodium of either host or donor character, depending on the combination of surviving graft and regenerating host regions. It is interesting that host-type regeneration occurred with much higher frequency in tarsus to upper arm transplants (76%) than in wrist to thigh transplants (21%). We have been able to discover no obvious reasons for this difference.

The present study reveals several advantages and disadvantages to each of the marking methods employed to determine the origin of regenerated structures. The use of forelimb-hindlimb morphological differences in autografts is advantageous due to the ease of doing large numbers of transplants which survive without immunorejection. However, this marking system has two major limitations. First, it cannot by itself indicate the exact cellular consti- tution of the intercalary regenerate. Thus, it cannot rule out the possibility that a substantial number of graft cells mix with host cells in the intercalary regenerate, but that the pattern of the intercalated structures is controlled by the host field. Second, partial graft resorption and the cooperation of host cells and surviving graft cells can give rise on the tissue level to mistakes in determining the origin of individual parts of the autopodium. These limitations are substantially overcome by the use of species-specific size differences in heterografts between A. mexicanum and A. maculatum, but the frequency of good development of the graft is somewhat reduced by an en- hanced frequency of immunorejection. Fur- thermore, A. madatum larvae are availa- ble only during the spring months.

Recombinant homograft limbs of dark and white coloration have been created in axolotls by several investigators for the pur- pose of tracing the origin of regenerate tissues (Wallace et al., 1974; Maden and Wal- lace, 1975; Tank, 1978; Wallace and Watson,

274 DEVELOPMENTAL BIOLOGY VOLUME 79, 1980

1979; Maden, 1980). These color differences are very useful, but are not entirely reliable by themselves becatise they are confined to the skin and tunics of the blood vessels. Thus, as discussed above, the epidermal wound-healing patterns at the graft-host junction may sometimes produce pigmentation patterns which are at odds with the morphological patterns in the same limb. Furthermore, the mesenchyme of a graft may partially or totally resorb to be replaced by host mesenchyme, while its epidermis survives, which also can lead to misinterpretation of origins if pigmentation alone is relied upon (see also Stocum, 1980).

The most precise way in which to trace the origins of regenerate structures is to make recombinant diploid-triploid limbs. Ploidy has long been in use as a cellular marker and has been especially utilized in limb regeneration studies to trace the redif- ferentiative fate of blastema cells derived from the various limb tissues (Hay, 1952; Steen, 1968,197O; Namenwirth, 1974; Dunis and Namenwirth, 1977). By analysis of the frequency distribution of nucleolar number, the cellular composition and degree of cell mixing in host and donor tissues can be determined. Our results with this marker demonstrate that intercalated skeletal segments are derived solely from the host, with cell mixing occurring only at the graft-host junction. The major disadvantages of this method are that triploid animals are dfi- cult to make in quantities sufficient for routine experimental work, and the counting of nucleoli is extremely time consuming, besides requiring the preparation of sec- tioned material. Because the nucleoli do not all lie in the plane of section and since nucleoli fuse in some cells (Dearing, 1934), it is necessary to count nucleoli in large samples of cells.

Since the results obtained by the use of the diploid-triploid markers are in good agreement with those based on size differences in heterografts or based on a combination of morphological and pigmentation markers in homografts of forelimb blaste-

mas to hindlimb stumps, we believe that these markers are expedient, yet reliable substitutes for the cellular marker in determining the derivation of structures in regenerating amphibian limbs. Since chronic immunorejection also occurs in both heterografts and homografts, and is confined to graft-derived structures, it also offers an additional marker in the cases where it occurs.

Research supported by National Science Founda- tion Grant BMS 71-015 79 A01 and National Institutes of Health Grant HD 12659 to D.L.S.

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The origin of skeletal structures during intercalary regeneration of larval Ambystoma limbs1, 2

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