Independence of Myogenesis from Chondrogenesis:

Retinoic acid induces extensive changes in skeletal development without conrurrently affecting the morphogenesis of the musculature in the regenerating limbs of Ambystoma larva¹

Department of Visual Sciences, School of Optometry and The Medical Sciences
Program, School of Medicine,
Indiana University, Bloomington, Indiana 47405 USA

Adapted from an article published under the title, Effects of retinoic acid on the muscle patterns produced during forelimb regeneration in larval salamanders (Ambystoma), in Cytobios 66:41-61 (1991).²

key words: retinoic acid, retinoids, vitamin A, regeneration, limb regeneration, muscle, muscle pattern, musculature, myogenesis, salamander, axolotl, Ambystoma

Abstract

After systemic treatment with retinoic acid (RA), A. opacum and A. punctatum larvae regenerated forelimbs with a wide variety of skeletal and gross anatomical abnormalities. Yet the musculatures within the RA-treated limb regenerates were normal even in instances where the cartilages were deformed beyond recognition as components of the limb skeleton.
RA is known to induce reduplication of limb structures, sometimes entire segments thereof. When the latter condition occurred in the present study, the corresponding replicates exhibited limb musculatures that were perfect down to minute details, yet of opposite bilateral symmetry. The data:

attest to the independence of myogenesis and chondrogenesis during limb regeneration.
unmasked an otherwise hidden potential within post-embryonic salamander limb tissue for manufacturing a musculature of the opposite side of the body.
generate, as corollary of the latter, the thesis that cells of the musculature of the limb regenerate are descendents of developmentally uncommitted cells.

Introduction

In limb regeneration among salamanders, retinoic acid (RA) can

distort skeletogenesis;
transmogrify the new part's external form;
stimulate the development of supernumerary cartilages (and bones in adult animals); and
promote the reduplication of entire segments -- fingers, hands, wrists, forearms, arms and shoulder elements (or toes, feet, ankles, legs and thighs in the hind limb).

(See Maden et al 1985; Stocum and Thoms, 1984; Thoms and Stocum, 1984; Ragsdale et al 1989). This property of RA permits the testing of an hypothesis advanced some years ago to the effect that myogenesis and chondrogenesis are mutually independent during limb regeneration (Pietsch, 1962a). The evidence for the independence hypothesis came from experiments in which muscle differentiation was caused to vary while skeletogenesis was held constant. RA permited the experimental formulation of the logical converse: Given a changed skeletogenic outcome, would the RA-treated limb regenerate's muscles concomitantly change or proceed to a normal morphogenic conclusion? The independence hypothesis predicted the latter alternative.

While the opportunity presented itself, comparisons were made of the muscle patterns within reduplicated members with a view to their bilateral symmetry. The question motivating the latter was whether, among of the post-embryonic salamander tissues, cells exist with an ontogenic potentiality for manufacturing the opposite limb's musculature.

Materials and methods

The principal experimental subjects were A. opacum and A. punctatum (maculatum) larvae brought into the laboratory as gastrulae and monitored through the subsequent Harrison stages so as to insure (by appropriate culling) the developmental homogeneity of the animal stock. Ancillary experiments (described below) were conducted with 35-40 mm white (non-albino) A. mexicanum (axolotl) larvae spawned at the Indiana University Axolotl Colony. Ambystoma are highly cannibalistic, especially towards appendages. To avoid uncertainties about the stock (whether prospective amputees bore natural or regenerated limbs) and to insure a uniform starting point with each experiment, prospective subjects were individually segregated, and thus maintained, at Harrison stage 43 (see Rugh, 1962); i. e., prior to the onset of free feeding. The precautions in question were motivated by the fact that nerve patterns in regenerated limbs are not identical to those in the natural limb (see Piatt 1957). Nerves can influence regenerative myogenesis (Pietsch, 1962b).
All animals were kept in 5 percent Holtfreter's solution, changed daily, and daily were fed newly hatched brine shrimp. With a view, ultimately, to minimizing photic degradation of RA in the test subjects, while at the same time maintaining a constant environment for control and experimentals, all animals, except a special control subgroup, were kept in capped amber bottles and were handled in low intensity illumination (after considerable preliminary testing of the adequacy of the methods). The special control animals were kept individually in clear finger bowls; they showed no differences from the controls in amber bottles. Nociceptive procedures (amputations and injections) were carried out after animals had been narcotized in 1:5000 MS 222.
A. opacum and A. punctatum larvae were 30-32 mm in body length when subjected to bilateral amputation through the neck of the humerus, bilateral in part for economy and efficiency but also for independently assessing individual variations, as will be explained with results; the two limbs of a given animal were amputated seconds apart and, in a given run, all limbs were amputated within less than 15 minutes. The plane of amputation, coinciding with the juncture of the free limb and the body, was selected partly because it could be quickly and precisely duplicated in a large number of subjects but also to involve as much of the appendage in regeneration as possible. The principal subjects had a wet (but blotted) body weight of 0.35-0.50 g at the time of treatment. The amputated limbs were fixed and examined to confirm the plane of amputation as well as to analyze the natural (vis-à-vis regenerated) musculature. Except for a group of untreated controls, the amputees received a single 1.0 ul intracoelomic injection of either dimethylsulfoxide (DMSO) or DMSO-dissolved all-trans RA (Sigma XX), using a microinfusion apparatus and operative procedures detailed elsewhere (Pietsch, 1987). A single batch of DMSO, stored at room temperature, was used throughout. (Refrigerated DMSO crystallizes and redissolves with some difficulty.³) Aliquots of one large lot of RA (Sigma 104F-0135) were used in all experiments, each aliquot having been stored preweighed and dry in a sealed ampoule, in the dark at -30 oC, until a few minutes before administration. (DMSO solutions of RA show major spectral changes upon thawing. DMSO crystallization is sufficiently exergonic to drive redox reactions among the retinoids.) The dosage of RA was 125 ug/g body weight, a level estimated from prior data (op. cit.) as the amount that, following intracoelomic injection, would create a "treatment interval" of at least 14 days.
Concerning the treatment interval, RA forms a bright yellow ppt in coelomic (peritoneal) fluid. Spectrophotmetric comparisons of this ppt with freshly dissolved aliquots of its parent batch indicated that the ppt one observes in the body cavity is indeed RA (op. cit.). Thus, visible through the body wall, the yellow ppt is a positive indicator of maximal intracoelomic RA treatment in progress at that moment. However, to avoid exposing the principal subjects to the bright light of the dissecting microscope during the putative treatment interval a parallel monitor series was run with the aforementioned white axolotl larvae (whose tissues are virtually transparent and whose green gall bladder provided a highly contrastable background for the brilliant yellow of the ppt); the right forelimb of each white axolotl was amputated through the humeral neck and the retardation of the growth of its blastema was used to guarantee efficacy (op. cit. for rationale).
Principal subjects were divided into groups with each animal receiving a single treatment of either RA or DMSO at one of the follows days, postamputation: 0, 10, 14 or 21 days. Two important sets of facts were taken into account in choosing the subsequent incubation period. First, RA invariably retarded the growth of the blastema (op. cit.), often reversibly but sometimes permanently. Second, under the conditions of this laboratory, limb regeneration among larvae of the age and species employed is complete in approximately 30 days. With these facts in mind, the principal subjects were kept alive until either day 108 or day 114, post-amputation; i. e., incubation periods of durations sufficient to allow for complete regeneration in the face of any transitory effects.
For sacrifice, animals were anesthetized in MS 222 and, when unconscious, transferred to Bouin's fixative. Specimens were first examined in toto under the stereoscopic microscope. Then the entire limb, including the girdle, was dissected and processed for paraffin embedding. The limb skeleton of Ambystoma larvae is cartilaginous, obviating the need to demineralize the tissue for sectioning. Most limbs were oriented (under the stereoscopic microscope) for serial transverse sectioning though at least the wrist and forearm and, when possible (in the face of reduplications and distortions) the hand. After bending the elbow 90^o, the upper arm was serially sectioned, longitudinally in some instances, and transversely in others. Slides were stained with Mallory's trichrome procedure (after which collagen appears blue and muscle tissue red). No species differences were observed. Therefore, for the sake of simplicity the data from A. opacum and A. punctatum will be combined for presentation.

Results

1. Minimal Dosage Interval

In the white axolotls used to monitor dosage, the yellow RA ppt was visible for some 14-17 days, during which time regeneration of the right forelimb (the bioassay) was severely retarded. Thus, considering the treatment schedule employed in the principal experiments, no phase of regeneration escaped the effects of RA, when the groups are considered collectively.

2. Skeleton and External Form

The effects of RA on skeletogenesis were basically similar to those reported in the literature (see especially Maden et al, 1985; Thoms and Stocum, 1984). Thus only an overall summary on the macroscopic findings will be presented here (see Table 1).

No regenerated limb of either the untreated or the DMSO-treated control animals showed any skeletal abnormalities or distortions of macroscopic form. Among the limbs of animals treated with RA on the day of amputation (day 0 in Table 1), 62.5 percent showed external abnormalities; those with no outwardly obvious malformations, upon subsequent histological examination, without exception, showed some form of skeletal deformity. With RA treatment at day 10, over 72 percent of the cases had an external abnormality. Again, microscopic examination revealed skeletal malformations in the balance of the cases. All subjects treated at either 14 or 21 days showed macroscopically visible malformations, and histological sections revealed additional anomalies among the cartilages.

In sum, skeletogenesis failed to reach a completely normal outcome in a single RA-treated regenerate during the entire investigation.

Now considerable individual variation attended the types of malformations, far more than had been anticipated a priori, the aberrations ranging from syndactyly to supernumerary digits to segmental reduplications (Figs. 1-8). Compound anomalies were not at all rare (note Figs. 5 and 6). With one relatively minor exception (see next paragraph), no obvious correlation existed between the type of abnormality and the day of treatment. That variability was not attributable to protocol was attested to by the lack of correlation between contralateral regenerates from the same animal (with amputations within seconds apart and identical systemic RA treatment): the regenerate of one side might terminate in a conical structure while that of the other could exhibit two or even three forearms, wrists and hands with varying numbers of fingers. Or one side might show a two- or three-fingered hand (the normal number is 4) while the other might have two or even three hands, the hands themselves with their own intrinsic abnormalities. (See also Figures 4 and 5.) The crucial point here is that the observed variability is a function of the system's response to RA.

The exceptional condition involved hyperplasia of the shoulder (see Figs. 7, 8). All RA-treated specimen, irrespective of the day of treatment, exhibited enlarged shoulders and supernumerary girdle parts were evident upon microscopic examination (Fig. 9). But with treatment at days 14 or 21 the proximal arms bulged conspicuously and were macroscopically obvious in each RA-treated specimen.

3. Control Muscle Patterns

The criteria for the analysis of muscle patterns may be found in Blount (1935), and Piatt (1956, 1957); see also Grim and Carlson (1974). A brief summary description of the highlights of the natural limb musculatures is presented here for the reader's convenience. Diagrammatic representations may be found in Figures 10-12.

The girdle musculature was too complex for critical analysis in the 2-dimensional array presented by microscopic sections. However, distal to the insertions of the scapular and pectoral muscles, sections of the arm, whether longitudinal or transverse, presented three easily identifiable muscles (using Blount's terminology):

anconeus (triceps), dorsally;
coracobrachialis, ventromedially;
humero-antibrachialis (homologue of the human biceps brachii and brachialis), ventrolaterally.

The small but constant and highly recognizable ulnocarpalis muscle has furnished an invaluable landmark in cross sections of the Ambystoma forearm and wrist for other investigations (see Piatt, 1956; 1957; Pietsch, 1961a, b; 1962a, b), and the present experiments were no exception. The ulnocarpalis runs parallel and ventral to most of the ulna and, passing into the wrist, inserts into the deep fascia (mesenchymal in the larva) on the volar aspect of the middle row of carpals. Having identified the ulnocarpalis, the observer can immediately distinguish the ulna from the radius (otherwise impossible without serially tracing), quickly pick out the obliquely oriented pronator quadratis and confidently stipulate the flexor versus extensor surface of the forearm. The observer can then ascertain the extensor carpi ulnaris, extensor digitorum (communis) and extensor carpi radialis, dorsally, and the flexor carpi ulnaris, palmaris longus and flexor carpi radialis, ventrally.

Muscles were analyzed in the serial sections of 4 untreated and 27 DMSO-treated control regenerates; each one exhibited the cross sectional anatomy described in the previous paragraph for the natural limbs (Figs. 13-16).

4. Muscle Patterns Following RA Treatment

Of 62 RA-treated regenerates, representing treatment at various postamputation days, initial scanning of the slides revealed that each had developed appreciable quantities of skeletal muscle tissue. In instances of severe ankylosis and/or spasticity, resulting from skeletal and joint deformities, muscles often exhibited disuse atrophy. Still, even in the latter instances, the muscle fibers, readily identified as such, were organized into discrete fascicles. In no case was muscle present in the form of whorls or clumps or random fleshy masses (cf. Pietsch 1961b; 1962a, b). Initial inspections of slides sometimes produced the misconception, in 2-dimensions, that a shoulder muscle was an abnormal muscle mass. But serial tracing invariably showed the latter muscle to be attached to a girdle element (scapula, coracoid or ) or to the proximal end of the humerus. Numerous instances were encountered where hyaline cartilage tissue existed without muscle. But muscles were always associated with skeletal elements (cf. op. cit.).

Each specimen exhibited the main muscles of the upper arm: anconeus, coracobrachialis and humero-antibrachialis (Fig. 17). In some instances, when the cartilage was severely gnarled or elements were fused, it was the muscles that made it possible to tell that the segment was indeed arm or forearm.

When a forearm region was present (54 cases) the main features of the antibrachial musculature were unmistakable even with extensive skeletal abnormalities within the same microscopic field (Figs. 18-25). All regenerated forearms and wrists of each RA-treated animal exhibited an ulnocarpalis and pronator quadratus.

If a hand was present, the interossei were readily identifiable between the metacarpals; the superficial and deep palmar muscles, ventrally; and the heads of the humero-metacarpalis, dorsally (Figs. 26 and 27).

5. Muscles in Reduplicated Members

Special attention was directed towards reduplicated structures, when present. Bizarre orientation often made specific morphological identifications problematical . However, when the supernumerary members lie parallel, and it was possible to judge and compare the morphology of both, the musculatures exhibited at least some portions that were mirror images of each other, down to the most minute details. In one favorably oriented specimen, two arms had been produced. Fused along their ventromedial aspects, the members of this specimen partially shared the bellies of two coracobrachialis muscles; but it was as though the two anconeus and two humero-antibrachialis muscles were on the apposable pages of an open book (see Fig. 28).

Figures 29 and 30 show an excellent example of another reduplicated specimen, but with two forearms, wrists and hands. The replicates, fused each other dorsally and, sharing the proximal one seventh of the extensor digitorum, were within 150 um of being in perfect proximo-distal register. Using the ulnocarpalis muscles as a guide, one could serially trace the specimen and identify all other antibrachial muscles, as well as the intrinsic musculature of the wrist and hand. Indeed, the out-of-register of the two members was estimated by finding the termination of one ulnocarpalis and counting the 15 sections (10 um each) distally to the insertion of the other. Unfortunately, too few replicated specimens were available to establish any trends as to the line of fusion or planes from which the reduplicated segments emerged.

Discussion

Independence

The results clearly show that systemic retinoic acid treatment can be sufficient to induce drastic distortions in the cartilaginous skeleton of the regenerated limb without scrambling the regenerate's muscle pattern. Muscle patterns in the limb regenerate are far from immutable (Pietsch, 1961b, 1962a, b). The dosing schedules in the present investigation extended well into the periods before and after which myogenesis is self-differentiating (Pietsch, ibid). Therefore, the normally organized limb muscles cannot be explained simply by assuming either that muscle is resistant to change or that the RA treatment regimen missed critical myogenic periods.

Previously (Pietsch, 1962a), muscle in the limb regenerate changed while cartilage remained constant, and the conclusion was reached that the morphogenic aspects of myogenesis and chondrogenesis proceed independently. The present findings validate the logical converse, chondrogenesis varying while myogenesis remained unchanged. Thus the independence hypothesis passed the pragmatic test to which it was subjected: it worked.

Myogenesis and chondrogenesis appear also to be mutually independent in embryonic systems (Swalla and Solrush, 1986). Thus the persistence of the limb muscle pattern in the present experiments would seem to be a manifestation of a principle of development that transcends salamander limb regeneration, per se. An important corollary of the latter, articulated in cellular terms, is that genes and cytoplasmic factors used for one set of events can be activated or repressed or mutated without necessarily activating or repressing or mutating the other, and vice versa.

Morphogenic Potency of Future Muscle Cells

The existence of normally fasciculated but symmetrically opposite muscles within supernumerary limb segments suggest that the regenerate's musculature is of epigenic and pluripotential origin (compare with Pietsch, 1961a). It would appear that the cellular forerunners of the new muscle in question came from an ontogenically indeterminate pool; i. e., the developmental fate of at least some cells in larval limb tissue is not pre-programmed. It is not totally out of the question, however, that one or both mirror image opposites represent de novo limb development rather than regeneration, per se. Retinoic acid is a known morphogen with a capacity to activate homeobox gene clusters (Mavilio et al 1988). The distinction, regeneration versus de novo development, is not trivial in that the processes leading up to the cell source of the two could be very different (e.g., dedifferentiation versus embryonic rests), and the different events could conceivably involved the genetic apparatus of the cells in interestingly unlike ways. Multiple receptors for RA (RARs) have been identified during limb regeneration in the newt (Ragsdale et al 1989). Multiple RARs suggest the possibility that a variety of particular routes may be taken to the same ultimate end, a possibility worth considering as the facts accumulate. Whatever, the present data show unequivocally that a hidden potentiality for a complete contralateral musculature exists within post-embryonic salamander limb tissue. Retinoic acid unmasked the latter potentiality, although by mechanism not evident in the data.

Pacifici et al (1980), working in tissue culture with embryonic chick limb bud cells, found the formation of muscle fibers to be resistant to levels of Vitamin A that inhibited the differentiation of cartilage. Do the present results reflect insensitivity of myogenesis to the retinoids? In stimulating the development of mirror image opposite musculatures, RA obviously affected myogenesis. But even when their symmetry belonged to the other side of the body, the muscles in the replicates were an integral part of a readily recognizable whole. The effects on muscle were an indirect result of RA having induced the differentiation an entire limb segment, including its musculature, without jumbling the mechanisms responsible for the internal attributes of muscle pattern. (The logic is akin to the difference between a person's dancing a jig and his or her simultaneous rotation around the sun by virtue of being earthbound.) RA's effects on muscle were quite unlike those on cartilage, and even with the caveat just discussed the findings are in line with the observations of the investigators last cited.

Acknowledgment

This is to thank Jacque E. Kubley for printing and mounting the photographs and for rendering the final versions of the drawings. I am grateful to the Indiana University Axolotl Colony, Susan Duhon, Manager for the kind gift of white axolotls. I am also indebted to my colleague Dr. Sally Hegeman for her insight into pharmacology and for her encouragement during the conceptualization phase of the project.

References

BLOUNT I. W. H. 1935. The anatomy of normal and reduplicated limbs in amphibians with special reference to musculature and vascularization. J. Exp. Zool. 69 407-458.

GRIM M. and Carlson, B. M. 1974. A comparison of morphogenesis of muscles of the forearm and hand during ontogenesis and regeneration in the axolotl Ambystoma mexicanum. I. Anatomical description of muscles of the forearm and hand. Z. Anat. Entwickl.-Gesch. 145 137-148.

MADEN M. Keeble S. and Cox R. A. 1985 The characteristics of local application of retinoic acid to the regenerating axolotl limb. Roux's Arch. Dev. Biol . 194 228-235.

MAVILIO F. Simeone A. Boncinelli E. and Andrews P. W. 1988 Activation of four homeobox gene clusters in human embryonic carcinoma cells induced to differentiate by retinoic acid. Differentiation 37 73-79.

PACIFICI M. Cossu G Molinaro M. and Tato F. 1980 Vitamin A inhibits chondrogenesis but not myogenesis. Exp. Cell Res. 129 469-474.

PIATT J. 1956. Studies on the problem of nerve pattern. I. Transplantation of the forelimb primordium to ectopic sites in Amblystoma. J. Exp. Zool. 131 173-202.

PIATT, J. 1957. Studies on the problem of nerve pattern. III. Innervation of the regenerated forelimb in Amblystoma. J. Exp. Zool. 136 229-247.

PIETSCH P. 1961a. Differentiation in regeneration. I. The development of muscle and cartilage following deplantation of regenerating limb blastemata of Amblystoma larvae. Dev. Biol. 3 255-264.

PIETSCH P. 1961b. The effects of heterotopic musculature on myogenesis during limb regeneration in Amblystoma larvae. Anat. Rec. 141 295-304.

PIETSCH P. 1962a. Independence of chondrogenesis from myogenesis during limb regeneration in Amblystoma larvae. J. Exp. Zool. 150 119-128.

PIETSCH P. 1962b. The influence of spinal cord on differentiation of skeletal muscle in regenerating limb blastema of Amblystoma larvae. Anat. Rec. 142 169-178.

PIETSCH P. 1987. The effects of retinoic acid on mitosis during tail and limb regeneration in the axolotl larva, Ambystoma mexicanum . Roux's Arch. Dev. Biol . 196 169-175.

RAGSDALE C. W. Petkovich M. Gates P. B. Chambon P and Brockes J. P. 1989 Identification of a novel retinoic acid receptor in regenerative tissue of the newt. Nature 341 654-657.

RUGH R. 1962. Experimental Embryology. Minneapolis: Burgess

STOCUM D. L. and Thoms S. D. 1984. Retinoic acid-induced pattern completion in regenerating double anterior limbs of urodeles. J. Exp. Zool. 232 207-215.

SWALLA J . and Solrush M. 1986. The independence of myogenesis and chondrogenesis in micromass cultures of chick wing buds. Dev. Biol. 116 31-38.

THOMS S. D. and Stocum D. L. 1984. Retinoic acid-induced pattern duplication in regenerating urodele limbs. Dev. Biol. 103 319-328.

TABLE **Skeletal Abnormalities in Limb Regenerates**
Treatment (Day)¹	Treatment (type)	Regenerated Limbs (number )	Abnormal Limbs (number)²
0	none	6	0
0	DMSO	18	0
0	RA	24	15
10	DMSO	16	0
10	RA	22	16
14	DMSO	12	0
14	RA	10	10
21	DMSO	8	0
21	RA	12	12
¹post-amputation ²Externally visible; all RA-treated cases showed some skeletal abnormalities upon histological examination.

Figures

Figures 1-8 Macrographs of Bouin-fixed, regenerated forelimbs from RA-treated Ambystoma larvae. The limbs in Figures 1 and 2 were from an A. punctatum treated at day 0. The former has an extra digit (see arrow) but otherwise looks normal. Although the limb in Figure 2 was rated normal upon macroscopic examination, histological analysis revealed an unsegmented cartilaginous slab in lieu of the proximal row of discrete carpals and the distal ends of the ulna and radius (normally connected by a mesenchymal interosseous membrane). Figure 3 shows a conical spike growing off the regenerated upper arm (brachium) of an A. opacum treated at day 10; conical masses (of unpredictable length) were a regular occurrence among RA-treated specimens, but their attributes could not be correlated with day of treatment. Figures 4 and 5 illustrate varied effects of RA on limb regenerates of the very same animal, in this case an A. punctatum treated at day 0; note that while the specimen in Figure 4 had only two fingers (the normal number is four), that of Figure 5 featured a complex of abnormalities : part of an extra hand, an independent supernumerary digit and, additionally, a massive conical growth (tiny arrow) off its elbow; the cone turned out (microscopically) to be a brachydactylic supernumerary arm.
Figure 6 shows three limbs (go here for more contrast) grown from the same stump of an A. punctatum treated at day 0. Figures 7 and 8 are the hypertrophied shoulders described in the text; both subjects were A. opacum larvae, the former RA-treated on day 10 and the latter on day 14. Swollen shoulders invariably contained supernumerary pectoral girdle elements (see Fig. 9).

Figure 9. Micrograph of an hypertrophied shoulder of an RA-treated A. punctatum (day 10). H and H' are humeral heads; G and G' are glenoid fossae. Primary magnification, 40 X.

Figure 10. Diagram of the Ambystoma arm (brachium) in cross section.

Figure 11. Diagram of the Ambystoma forearm (antibrachium) muscles in cross section.

ECR extensor carpi radialis FCU flexor carpi ulnaris

ECU extensor carpi ulnaris P plamaris longus

ED extensor digitorum (communis) PQ pronator quadratus

FCR flexor carpi radialis UC ulnocarpalis

Figure 12. Diagram of the Ambystoma hand (manus) in cross section. H= humero-metacarpalis (tendon), I= interosssei, M= metacarpal, PS= palmaris superficialis, PP=palmaris profundus

Figure 13. Regenerated distal forearm of an untreated control animal. U is the ulna, R is the radius, UC is the ulnocarpalis muscle, PQ is the pronator quadratus; see figure 11 for additional orientation. Primary magnification, 40 X.

Figure 14 Regenerated proximal wrist of an untreated control animal. U is the ulnare carpal, R is radiale, I is the intermediale UC is the ulnocarpalis muscle, PQ is the pronator quadratus. The muscle pattern in the proximal wrist is an extension of the morphology found in the distal forearm. Primary magnification, 40 X.

Figure 15. Regenerated distal forearm of a DMSO-treated control animal. Go to figure 11 for orientation. Primary magnification, 40 X.

Figure 16. Regenerated proximal wrist of a DMSO-treated animal. Compare with Figure 11. Primary magnification, 40 X.

Figure 17. The regenerated left arm of an RA-treated subject. A is the anconeus, C & H represent the coracobrachialis and humero-antibrachialis muscles, respectively; compare with Figure 10. Primary magnification, 40 X.

Figures 18 and 19. Wrist-forearm transition zone of a DMSO-treated control presented for close comparison with next two photographs; same abbreviations as in Figures 12 and 13. Primary magnification, 40 X.

Figures 20 and 21. Wrist-forearm transition region of an RA-treated regenerate. The cartilages have fused into a single mass but the muscle patterns remain identical to those of the controls; for additional orientation see figure 11. Primary magnification, 40 X.

<---Figures 22-24. Regenerated forearms of RA-treated subjects; same abbreviations as in Figure 13; for additional orientation, see Figure 11. The musculatures indistinguishable from the controls. Primary magnification 40 X.

Figure 25. Wrist-forearm transition zone of an RA-treated regenerate with compound skeletal abnormalities visible at the same cross-sectional level: an aberrant extra set of digits; also what appears to be an extra carpal dorsal to the radiale (R). The ulnocarpalis (UC) serves to orient the observer for identification of the intermediale cartilage (I) and the pronator quadratus muscle (PQ). Primary magnification, 40 X.

Figures 26 and 27. DMSO-treated control and RA-treated hands, respectively. H-M designates the small Humero-Metacarpalis muscles (see Fig. 12); IO, the interossei of the hand; M the metacarpals; PP the palmaris profundus; and PS the palmaris superficialis. The treated and control hand muscle patterns exhibit no morphological differences. Primary magnification, 40 X.

Figure 28. Reduplicated arm of an RA-treated subject. H and H' are humeri. HA designates the mirror image humero-antibrachii muscles. The A's point to two bilaterally symmetrical anconeus muscles. A few sections distal to the one in the photograph the two replicates separated into free members, similar to the specimen in fig. 6. Primary magnification, 40 X.

Figure 29. Macrograph of an RA-treated specimen exhibiting bilateral replication of the forearm and hand. In this specimen both the skeleton and musculature of the replicates were equal but of opposite symmetry. A section of this specimen is shown in Figure 30.[For more about this specimen, go here!]

Figure 30. Section through the same specimen as in Figure 29. UC's designate the ulnocarpalis muscles and EX the extensor digitorums. The two members still share the same skin in this section but become completely free more distally (see Fig. 29). These two members are some 150 uM out of perfect register. Nevertheless, many of the same muscles can be seen in a given cross section, as in the present figure. Serial inspection revealed that both members possessed identical but opposite forearm and wrist muscles. The subject was an A. opacum larva treated with RA 10 days after amputation. Primary magnification, 40 X. [For additional information about this specimen, go here!]

"Monitor"

The bright yellow spot is precipitated retinoic acid (RA) in the coelomic cavity of an albino axolotl. The dark green mass above the ppt is the gall bladder, which lent contrast to the RA. The albinos were selected as "monitor" subjects because the RA could be seen at a glance through their transparent ventral abdominal wall. Monitor animals such as this were included with principal experiments as a means of tracking dosage on the premise that,as long as ppt remained in the coelom the subject was receiving the maximum systemic dose of RA. Note that the animal lacks a right forelimb. The latter had been amputated; its regeneration, held in check until the ppt disappeared, served provided a bioassay (see Pietsch, 1987).

Multiple Limbs

Here white arrows point to the outlines of three distal supernumerary limb segments. The black arrow points to conical mass on the shoulder that, upon histological examination, turned out to be a badly distorted partial fourth limb.

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ECR	extensor carpi radialis	FCU	flexor carpi ulnaris
ECU	extensor carpi ulnaris	P	plamaris longus
ED	extensor digitorum (communis)	PQ	pronator quadratus
FCR	flexor carpi radialis	UC	ulnocarpalis