The regenerating limb blastema was transplanted in place of the eye in Amblystoma larvae. Skeletal muscle with abnormal patterns developed around the blastema-derived cartilages. When a narrow band of limb stump was left attached to the transplanted blastema, reforming muscles of the limb and the orbit appeared to overgrow each others territory and mix. The latter regenerates developed muscles of mixed morphological patterns.This study was designed and executed to gain insight into myogenesis during regeneration of the salamander's limb. Inquire was made into the relationship between preexisting and newly differentiated muscle. In previous investigations, blastema cells, experimentally isolated from stump tissues, produced organized cartilages but not muscles (Pietsch, '61b). If a narrow band of stump was left at the base of a transplanted blastema then muscles did develop, and the morphological distribution of fibers was limb-like in minute detail. It was inferred that the removal of the blastema from the stump, in effect, removed factors indispensable for the development of a limb musculature.
In tail regeneration, S. Holtzer ('56) demonstrated that the quantity of muscle developed was in direct proportion to the amount preexisting in the tail stump: Reduction in muscle on one side of the tail led to a corresponding unilateral diminution in regenerated muscle, confined to the same side. This information coupled with observations on transplanted limb regenerates led to a reconsideration of the hypothesis that myogenesis during limb regeneration is causally related to preexisting muscle (see Morgan '01). The hypothesis was put to a simple test by causing regenerating limb blastemas to differentiate in an environment containing heterotopic muscle (viz. muscles of the orbit). If the hypothesis is valid, transplanted blastemata should produce limbs with appreciable amounts of muscle but showing patterns that typify the host rather than the donor site.
Of the 4 specimens lacking in skeletal muscle, three represented experiments with 11-day and one with an 8-day blastema. In these 4 cases no extraocular muscles were found in the orbit.
Patterns assumed by muscle fibers in transplants were examined and compared with muscular morphology in normal limb regenerates. Attention was focused on the antibrachium where the pattern is simple but nevertheless sufficiently variegated for morphological analysis. The anatomy of Amblystoma
limb muscles has been described by Blount ('35) and is very well illustrated in Piatt's drawings (see '57 for additional references). Therefore only a summary is presented here (fig. 3). Quite characteristic of antibrachium is the small ulnocarpalis muscle which runs through the forearm into the wrist and occupies a position parallel and ventral to the ulna. This small constant muscle identified, it is then possible to orient an entire cross section of the forearm.
Of the 25 orbital transplants with muscle, one showed fibers arranged according to the plan of forelimb musculature. In the other 24, redoubled examination failed to reveal a single muscle that characterized any segment of the limb (cf. figs 5 and 6). In the one exceptional case, the limb had developed from a transplanted 15-day blastema. In the distal antibrachial region, near the wrist, muscle pattern was similar, though not identical, to that in normally regenerated limbs. Ulnocarpalis and wrist flexors and extensors were easily identified. However, less than 200 u proximally, in this very same specimen, the limb-like pattern was given over to one that is not encountered in any segment of Amblystoma appendage, natural or regenerated (figs. 7 and 8).
First, it was necessary to see if limb and extraocular muscle could overgrow the same territory. Thus, previously uninjured limb was amputated below the elbow and transplanted in place of eye. Six specimens, subsequently, were examined and in each the antibrachial musculatures no longer presented the anatomical features that typify normal limbs. Distally, in the writs of each specimen and thus far removed from the host-donor interface, it was possible to identify ulnocapralis.
Was the obscuring of the muscle pattern a function of some unknown general conditions in the orbit (e. g. reduced blood supply, foreign nerves)? This possibility was tested by orbital transplantation of limb removed at the head of the humerus. Thus there would intervene approximately 1.5 mm of tissue between donor antibrachial and host extraocular muscles. Hands, wrists and forearms of the cases studied (5) showed musculatures indistinguishable from those of intact limbs. However, from the elbow proximally, there was progressive loss of limb pattern, and at the junction with the host tissue, it was impossible to identify any muscles indigenous to the arm (viz., humero-antibrachialis, anconeus, coracobrachialis complexes).
With the aforementioned information available, 11-day blastemata with less than 1 mm of stump attached were transplanted to the orbit. Six cases were studies and in each the muscle of the transplant stump had lost the characteristic limb pattern. No specific limb muscles were observed in proximal regions of regenerates, but distally, typical antibrachial muscles were found amid or alongside irregular muscle masses which did not conform to limb morphology ( fig. 4); i. e. the muscle pattern was mixed.
The causal relationship between preexisting and regenerate muscle is further supported by the fact that when blastema with stump attached was implanted into dorsal fin, typical musculatures developed (Pietsch, '61b). The fin and orbit differ in the critically important respect that the former does not possess skeletal muscle. The important thing at the moment seems to be that whatever mediates myogenesis operates simultaneously at the histogenic and morphogenic levels and as a concomitant of muscle already present. This may be true of the embryo as well (see Hall, '50). The question is thus raised, is myogenesis concurrently histogenic and morphogenic? Are the mechanism underlying the synthesis of contractile protein molecules separable or inseparable from those bestowing morphogenic order on muscle of a given body region?
An ancillary but interesting observation during this study was that cartilages were always elements of the limb skeleton. Mixing of stump musculatures did not alter the outcome of chondrogenesis. Pietsch (unpublished) amputated orbitally transplanted limbs and observed that chondrogenesis was not altered in regenerated portions despite modifications in musculature. This indicates that myogenesis and chondrogenesis are independent events and that myogenic and chondrogenic cells possess, from the outset of regeneration, specific, highly organized developmental mechanisms.
Hall, E. K. 1950 Experimental modifications of muscle development in Amblystoma punctatum. Ibid., 113: 355-377
Holtzer, S. 1956 The inductive activity of the spinal cord in urodele tail regeneration. J. Morph., 99: 1-39
Levander, G. 1956 Induction phenomena in regeneration of striped muscle. Ark> Zool. 8, 565-577.
Morgan, T. H. 1901 Regeneration. The MacMillian Co., New York.
Naville, A. 1922 Histogenèse et régénération du muscle chez les Anoures. Arch. Biol., 32: 37-171.
O'Steen, W. K. and B. E. Walker 1961 Radioautographic studies of regeneration in the common newt. II. Regeneration of the forelimb. Anat. Rec. 139:547-556.
Piatt, J. 1957 Studies on the problem of nerve pattern. III. Innervation of the regenerated forelimb in Amblystoma. J. Exp. Zool. 136: 229-264.
Pietsch, P. 1961a The effects of colchicine on regeneration of mouse skeletal muscle. Anat. Rec. 139: 167-172. Pietsch, P. 1961b Differentation in regeneration. I. The development of muscle and cartilage following deplantation of regenerating limb blastemata of Amblystoma larvae. Develop. Biol., 3: 255-264.
Stockdale, F. E. and H. Holtzer 1960 The formation of multinucleated myotubes. Anat. Rec. 138: 384.
Fig. 1. Homologous spleen graft in the orbit. Specimen fixed three weeks after
transplantation. Three bundles of muscle extend toward the transplanted lienal
tissue in the center of the photograph. Magnification 150 X.
Fig. 2. Portion of a limb that developed from a stumpless 11-day blastema
transplanted to the orbit. Specimen was fixed 30 days after transplantation.
Skeletal muscle, indicated by the arrow, extends from the orbital wall (O) to
blastema-derived cartilages (C). Magnification 100 X.
Fig. 3. Schematic representation of the antibrachium in cross section. The
most prominent feature of this region is the small but constant ulnocarpalis
muscle (UC) which lies ventral an parallel to the ulna (U) throughout most of
the antibrachium. Pronator profundus and interosseus muscles (PR & 1)
often form a continuous mass of obliquely directed fibers running between the
ulna and radius (R). More distally, towards the wrist, the plane occupied by
PR & 1 is taken over by the palmaris profundus
(see Fig. 5); the
delicate, almost imperceptible interosseous membrane is supplanted by a carpal
(the intermediale) which projects upwards between the distal ends of the radius
and ulna. Relative sizes of muscles vary somewhat depending on the exact antibrachial
level, and in older larval limbs the long flexors and extensors
present two or three well-fasciculated heads. Identification of both cartilages and muscles is
facilitated in cross sections by first finding and identifying the ulnocarpalis, then the ulna and then relating other structures to them.
Fig. 4.
Phase contrast photomicrograph of an antibrachium developed following
transplantation to the orbit of an 11-day blastema with stump attached. The
arrow indicates the ulnocarpalis lying ventral and parallel (above and left in the photo) to the ulna (U).
The flexor carpi ulnaris lies superficial and ventromedial to the ulnocarpalis.
Other muscle masses cannot be identified as specific muscles. This is an
example of the "mixed" musculature observed after stump tissue remained
attached to the transplanted blastema. Magnification 150 X.
Fig. 5. Transverse section through the proximal portion of the wrist of a
normally regenerated forelimb 6 weeks after the amputation just above the
elbow. The ulnocarpalis muscle is indicated by an arrow. Lateral to the
ulnocarpalis and at roughly the same depths is the palmaris profundus; the
latter muscle occupied the same plane as the pronator profundus and
interosseous in the antibrachium (see fig. 3). The palmaris superficialis is
still a prominent fleshy mass at this level, as is the extensor digitorum.
Flexors and extensors of the wrist have tapered toward their respective
insertions. Magnification 150 X.Fig. 6. Proximal wrist region developed following transplantation of a stumpless 11-day blastema to the orbit. The three proximal carpals are surrounded by a continuous mass of skeletal muscle tissue showing none of the morphogenic features of the musculature a normally regenerated limb. Compare with figure 5. Extra ocular muscles may be seen on the extreme right of the photograph. The union of extraocular muscle tissue, per se, and that of the regenerate is about 0.5 mm proximal to this section. Magnification 150 X.
Fig. 7. Section through the distal antibrachium of a specimen developed from a
transplanted stumpless 15-day blastema. The ulnocarpalis muscle (arrow) lies
in its typical position ventral to the ulna (U). With the latter as a
reference, the flexor carpi ulnaris can be identified ventral to the
ulnocarpalis. The crescent-shaped muscle superficial to the ulna is the
extensor carpi ulnaris. The muscle masses on the radial side of the section
care not typical of the limb. Compare with figure 3. Magnification 150 X.Fig. 8. Same as in figure 7 but 200 microns proximally. The forelimb muscle pattern is not evident at this level. Compare with figure 3 and figure 7. Magnification 150 X.
**Present institution: Indiana University, Bloomington, Indiana, USA