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The influence of spinal cord on differentiation of skeletal muscle in regenerating limb blastema of Amblystoma larvae.

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The Influence of Spinal Cord on Differentiation of
Skeletal Muscle in Regenerating Limb Blastema
of Amblystoma Larvae'
Department of A n a t o m y , Uidversity of Pennsylvania a n d B o w m a n Gray
School of Medicine, W a k e Forest College
Regeneration of the salamander limb is
a phenomenon attended by a large, conjectural literature. Not a few text writers have
abstracted from technical writings the belief that regenerated limb is the product of
differentiation within a primitive, indiff erent mesenchymal aggregation (viz. the SOcalled blastema). Decisive evidence in support of that opinion is lacking. However,
one need only examine the bulk of more
recent articles to see that the belief under
consideration constitutes a major premise
upon which are built the main lines of
thinking concerning regeneration.
Pietsch ('60a, b, '61b) performed a number of experiments which suggest that the
blastema does not behave like an aggregation of indifferent mesenchyme. Blastemata of various ages were cultivated in
chambers in the dorsal fin of Amblystoma
larvae. In some cases stump was left attached at the base of the blastema; in
others stump was excluded. With stump
present limb skeleton and musculature differentiated. With stump absent typical
limb cartilages developed but musculatures
did not. The blastema from the outset of
its history as a discrete entity failed to behave as though developmentally neutral or
indifferent. However, there was no evidence that the musculature arose from
blastema cells. In some of the transplants
just mentioned an occasional myogenic
cell was identified by the presence in it of
one or two myofibrils. Might these and perhaps other myogenic cells produce multinucleated muscle fibers? In other words, are
there cells in the blastemal aggregation
capable of forming muscle?
An experimental approach was indicated
by the findings of other investigators, who,
using a variety of materials and methods,
have demonstrated that spinal cord enhances myogenesis in systems that contain
rnyoblasts (Holtzer and Detwiler, '53;
Holtzer, Holtzer and Avery, '55; Aveiy,
Chow and Holtzer, '56; Holtzer, '56; Muchmore, '58). There was good reason to suspect that if myogenically competent cells
enter into the formation of the blastema,
these would respond to the presence of
spinal cord by producing skeletal muscle
fibers. Towards this end, blastemata and
segments of spinal cord were transplanted
together to the dorsal fin.
Amblystoma opacum and A. punctatum
larvae, 30 mm in length, were used. Aquarium room temperature was kept at about
20°C. Limbs were amputated through the
proximal one-third of the arm. Six, 7, 8,
11 or 15 days after amputation a given
blastema was removed from its stump and
auto-transplanted singly in a tunnel
reamed into the gelatinous dorsal fin connective tissue (see procedure in Weiss, '50).
A 1.5 mm segment of anterior spinal cord
was homo-transplanted with each blastema. Spinal cord-donor and host animals
were siblings.
Specimens were preserved in Bouin's
fluid 30 days after transplantation. Processed routinely for paraffin sectioning at
10 u, specimens were stained in iron hematoxylin and examined histologically for
muscle. Some tissues were silver stained,
but this did not prove of significant value
to the study. Cases in which either blastema or spinal cord had degenerated were
excluded from analysis.
1 Supported by grants from N.I.H.
2 Present address: Department of Anatomy, School
of Medicine, University of Buffalo, Buffalo 14, N e w
Experiments also were performed in
which either spinal cord or blastema was
transplanted alone. In the former, the only
foreign tissue in the fin was the transplanted piece of spinal cord. Blastema by
itself responded as previously reported (see
Pietsch, '61b), namely by producing limb
cartilages but no skeletal muscle fibers.
These experiments shall not be considered
The term blastema is used to denote the
cone of new tissue observed under the dissecting microscope between the fifth and
sixteenth days following limb amputation.
Under conditions of this laboratory regeneration is usually complete within a
month's time, but definitive tissues supplant blastema cells by about day 16.
Cartilage developed in each of the specimens examined. There was no indication
that spinal cord enhanced or impaired
ability of blastema cells to produce limb
Transplanting 6-day blastema (blastema
developed 6 days after amputation) in company with spinal cord resulted in development of mature skeletal muscle fibers in
approximately one-third of the cases (see
Myogenesis following transplantation of blastem1
plus spinal cord
Age of
in postamputation
table). In experiments with spinal cord
and 7-day blastema, 12 of 29 cases exhibited newly differentiated skeletal muscle. Blastemata of more advanced age at
time of transfer to the dorsal fin chamber
invariably produced muscle fibers in response to spinal cord (see table, text fig. 1).
The 6- and 7-day cases lacking in muscle were re-examined at high magnification. Three specimens in each series
showed an occasional myofibril-containing
cell in the immediate vicinity of newly differentiated limb cartilages. Several re-examinations failed to reveal even this sign
of muscle in the remaining cases (note last
column in table).
Fig. 1 Low power photomicrograph showing spinal cord and blastema-derived skeletal
muscle (lower left). In the operation fin epithelium (note arrow) was punctured and gelatinous fin connective tissue reamed out to accommodate blastema and a n anterior segment
of spinal cord. In this specimen a transplanted 11-day blastema produced the muscle seen
in the photograph. There is a small, two or three cell focus of cartilages (lower arrow) in the
muscle q a s s . Antibrachial skeletal elements were prominent in other sections of this specimen (see fig. 2 ) .
Multinucleated fibers in the transplants
were narrow, cross-striated bundles of myofibrils similar in appearance and size to
muscle fibers in newly regenerated larval
limbs. All regions of the sarcomere were
Rough estimates were made of amounts
of muscle relative to that found in normal
limb regenerates. Lacking other criteria
for comparing transplant and normal regenerates, it was postulated that amount
of cartilage would bear an approximate relationship to overall limb size. Specimens
were chosen in which antibrachial cartilages appeared in transverse section. Images were projected onto standard index
cards at 70 X and areas of muscle a-nd
cartilage traced. Traced areas were cut
out, washed in acetone, dried and weighed.
Muscle-cartilage ratios were computed
from these weights. In a case such as that
photographed in figure 2, the relative
amount of muscle is about 75% that found
in a normal regenerate of the same age.
Further quantification of data concerning
volumes of muscle was not possible owing
to absence of suitable frame of reference.
However, the crude estimates employed
indicated that amounts of muscle ranged
from about 40 to 100% of normal.
Muscle fibers that differentiated in transplant assumed any of three relationships to
newly developed cartilages: ( a ) uniformly
distributed around cartilages (figs. 2 and
4 ) ; (b) concentrated in one to several
clumps at one side of cartilages (figs. 3
and 5 ) ; ( c ) in clusters of varying sizes
located 50 to 300 p away from cartilages.
Distribution of the fibers could not be predicated on the basis of age of blastema at
transplantation. It is also noted that combinations of the three mentioned configurations were sometimes encountered (as in
the case represented in text fig. 1 and
fig. 2).
While appreciable in amount, and
though sometimes parceled into two or
more clumps, in not a case was there an
arrangement of muscle tissue according
to the patterns that characterize forelimb
musculature (compare figs. 2-5 with those
in Piatt, '57; see also fig. 1in Pietsch, '61b).
In the experiments comprising this study
blastema cells isolated from stump tissues
developed multinucleated skeletal muscle
fibers as a consequence of unknown spinal
cord influences. Without spinal cord in the
environment of an isolated blastema myogenesis failed (see Pietsch, '60b, '61b). On
the other hand, if a narrow band of stump
were left attached to the blastema, or if the
aggregation were relocated in a site containing muscle, myogenesis then would
proceed (see also Pietsch, '60a). In limb
regenerates muscle differentiated in the
presence of pre-existing muscle bears the
pre-existing morphological organization; if
muscle in the environment is limb-like, the
new muscle pattern is limb, if non-limb the
new muscle tissue is arranged non-limb,
and, if mixed, regenerate fibers are correspondingly mixed in their interrelationships (Pietsch, in press). However, muscle produced by blastema cells in response
to spinal cord failed to exhibit a plan of
distribution found anywhere in the limb.
Thus, the net result of introducing blastema to spinal cord or pre-existing muscle
is similar from the standpoint of tissue
produced, but different morphogenically.
This fact introduces reasonable doubt that
the cells which responded to spinal cord
would have produced limb musculature
had regenerates differentiated in their normal locations. It is inferred from this infoimation that specific myogenic mechanisms in spinal cord are not identical to
those mediated by pre-existing muscle.
Though there is good evidence that muscle plays an important role in regenerative
myogenesis (see also Holtzer, '56 for tail
regeneration) and despite atypical morpliogenesis observed in this study, any proffered explanation of limb regeneration
must take into account the presence of
myogenically competent cells among the
blastemal aggregation.
Using different mesenchymal systems,
several other workers have observed
marked increase in myogenesis as a response to spinal cord (see reference in introduction). These workers concur that
the effect is enhancement of proliferation
in contradistinction to de nouo production
of myoblasts. Experimental conditions
necessary to decide on this point would
have to satisfy the following: ( a ) the test
system must be free of cells with myofibrils
or contractile protein molecules; (b) the
system must contain myogenically competent cells; i.e., cells able to form muscle
given appropriate circumstances; ( c ) the
system must not give rise to muscle spontaneously under conditions of isolation.
These conditions may have been met in the
present study. De Haan ('56) and Laufer
('59) have demonstrated immunologically,
that contractile proteins are absent from
the blastema until relatively late in development. Hay ('59), with electronmicroscopy,
has noted absence of mgofibrils in early
stage aggregations. Considered in light of
the evidence just mentioned it would seem
that spinal cord stimulated de nouo myodifferentiation among certain blastema
cells. An important question appears to
be, what constitutes a myogenically competent cell? It is doubted that such a cell
is developmentally indifferent even in the
embryo (e.g., see evidence of Holtzer and
Detwiler, '53). Electron microscopy and
treatments with antibodies against actin or
myosin give excellent phenomenological information. However, neither method has
provided evidence that parent cells of muscle are potentially unspecialized or developmentally uncommitted. It seems quite possible that blastema cells which formed the
muscle observed in this study possessed
the essential requirements for myogenesis
but that it took something like increased
proliferation to "trigger" synthesis of new
contractile protein molecules.
Removing the 6-day blastema to exclusion of all subjacent stump tissue must be
questioned. An attempt was always made
to leave some blastema cells on the stump
as a means of checking against contamination. If this were not always achieved as might well have been the case owing to
small size of the blastema at 6 days - then
it is quite possible that muscle formed from
6-day blastema came from myoblasts of
stump origin. Contamination, however,
does not explain the rise between days 6
and 7 in number of cases exhibiting skeletal muscle.
Because spinal cord does seem to stimulate mitotic activity (see Overton, '55) and
because mitosis is an important concomitant of muscle regeneration elsewhere (see
Pietsch, '61a) it seems worth considering
the possibility that chemical events leading
to myofibril formation are related to cell
division, perhaps to alterations in division
rates. Pietsch ('61a), working with mammalian skeletal muscle regeneration, obtained evidence indicating altered mitotic
rates in wound coagulum cells prior to myotube formation. When these more rapidly
dividing cells were arrested in division
(with colchicine) regenerating myotubes
did not develop.
In order to test for the presence of myogenically competent cells in the regenerating limb blastema, aggregations of varying
ages were transplanted to the dorsal fin in
company with segments of anterior spinal
cord. Histological examination, 30 days
later, revealed mature skeletal muscle fibers in experiments with blastemata of all
ages. This was true in 5 of 16 cases using
6-day blastema, 1 4 of 29 experiments with
7-day blastema, and in every case using a
blastema of more advanced stage. Pattern
of fibers did not suggest limb morphology.
While myogenically competent cells appear
to contribute to the blastema, doubt exists
that these are the same cells which would
have produced muscle in the normal limb
Avery, G., M. Chow and H. Holtzer 1956 An
experimental analysis of the development of the
spinal column. V. Reactivity of chick somites.
3. Exp. Zool., 132: 4 0 9 4 2 6 .
De Haan, R. L. 1956 The serological determination of developing muscle protein in the
regenerating limb of Amblystoma mexicana.
Ibid., 133: 73-86.
Hay, E. D. 1959 Electron microscopic observations of muscle dedifferentiation in regenerating
Amblystoma limbs. Develop. Biol., 1 ; 555-585.
Holtzer, H., and S. R. Detwiler 1953 An experimental analysis of the development of the
spinal column. 111. Induction of skeletogenous
cells. J. Exp. Zool., 123: 335-370.
Holtzer, H., S. Holtzer and G. Avery 1955 An
experimental analysis of the development of the
spinal column. IV. Morphogenesis of tail
vertebrate during regeneration. Ibid., 96: 145172.
Holtzer, S. 1956 The inductive activity of the
spinal cord in urodele tail regeneration. J.
Morph., 99: 1-39.
Laufer, H. 1959 Immunochemical studies of
muscle proteins in mature and regenerating
limbs of the adult newt, Tritzirus uiridescens.
J. Embryo]. exp. Morph., 7: 4 3 1 4 5 8 .
Muchmore, W. B . 1958 The influence of embryonic neural tissues on differentiation of
striated muscle in Amblystoma. J. Exp. Zool.,
139: 181-188.
Overton, J. 1955 Mitotic responses i n amphibian epidermis to feeding and grafting. Ibid.,
130: 433-484.
Piatt, J. 1957 Studies on the problem of nerve
pattern. 111. Innervation of the regenerated
forelimb in Amblystoma. Ibid., 136: 229-247.
Pietsch, P. 1960a The development of muscle
and cartilage i n deplanted regenerating limb
blastemas of Amblystoma larvae. Diss. Abstr.,
31: 723.
1960b The differentiation of limb blastemas deplanted into the dorsal fin of Amblystoma. Anat. Rec., 136: 258.
1961a The effects of colchicine on regeneration of mouse skeletal muscle. Ibid., 139:
1961b Differentiation i n Regeneration.
I. The development of muscle and cartilage following deplantation of regenerating limb blastemata of Amblystoma larvae. Develop. Biol., 3:
Weiss, P. 1950 The deplantation of fragments
of nervous system jn amphibians. I. Central
reorganization and the formation of nerves. J.
Exp. Zool., 113: 397-462.
Antibrachial cartilages surrounded by a n uninterrupted collar of skeletal muscle tissue. Specimen is the same one photographed in text
figure 1, but through a different section. In this case, a n 11-day blastema was transplanted along with spinal cord. Spinal cord is located
several hundred microns from this section. Muscle is in direct contact
with fin connective tissue (Fin C.T.). Transplant epithelium did not
survive. Magnification 100 X.
3 Cartilage and muscle that developed from a 6-day blastema transplanted in company with spinal cord. Muscle occupies the right and
center of the photograph and assumes the form of a single massive
clump. Fibers come into contact with piece of cartilage left of photograph at a n angle of about 45'. This tends to accentuate muscle
nuclei giving mass a denser appearance than is actually the case.
Fiber diameters are of about the same magnitude as those in the intact
limb. Magnification 100 X.
Paul Pietsch
Cartilages (arrow at left and lower center) wrrounded by massive
sworl of skeletal muscle. Muscle and cartilages were derived from a
transplanted 7-day blastema. Spinal cord occupies the upper right
hand quadrant of photograph. Magnification 100 X.
Cartilage and muscle derived from a transplanted 11-day blastema in
response to presence of spinal cord. The material under the black
lines is epithelial debris, which like the cartilage in center of field, is
surrounded by sworling tufts of skeletal muscle fibers. Fin epithelium
may be seen at lower and left edges of photograph. Magnification
100 x.
Paul Pietsch
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spina, skeletal, limba, muscle, amblystoma, cord, differentiation, larvae, blastema, regenerative, influence
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