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Effects of blastema transplantations on regeneration processes of limbs in Amblystoma larvae.

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EFFECTS O F BLASTEMA TRANSPLANTATIONS ON
REGENERATION PROCESSES O F LIMBS
I N AMBLYSTOMA LARVAE
OSCAR E. SCHOTTE AND NARGARET HARLAND
Department of Biology, Amherst College, Massachusetts
EIGHT FIGURES
INTRODUCTION
Studies of regeneration in both Urodela (Butler, '33;
Puckett, '36 ; Thornton, '38a and '38b) and in Anura (Schott6
and Harland, '43a) have clearly shown that amputation of a
limb elicits profound changes in the tissues at or beneath the
amputation surfaces. There is first an important macroscopically invisible phase, the phase of dediff erentiation, consisting essentially of a loss of histological characteristics of
all the formed elements of the limb. With the appearance of
the blastema, composed mostly of dedifferentiated elements
from the amputation area, the macroscopically visible phase
of regeneration begins, characterized by growth and differentiation of new structures.
On the strength of convincing experimental evidence, Butler
and Puckett ('40) have suggested that in some way, the
appearance of a blastema causes the dedifferentiation processes to subside. Conversely then, the absence of a blastema,
should result in disturbances in the cellular equilibrium between dedifferentiation and regeneration proper. Such a condition was obtained in experiments on urodele larvae (Schott6
and Butler, '41; Butler and Schott6, '41) and on anuran tadpoles (8chottG and Harland, '42 and '43b), where macroscopical and histological observations concurred to show that
no blastema ever forms in nerveless amputated amphibian
165
166
OSCAR E. S C H O T T ~AND MARGARET HARLAND
limbs. Consequently, if after amputation, limbs were maintained nerveless for any length of time, progressive dedifferentiation took place resulting in resorption of entire limb
segments and, in some extreme cases, even of whole limbs.
Related to these facts is the interesting discovery of Thornton
('43) that an agent which stops proliferation, such as
colchicine, is also able to prevent blastema formation, the
result being continuous dedifferentiation with subsequent
resorptions.
After these histological studies had enabled the above
authors to correlate the normal sequence of the early phases
of regeneration with the presence of a blastema, an experimental demonstration of such a correlation was provided
by the transplantation of young blastemas on denervated
amputated limbs of larval Urodela (Schott6, Butler and Hood,
'41). I n these experiments the authors were able to show that
a young blastema transplanted on a freshly amputated and
denervated limb stump actually prevented the mentioned extreme dedifferentiations.
However, the positive outcome of the above experiment
could offer only suggestive evidence for an interpretation of
the mechanism of action of a transplanted blastema. For all
we know, the transplanted blastema might produce the same
effect in many different ways, possibly totally unrelated to
the dedifferentiation phase of regeneration. In the absence
of adequate histological confirmation it was not possible to
determine whether the blastema had merely slowed down the
dediff erentiation processes or suppressed them altogether.
Furthermore, denervated limbs representing, as it were
pathological conditions differing extremely from those found
in normal limbs, might not have been too happy a choice for
the experimental investigation of the role of the blastema
as a controlling agent of the early dedifferentiation processes.
If Butler's interpretation of the role of the blastema as an
agent regulating the extent of dedifferentiation in normal
regeneration reflects actual processes, then it was to be espected that the transplantation of a young blastema onto a
E F F E C T S O F BLASTEMA T R A N S P L A N T A T I O N S
167
normal limb immediately after amputation should so interfere with the early phases of regeneration as to cause the
suppression of the dedifferentiation phase altogether. F o r
that reason, the method chosen to test anew Butler’s hypothesis was the autoplastic or homoplastic transplantation of an
early limb blastema on freshly amputated limbs combined with
a systematic histological investigation of its effects on the
tissues of the stump.
MATERIALS A N D METHODS
The material used for this research consisted chiefly of
young Amblystoma larvae collected from ponds in the vicinity
of Amherst, and of a few larvae of A. opacum raised from
eggs in the laboratory. At the time of operation, these larvae
varied in length from 18 to 55 mm., the majority of the experimental specimens measured, however, from 25 to 35 mm.
Blastemas were secured either from another limb of the host
itself, or from limbs of a different donor : in autoplastic transplantations blastemas were secured from the zeugopode region
of a previously amputated right limb; in homoplastic transplantations, either a fore or a hind limb amputated several
days previously served as blastema donor. The age of the
transplanted blastemas varied from 5 to 9 days.
Operative t e c h w i p e . Both larvae, the donor with its
blastema and the host, under narcosis in M.S. 1:5000, are
placed next to each other in a 15-cm. petri dish, on gauze
moistened with a mixture of M.S. solution and saline. All
the operations are, of course, performed under the dissecting
microscope. The left arm of the host animal, together with
the right control arm, are then amputated through the stylopode at the same rather distal level. The blastema transplant
is rendered more visible by staining it slightly in a Nile Blue
Sulfate solution about 5 minutes before the transplantation. We have found that this superficial stain with Nile
Blue is sufficiently strong to enable the distinction of the
transplant from the host tissue for the duration of the experiment (not longer than 9 days).
168
OSCAR E. S C H O T T ~ AND MARGARET HARLAND
The blastema is removed from the donor limb with iridectomy scissors, and placed on top of the amputated stump
with the aid of watchmaker forceps. I n order to maintain
the transplant in position, so as to allow its healing to the
fresh amputation surface, the left upper arm is kept for
several hours in a n upright position with the aid of small
pieces of silk thread serving as supports. It is advisable, at
this stage, to expose limb and transplant to the warmth of the
operation lamp until limb and blastema shrink slightly through
drying. After this the animal, still narcotized in half strength
M.S. solution, is kept for at least 24 and better 48 hours, in
the refrigerator a t about 10°C. The larvae are then released
and examined under a dissecting microscope for the presence
of the transplant. The operation is successful in about 40%
of the cases.
Fixation for histological study was done between 3 and 9
days after the transplantation of the blastema and the amputation of the right control limb. Sections were made at 7 mu,
and always stained in Harris’ Hematoxylin, with Orange G
as counterstain.
EXPERIMENTAL RESULTS
I n an experiment in which the action of a transplanted
blastema on the tissues of the amputation surface is being
investigated, it is of first importance t o determine the nature
and the intimacy of the contact ultimately effected between
the transplant and its supporting stump. I n that respect
several possibilities can arise: (1) the blastema covers the
amputation surface only partially ; (2) the blastema, originally
well placed, has subsequently become displaced ; (3) the blastema becomes resorbed after a short time; (4) the blastema,
although fitting the amputation surface perfectly at first, does
not succeed in taking root and is eventually displaced by a
new blastema; ( 5 ) the blastema fits perfectly, remains in that
position for the duration of the experiment and does not
become resorbed.
EFFECTS O F BLASTEMA TRANSPLANTATIONS
169
Roughly, all these possibilities can be included in two categories-the partially successful, (points 1-4) and the wholly
successful transplantation experiments (point 5 ) .
I , Effects of blastemas covering the arnputatiou
surface partially or only temporarily
There were nineteen cases in which the above poor fits
or ultimate resorptions of blastemas occurred. From opr
records and from the original camera lucida drawings, the
following examples can be briefly described.
1
2
Fig. 1 Camera lucida drawing of left upper arm from a 25-mm. Amblystoma
opacum larva 3 days after autoplastic transplantation of an 8-day-old blastema
Fig. 2a and 2b Camera lucida drawings of left upper arm from a 24-nun. A.
punctatum larva. a, 3 days after autoplastic transplantation of 8-day blastema ;
b, 3 days later, blastema in the process of being walled off.
I n case H B 3 represented in figure 1, it will be seen that
the blastema is fixed on the dorsal side of the protruding
humerus, covering neither the whole of the amputation surface nor the amputated humerus.
I n the two successive drawings of case H B 13, another
possibility is presented. Here at first (fig. 2a) the transplantation seemed perfectly successful, the whole of the amputation surface being covered by the blastema, and the shaft
of the humerus apparently situated in the midst of the
blastema tissue. Two days after the operation, however
(fig. 2b), the transplant acquired a spherical appearance, and
170
OSCAR E.
SCHOTTB
AND MARGARET HARLAND
became eventually constricted at its base by the healing
over epidermis of the amputation surface. Although no
further modification in the relationship between blastema
and amputation stump was observed, it could be seen, on
section, that no fusion between the proximal tissues of the
blastema and the old tissues of the stump had occurred. Moreover, the blastema epidermis had rounded up into a perfect
sphere separating the transplant entirely from the tissues
of the stump.
Fig. 3a and 3b Camera lucida drawings of l ef t arm of 22-mm. A.
punctatum larva. a, 3 days after homoplastic transplantation of 8-day blastema;
b, 2 days later.
I n the case of HB 16 anotber possibility can be illustrated.
Here, the blastema at first fitted (fig. 3a) perfectly over the
amputation surface and over the amputated humeral shaft,
but due to the usual contractions, after amputation, of the
soft constitutents of the limb, the transplant was drawn away
into a lateral position, causing the shaft of the humerus to
break through the tender epidermis of the blastema (fig. 3b).
On section it was foufid that 7 days after the experiment, the
humerus remained still free of epidermal covering.
I n case HB 103, the resorption of a well-situated transplant
can be observed. Figure 4a shows the usual aspects of a
successful transplantation, but 3 days later (fig. 4b) we found
an extraordinary regression of the transplant which we
attribute to some injury. Eventually, the blastema became
E F F E C T S O F BLASTEMA TRANSPLANTATIOKS
1i1
Fig. 4 a 4 c Camera lucida drawings of 3 stages of autoplastic transplantation
of 7-day blastema on left upper arm of A. punctatum larva of 23 mm. a, 2 days
after transplantation; b, 2 days later; c, 9 days after transplantation.
difficult to distinguish, and the usual phases of dedifferentiation could be seen through the transparent tissues to occur
beneath the receding transplant. As a result, 9 days after
transplantation (fig. 4c) a well formed blastema was seen, in
no way different from the one which had developed on the
simply amputated control limb. There is little doubt that,
in this case, an entirely new blastema was formed from the
stump and that the transplant vanished totally.
A final example of an only partially successful blastema
transplantation was observed in case HB 112. The originally
large transplant of figure 5a was seen to retract considerably
4 days later (fig. 5b). From then on, however, the easily
visible Nile Blue stained transplant remained unmodified for
several days. When 7 days after the transplantation, a last
Fig. 5a-5e Camera lucida drawings of 3 stages of homoplastic transplantation
of 7-day regenerate on left upper arm of a 31-mm. A. punctatum larva. a, 2 days
after transplantation; b, 2 days later; c, 7 days after transplantation.
172
OSCAR E. S C H O T T ~ AND MARGARET HARLAND
drawing of this case was made (fig. 5c), the still distinguishable transplant was found situated over the tip of a much
larger regenerate, which meanwhile had formed, obviously,
from the amputation tissues.
The other fourteen cases included in this section all enter
into one or the other of the above categories, and they all
showed the same effects-the Mastema either grew in sidewise or was shifted subsequently into a sidewise position, or
else it became partially or completely resorbed. In each instance the stu*mp tissues had formed another, entirely new
blastema, except in those rare cases in which the humerus
was protruding through the epidermis.
The histological verification of all nineteen cases showed
invariably the same picture, namely that the dedifferentiation
processes were found to be quite identical in both limbs. I n
other words, misplaced or readily resorbed transplanted
blastemas do not exert any influence whatsoever on the early
regeneration phases of the tissues in the amputation area.
I I . E f e c t s of successfully transplamted blastemas on
dediferentiation processes of amputated 2imbs
I n twenty cases the transplanted blastemas not only covered the amputation surface entirely, but they also remained
attached to the stump without appreciable resorption until
the end of the experiment (from 5 to 9 days).
Since most of our larvae were young, many changes within
the limb, after amputation and blastema transplantation,
could be observed on the living limb as a result of its transparency, as previously described by Schott6 and Butler ( '41).
The differences between the two limbs, discernible particularly in their humeri, were especially striking in the case of
HB 79, (autoplastic transplantation of 8-day regenerate on
left limb, right limb simply amputated), figures 6a to 6f,
(see explanations of figures).
It can be seen clearly from these figures that while in the
right limb, without a transplant, the dedifferentiation processes were progressive up to the ninth day, in the left limb,
EFFECTS O F B L A S T E M A T R A N S P L A N T A T I O N S
\
a
173
h
f
Fig. Sa-Gf Camera lueida drawings of left upper arm with transplant
(a, G and e ) and of amputated right control arm (b, d a n d f ) in a n Amblystoma
punetaturn larva of 30 mm. a, left limb 3 days after transplantation; b, right
limb 3 days a f t e r amputation; the dots a t the t i p indicate either small bload
sinuses or beginning dedifferentiation processes; c, left limb with blastema 5 days
after transplantation; d, right control limb 5 days a f t e r amputation in the
midst of active dedifferentiation processes with beginnings of blastema formation ;
e, blastema on left limb, 7 days a f t e r transplantation shows traces of beginning
differentiation ; f , right limb 7 days a f t e r transplantation showing emptied
humeral shaft, wavy perichondral sheath and well-developed blastema.
174
OSC.\I:
E. S C H O T T h AX11 MARGARET H A R L A N I )
tlie arriputation surface of which mas covered with a blastema,
110 similar breaking down of the humerus was observed. The
comparison between right and left is particularly noticeable
5 and 7 days from the beginning of the experiment. Ninc
clays after amputation, ivliile tlie humeriis of the right limb
~ ) i * c w n superficially
ts
tlie aspect of a liollow tube, on the left,
tlie shaft of the humerus, protiwding into tlie blastema, sl~oms
oiily the slightest signs of dedifferentiation. The histological
investigation confirms the above morphological observations
completely. Tliere a r e only very slight changes in tlic aspect
of the cartilage at the distal tip of the left humerus, ~ l l i l e
iicai.ly the whole of the perichondral shaft of the control
liiime~~ns
was empty matrix and showed aspects very similar
t o tlie ones represented in figure 8 of the next case.
In case IIB 104 (Amblystoma purictatum larva, 32-mm.
ti~aiisplantatioiiof a i-day regenerate, fixation of both limbs 8
c h i p after ti.ansplantation and amputation), tlie records of
tlie drawings of both limbs of this case indicate optimal conditions for tlic experiment-a
fairly large blastema 1)laced
squarely on top of the amputation surface of the left limb
and remaining there for the duration of the experiment; on
llie control side, the young blastema became apparent at the
usual sixth day after amputation. A t the time of fixation,
the transplanted hlastema had elongated a little, but it was
ascertained from its still persisting Nile Blue stain that tlie
traiisplant was not replaced by a new blastema.
A comparison of the sections is instructive. In figure 7
the left limb shows the presence of a still undifferentiated.
although quite large, blastema. The extreme distal tip of the
liumeral shaft is not totally devoid of features occurring in
early dedifferentiation processes, but this affects only a small
fraction of the humerus. The rest of the cartilage within the
perichondral bone is still firmly embedded in its own matrix,
no conspicuous vacuolation had occurred, and in all other
i ~ s p e c t sthe skeletal structures a r e rather normal. On the
right side (fig. 8), on the contrary, the humerus presents an
entirely different aspect. This structure has been practically
EFFECTS O F BLASTEMA TItANSP1,ANTATIOSS
175
emptied of its cartilaginous coiltent, tlie matrix has bccii
dissolved along its whole length, arid tlie cells visible in tlic
middle of the cnipt;v pei*icliondral slicatli a r e flee cells \vliicli
to all intents and 1)urposes have lost tlic characteristics of
cartilagiiions cells. The emptying of tlie humerus is furtlicr
iiiidcrlinecl by the wavy structure of the pericliondral sheath,
which has obviously lost its iiinei* bupport. h i brief, tliis
Fig. i
Fig. X
Longitutlinal section of left liiiil) of w s t ' HI3 104. X 60.
Loiigitntliilal seetioil of riglit control linib of case HI: 104. X 60.
liumcixs represents tlie typical features of a skeletal structure
in tlie iiiidst of dedif'ferentiation processes, a s was so well
described by Butler in '33 and again in '41 (Butler and
Kchottd).
Anioiiq tlie twenty cases there '~vei'c seven in which tlie
caompai-isonbetween tlic limb carrying a well placed blastema
arid tlic simply amputated control limb, presented similar
very striking differences, in their degrees of dedifferentiation.
I n seven otlier cases the compai*isoii between the dedifferen-
1'76
OSCAE E. S C H O T T ~ A N D MARGARET HARLAND
tiation phases of the two sides of the animal was suggestive,
altliougli not altogether conclusive. Finally, in six cases, no
noticeable differences could be observed, 011 section, iii limb
stump tissues adjacent to a blastema transplant as compared
to those of the control limb.
The careful investigation, from oui' records, of possible
( w u s e s wliicli would explain the alxence of ariy modification
iii tlie dedifferciitiation phases on limbs carrying a I>lastema
ti*ansplant was not very satisfactory. In all these cases, it is
true, the blastema varied in age, but this was true also of the
m o w successful cases.
There are, liowevei*, many factors wliicli might affect the
ti~ansplants.There is, f o r instance, no means of ascertaining
a t what precise moment tlie transplanted blastema took root
on the amputation stump. It is common observation that the
amputation surfaces of different individuals present different
amounts of hemorrhage, and often oiic finds 011 section
large blood sinuses which could, in our opinioii, interfere successfully with the early fusion of tlie traiisplanted blastema.
'lire must consider, also, the possible action of lilastcmas of
clifercnt ages, obtained, in addition, from donors of various
oiigins and ages.
Anotlier cause of error, to wllicl1 not enougli attention mas
])aid at the tinie these operations were performed was discovered while studying the sectims. In several cases, pieces
of debris of humeral shaft which obviously liad been removed
from the donoi. a r m \{-ere found a t tlie basis of the blastemw
ti*ansplant. While perichondral pieces are easy t o detect, it is
mucli more difficult to become aware of possible timisplaiitations of the soft constituents of the limb, R fact which thus
multiplies the causes of error. F r o m what we kriow a t preseut
it is unlikely that a blastema, with an al)preciable amount of
c.lenients from its own base, would affect tlie dediffei-entiation
pi-ocesses of the stump on
it was timisplanted. Tndeetl,
Scliott6, Butlei. and Hood ( '41) have shown that transplanted
older blastemas showing beginnings of histogenetic and morpliogenetic differentiations a r e unable to check dediflf'erentia~7liicli
EFFECTS O F BLASTEMA TRANSPLANTATIONS
17i
tion processes of denervated amputated limbs. Moreover, when
placed on a stump in the process of active regression, they
become involved in the resorption processes of their new base
and ultimately disappear themselves.
CONCLUSIONS
From the foregoing experiments it can be seen that while
the influence of badly fitting or resorbing blastema transplantations was, in nineteen cases, absolutely nil with respect to
the dedifferentiation phases on the neighboring stump, in the
twenty cases in which the blastemas were found to fit the
amputation surface, pronounced effects were obtained in over
two-thirds of the cases. Although these effects were not identical in all the positive cases, in a third of them the differences
in the extent of dedifferentiation in limbs with a transplant
as compared to a limb without a transplant were so pronounced that no doubt in the mind of the observer was left
as t o an effective interference with the normal regeneration
processes.
It is noteworthy that among the unsuccessful transplants
described in the first section, there mere many cases in which
the blastema a t first fitted well over the amputation surface,
and was only subsequently displaced. I n six other cases of that
series, there were excellent fits, but the blastemas became
resorbed precociously. I n these instances no effects of the
transplant were observed. This seems to indicate that the
expected action of the blastema cannot manifest itself if
merely a transitory contact has been established and that,
furthermore, perfect contiguity between cells of the blastema
and the tissues of the amputation surface must exist.
Such conditions, detectable in some cases, undetectable or
not understood in others, point to the probable explanation
of cases described in the second section in which apparently
healthy blastemas did yet not exert any appreciable effect on
the dedifferentiation processes of stumps on which they were
perfectly well placed.
178
OSCAR E. S C H O T T ~AND MARGARET HARLAND
Whether or not the negative cases of these series can be
accounted for by these hypothetical causes, there is no doubt
that the problem of the role of the blastema as a controlling
agent of the early phases of regeneration deserves further
attention. New experiments with improved techniques are
being undertaken a t present in this laboratory to clarify this
difficult and vastly important question.
In spite of the above mentioned reservations and even
though this research is considered to be only in an advanced
stage of preliminary investigation, these experiments strongly
suggest that, under ideal conditions, a well placed young
blastema is capable of actually suppressing the dedifferentiation phase altogether. Butler’s view of the role of the blastema
as an agent regulating the cellular interactions of early regeneration thus finds new confirmation.
SUMMARY
The influence of the blastema on the dedifferentiation phase
of early regeneration was studied by the method of autoplastic
and homoplastic transplantation of young blastemas onto
freshly amputated limbs of Amblystoma larvae. The blastema
was transplanted to the stump of the distal or middle upper
arm of the left limb, the right arm, without a transplant,
being simultaneously amputated for control purposes. After
study of the behavior of the transplanted blastema and systematic observation of the macroscopical changes in both limbs,
the larvae were fixed for histological comparison of the two
limbs.
The results differed sharply according to the way the transplanted blastemas established themselves on the amputation
surfaces of the limb: a ) I n nineteen cases in which the
blastema transplant covered the amputation surface only partially, became ultimately displaced or resorbed, no differences
could be detected in the dedifferentiation phases of the two
limbs. I n other words, misplaced or resorbing blastemas do
not influence the dedifferentiation processes at all. b ) Tn
the twenty cases in which the blastemas fitted the amputation
EFFECTS OF BLASTEMA TRANSPLANTATIONS
179
surface perfectly and remained there for the duration of the
experiment noticeable effects were observed in more than two
thirds of the cases; in seven cases, particularly, rery conspicuous differences in the status of dedifferentiation of the
tissues of the stump were observed on the side carrying a
blastema as compared with the simply amputated control limb.
I n six cases, however, the successful transplantation of a
blastema seemed not to affect the extent of the dedifferentiation processes of the amputated limb. Possible causes affecting the results in the negative cases are being discussed.
I n spite of the fact that some experimental conditions still
escape control, it is concluded that a young blastema transplanted onto a freshly amputated limb is capable, by its presence, of actually suppressing the dedifferentiation phase of
regeneration.
LITERATURE CITED
BUTLER,
E. G. 1933 The effects of x-radiation on the regeneration of the forelimb of Amblystoma larvae. J. Exp. Zool., vol. 65, pp. 2i1-303.
Bu’rLm, E. G., A N D W. 0. PUOKETT
1940 Studies on cellular interaction during
limb regeneration in Amblystoma. J. Exp. Zool., vol. 84, pp. 223-239.
BUTLER,E. G., AND 0. E. SCHOTTJ?
1941 Histological alterations in denervated
non-regenerating limbs of urodele larvae. J. Exp. Zool., vol. 88, pp.
307-341.
PUCKETT,
W. 0. 1936 The effects of x-radiation on limb development and
regeneration i n Amblystoma. J. Morph., vol. 59, pp. 173-213.
AND E. G. BUTLER1941 Morphological effects of denervation
and amputation of limbs in urodele larvae. J. Exp. Zool., vol. 87, pp.
279-322.
SCHOTTE,
0. E.,
SCHOTT~,
0. E., E. G. BUTLER
AND R. T. HOOD 1941 Effects of transplanted
blastemas on amputated nerveless limbs of urodele larvae. Proc. SOC.
Exp. Biol. and Med., vol. 48, pp. 500-503.
sCHOTT%,
0. E., A N D M. HARLAND1942 Influence of spinal nerves on the regeneration of hind limbs of Anuran tadpoles. A n at . Rec., vol. 82,
p. 445. (Abstract)
1943a Amputation level and regeneration in limbs of late Rana
clamitans tadpoles. J. Morph., 101. 73, pp. 339-363.
1943b Effects of denervation and amputation of hind limbs in
Anuran tadpoles. J. Exp. Zool., vol. 93, pp. 453-493.
180
OSCAR E.
THORNTON,
C. S.
SCHOTTB
AND MARGARET HARLAND
1938a The histogenesis of muscle in the regenerating forelimb
of larval Amblystoma punctatum. J. Morph., vol. 62, pp. 1 7 4 7 .
1938b The histogenesis of the regenerating forelimb of larval
Amblystoma after exarticulation of the humerus. J. Morph., vol. 62,
pp. 219-241.
1943 The effect of colchicine on limb regeneration in larval Amblystoma. J. Exp. Zool., vol. 92, pp. 281-295.
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