/ . Embryol exp. Morph. Vol. 49, pp. 243-258, 1979
Printed in Great Britain © Company of Biologists Limited 1979
243
Duplicated axolotl regenerates
By H. WALLACE 1 AND A. WATSON 1
From the Department of Genetics, University of Birmingham
SUMMARY
Three series of palette stage regenerates were prepared by amputating both arms of
juvenile axolotls in the mid-forearm, above the elbow, or close to the shoulder. Within each
series, excised regenerates were replaced in their original orientation (as a control) or rotated
at 90° intervals about the proximodistal axis, or were transplanted to the contralateral arm
with identical rotations. Control grafts occasionally caused the formation of a single extra
digit. AH experimental rotations provoked duplicate or triplicate structures, ranging from
accessory digits to extra forearms. Shoulder level grafts were subject to a pronounced correctional derotation and yielded a variable proportion of duplications. Forearm and elbow-level
rotations invariably yielded duplications, which therefore result from an axial discrepancy
rather than complete axial opposition between graft and host. These results are incompatible
with the clockface model of positional information and demand a modification of other
current models. The recorded frequency and orientation of duplications suggest that a limb
contains at least two polarized transverse axes which cannot be respecified during regeneration. A substantial discrepancy on either axis reduces the normal regulative interaction
between graft and host, allowing either of them to regenerate independently.
INTRODUCTION
Considering that the determination of transverse axes in amphibian limbbuds and regenerates was established fairly reliably some 50 years ago, there
has been a remarkable resurgence of interest in the subject lately. Axial determination has usually been tested by rotating tissue through 180° to reverse an
axis relative to that of adjacent tissue. Either the anteroposterior (AP) or
dorsoventral (DV) axis can be reversed by transplanting a limb-bud orthotopically to the contralateral flank; both axes are reversed (APDV) by replacing
it in its original site after 180° rotation. Regenerates can be transplanted to limb
stumps to effect the corresponding axial reversals. By means of such transplantations, Harrison (1925) and Swett (1927) demonstrated the successive
determination of AP and DV axes in the limb primordium and Lodyzenskaja
(1930) showed that these axes remained determined throughout regeneration.
In addition to the basic criterion of determination, that the graft should maintain its original axes and thus appear disharmonic to its new surroundings,
these experiments revealed that an axial reversal usually provoked the growth
1
Authors' address: Department of Genetics, University of Birmingham, PO Box 363
Birmingham B15 2TT U.K.
244
H. WALLACE AND A. WATSON
of accessory limb structures which duplicated the graft. Since the control
operation of replacing a limb-bud or regenerate without rotation practically
never caused such duplication, it has become an accepted practice to assess
axial confrontations by the production of duplications (e.g. Abeloos & Lecamp,
1931). This latter criterion permits axial determination to be tested in different
tissues of the limb, as explored in recent studies. Axial reversal of limb skin or
muscle prior to amputation through the grafted tissue often results in a partially
duplicated regenerate, variously interpreted as indicating a dominant AP axis
(Carlson, 1974, 1975), AP and DV axes of equivalent status (Lheureux, 1972,
1975) or the presence of additional polarized transverse axes (Lheureux, 1977).
Axial reversals of growing regenerates and mature hands have led Bryant &
Iten (1976, 1977) to abandon the classical axes in favour of a more abstract
'clockface' model, which postulates a continuous gradation of positional values
around the limb circumference.
The consistent occurrence of duplicated and triplicated regenerates following
axial reversal, among four species of urodeles in the cases cited above, is somewhat obscured by other experimental differences. The principal difference is
related to the site of operation along the arm, for accessory structures may arise
close to that site or a considerable way further down and then only duplicate
distal regions of the arm. Consequently, duplications have been variously
described as fan-hands, expanded or double hands, multiple hands or supernumerary (fore)arms, although they are agreed to be merely proximodistal
modulations of the same fundamental response. This response has been interpreted almost invariably as an interaction between determined axes or positions
of graft and host which have been experimentally opposed. The several current
models of morphogenesis based on this are essentially different formulations
of the axial conflict or opposition required to provoke duplications. While
searching for some means of distinguishing between these models, we realized
their proponents had neglected to demonstrate the necessity of complete axial
reversal by testing the effect of smaller angular displacements. In fact, a 90°
rotation of a forearm regenerate had been reported to cause duplications in
adult Triturus alpestris (Schwidefsky, 1935) and does so as efficiently as 180°
axial reversals in larval Pleurodeles waltlii (Wallace, 1978). The present report
records a more extensive study of the duplications caused by rotating regenerates on the forearms and upper arms of juvenile axolotls. The occurrence of
duplications following any substantial rotation, amply confirmed here,
eliminates the clockface model and places constraints on the applicability of
more conventional models of axial determination.
MATERIALS AND METHODS
All operations were performed on late cone to early palette stage regenerates, because the orientation of their flattened tips is easily recognized, and
because previous investigators have found that their transverse axes are fully
Duplicated axolotl regenerates
90°
18O01
245
270°
Fig. 1. Diagram of the operations. S is an end-on view of a left limb stump showing
the cardinal axes AP and DV. Above it is the blastema from the same arm, b.
Ipsilateral grafts consist of replacing b on S at successive clockwise rotations at 90°
intervals shown in the upper row of figures. Contralateral grafts consist of bringing
a right arm blastema round to fit on S (so that it now has a new orientation, d)
and imposing identical rotations shown in the lower row of figures. The unrotated
control and conventional axial reversals AP, DV and APDV are thus depicted by
letters b, d, p and q; the novel rotations are 90° (palm forward) and 270° (palm back)
Stars mark the position of accessory growths predicted by the clockface model, according to the rules stipulated by Bryant & Iten (1976).
determined. Three series of regenerates were obtained by amputating both arms
of juvenile axolotls {Ambystoma mexicanwn) at different levels:
Series I, through the mid forearm of 80 mm long specimens;
Series II, just above the elbow of 80-90 mm specimens;
Series III, close to the shoulder of 90-100 mm specimens, some of which had
been used before in series I.
The specimen was anaesthetized in 0-05-0-1% MS222 and supported on
moist tissue during the operation. An entire regenerate was excised and promptly
replaced on the same arm stump or transferred to a contralateral arm stump
at a predetermined rotation, to occupy a site from which a regenerate of identical
age and level had just been removed. No exchanges were made between different
series and even minor shifts along the arm were avoided as far as possible.
Shoulder level operations could only be performed on one side at a time, and
the other arm was used 2 or 3 days later. The contralateral exchanges of
series III were thus homografts between sibs. Series I and II were all autografts
performed on both arms of specimens lying on their backs. Within each series,
regenerates were rotated at successive 90° intervals on both ipsilateral and
contralateral arm stumps to obtain ten examples for each of the eight rotational
classes (Fig. 1). The results are based on these 240 successful operations and a.
few additional homografts between dark and white specimens.
246
H. WALLACE AND A. WATSON
Initial healing of the graft occurred in air while the host was mostly covered
by moist tissue. The specimens were immersed-in 0-005-0-01 % MS222 about
an hour later and thus kept placid overnight. Protracted exposure to previously
used or stored anaesthetic solutions sometimes caused paralysis and death,
but a freshly prepared and neutralized solution proved satisfactory. After
checking graft retention and a resumed circulation 2 or 3 days later, the arms
were only inspected a few times until scored at least 6 weeks after the operation.
Most arms were preserved in formalin, stained with Victoria blue and cleared
in methyl benzoate for later analysis of the skeleton.
RESULTS
(1) General observations. Late cone to early palette stage regenerates have
a characteristic asymmetry which allows their orientation to be determined
during transplantation. The dorsoventrally flattened hand-plate is turned up
at the tip which corresponds to the first and second digits on the anterior side
of the midline. The transverse axes of the arm defined in this way bear no
constant relationship to the main body axes, as pointed out by Faber (1960).
The AP axis of the forearm is parallel to the cephalocaudal axis when the arm is
extended sideways, but perpendicular to it when the palm of the hand rests
against the flank. The AP axis of the upper arm lies in an intermediate position,
displaced by about 60° from the cephalocaudal axis. As explained in Fig. 1,
the ventral surface or palm of the regenerate was arbitrarily defined as facing
downward and the successive rotations made it face forward (90°), upward
(180°), or backward (270°) for both ipsilateral and contralateral operations.
The regenerates generally became gorged with blood 2 or 3 days after transplantation and some blood circulation could be detected in them a few days
later. Regenerates which had been replaced without rotation showed the most
rapid recovery in this respect, but even their growth was delayed for a week or
two. Most of the rotated grafts became smaller symmetrical cones before
resuming growth. When these grafts had returned to an early palette stage, 2 or
3 weeks after the operation, many had a normal appearance showing either the
imposed rotation or a relatively normal orientation ascribed to correctional
derotation. Other grafts, however, were already bifurcated with incipient
duplications which obscured their orientation. One or two accessory blastemata
usually appeared later on grafts which had not derotated completely, either
at the base of the graft or displaced towards its tip. The majority of limbs with
rotated grafts thus eventually carried duplicate or triplicate structures distal
to the site of operation, which only dictated the maximum extent of duplication.
Series I forearm grafts never produced complete duplicate hands, for instance,
but usually resulted in enlarged hands with 6-16 carpal cartilages and up to 11
digits often arranged in two or three groups (Fig. 2A). Series II elbow level
grafts produced similar partial duplications of digits and carpals (Fig. 2D), or
complete duplications of the entire hand with a distal split of the radius or
247
Duplicated axolotl regenerates
5 mm
D
Fig. 2. (A-C) Cleared whole-mounts showing different types of duplications
following 270° rotation of contralateral regenerates. (A) Complex hand after
operating in the forearm; (B) multiple forearms after operating above the elbow;
(C) multiple forearms after operating near the shoulder. (D) complex hand
following a 90° ipsilateral rotation above the elbow.
ulna, or even completely duplicated forearms (Fig. 2B). Duplications arising
from shoulder level grafts (Series III) usually originated as a distal bifurcation
of the humerus and involved the entire forearm, hand and digits (Fig. 2C). A
few of them, however, emerged lower down the arm as limited duplications of
fingers or hands. This variable extent of duplication is roughly classified here
by describing the entire complex derived from graft and host arm as possessing:
(a) a normal hand with four digits and about the typical number of nine
carpals;
248
H. WALLACE AND A. WATSON
Table 1. Numbers of arms showing different types of duplication,
from a total of ten arms at each rotation in each series
Contralateral graft
rotation
Ipsilateral graft
rotation
Type of arm
(no. of digits)
<-
i
0°
Normal
Series I - mid-forearm
Simple hand (4)
Complex hand (5-11)
Series II - above elbow
Simple hand (4)
Complex hand (5-9)
Multiple hand (8-14)
Multiple forearm (8—12)
Series III - near shoulder
Simple hand (4)
Complex hand (5-7)
Multiple hand (8-11)
Multiple forearm (8-12)
9
1
180°
90°
Pf APDV
0
10
0
10
270°
Pb
0
10
•
0°
AP
1*
9
90°
Pf
180°
DV
270°
pb
0
10
1*
9
0
10
8
2
0
1*
7
2
0
7
3
1*
4
4
0
4
5
1*
3
5
0
2
6
0
4
4
0
0
0
1
1
1
2
2
10
0
0
0
10
0
0
0
5
0
1
4
8
0
1
1
0
1
0
9
2
0
0
8
5
0
0
5
4
3
1
2
•Anatomically composite ^i-digit hands. These and all other simple hands conformed to
the AP axis of the host arm.
pf, palm forward; pb, palm back
(b) a complex hand with five or more digits, often with extra carpals;
(c) multiple hands containing two or three distinct groups of carpals and
digits;
(d) multiple forearms comprising two or three complete forearms each of
which usually ended in a normal hand. Sometimes however, one of a pair of
forearms ended in a complex or multiple hand. Table 1 uses this classification
to display the nature of the duplications encountered and thus substantiate
a point made in the introduction. A duplication may arise at any position distal
to the graft-host junction and only contains structures distal to the arm level
where it arose. In other words, categories (b), (c) and (d) of the above classification are equivalent manifestations of the response to the operation and all are
duplications by definition.
(2) Frequency of duplicated arms. Each rotational group of each series is
represented by ten arms in Table 1, which thus shows directly the relative frequencies of normal arms with4-digit hands and of the various kinds of duplicated
arms described above. Series I produced the clearest evidence of the effect of
rotation, for all these rotated forearm grafts provoked accessory digits. Apart
from two 4-digit hands which each contained two graft digits and two accessory
digits (distinguished by their orientation), all these arms could be classified
uniformly as possessing complex hands. The control operation of replacing a
Duplicated axolotl regenerates
249
Table 2. Mean number of digits present on each graft and accessory hand
Ipsilateral graft rotation
Type of
duplication
Graft digits
Complex hands
Multiple hands
Extra forearms
Accessory digits
Complex hands
Multiple hands
Extra forearms
<
Contralateral graft rotation
f
0°
90°
180°
270°
0°
90°
180°
270°
4-0*
—
—
2-7
3-5
—
2-5
40
3-8
2-2
3-2
40
2-4
3-4
40
2-9
3-2
3-7
2-3
3-2
3-6
2-6
3-2
40
21
3-3
—
2-3
3-4
40
2-2
2-4
3-7
2-7
3-6
40
2-2
3-9
3-9
2-4
3-8
3-8
2-5
3-5
40
1.0*
—
—
* The orientation of these digits does not reveal their origin, but see Section 4.
regenerate without rotation resulted in normal 4-digit hands, except for one
hand which carried a small fifth digit. Series II also provided a fairly clear
contrast between the control operation and the experimental rotations. Two
control hands possessed five digits but the rest were completely normal. All
the rotations provoked accessory structures, ranging from one or two accessory
digits flanking a reduced graft hand to large extra forearms. The majority of
these elbow-level rotations, however, produced complex or multiple hands.
Series III conformed to the previous pattern of results for the control operation,
where no duplications occurred, but normal 4-digit hands were also encountered
at most rotations performed at the shoulder. These were graft hands which had
undergone correctional derotation to match the AP axis of the host arm.
Ipsilateral grafts derotated to form apparently normal arms but contralateral
grafts assumed the orientation expected after a dorsoventral inversion, with
the hand resting palm upward. The remainder of these shoulder level grafts
showed much less derotation and provoked duplications, ranging from accessory
digits to the more typical extra forearms. The latter appeared most frequently
for contralateral grafts, perhaps only because they could not derotate to match
both postulated axes of the host arm. Derotation also occurred in series I and II
but only exceeded 60° in the few cases noted as anatomically composite hands
in Table 1.
Table 1 shows that all the rotations tested were capable of provoking duplications and suggests they were equally efficient at doing so, when the effect of
derotation is discounted. Assuming the imposed rotations were only accurate
to about 20° and were perhaps subject to further displacement during initial
healing, these results imply that any substantial axial discrepancy between host
and graft is likely to cause some form of duplication.
(3) Characteristics of duplications. The duplicated arms contained up to
three accessory structures of variable size and shape in addition to that formed
250
H. WALLACE AND A. WATSON
Table 3. Number of duplications arising at different positions marked by
the nearest pole of the host arm axes, for 10 arms at each rotation
Series I-forearm
Imposed
rotation
r
Series Ill-shoulder
Series II-elbow
r-
A
A
P
^
A
P
D V Total A
9
9
0
0
0
0
0
0
18
19
19
6
9
7
7
8
7
3
2
4
3
2
1
19
21
19
0
1
2
19
19
D
A
t
V 'Total A
1
P
D
V Total
0
1
1
0
5
0
0
1
1
0
0
1
0
7
3
3
7
10
7
0
1
0
0
13
15
3
2
3
10
1 5 3
26 10
7
11
59
Ipsilateral
grafts
90°
180° (APDV) 10 9
10
9
270°
Contralateral
grafts
10 10
0° (AP)
90°
180° (DV)
270°
Totals
8
6
7
60
8
0
0
2
20
18
9
7
8
7
1
3
8
2
1
17
2
1
4
8
15
2
8 1 2
61
3
5
18
129
6 5
46 43
3
20
4
21
18
130
2
16
N.B. The total columns approach two duplications/arm, except where reduced by
derotation in series III. The excess of A and P duplications is most marked after AP and
APDV grant rotations but is evident throughout series I.
by the graft. Counting the number of digits offered an objective means of
testing whether or not some rotation produced more complete accessory
structures than the others. This test is applicable to all three series, for the
orientation of digits in a complex hand distinguishes between those derived
from the graft and the accessory ones of uncertain origin. Such an analysis
revealed no difference between rotations but emphasized an unexpected feature
of the results, a correlation between the numbers of graft and accessory digits.
Complex hands from all series almost invariably contained Jess than four graft
digits, multiple hands often had four graft digits and complete graft forearms
usually did so (Table 2). The digits of the corresponding accessory structures
tended to increase in the same order and, although not counted accurately, the
total number of carpal cartilages showed a similar increasing pattern. Contralateral grafts seemed to produce more accessory digits than ipsilateral grafts,
but no particular rotation could be identified as consistently different from the
others and all were markedly more effective than the control operations (Table 2).
This similarity reinforces the conclusion that all rotations tested were equivalent in reducing the number of graft digits and provoking the formation of
accessory structures. The correlation between the completeness of the graft and
accessory structures perhaps indicates that neighbouring blastemata can only
achieve a normal structure after undergoing sufficient distal growth to diverge
from each other. Series I offered the least opportunity for such growth. These
rotated forearm grafts only formed two or three digits on average and were
Duplicated axolotl regenerates
251
usually flanked by two groups of accessory digits which did not precisely
complement or duplicate the defective graft structure in most cases.
Accessory structures occurred in all possible positions relative to the transverse axes of the host arms, but most frequently at certain positions which
could be related both to the rotation imposed on the graft and to a constant
host influence. The preferred positions were anterior and posterior in all series
and at almost all rotations, almost exclusively so after AP and APDV axial
reversals (Table 3). Dorsal and ventral duplications appeared more commonly
after 90° and 270° rotations and were found most consistently after DV axial
reversal. Although this analysis suggests the AP axis is somehow stronger or
more effective than the DV axis, it may be a spurious comparison. The elliptical
cross-section of the forearm probably favours the appearance of duplications
at the poles of the relatively long AP axis, and the arrangement of muscles
and nerves in the upper arm could have a similar effect. The total number of
duplications fell in the range of 15-21 for 10 arms at each rotation of series I
and II (Table 3), reflecting the common occurrence of triplicate arms. The two
accessory structures of these arms usually emerged from opposite sides of the
main axis, but sometimes arose in adjacent quadrants as reported by Maden &
Turner (1978).
The flexure of a single digit reveals its DV orientation but the AP axis can
only be deduced from groups of digits. Consequently the orientation of duplications could not be assessed completely for the smallest complex hands, besides
being subject to error through the distorted growth of accessory forearms. When
both transverse axes were evident, the duplication could be characterized as a
left or right hand even if its orientation was abnormal. The vast majority of
assessable duplications conformed to the laterality or handedness of the host
limb, being harmonic in Harrison's terminology. An exceptional class of duplications occurred on triplicate arms following ipsilateral graft rotations. Here
one duplication was harmonic but the other one was disharmonic, of opposite
handedness to both the host and the graft, but a mirror image of the graft hand.
Disharmonic duplications were present on many but not. all of the best developed
triplicate arms at each of the ipsilateral rotations. These duplications have been
reported previously only after APDV axial reversals, first by Bryant & Iten
(1976) who stated they arose from the PD quadrant of the host arm, then by
Maden & Turner (1978) who denied that they arose in any constant position.
We have recorded them at roughly the P, D, and V poles and in the AV and
PD quadrants, without perceiving any relationship between these positions
and the graft rotation.
(4) Homografts. The obvious advantage of exchanging grafts between dark
and white specimens eventually overcame our inclination to restrict this study
to autografts. The axolotls used here were a backcross generation from a former
intercolony cross, and were expected to show the segregation of about seven
incompatibility factors (Maden & Wallace, 1975). The upper arm contralateral
252
H. WALLACE AND A. WATSON
Fig. 3. Examples of homografts between dark and white sibs. The boundary
between dark (cross-hatched) and white tissue coincided with the edge of graft
vasodilation in each case. All are dorsal views at the same magnification. Top
row, a left hand 2, 3 and 6 months after grafting an ipsilateral palette rotated 90°.
The graft was rejected and replaced by host digits. Bottom row, complex feet 2
months after reciprocal AP palette grafts. Both grafts only formed four toes, but
the white graft also contributed accessory digits to the anterior and posterior
duplications.
grafts of series III were homografts between white sibs, merely as a matter of
convenience. These grafts formed complete sets of digits and provoked wellgrown duplications before any rejection was noticed. Six or more weeks after
transplantation, however, many of the grafts became inflamed with vasodilation and a slow circulation. About half of the grafts contained areas of haemorrhage at the time they were fixed.
Realizing that rejection would not interfere with the gross results of this
experiment, we performed several additional rotations using palette stages
exchanged between dark and white sibs. The alternative criteria of pigmentation
Duplicated axolotl regenerates
253
or vasodilation served to distinguish between the host or graft origin of digits
in complex hands, as depigmentation is one of the earliest signs of rejection.
These criteria were found to agree well with an assessment of the digit origin
based on its orientation, and thus confirmed the preceding account in four
important respects. Firstly, unrotated control homografts produced a complete
set of four digits with graft colouration and orientation in all three successful
operations. This provides a rough control value for the data in Table 2 which
could not be based on orientation alone, because these normal hands perpetuate
the orientation shared by host and graft. Secondly, graft-orientated digits of
complex hands were sometimes completely destroyed while the adjacent
accessory digits persisted. The rejected digits were not replaced until 3
months after their complete disappearance (Fig. 3), or were not replaced at all.
Thirdly, the majority of harmonic duplications showed both the orientation
and coloration of the host arm although some accessory digits were derived
from graft tissue in 10/29 cases (Fig. 3). Most of the duplications recorded
in the previous series may also be presumed to be derived from host tissue,
either mainly or exclusively. Lastly, the only disharmonic duplication encountered was a mirror image extension from the base of the APDV graft hand.
Both clusters of digits remained unpigmented and showed a mild vasodilation,
in contrast to two adjacent dark digits produced from the host arm. Some
harmonic duplications are probably also formed in this way, but we have not
obtained an example from the few contralateral homografts to confirm our
suspicion.
DISCUSSION
The experiments reported here were deliberately confined to regenerates
with fully determined axes and unaltered proximodistal position for reasons
outlined below. There has been considerable argument about when the transverse axes become determined during limb regeneration. Milojevic (1924) and
Schwidefsky (1935) both concluded that the transverse axes of any stage up to
the cone blastema could be respecified to conform to those of a host arm stump,
but neither investigator excluded the possibility that such young blastemata
were eroded and replaced by host tissue. Iten & Bryant (1975) initially reached
the same conclusion because they considered the extra digits of expanded and
double hands must indicate a partial respecification of the reversed AP graft
axis. When it is realized these are accessory digits, duplications by definition
and now universally accepted as consequences of a conflict between determined
axes (cf. Bryant & Iten 1977), the expanded and double hands actually demonstrated a determined AP axis in relatively young blastemata. These results
thus supported those of Lodyzenskaja (1930) who deduced the presence of
determined axes as early as two days after amputation from the duplications
following AP, DV and APDV axial reversals. Analogous axial reversals of skin
or muscle prior to amputation also provoke duplications within the spectrum
of structures described here, convincing us there is no period during regenera17
EMB49
254
H. WALLACE AND A. WATSON
tion when these tissues lack axial determination. In order to avoid criticism on
this score, however, we operated on later regenerates with a visible axial pattern
and with fully determined axes according to all the cited investigations.
Most of the duplications arose some way down the arm from the site of our
operations, as though emerging from the graft regenerate. A similar distal
displacement can be discerned in many reports of analogous experiments and
seems to be a general feature of both transplanted regenerates and of regeneration following axial reversal of limb tissues. Dark-white homografts demonstrated that the boundary between host and graft tissue had also shifted distally
to about the same extent as the base of the duplications (cf. Tank, 1978). This
indicates that the base of the graft must be eroded to a variable extent and
replaced by a limited intercalary regeneration of the host arm, even though.a
loss of graft cells is not readily detected (Iten & Bryant, 1975; Stocum, 1975;
Stocum & Melton, 1977). Shifting a distal regenerate to a more proximal position is known to cause extensive intercalary regeneration which somehow
influences the frequency and nature of the duplications provoked by rotated
grafts (Abeloos & Lecamp, 1931, Bryant & Iten, 1977). We attempted to minimize this complication by avoiding gross proximodistal shifts of the grafts.
The experiments cited previously involved complete reversals of AP, DV or
both axes. The present results confirm that axial reversals regularly provoke
duplications unless compensatory derotation has anulled the operation. Also
confirming previous results, the control operation of replacing a regenerate
without rotation rarely produces any duplication, never more than a single extra
digit. Conceding that such replaced grafts fit the host wound surface more
closely than most rotated grafts might arouse some suspicion that an uncovered
wound surface could be responsible for the observed duplications. A wound
probably is necessary but it is not a sufficient condition for accessory blastemata,
as the following examples testify. Lodyzenskaja (1930) routinely placed small
blastemata on large wound surfaces but only obtained duplications from axially
reversed grafts. Schwidefsky (1935) deliberately displaced some grafts without
rotating them, to obtain only normal or hypomorphic hands. All subsequent
investigators considered the control operation an adequate one, and most of
them ascribed duplications to a conflict between diametrically opposed transverse axes. Schwidefsky (1935), however, also reported the occurrence of
duplications following a 90° rotation of an ipsilateral graft. The present results
provide a systematic confirmation of this rather casual and neglected observation. The four possible 90° rotations provoke duplications at three positions
along the arm, and do so quite as efficiently as complete axial reversals. That
is also true of forearm regenerates in larval Pleurodeles (Wallace, 1978). Even
smaller rotations produce duplications on axolotl upper arms (Maden & Turner,
1978). All these results demonstrate that duplications result from an axial
discrepancy and belatedly establish the rule formulated by Abeloos & Lecamp
(1931): duplications arise when graft and host axes do not coincide.
Duplicated axolotl regenerates
255
Table 4. Predicted presence (+) or absence (—) of duplications at the
rotations investigated
Ipsilateral rotations
Model used for
the prediction
Clockface
Axial conflict on:
AP axis only
AP and DV axes
Axial discrepancy on:
AP axis only
AP and DV axes
Actual results
-
_
—
_
*
A
180°
APDV
270°
pb
0°
AP
90°
pf
180°
DV
>
270°
pb
+
-
+
+
+
+
—
+
+
_
—
+
+
—
+
_
—
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
90°
pf
-
—
—
Contralateral rotations
A
,
0°
Normal
,
_
_
The impact of this discovery is revealed in Table 4 which compares our results
to predictions based on different models of blastemal morphogenesis. Each of
these models was devised to account for duplications following complete axial
reversals, intended to produce axial conflicts or the equivalent interaction
between opposite positions of the clockface model. None of the models actually
predicted the occurrence of duplications following 90° or 270° ipsilateral
rotations, so they must all be modified or rejected. The clockface model is
incapable of sufficient distortion to account for duplications and triplications
at all the rotations tested, so we need not consider it further. The other models
which mainly differ in the number of postulated axes, can be adapted to accommodate the conclusion that any substantial axial discrepancy is sufficient to
provoke duplications and then correctly predict the results of ipsilateral graft
rotations. The contralateral grafts, however, permit a further distinction between
these models. A single AP axis would generate duplications at all the rotations
except the contralateral DV inversion (Table 4), which is why Carlson (1974,
1975) adopted this model. Other investigators have recorded duplications at this
particular rotation and consequently advocated a second DV axis. The contralateral rotations can be envisaged as a systematic search for a single transverse
axis. If the rotations had been precisely at successive 90° intervals, one of them
might have failed to generate any duplications because it failed to deflect a
single orthogonal (AP or DV) or diagonal axis. Since the rotations were
imprecise and subject to derotation, it seems likely that one contralateral
configuration would have indicated the presence of a single transverse axis
along any diameter by a reduced frequency of duplications. The results of series
III might be interpreted in this way but those of series I and II (Table 1) are not
compatible with the notion of a single transverse axis. Any model specifying
two axes would satisfy these results, which provide no basis for determining the
position of the axes or for postulating additional axes. We have apparently
17-2
256
H. WALLACE AND A. WATSON
succeeded in vindicating the common sense assumption (cf. Harrison, 1925)
of two arbitrary transverse axes which may be conveniently thought of as
corresponding to the AP and DV alignments of the hand.
Although duplications arise from a discrepancy between host and graft
axes, it is difficult to justify the prevalent assumption that they are caused by an
interaction of discordant axes. It seems more plausible to envisage the only
interaction as occurring when the graft and host axes are approximately aligned,
a regulation which prevents any regenerative outgrowth from the wounded
region. A substantial axial discrepancy could prevent that interaction and thus
allow new regeneration from both the host and the graft. Even a rotated graft
probably acts as a mechanical block to normal regeneration of the host arm,
which thus produces one or two lateral regenerates on either side of the graft.
In addition to these, an adequately innervated graft is surely capable of regenerating a mirror image of itself from its base, in the manner of a proximodistally
reversed arm (Monroy, 1942). These premises are sufficient to account for the
observed numbers, orientation and handedness of duplications without postulating any modification of the transverse axes of graft or host (cf. Milojevic &
Hoffman, 1926). In some instances, graft and host join forces to produce a
chimaeric duplication which emerges at a point of maximum axial discrepancy.
Assuming no morphogenetic interaction other than regulation within the
chimaeric blastema, contralateral grafts would still produce harmonic duplications in this way (Fig. 3) but ipsilateral grafts should generate chimaeric duplications with anomalous axes, such as the double posterior one reported by
Maden & Turner (1978).
Many of the characteristic features of duplications noted here have been
described in earlier investigations using axolotls and newts. These previous
reports naturally emphasized some features more than others, sometimes
concentrating on 'well-formed' supernumerary arms by either ignoring or
misinterpreting the accessory digits which resulted from identical operations.
These are both duplications and our results show them to be proximodistal
modulations of the response to graft rotation. The total frequency of duplicated
arms has usually been recorded as sufficiently large, after axial reversals, that
perhaps only derotation prevented it reaching 100 % as suggested by Maden &
Turner (1978). Derotation and a concomitantly reduced frequency of duplication is much more evident after shoulder level operations than after elbow or
forearm operations. This observation reconciles the virtually complete yield of
complex hands recorded by Lodyzenskaja (1930) with the lower yields of more
striking duplications obtained after upper arm axial reversals by other investigators. The predominance of anterior and posterior duplications found here is
also in excellent agreement with the results obtained by Lodyzenskaja (1930),
although we suspect derotation influences these positions which may be more
closely related to the final graft orientation than to the imposed rotation.
Bryant & Iten (1976) and Tank (1978) reported duplications only at restricted
Duplicated axolotl regenerates
257
positions after axial reversals, but Maden & Turner (1978) found them to occur
in any position after APDV reversals. An independent means of establishing
the origin of a duplication might well clarify our interpretation of its position
and orientation. Several investigators have used differently coloured homografts for this purpose, without explicitly endorsing the reliability of the technique. Our own limited experience of homografts suggests that the graft and
host often regenerate independently of each other to produce duplications
with a predictable orientation but variable position. Finally, our observation
that contralateral grafts tend to derotate so as to conform to the host AP axis
is apparently unprecedented. We have not attempted to explain it but would
welcome an independent confirmation or denial of the phenomenon, either
for regenerates or limb-buds. Nicholas (1924) discovered that limb-buds rotated
through 90° or 270° form duplications and tend to derotate by the shortest
route, but confined his study to ipsilateral rotations. We should have anticipated
that rotated regenerates would behave in an identical manner.
REFERENCES
M. & LECAMP, M. (1931). Sur la production de formations anormales et multiples
dans les membres du Triton par transplantation de regenerats. C. r. hebd. Seanc. Acad. Sci.,
Paris 192, 639-641.
BRYANT, S. V. & ITEN, L. E. (1976). Supernumerary limbs in amphibians: experimental
production in Notophthalmus viridescens and a new interpretation of their formation.
DevlBiol. 50, 212-234.
BRYANT, S. V. & ITEN, L. E. (1977). Intercalary and supernumerary regeneration in regenerating and mature limbs of Notophthalmus viridescens. J. exp. Zool. 202, 1-15.
CARLSON, B. M. (1974). Morphogenetic interactions between rotated skin cuffs and underlying stump tissues in regenerating axololl forelimbs. Devi Biol. 39, 263-285.
CARLSON, B. M. (1975). The effects of rotation and positional change of stump tissues upon
morphogenesis in the regenerating axolotl limb. Devi Biol. 47, 269-291.
FABER, J. (.1960). An experimental analysis of regional organisation in the regenerating forelimb of the axolotl {Ambystoma mexicanuni). Archs Biol., Liege 71, 1-72.
HARRrsON, R. G. (1925). The effect of reversing the medio-lateral or transverse axis of the
fore-limb bud in the salamander embryo {Amblystoma punctatuni). Wilhelm Roux Arch.
EntwMech. Org. 106, 469-502.
ITEN, L. E. & BRYANT, S. V. (1975). The interaction between the blastema and stump in the
establishment of the anterior-posterior and proximal-distal organisation of the limb
regenerate. Devi Biol. 44, 119-147.
LHEUREUX, E. (1972). Contribution a l'etude du role de la peau et des tissus axiaux du membre
dans le declenchement de morphogeneses regeneratrices anormales chez le triton Pleurodeles waltlii Michah. Annls Embryol. Morph. 5, 165-178.
LHEUREUX, E. (1975). Nouvelles donnees sur les roles de la peau et des tissus internes dans
la regeneration du membre du triton Pleurodeles waltlii Michah. (Amphibien Urodele).
Wilhelm Roux Arch. EntwMech. Org. 176, 285-301.
LHEUREUX, E. (1977). Importance des associations de tissus de membre dans le developpement des membres surnumeraires induits par deviation de nerf chez le triton Pleurodeles
waltlii Michah. /. Embryol. exp. Morph. 38, 151-173.
LODYZENSKAJA, V. I. (1930). La transplantation des bourgeons de regeneration des extremites de l'axolotl. Trav. Lab. Zool. exp. Morph. Anim. 1, 61-120.
MADEN, M. & TURNER, R. N. (1978). Supernumerary limbs in the axolotl. Nature, London
273, 232-235.
ABELOOS,
258
H. WALLACE AND A. WATSON
M. & WALLACE, H. (1975). The origin of limb regenerates from cartilage grafts.
Acta Embryol. exp. 1975, 77-86.
MILOJEVIC, B. D. (1924). Beitrage zur Frage iiber die Determination der Regenerate. Wilhelm
Roux Arch. EntwMech. Org. 103, 80-94.
MILOJEVIC, B. D. & HOFFMAN, S. (1926). Le deboublement des extremites chez Rana
esculenta a la suite de la transplantation des bourgeons primares des pattes posterieures.
C. r. Seanc. Soc. Biol. 95, 981-983.
MONROY, A. (1942). La rigenerazione bipolare in segmenti di arti isolati di Triton crist.
Arch. ital. Anat. Embryol. 48, 123-142.
NICHOLAS, J. S. (1924). Regulation of posture in the fore limb of Amblystoma punctatum.
J. exp. Zool. 40, 113-159.
SCHWJDEFSKY, G. (1935). Entwicklung und Determination der Extremitaten Regenerate bei
den Molchen. Wilhelm Roux Arch. EntwMech. Org. 132, 57-114.
STOCUM, D. L. (1975). Regulation after proximal or distal transposition of limb regeneration
blastemas and determination of the proximal boundary of the regenerate. Devi Biol. 45,
112-136.
STOCUM, D. L. & MELTON, D. A. (1977). Self-organisational capacity of distally transplanted
regeneration blastemas in larval salamanders. /. exp. Zool. 201, 451-461.
SWETT, F. H. (1927). Differentiation of the amphibian limb. J. exp. Zool. 47, 385-435.
TANK, P. W. (1978). The occurrence of supernumerary limbs following blastemal transplantation in the regenerating forelimb of the axolotl, Ambystoma mexicanum. Devi
Biol. 62, 143-161.
WALLACE, H. (1978). Testing the clockface model of amphibian limb regeneration. Experientia
34, 1360-1361.
MADEN,
{Received 27 July 1978, revised 11 September 1978)