/ . Embryol. exp. Morph. Vol. 70, pp. 197-213, 1982
Printed in Great Britain © Company of Biologists Limited 1982
197
Axial organization of the regenerating limb:
asymmetrical behaviour following
skin transplantation
By M. MADEN 1 AND K. MUSTAFA
From the Developmental Biology Division,
National Institute for Medical Research, London
SUMMARY
An extensive series of skin grafting operations has been performed to investigate axial
organization in the regenerating axolotl limb. Semicircular cuffs of skin from either anterior,
posterior, dorsal or ventral surfaces were exchanged between right and left limbs thereby
creating limbs with double anterior, double posterior, double dorsal or double ventral
skin, all with normal internal tissues. Both fore and hindlimbs were used at both upper and
lower limb levels. Following amputation through the grafted region the resulting regenerates
were analysed both by whole-mount cartilage staining to observe the pattern of digits and
by serial sectioning to observe the pattern of muscles. There were clear asymmetries in
ability to produce duplications - posterior to anterior grafts resulted in a consistently high
frequency of digital duplications, whereas anterior to posterior grafts produced very few.
Similarly, dorsal to ventral grafts resulted in a good frequency of muscle duplications,
whereas ventral to dorsal grafts did not. Such asymmetrical behaviour is not predicted by
most models involving local cell:cell interactions and the significance of the results for
theories of pattern formation is discussed.
INTRODUCTION
The analysis of pattern formation in regenerating limbs has, like other
developmental systems, relied on techniques of transplanting tissues and
observing the resultant disruption of pattern. For example, when regeneration
blastemas are grafted to contralateral limb stumps to reverse either one of the
transverse axes of the limb or grafted upside down on the same stump to
reverse both transverse axes, supernumerary limbs are induced (Iten & Bryant,
1975; Bryant & Iten, 1976; Tank, 1978 a; Wallace & Watson, 1979; Maden,
1980; Tank, 1981; Maden, 1982). Details of the structure and handedness of
such supernumerary limbs has provided many insights into the way in which
the transverse axes of the limb are organized (French, Bryant & Bryant, 1976;
Stocum, 1980; Slack, 1980a; Maden & Mustafa, 1982)
Another method of analysing axial organization which has the potential of
1
Author's address: Division of Developmental Biology, National Institute for Medical
Research, The Ridgeway, Mill Hill, London NW7 1AA U.K.
198
M. MADEN AND K. MUSTAFA
providing more precise information, as evidenced by work on the insect leg
(French, 1978; 1980), is to transplant individual tissues to various positions on
the limb circumference. Thus the roles of skin, muscle and bone have been
investigated (Carlson, 1974; 1975a, b\ Tank, 1979) with the result that skin
seems to be the most morphogenetically active component. The greater precision
of such a method has revealed significant differences between the general
conclusions from blastemal transplantations and tissue transplantations. Firstly,
concerning the anteroposterior axis, a difference in the power of anterior and
posterior skin to induce supernumerary limbs has been demonstrated. Slack
(19806) grafted anterior half circumferences of skin to the posterior side and
obtained normal limbs after amputating through the graft, whereas the converse
(posterior to anterior) resulted in mirror-imaged supernumerary limbs. No
such asymmetry had previously been suspected because blastemal transplantations involving the anteroposterior axis produce two supernumeraries, one at
the anterior pole and one at the posterior pole. Secondly, concerning the
dorsoventral axis, Carlson (1974) grafted skin from one limb to the contralateral side, thereby inverting only the dorsoventral axis, and obtained normal
limbs. Similarly, grafting dorsal skin to ventral or vice versa, analogous to
Slack's experiments on the anteroposterior axis, produced normal limbs too
(Carlson, 1974; Tank, 1979). This prompted Carlson to conclude that the
dorsoventral axis is not polarized in the regenerating axolotl limb. This contention is in stark contrast to the results from blastemal transplantation where
inversion of the dorsoventral axis produces supernumerary limbs (Tank, 1978 a;
Maden, 1980; 1982).
Now, much information on the organization of the dorsoventral axis has
been gained by serially sectioning supernumerary limbs to examine muscle
patterns (Maden, 1980, 1982; Maden & Mustafa, 1982). It is conceivable that
the apparently normal limbs obtained in the experiments of Carlson and Tank
although not producing supernumeraries, have abnormal muscle patterns
analogous to the mirror-imaged cartilage patterns revealed in the anteroposterior axis. Therefore, in an effort to resolve the above contradictions and
to re-examine the effects on the dorsoventral axis, we carried out similar skin
grafting operations. Both upper and lower, fore and hindlimbs of axolotls were
used and the resulting regenerates both stained for cartilage and serially
sectioned. The results reveal that the dorsoventral axis is indeed affected by
skin grafts and that asymmetries in both axes exist. The significance of these
observations for theories of pattern formation is discussed.
MATERIALS AND METHODS
The experiments were performed on 70-80 mm axolotls, Ambystoma mexicanum, under MS222 anaesthesia. Half-circumference segments of skin were
exchanged between contralateral pairs of limbs and sutured in place with
Axial organization of the regenerating limb
la
lb
lc
Id
199
Fig. 1. Diagram showing the experimental design. In the upper diagram exchange
of anterior and posterior half-skin cuffs between left and right upper forelimbs is
shown, resulting in left limbs having double anterior skin and normal internal
tissues (a) and right limbs with double posterior skin and normal internal tissues
(b). In the lower diagram a similar exchange between dorsal and ventral half-skin
cuffs is shown, resulting in limbs with double dorsal skin (c) or double ventral
skin (d). These four operations were also performed at the lower forelimb, upper
hindlimb and lower hindlimb levels.
0-2 Ethilon monofilament suture thread (Ethicon Ltd.). Four categories of
operations were produced: double anterior - Series l - ( F i g . la), double
posterior - Series 2 - (Fig. 1 b), double dorsal - Series 3 - (Fig. 1 c) and double
ventral - Series 4 - (Fig. 1 d), and each performed at four different levels:
upper forelimb, lower forelimb, upper hindlimb and lower hindlimb. Ten
animals were used for each series making 160 operations in all.
200
M. MADEN AND K. MUSTAFA
One week after operating, when the grafts had healed perfectly in place,
limbs were amputated through the grafted region. They were allowed to regenerate for at least 8 weeks and the resulting regenerates fixed in neutral
formalin. They were stained in Victoria Blue to reveal cartilage elements.
Regenerates from the double dorsal and double ventral series were then prepared
for sectioning at 10 ptm and the slides stained with haematoxylin and eosin.
Muscle patterns were analysed as previously described (Maden, 1980, 1982).
RESULTS
Cartilage patterns - the anteroposterior axis
Series 1. Anterior skin -> posterior
Forelimbs. Grafting anterior skin in place of posterior skin had no significant
effect on the cartilage structure of the regenerates whether the operations were
performed at upper or lower forelimb levels (Table 1, rows 1 and 2); two
regenerates from the lower limb series had a bifurcated digit 4 resulting in a
few extra phalanges and the remainder were indistinguishable from normal
limbs (Fig. 2).
Hindlimbs. The effects of grafting anterior skin to posterior were more
noticeable in the hindlimb. At the upper hindlimb level four regenerates were
normal (five digits, Fig. 3), three were hypomorphic and three had supernumerary elements (Table 2, row 1). The three cases with supernumerary
elements each had an extra digit 3 on the posterior side of the limb (Fig. 4).
At the lower hindlimb level there were no normal regenerates (Table 2,
row 2). Three had extra digits on the posterior side, as in Fig. 4 and the
remaining six were hypomorphic. Each of these hypomorphics had three
digits (Fig. 5) and a reduced number of tarsals. Unlike those at the upper
limb level where the hypomorphics simply had digits missing, their pattern
of digits here was not normal - they were clearly not digits 1, 2 and 3 because
digit 3 has four phalanges (see Fig. 3). They were therefore identified as digits
1, 2 and 2 or 1, 2 and 1. In some cases the pattern of tarsals looked mirrorimaged and thus it was concluded that these six hypomorphic regenerates
were double anterior in structure. Thus the frequency of duplication in this
series was 100%.
Thus, in the forelimb, anterior to posterior skin grafts had no significant
effect. In the hindlimb supernumerary digits were produced. Clear mirrorimaged reduplicates only occurred in the lower hindlimb series and then only
consisted of three digits.
Series 2. Posterior skin -> anterior
Forelimbs. In contrast to the previous series, grafting posterior skin in place
of anterior skin had a major effect on regenerate morphology (Table 1, rows
3 and 4). At the upper arm level, seven out often cases produced supernumerary
A->P
A-*P
P-»A
P^A
D-*V
D-»V
V-»D
V->D
Operation
Cartilage structure of regenerates in terms of digit number
10
10
4*
10
10
9
8
9
8
* One of these limbs had supernumerary forearm elements and carpals, but only 4 digits.
Surviving
cases
Upper
Lower
Upper
Lower
Upper
Lower
Upper
Lower
Hindlimb
level
A->P
A-*P
P->A
P->A
D-*V
D-»V
10
9
10
9
9
9
9
9
Surviving r—
Operation
cases
1
1
—
6
9
7
7
2
4
3
1
—
1
1
—
1
3
—
—
—
—
1
1
—
1
2
4
—
—
1
1
Cartilage structure of regenerates in terms of digit number
(The normal number of digits in the hindlimb is 5.)
—
—
4
1
—
—
10
Table 2. Cartilage structure of the regenerates from four types of skin grafting operation in upper and lower hindlimbs
Lower
Upper
Lower
Upper
Lower
Upper
Lower
Upper
Forelimb
level
(The normal number of digits in the forelimb is 4.)
Table 1. Cartilage structure of the regenerates from four types of skin grafting operation in upper and lower forelimbs
s
I"
Ǥ
$
3'
202
M. MADEN AND K. MUSTAFA
2
4
3/4
3
3
Axial organization oj the regenerating limb
203
structures with limbs ranging from five to nine digits and at the lower arm
level the frequency was even higher - nine out of ten.
Some of the upper arm supernumeraries were mirror-imaged limbs in the
anteroposterior axis as one might expect (see Fig. 6). However, the majority
were more complicated in that the extra elements were displaced ventrally
(never dorsally). This resulted in a boomerang-shaped limb (viewed end on)
with the ventral surfaces of the normal and supernumerary hands facing each
other. Presumably this represents a flat mirror-imaged duplicate which has
been twisted during development.
Regenerates from lower arm amputation levels were more uniform, all being
simple mirror-images in the anteroposterior axis. The nine supernumeraries
fell into the categories described by Slack (1980Z?) namely, duplicates with a
digit pattern of 4 3 2 1 2 3 4 (Fig. 6) or 4 3 2 2 3 4, partial duplicates lacking
one posterior extremity, e.g. 3 2 1 2 3 4 (Fig. 7) or duplicates with serial
repetition, such as 2 2 1 2 3 4. There were no hypomorphic limbs and one
was normal.
Hindlimbs. In the hindlimb too, posterior to anterior skin grafts had a profound effect upon regenerate morphology (Table 2, rows 3 and 4). At the
upper level eight out of ten limbs produced supernumeraries ranging from
seven to ten digits and at the lower level every limb was reduplicated with six
to nine digits.
The upper level regenerates were nearly all double limbs with nine or ten
digits (Fig. 8). Half of them were complicated, as in the case of the forelimbs,
by digits being displaced ventrally with the ventral surfaces of the two limbs
facing each other. The remainder were straight-forward mirror images as
described above, that is with a digital pattern of 5 4 3 2 1 2 3 4 5 or partial
duplicates lacking one posterior extremity or duplicates with serial repetition.
This same pattern of duplication was found in lower hindlimb regenerates
with half of the regenerates having digits displaced in the dorsoventral axis
and the remainder with normal duplications (Fig. 9).
Fig. 2. Typical forelimb regenerate from Series 1 - grafting anterior skin to
posterior. After amputating either through the upper or lower forelimb levels
regenerates were normal as shown here. Digit numbers are marked, x 14.
Figs. 3-5. Three types of hindlimb regenerate from Series 1 - grafting anterior skin
to posterior.
Fig. 3. Normal regenerate from the upper hindlimb level. Digit numbers are
marked, x 14.
Fig. 4. Regenerate from the upper hindlimb level with one extra digit on the
posterior side (left) making six digits in all. The two posterior digits could be
either digits 3 or 4, digit 5 being absent, x 14.
Fig. 5. Hypomorphic regenerate from the lower hindlimb level with only seven
tarsals and three digits. The mirror-image nature of the regenerate is apparent,
particularly the digits which were assigned the numbers 1, 2, 1. x 16.
204
M. MADEN AND K. MUSTAFA
7
4
4
8
Figs. 6-9. Regenerates from Series 2 - grafting posterior skin to anterior.
Fig. 6. Lower forelimb level duplicated regenerate with a digital pattern of
4 3 2 1 2 3 4. x 16.
Fig. 7. Lower forelimb level partial duplicate with one posterior extremity missing,
making a digital pattern of 3 2 1 2 3 4. x 16.
Fig. 8. Upper hindlimb level full duplicate with a digital sequence of 5 4 3 2 1 2 3 4
and a very small 5th digit, x 14.
Fig. 9. Lower hindlimb level duplicate with a digital sequence of 5 4 3 2 2 3 4 5.
xl4.
Axial organization of the regenerating limb
205
11
10
Fig. 10. Regenerate from Series 3 - dorsal skin to ventral. At both upper and lower
forelimb and hindlimb levels the regenerates were mostly normal. The normal
regenerate shown here is from the upper forelimb level, x 14.
Fig. 11. Regenerate from Series 4-ventral skin to dorsal. As in Series 3 most
regenerates were normal, that shown here is also from the upper forelimb level.
xl4.
Thus, in the forelimb and hindlimb, grafting posterior skin to anterior has
a major effect on pattern formation in the regenerates, causing the production
of mirror-imaged duplicated limbs with up to ten digits in 70-100% of the
cases. Clearly then, there is an asymmetry between this situation and the
converse graft of anterior skin to posterior described in Series 1 where the
vast majority (85 %) of regenerates were normal or hypomorphic. Those that
were mirror-imaged duplicates were hypomorphic too.
Series 3. Dorsal skin -» ventral
Forelimbs. Grafting dorsal skin in place of ventral skin had very little effect
on the cartilage structure of the regenerates whether the operations were
performed in the upper arm or lower arm (Table 1, rows 5 and 6). Of the
regenerates, 88% (15 out of 17) were normal (Fig. 10). The hypomorphic
case recorded in Table 1 was one which produced a two-digit anterior half
206
M. MADEN AND K. MUSTAFA
limb and the hypomorphic case was a limb with an extra digit 4 which protruded
ventrally below the normal one.
Closer scrutiny of these supposedly normal regenerates revealed slight abnormalities in carpal shape and in many cases digits bent dorsally instead of
ventrally.
Hindlimbs. Similar results were obtained on the hindlimb (Table 2, rows 5
and 6). Of upper and lower regenerates, 83 % (15 out of 18) were normal, one
limb had one extra digit and two were hypomorphic.
Series 4. Ventral skin -> dorsal
Forelimbs. Grafting ventral skin in place of dorsal skin also had no effect
on the cartilage structure of the regenerates from either upper or lower arm
levels (Table 1, rows 7 and 8). 100% of the regenerates were normal (Fig. 11).
Hindlimbs. Of the hindlimbs, 78 % (14 out of 18) were normal. Three limbs
produced extra digits, all on the anterior edge of the regenerate and displaced
in the dorsoventral axis. Even the two with eight digits had poorly developed
supernumerary digits which could not be identified.
From the results of the last two series we might conclude, as Carlson (1974)
did, that the dorsoventral axis of the regenerating limb of axolotls is not
polarized. However, the above method of scoring cartilage elements is only
concerned with the anteroposterior axis which was not altered by such operations.
Fig. 12. Muscle patterns in the normal forelimb at the metacarpal level. On the
dorsal surface, above each metacarpal is a small, crescent-shaped muscle, the
extensor digitorum brevis (arrows). On the ventral surface is a large mass of muscle
composed of about 16 separate muscles fused together. The ventral muscles of digit
4 (far left) have split off from the main body of muscle in preparation for the
separation of digit 4. x 60.
Fig. 13. Muscle patterns in a regenerate from Series 3 - dorsal skin to ventral.
In contrast to Fig. 12 it can be seen that instead of a large mass of muscle on
the ventral surface there are four crescent-shaped extensores digitorum breves which
mirror-image those present as normal on the doisal surface. This limb is clearly
double dorsal in structure, x 55.
Fig. 14. Muscle patterns in another regenerate from Series 3. Here the normal
complement of four extensores digitorum breves are on the dorsal surface of the
metacarpals. On the ventral surface of the two digits on the left there are also
e.d.b. present (arrows). Beneath the other two digits are normal ventral muscles
(v). Thus the left half of this regenerate is mirror-imaged (double dorsal) and the
right half is normally asymmetric, x 50.
Fig. 15. Regenerate from Series 4-ventral skin to dorsal. Here the only asymmetrical digit is the far right one with an e.d.b. (arrow) on the dorsal surface and
ventral muscles underneath. The remaining three digits have ventral muscles on
both the ventral and dorsal surfaces which fuse in the mid-line to completely surround the digits in muscle. No e.d.b. are present in those three double ventral
digits, x 52.
Axial organization of the regenerating limb
CO
C\J
207
208
M. MADEN AND K. MUSTAFA
Table 3. Muscle patterns in regenerates from Series 3 and 4
(The reduplicated cases are scored below according to the number of
individual digits in the limb with reduplicated muscle patterns.)
Pattern of muscles
Number of reduplicated digits
Level
Fore
Upper
Lower
Upper
Lower
Upper
Lower
Upper
Lower
Hind
Fore
Hind
D-*V
D-»V
D-*V
V-»D
V->D
V->D
9
8
9
9
9
8
9
9
2
7
5
4
7
7
00 00
Limb
Surviving
Operation
cases
Normal
5
4
3
2
1
—
2
—
3
—
—
—
—
2
1
1
1
2
—
2
1
2
2
1
1
—
—
—
1
1
Any dorsoventral pattern duplication, equivalent to anteroposterior reduplication, would not be apparent unless a separate supernumerary was generated.
Consequently the dorsoventral organization of these regenerates, in terms of
their muscle patterns, was examined in serial sections.
Muscle patterns - the dorsoventral axis
The normal muscle pattern of axolotl limb has been described before, using
this technique of reconstructed serial sections (Maden, 1980a, b). A detailed
description will therefore not be repeated here; suffice it to show a typically
normal section through the metacarpal level in Fig. 12. The ventral surface
of the hand is characterized by a group of about 16 muscles (Grim & Carlson,
1974) together forming a solid mass of muscle, while the dorsal muscles have
separated into four distinct extensores digitorum breves. These dorsal muscles
have a characteristic crescent shape and sit on the top of the carpals and
digits.
Series 3. Dorsal skin -> ventral
Forelimbs. Despite the fact that all the upper arm regenerates looked normal
when stained for cartilage, seven out of nine had varying degrees of duplication
in their pattern of muscles (Table 3, top row). Three were perfect double
dorsal limbs (Fig. 13), identical in structure to those that have been previously
described (Maden, 1980a, b), that is with crescent-shaped extensores digitorum
breves on both sides of the limb. The ventral surface is totally devoid of the
mass of muscle normally present. The remaining four limbs had partial duplication, that is either half of the limb (two digits) was double dorsal and the
other half normal (Fig. 14) or just one digit had duplicated muscle patterns.
Axial organization of the regenerating limb
209
At the lower arm level the clear effect seen above was not repeated. Here
only one limb showed any sign of duplication Table 3, row 2) and that had
only one digit affected even then.
Hindlimbs. At the upper hindlimb level, four of the nine regenerates had
duplicated muscle patterns (Table 3, row 3) one of which was fully double
dorsal, the other three partial. At lower levels, five out of the nine regenerates
had double dorsal muscle patterns (Table 3, row 4), all of which were partially
duplicated.
Thus, after grafting dorsal skin to ventral, three of the levels tested gave
between 45 % and 80 % of double dorsal regenerates, the exception being the
lower forelimb level.
Series 4. Ventral skin -> dorsal
Forelimbs. Only three limbs out of a total of 17 showed any signs of muscle
duplication at the upper and lower forelimb levels (Table 3, rows 5 and 6).
The duplications produced in this series were such that double ventral muscle
patterns were produced. There were no extensores digitorum breves present,
instead large expanses of muscle both dorsally and ventrally which fused in
the mid-line to surround the digits in solid muscle (Fig. 15). One limb had
three digits with double ventral muscle (Fig. 15) and the other two had just
one digit duplicated.
Hindlimbs. In the hindlimbs too the frequency of duplication was very low
with only 2 limbs out of 16 showing any signs (Table 3, rows 7 and 8). One
regenerate had one digit with double ventral muscles and the other had two
digits so duplicated.
DISCUSSION
It is apparent from the results presented above that there are distinct asymmetries in behaviour following the grafting of skin from one side (either
anterior, posterior, dorsal or ventral) to the other. Posterior skin grafted to
anterior gave a very high frequency of duplicated regenerates while the converse operation gave a very low frequency. Similarly, dorsal skin grafted to
ventral gave a moderate frequency of duplicated muscle patterns while the
converse did not. To assist in further comparisons, the frequency of duplication
in each series is recorded in Table 4 where the frequency is not calculated
simply in terms of the number of limbs affected, but rather by considering
each individual digit. This provides a valid comparison between the anteroposterior axis and the dorsoventral axis in terms of their ability to cause
duplications. Thus Table 4 reveals that posterior skin has the greatest ability
to do so, followed by dorsal skin, anterior skin and lastly ventral skin. It is
comforting to discover that this conclusion is exactly the same as that reached by
Lheureux (1975). He performed skin grafts on Pleurodeles forelimbs such that only
210
M. MADEN AND K. MUSTAFA
Table 4. Frequency of reduplication in each series scored in terms of the
proportion of the total number of digits regenerated that were reduplicated
Limb
Level
Operation
Fore
Upper
Lower
Upper
Lower
Upper
Lower
Upper
Lower
Upper
Lower
Upper
Lower
Upper
Lower
Upper
Lower
P-^A
P-*A
P^A
P->A
D->V
D->V
D->V
D-»V
A->P
A-*P
A->P
A-*P
V->D
V-*D
V-*D
V->D
Hind
Fore
Hind
Fore
Hind
Fore
Hind
Average
Total
Total
reRenumber of number of
digits
digits re- duplication duplication
regenerated duplicated
(%)
(%)
39
38
50
42
34
33
40
45
40
40
44
33
36
32
50
47
11
23
29
25
16
1
7
9
0
0
3
11
4
1
2
1
28
60
58
60,
47
3
17-5
20
0
0
7
33.
11
3
4
2.
52
22
10
one polar quality (dorsal, ventral, anterior or posterior) was present at the
amputation plane by rotating strips of skin through 90°. The ability to induce
supernumerary elements was highest with posterior skin (49%), then dorsal
(28%), anterior (18%) and least with ventral skin (7%): even the frequencies
are virtually identical to those in Table 4. Similarly the highest frequency of
hypomorphic limbs was found with anterior skin, exactly as reported here.
However, it is important to note that Lheureux's analysis was performed only
on cartilage elements in the regenerates and not on muscle patterns. Perhaps
Pleurodeles are more efficient at producing distinct supernumeraries after
dorsoventral inversions than axolotls.
The analysis of muscle patterns in the regenerates has provided important
new data from the supposedly' normal' limbs. Had the frequency of duplication
simply been recorded in terms of cartilage elements then the above results
would have confirmed Carlson's conclusion that the dorsoventral axis of the
regenerating axolotl limb is not polarized. There is no reason to suppose that
a mirror-imaged muscle pattern is not an equally valid duplicate to a mirror
image in the anteroposterior axis revealed by whole-mount cartilage staining.
It would be equivalent to an AP duplicate with a digital sequence of 4 3 3 4.
Thus the dorsoventral axis is polarized in the regenerating axolotl limb, but
Axial organization of the regenerating limb
211
not to the same degree of'strength' as the anteroposterior axis when measured
in terms of degree of duplication (Table 4).
The scope of these experiments, in performing them on both fore and
hindlimbs at upper and lower limb levels, was such that some inconsistencies
were bound to occur, yet these were few. In Series 1 (anterior skin -> posterior)
all forelimb regenerates were normal, but very few hindlimbs were. Only at
the hindlimb lower level were any mirror-image duplicates found and they
only consisted of three digits. In Series 3 (dorsal skin -> ventral), at the forelimb lower level only one muscle reduplicate appeared in contrast to the good
frequency at the other three levels. On the other hand, Series 1 and 4 were
entirely consistent, the former resulting in a uniformly high frequency of good
five- to 10-digit duplicates and the latter being uniformly inert.
Consistency with other results is variable. The results of Lheureux (1975)
have already been mentioned and those of Slack (19806) are also identical.
However, inconsistencies arise in considering the results of Carlson (1974)
and Tank (1979). The fact that neither author reported duplicates (or very
few) after dorsal or ventral skin grafting has been resolved in that they did
not section the regenerates to examine muscle patterns. More significant is the
lack of any difference between anterior and posterior skin in their ability to
induce duplication (Carlson, 1974) and yet another type of result, the high
proportion of no regeneration after such operations (Tank, 1979); both remain
unresolved.
What conclusions can be drawn from the distinct asymmetries between the
opposite sides of the two transverse axes of the regenerating limb after skin
grafting? Models involving straight-forward local cell-cell interactions are incapable of explaining such asymmetries. In the polar coordinate model (French,
Bryant & Bryant, 1976) for instance, transplanting tissue to the other side of
the circle, e.g. from 9 to 3, should not produce a different result from the
converse operation, from 3 to 9, when it interacts with the underlying internal
tissues. Both should intercalate the same missing values, but this is not what
happens in practice. Furthermore, it is not appropriate to explain the asymmetries in terms of the results of experiments on double half limbs (Bryant &
Baca, 1978; Stocum, 1978; Tank, 19786). In this case two half limbs of the
same pattern (e.g. two anterior halves or two posterior halves) are constructed
and on amputation, double posteriors regenerate more pattern than double
anteriors. Whilst initially this result seems similar, the two are not readily
comparable at all because in the double-half limb experiments there are no
points of incongruity around the limb circumference and the regenerates, not
surprisingly, rarely produce more than the normal number of digits in total
(even though they may be mirror imaged) and usually considerably less. In the
experiments reported here, generating positions of maximum incongruity was
precisely the point and the subsequent regeneration involves the production of
supernumerary elements - indeed some regenerated almost two whole limbs
212
M. MADEN AND K. MUSTAFA
(Figs. 6-9). Clearly such supernumerary tissue must be produced by the interaction between the grafted skin and the underlying internal tissues.
The only model which addresses this question of asymmetrical behaviour is
that proposed by Slack (19806) following his anterior and posterior skin
grafting experiments. This considers the anteroposterior axis in terms of a
series of territories in which sets of biochemical switches are either on or off.
At the posterior edge all the switches are on, at the anterior edge all are off.
The dominance of higher switches over lower switches so that any posterior
territory contains the information for making all the more anterior territories,
but not vice versa, explains the anteroposterior asymmetry. This concept can
easily be supplemented by proposing a similar scheme which operates in the
dorsoventral axis with the region where all the switches are on at the dorsal
edge and all the switches off at the ventral edge. Furthermore variation in the
precise location of territories within the cross section of the limb could explain
the differences in behaviour between different levels of the same limb when the
same graft has been performed (e.g. Series 1 and 3) and the differences between
fore and hindlimbs (e.g. Series 1 and 3). Clearly, many more tests of competing
models of pattern formation during limb regeneration need to be performed
before any be accepted wholeheartedly, but the asymmetrical behaviour of skin
grafts revealed above severely strain the credulity of some.
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{Received 27 November 1981, revised 15 February 1982)