J. Embryol. exp. Morph. 82, 217-239 (1984)
Printed in Great Britain © The Company of Biologists Limited 1984
217
Regeneration of surgically created mixed-handed
axolotl forelimbs: pattern formation in the
dorsal—ventral axis
By NIGEL HOLDER AND CHARLESTON WEEKES
Anatomy Department, King's College, Strand, London WC2R2LS,
U.K.
SUMMARY
The regeneration of surgically created mixed-handed limb stumps is examined in the
axolotl. Operations were performed in the lower arm and upper arm regions and grafts were
allowed to heal for approximately one month prior to amputation or were amputated
immediately. In the lower arm group both anterior and posterior limb halves were inverted,
whereas only posterior halves were inverted in the upper arm group. Almost all the limbs
regenerated were normal in the anterior-posterior axis, whereas a range of limb types were
found when the dorsal-ventral axis was analysed using the metacarpal muscle pattern and
epidermal Leydig cell number as positional markers. The carpal and forearm muscle patterns
were also analysed in order to assess whether the pattern determined from analysis at the
metacarpal level reflected that seen at more proximal levels. The results are discussed in terms
of the possible role of cell contribution from the stump to the blastema and the relevance of
the study to models of pattern regulation.
INTRODUCTION
Pattern regulation during limb regeneration is standardly discussed in terms
of the three cardinal limb axes; the anterior-posterior axis, the dorsal-ventral
axis and the proximal-distal axis. The recreation of positional values within the
blastema has been studied by various types of tissue-grafting operations which
affect cellular position with respect to one or more of these axes (Tank & Holder,
1981). In this paper we further analyse pattern regulation in the dorsal-ventral
axis in the light of recent advances. Unlike the anterior-posterior axis, the
dorsal-ventral appears not to rely on a principle of continuity in that limb
patterns bearing clear anatomical discontinuities in this axis are readily obtained
after certain grafting experiments. To our knowledge no case exists in the
literature where comparable discontinuities have been observed in the anteriorposterior axis following many different types of surgical manipulation.
The discontinuities in the dorsal-ventral axis have been identified in supernumerary limbs following 180° ipsilateral blastema rotations (Maden, 1980,
1982, 1983; Maden & Mustafa, 1982; Tank, 1981; Papageorgiou & Holder,
1983), and in supernumerary outgrowths formed following skin transplantation
and nerve deviation (Reynolds, Holder & Fernandes, 1983; Maden & Holder,
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N. HOLDER AND C. WEEKES
1984). Four basic categories of limb anatomy have been identified in these types
of supernumerary limbs, two of which bear anatomical discontinuities. These are
part symmetrical-part asymmetrical patterns and mixed-handed limbs in which
one part of the pattern is inverted in the dorsoventral axis with respect to the
remainder. The remaining limb types found in these supernumeraries are either
normal or completely symmetrical double dorsal or double ventral limbs.
Neither of these limb types bear anatomical discontinuities.
The existence of discontinuities poses severe problems for any model based on
a principle of continuity, irrespective of the method for creating continuous
positional values. For this reason we have performed a series of experiments
designed to further elucidate the mechanisms of pattern regulation in the dorsalventral axis. In this study we have analysed the regenerative ability of surgically
created mixed-handed limbs. As the result of surgical construction we know what
the initial structure of any outgrowth will be, unlike the situation with supernumeraries. The times of graft healing prior to amputation and the limb levels
at which operations were performed were also varied to test the specific predictions of the shortest arc intercalation model for the control of pattern regulation
and distal outgrowth (Bryant, 1978; Bryant & Baca, 1978; Bryant et al. 1982;
Holder et al. 1980). The results demonstrate a number of points relating to the
maintenance and position of discontinuities in the dorsal-ventral axis, the complicated diversity of limb anatomies which form following pattern regulation in
this axis and the relationship between this axis and the anterior-posterior axis.
MATERIALS AND METHODS
General
All experiments were performed on larval axolotls (Ambystoma mexicanwn)
which were spawned in the colony at King's College. Animals were kept in
individual plastic containers in standing tap water throughout the course of the
experiment and were fed twice a week on chopped heart. All animals were
anaesthetized in MS222 during surgery or harvesting of the limbs. The animals
were between 55 mm and 85 mm in length.
Experimental
Operations were performed on either upper or lower arms. The exact surgical
procedure will be described for both.
1. Lower arm operations
In experimental cases the limb was split between digits 2 and 3 and a cut made
up to the elbow between the radius and ulna. In one group the anterior half of
the limb distal to the elbow was removed by disarticulating the radius and cutting
the anterior tissue free. In a second group the posterior half (digits 3 and 4) was
Regeneration of mixed-handed axolotl forelimbs
v D
D
219
v
V D
Fig. 1. Diagrammatic representations of the operations performed. A. Lower arm
operations. B. Upper arm operations. In both diagrams the dashed region in the end
on views of the amputated limbs represent the grafted tissue. A, anterior; P, posterior; D, dorsal; V, ventral; r, radius; u, ulna; h, humerus.
removed after disarticulation of the ulna. The corresponding procedure was carried out on the contralateral limb and the separated pieces were exchanged. The
grafts were then realigned on the opposite limb with the anterior-posterior and
proximal-distal axes of graft and host in harmony and the dorsal-ventral axes
misaligned (Fig. 1A). The grafts were then sutured in place using fine thread.
Sutures were placed at the proximal end of the graft and in the dorsal and ventral
midline. The limbs were either amputated immediately following the surgery or
after about a month of healing (between 25 and 35 days after initial surgery). In
both instances, amputation was performed at the mid-forearm level, leaving at
least 2 mm of forearm stump with a dorsal-ventral discontinuity (Fig. 1A).
Control operations involved following the same surgical procedure but the
anterior or posterior halves of the lower limb were sutured back into their normal
position without exchange to the contralateral side. These limbs were amputated
either immediately after surgery or after a month of healing.
2. Upper arm operations
Only posterior tissue was exchanged in this group. The operations involved
cutting out the posterior half of the upper arm. Care was taken to leave the
forearm flexor nerve and its adjacent blood vessel intact in the mid-ventral line,
and the muscles were removed from the posterior edge of the humerus, leaving
it intact in the host limb. The dorsal anconaeus muscle was split into two parts
and the triceps muscle was included in the graft. Once removed, the graft was exchanged with the corresponding posterior upper limb half from the contralateral
220
N. HOLDER AND C. WEEKES
limb. The grafts were aligned in the anterior-posterior and proximal-distal axes
and misaligned in the dorsal-ventral axis (Fig. IB). The grafts were sutured in
place at the proximal end and in the dorsal and ventral mid-lines. Limbs were then
amputated either immediately after surgery or after one month of healing.
Control operations involved the same surgical procedure but the grafts were
replaced and sutured into their normal positions without contralateral exchange.
Control operated limbs were either amputated immediately or after one month
of healing.
All operated animals were observed at weekly intervals to assess the survival
of the graft.
3. Analysis of limb anatomy
All experimental and control operated limbs were removed from the animal
after 6 to 8 weeks of regeneration. They were fixed in Bouin's fluid and decalcified in EDTA before being dehydrated, stained with Victoria blue and cleared.
The skeletons of all limbs were then drawn using a camera lucida. The limbs were
then returned to absolute alcohol and processed for wax embedding. Serial 10 [im
transverse sections were cut and stained with haematoxylin and eosin. The
muscle pattern of each limb was then recorded by examining sections at the plane
of the metacarpals, carpals and mid-forearm.
In addition to the musle patterns, the dorsal-ventral asymmetry of Ley dig cell
number in the epidermis was assessed at the metacarpal level. Ley dig cells are
large secretory cells which are commonly found in the epidermis of urodeles (see
Kelly, 1966, and Hay, 1961, for detailed accounts of their structure and possible
function). In order to analyse Ley dig cell number, a series of unoperated control
limbs were fixed, wax embedded and sectioned to assess the normal epidermal
anatomy in the forelimb. Leydig cell asymmetries were quantitatively assessed
by counting the number of Leydig cells in dorsal and ventral epidermis and
expressing this number as a percentage of total epidermal cell number for dorsal
and ventral limb regions.
Fig. 2. Aspects of normal limb anatomy. (A) Camera-lucida drawing of a dorsal
view of a normal forelimb regenerate showing the skeleton in a Victoria-blue-stained
specimen. The dashed lines show the approximate levels at which transverse sections
were analysed in order to examine muscle patterns and epidermal character. Magnification xlO. (B) Camera-lucida drawing of a transverse section at the metacarpal
level of a normal limb. The ventral muscles are marked with asterisks. Magnification
x38. (C) Light micrograph of the dorsal epidermis of a normal limb at the metacarpal
level. The relatively thick epidermis contains many Leydig cells (L) with scattered
epidermal cells (e). A clear basement membrane (bm) is seen and a region of an ebd
muscle. Magnification x 108. (D) Light micrograph of the ventral epidermis from the
same section as C shown at the same magnification. The epidermis is clearly thinner,
contains fewer Leydig cells and relatively greater numbers of other epidermal cells.
h, humerus; r, radius; u, ulna; r', radiale; u', ulnare; i, intermedium; m, metacarpal;
A, anterior; P, posterior; D, dorsal; V, ventral.
Regeneration of mixed-handed axolotl forelimbs
• „.»
221
*
Q
EMB82
222
N. HOLDER AND C. WEEKES
RESULTS
A. Normal limb anatomy
The skeleton, muscles and Leydig cell numbers were used to assess the pattern
of the regenerated limbs. The skeleton was used to examine the normality of the
anterior to posterior axis. The crucial features of the forelimb skeleton have been
outlined on numerous occasions (see for example, Tank & Holder, 1978) and a
normal limb skeleton is shown in Fig. 2A.
The muscle pattern at the metacarpal level has been used as a dorsal-ventral
marker in several recent studies (see description by Maden 1980,1982). In these
previous experiments the muscle patterns were examined mostly in supernumerary limbs (Maden, 1980,1982; Maden & Mustafa, 1982; Tank, 1981; Reynolds
et al. 1983; Papageorgiou & Holder, 1983) where more proximal levels are
unclear due to fusion of the supernumeraries with the host limb. In the present
series of experiments, because the experimental limbs were direct outgrowths
from an operated stump, we have assessed the muscle patterns at the carpal and
mid-forearm levels for the first time as markers for dorsoventrality in amphibian
limbs. For this reason a brief account of the muscle patterns at these more
proximal limb levels is needed. The normal anatomy of the muscles of the
forearm and hand of the axolotl forelimb has been described in detail by Grim
& Carlson (1974).
The patterns of muscles in the metacarpal region are clearly dissimilar in the
dorsal and ventral positions. The important difference is that in the ventral
region there are a complicated series of five separate muscles which are continuous across the anterior-posterior axis whereas dorsally four separate muscles
occur, one associated with each metacarpal (Fig. 2B, and Maden, 1980, 1982).
At the proximal carpal level the muscle pattern on the dorsal side of the carpals
is similarly less complicated than that on the ventral side. There are two muscles
dorsally and six muscles ventrally. The names and positions of these muscles are
Fig. 3. Normal muscle patterns at the proximal carpal and mid forearm levels. (A)
A transverse section at the proximal carpal level stained with haematoxylin and
eosin. Magnification x34. (B) Camera-lucida drawing of the section shown in A,
giving the names of the muscles. The abbreviations are: (1) extensor muscles; edc,
extensor digitorum communis; ad 1, abductor minimi 1. (2) flexor muscles; ps, palmaris superficialis; uc, ulno carpalis; pp 1,2,3, palmaris profundus muscles; abdm,
abductor brevis digiti mimimi. The carpals are radiale, intermedium and ulnare from
anterior to posterior (see Fig. 2A). (C) Transverse section at the mid-forearm level
stained with haematoxylin and eosin. Magnification x32. (D) Camera-lucida drawing of the section shown in C giving names of the muscles. The abbreviations are: (1)
extensor muscles; edc, extensor digitorum communis; ecr, extensor carpi radialis; ear,
extensor antebrachii radialis; eacu, extensor antebrachii carpi ulnaris. (2) flexor
muscles; ps, palmaris superficialis; uc, ulno carpalis; facr, flexor antebrachii et carpi
radialis; feu, flexor carpi ulnaris; pq, pronator quadratus. D, dorsal; V, ventral; A,
anterior; P, posterior; r, radius; u, ulna.
Regeneration of mixed-handed axolotl forelimbs
223
224
N. HOLDER AND C. WEEKES
Table 1. Ley dig cell/epidermal cell ratio
Dorsal epidermis
Ventral epidermis
General
epidermal cells
Leydig cells
Total
Ratio
409 ±45*
525 ±79
194 ±26
80 ±26
603 ± 65
605 ±91
0-47 ±0-04
0-15 ±0-02
*±S.D.
shown in Fig. 3A,B. The forearm muscle pattern is more complex. The main
complicating feature is the change in the pattern which occurs at different
forearm levels. For this reason the mid-forearm was selected as the position to
assay the pattern (Fig. 3C,D). This region was easily located because of the
presence of a ventral muscle concerned with pronating the limb, the pronator
quadratus (pq), which runs between the radius and ulna in the mid-third of the
forearm. In transverse sections the fibres of the muscle are cut tangentially
making its identification straightforward (see Fig. 6D). The remaining flexor and
extensor muscles in this mid-forearm position are also readily identifiable by
their individual shape and their position relative to the other muscles and to the
radius and ulna. Dorsally just four muscles are found, whereas five muscles occur
in the ventral flexor group. The names and positions of these muscles are shown
in Fig. 3.
The third marker used to assay dorsoventrally is the presence of Leydig cells
in the epidermis. At the metacarpal level more Leydig cells are found in the
dorsal epidermis than the ventral epidermis. This difference has been quantified
by counting Leydig cells in sections at this level in seven normal limbs, and
expressing Leydig cell number as a ratio of the total epidermal cell count in
dorsal and ventral epidermis. The results are presented in Table 1. At the light
microscope level the remaining epidermal cells appear to be histologically
similar and no attempt was made to further categorize them. In Table 1 these
cells are referred to as general epidermal cells. It can be seen that closely similar
numbers of cells are found in dorsal and ventral epidermis but approximately
three times as many of the total epidermal cells are Leydig cells in the dorsal
epidermis. This marker is extremely specific and the numbers are statistically
significantly different. The clear structure of the Leydig cells allows them to be
easily visualized and because of their large size relative to the remaining
epidermal cells the dorsal epidermis is noticeably thicker than the ventral
epidermis (Fig. 2C,D). This feature alone often allows identification of dorsal
epidermis after a mere glance at an appropriate section. Unfortunately, the
significant difference in Leydig cell number at the metacarpal level disappears
at more proximal levels where many Leydig cells are found in dorsal and ventral
epidermis.
Regeneration of mixed-handed axolotl forelimbs
225
Table 2. The anatomy of the a-p axis
Healing
Level of
time
amputation (days)
Lower arm
Lower arm
Upper arm
Upper arm
0
30
0
30
Total
Normal
18
13
16
18
10
14
10
Extra
digits
Normal
digits
No.
Abnorm. Reduced* Spikes/t
forearm digit no. Half limbs regn.
* Normal forearm but only three digits.
t Abnormal forearm and reduced digits.
B. Anatomy of experimental limbs
The features of the anterior-posterior and dorsal-ventral axes will be presented in turn.
1. The anterior-posterior axis
The anatomy of the a-p axis was assessed from Victoria-blue-stained whole
mounts. It was normal in the great majority of cases, in both upper arm and lower
arm operations following immediate amputation and amputation after
approximately one month of graft healing. Of a total of 65 operated limbs in all
groups 52 (80 %) had an essentially normal a-p pattern in the hand (Table 2).
No clear differences were evident in the lower arm amputations between the
anterior (12 cases) and posterior (19 cases) operated groups, so this variation has
not been entered in Table 2.
In the lower arm amputations, principally in the immediate amputation group,
the forearm elements were often abnormal even though a normal hand was
formed. The forearm abnormalities involved fusion of the radius and ulna
proximally with an essentially normal elbow (eight of the ten cases) or an abnormal elbow (two of the ten cases). In contrast, when a normal hand was formed
following amputation in the upper arm the forearm was invariably normal (Table
2). In just two cases, both in the lower arm amputated groups, only three digits
were formed in an otherwise normal limb.
Two categories of limbs were found only in the upper arm groups. These were
non-regenerates, where no outgrowth occurred following amputation (two
cases, both in the 30-day healing group), and limbs which had either a single
forearm element and one or two digits, or two forearm elements with one or two
digits (Table 2).
2. The dorsal-ventral axis
A range of limb types was identified following the analysis of muscle and epidermal markers at the metacarpal level of the regenerates. The results of this analysis
226
N . HOLDER AND C. WEEKES
Table 3. Muscle patterns of lower arm operated limbs
Digit number
Case
Healing time
(days)
1
2
3
4
Category
D
V
V
D
V
D
V
D
D
V
V
D
V
D
V
D
V
D
V
D
V
1
D*V
V
D*V
D
V
D
V
V*D
V
D
V
V*D
V
D
V
D
D
D
D
V
35
D *
D *
30
V
V
V
D
V
D
V
V
D
V
D
V
V
D
V
D
V
D
V
D
V
D
D*V
V
D*V
V
D*V
V
V
V
V
V
V
D
V
D
V
D
V
D
V
D
V
V
Anterior side inverted.
1
0
2
35
3
25
4
25
5
34
v *
D *
6
7
8
0
D *
D *
v *
vV *
vV *
2b
2b
2b
2c
3b
5a
5b
Posterior side inverted.
9
30
10
30
11
30
12
30
13
30
14
0
15
16
17
18
19
20
21
22
0
0
0
0
D
V
D
V
D
V
D
V
D
V
D
V
D
V
D *
v *
D*V
V*D
D*V
V *
D*V
v *
D*V
v *
D
V
D
V
D
D
V
V
D
D *
V
V
D
D *
V
V
D
V
30
D
0
V
D
V
0
D *
0
V
D
V
* represents a point of discontinuity.
D *
V
V
V
D *
V
D *
V
V
V
V
V*D
V
v *
V
V
V
V
V
V
V
V
V
V
D
V
V
D
V
D
2a
2a
2b
2b
2b
3a
3a
3a
3a
3a
3b
5a
5b
5b
Regeneration of mixed-handed axolotl forelimbs
227
Table 4. Dorsal/ventral symmetry of upper arm operated limbs
Digit muscle patterns
Case
Healing time
(days)
1
2
3
4
Category
D
V
D
V
D
V
D
V
D
V
D
V
D
V
D
V
V
V
D
D
D
V
D
V
D
V
D
V
D
V
D
V
D
V
D
V
D
V
D
V
D
V
D
V
D
V
D
V
D
V
D
V
D
V
D
V
D
V
V
D
V
D
V
V
V
D
V
D
1
Posterior side inverted.
1
0
2
0
3
0
4
0
5
30
6
0
7
0
8
30
9
30
10
30
11
12
13
14
15
16
17
0
0
0
30
0
0
0
19
30
20
0
D
21
0
22
30
30
23
30
24
30
D
V
D*V
V
V
v *
D *
D
D
v *
D
V *
D
v *
D
v *
D
D
V
D
V
D
18
D *
V *
v *
vD *
D
D
D
V
V
D
V
D
V
V
D
D *
vV *
V
V
D
V
D
D
D
D
D
D
D
D
D
D *
D
D
D
D
D
D
D
1
1
2a
2c
3a
3a
3a
3a
3a
3a
3a
D
3b
D
D
D
D
D
D
D
4
D
D
D
D
V
V
V
D
v *
D
D
V
V
D *
* represents a point of discontinuity.
V
1
V
V
V
V
V
V
D
D
D
D
D
D
V
D
V
D
V
D
V
D
D
D
D
D
•
D
1
V
V
D *
V
D *
V
D
V *
D
D
V
V
V
3b
3b
3b
3c
3c
4
4
5b
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N. HOLDER AND C. WEEKES
are shown for the lower arm operations in Table 3 and the upper arm operations in
Table 4. Following the description of the different types of limbs their frequency of
formation in the different experimental groups will be presented. Finally, the
results of the analysis of forearm and carpal muscle patterns will be discussed.
a. Extra skeletal structures. Of the 65 experimental limbs five (8 %) produced
extra skeletal structures in the dorsal-ventral plane. All of these involved the
formation of extra digits which emerged on the ventral stump side of the outgrowth at the metacarpal level. One of these cases was from a lower arm amputation (case 13, Table 3) and four were from upper arm amputations (cases 2, 6,
9 and 19, Table 4). All except one were single extra digits and case 6 in Table 4
had two clearly defined digits beneath the inverted digits 3 and 4.
b. Categories of limb types identified from sections. Only limbs with four
digits in the anterior-posterior axis and an otherwise normal or near normal
anterior-posterior anatomy were sectioned. Of these seven lower arm operated
limbs failed to process correctly following Victoria blue staining. As a result, 22
out of 31 lower arm amputations and 24 out of 32 upper arm amputations were
analysed. These sectioned limbs fell into five basic anatomical classes as determined by the muscle and epidermal markers at the mid-metacarpal level. These
limb types are described below. The anatomies of each individual limb for lower
arm and upper arm groups are given in Tables 3 and 4, the five classes of limb
types are represented diagrammatically in Fig. 4 and examples of each class of
abnormal anatomy are shown in Fig. 5.
Type 1. Normal limbs
The anatomy of normal limbs has already been described. An example is
shown in Fig. 2B.
Type 2. Mixed-handed limbs
Three subclasses of mixed-handed limbs were identified depending on the
position of the anatomical discontinuity, (a) In the first of these the pattern was
exactly that which was created by surgery, with the line of discontinuity clearly
located between digits 2 and 3, (Fig. 5A). (b) In some cases the line of discontinuity ran at an angle from dorsal to ventral so that one digit bore a hybrid muscle
which changed form from ventral to dorsal. This distinction was coupled with a
change in epidermal character at the corresponding anterior-posterior position,
(c) The line of discontinuity shifted one complete digit width in this group so that
three digits had one polarity and one digit was inverted.
Type 3. Part normal/part symmetrical limbs
Again, three sub classes of such limbs were seen which were distinguished by
the position of the discontinuity. Thus, the symmetrical region involved one, two
or three digits with the remainder of the pattern being asymmetrical. The
symmetrical region involved either dorsal or ventral character, (Fig. 5B,C).
Regeneration of mixed-handed axolotl forelimbs
Type 1
Type 2
Type 3
Type 4
Type 5
229
TypeS
Fig. 4. Schematic representations of the categories of regenerates seen with
reference to the anatomy of the dorsal-ventral axis at the metacarpal level. Five
classes are identified based on muscle patterns and epidermal character. Representative sections of each class and some of the sub-classes described in the text are
shown. The dashed regions represent dorsal and the clear regions ventral and the
small circles are metacarpals.
Type 4. Symmetrical limbs
These limbs showed a completely symmetrical character involving either
double dorsal or double ventral symmetry (Fig. 5D).
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N. HOLDER AND C. WEEKES
D
D
Fig. 5. Camera-lucida drawings of specimens from the experimental series showing
the skeletons and metacarpal level muscle patterns of the same limb. Abbreviations
in all drawings are; A, anterior; P, posterior; D, dorsal; V, ventral. Numbers 1-4
refer to digit numbers, see Fig. 2A. Magnifications of skeletal preparations x9, of
sections x30. (A) A mixed-handed pattern with the discontinuity between digits 2
and 3, (type 2a). This limb regenerated after immediate amputation of a right upper
arm operation in which the posterior side was inverted (case 6, Table 4). (B) A part
double ventral/part normal pattern with the posterior two digits symmetrical (type
3a). This limb regenerated after immediate amputation of a right lower arm operation in which the posterior side was inverted (case 17, Table 3). (C) A part double
dorsal/part normal pattern with the posterior two digits symmetrical (type 3a). (D)
A completely symmetrical double dorsal limb (type 4) regenerated following immediate amputation of an operated left upper arm (case 21, Table 4). (E) A limb with
a symmetrical ventral region flanked by asymmetrical regions of the same polarity
(type 5a). This limb regenerated following immediate amputation of a left lower arm
operation in which the posterior side was inverted (case 20, Table 3). (F) A limb with
a symmetrical ventral region flanked by asymmetrical regions of opposite polarity
(type 5b). This limb regenerated following immediate amputation of a left lower arm
in which the posterior side was inverted (case 22, Table 3).
Regeneration of mixed-handed axolotl forelimbs
D
231
D
Type 5. Limbs with a symmetrical region located between two asymmetrical
regions
Two sub-classes of this pattern were found and ventral symmetry was identified in all cases. In the first a double ventral region was flanked by two
asymmetrical regions of the same polarity (Fig. 5E), whereas the second type
showed flanking regions of opposite polarity (Fig. 5F). These types of limbs have
not been described previously.
c. The frequency of formation of limb types. The frequency of formation of
the five limb types varied only slightly between forearm and upper arm groups
(Table 5). In the upper arm groups part normal part symmetrical limbs were most
frequent (54 %), with all three subclasses of this type represented. All classes
were represented in the upper arm operations. In contrast, mixed handed limbs
were most frequent in the lower arm operated group (41 %) and part normal/
part symmetrical patterns were also commonly found (32%). No completely
symmetrical limbs were formed in the lower arm group.
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N. HOLDER AND C. WEEKES
Table 5. Frequency of limb types found in the regenerates with reference to level
of amputation
Lower arm
Upper arm
Limb type*
Total
%
1
2
3
4
5
5
2
13
3
1
21
8
54
13
4
1
9
7
0
5
Totals:
24
100
22
Total
%
4
41
32
0
23
100
* With reference to the categories shown in Fig. 4.
Table 6. Frequency of limb types found in the regenerates with reference to time
of graft healing
Immediate amputation
30 day amputation
Limb type*
Total
%
Total
1
2
3
4
5
5
2
13
1
3
21
8
54
4
13
1
9
7
2
3
4
41
32
9
14
Totals:
24
100
22
100
%
* With reference to the categories shown in figure 4.
Similar frequencies of limb types are seen if immediate and 20-day periods of
healing are compared as pooled data from lower arm and upper arm groups
(Table 6). Thus, after immediate amputation, part normal/part symmetrical
patterns are the most frequent (54 %) whereas, following graft healing for 30
days, mixed handed (41 %) and part normal/part symmetrical limbs (32 %) are
the commonest types regenerated.
Fig. 6. Examples of abnormal carpal and forearm muscle patterns. (A) Light
micrograph of a section cut at the proximal carpal level of a limb identified as double
dorsal at the metacarpal level (case 22, Table 4). Magnification x32. (B) A camera
lucida drawing of the same section identifying the muscles. Abbreviations as for Fig.
3A,B. (C) Light micrograph of a section cut at the mid forearm level of a limb
identified as double ventral at the metacarpal level (case 23, Table 4). Magnification
x32. (D) Higher power view of the section in C showing the two copies of pq.
Magnification X109. Abbreviations as for Fig. 3C,D.
Regeneration of mixed-handed axolotl forelimbs
233
234
N. HOLDER AND C. WEEKES
d. The effect of grafting on the anatomy of regenerates. One consistent aspect
of the results is the maintenance of anatomy and polarity in the host, unoperated,
side of the limb pattern and the variability of the operated side in the regenerated
patterns. When the eventual patterns are examined the extreme lateral or medial
digit on the unoperated side (digit 1 for posterior inversions and digit 4 for
anterior inversions) is normal in terms of anatomy and polarity in 100 % of the
lower arm operations and 79 % (19 out of 24) of the upper arm operations. In
clear contrast, the appropriate extreme medial or lateral digit on the operated
side shows an inverted asymmetrical anatomy in 54 % (12 out of 22 cases) in
lower arm operations and 20 % (5 out of 24 cases) in upper arm operations. The
digit in the middle of the pattern in the unoperated side show less stability due
to local variations in position and extent of the anatomical discontinuity.
The invariability of the host side of the limb is not absolute and five cases occur
in the upper arm group which show alterations in anatomy of digit 1. Of these,
three were totally symmetrical limbs (cases 21—23, Table 4) and two were part
normal/part symmetrical limbs in which digits 1 and 2 were symmetrical and the
operated side regenerated an inverted asymmetric pattern (cases 10 and 11,
Table 4).
e. Analysis of carpal and forearm muscle patterns. The object of analysing
muscle patterns at more proximal levels was to establish whether the patterns
determined by analysis at the metacarpal level reflected the anatomy of the
whole regenerate. In addition, any alterations in the position of discontinuities
down the proximal-distal axis could be established. In the following discussion
the limb types are discussed with reference to their anatomy at the metacarpal
level.
In general, the carpal and forearm muscle patterns are not as informative as
the more distal muscle pattern. This is particularly true of the mixed-handed
patterns where the forearm musculature is extremely complex. However,
normal and totally symmetrical limbs and part normal/part symmetrical patterns
show clearly interpretable muscle patterns at more proximal levels.
The most straightforward cases are the normal or totally symmetrical
regenerates. In the upper arm group, all of the five normal cases showed normal
muscle patterns at carpal and mid-forearm levels. These patterns are indistinguishable from those shown in Fig. 3. The three totally symmetrical cases also
showed symmetrical muscle patterns at the carpal and forearm levels. The symmetry at these more proximal levels was appropriate for that seen at the digits.
Examples of symmetrical carpal and forearm muscle patterns are shown in Fig.
6. These observations clearly show that the normal and totally symmetrical cases
have this anatomy at least from the mid-forearm level and are likely, therefore,
to show this anatomy to the amputation plane.
Part normal/part symmetrical patterns with two or three symmetrical digits
are more complicated at proximal levels. In the upper arm group nine limbs of
this type showed distinct symmetry in the carpals and forearm. In the seven
Regeneration of mixed-handed axolotl forelimbs
235
examples with dorsal symmetry five showed dorsal symmetry on the appropriate
side in the forearm and thepg, uc and feu were missing (for muscle patterns and
muscle names see Fig. 3), and the opposite side was asymmetrical. Thus the
proximal levels again reflect the pattern seen in the digits. One case (number 14,
Table 4) had a normal forearm but the carpals appeared to be symmetrical
double dorsal, leading to the part normal/part symmetrical hand. This limb
showed clear movement of lines of discontinuity down the proximal-distal axis.
The remaining case with double dorsal symmetry was uninterpretable. The two
limbs with part ventral symmetry both showed duplication oiabdm at the carpals
with an otherwise normal muscle pattern and one showed two copies of pq at the
forearm level with an otherwise normal pattern. In the upper arm group three
limbs showed part normal/part asymmetrical patterns with only one double
ventral digit (type 3d). In all cases (16-18, Table 4) the forearm muscle patterns
were clearly normal, yet the carpal pattern reflected the discontinuity in two
cases with abdm being duplicated on the posterior side with an otherwise normal
pattern.
In the lower arm groups the single normal limb had a clearly normal carpal and
forearm musculature. The part normal/part symmetrical cases invariably involved double ventral symmetry. Of the five cases with greater than one symmetrical digit, three had two copies of the pq muscle (Fig. 6C), and three had
duplicated sets of uc and feu at the forearm level and all had two copies of abdm
at the carpal level.
The duplication of the pq muscle also occurred in many of the mixed-handed
patterns in both upper and lower arm regenerates. This observation, even in the
absence of clarity in surrounding muscles, indicates that the discontinuity in such
limbs has structural consequences in the dorsal-ventral axis at more proximal
levels.
C. Controls
A total of 24 control operations were performed. These include 16 in the lower
arm group, with eight cases amputated immediately and eight amputated after
30 days of healing. At each healing time four operations involved removing and
replacing the anterior side and four the posterior side. Eight cases were analysed
in the upper arm group, four of which were amputated immediately and four
amputated after 30 days of healing. In all cases the a-p axis was essentially
normal as assessed from Victoria blue stained wholemounts and the d-v axis was
also normal at metacarpal, carpal and forearm levels.
DISCUSSION
The results presented in this study provide several insights into the mechanism
of pattern regulation in the dorsal-ventral axis. The advantage of examining
regeneration from surgically constructed mixed-handed limb stumps is that the
236
N. HOLDER AND C. WEEKES
spatial relationships of cells in the stump prior to amputation are clearly identified. This is in contrast to supernumerary limbs produced following 180°
ipsilateral blastema rotation where the eventual anatomy of the extra limbs is
used to retrospectively determine the events which led to their formation
(Maden & Mustafa, 1982; Maden, 1983). It is reassuring, therefore, that the
principles of pattern regulation derived from these two types of grafting experiment are similar. Two basic points can be made from the results of the present
study. (1) It is clear that a number of anatomical limb types can regenerate
following amputation of mixed-handed limb stumps. Of the five basic types
identified (Fig. 4) four have been described in supernumeraries produced either
from blastemal rotation (Maden, 1980, 1982, 1983; Maden & Mustafa, 1982;
Papageorgiou & Holder, 1983) or nerve deviation and skin grafting (Reynolds
et al. 1983; Maden & Holder, 1984). These are normal limbs, mixed-handed
limbs, part normal/part symmetrical limbs and completely symmetrical limbs.
In addition, we have identified a new class of anatomy where a symmetrical
region lies centrally in the pattern and is flanked by asymmetrical digits (Table 5
and Figs 4, 5E,F). (2) Anatomical discontinuities where dorsal and ventral cells
lie adjacent to one another occur in many of the limbs regenerating from mixedhanded stumps. During regeneration it appears that little interaction occurs
between normally disparate cells in the dorsal-ventral axis, although, in a very
few cases (8 % of operated limbs) extra digits appeared on the ventral side of the
outgrowth.
A striking feature of the results is that the anatomy of the regenerates on the
grafted side of the limb is far more variable than the anatomy of the unoperated
side (Tables 3 and 4). This is evident irrespective of whether the anterior or
posterior limb region is inverted. The significance of this observation with respect to the patterning mechanism is unclear at the present time, however, the
variability of regeneration of grafted tissue has been noted previously following
the amputation of surgically created double anterior and double posterior limbs
(Tank & Holder, 1978; Holder et al. 1980; Holder, 1981). In these cases half
limbs regenerate with a frequency of approximately 25 % and the regenerate
invariably arises from the unoperated side of the stump. This result suggests that
dedifferentiation and cell contribution to the blastema maybe severely affected
in the grafted tissue and this conclusion is supported by the histological appearance of tissue in the grafted limb region where the muscle and general organization of other tissues is clearly disrupted (Tank & Holder, 1978). It is possible,
therefore, that the pattern of cell contribution from different parts of the stump
plays a role in the production of the different limb types produced in the present
study. A complicating factor here is the appearance of the regenerates from
control grafts which invariably have a normal d-v anatomy. It is possible that cell
contribution from the stump is a crucial part of the pattern regulation process and
efforts to study this aspect of regeneration are under way at the present time.
The results in this paper have a bearing on two formal models for pattern
Regeneration of mixed-handed axolotl forelimbs
237
regulation. The first of these is the boundary model (Meinhardt, 1983) which
proposes that two boundaries or discontinuities exist in the normal limb, one in
each of the transverse axes. Meinhardt has suggested that these two boundaries
must intersect before distal outgrowth will occur. The model does not discuss the
nature of the distinction between dorsal and ventral cells but the notion of a
boundary between cells in these limb regions in a normal limb is supported by
the appearance of limbs bearing clear discontinuities in this and other studies
(see also Maden, 1983). Such discontinuities would represent a shift in position
of the boundary in abnormally patterned limbs. However, the regeneration of
symmetrical double dorsal and double ventral limbs and limbs which are part
symmetrical on the host posterior side pose problems for the boundary model
because, presumably, no d-v boundary is present in such symmetrical regions and,
as a result, the interaction between the a-p and d-v boundaries necessary for limb
outgrowth cannot occur. Nonetheless, limbs with such patterns do regenerate.
Thus, although the idea of a discontinuity in the d-v axis in normal limbs is
attractive the formulation of the boundary model as it stands is problematical.
The second formalism to be discussed is the polar coordinate model (Bryant
et al. 1981). Any model which predicts the smoothing out of discontinuities and
the subsequent appearance of extra structures fails to explain the maintenance
of pattern discontinuities. At face value, therefore, the results presented here
cannot be explained by the polar coordinate model. However, this conclusion
may be thought naive because, as has been discussed previously (Bryant et al.
1982; Holder et al. 1980; Holder, 1981), local cell-cell interactions may be
constrained by healing modes which may be governed by wounds or field shape.
Such arguments have been used to explain the differential regeneration of
symmetrical double posterior and double anterior limbs following amputation
after various periods of graft healing and the expansion of the a-p axis following
amputation of double posterior limbs at different limb levels (Holder et al. 1980).
In brief, at short healing times cell contact is prevented by the wound, but, as
healing continues cell contact ensues. In terms of the present experiments,
therefore, a lack of interaction between dorsal and ventral cells at the sites of
discontinuity maybe expected when mixed-handed limbs are amputated immediately after surgery but extra structures would be expected when such limbs are
amputated after a month of healing. In fact, extra structures are produced at a
very low frequency at both time periods. This result is consequently not consistent with a healing constraint governing cell-cell contact. Similarly, expansion
of the a-p axis was originally explained by a preferential dorsal to ventral healing
mode at the forearm level (Holder etal. 1980; Bryant et al. 1982) and the elliptical shape and cell numbers separating the cardinal axes in the lower arm blastema
are consistent with this notion (Holder, 1981; Holder & Reynolds, 1983,1984).
However, such a biased mode of cell contact between dorsal and ventral cells
would preclude the regeneration of symmetrical double dorsal and double
ventral limbs distal to the elbow because cells with like positional values would
238
N. HOLDER AND C. WEEKES
contact and intercalation would cease. This is exactly the argument used to explain
why symmetrical double anterior and double posterior limbs fail to regenerate at
long healing times in upper arm amputations (Holder et al. 1980; Bryant et al.
1982). It is evident from the results presented here and those from experiments
involving 180 ° blastema rotation (Maden & Mustafa, 1982) that such symmetrical
limbs can and do regenerate distally complete limbs which have a normal a-p axis.
We must conclude that the arguments of the shortest arc intercalation model
(Bryant, 1978; Bryant & Baca, 1978) which underlies the proposals of the polar
coordinate model with respect to amphibian limbs are inconsistent with both a lack
of a healing time effect in the regeneration of mixed handed limbs and the
regeneration of symmetrical double dorsal and double ventral limbs.
Finally, the results presented in this paper demonstrate that, under the conditions imposed by this particular grafting procedure, dorsal-ventral discontinuities are stable. However, this is apparently not the case when the dorsalventral axis is misaligned following contralateral blastema exchanges which
results in the formation of supernumerary limbs at dorsal and ventral positions
in the graft-host junction (Bryant & Iten, 1976; Tank, 1978). The reason for this
difference is unclear at the present time, but the issue will be discussed in detail
in a subsequent paper which will describe regeneration from limb stumps bearing
discontinuities in the anterior-posterior axis which are surgically created in a
manner comparable to mixed-handed stumps.
It is a pleasure to thank Malcolm Maden, Rosie Burton, Nigel Stephens and Peter Wigmore
for various comments and criticisms during the course of this work, which was supported
financially by the SERC.
REFERENCES
S. V. (1978). Pattern regulation and cell commitment in amphibian limbs. In The
Clonal Basis of Development, (eds S. Subtelney & I. Sussex). 36th symp. Soc. Devi Biol.
pp. 63-82. New York: Academic Press.
BRYANT, S. V. & ITEN, L. E. (1976). Supernumerary limbs in amphibians: Experimental
production in Notophthalmus viridescens and a new interpretation of their formation. Devi
Biol. 50, 212-234.
BRYANT, S. V. & BACA, B. A. (1978). Regenerative ability of double-half and half upper arms
in the newt, Notophthalmus viridescens. /. exp. Zool. 204, 307-324.
BRYANT, S. V., FRENCH, V. & BRYANT, P. J. (1981). Distal regeneration and symmetry. Science
212, 993-1002.
BRYANT, S. V., HOLDER, N. & TANK, P. W. (1982). Cell-cell interactions and distal outgrowth
in amphibian limbs. Amer. Zool. 22, 143-151.
GRIM, M. & CARLSON, B. M. (1974). A comparison of morphogenesis of muscles of the
forearm and hand during ontogenesis and regeneration in the axolotl (Ambystoma
mexicanum). 1. Anatomical description of muscles of the forearm and hand. Z. Anat.
EntwGesch. 145, 137-148.
HAY, E. D. (1961). Fine structure of an unusual intracellular supporting network in the Leydig
cells of Ambystoma epidermis. J. biophys. biochem. Cytol. 10, 457-463.
HOLDER, N. (1981). Pattern formation and growth in the regenerating limbs of urodelean
amphibians./. Embryol. exp. Morph. 65, 19-36.
BRYANT,
Regeneration of mixed-handed axolotl forelimbs
239
N. & REYNOLDS, S. (1983). Morphogenesis of the regenerating limb blastema of the
axolotl: Shape, autonomy and pattern. In Limb Development and Regeneration. Progress
in Clinical and Biological Research, Vol. 110A, pp. 477-490. New York: A. Liss.
HOLDER, N. & REYNOLDS, S. (1984). Morphogenesis of the amphibian limb blastema: The
relationship between pattern and form. J. Embryol. exp. Morph. 79, 165-181.
HOLDER, N., TANK, P. W. & BRYANT, S. V. (1980). Regeneration of symmetrical forelimbs
in the axolotl, Ambystoma mexicanum. Devi Biol. 74, 302-314.
KELLY, D. E. (1966). The Leydig cell in larval amphibian epidermis, fine structure and
function. Anat. Rec. 154, 685-700.
MADEN, M. (1980). Structure of supernumerary limbs. Nature 286, 803-805.
MADEN, M. (1982). Supernumerary limbs in amphibians. Amer. Zool. 22, 131-142.
MADEN, M. (1983). A test of the predictions of the boundary model regarding supernumerary
limb structure. /. Embryol. exp. Morph. 76, 147-155.
MADEN, M. & HOLDER, N. (1984). Axial characteristics of nerve induced supernumerary limbs
in the axolotl. Wilhelm Roux. Arch, devl Biol. (in press).
MADEN, M. & MUSTAFA, K. (1982). The structure of 180 degree supernumerary limbs and a
hypothesis of their formation. Devi Biol. 93, 257-265.
MEINHARDT, H. (1983). A boundary model for pattern formation in vertebrate limbs.
J. Embryol. exp. Morph. 76, 115-137.
PAPAGEORGIOU, S. & HOLDER, N. (1983). The structure of supernumerary limbs formed after
180 degree blastemal rotation in the newt Triturus cristatus. J. Embryol. exp. Morph. 74,
143-158.
REYNOLDS, S., HOLDER, N. & FERNANDES, M. (1983). The form and structure of supernumerary hindlimbs formed following skin grafting and nerve deviation in the newt, Triturus
cristatus. /. Embryol. exp. Morph. 77, 221-241.
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.
TANK, P. W. (1981). Pattern formation following 180 degree rotation of regeneration
blastemas in the axolotl, Ambystoma mexicanum. J. exp. Zool. 217, 377-387.
TANK, P. W. & HOLDER, N. (1978). The effect of healing time on the proximodistal organisation of double half forelimb regenerates in the axolotl, Ambystoma mexicanum. Devi Biol.
66, 72-85.
TANK, P. W. & HOLDER, N. (1981). Pattern regulation in the regenerating limbs of urodelean
amphibians. Q. Rev. Biol. 56, 113-143.
HOLDER,
(Accepted 19 March 1984)