/. Embryol. exp. Morph. Vol. 58, pp. 265-288, 1980
Printed in Great Britain © Company of Biologists Limited 1980
265
Morphogenetic properties of the skin in
axolotl limb regeneration
By JONATHAN M. W. SLACK 1
From the Imperial Cancer Research Fund,
Mill Hill Laboratories, London
SUMMARY
A study has been made of the morphogenetic properties of anterior and posterior skin
from the lower forelimb of the axolotl. The basic experiment consisted of a graft of a half
cuff of skin from a donor to a host limb followed by a 2-week healing period, amputation
through the graft, and a study of the resulting regenerate. Limbs with double posterior
skin formed double posterior regenerates and, in contrast, limbs with double anterior skin
formed normal or slightly hypomorphic regenerates. Posterior skin from post-metamorphic
animals had a similar but weaker effect to that from ordinary axolotls.
Immunological rejection of allografts could be completely avoided if the donor limb was
transplanted to the flank of the host when both were at the stage of tail-bud embryos, and
the skin graft was later carried out between the supernumerary limb and one of the host
limbs. This technique was used to show that immunological rejection does not affect the
formation of duplicates from the limbs with double posterior skin, and to facilitate the
studies of the cellular provenance of the regenerate.
The cellular composition of duplicate regenerates was studied by using both triploid
donors and triploid hosts. It was shown that the posterior side of the duplications consisted
wholly of host tissue and the anterior side consisted of mixed donor and host tissue. Formation
of the duplicated regenerate therefore seems to involve positional reprogramming of both
donor and host tissues together with metaplasia of the donor tissue.
It was not possible to inhibit the duplication-inducing property of posterior skin by
treatment with a variety of enzymes.
A model based on the serial threshold theory of regeneration is advanced to explain the
results. This model successfully accounts for the observed non-equivalence of anterior and
posterior skin, and also explains the different regeneration behaviour of anterior and
posterior half limbs, the limited regeneration of double anterior limbs, and the pattern
expansion and contraction shown by regenerates from double posterior limbs.
INTRODUCTION
The axolotl is an animal which can regenerate its limbs after amputation
and in which the spatial arrangement of structures in the regenerate is restored
with very high fidelity. This formation of a complex spatial pattern distinguishes
true regeneration from other phenomena such as the hypertrophy of the mammalian liver after a part has been removed, or the regrowth of nerve axons
1
Author's address: Imperial Cancer Research Fund, Mill Hill Laboratories, Burtonhole
Lane, London, NW7 IAD, U.K.
266
J. M. W. SLACK
down a pre-existing tract. It poses a specific biological problem: how do the
cells of the regeneration blastema 'know' which structures to turn into and
in what relative order these should be arranged ?
Certain theorists of regeneration have postulated that the cells in each
region of tissue in a differentiated limb, or any other organ capable of regeneration, are labelled with 'positional values' which vary in a continuous
way across the organ (Wolpert, 1971; Bryant & Iten, 1976; Maden, 1977;
Lheureux, 1977). According to these authors, when cells near the amputation
surface dedifferentiate and divide to form the blastema, their positional values
are erased and are recomputed in such a way that a complete set is reformed
which is continuous with the positional values represented in the stump. The
pathway of cytodifferentiation to be followed by each group of blastemal cells
is then selected in accordance with their new positional value, irrespective of
their cytological type in the previous limb.
In a theoretical paper (Slack, 1980a) I have argued that these positional
values are made up of combinations of ' o n ' and 'off' states of a set of biochemical switches, and that the arrangement of these combinations, or 'codings'
is such that the structure of the missing parts can be computed from the
codings represented at the amputation surface without any long range interactions with the remaining part of the organ. If this view is accepted then the
problem of pattern formation can be rephrased as: 'What is the relation
between the anatomical position and the coding and what are the rules for
altering codings during regeneration?' One method of attacking this problem
experimentally is to alter the arrangement of tissues in the organ in order to
provoke the regeneration of an abnormal pattern. If a normal limb is amputated
then the pattern of the regenerate is the same as the original, but this is not
generally true of abnormal limbs generated by embryonic manipulation or by
surgery on adults (Swett, 1924; Newth, 1958). If enough reliable data can be
collected on the relationship between starting patterns and final patterns we
might be able to deduce the number and arrangement of codings and the rules
for their interconversion, and once the rules are known it may become possible
to make informed guesses about the biochemical nature of the codings.
For some years it has been known that grafts of skin from one part of the
limb to another can derange the pattern of a regenerate which is formed after
amputation through the grafts (Droin, 1959; Rahmani, 1960; Lheureux, 1972)
and the active component is known to be the dermis rather than the epidermis
(Carlson, 1975). In the present paper a systematic comparison is made between
anterior and posterior skin with respect to their morphogenetic properties.
These two surfaces of the limb are indistinguishable histologically but it is
concluded that their codings differ and that the posterior edge has more
switches on than the anterior edge. This type of difference between tissues of
the same histological type but different position in the body has been called
'non-equivalence' by Lewis & Wolpert (1976).
Properties of the skin in axolotl limb regeneration
267
The provenance of the different cells in the compound regenerates has been
investigated using both triploid donors and triploid hosts, and a new method
of ensuring immunological compatibility between cytologically-labelled grafts
and hosts has been introduced by growing an embryonic limb rudiment of one
ploidy on the flank of an embryo of the other ploidy.
In the Discussion a set of rules is proposed based on the serial threshold
theory which can explain both the new results presented here, and also some
previous experimental results published by myself and by other authors.
MATERIALS AND METHODS
The basic graft which was used in these experiments was the transplantation
of a half cuff of skin from one surface (anterior or posterior) of the lower
forelimb to the other. The animals were axolotls of length 10-15 cm which
were obtained either by natural or by artificial matings (the artificial mating
procedure is given in Slack & Forman, 1980). They were allowed to develop
in individual plastic containers to avoid cannibalism with its associated risk of
the displacement of limb tissues. Up to 4-5 cm in length they were kept in
20 % Steinberg solution made up with glass-distilled water and fed daily on
brine shrimps. Above this size they were transferred to tap water and fed
three times per week, first on Tubifex and later on minced lambs' hearts with
supplementary vitamins and minerals. The aquarium temperature was 20 °C
giving a water temperature of 18 °C.
The animals were anaesthetised in 1/2000 MS222 (Sandoz) in tap water. The
skin on one side (anterior or posterior) of the lower forelimb was removed with
iridectomy scissors and fine forceps. It was transferred to a dish containing
'normal amphibian medium' (Slack & Forman, 1980) and examined to ensure
that no muscle was adhering. A similar-sized piece of skin was removed from
the host limb and discarded, and the graft was attached with its proximodistal
and dorsoventral axes the same as the host and secured with four sutures, one
at each corner (tied with 'Ethilon', W2814 Ethicon Ltd.). The host animals
were returned to tap water and were allowed to recover in the dark at 10 °C
for two days before being returned to the aquarium. Two weeks after the
graft, both forelimbs were amputated through the mid-zeugopodium; in the
case of the experimental limb this was approximately through the centre of the
graft. Regeneration was complete after another 6-8 weeks, and both limbs
were amputated through the upper arm and prepared for examination.
In some cases the donors or hosts were not just ordinary axolotls but had
received some special treatment. Triploid animals were made as follows (see
Namenwirth, 1974). Thirty minutes after artificial fertilization eggs were heated
to 36 °C for 10 min which drives the second polar body back into the eggs.
The larvae were allowed to hatch and screened for triploidy by examination
of squashes of small pieces of tail tip by Nomarski interference microscopy
l8
EMB 58
268
J. M. W. SLACK
Fig. 1. Axolotl larval tail-tip cells viewed by Nomarski differential interference
microscopy, (a) Diploid, {b) triploid. Scale bars indicate 20 /im.
(Fig. 1). According to Fankhauser & Humphrey (1943), the number of nucleoli
per cell in the axolotl corresponds to the ploidy, so that normal larvae have
two nucleoli per nucleus and triploids have three. This method yielded about
50 % triploids among the surviving larvae and also about 0-5 % uni-nucleolate
cases which were presumed to be haploids. There was a small amount of
mortality among the triploid larvae but those which survived and grew appeared
to be identical to diploids, although a few which were grown to sexual maturity
proved to have abnormal gonads; the females had rudimentary ovaries and
the males had apparently normal testes but were sterile.
Animals bearing supernumerary limbs were prepared by grafting an extra
limb rudiment from one stage-34 embryo to the flank of another (Slack, 1977,
for updated grafting procedures see Slack, 19806). These animals were reared
in the same way as the normal ones and the skin grafts were later carried out
between the supernumerary limb and one of the host limbs. The reason for
this is that a supernumerary limb grown from a limb rudiment transplanted
to the flank of a host embryo is later tolerated by the immune system of the
host and it is therefore possible to prepare animals in which the supernumerary
is triploid and the host diploid. So the fate of donor cells in the combination
can be followed at any subsequent time without the complication of immunological rejection (Fig. 2).
Properties of the skin in axolotl limb regeneration
269
Fertilized eggs — some
heated to induce triploidy
36°
5—6 days
Diploid axolotl
with supernumerary
triploid limb
Triploid limb rudiment
grafted to diploid host at
tailbud stage
6—8 weeks
2 weeks
Graft posterior skin of
supernumerary to anterior
of host forelimb
Amputate
through
graft
Study cellular
composition of
duplicated regenerate
Fig. 2. Protocol for the experiments involving immunologically tolerant hosts.
Metamorphosed animals, here called ' efts', were prepared by giving repeated
injections of L-thyroxine dissolved in 'normal amphibian medium' into the
dorsal musculature of 10 cm axolotls. Injections were given three times a week,
the dose being varied between 2 and 5 fig depending on the visible pace of
metamorphosis. The gills and tail fin were usually resorbed after about 3 weeks
18-2
270
J. M. W. SLACK
after which the injections were stopped. Metamorphosis was judged to be
complete when the skin pattern of white spots on a black background was
fully developed. This was 6 to 8 weeks after commencement of the injections.
Limbs were prepared for examination either as whole mounts or as histological sections, the latter being necessary to locate the triploid tissue in
triploid-diploid combinations. For whole mounts the limbs were fixed in 4 %
formaldehyde, 1 % CaCl2, 50 mM-Tris pH 7-0 overnight. They were bleached,
where necessary, by exposure to Mayer's bleach overnight, followed by H 2 O 2
(100 vol.)/distilled water/alcohol 20:10:70 until white. They were equilibrated
in 1 % HC1 in 70 % alcohol and stained for 1 h in 1 % Victoria Blue 4R (Lambs)
in the same solution. They were dehydrated and cleared in Oil of Wintergreen.
Limbs were classified into one of the following groups:
Normal. Hands bear four digits, digital formula I, II, III, IV. The first,
second and fourth have two and the third digit has three phalanges. The usual
complement of carpals is eight (three proximal, two central, three distal) but
limbs are still classified as normal if adjacent carpals are fused. Most control
regenerates have the radiale fused to the radius.
Hypermorphic. These contain all the normal structures plus some additional
ones. e.g. I, II, III, IV, IV.
Hypomorphic. These contain only some of the normal structures, e.g. I-II-IH.
Duplicate. Hands have variable numbers of elements but these comprise
two sets of posterior structures arranged around a longitudinal axis of mirror
symmetry. In this work most have five or six digits with their associated carpals,
e.g. iv, iir, ir, II, in, iv.
Partial duplicates. These are similar to duplicates but lack one posterior
extremum, e.g. Ill', II', II, III, IV.
Duplicate with serial repetition. Similar to duplicates but with one or more
elements repeated away from the axis of symmetry, e.g. IV, IV, III', III', II',
II, III, IV.
Representative examples of each type are shown in Fig. 3.
For histology the limbs were fixed in 4 % glutaraldehyde 0-1 M sodium
phosphate pH 7-4 overnight at 4 °C, washed in buffer, and decalcified in
5%EDTA in 0-1 M sodium phosphate pH 7-4 for several days. They were
dehydrated, embedded via xylene in 58 °C wax, and sectioned at 15 /on. The
sections were brought to water and incubated for 1 h at 37 °C in 0-2 mg/ml
DNAase in 30 mM-MgSO4, 10 mM Hepes pH 7-4. This removes most of the
nuclear DNA from muscle and cartilage cells and allows the nucleoli to be
visualised more easily (Namenwirth, 1974). They were stained in Unna-Pappenheim stain with double pyronin (0-2 % methyl green, 0-125 % pyronin in 0 1 M
sodium acetate pH 4-8), dehydrated in graded acetones and mounted in DPX.
Although most cells in a triploid animal have three nucleoli, all three may
not be seen in a particular section if part of the nucleus is in the adjacent
section. So the proportion of trinucleolate cells seen is always an underestimate
(1) Anterior skin -> posterior
(2) Posterior skin -> anterior
Comprising
Allografts
Tolerant hosts
(3) Anterior skin -»• anterior
(4) Posterior skin -> posterior
(5) Posterior skin removed
(6) Eft posterior skin -> anterior
(7) Eft limb amputated
Total
Operation
6
7
12
8
6
78
15
22
11
11
Cases
0
0
6
7
12
0
6
6
0
0
0
Normal
0
0
0
2
0
2
1
0
1
Hypermorph
Double anterior duplicate.
0
0
0
3
0
6
0
Hypomorph
0
0
0
0
0
0
1
0
1
Other
Table 1. Classification of structures of regenerated limbs
0
0
0
0
0
0
2
2
0
Partial
duplicate
0
0
0
2
0
1*
12
7
5
0
0
0
1
0
0
6
2
4
Duplicate
with serial
Duplicate repetition
to
1
SIS'
OS
5
272
J. M. W. SLACK
(a)
Fig. 3. Types of regenerate formed in these experiments, (a) normal, (6) hypomorphic, (d) duplicate, (e) partial duplicate, (/) duplicate with serial repetition. Scale
bars represent 2 mm.
of the proportion of triploid cells in the tissue. In order to make quantitative
comparisons it is necessary to examine the same tissue type, since larger nuclei
are less likely to lie entirely within a section, and to control the orientation of
the organ relative to the section since for non-spherical nuclei orientation also
affects the chances of finding the whole nucleus in a section.
RESULTS
Structure of compound regenerates
In Table 1 are shown the structures of regenerates which were formed
following a skin graft, a two week healing period, and amputation through the
graft. The limbs with double anterior skin regenerated normal or hypomorphic
Fig. 4. Histology of skin, (a) Normal axolotl skin. Epi, epidermis; L, Leydig cell; BM, basement membrane; Der, dermis;
M, melanophore; Muse, muscle, (b) axolotl skin allograft infiltrated by small mononuclear cells, (c) eft skin. Epi, epidermis; Der, dermis; Muse, muscle; Gl, mucus gland. Scale bars indicate 50 fim Sections are 6/wn thick and stained with
haematoxylin and Biebrich scarlet.
to
274
J . M . W . SLACK
limbs, the latter lacking the posterior parts. In contrast, limbs with double
posterior skin regenerated duplicates with double posterior symmetry.
Some control experiments were carried out in which anterior skin was
grafted anteriorly and posterior skin grafted posteriorly. These all gave normal
regenerates after amputation through the graft. Normal regenerates were also
formed when the posterior half cuff was removed and not replaced, the wound
simply being allowed to heal for two weeks before amputation.
Since one of the objects of these experiments was to follow the fate of cells
in marked grafts, operations had to be carried out between different animals
rather than between right and left limbs of the same animal. So it was of some
importance to determine whether immunological rejection of the graft had
any bearing on the morphogenetic phenomena. Rejection of skin grafts in
urodeles has been investigated by Cohen (1971), who describes it as a slow
process which lasts many weeks and depends on several weak histocompatibility
loci. Rejection in the present series was detectable in the dissecting microscope
by the destruction of graft melanophores, and in histological sections by
infiltration of the grafts by small mononuclear cells (Fig. 4 b). No rejection
was apparent in sections at the time of amputation, which was 2 weeks after
the graft, or at the stage of dedifferentiation one week later. After 6-8 weeks
of regeneration the degree of rejection varied in individual cases from very
slight to very extensive. In the series of grafts in which double posterior skin
was assembled, eleven cases were carried out between different individuals, and
eleven cases using donor limbs which had been originally grafted to the flank
of the host at the stage of the tailbud embryo following the protocol of Fig. 2.
The former group of regenerates showed various degrees of rejection of the
donor tissue while the latter group showed no rejection at all, judged either
by gross inspection or by histology. Since the structures of the regenerates
obtained in the two series were not significantly different it can be concluded
that in these experiments immunological rejection neither potentiates nor
inhibits the formation of duplications. However, it is to be expected that the
effect of the graft would be inhibited if a long enough healing period were
allowed between the graft and the amputation, because eventually the grafted
skin would be completely destroyed.
It was thought to be of some interest to examine some metamorphosed
axolotls (efts), since in other amphibia regenerative ability often falls off at
metamorphosis (Scadding, 1977) and the eft has skin with a quite different
histological structure, notably a thick dermis containing huge mucous glands
(Fig. 4 a, c). A number of grafts were carried out in which posterior forearm
skin from the eft was grafted to the anterior forearm of an axolotl and the
usual protocol followed thereafter. The regenerates showed a range of structures
intermediate between those obtained from the double anterior and double
posterior skins, with a few duplications and some minor abnormalities (Table 1).
In this group it did seem as though the cases which did not form duplicates
Properties of the skin in axolotl limb regeneration
275
were those showing the most graft rejection. The eft donors had their legs
amputated at the proximal limit of the graft and these regenerated normally,
although both healing and regeneration were about three times slower than
for a similar size axolotl. This small series is perhaps not conclusive, but it
could indicate that the codings are still present after metamorphosis but that
interactions between eft and axolotl skin occur less readily than between
axolotls.
Provenance of cell in duplicates
In order to interpret the morphology of compound regenerates in terms of
their formation it is important to know the cellular composition of the different
parts. If it can be shown that formerly anterior host tissue has contributed
to posterior structures, then this means that the tissue in question has been
reprogrammed in terms of its positional coding. If it can be shown that graftderived cells contribute to tissue types not present in the graft then this means
that metaplasia has occurred.
In the present work triploidy has been used as the marker and a number
of cases have been examined histologically in which either the donor or the
host was triploid. All these experiments involved grafts of posterior skin to
the anterior. Altogether, seven cases of diploid grafts to triploid hosts were
examined histologically, and sixteen cases of triploid grafts to diploid hosts.
Of the latter, seven cases were performed with tolerant hosts following the
protocol of Fig. 2.
Where the host was triploid and the donor skin diploid the results were as
follows. On the posterior side of the duplication, which was the side away
from the grafts, all tissues contained abundant trinucleolar cells with frequencies
similar to those in triploid control limbs. On the anterior side, there were
many trinucleolar cells in the epidermis and connective tissue but their frequency
in the cartilages fell off from the mirror plane to the anterior digit IV (see
Table 2 and Fig. 5/).
Where the host was diploid and the donor skin triploid nine grafts were
made to non-tolerant hosts. In four of these the regenerates were examined
eleven days after the amputation which is the stage of dedifferentiation. They
showed no immunological rejection and had abundant trinucleolar cells on
the anterior side of the apical ectodermal cap. A few trinucleolar cells were
also found among the dedifferentiated mesenchymal cells on the anterior side
(Fig. 5 c). In the other five cases the regenerates were examined after 6-8 weeks.
All of these showed extensive graft rejection and only a few trinucleolar cells
could be recognised because of the extensive degradation of the tissues. Those
that were found were present in the epidermis, muscle and cartilage on the
anterior side of the duplication. In the seven cases in which the hosts were
tolerant there was no rejection and in most but not all cases many more
trinucleolar cells were visible. They were abundant on the anterior side of the
2/z
FL15R
FL22R
FL17R
T15R
T19R
T29R
In
Donor
Case
3«
3«
In
In
2n
3/7
3«
3«
Host
[ost
IV
00
6
14
14
35
26
35
66
6
55
6
6
5
f
Digit
number
00
23
10
10
25
26
26
III'
A
Anterior side
22
0
44
28
II'
IF
35
1
0
27
27
39
II
IV
38
38
36
24
0
0
0
III
34
34
42
28
0
0
0
A
Posterior side
Table 2. Percentage of trinucleolar cells found in the metacarpals of double posterior duplications
^
.
<
ON
to
Properties of the skin in axolotl limb regeneration
211
Fig. 5. (a) Cartilage cells in a triploid limb, (b) muscle cells in a triploid limb,
(c) a trinucleolar cell in the mesenchymal part of the early blastema resulting
from a triploid skin graft on a diploid host, (d) a trinucleolar cartilage cell in the
anterior part of the duplicate regenerate formed from a triploid skin graft on
a diploid host, (e) a trinucleolar muscle cell in the anterior part of a similar regenerate. (/) a trinucleolar cartilage cell anterior to the midline in a duplication
formed from a diploid graft to a triploid host. Nucleoli are indicated by arrows,
scale bars represent 10 /*m.
mirror plane, particularly in digits III' and I V . Particularly large numbers
were found in the cartilages, but they were also found in muscle cells, connective
tissue and epidermis (Fig. 5d, e and Table 2).
It seems therefore that the posterior side of the duplication is composed
wholly of host tissue and the anterior side of a mixture of donor and host
tissue. With respect to metaplasia, the present results confirm those of Dunis &
Namenwirth (1967) which showed that descendent cells from a skin graft
could become incorporated into the muscle and cartilage of the regenerate.
4
4
4
4
6
5
5
6
11
/?-Galactosidase
Deoxyribonuclease
Fucosidase
Glucuronidase
iV-acetylglucosaminidase
iV-acetylneuraminidase
Pronase
Ribonuclease A
Trypsin
2/t/ml
1 mg/mg
100 mu/ml
1000 u/ml
100 mu/ml
10 u/ml
10 mg/ml
1 mg/ml
10 mg/ml
concentration
D
PD
D
D
O, PD
D
H
PD
H, H
1
D
N
D
D
DSR
h,D
H
PD
H, h, O, D,
DSR
1:10
A
D
PD
DSR
D
N, D
D
D
D,D
N, D
1:100
D
D
D
D
DSR
PD
D
D,D
h, PD
1: 1000
1:10000
Each letter in the table represents a single case: D duplication, PD partial duplication, DSR duplication with serial repetition,
N, Normal; h, hypomorphic, H, hypermorphic; O, other.
49
Cases
Enzyme
Dilutions
Table 3. Structure of regenerates formed after enzyme treatment of posterior skin cuff
to
oo
Properties of the skin in axolotl limb regeneration
279
Distal
Apical ectodermal cap
Posterior
Anterior
Stump
• Epidermis
Proximal
Fig. 6. Diagram of a regenerating left limb viewed from the dorsal side. The
following figures depict the region enclosed by the dashed line.
It therefore seems as though formation of the duplication involves extensive
positional reprogramming of host tissue and some reprogramming together
with metaplasia of donor tissue.
Enzyme treatment of posterior grafts
The reprogramming ability of posterior but not anterior skin implies that
the former contains something which the latter lacks. It was thought that if the
substances which embody the codings were present in the extracellular matrix
as suggested by Tank & Holder (1979), then perhaps they could be destroyed
by enzymic treatment without destroying the cells. Accordingly posterior halfskin cuffs were soaked in various concentrations of nine enzymes for 1 h at
room temperature before being grafted to the anterior side of host limbs. The
grafts were allowed to heal for 2 weeks and the limb amputated through the
graft. In this experiment, a positive result would consist of normal limbs
growing after high dose treatment and duplications growing after low-dose
treatment, a negative result would consist of duplications growing after any
dose. The results are shown in Table 3. There is perhaps a slight tendency for
high concentrations of the proteases to inhibit the formation of duplications,
but the really remarkable thing is that the treatments have so little effect.
There are so many possible reasons for this failure that the experiment cannot
be regarded as informative and it is mentioned here solely to record a negative
result.
280
J. M. W. SLACK
Distal
00111 y
(00001
11111 ^
t'ooi 1 A
C00001
01111 S——TOO011
11111 >
0)0111 3
C00001
/=oo
Fig. 7. Time course of specification of the blastema during normal regeneration.
DISCUSSION
The results presented in this paper show that anterior and posterior skin
are nonequivalent. A graft of posterior skin to the anterior followed by
amputation results in the formation of double posterior duplications in which
there is some reprogramming of host tissue and some metaplasia and reprogramming of donor tissues. The converse graft results in the formation of
normal or hypomorphic regenerates.
These and other results will now be discussed in terms of the serial threshold
theory of regeneration (Slack, 1980). According to this theory pattern formation
in regeneration can be accounted for by assuming that the differentiated organ
is partitioned into territories which consist of groups of cells plus their associated
extracellular matrix. These territories are coded in such a way that all of a set
of biochemical switches are on at one end and successive territories across the
organ each have one more switch off. It is assumed that the 'on' state of a
switch corresponds to the presence of a particular substance, the switch
product, and that the 'off' state corresponds to its absence. So each territory
contains the information for making all the territories further down in the
sequence in much the same way that each of a stack of Russian dolls contains
all of the smaller dolls in the set. In the case of the anteroposterior axis of the
amphibian limb, the results suggest that the posterior edge should be identified
as the region in which all the switches are on.
In order to explain the mechanism of regeneration one has to do more than
Properties of the skir in axolotl limb regeneration
281
Fig. 8. Predicted regenerate following a graft of posterior skin to the anterior.
Fig. 9. Predicted regenerate following a graft of anterior skin to the posterior.
identify the arrangement of the codings: it is necessary also to explain how
a blastemal territory can become respecified by its surroundings, and this can
be done by postulating two simple relationships between the states of the
switches in neighbouring blastemal territories. The first, which is necessary
for the spread of pattern information through the tissue, is the dominance of
higher over lower switches such that the product of a particular switch turns
on all the lower switches in the sequence but has no effect on the higher ones.
The second is a spatial averaging property which causes each switch in each
territory to tend to adopt the same state as in the majority of neighbouring
territories. This sort of diffusion process necessarily accompanies situations of
intercellular communication.
It is assumed that there is an early stage immediately following dedifferentiation during which the cells of the blastema have all their switches off. The
pattern of the regenerate is then controlled by the codings of the most distal
layer of territories in the stump because the switch products present in this
region can influence the codings adopted by the neighbouring blastemal territories. These can then in turn influence their neighbours and so the blastema
as a whole will pass through a sequence of unstable states until a final stable
arrangement of codings has been set up. This stable arrangement corresponds
to a 'determined' blastema, although since it would still be possible to change
the codings at this stage by surgical rearrangement the determination is not
irreversible.
282
J. M. W. SLACK
Fig. 10. Predicted regenerate following a graft of anterior skin to the posterior
with removal of the most posterior host territory.
The process will be illustrated by a series of diagrams which represent the
blastema viewed from the dorsal aspect as shown in Fig. 6. Each territory
will be shown as a hexagon and its coding by a group of binary digits. The
' o n ' state of each switch is shown as ' 1 ' and the 'off' state as ' 0 ' . An empty
hexagon represents the coding 00000, which means that all the switches are
off. In the diagrams the lowest row of hexagons belong to the most distal layer
of stump tissues next to the blastema and since these are differentiated tissues
their codings cannot be changed. The remainder of the hexagons represent the
mesenchymal part of the blastema. In reality the blastema would be growing
at the same time as these territories are being specified but in order to keep
the presentation simple the process is depicted as though the blastema has
a fixed size and the specification proceeds stepwise from the proximal edge.
In order that the predicted sequence of events be unambiguous it is necessary
to put the principles of serial dominance and spatial averaging into a precise
form, and this can be done as follows with (1) and (2) expressing the former
and with (3) expressing the latter.
(1) The rth switch is turned on in a territory (hexagon) with no time delay
if the i+l th switch is on in that territory. This ensures that the sequence of
switches which is on in a territory is uninterrupted.
(2) The i th switch is turned on at time t if the i + 1 th switch was on at
time t-\ in one or more of the neighbouring territories. This ensures that
positional information spreads through the blastema and that a series of
decreasing codings is set up along the axis in question. A special rule is
necessary for the top switch in the sequence (the wth) because it has no higher
product which can turn it on. It seems reasonable to suppose that it can be
turned on by its own product in territories adjacent to a lateral edge. Therefore:
the nth switch is turned on in edge territories at time t if it was on in one or
more neighbouring territories at t-\.
(3) In the absence of influences from higher switches, the state of the i th
switcji at time t is adjusted to be the same as that in the majority of the neighbouring territories at t-l. This means that the boundaries between regions
tend to be straightened and to lie perpendicular to the axis. Since hexagons
Properties of the skin in axolotl limb regeneration
283
Anterior half
t=0
t=
Posterior half
Fig. 11. Predicted regenerates from anterior and posterior half limbs.
pack with a coordination number of six, 'majority' here means four or more
out of six, or in the case of edge territories, three out of five, three out of four
or two out of three.
The operation of the model is depicted in Fig. 7 for normal regeneration.
At t = 0 only the row of stump territories have codings and the blastemal
territories have all their switches off. By t = 1 the first row of blastemal
territories has become specified, although not necessarily with their final codings.
A later intermediate stage is shown at t = 3, and by t = 5 a configuration of
codings is reached which is stable in the sense that it does not spontaneously
change thereafter. This is of course the normal pattern in which all switches
are on at the posterior edge and each territory towards the anterior has one
more switch off.
In this model the normal pattern is not the only stable pattern and it is
to be expected that a rearrangement of the stump territories may lead to the
regeneration of a stable but abnormal pattern. If an extra posterior territory
is added to the anterior edge, as shown in Fig. 8, then the same set of rules
generate a double posterior duplication. This is the result obtained in the
experimental section of this paper when a posterior half cuff of skin is grafted
to the anterior side and the combination later amputated through the graft.
Furthermore, if only the right hand column of blastemal territories is regarded
as being composed of cells derived from the graft then the cellular composition
19
EMB 58
284
J. M. W. SLACK
t =0
t = <*>
Fig. 12. Pattern contraction in the regenerate formed from a
double anterior limb.
indicated by the model is also roughly similar to that found in reality with
a large contribution of donor tissue at the anterior edge but with much of the
duplicate being formed from host tissue. The widespread metaplasia of donor
tissues found experimentally indicates that the clonal origin of cells is not
relevant to the new codings which they acquire in the blastema, and so this
factor is not included in the model.
The converse experiment of grafting anterior skin to the posterior and later
amputating through the graft is simulated in Figs 9 and 10. In Fig. 9 an extra
anterior territory is added to the posterior edge of the stump. This has little
effect on the regenerate which develops a normal pattern slightly twisted
towards the operated side. In Fig. 10 the graft replaces the most posterior
territory of the stump, and this gives rise to a hypomorphic regenerate lacking
the most posterior structures. Reference to Table 1 will show that the normal
limb and the hypomorph are the two most common outcomes of this experiment.
In Table 1 it is also shown that the simple removal of the posterior skin leads
to normal regeneration. This result is consistent with the model if it is assumed
that during the healing period the gap is filled by posterior tissue from the
proximal and distal edges of the wound.
The application of the serial threshold theory to the amphibian limb in the
form of the present model also allows the explanation of a number of related
experiments by myself and others which have not been satisfactorily explained
in the past:
Half limb regeneration. The morphogenetic potency of anterior and posterior
half limbs has been studied by Goss (1957) and Maden (1979). The posterior
half from the upper arm will frequently regenerate an entire hand whereas
the anterior half forms a regenerate consisting only of a few anterior structures.
This situation is represented in Fig. 11 by a full size blastema with only a
partial set of stump territories, and it may be seen that the posterior half contains
information which initiates a course of events close to the normal while the
anterior half does not. If the posterior blastema were only of half size in terms
of territory number then it should form a posterior half regenerate, as found
by these authors for half lower arms.
Properties of the skin in axolotl limb regeneration
285
(b)
t=0
Fig. 13. (a) Pattern contraction in the regenerate from a double posterior limb
where the number of territories across the blastema is less than that in the stump.
(b) Pattern stability in the regenerate from a double posterior limb in which the
number of territories across the blastema is the same as that in the stump, (c)
Pattern expansion in the regenerate from a double posterior limb in which the
number of territories across the blastema is greater than that in the stump.
Pattern contraction of double anterior limbs. Double anterior limbs have been
constructed surgically by Bryant & Baca (1978), Stocum (1978) and Tank
(1978). They show little if any capacity for regeneration and the structures
which are formed are of anterior character (Fig. 12).
Pattern contraction and expansion of double posterior limbs. Bryant (1976)
showed that surgically constructed double posterior limbs would regenerate
few if any structures. Slack & Savage (1978) described regeneration behaviour
of embryonically produced double posterior limbs and found good regeneration
19-2
286
J. M. W. SLACK
with a slight tendency for pattern contraction and also a number of cases of
expansion. Tank & Holder (1978) partly resolved this conflict of results by
showing that contraction of regenerates from surgically constructed limbs
became more acute the longer the healing time allowed between the graft and
the amputation. The exact course of events during healing is not known, but
it seems likely that cell death, cell division, revascularisation and changes in
the extracellular matrix would all affect the diffusion of the active factors. If
diffusion constants were reduced then the size of the territories would be
reduced and more territories would be established in a blastema of given size.
If diffusion constants were increased then less territories would be established.
Such changes will thus be represented here by changes in territory number
across the blastema.
In Fig. 13A-C are shown cases in which the blastema narrows, keeps the
same width and widens. The first shows a severe contraction in which three
types of territory are eliminated from the midline of the regenerate. The second
shows a retention of all the types of territory present in the stump, and the
third shows the addition of two extra types of territory in the midline. This is
exactly the type of behaviour observed by Slack & Savage (1978). In this study
we found that expansion and contraction always involved the addition or
subtraction of elements at the centre of the pattern, and that the new elements
were always neighbours of the ones already present. Furthermore branching
of midline cartilages could occur and could be either proximally or distally
directed. Both types of branching are shown by the group of territories coded
01111 in Fig. 13 C: these are separated in the stump, then coalesce in the proximal
part of the blastema and diverge again more distally.
Reprogramming of blastemas. Iten & Bryant (1975) showed that very early
blastemas could be reprogrammed in the anteroposterior axis if they were
inverted on the contralateral stump, later blastemas were partially reprogrammed
and produced hypermorphs and duplicates, and later blastemas still could
produce complete supernumerary hands from the incongruent junctions.
According to the present model, the very early blastema is completely bland,
the intermediate blastema has some territories specified but is still labile, and
the later blastema is fully determined. In the last case the formation of a
supernumerary limb requires a 'de-determination' at the junction and the
formation of an intercalary blastema with polarity depending on the codings
of the adjacent territories.
The idea which lies at the heart of the serial threshold theory is that a region
of tissue can regenerate structures with all the 'lower' codings but not those
with the 'higher' ones. This property is shown not only in the anteroposterior
axis of the amphibian limb but also in the distal regeneration of vertebrate
and arthropod appendages and in the posterior regeneration of worms (see
Slack, 1980#). The widespread occurrence of this type of phenomenon must
suggest the possibility of a common biochemical basis for the incremental
Properties of the skin in axolotl limb regeneration
287
codings in all such cases. It is therefore unfortunate that to date most developmental biochemistry has concentrated on the differences between different
tissues rather than on the differences between the same tissue in different
places.
I should like to thank Shirley Williams for technical assistance, Bob Bloomfield for
supervising the axolotls and John Cairns for his interest in serial thresholds.
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{Received 8 February 1980 revised 13 March 1980)