morphogenetic properties of the skin in axolotl limb ... · morphogenetic properties of the skin in...
TRANSCRIPT
/ . Embryol. exp. Morph. Vol. 58, pp. 265-288, 1980 2 6 5Printed in Great Britain © Company of Biologists Limited 1980
Morphogenetic properties of the skin inaxolotl limb regeneration
By JONATHAN M. W. SLACK1
From the Imperial Cancer Research Fund,Mill Hill Laboratories, London
SUMMARYA 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 halfcuff of skin from a donor to a host limb followed by a 2-week healing period, amputationthrough the graft, and a study of the resulting regenerate. Limbs with double posteriorskin formed double posterior regenerates and, in contrast, limbs with double anterior skinformed normal or slightly hypomorphic regenerates. Posterior skin from post-metamorphicanimals had a similar but weaker effect to that from ordinary axolotls.
Immunological rejection of allografts could be completely avoided if the donor limb wastransplanted to the flank of the host when both were at the stage of tail-bud embryos, andthe skin graft was later carried out between the supernumerary limb and one of the hostlimbs. This technique was used to show that immunological rejection does not affect theformation of duplicates from the limbs with double posterior skin, and to facilitate thestudies of the cellular provenance of the regenerate.
The cellular composition of duplicate regenerates was studied by using both triploiddonors and triploid hosts. It was shown that the posterior side of the duplications consistedwholly of host tissue and the anterior side consisted of mixed donor and host tissue. Formationof the duplicated regenerate therefore seems to involve positional reprogramming of bothdonor and host tissues together with metaplasia of the donor tissue.
It was not possible to inhibit the duplication-inducing property of posterior skin bytreatment with a variety of enzymes.
A model based on the serial threshold theory of regeneration is advanced to explain theresults. This model successfully accounts for the observed non-equivalence of anterior andposterior skin, and also explains the different regeneration behaviour of anterior andposterior half limbs, the limited regeneration of double anterior limbs, and the patternexpansion and contraction shown by regenerates from double posterior limbs.
INTRODUCTION
The axolotl is an animal which can regenerate its limbs after amputationand in which the spatial arrangement of structures in the regenerate is restoredwith very high fidelity. This formation of a complex spatial pattern distinguishestrue regeneration from other phenomena such as the hypertrophy of the mam-malian liver after a part has been removed, or the regrowth of nerve axons
1 Author's address: Imperial Cancer Research Fund, Mill Hill Laboratories, BurtonholeLane, London, NW7 IAD, U.K.
266 J. M. W. SLACK
down a pre-existing tract. It poses a specific biological problem: how do thecells of the regeneration blastema 'know' which structures to turn into andin what relative order these should be arranged ?
Certain theorists of regeneration have postulated that the cells in eachregion of tissue in a differentiated limb, or any other organ capable of re-generation, are labelled with 'positional values' which vary in a continuousway across the organ (Wolpert, 1971; Bryant & Iten, 1976; Maden, 1977;Lheureux, 1977). According to these authors, when cells near the amputationsurface dedifferentiate and divide to form the blastema, their positional valuesare erased and are recomputed in such a way that a complete set is reformedwhich is continuous with the positional values represented in the stump. Thepathway of cytodifferentiation to be followed by each group of blastemal cellsis then selected in accordance with their new positional value, irrespective oftheir cytological type in the previous limb.
In a theoretical paper (Slack, 1980a) I have argued that these positionalvalues are made up of combinations of 'on ' and 'off' states of a set of bio-chemical switches, and that the arrangement of these combinations, or 'codings'is such that the structure of the missing parts can be computed from thecodings represented at the amputation surface without any long range inter-actions with the remaining part of the organ. If this view is accepted then theproblem of pattern formation can be rephrased as: 'What is the relationbetween the anatomical position and the coding and what are the rules foraltering codings during regeneration?' One method of attacking this problemexperimentally is to alter the arrangement of tissues in the organ in order toprovoke the regeneration of an abnormal pattern. If a normal limb is amputatedthen the pattern of the regenerate is the same as the original, but this is notgenerally true of abnormal limbs generated by embryonic manipulation or bysurgery on adults (Swett, 1924; Newth, 1958). If enough reliable data can becollected on the relationship between starting patterns and final patterns wemight be able to deduce the number and arrangement of codings and the rulesfor their interconversion, and once the rules are known it may become possibleto 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 thelimb to another can derange the pattern of a regenerate which is formed afteramputation 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 betweenanterior and posterior skin with respect to their morphogenetic properties.These two surfaces of the limb are indistinguishable histologically but it isconcluded that their codings differ and that the posterior edge has moreswitches on than the anterior edge. This type of difference between tissues ofthe 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 267The provenance of the different cells in the compound regenerates has been
investigated using both triploid donors and triploid hosts, and a new methodof ensuring immunological compatibility between cytologically-labelled graftsand hosts has been introduced by growing an embryonic limb rudiment of oneploidy on the flank of an embryo of the other ploidy.
In the Discussion a set of rules is proposed based on the serial thresholdtheory which can explain both the new results presented here, and also someprevious experimental results published by myself and by other authors.
MATERIALS AND METHODS
The basic graft which was used in these experiments was the transplantationof a half cuff of skin from one surface (anterior or posterior) of the lowerforelimb to the other. The animals were axolotls of length 10-15 cm whichwere obtained either by natural or by artificial matings (the artificial matingprocedure is given in Slack & Forman, 1980). They were allowed to developin individual plastic containers to avoid cannibalism with its associated risk ofthe displacement of limb tissues. Up to 4-5 cm in length they were kept in20 % Steinberg solution made up with glass-distilled water and fed daily onbrine shrimps. Above this size they were transferred to tap water and fedthree times per week, first on Tubifex and later on minced lambs' hearts withsupplementary vitamins and minerals. The aquarium temperature was 20 °Cgiving a water temperature of 18 °C.
The animals were anaesthetised in 1/2000 MS222 (Sandoz) in tap water. Theskin on one side (anterior or posterior) of the lower forelimb was removed withiridectomy scissors and fine forceps. It was transferred to a dish containing'normal amphibian medium' (Slack & Forman, 1980) and examined to ensurethat no muscle was adhering. A similar-sized piece of skin was removed fromthe host limb and discarded, and the graft was attached with its proximodistaland dorsoventral axes the same as the host and secured with four sutures, oneat each corner (tied with 'Ethilon', W2814 Ethicon Ltd.). The host animalswere returned to tap water and were allowed to recover in the dark at 10 °Cfor two days before being returned to the aquarium. Two weeks after thegraft, both forelimbs were amputated through the mid-zeugopodium; in thecase of the experimental limb this was approximately through the centre of thegraft. Regeneration was complete after another 6-8 weeks, and both limbswere amputated through the upper arm and prepared for examination.
In some cases the donors or hosts were not just ordinary axolotls but hadreceived some special treatment. Triploid animals were made as follows (seeNamenwirth, 1974). Thirty minutes after artificial fertilization eggs were heatedto 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 examinationof 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 interferencemicroscopy, (a) Diploid, {b) triploid. Scale bars indicate 20 /im.
(Fig. 1). According to Fankhauser & Humphrey (1943), the number of nucleoliper cell in the axolotl corresponds to the ploidy, so that normal larvae havetwo nucleoli per nucleus and triploids have three. This method yielded about50 % triploids among the surviving larvae and also about 0-5 % uni-nucleolatecases which were presumed to be haploids. There was a small amount ofmortality among the triploid larvae but those which survived and grew appearedto be identical to diploids, although a few which were grown to sexual maturityproved to have abnormal gonads; the females had rudimentary ovaries andthe males had apparently normal testes but were sterile.
Animals bearing supernumerary limbs were prepared by grafting an extralimb rudiment from one stage-34 embryo to the flank of another (Slack, 1977,for updated grafting procedures see Slack, 19806). These animals were rearedin the same way as the normal ones and the skin grafts were later carried outbetween the supernumerary limb and one of the host limbs. The reason forthis is that a supernumerary limb grown from a limb rudiment transplantedto the flank of a host embryo is later tolerated by the immune system of thehost and it is therefore possible to prepare animals in which the supernumeraryis triploid and the host diploid. So the fate of donor cells in the combinationcan be followed at any subsequent time without the complication of immuno-logical rejection (Fig. 2).
Properties of the skin in axolotl limb regeneration 269
Fertilized eggs — some
heated to induce triploidy
36°
5—6 days
Diploid axolotlwith supernumerarytriploid limb
2 weeks
Triploid limb rudimentgrafted to diploid host attailbud stage
6—8 weeks
Graft posterior skin ofsupernumerary to anteriorof host forelimb
Amputatethroughgraft
Study cellularcomposition ofduplicated regenerate
Fig. 2. Protocol for the experiments involving immunologically tolerant hosts.
Metamorphosed animals, here called ' efts', were prepared by giving repeatedinjections of L-thyroxine dissolved in 'normal amphibian medium' into thedorsal 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 ofmetamorphosis. 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 becomplete when the skin pattern of white spots on a black background wasfully developed. This was 6 to 8 weeks after commencement of the injections.
Limbs were prepared for examination either as whole mounts or as histo-logical sections, the latter being necessary to locate the triploid tissue intriploid-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 H2O2
(100 vol.)/distilled water/alcohol 20:10:70 until white. They were equilibratedin 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 usualcomplement of carpals is eight (three proximal, two central, three distal) butlimbs are still classified as normal if adjacent carpals are fused. Most controlregenerates have the radiale fused to the radius.
Hypermorphic. These contain all the normal structures plus some additionalones. 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 mirrorsymmetry. 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 posteriorextremum, e.g. Ill ' , II', II, III, IV.
Duplicate with serial repetition. Similar to duplicates but with one or moreelements 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 in5%EDTA in 0-1 M sodium phosphate pH 7-4 for several days. They weredehydrated, embedded via xylene in 58 °C wax, and sectioned at 15 /on. Thesections were brought to water and incubated for 1 h at 37 °C in 0-2 mg/mlDNAase in 30 mM-MgSO4, 10 mM Hepes pH 7-4. This removes most of thenuclear DNA from muscle and cartilage cells and allows the nucleoli to bevisualised more easily (Namenwirth, 1974). They were stained in Unna-Pappen-heim stain with double pyronin (0-2 % methyl green, 0-125 % pyronin in 01 Msodium acetate pH 4-8), dehydrated in graded acetones and mounted in DPX.
Although most cells in a triploid animal have three nucleoli, all three maynot be seen in a particular section if part of the nucleus is in the adjacentsection. So the proportion of trinucleolate cells seen is always an underestimate
5T
able
1.
Cla
ssif
icat
ion
of s
truc
ture
s of
reg
ener
ated
lim
bs
Ope
ratio
nH
ypo-
H
yper
-C
ases
m
orph
N
orm
al
mor
phO
ther
Dup
licat
ePa
rtia
l w
ith s
eria
ldu
plic
ate
Dup
licat
e re
petit
ion
OS SI- S'
(1)
Ant
erio
r sk
in -
> p
oste
rior
(2)
Post
erio
r sk
in ->
ant
erio
rC
ompr
isin
gA
llogr
afts
Tol
eran
t ho
sts
(3)
Ant
erio
r sk
in -»
• an
teri
or(4
) Po
ster
ior
skin
->
pos
teri
or(5
) Po
ster
ior
skin
rem
oved
(6)
Eft
pos
teri
or s
kin
-> a
nter
ior
(7)
Eft
lim
b am
puta
ted
Tot
al
15 22 6 7 12 8 6
11 11
6 0 0 0 0 3 0
0 0
6 0 6 7 12 0 6
0 0
2 1 0 0 0 2 0
0 1
0 1 0 0 0 0 0
0 1
0 2 0 0 0 0 0
2 0
1* 12 0 0 0 2 0
7 5
0 6 0 0 0 1 0
2 4
78D
oubl
e an
teri
or d
uplic
ate.
1 to
272 J. M. W. SLACK
(a)
Fig. 3. Types of regenerate formed in these experiments, (a) normal, (6) hypo-morphic, (d) duplicate, (e) partial duplicate, (/) duplicate with serial repetition. Scalebars represent 2 mm.
of the proportion of triploid cells in the tissue. In order to make quantitativecomparisons it is necessary to examine the same tissue type, since larger nucleiare less likely to lie entirely within a section, and to control the orientation ofthe organ relative to the section since for non-spherical nuclei orientation alsoaffects 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 formedfollowing a skin graft, a two week healing period, and amputation through thegraft. The limbs with double anterior skin regenerated normal or hypomorphic
Fig
. 4. H
isto
logy
of
skin
, (a
) N
orm
al a
xolo
tl sk
in. E
pi,
epid
erm
is;
L,
Ley
dig
cell
; B
M,
base
men
t m
embr
ane;
D
er,
derm
is;
M,
mel
anop
hore
; M
use,
m
uscl
e,
(b)
axol
otl
skin
allo
graf
t in
filtr
ated
by
smal
l m
onon
ucle
ar
cells
, (c
) ef
t sk
in.
Epi
, ep
i-de
rmis
; D
er,
derm
is;
Mus
e, m
uscl
e; G
l, m
ucus
gla
nd.
Scal
e ba
rs i
ndic
ate
50 fi
m S
ectio
ns a
re 6
/wn
thic
k an
d st
aine
d w
ithha
emat
oxyl
in a
nd B
iebr
ich
scar
let.
to
274 J .M.W. SLACK
limbs, the latter lacking the posterior parts. In contrast, limbs with doubleposterior skin regenerated duplicates with double posterior symmetry.
Some control experiments were carried out in which anterior skin wasgrafted anteriorly and posterior skin grafted posteriorly. These all gave normalregenerates after amputation through the graft. Normal regenerates were alsoformed when the posterior half cuff was removed and not replaced, the woundsimply being allowed to heal for two weeks before amputation.
Since one of the objects of these experiments was to follow the fate of cellsin marked grafts, operations had to be carried out between different animalsrather than between right and left limbs of the same animal. So it was of someimportance to determine whether immunological rejection of the graft hadany bearing on the morphogenetic phenomena. Rejection of skin grafts inurodeles has been investigated by Cohen (1971), who describes it as a slowprocess which lasts many weeks and depends on several weak histocompatibilityloci. Rejection in the present series was detectable in the dissecting microscopeby the destruction of graft melanophores, and in histological sections byinfiltration of the grafts by small mononuclear cells (Fig. 4 b). No rejectionwas apparent in sections at the time of amputation, which was 2 weeks afterthe graft, or at the stage of dedifferentiation one week later. After 6-8 weeksof regeneration the degree of rejection varied in individual cases from veryslight to very extensive. In the series of grafts in which double posterior skinwas assembled, eleven cases were carried out between different individuals, andeleven cases using donor limbs which had been originally grafted to the flankof 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 thedonor tissue while the latter group showed no rejection at all, judged eitherby gross inspection or by histology. Since the structures of the regeneratesobtained in the two series were not significantly different it can be concludedthat in these experiments immunological rejection neither potentiates norinhibits the formation of duplications. However, it is to be expected that theeffect of the graft would be inhibited if a long enough healing period wereallowed between the graft and the amputation, because eventually the graftedskin would be completely destroyed.
It was thought to be of some interest to examine some metamorphosedaxolotls (efts), since in other amphibia regenerative ability often falls off atmetamorphosis (Scadding, 1977) and the eft has skin with a quite differenthistological structure, notably a thick dermis containing huge mucous glands(Fig. 4 a, c). A number of grafts were carried out in which posterior forearmskin from the eft was grafted to the anterior forearm of an axolotl and theusual protocol followed thereafter. The regenerates showed a range of structuresintermediate between those obtained from the double anterior and doubleposterior 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 275were those showing the most graft rejection. The eft donors had their legsamputated at the proximal limit of the graft and these regenerated normally,although both healing and regeneration were about three times slower thanfor a similar size axolotl. This small series is perhaps not conclusive, but itcould indicate that the codings are still present after metamorphosis but thatinteractions between eft and axolotl skin occur less readily than betweenaxolotls.
Provenance of cell in duplicates
In order to interpret the morphology of compound regenerates in terms oftheir formation it is important to know the cellular composition of the differentparts. If it can be shown that formerly anterior host tissue has contributedto posterior structures, then this means that the tissue in question has beenreprogrammed in terms of its positional coding. If it can be shown that graft-derived cells contribute to tissue types not present in the graft then this meansthat metaplasia has occurred.
In the present work triploidy has been used as the marker and a numberof cases have been examined histologically in which either the donor or thehost was triploid. All these experiments involved grafts of posterior skin tothe anterior. Altogether, seven cases of diploid grafts to triploid hosts wereexamined histologically, and sixteen cases of triploid grafts to diploid hosts.Of the latter, seven cases were performed with tolerant hosts following theprotocol of Fig. 2.
Where the host was triploid and the donor skin diploid the results were asfollows. On the posterior side of the duplication, which was the side awayfrom the grafts, all tissues contained abundant trinucleolar cells with frequenciessimilar to those in triploid control limbs. On the anterior side, there weremany trinucleolar cells in the epidermis and connective tissue but their frequencyin the cartilages fell off from the mirror plane to the anterior digit IV (seeTable 2 and Fig. 5/).
Where the host was diploid and the donor skin triploid nine grafts weremade to non-tolerant hosts. In four of these the regenerates were examinedeleven days after the amputation which is the stage of dedifferentiation. Theyshowed no immunological rejection and had abundant trinucleolar cells onthe anterior side of the apical ectodermal cap. A few trinucleolar cells werealso 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 cellscould be recognised because of the extensive degradation of the tissues. Thosethat were found were present in the epidermis, muscle and cartilage on theanterior side of the duplication. In the seven cases in which the hosts weretolerant there was no rejection and in most but not all cases many moretrinucleolar cells were visible. They were abundant on the anterior side of the
to ON
Tab
le 2
. Per
cent
age
of t
rinu
cleo
lar
cell
s fou
nd
in t
he m
etac
arpa
ls o
f do
uble
pos
teri
or d
upli
cati
ons
Cas
e D
onor
H
ost
num
ber
IV
III'
II
' II
II
I IV
. <
FL
15
R
2/z
3«
6 0
0 4
27
34
38
^F
L2
2R
FL
17
R
In
3«
5 14
10
35
T15
RT
19R
T29
R
[ost
3« 3/7
3« In In 2n
Dig
itnu
mbe
r
6 6 5 6 6 5
f
IV
0 6 14 35 26 35
Ant
erio
r si
deA
III' 0 23 10 25 26 26
IF
4 28 22 0
II 27 39 1 0
Pos
teri
or s
ide
A III 34 42 28 0 0 0
IV 38 36 24 0 0 0
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 resultingfrom a triploid skin graft on a diploid host, (d) a trinucleolar cartilage cell in theanterior part of the duplicate regenerate formed from a triploid skin graft ona diploid host, (e) a trinucleolar muscle cell in the anterior part of a similar re-generate. (/) a trinucleolar cartilage cell anterior to the midline in a duplicationformed 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 IV. Particularly large numberswere found in the cartilages, but they were also found in muscle cells, connectivetissue and epidermis (Fig. 5d, e and Table 2).
It seems therefore that the posterior side of the duplication is composedwholly of host tissue and the anterior side of a mixture of donor and hosttissue. With respect to metaplasia, the present results confirm those of Dunis &Namenwirth (1967) which showed that descendent cells from a skin graftcould become incorporated into the muscle and cartilage of the regenerate.
/?-G
alac
tosi
dase
Deo
xyri
bonu
clea
seFu
cosi
dase
Glu
curo
nida
seiV
-ace
tylg
luco
sam
inid
ase
iV-a
cety
lneu
ram
inid
ase
Pro
nase
Rib
onuc
leas
e A
Try
psin
4 4 4 4 6 5 5 6 11
2/t/
ml
1 m
g/m
g10
0 m
u/m
l10
00 u
/ml
100
mu/
ml
10 u
/ml
10 m
g/m
l1
mg/
ml
10 m
g/m
l
D PD
D D O,
PD
D H PD
H,
H
D N D D DS
Rh
,DH P
DH
, h,
O, D
,D
SR
D PD
DSR
D N,
DD D D
,DN
, D
D D D D DSR
PD
D D,D
h, P
D
to oo
Tab
le
Enz
yme
3.St
ruct
ure
Cas
es
of r
egen
erat
es fo
rmed
af
ter
conc
entr
atio
n 1
enzy
me
trea
tmen
t
1:10
of p
oste
rior
ski
n
Dilu
tions
A
1:10
0
cuff 1:
1000
1 :10
000
49
Eac
h le
tter
in th
e ta
ble
repr
esen
ts a
sin
gle
case
: D d
uplic
atio
n, P
D p
arti
al d
uplic
atio
n, D
SR
dup
licat
ion
with
ser
ial r
epet
ition
,N
, N
orm
al;
h, h
ypom
orph
ic,
H,
hype
rmor
phic
; O
, ot
her.
Properties of the skin in axolotl limb regeneration 279Distal
Posterior
Apical ectodermal cap
Stump
Anterior
• Epidermis
Proximal
Fig. 6. Diagram of a regenerating left limb viewed from the dorsal side. Thefollowing figures depict the region enclosed by the dashed line.
It therefore seems as though formation of the duplication involves extensivepositional reprogramming of host tissue and some reprogramming togetherwith metaplasia of donor tissue.
Enzyme treatment of posterior grafts
The reprogramming ability of posterior but not anterior skin implies thatthe former contains something which the latter lacks. It was thought that if thesubstances which embody the codings were present in the extracellular matrixas suggested by Tank & Holder (1979), then perhaps they could be destroyedby enzymic treatment without destroying the cells. Accordingly posterior half-skin cuffs were soaked in various concentrations of nine enzymes for 1 h atroom temperature before being grafted to the anterior side of host limbs. Thegrafts were allowed to heal for 2 weeks and the limb amputated through thegraft. In this experiment, a positive result would consist of normal limbsgrowing after high dose treatment and duplications growing after low-dosetreatment, a negative result would consist of duplications growing after anydose. The results are shown in Table 3. There is perhaps a slight tendency forhigh 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 cannotbe regarded as informative and it is mentioned here solely to record a negativeresult.
280 J. M. W. SLACK
Distal
00111 y (0000111111 ^ t'ooi 1 A C00001
01111 S——TOO01111111 > 0)0111 3 C00001
/ = o o
Fig. 7. Time course of specification of the blastema during normal regeneration.
DISCUSSION
The results presented in this paper show that anterior and posterior skinare nonequivalent. A graft of posterior skin to the anterior followed byamputation results in the formation of double posterior duplications in whichthere is some reprogramming of host tissue and some metaplasia and re-programming of donor tissues. The converse graft results in the formation ofnormal or hypomorphic regenerates.
These and other results will now be discussed in terms of the serial thresholdtheory of regeneration (Slack, 1980). According to this theory pattern formationin regeneration can be accounted for by assuming that the differentiated organis partitioned into territories which consist of groups of cells plus their associatedextracellular matrix. These territories are coded in such a way that all of a setof biochemical switches are on at one end and successive territories across theorgan each have one more switch off. It is assumed that the 'on' state of aswitch corresponds to the presence of a particular substance, the switchproduct, and that the 'off' state corresponds to its absence. So each territorycontains the information for making all the territories further down in thesequence in much the same way that each of a stack of Russian dolls containsall of the smaller dolls in the set. In the case of the anteroposterior axis of theamphibian limb, the results suggest that the posterior edge should be identifiedas 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 howa blastemal territory can become respecified by its surroundings, and this canbe done by postulating two simple relationships between the states of theswitches in neighbouring blastemal territories. The first, which is necessaryfor the spread of pattern information through the tissue, is the dominance ofhigher over lower switches such that the product of a particular switch turnson 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 eachterritory to tend to adopt the same state as in the majority of neighbouringterritories. This sort of diffusion process necessarily accompanies situations ofintercellular communication.
It is assumed that there is an early stage immediately following dedifferenti-ation during which the cells of the blastema have all their switches off. Thepattern of the regenerate is then controlled by the codings of the most distallayer of territories in the stump because the switch products present in thisregion can influence the codings adopted by the neighbouring blastemal terri-tories. These can then in turn influence their neighbours and so the blastemaas a whole will pass through a sequence of unstable states until a final stablearrangement of codings has been set up. This stable arrangement correspondsto a 'determined' blastema, although since it would still be possible to changethe codings at this stage by surgical rearrangement the determination is notirreversible.
282 J. M. W. SLACK
Fig. 10. Predicted regenerate following a graft of anterior skin to the posteriorwith removal of the most posterior host territory.
The process will be illustrated by a series of diagrams which represent theblastema viewed from the dorsal aspect as shown in Fig. 6. Each territorywill be shown as a hexagon and its coding by a group of binary digits. The'on ' state of each switch is shown as ' 1 ' and the 'off' state as '0 ' . An emptyhexagon represents the coding 00000, which means that all the switches areoff. In the diagrams the lowest row of hexagons belong to the most distal layerof stump tissues next to the blastema and since these are differentiated tissuestheir codings cannot be changed. The remainder of the hexagons represent themesenchymal part of the blastema. In reality the blastema would be growingat the same time as these territories are being specified but in order to keepthe presentation simple the process is depicted as though the blastema hasa fixed size and the specification proceeds stepwise from the proximal edge.
In order that the predicted sequence of events be unambiguous it is necessaryto put the principles of serial dominance and spatial averaging into a preciseform, and this can be done as follows with (1) and (2) expressing the formerand with (3) expressing the latter.
(1) The rth switch is turned on in a territory (hexagon) with no time delayif the i+l th switch is on in that territory. This ensures that the sequence ofswitches 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 attime t-\ in one or more of the neighbouring territories. This ensures thatpositional information spreads through the blastema and that a series ofdecreasing codings is set up along the axis in question. A special rule isnecessary for the top switch in the sequence (the wth) because it has no higherproduct which can turn it on. It seems reasonable to suppose that it can beturned 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 ormore neighbouring territories at t-\.
(3) In the absence of influences from higher switches, the state of the i thswitcji at time t is adjusted to be the same as that in the majority of the neigh-bouring territories at t-l. This means that the boundaries between regionstend 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 halfFig. 11. Predicted regenerates from anterior and posterior half limbs.
pack with a coordination number of six, 'majority' here means four or moreout of six, or in the case of edge territories, three out of five, three out of fouror 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 blastemalterritories have all their switches off. By t = 1 the first row of blastemalterritories 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 ofcodings is reached which is stable in the sense that it does not spontaneouslychange thereafter. This is of course the normal pattern in which all switchesare on at the posterior edge and each territory towards the anterior has onemore switch off.
In this model the normal pattern is not the only stable pattern and it isto be expected that a rearrangement of the stump territories may lead to theregeneration of a stable but abnormal pattern. If an extra posterior territoryis added to the anterior edge, as shown in Fig. 8, then the same set of rulesgenerate a double posterior duplication. This is the result obtained in theexperimental section of this paper when a posterior half cuff of skin is graftedto the anterior side and the combination later amputated through the graft.Furthermore, if only the right hand column of blastemal territories is regardedas being composed of cells derived from the graft then the cellular composition
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284 J. M. W. SLACK
t = 0 t = <*>
Fig. 12. Pattern contraction in the regenerate formed from adouble anterior limb.
indicated by the model is also roughly similar to that found in reality witha large contribution of donor tissue at the anterior edge but with much of theduplicate being formed from host tissue. The widespread metaplasia of donortissues found experimentally indicates that the clonal origin of cells is notrelevant to the new codings which they acquire in the blastema, and so thisfactor is not included in the model.
The converse experiment of grafting anterior skin to the posterior and lateramputating through the graft is simulated in Figs 9 and 10. In Fig. 9 an extraanterior territory is added to the posterior edge of the stump. This has littleeffect on the regenerate which develops a normal pattern slightly twistedtowards the operated side. In Fig. 10 the graft replaces the most posteriorterritory of the stump, and this gives rise to a hypomorphic regenerate lackingthe most posterior structures. Reference to Table 1 will show that the normallimb 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 leadsto normal regeneration. This result is consistent with the model if it is assumedthat during the healing period the gap is filled by posterior tissue from theproximal and distal edges of the wound.
The application of the serial threshold theory to the amphibian limb in theform of the present model also allows the explanation of a number of relatedexperiments by myself and others which have not been satisfactorily explainedin the past:
Half limb regeneration. The morphogenetic potency of anterior and posteriorhalf limbs has been studied by Goss (1957) and Maden (1979). The posteriorhalf from the upper arm will frequently regenerate an entire hand whereasthe 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 apartial set of stump territories, and it may be seen that the posterior half containsinformation which initiates a course of events close to the normal while theanterior half does not. If the posterior blastema were only of half size in termsof territory number then it should form a posterior half regenerate, as foundby 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 limbwhere 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 thenumber 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 thenumber of territories across the blastema is greater than that in the stump.
Pattern contraction of double anterior limbs. Double anterior limbs have beenconstructed surgically by Bryant & Baca (1978), Stocum (1978) and Tank(1978). They show little if any capacity for regeneration and the structureswhich 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 regeneratefew if any structures. Slack & Savage (1978) described regeneration behaviourof embryonically produced double posterior limbs and found good regeneration
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286 J. M. W. SLACK
with a slight tendency for pattern contraction and also a number of cases ofexpansion. Tank & Holder (1978) partly resolved this conflict of results byshowing that contraction of regenerates from surgically constructed limbsbecame more acute the longer the healing time allowed between the graft andthe amputation. The exact course of events during healing is not known, butit seems likely that cell death, cell division, revascularisation and changes inthe extracellular matrix would all affect the diffusion of the active factors. Ifdiffusion constants were reduced then the size of the territories would bereduced 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 numberacross the blastema.
In Fig. 13A-C are shown cases in which the blastema narrows, keeps thesame width and widens. The first shows a severe contraction in which threetypes of territory are eliminated from the midline of the regenerate. The secondshows a retention of all the types of territory present in the stump, and thethird shows the addition of two extra types of territory in the midline. This isexactly the type of behaviour observed by Slack & Savage (1978). In this studywe found that expansion and contraction always involved the addition orsubtraction of elements at the centre of the pattern, and that the new elementswere always neighbours of the ones already present. Furthermore branchingof midline cartilages could occur and could be either proximally or distallydirected. Both types of branching are shown by the group of territories coded01111 in Fig. 13 C: these are separated in the stump, then coalesce in the proximalpart of the blastema and diverge again more distally.
Reprogramming of blastemas. Iten & Bryant (1975) showed that very earlyblastemas could be reprogrammed in the anteroposterior axis if they wereinverted on the contralateral stump, later blastemas were partially reprogrammedand produced hypermorphs and duplicates, and later blastemas still couldproduce 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, andthe later blastema is fully determined. In the last case the formation of asupernumerary limb requires a 'de-determination' at the junction and theformation of an intercalary blastema with polarity depending on the codingsof the adjacent territories.
The idea which lies at the heart of the serial threshold theory is that a regionof tissue can regenerate structures with all the 'lower' codings but not thosewith the 'higher' ones. This property is shown not only in the anteroposterioraxis of the amphibian limb but also in the distal regeneration of vertebrateand arthropod appendages and in the posterior regeneration of worms (seeSlack, 1980#). The widespread occurrence of this type of phenomenon mustsuggest the possibility of a common biochemical basis for the incremental
Properties of the skin in axolotl limb regeneration 287codings in all such cases. It is therefore unfortunate that to date most develop-mental biochemistry has concentrated on the differences between differenttissues rather than on the differences between the same tissue in differentplaces.
I should like to thank Shirley Williams for technical assistance, Bob Bloomfield forsupervising the axolotls and John Cairns for his interest in serial thresholds.
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BRYANT, S. V. & ITEN, L. E. (1976). Supernumerary limbs in amphibians: their experimentalproduction in Notophthalamus viridescens and a new interpretation of their formation.DevlBiol. 50, 212-234.
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LHEUREUX, E. (1977). Importance des associations de tissus de membre dans le developpe-ment des membres surnumeraires induits par deviation de nerf chez le Triton PleurodelesWaltlii Michah. / . Embryol. exp. Morph. 38, 151.
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SLACK, J. M. W. (19806). Regulation and potency in the limb rudiment of the axolotlembryo. / . EmbryoL exp. Morph. (In the Press).
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{Received 8 February 1980 revised 13 March 1980)