J. Embryol. exp. Morph. 86, 247-269 (1985)
Printed in Great Britain. © Company of Biologists Limited 1985
247
Normal fates and states of specification of different
regions in the axolotl gastrula
J. HERMAN CLEINE AND JONATHAN M. W. SLACK
Imperial Cancer Research Fund, Mill Hill Laboratories, Burtonhole Lane,
London NW7IAD, U.K.
SUMMARY
A fate map was constructed for four regions of the early gastrula of Ambystoma mexicanum
using orthotopic grafts from donors labelled with FLDx (fluoresceinated-lysinated-dextran). The
region around the animal pole gave rise to epidermis only and did not include prospective neural
plate. The dorsal marginal zone contributed to cephalic endoderm and to the whole length of the
axial mesoderm (notochord and somites), the lateral marginal zone to lateroventral and somitic
mesoderm, and the ventral marginal zone to lateroventral mesoderm. It was found that the dorsal
marginal zone contributed relatively more to the anterior regions of the mesodermal mantle and
the ventral marginal zone more to its posterior parts.
The same regions of the gastrula and also vegetal yolky tissue were cultured as explants and
labelled with tritiated mannose. Their glycoprotein synthesis pattern was compared to those of
the neurula tissues to which they contribute in vivo. Animal pole explants synthesized large
amounts of the epidermis-specific marker epimucin. Dorsal marginal zone explants did not
synthesize epimucin but did make amounts of S2 and S6 indicative of mesoderm, as well as the
notochord-specific markers S2-2 and S3-2. Lateral marginal zone explants showed the same
pattern as the dorsal marginal zone including the two notochord-specific markers, although they
do not contribute to notochord in vivo. Ventral marginal zone explants were more variable in
their behaviour. Yolky tissue from the vegetal hemisphere of the gastrula or the archenteron floor
of the neurula synthesized mainly polydisperse material of high molecular weight rather than
discrete glycoproteins.
The results indicate that at the early gastrula stage states of specification exist which correspond
to the three germ layers, ecto-, meso- and endoderm.
The ectodermal specification of animal pole explants is quite robust and cannot easily be
changed by variation of the culture conditions. However treatment with a concentrated pellet of
vegetalizing factor does induce a change to mesodermal specification, which is clearly detectable
in the pattern of glycoprotein synthesis. Similar inductive interactions between different regions
of the early embryo are thought to occur during normal development.
INTRODUCTION
A fate map shows what each region of an embryo will become in normal development and a specification map shows what each region will become if it is cultured
in isolation. If the two maps are identical at the stage in question the embryo is
described as a mosaic. If the two maps differ then the embryo is described as
regulative and it is supposed that the difference arises because interactions between
the parts at a later developmental stage are necessary for their correct specification.
Key words: Axolotl, gastrula, fate map, cell lineage label, glycoprotein synthesis, specification.
248
J. H. CLEINE AND J. M. W. SLACK
In this paper we report fate mapping and isolation experiments on early gastrulae
of the axolotl, Ambystoma mexicanum.
Conceptually, such studies are not new. The classical fate maps for both urodele
and anuran embryos were published by Vogt (1929) and a map for the axolotl
specifically by Pasteels (1942). Isolation studies on early gastrulae of the axolotl and
other species were performed by Holtfreter (1938a, b) and many similar studies
have been carried out since then. However a quantum jump in technology has taken
place since this earlier work and we feel we have a duty to repeat the most important
classical experiments using modern techniques to find whether the interpretations
presented in embryological textbooks (e.g. Slack, 1983) are correct or not.
All amphibian embryo fate maps until 1978 were established by the method of
vital staining. This is not entirely satisfactory since the dyes have a tendency to
spread and fade as development proceeds. In the last few years a number of new
cell lineage labels have been introduced (reviewed Slack, 1984a) which cannot be
passed between cells and which allow visualization of a single labelled cell surrounded by unlabelled ones, or vice versa. In our experiments we have used orthotopic grafts from embryos uniformly labelled with fluoresceinated-lysinateddextran (FLDx; Gimlich & Gerhart, 1984) to unlabelled hosts.
All amphibian embryo isolation experiments until 1984 involved in vitro culture
of the explants until terminal differentiation had occurred followed by examination
of histological sections and scoring for the presence of various differentiated cell
types. This is unsatisfactory because the observed events are at the end of a long
chain of causality with respect to the events of interest, i.e. specification of regions
in the gastrula. It is very unlikely that cells in the early gastrula are being specified
as future neurons, fibroblasts, myoblasts or erythrocytes, and much more likely that
their commitments at this stage are to geographical regions rather than particular
histological cell types. Now however it is possible using a variety of biochemical and
immunological techniques to observe the behaviour of explants long before terminal differentiation occurs. In this study we have used the synthesis of high molecular
weight glycoproteins as criteria since a previous study showed considerable regional
specificity of synthesis patterns of this class of molecule during neurulation (Slack,
19846).
The results show that a certain revision of the classical fate map is necessary,
particularly in relation to the boundary between prospective neural plate and
epidermis. In fact the results are rather similar to those previously obtained in this
laboratory for Xenopus laevis (Smith & Slack, 1983; Slack, Dale & Smith, 1984)
suggesting that urodele and anuran fate maps are not as different as has sometimes
been thought. The isolation studies show that the gastrula is less mosaic than that
of Xenopus; three different zones can be distinguished giving ectodermal, mesodermal and endodermal type development. Variations in ion balance or addition of
particular substances to the medium do not respecify the explants, as has sometimes
been claimed (Barth & Barth, 1974; Lovtrup & Perris, 1983), but treatment with
vegetalizing factor (Tiedemann, 1976) can mesodermalize ectoderm explants.
Fate and specification in the axolotl gastrula
249
MATERIALS AND METHODS
Labelling of donor eggs with FLDx
Eggs of the axolotl (Ambystoma mexicanum) were obtained and fertilized in vitro as described
by Mohun, Tilly, Mohun & Slack (1980). Embryos were decapsulated and demembranated with
fine watchmaker's forceps. Stages are according to Bordzilovskaya & Detlaff (1979).
Embryos to be used as donors were injected before first cleavage with 70 nl FluoresceinLysine-Dextran (FLDx: 100|Ug/ml in water) prepared as described by Gimlich & Gerhart
(1984). Injections were carried out by using a Burleigh Inchworm to drive a 10 [A pressure-tight
syringe connected to a liquid-filled glass micropipette. During and after the injection embryos
were kept in 5 % NAM with 5 % w/v Ficoll so as to lower the hydrostatic pressure in the
perivitelline fluid (Kirschner & Hara, 1980). NAM is 'Normal Amphibian Medium' (Slack,
19846).
Grafting
Operations were carried out on stage-10 embryos (onset of gastrulation) using tungsten needles
and hair loops. Embryos were transferred to 5 cm plastic petri dishes coated with a 2 mm thick
layer of 2 % agar (Difco Noble agar). The dishes contained 50 % NAM in which the calcium
concentration was lowered to 120 [M, and to which 1 % w/w Ficoll was added. Although wound
healing is relatively slow in low calcium saline, this medium prevents the explants from becoming
deformed or curling up (Nakatsuji & Johnson, 1984) and makes manipulation and insertion of
the graft easier. After having received the graft the host embryos were put into wells made in the
agar and allowed to develop to stage 23-26. Approximately two hours after operation the
medium was replaced by 5 % NAM to facilitate normal gastrulation. Note that in dilutions of
NAM, only the salts are diluted, the phosphate and antibiotic remaining full strength.
Histology
Embryos were fixed in 4 % paraformaldehyde in 70 % PBSA for 18-24 hs at 4°C, rinsed in
70 % PBSA, dehydrated in an ethanol/butanol series, embedded in paraffin wax (m.p. 56 °C) and
sectioned at 10 pm. Rehydrated sections were stained with DAPI (4,6-diamidino-2-phenyl indole, 1 jug/ml) for 10 minutes, rinsed in tap water, dehydrated through ethanols, cleared in xylene
and mounted in DPX. The sections were scored using a Zeiss photomicroscope with
epifluorescence optics, labelled areas being assessed visually on every tenth section through the
length of the specimen. The accuracy of this method was checked by making camera-lucida
drawings on graph paper and counting squares and it was found to be accurate to about 10 %.
Results will be presented in terms of a normalized anteroposterior distance. So for example
'0-4-0-6' means the region from 40-60 % embryo length from the snout.
Labelling of explants with tritiated mannose
Parts of early gastrulae were excised with electrolytically sharpened tungsten needles and
cultured as explants in NAM until control embryos reached stage 13-14. They were then cut into
small pieces to facilitate access of the label, transferred to plastic scintillation vial inserts and
incubated with 50/iCi tritiated mannose in approximately 0-3 ml NAM until controls reached
stage 23-26. After incubation the tissue was rinsed twice with NAM, drained and stored frozen
at - 70 °C. There is some relocation of 3 H from mannose to amino acids during the labelling
period but the principal bands visible on the gels are known to be glycoproteins because they bind
specifically to lentil lectin Sepharose.
In cases were the animal pole explants were treated with a substance or with a medium other
than NAM, this treatment was given in the initial incubation, i.e. while control embryos were
undergoing gastrulation.
Vegetalizing factor was supplied by Prof. H. Tiedemann. It was Fraction E-5 diluted 1:1 with
gamma globulin and was wrapped in sandwiches composed of two explants from the animal pole.
250
J. H. CLEINE AND J. M. W. SLACK
Materials
Ficoll (Ficoll-400) Pharmacia.
DAPI (4,6-diaminidino-2-phenyl indole dihydrochloride) Boehringer.
2,6-[3H]mannose (>30Ci/mmol) Radiochemical Centre Ltd., Amersham.
Cyclic AMP, free acid, Sigma.
dextran sulphate (Afr 500000) Pharmacia.
micrococcal nuclease (NFCP) Worthington.
DNase (DPFF) Worthington.
RNase (RASE) Worthington.
PMSF (phenyl methyl sulphonyl fluoride) Sigma.
Preparation of a soluble protein fraction from explants
Each sample of tissue was homogenized with 100//I MNS (0-2M-sucrose, 5mM-Tris-HCl
pH 8-8,1 mM CaCk, 25 jug/ml micrococcal nuclease) in a 2 ml Dounce homogenizer with B pestle,
rehomogenized with 10/d DR (lmg/ml DNase, 0-5mg/ml RNase 0-5M-Tris Cl pH7-0) and
rehomogenized again with 0-4ml HMP (0-2M-sucrose, 10mM-sodium phosphate pH7-5, lmMCaCb, 1 mM-MgCh, 2mM-PMSF). The homogenate was spun at low speed (45 seconds at Mark
1, Gallenkamp bench centrifuge) to remove yolk granules. The supernatant was decanted, the
yolk pellet resuspended in another 0-5 ml HMP and spun at low speed once more. Both supernatants were pooled, 0-1 ml was removed for protein estimation by the Folin reaction and to the
remaining 0-9 ml supernatant 0-1 ml 10% SDS was added, which dissolves all structures apart
from melanin granules. This was then spun for 5 minutes in a microfuge (Beckmann-8700g). For
gel electrophoresis the samples were concentrated by dialysing against 0-25 % SDS in 3 mM-TrisHC1 pH6-8, lyophilizing and dissolving in 50//1 of 5% /S-mercaptoethanol, 10% sucrose and
0-002 % bromophenol blue. This was boiled for 1 minute and microfuged for 4 minutes. 5 jul of
the supernant was kept for counting and the remainder stored at - 70 °C until being used for
electrophoresis. Both pellets contained little radioactivity (2-3 % of that in the soluble fraction)
and are not considered further.
Gel electrophoresis
Portions of each sample containing equal counts (6000-15000 c.p.m.), but not exceeding
100 i*g protein per track, were loaded onto 4-8% SDS polyacrylamide gradient gels and
electrophoresed at constant current until the bromophenol blue reached the bottom. Gels were
fixed in 30% methanol and 10% acetic acid, stained with 0-05% Coomassie blue, and
fluorographed by the method of Bonner & Laskey (1974).
RESULTS
Fate mapping experiments
FLDx-labelled embryos were examined to confirm that all parts became labelled
following in j ection. From this it can be concluded that all labelled tissue in the grafted
embryos is derived from the injected donor, and all unlabelled tissue from the host.
Orthotopic grafts with labelled donor tissue onto unlabelled recipients were
carried out as illustrated in Fig. 1. Both donor and hosts were early gastrulae (stage
10). Healing of the graft was generally good but not all host embryos developed
normally. In particular embryos with a dorsal marginal zone (DMZ) graft were
prone to abnormal gastrulation apparently because invagination in the graft was not
integrated with that of the surrounding tissue. Other embryos sometimes developed
wrinkles and deformities of the grafted area which in certain cases led to a severely
Fate and specification in the axolotl gastrula
251
abnormal appearance. Only embryos that showed a normal external morphology
were used for further analysis. Likewise, after examination of the sections, data
were taken only from embryos with an internally normal gross anatomy, although
slight deviations were tolerated such as small areas of thickened epidermis in the
case of AP grafts and minor asymmetries of the axial system in embryos with a
FLDx-labelled donor
Unlabelled recipients
VMZ
Fig. 1. Orthotopic grafting experiments. A-C indicate positions and sizes of the grafts,
D-G indicate the appearance of the recipients.
252
J. H. CLEINE AND J. M. W. SLACK
B
"*:A-
,•*./
Fig. 2. General appearance of orthotopic grafts at stages 23-26.
(A, B) DMZ graft, dark field and FLDx fluorescence.
(C, D) VMZ graft, dark field and FLDx fluorescence.
(E, F) AP graft, DAPIfluorescenceand FLDx fluorescence.
(G, H) LMZ graft, DAPIfluorescenceand FLDx fluorescence. Scale bar, 0-2mm.
Fate and specification in the axolotl gastrula
G
253
H
DMZ graft. About ten embryos at the head-extension stage (stage 23-26) were
examined in each group (AP, DMZ, VMZ, LMZ graft).
Transverse sections through each of the four types of grafted embryo are shown
in Fig. 2 to show their general appearance. The detailed results are presented in Figs
3-6 in terms of the average proportion of each structure labelled at each anteroposterior level of the body, and a reconstruction of an archetypal case of each type
of graft is shown in Fig. 7.
254
J. H. CLEINE AND J. M. W. SLACK
AP grafts
Ten recipients of AP grafts were examined for distribution of the label
throughout the embryo. Most of the label was located in the ventrolateral epidermis
of the anterior half (Figs 2,3 and 7 A) endodermal and mesodermal structures being
completely unlabelled. In two embryos some label was also found in the anteriormost parts of the forebrain whereas the other eight showed no labelled neural tissue
at all. The proportion of labelled neural structures was much less than expected on
the basis of the existing fate map of the axolotl gastrula (Pasteels, 1942). According
to this the upper boundary of the prospective neural plate extends mediolaterally
from the animal pole to the lateral marginal zone, which implies that roughly half
of each graft would consist of presumptive neural tissue. Our results indicate that
this is not the case. Therefore we suspect that the upper boundary of the prospective
neural plate rather runs about halfway between the animal pole and the DMZ, as
in Xenopus and other Anura (Keller, 1975).
A certain amount of labelled debris was found in the ventrolateral part of the
endoderm in the region 0-1-0-7 along the anteroposterior axis (see Fig. 2F). This
debris is likely to have been sloughed off from the edges of the graft into the
blastocoel which moves ventrally during gastrulation. Debris can easily be distinguished from tissue by examining the sections under DAPI fluorescence to
visualize the cell nuclei.
10
Forebrain
Epidermis
Whole embryo
50
75
Level along anteroposterior axis (%)
100
Fig. 3. Orthotopic graft of animal pole tissue. The histograms show the proportion of
tissues labelled at each level along the anteroposterior axis. The bars are averages for
all the cases.
255
Fate and specification in the axolotl gastrula
70
Notochord
60
u
50
40
•B 30
Io
o.
Somitic mesoderm
a 20
Lateroventral mesoderm
0L_
40 r
.2 30
Endoderm
i 20
22
10
I
a.
20
Whole embryo
10
0
25
50
75
Level along anteroposterior axis (%)
Fig. 4. Orthotopic graft of dorsal marginal zone tissue.
100
256
J. H. CLEINE AND J. M. W. SLACK
Taken together our results indicate that the animal pole region gives rise only to
epidermis. The two cases in which a small amount of neural tissue is derived from
it as well is likely to reflect some variability in accuracy of the operation rather than
differences in fate maps between individual embryos.
DMZ graft
Ten embryos with a DMZ graft were examined. Most of the label was found in
the head and pharynx region (Figs 4, 7B) in particular in the anterior pharyngeal
endoderm. It should however be emphasized that pharyngeal endoderm, head
mesoderm and prechordal mesoderm form a continuous mass without any clear
demarcations between them (Adelmann, 1932). We have called all this tissue
'endoderm' since that is quantitatively its major component, but some mesoderm
is also included in this region.
Of the structures that arose from the graft the notochord was proportionally
the most-heavily labelled. In three embryos the anterior 80% of its length was
completely labelled, in the others most of the label was found in its anterior
part, gradually declining in the posterior direction. On average the notochord
was heavily labelled in the region 0-2-0-9 (Fig. 4). The proportion of labelled
somitic mesoderm was moderate, on average from 18% in its anterior region
to about 5 % in the middle and posterior regions. But in the three embryos with
the heavily labelled notochords, mentioned above, up to 40 % of the anterior
somitic mesoderm was labelled. In two of these embryos a small amount of
anterior lateroventral mesoderm adjacent to the somitic mesoderm was also
labelled.
Summarizing we can say that the DMZ, apart from contributing mainly to
anterior endoderm, gives rise to axial mesoderm along the whole length of the
body, in particular to notochord and to a lesser extent to somitic mesoderm.
Ventral marginal zone grafts
In this group nine embryos were examined. Most of the label was confined to the
posterior half of the body (Figs 5, 7C). The lateroventral mesoderm was proportionally the most-heavily labelled with more than 15 % of labelled tissue in the
region 0-6-0-8. Label extended throughout a considerable arc of tissue with extensive mixing of labelled and unlabelled cells. So as in Xenopus, the VMZ undergoes
substantial dorsal convergence during gastrulation. The posterior endoderm was
also heavily labelled with a peak around 0-8.
In three embryos a very small amount (less than 4 % in any region) of somitic
mesoderm in the trunk was also labelled. Three other embryos showed a small area
of labelled epidermis just anterior to the blastopore. It is likely that in these cases
the upper edge of the graft also contained some ectoderm. That this occasionally
happens is not surprising since the marginal zone of presumptive mesoderm is
narrowest at the ventral side of the gastrula. No label was found in the notochord
of any embryo in this group.
Fate and specification in the axolotl gastrula
10 r
257
Somitic mesoderm
0-25
0-5
Level along anterc) posterior axis
0-75
Fig. 5. Orthotopic graft of ventral marginal zone tissue.
Summarizing we can say that a ventral marginal zone graft (VMZ-graft), gives
rise to lateroventral mesoderm and posterior endoderm. The former is somewhat
anterior to the latter as we would expect from the separation of germ layers which
occurs at the ventral blastopore lip during gastrulation.
Lateral marginal zone grafts
Nine embryos were studied in this group. Each received one graft to the left
or right side taken from the same side of the donor. Although the grafts were
unilateral the results are still given as proportions of the whole transverse section,
so the figures would be about double if they reflected the contributions from both
sides.
The total label was around 5 % of section area from 0-15-0-9 along the anteroposterior axis (Figs 2,6). This is made up of substantial contributions to the somites,
the lateral plate mesoderm and the endoderm. The contribution to the notochord
came from a single case which may perhaps have been a slightly misplaced graft.
The peak contribution to somites is around 0-3, to the lateral plate around 0-5-0-6
and to the endoderm around 0-8, indicating a similar separation between endoderm
and mesoderm during invagination as apparent for the VMZ.
In terms both of dorsoventral and anteroposterior contributions the LMZ can
thus be regarded as intermediate between DMZ and VMZ.
258
J. H. CLEINE AND J. M. W. SLACK
Notochord
Somitic mesoderm
0-25
0-5
Level along anteroposterior axis
0-75
Fig. 6. Orthotopic graft of lateral marginal zone tissue.
Synthesis of glycoproteins by gastrula explants
The same four types of explant, and also explants from the vegetal pole, were
cultured in NAM and labelled with pHJmannose as described in Materials and
Methods. After labelling the explants were processed for gel electrophoresis and
the patterns of glycoprotein synthesis visualized by fluorography. These patterns
were compared with each other and with explants from neurulae which were dissected from stage-14 embryos and also labelled until controls reached stage 23-26.
It has previously been shown that regions of the neurula show distinctive differences
in the synthesis of high molecular weight glycoproteins and that certain bands can
serve as markers for epidermis, notochord and total mesoderm. In Fig. 8 tracks
6-10 these controls show that only epidermis makes epimucin (track 7), only
notochord makes S2-2 and S3-2 (track 8) and notochord and dorsal mesoderm make
more S2 and S6 than other regions.
The first problem was to determine the robustness of this procedure as a test of
specification. Many workers refuse to believe in a 'neutral medium' which does not
have any influence on the developmental pathway of the tissue. However the
present procedure allows not only a comparison between in vitro and in vivo
behaviour, but also enables every component of the medium to be varied to ensure
that it is not having an instructive effect.
Fig. 7. Serial reconstructions of stage 23-26 embryos indicating the position of labelled
tissues (shaded). Four representative cases are shown. (A) AP graft; (B) DMZ graft;
(C) VMZ graft; (D) LMZ graft
Fate and specification in the axolotl gastrula
259
260
3
J. H. CLEINE AND J. M. W. SLACK
1
2
f
3
4
MM
5
M
6
7
8
9
10
> •
1C
S 1 —
>••/•!/'
S2 —
S2.2S3-CZ
205
S3.2S4 —
S5
-116
— 97
S6
I
— 66
Fig. 8. Pattern of glycoprotein synthesis from the gastrula regions shown in Fig. 1.
Equal counts loaded on each track.
Track 1, animal pole; 2, DMZ; 3, VMZ; 4, LMZ; 5, vegetal pole.
Tracks 6-10 labelled regions from neurulae. Track 6, neural plate; 7, epidermis; 8,
notochord; 9, dorsal mesoderm; 10, yolk mass. Designations of bands on left, relative
molecular mass markers on right.
In preliminary experiments we found that total radioactive incorporation of
[ C] glucose into TCA-insoluble material was unaffected by omission of K + or
Mg 2+ , use of NaCl at x 0-5 or x 1 normal strength and pH of 7-5 or 8-5. Omission
of Ca2+ caused the explants to disaggregate, and NaCl at x 0-25 or x 1-5 normal
depressed incorporation. Examination of the glycoprotein patterns on 4-8 % gels
of animal pole explants labelled with pHJmannose showed that the pattern looked
the same following all these regimes, even those in which the total incorporation
was depressed. We conclude that NAM, and also similar amphibian salines of
x 0-5-1 isotonicity such as Holtfreter, Niu-Twitty, de Boer, or Ringer solutions are
indeed neutral culture media.
14
Fate and specification in the axolotl gastrula
261
States of specification
The AP explants always synthesized epimucin visible as a major band (Fig. 8
track 1). Epimucin is an epidermis-specific marker in the neurula and its formation
shows that extensive amounts of epidermis arise in these explants. However the
explants do not show exactly the same behaviour as neurula epidermis in that they
make more of all the other species. This was noted in a previous study (Slack, 1984c)
in which it was shown by electron microscopy that, for incubation periods used
here, only the outer cell layer differentiates into epidermis.
DMZ explants did not make epimucin but did make amounts of S2 and S6
comparable to neurula notochord. They also made two notochord-specific species
S2-2 and S3-2 (Fig. 8 track 2).
LMZ explants showed in all experiments exactly the same synthesis pattern as
DMZ explants, including the presence of the S2-2 and 3-2 notochord markers (Fig.
8 track 4). This represents a major disjunction between normal fate and specification since the LMZ did not contribute to notochord in the fate mapping experiments
except in a single case.
The VMZ explants showed more variable behaviour in some experiments
resembling the other marginal zone explants (as in Fig. 8 track 3) and in others
resembling the AP explants. This suggests that what we call ventral marginal zone
lies near the junction of two differently specified regions whose boundary may not
be completely fixed if mesoderm induction is still going on at the time of explantation. Hence apparently similar pieces of tissue from different batches of embryos
may show different behaviours.
Explants of yolky tissue from around the vegetal pole synthesized little in the way
of discrete glycoprotein species, most of the radioactivity being in high relative
molecular mass poly disperse material (Fig. 8 track 5). The same behaviour is shown
by the yolk mass from neurulae (track 10). It is worth emphasizing that the yolk is
not metabolically inactive in other respects. When labelled with [35S]methionine it
shows numerous labelled bands, which are in fact then the same as for all other
regions of the neurula.
In summary, thefivetypes of explant which have been examined show three types
of behaviour which may be described as epidermal, notochordal and yolk type in
terms of neurula-stage tissue types. It seems reasonable to identify these behaviours
with specification for each of the three germ layers: ectoderm, mesoderm and
endoderm.
Treatment of animal pole tissue with chemicals
Certain pure chemical substances have been claimed to have a vegetalizing or
mesodermalizing effect when applied to isolated ectoderm. Among these are
lithium chloride (Masui, 1960, 1962), cyclic nucleotides and negatively charged
polymers (Ldvtrup & Perris, 1983), as well as inorganic ions (Barth & Barth, 1974).
We felt that we should reexamine these claims now that a more objective test of
262
J. H. CLEINE AND J. M. W. SLACK
1
2
3
4
5
6
7
8
9
1CT3
S1
205
S5
— 116
— 97
S6
— 66
Fig. 9. Patterns of glycoprotein synthesis in ectoderm explants treated with substances.
Track 1, AP control; track 2, DMZ control; track 3, NAM pH8-5; track 4, LiCl
11 mM; tracks 5 & 6, cAMP 5jUM & ljUM; tracks 7, 8 & 9, dextran sulphate 1/xg/ml,
/
/
specification is available. Animal pole explants were incubated in various concentrations of LiCl (substituting for NaCl in NAM), of cyclic AMP and of dextran
sulphate. They were treated while host embryos were traversing stages 10-14 (gastrulation) and then labelled in exactly the same way as the explants described above.
The lithium proved lethal at concentrations of 110 and 55 mM but apart from this
none of the treatments made any difference to specification (Fig. 9), all the surviving AP explants behaved like controls and not like marginal zone explants.
Mesodermal induction
According to the work of Nieuwkoop and others (Nieuwkoop, 1969, 1973) the
first interaction in amphibian development is an induction of an annular mesodermal rudiment from the animal hemisphere under the influence of the vegetal
Fate and specification in the axolotl gastrula
263
hemisphere. We have made several attempts to duplicate this result by showing a
mesodermal glycoprotein synthesis pattern in combinations of animal pole with
vegetal pole explants. Our conclusion is that the induction is demonstrable using
ectoderm from stage 10 or stage 7, but only just. Evidently it is not possible using
this experimental design to suppress epidermal differentiation entirely in the
animal pole component. So the combinations always make epimucin. Also the
different synthesis patterns described above are largely due to quantitative differences in S2 and S6 and such differences will be less apparent in combinations which
are part epidermal and part mesoderm. The only conclusive proof of mesodermalization is the identification of the notochord specific bands S2-2 and S3-2, and
3
10"3
10 "
— S1
205 —
-S2.2
3-S3"
-S3.2
- S4'
-
205
S5
116—
— 116
97—
66—
S6
— 97
— 66
Fig, 10. Mesoderm induction in ectoderm explants.
(A) AP-VP combination. Track 1, combination; track 2, AP control; track 3, DMZ
control.
(B) Vegetalizing factor. Track 1, AP control; track 2, AP treated with vegetalizing
factor; track 3, DMZ control.
264
J. H. CLEINE AND J. M. W. SLACK
this is not easy against a considerable polydisperse background following sugar
labelling. Our results show that in some cases these bands were visible in the
AP-VP combinations (Fig. 10) and in others they were not. We feel that this is
evidence in favour of the reality of mesodermal induction but it is not decisive.
We have also studied animal pole explants which were treated with 'vegetalizing
factor' supplied by H. Tiedemann, see Materials and Methods. Tissue from stage7 and stage-10 embryos was used and in this case the epimucin synthesis was
suppressed partially or completely, and the pattern of synthesis was altered to
resemble that shown by explants of marginal zone (Fig. 10). Evidently a concentrated factor can produce a more complete transformation than the vegetal pole
tissue itself.
DISCUSSION
Fate map
The marking experiments presented here are not intended to construct a complete new fate map for the axolotl but rather to map certain regions which are of
particular interest in connection with experiments on induction. This is why the
animal pole region and parts of the marginal zone were selected for study.
The term 'marginal zone' refers to that region of the gastrula destined to invaginate actively during gastrulation. It is not of course possible to see the extent
of the marginal zone at stage 10 and the results of the fate mapping experiments
suggest that our grafts overlapped the vegetal boundary of the marginal zone and
included some prospective endoderm.
The use of a high molecular weight lineage labels has several advantages over the
previous method of vital staining. The label is sharp and unambiguous and does not
fade or spread from cell to cell. Like previous experiments on Xenopus (Jacobson,
1982, 1983; Smith & Slack, 1983) the results show more mixing of labelled with
unlabelled cells than was suspected from vital staining. However despite this there
is a clear topographic mapping from the head-extension stage back onto the early
gastrula stage showing that the movements of gastrulation and neurulation are
coherent, with cell mixing occurring only in the short range.
Although all of the cases are included in Figs 3-6, when we analyse the results
we ascribe a different significance to the low percentage contributions depending
on whether they represent a small contribution in every case, or a large contribution
in a minority of cases. The latter results are of course more likely to result from
slight variations in the grafting procedure than from variations in fate map between
individual embryos. So we conclude that in normal development the region of the
animal hemisphere indicated in Fig. 1 contributes only to epidermis, this being
located anteroventrally at later stages, and that there is no contribution either to
neural plate or to mesoderm.
We conclude that dorsal, lateral and ventral marginal zone map respectively to
notochord, somite and lateroventral parts of the mesodermal mantle of later stages.
Fate and specification in the axolotl gastrula
265
The results show that a very considerable dorsal convergence occurs, so a VMZ
explant of about 50° partial circumference in the stage-10 embryo expands to
populate the whole lateral plate/blood island region which occupies about 270° of
the circumference at stage 23-26. Likewise a 50° LMZ piece expands to about 90°
whereas a 50° DMZ piece contracts to about 30°. The fact that these angles add up
to more than 360° indicates the degree of overlap between prospective regions due
to interpenetration of cells during the dorsal convergence movements.
If a particular tissue type, such as somite, is considered at a particular transverse
section plane, then it is clear that much is unaccounted for. In other words the
ordinates of Figs 4-6 do not add up to 100 %. The reasons for this are probably
threefold. Firstly there are gaps between the regions mapped totalling perhaps 160°
around the circumference. Secondly, there may be prospective somite in the host
to the animal pole side of the grafts, although this is perhaps less likely for the
ventral grafts among which three cases overlapped the prospective epidermis.
Thirdly, it is possible that some cells are lost as a result of damage at the cut edges
of both graft and host.
Dorsoventral levels of the marginal zone thus map to different dorsoventral
levels of the mesodermal mantle, but they also map to different anteroposterior
levels. This is clearly apparent from the Figs 3-6 'whole embryo' histograms: the
DMZ populates the anterior half of the body, the LMZ populates most of the body
length except the extreme ends and the VMZ populates the posterior half. In this
regard there is also a clear slippage between the labelled mesoderm and endoderm:
in the DMZ grafts the endoderm is anterior to the mesoderm, and in the LMZ and
VMZ grafts the endoderm is posterior to the mesoderm. This is entirely to be
expected on the basis of our understanding of the gastrulation movements. The
dorsal invagination is an inpushing of both endoderm and mesoderm and results in
the formation of the archenteron cavity. It can be visualized as similar to the
deformation of a rubber ball by pushing in at one point. However the lateroventral
invagination is an anterodorsal migration of mesoderm only between the other
germ layers. Since the prospective mesoderm is at least partly on the surface at stage
10 (Smith & Malacinski, 1983) there must be a rupture between endoderm and
mesoderm along the line of the lateroventral blastopore lip, and this is in fact clearly
visible when axolotl embryos are dissected during late gastrula or early neurula
stages.
Perhaps the most remarkable aspect of the results is how similar they are to those
from comparable experiments on Xenopus (Smith & Slack, 1983; Slack et al. 1984
and unpublished results). In recent years various authors have highlighted different
aspects of the fate maps of anurans and urodeles (e.g. Brun & Garsen, 1984; Smith
& Malacinski, 1983) but the topographic mapping of the regions considered here
by orthotopic grafting is essentially identical.
The principal respects in which our results are at variance with previous urodele
fate maps (Vogt, 1929; Pasteels, 1942; Nakamura, Hayashi & Asashima, 1978) are
twofold. Firstly the prospective boundary between epidermis and neural plate
266
J. H. CLEINE AND J. M. W. SLACK
cannot run near the animal pole as usually shown. Although we have not attempted
to map the neural plate we suspect that the prospective regions will be found in a
supraequatorial position on the dorsal side, as in Xenopus and other anura (Keller,
1975). Secondly there is the marked projection of dorsal marginal zone to anterior
mantle and ventral marginal zone to posterior mantle. This should perhaps be
expected on the basis of our normal understanding of the gastrulation movements
but is not emphasized in the published fate maps in which the future anteroposterior
axis is considered to correspond approximately with the animal-vegetal axis of the
gastrula.
Specification
Previous studies of specification have involved long-term culture followed by the
scoring of the mixtures of differentiated cells which arise in the explants as 'ectodermaF, 'mesodermal' etc. Although this method has given us much useful information it is subject to various objections: it is difficult to be confident that the culture
medium is truly neutral; the long culture period allows secondary and tertiary
interactions to occur which may obscure the original specification; explants always
contain undifferentiated tissue; and, finally results are normally presented in a form
which ignores the proportions of tissues within the explants.
The use of early biosynthetic properties circumvents all these problems: we can
ensure the neutrality of the medium; we look only at early events; and the variations
within and between explants are automatically averaged when they are
homogenized. On the other hand it must be admitted that the regional markers
which we presently know about do not allow as clear and qualitative a discrimination between the regions as we would like. The neurula tissues which were studied
as controls gave the same results as those reported previously, viz epidermis makes
epimucin, all mesodermal explants make a lot of S2 and S6, notochord makes S2-2
and S3-2, and the yolk mass makes mainly high molecular weight poly disperse
material. No unique marker, or even special quantitative behaviour was observed
in the neural plate. It should be emphasized that in these studies we make no claims
about the absolute amounts of each molecular species since we do not know the
specific activities of mannose and other precursor substances in vivo. All we claim
is that the distribution of label across the bands is characteristic of the different
regions.
The results show that three types of behaviour are displayed by the isolated
gastrula tissues, and that these approximate to epidermis, notochord and yolk mass
in the neurula although in no case show the specific features as clearly as the
dissected neurula tissues. It seems reasonable to identify these states of specification with the traditional germ layers: ectoderm, mesoderm and endoderm respectively.
Despite the theoretical shortcomings of the histological method for assessing
specification, our results are in fact the same as the classic histological studies of
Holtfreter (1938a; Holtfreter & Hamburger, 1955) insofar as he found that the
Fate and specification in the axolotl gastrula
267
whole marginal zone except for the extreme ventral part could give rise to
notochord in explants. Similar results were obtained by Koebke (1977) and Slack
(unpublished). In other words the portion of the circumference which gives rise to
notochord in isolates (perhaps 270°) greatly exceeds the area which does so in
normal development (perhaps 30°). This situation is not found in the early anuran
gastrula where the specification and fates of different parts of the marginal zone are
at least similar if not identical (Holtfreter, 1938&; Slack & Forman, 1980).
The most plausible explanation is perhaps that in the axolotl at stage 10 there
exists a mesodermal rudiment as an annulus of cells around most or all of the
equator which has arisen as a result of mesodermal induction and which is not
internally regionalized. The difference between dorsal and ventral lies in some
slight bias or gradient, perhaps inherited from the fertilized egg, which guarantees
that the dorsal extremum will be the first to become determined as a notochord
rudiment and that this will then inhibit the appearance of dorsal centres elsewhere.
This is the serial diversion theory of Cooke (1982, 1983). The ability of axolotl
marginal zone to dorsalize VMZ explants from Xenopus does indeed die off in a
graded fashion from dorsal to ventral rather than being sharply localized in the
prospective notochord region (Slack & Forman, 1980).
The animal pole, or ectoderm, explants are the usual test tissues for experiments on
mesodermal induction. Previous study of the biosynthetic behaviour of these explants (Slack, 19846) showed no mesoderm-type behaviour on culture in NAM. The
present study has made us more confident in the reality of induction by showing that
the ectoderm is unaffected by a variety of alterations in the medium and by LiCl,
cAMP and dextran sulphate all of which have been claimed from time to time to have
mesodermalizing effects (Masui, 1961; Ogi, 1961; Englander & Johnen, 1967;
Ltfvtrup&Perris, 1983;Barth&Barth, 1974). This means that the ectodermal specification of the tissue is quite robust and not easily altered by non-specific stimuli.
By contrast the vegetalizing factor of Tiedemann (Born et al. 1972; Tiedemann,
1976) had a profound effect and we therefore think that this factor should be taken
more seriously by the scientific community than it has been in the past.
Animal-vegetal combinations following the design of Nieuwkoop (1969) and
Nakamura, Takasaki & Ishihara (1971) also show mesodermal induction, although
not in all experimental series. We feel here that the negative results are probably
due to the fact that only a small proportion of the ectoderm becomes mesodermalized and that the notochord-specific bands cannot be seen against the considerable poly disperse background obtained after labelling with 3H-sugars.
Our general conclusion from both sets of experiments is that the results obtained
by workers in the interwar period are remarkably good considering the technical
limitations under which the work was done. The use of more discriminating
techniques allows us to make corrections on matters of detail but in general the
classical account of early amphibian development remains valid.
We thank Jim Smith and Les Dale for preparing the FLDx.
268
J. H. CLEINE AND J. M. W. SLACK
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(Accepted 5 November 1984)