/. Embryol. exp. Morph. 78, 299-317 (1983)
Printed in Great Britain © The Company of Biologists Limited 1983
299
Dorsalization and neural induction: properties of the
organizer in Xenopus laevis
By J. C. SMITH 1 AND J. M. W. SLACK 1
From the Imperial Cancer Research Fund, Mill Hill Laboratories, London
SUMMARY
We have studied the action of the organizer in Xenopus laevis using grafts labelled with
horseradish peroxidase (HRP). Orthotopic grafts of the dorsal marginal zone (the organizer)
from an HRP-labelled embryo into an unlabelled host showed that this region contributes to
the anterior archenteron wall, to the entire craniocaudal extent of the notochord and to a few
cells in the somites. Little or no contribution was made to the neural tube. Orthotopic grafts
of the ventral marginal zone (the tissue that responds to a grafted organizer) indicated that it
only contributes to the posterior half of the embryo. Within this region it spreads around the
entire ventrolateral mesoderm, occasionally contributing a few cells to the somites. The
posterior endoderm was also heavily labelled.
When the dorsal marginal zone from an HRP-labelled embryo was inserted into a slit cut
in the ventral marginal zone of an unlabelled host a mirror-symmetrical double-dorsal
duplicated embryo resulted, in which only the notochord and a few cells in the somites of the
secondary embryo were derived from the graft. The bulk of the secondary somites was,
therefore, derived from host ventral marginal zone tissue which normally makes very little
contribution to the somites. This indicates that host ventral marginal zone becomes dorsahzed
by the graft. The neural tube of the secondary embryo was also unlabelled, showing that it was
induced by the influence of the graft on the overlying ectoderm, which normally forms ventral
epidermis.
We have also grafted ventral marginal zone tissue into a slit cut into the dorsal marginal zone
of a host embryo. HRP-labelled tissue was grafted into an unlabelled embryo and vice versa.
This graft did not produce a double ventral embryo and this reinforces the traditional view that
the dorsal marginal zone is a special signalling region. Instead, the resulting embryos usually
had a twinned notochord with the graft tissue in between, differentiated as somite. This
confirms that juxtaposing ventral and dorsal marginal zone 'dorsalizes' the ventral tissue but
does not affect the dorsal tissue which differentiates, as usual, as notochord. Thus, our results
allow us to conclude that the organizer mediates two distinct interactions in bringing about the
formation of duplicated embryos. Thefirstis dorsalization of adjacent ventral mesoderm and
the second is the induction of neuroepithelium from ectoderm overlying the new archenteron
roof.
INTRODUCTION
The organizer graft, first described by Spemann & Mangold (1924), features
in many textbooks of developmental biology but there is a surprising vagueness
in modern accounts concerning the exact nature of the graft and the composition
1
Authors' address: Imperial Cancer Research Fund, Mill Hill Laboratories, Burtonhole
Lane, London NW7 IAD, U.K.
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J. C. SMITH AND J. M. W. SLACK
of the resulting duplicated embryos with respect to graft and host tissue. There
are various reasons for this, among which one might list the small number of
cases described in the original paper, the poor cellular marker which was available at that time (pigmentation of different species) and a tendency for later
workers to concentrate solely on neural induction which is the most obvious
external consequence of the experiment but not the primary event.
In some respects it is astonishing that so little work has been done in recent
years on the problem of the organizer since it is clear that what is at stake is one
of the central questions of embryology, namely: what are the processes which
control the emergence of the vertebrate body plan from an apparently uniform
sheet of cells? In this paper we describe an analysis of the organizer using grafts
labelled with horseradish peroxidase. This allows single cell resolution in assessing the provenance of structures and allows us to conclude that the organizer
phenomenon reflects two distinct inductive interactions: a primary dorsalization
of ventral mesoderm and a secondary induction of neuroepithelium by the new
archenteron roof.
MATERIALS AND METHODS
Eggs were obtained from female Xenopus laevis injected 18 h previously with
500i.u. human chorionic gonadotrophin ('Pregnyl': Organon). They were fertilized using the macerated testes of a sacrificed male. Typically, over 95 % of
these eggs would rotate and over 90 % would commence cleavage. As soon as
the eggs had rotated, ten to thirty were dejellied, either manually or chemically
(Gurdon, 1974). Manually dejellied embryos were transferred to full-strength
'normal amphibian medium' (NAM: Slack & Forman, 1980) in dishes coated
with 1 % agar (Noble agar, Difco Laboratories). Chemically dejellied embryos
were transferred to the same medium but with the addition of Ficoll type 400
(Pharmacia) to 5 %. Manually dejellied embryos retained a little jelly and this
prevented leakage of cytoplasm after injection of HRP. Chemically treated
embryos had no jelly left but the Ficoll removes fluid from the perivitelline space
and this prevents leakage by reducing the pressure exerted on the egg (Newport
& Kirschner, 1982).
The embryos were injected with 20 nl of 0-25 %, 0-3 % or 0-5 % horseradish
peroxidase (HRP: Boehringer) before the first cleavage. The HRP solution was
prepared by dissolving a weighed amount (around 0-5 mg) in 50 mM-NaCl, 5 mMpotassium phosphate pH7-5, giving a final volume of 0-1-0-2 ml. This was
dialysed against the same solvent for 1-2 h at room temperature using a BRL
Microdialysis System. It was then centrifuged to remove particulate matter and
kept at 4 °C until required. The solution was always used the same day. Injections
of HRP were made near the vegetal pole of the fertilized eggs using glass
micropipettes with outside tip diameters of about 15 jum. Injected eggs and uninjected synchronous sibling controls were then incubated overnight at 15 °C.
Organizer in Xenopus laevis
301
DMZ
VMZ
DMZ
VMZ
I
lmm
Fig. 1. The regions used for grafting. (A) Early Xenopus gastrula (stage 10) viewed
from the vegetal pole. (B) A section in the plane indicated in (A), (after Keller,
1981). DMZ, dorsal marginal zone; VMZ, ventral marginal zone; AP, animal pole;
VP, vegetal pole.
The next day, operations were carried out on embryos in which the dorsal
blastopore lip had just become externally visible (stage 10, Nieuwkoop & Faber,
1967; see Fig. 1). They were performed in full-strength NAM using hand-ground
forceps, hair loops and electrolytically sharpened tungsten needles. There were
four types of operation, shown in Fig. 2. First, the dorsal marginal zone (consisting of a wedge 30-35° wide) from an HRP-labelled embryo was grafted to
replace that of an unlabelled embryo. Secondly, the ventral marginal zone from
an HRP-labelled embryo was grafted orthotopically to an unlabelled embryo.
Thirdly, the dorsal marginal zone from an HRP-labelled embryo was inserted,
in the correct animal-vegetal polarity, into a slit cut in the ventral marginal zone
of an unlabelled host. Finally the ventral marginal zone from an HRP-labelled
embryo was inserted, also with the correct animal-vegetal polarity, into a slit cut
in the dorsal marginal zone of an unlabelled host. After the operations the
embryos were allowed to heal and the medium was gradually changed to 1/10
strength NAM to prevent exogastrulation.
The embryos were allowed to develop for 24-48 h at 15°, 18° or 22 °C until
they reached stage 25-32 (Nieuwkoop & Faber, 1967). At stages later than this
some cells with endogenous peroxidase activity begin to appear in the embryo.
They were fixed for 2-5-3 h in 1 % paraformaldehyde, 2-5 % glutaraldehyde in
0-1 M-sodium phosphate pH7-5. Then they were washed in 5 % sucrose in the
same buffer and stored in this solution at 4°C. To prepare frozen sections the
embryos were immersed in 15 % sucrose in phosphate buffer for 2h at 4°C and
then in 15 % gelatin, 15 % sucrose in phosphate buffer for 10 min at 37 °C. They
were frozen in a fresh sample of this solution and transverse serial sections were
J. C. SMITH AND J. M. W. SLACK
Fig. 2. The four grafts. (A) The DMZ from an HRP-labelled embryo replaces that
of an unlabelled host. (B) The VMZ from an HRP-labelled embryo replaces that of
an unlabelled host. (C) The DMZ from an HRP-labelled embryo is inserted into a
slit cut in the VMZ of an unlabelled host. (D) The VMZ from an HRP-labelled
embryo is inserted into a slit cut in the DMZ of an unlabelled host.
cut at 25 [im in a cryostat. The sections were mounted on subbed slides, allowed
to dry overnight over anhydrous calcium chloride and reacted for peroxidase by
the method of Mesulam (1976). They were then stained with methyl green and
orange G and mounted in DPX.
RESULTS
A preliminary series of experiments established that injecting 20 nl of between
0 • 25 % and 0 • 5 % HRP into the vegetal pole of fertilized eggs gave the best results.
Organizer in Xenopus laevis
303
Over 75 % of the embryos injected in this way developed to stage 32 and beyond
and when these embryos were fixed and sectioned all the cells were labelled. The
labelling was intense and uniform throughout ectodermal and mesodermal structures and usually somewhat lighter in the endoderm, probably because of the
large volume occupied by yolk granules in these cells. The label was always
detectable in all regions by bright-field illumination and particularly pronounced
when viewed through crossed polaroids (Illing & Wassle, 1979; Fig. 3). The
Pi
3A
—
B
Fig. 3. Sections through an HRP-labelled (A, B) and a control (C, D) embryo. A
and C are viewed in bright-field and B and D through crossed polaroids. Notice that
pigmented cells in C are not birefringent. Scale bar is 100 /mi.
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J. C. SMITH AND J. M. W. SLACK
specimens were routinely examined by both illumination systems because the use
of crossed polaroids was a convenient way to distinguish between heavily
labelled graft cells and pigment cells, which are not birefringent. Smaller injection volumes than 20 nl often resulted in a non-uniform distribution of label and
concentrations of HRP greater than 1 % were invariably fatal.
Hirose & Jacobson (1979) have demonstrated that HRP is suitable for labelling individual blastomeres in Xenopus embryos because the enzyme is confined
to the injected cell, is distributed throughout that cell, and is transmitted to its
progeny. We have confirmed these results, but felt that it was also necessary to
show that cells cannot become labelled by uptake of HRP from a labelled region
which dies and disintegrates, since this might be a problem in either injection or
grafting experiments.
To discover whether this occurs one wants to know if, at appropriate concentrations, a cell can ingest HRP from the surrounding medium. Accordingly,
Xenopus gastrulae were cut in half and incubated for 24 h at 22 °C in 1/10
strength NAM containing 0, 0-0005, 0-001, 0-0025, 0-005, or 0-0125% HRP.
They were then fixed, sectioned and reacted for HRP.
At all concentrations of HRP small patches of reaction product were seen
extracellularly in the yolky endoderm and these probably represented medium
which became trapped in the embryo when it healed and was retained by fixation. Intracellular staining only occurred at the two highest concentrations of
HRP and this was very faint indeed, being only just visible with crossed polaroids
and invisible with bright-field optics. Since in the worst possible case the concentration of HRP surrounding an unlabelled cell as a result of death of neighbouring labelled cells would be equivalent to the concentration of HRP in the
injected pre-cleavage embryo, which is about 0-003 %, we can be confident in the
results that follow that a cell which appears labelled is derived from the graft and
one which appears unlabelled is from the host.
Normal fate of the dorsal marginal zone
The normal fate of the dorsal marginal zone was established by orthotopic
grafts between an HRP-labelled donor embryo and an unlabelled host (see Fig.
2). Thirteen such grafts were analysed. A camera-lucida reconstruction of a
typical specimen and a section through the midtrunk of this embryo are shown
in Fig. 4.
Much of the label ended up in the notochord. Usually the entire craniocaudal
extent was labelled but in four cases the caudal third was unlabelled. There was
Fig. 4. Analysis of an unlabelled embryo which had its dorsal marginal zone
replaced by the DMZ from an HRP-labelled embryo. (A), Drawings of transverse
sections through the embryo at intervals of 350 [tm. Stipple shows HRP-labelled
cells. (B), Transverse section through the trunk, bright field. (C), The same section
viewed through crossed polaroids. The notochord is labelled, and so are a few cells
in the somites. Scale bar in (A) is 0-5 mm, in (B) and (C), 200jum.
Organizer in Xenopus laevis
B
305
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J. C. SMITH AND J. M. W. SLACK
also a substantial contribution to the anterior archenteron wall. In the trunk
region there was a limited contribution to the somites but no labelling of
pronephros, lateral plate or blood islands. There were a few labelled cells in the
archenteron wall and one or two in the neural tube.
These results are in good agreement with those of Keller (1975, 1976) using
vital staining. Because of the diffusibility of vital dyes between cells it has been
hard to assess the degree of cell mixing in previous work. The present results
show clearly that the dorsal marginal zone remains as a coherent mass of cells in
the dorsal midline throughout gastrulation and neurulation.
Normal fate of the ventral marginal zone
The normal fate of the ventral marginal zone was also established by orthotopic grafting (Fig. 2). Eleven grafts were analysed and a camera-lucida reconstruction and a section through the midtrunk of a typical case are shown in Fig.
5. In all cases labelled tissue was only visible in the posterior half of the embryo,
in which region it made up the bulk of the ventral and lateral plate mesoderm and
also contributed substantially to the endoderm. In three cases the labelled cells
in the mesoderm were confined to the lateral plate alone but in the rest a few graft
cells were also seen in the somites. However, this contribution was very limited;
less than 1 % of the somitic tissue in the posterior half of the embryo being
labelled. Occasionally some cells wrapping around the posterior portion of the
notochord were labelled. No contribution by the ventral marginal zone was made
to the notochord itself or to the neural tube but in five cases a few cells in the
posterior epidermis were labelled. The results show that the behaviour of the
ventral marginal zone is strikingly different from that of the dorsal marginal
zone. Instead of contributing to the whole craniocaudal extent of the embryo it
contributes only to the posterior half, and instead of remaining as a tight
coherent cell mass in the midline, it spreads out over the whole ventrolateral
region of the cross section. These results agree substantially with those of Keller
(1975,1976), but also show that there is mingling of labelled and unlabelled cells.
Evidently the ventral tissue does not remain coherent as its cells move dorsally.
The organizer graft
Grafts were made of the dorsal marginal zone from a labelled embryo to a slit
cut in the ventral marginal zone of an unlabelled host (Fig. 2). The graft was
made in this way, rather than by replacing a piece of ventral host tissue, in order
to avoid removing the very region that was fate-mapped above. Usually this graft
Fig. 5. An unlabelled embryo which had its ventral marginal zone replaced by the
VMZ from an HRP-labelled embryo. (A), Drawings of transverse sections as in Fig.
4. (B), A transverse section through the trunk, bright field. (C), The same section
viewed through crossed polaroids. The lateral plate is labelled and so is a single cell
near the somites. Scale bar in (A) is 0-5 mm, in (B) and (C), 200 jum.
Organizer in Xenopus laevis
B
307
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J. C. SMITH AND J. M. W. SLACK
resulted in the formation of a mirror symmetrical, double dorsal duplicated
embryo (Fig. 6B). Eleven cases were analysed in detail. The secondary axis of
one of the embryos was poorly developed, and this is not considered further. In
the remaining ten, the notochord of the secondary embryo was completely
labelled and a few cells in the adjacent somites were also derived from the graft
(Fig. 7). The proportion of labelled cells in the somites of these secondary
embryos was probably greater than that in the somites of embryos that had
received orthotopic grafts of HRP-labelled dorsal marginal zone. However, this
difference was only slight and we do not think it constitutes evidence for
'ventralization' of the dorsal marginal zone by its new, ventral, environment.
The important point is that the somites of the secondary embryos were substantially derived from host ventral marginal zone tissue, which in normal development donates very few cells to the somites. So it seems that the host ventral
marginal zone becomes dorsalized by the graft. The neural tube of the secondary
embryo was also unlabelled, showing that it must have been induced by the
influence of the graft on the overlying ectoderm, which normally forms ventral
epidermis. In only one case had any labelled cells migrated into the host embryo's axial structures and they were very few in number (see Fig. 7).
JW •&*!„<
6A
X
B
Fig. 6. (A) A normal Xenopus embryo, stage 30. (B) A duplicated embryo following
an organizer graft. (C) An abnormal embryo resulting from a graft of ventral marginal zone tissue to the dorsal marginal zone. All scale bars are 1 mm.
Organizer in Xenopus laevis
309
Ventral marginal zone grafted to dorsal marginal zone
For the first time we have performed the reciprocal experiment to the organizer graft. That is, ventral marginal zone tissue was inserted into a slit cut in
f 4 i
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:
\
\
7A
B
D
Fig. 7. A section through a duplicated embryo formed by inserting an HRP-labelled
dorsal marginal zone into the VMZ of an unlabelled host. (A), Bright-field illumination. (B), Viewed through crossed polaroids. (C), Higher power of the notochord,
somites and neural tube of the secondary axis. (D), The same through crossed
polaroids. Notice that only the notochord and a few cells in the somites of the
secondary axis are labelled. There is one labelled cell in the host's axial structures
(arrow). (A), (B): Scale bar is 2 0 0 ^ . (C), (D): Scale bar is 50/an.
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J. C. SMITH AND J. M. W. SLACK
B
"——
Fig. 8. A section through an embryo with a twinned notochord formed by grafting
an HRP-labelled ventral marginal zone to the DMZ of an unlabelled host. (A),
Bright-field illumination. The two notochords are arrowed. (B), Viewed through
crossed polaroids. Scale bar is 200 ^m.
the dorsal lip of the blastopore (Fig. 2). The gross appearance of the experimental cases suggests that there are two possible outcomes since they either look
almost normal or, as shown in Fig. 6C, appear extremely abnormal, with the tail
curved over the back and spina bifida. However the histological analysis shows
that these types are actually quite closely related. Fourteen grafts of HRPlabelled ventral marginal zone were analysed in detail, five of which were of the
type illustrated in Fig. 5C and nine which were apparently normal. Of the five,
two were difficult to analyse but in the remaining three the notochord was
twinned anteriorly with the graft tissue in between. The two notochords diverged
posteriorly in the region of the externally visible abnormality. Of the nine
normal-looking embryos, eight also had twin notochords anteriorly with the graft
tissue lying in between, the difference being that here the twin notochords converged and fused towards the posterior end of the embryo. In two cases there
were some labelled cells in one of the notochords but in all the others both
notochords were entirely composed of host tissue. There were also labelled cells
in the endoderm and occasionally some in the neural tube (Fig. 8). Thus, this
graft does not produce a double ventral embryo and this reinforces the traditional view that the dorsal marginal zone has a specially privileged status as a signalling centre.
The shape of the cells between the two notochords suggested to us that they
Organizer in Xenopus laevis
9A
311
B
Fig. 9. A section through an embryo with a twinned notochord formed by grafting
an unlabelled ventral marginal zone to the DMZ of an HRP-labelled host. (A),
Bright-field illumination. (B), Viewed through crossed polaroids. (C), Higher power
of the twinned notochords. (D), The same through crossed polaroids. Notice large
amounts of graft-derived striated muscle between the two notochords. (A), (B):
Scale bar is 200pm. (C), (D): Scale bar is 50|um.
were somite cells but it was difficult to confirm this because cytological detail is
obscured by the HRP reaction product. So, to confirm their identity, grafts of
unlabelled ventral marginal zone tissue were made into the dorsal lips of host
embryos labelled with HRP. After 2 days, the embryos were fixed, sectioned and
processed in the usual way and lightly stained with picro blue-black before
mounting in Euparal. In nine out of eleven cases the notochord was twinned (Fig.
9). Viewed through crossed polaroids seven of these embryos contained substantial amounts of unlabelled striated muscle in the graft region (Fig. 9D). In one
case there were only wisps of birefringent material and in the other the cells were
all undifferentiated. Since the predominant cell type produced by the somites in
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J. C. SMITH AND J. M. W. SLACK
amphibia is muscle it is reasonable to conclude that the grafts have been entirely
converted into somite tissue. It is therefore clear that juxtaposing ventral and
dorsal marginal zone 'dorsalizes' the ventral tissue but does not affect the dorsal
tissue which differentiates, as usual, as notochord.
DISCUSSION
We have grafted tissue from embryos labelled with horseradish peroxidase
into unlabelled hosts in order to establish the normal fates of the dorsal and
ventral marginal zones of the early gastrula and to study the inductive interactions that occur during early development.
The fates of the dorsal and ventral marginal zones
Orthotopic grafts of the dorsal marginal zone of an HRP-labelled embryo into
an unlabelled host show that this region contributes to the entire craniocaudal
extent of the notochord. In the trunk a few cells in the somites also derive from
the dorsal marginal zone and in addition it contributes heavily to the anterior
archenteron wall. Orthotopic grafts of the ventral marginal zone indicate that it
only contributes to the posterior half of the embryo. Within this region it spreads
out around the entire ventrolateral mesoderm, occasionally contributing a few
cells to the somites. It also contributes substantially to the posterior endoderm
and to the odd cell in the epidermis. The dorsal and ventral marginal zones also
differ somewhat in their behaviour with respect to cell mixing. The dorsal region
remains as a coherent group of cells in the dorsal midline while the ventral region
becomes interspersed with unlabelled cells in the course of its expansion. These
observations are in good agreement with Keller's (1975,1976) obtained by vital
staining, and are also in accord with fate maps obtained for other anurans and
for urodeles (Vogt, 1929; Nakamura, 1942; Pasteels, 1942; Okada & Hama,
1945).
Induction
Notwithstanding the concentration of the earlier workers on neural induction
(misleadingly referred to as 'primary induction') it is now clear that the
vertebrate body plan comes into existence as a sequence of inductive interactions
(Gerhart, 1980; Slack, 1983). The first interaction is the induction of the
mesoderm from the animal hemisphere under the influence of the vegetal region
(reviewed by Nieuwkoop, 1973). From the earliest stage that the mesodermal
rudiment can be demonstrated there is a difference in the state of specification
between the dorsal mesoderm, and the ventral mesoderm, the former being the
future organizer (Nakamura, 1978).
The second interaction occurs within the mesoderm and involves its
regionalization along the dorsoventral axis under the influence of the organizer.
Organizer in Xenopus laevis
313
This has been demonstrated in vitro by Slack & Forman (1980) using interspecific
combinations and is clearly shown in vivo by the results of the present work.
When dorsal mesoderm is grafted to a ventral position the host tissue, which
normally forms lateral plate and blood islands, contributes heavily to the somites
of the secondary axis. When ventral tissue is grafted to a dorsal position it is again
converted to somite, occasionally with a small contribution to the twinned
notochord. Thus both experiments show that when dorsal and ventral marginal
zone tissues are juxtaposed the dorsal tissue continues on its normal course of
development while the ventral tissue forms structures of more dorsal character
than it would otherwise have done.
It might be argued that because of the extensive cell movement in the ventral
marginal zone, the unlabelled cells in the somites of the secondary axis following
the organizer graft have moved all the way from the host axis and not been
dorsalized at all. It is because of this possibility that we felt it necessary to
perform the reciprocal graft of ventral to dorsal marginal zone. In this case the
convergence movements of the host keep the graft as a compact mass in the
dorsal region and most or all of the graft cells became somite. There is therefore
no possibility of this result arising from long-range movements of precommitted
cells.
The conclusion that dorsal tissue remains dorsal while ventral tissue becomes
more dorsal is exactly that reached by Slack & Forman but is shown more clearly
here because it avoids the complication of using two species with different rates
of development.
It is therefore highly probable that in normal development a similar interaction
occurs and results in the formation of an ordered set of mesodermal structures
by the action of the prospective notochord (the organizer) on the remainder of
the marginal zone to form somite, kidney, lateral plate and blood islands in
dorsoventral sequence. The spectacular formation of mirror-symmetrical
double-dorsal duplications following the organizer graft suggests at first sight
that the signal from the dorsal region is a 'gradient' of the kind described in the
developing limb or insect egg (Tickle, Summerbell & Wolpert, 1976; Sander,
1976; Slack, 1977). However studies by Cooke (1981) on the proportions of
mesodermal parts following organizer grafts suggest that a simple diffusion
gradient could not explain the results.
The third well-established inductive interaction in early development is neural
induction, the formation of a neural tube by ectoderm brought into contact or
close proximity with the archenteron roof. Neural induction is clearly not a
simple process since each region of the archenteron roof induces a specific region
of the nervous system (Mangold, 1933; Horst, 1948) and it may be that a
multiplicity of chemical signals is involved. The reality of neural induction has
recently been questioned (Jacobson, 1982; see Smith, 1983) on the grounds that
HRP-labelled organizer grafts produce HRP-labelled neural tubes, so the apparent induction is actually self differentiation of the graft.
314
J. C. SMITH AND J. M. W. SLACK
This is clearly not the case in the present results. In the secondary axes
produced by organizer grafts the neural tubes are overwhelmingly of host origin
and therefore must arise by induction. It is most unlikely that they could have
arisen by migration of cells that would have participated in the host nervous
system because this would require movement of committed cells on a scale which
has never been observed. The failure to observe neural induction in Jacobson's
experiments may arise from two factors. First, the graft may have contained a
good deal of prospective neural plate together with the organizer tissue. This is
quite likely to have occurred with grafts of Xenopus organizers where the prospective neural plate extends further towards the vegetal pole on the dorsal side
of the embryo than it does in the urodeles on which the original organizer grafts
were done (Keller, 1975; Vogt, 1929; Pasteels, 1942). Secondly, if the grafts were
inserted into the blastocoel cavity instead of the ventral marginal zone then they
may not have been able to contact ventral ectoderm in time to exert their influence.
In the past, there has been some confusion between the two interactions
mediated by the organizer, dorsalization and neural induction, probably because
it is the same tissue which causes both effects. This confusion may be cleared up
by realizing that the two interactions occur at different times; dorsalization when
the organizer is a small group of cells in the late blastula and neural induction
H-t-4
Fig. 10. Hypothetical determined states present in the early embryo. A: animal; V:
vegetal; M: mesoderm; O: organizer; Ml, M2, M3 dorsoventral subdivisions of
mesoderm; 0 1 , 02, 0 3 , 04: craniocaudal subdivisions of archenteron roof; E:
epidermis; Nl, N2, N3, N4: craniocaudal subdivisions of neural plate. Arrows within
embryos indicate inductive interactions.
Organizer in Xenopus laevis
315
when it has elongated and occupies the entire craniocaudal extent of the archenteron roof.
Our conception of embryonic development involves each region of tissue
passing through a sequence of determined states as a result both of its intrinsic
dynamics and its interactions with other parts of the embryo. A memory of the
sequence of decisions makes up the 'coding' of the region, otherwise known as
the 'epigenetic address' (Stein, 1980) or 'positional value' (Wolpert, 1969). The
entire assemblage of codings is the 'second anatomy' of the organism (Slack,
1982) which, in a sense, is the cause of the externally visible anatomy which is
established as the tissues become histologically differentiated.
A possible sequence of interactions and second anatomies is shown in Fig.
10 which is in accord with our own and previous results. Note that dorsalization
and neural induction are shown as spatially and temporally distinct interactions.
In the intervening period, during gastrulation, the organizer changes from a
clump of cells with the uniform coding 'O' to a sheet with an array of codings
Ol—»O4 (the archenteron roof). The mechanism of this craniocaudal determination is unknown although it does seem closely associated with the process
of invagination (Slack, 1983). The events which we believe follow an organizer
graft are shown in Fig. 11. First, the mesoderm becomes specified to form a
mirror-symmetrical double-dorsal array of territories. Then the gastrulation
movements of each part occur at the normal time appropriate to its coding and
the craniocaudal sequence of territories is established in the usual way. Finally
each part of both archenteron roofs induces the corresponding part of the neural
Fig. 11. The events following an organizer graft. Codings as in Fig. 10.
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J. C. SMITH AND J. M. W. SLACK
tube from the ectoderm giving rise to a completely symmetrical double dorsal
vertebrate body plan.
We thank Dr John Gerhart and Dr John Heath for advice about use of HRP, Malcolm
Hawkins for help with sectioning, and Bob Bloomfield for looking after the Xenopus.
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(Accepted 25 July 1983)
Note added in proof: it has recently come to our attention that grafts of ventral
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BAUTZMANN,
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