/. Embryol. exp. Morph. Vol. 26, 3, pp. 543-570, 1971
Printed in Great Britain
543
Histological features of neural induction
in Xenopus laevis
by D. TARIN1
From the Department of Anatomy, University of Leeds
SUMMARY
It was first established by grafting experiments that neural induction occurs in Xenopus
laevis and that it is the mesoderm in the dorsal lip of the blastopore which normally exercises
this function. The subsequent histological work provided the following information:
At stage 10i mesodermal invagination was already well under way, in advance of the
formation of the archenteric cavity. This confirms the earlier observations of Nieuwkoop &
Florschutz(1950).
The first evidence of neural induction, thickening of the mid-dorsal ectoderm combined
with the development of an inner tier of columnar cells, occurred at stage 11£.
By stage .12 there was generalized thickening of the dorsal ectoderm and between stage 12£
and .13 the brain and spinal cord regions of the neural plate became distinguishable.
The dorsal mesoderm segregated into notochord rudiment and two lateral masses at stage
13 and the latter further subdivided into paraxial mesoderm and lateral plates by stage 14.
The margins of the neural plate were clearly distinguished from presumptive epidermis by
stage 15 and the median neural groove was also well marked.
In the next two stages the folding of the neural plate in the line of this groove proceeded
rapidly. The dorsoventral enlargement of the somites and the relative shrinkage of the notochord were considered to contribute to the mechanism of neurulation.
Regionalization of the brain into prosencephalon, mesencephalon and rhombencephalon
was in progress at stages 18 and 19.
These results indicate that induction consists of an initial activation of dorsal ectoderm
(generalized thickening) followed by gradual transformation of the neural plate to form the
different parts of the central nervous system (regionalization).
Intercellular metachromatic material was noted in various parts of the embryo. This was
most plentiful between stage 10£ and stage .13 and thereafter gradually decreased. It was the
only feature which persisted long enough to represent a possible inductive agent.
At all stages the archenteron was lined with a continuous layer of endoderm. This indicates
that the mode of formation of the gastro-intestinal tube in Xenopus is different to that in
urodeles. It further implies that the mesoderm is not present on the blastular surface prior to
gastrulation but lies in deeper layers.
INTRODUCTION
This investigation was performed as an essential accompaniment to a
programme incorporating electron microscopical, histochemical and transplantation techniques in the study of primary embryonic induction in Xenopus
laevis.
It was first necessary to establish that induction occurs in Xenopus and that it
1
Author's address: Department of Anatomy, University of Leeds, Leeds 2, U.K.
544
D. TARIN
is effected by the mesodermal cells migrating through the dorsal lip of the
blastopore. The process was next observed in progress by time-lapse cinematography (Tarin, Scott & Sharp, 1970; Sharp & Tarin, 1970) and these histological
observations were then performed to provide a basis for the interpretation of
results obtained by other techniques, which will be presented in later publications.
The work has been performed on Xenopus he vis because the eggs can be
obtained at will in any season and this allows greater flexibility in the planning
of experiments.
MATERIALS AND METHODS
Fertilized eggs were obtained by the injection of' Pregnyl' (Organon Laboratories) according to the scheme described by Brown (1970). These were washed
twice in tap water and once in distilled water and finally placed in a dish containing 10% Niu-Twitty medium. The eggs were then reared and staged
according to the instructions of Nieuwkoop & Faber (1967). For histological
studies, six specimens at each of stages 10i, 11, H i , 12, 13, 14, 15, 16, 17, 18, 19
(Nieuwkoop & Faber, 1967) inclusive, were selected with a binocular dissecting
microscope and fixed in 4% glutaraldehyde in cacodylate buffer, pH 7-35. The
eggs were embedded in agar to permit subsequent orientation in the embedding
medium by the technique described by Scott, Tarin & Sharp (1970). Next they
were washed overnight in cacodylate-sucrose mixture, post-fixed in osmium
tetroxide, dehydrated in a graded series of alcohols and embedded in Araldite.
For each stage, three specimens were oriented to permit sectioning in a coronal
plane and three in a sagittal plane. Thick sections (1-2 /im) were cut with an
LKB Ultratome III and stained with 1 % toluidene blue. This study of neural
induction was performed mainly on mid-sagittal and mid-coronal sections.
Grafting techniques (Fig. 1)
The operations were performed in Petri dishes half filled with blackened agar
produced by adding animal charcoal to molten agar and then allowing it to set.
The agar was covered with full strength Niu-Twitty medium which was used
because the concentrated solution promotes more rapid healing than the 10%
dilution used for standard rearing of embryos. Five embryos of stages 10^-11
were used as donors for transplantation of the dorsal lip of the blastopore. Each
was placed beside a recipient of similar age in an agar lined Petri dish and the
jelly coats removed with forceps. The eggs were then placed in shallow grooves
in the agar and the central portion of the dorsal lip of the donor excised with fine
glass needles. This was then placed in a depression of similar size created on
each recipient by excising a piece of tissue in the region of the prospective
lateral or ventral lip. The graft was held in position while healing with microneedles, controlled by a Leitz micromanipulator, for approximately half an
hour. This procedure was repeated for each pair of eggs and the operated
Histology of neural induction in Xenopus
545
embryos were then left in the full strength medium at room temperature until
the next day. A series of five control embryos, grafted with a second ventral
blastoporal lip, was also performed. It is important to point out that the culture
medium contained penicillin (200 i.u./ml) and streptomycin (200 i.u./ml) and that
the light source was covered with a heat filter. Measurement of the medium's
temperature with a continuous recording thermocouple showed that unless a
filter was present it rose to above 35 °C with fatal results for the embryos.
Fig. 1. Diagram illustrating the procedure for grafting («) the dorsal lip and (6) the
ventral lip of the blastopore. (Dorsal lip graft, H; excised area, • ; ventral lip
graft, S).
RESULTS
Grafting experiments. In all five cases the embryos which received grafts of a
second dorsal lip developed secondary nervous systems (Figs. 2-4). Histological
sections across one of these animals showed that the secondary axis consisted
not only of a well-formed neural tube, but also of a notochord and other associated structures (Fig. 3). The control embryos which received grafts of the
ventral lip showed no tendency to develop a second nervous system.
Histological observations on neural induction in normal embryos
Phase I. Stages 10*, 11, 1H and 12
During this phase the formation of the blastopore is completed and mesodermal cells stream through the rim of this structure into the interior of the
546
D. TARIN
Histology of neural induction in Xenopus
547
embryo. HistologicaUy it was evident that mesodermal imagination occurred
earlier and faster in the dorsal than in the ventral region. Prior to stage 12
coronal sections were difficult to interpret and yielded no really valuable
information. The description of stages 10-£, 11, and 11^- is therefore based on
sagittal sections alone.
Stage 10\
External appearance. Small shallow blastoporal groove and dorsal lip of
blastopore evident.
Histologicalfeatures. Although indentation of the surface to form the archenteron was only just beginning, dorsal mesodermal invagination was well under
way (Figs. 5, 6). These cells were rounded and loosely grouped but formed an
easily recognized second sheet of tissue (Fig. 6) distinct from the surface layer.
(For ease of description the latter will be referred to as the ectoderm although,
at this stage, it cannot strictly be classified as such because it contained cells
which later invaginated to form the endoderm.) The many large intercelluar
spaces in the mesoderm were mostly filled with metachromatic material. In
contrast the dorsal ectoderm was composed of tightly packed polygonal cells,
separated by small amounts of metachromatic material and it had a clear
boundary with the underlying tissues. It was also noted that the ectoderm was
A
Bx
B»
C
D
E
G
H
J
K
Lx
L«
M
KEY TO LABELLING OF FIGURES
N Notochord
0 Ventral aspect of embryo
P Primary (host) nervous system
Q Blastopore
R Metachromatic material
S Secondary (induced) nervous system
T Lateral plate mesoderm
U Prosencephalon
V Mesenchephalon
w Rhombencephalon
X Prechordal plate
Y Yolk plug
z Stomadeal membrane
Archenteron
Brain (host)
Brain (induced)
Blastocoele cavity
Dorsal aspect of embryo
Ectoderm
Gut cavity
Endoderm
Spinal cord
Paraxial mesoderm
Dorsal lip of the blastopore
Ventral lip of the blastopore
Mesoderm
F I G U R E S 2-4
Fig. 2. Xenopus laevis embryo with two nervous systems (x 180) - primary (P) and
secondary (S). The secondary neural axis resulted from grafting the embryo with an
extra dorsal lip of the blastopore.
Fig. 3. Histological section (x 400) across a similar embryo to that in Fig. 2 to show
the presence of two brains (BL and B»). Two notochords (M) are also visible. Note that
induced nervous systems (B») are commonly not so perfectly formed as normal ones
(Fig. 4).
Fig. 4. Section across a normal embryo for comparison with Fig. 3 (x 400).
35
E M B 26
548
D. TARIN
Histology of neural induction in Xenopus
549
thicker in the dorsal and ventral regions of the egg (Fig. 5) than over the blastocoele cavity where its cells were somewhat more loosely arranged.
The presence of 'flask-shaped' cells described earlier by Holtfreter (1943) and
by Perry & Waddington (1966) in the vicinity of the invaginating archenteron
was confirmed. Strong metachromasia was also observed in the intercellular
spaces around these.
Stage 11
External inspection showed lateral extension of the blastoporal groove so
that the lateral lips of the blastopore were present.
Histologicalfeatures. These were, in general, quite similar to those of stage-10-iembryos. Some differences were noted, however. Thus, for instance, the archenteron cavity was slightly deeper and the surface indentation in the ventral region
was just beginning. The ventral mesoderm was also beginning to invaginate and
there was more metachromatic material in the intercellular spaces in both dorsal
and ventral regions. In the dorsal lip, however, there was no real change in the
extent of mesodermal movement or the arrangement of cells in either the mesoderm or the ectoderm.
Stage 11}
Externally the blastoporal groove formed a circle around the yolk plug. The
dorsal, ventral and lateral lips of the blastopore were distinguishable.
Histological sections showed that the archenteric and mesodermal invagination was further advanced with the latter still being somewhat ahead (Fig. 7).
The mesodermal cells were rounded and irregularly arranged but were more
closely grouped in the dorsal than the ventral lip. The ectoderm was thickest in
the mid-dorsal area where an inner tier of low columnar cells could be distinFIGURES 5-8
Fig. 5. Sagittal section of stage-10£ embryo (x 110). The dorsal (Z>) and ventral (O)
'ectoderm' is thicker than that over the blastocoele (C). Mesodermal invagination
(A/) is well under way on the dorsal side.
Fig. 6. Detail of the dorsal lip of stage-10£ embryo (x 400), sagittal section. This is the
same embryo as shown in Fig. 5. The mesodermal invagination (M) is far in advance
of the archenteron (A) and has formed a separate sheet of tissue under the ectoderm
(£). The arrows mark the approximate boundary between this sheet and the endoderm. Note that the mesodermal cells are rounded and loosely packed.
Fig. 7. Dorsal lip of stage-11 \ embryo (x 360). The extent of the mesodermal invagination and its boundary with the endoderm are marked with arrows. In the middorsal region (asterisk) the ectodermal cells are tightly packed and the mesodermal
and ectodermal layers are in close apposition.
Fig. 8. Mid-dorsal region (x 660) stage 1 \\. The blastopore lies to the left but is not
visible in this picture. Note the presence of intercellular metachromatic material
(arrows) in the mesoderm and to a lesser extent in the ectoderm and at the ectomesodermal boundary. Towards the right (rostrally) there is a decrease in this
material and the dorsal ectoderm is thinner.
35-2
550
D. TARIN
100/mi
Histology of neural induction in Xenopus
551
guished. These cells were extremely tightly packed together and in the same area
the mesodermal cells were very closely applied to the deep surface of the ectodermal layer (Fig. 7). These new features were considered to be the first evidence
of nervous system induction.
Metachromatic material was abundant in the mesodermal layer between the
rim of the dorsal lip of the blastopore and the mid-dorsal region. More rostrally
it abruptly terminated as the ectoderm decreased in thickness (Fig. 8).
Stage 12
Externally the circular blastopore was contracting in size.
Sagittal sections showed that the invagination of the archenteric cavity and
of the mesoderm was quite advanced (Figs. 9,10). It seemed that the mesodermal
cells had probably almost reached the blastocoele, but in this area it was
difficult to distinguish them from endodermal ones, because the size and
appearance of the cells present were intermediate between the two types.
Comparison of the dorsal and ventral lips of the blastopore revealed that
they differed in two main respects. First, the arrangement of the mesodermal
cells in the dorsal lip was much tighter and more regular (Fig. 11). In fact, some
in the caudal end of the advancing mesodermal sheet were columnar in shape.
Secondly, metachromatic material was present both in the large inter-cellular
spaces of the dorsal mesoderm and also in the narrow clefts between cells in
the outer (ectodermal) layer. In the ventral lip, however, it was present only
between the mesodermal cells.
The ectomesodermal junction was quite distinct in both dorsal and ventral
regions and the degree of separation between the two layers varied both from
specimen to specimen and in different areas of the same specimen. Dorsally,
however (Fig. 10), the layers were usually closely apposed (never more than
10 /mi apart and frequently less) but when the space was large it nearly always
contained clumps of metachromatic material. Towards the lip of the blastopore
the ectomesodermal boundary became indistinct about 8-10 cells from the actual
rim. Further towards the rim, the cells became irregularly disposed although still
closely grouped, and it was assumed that organisation into layers took place in
this region as the mesodermal cells passed through.
It was found that the cells of the 'ectodermal layer' are fairly uniform in
F I G U R E S 9, 10
Fig. 9. Sagittal section, stage-1 2 embryo ( x 100). Survey picture showing the yolk plug
(Y) protruding between the dorsal and ventral lips of the blastopore (LY and L2).
Fig. 10. Dorsal part of same (stage- .12) embryo as Fig. 9 ( x 300). Invagination of the
archenteron cavity (A) has progressed so that it now extends under most of the
dorsal surface. Note the thickening of the ectoderm and the presence of columnar
cells in this region. The rostral limit of mesodermal invagination is vague but it
probably corresponds with the position marked by the asterisk. The ectomesodermal
junction is clearly defined and the gap between the two layers varies in size.
552
D. TARIN
appearance and it was therefore not possible to distinguish the prospective
mesodermal elements on morphological criteria alone. Once they had passed
into the mesodermal layer however they were identifiable by such features as
grouping, position in the embryo, relative size, etc.
Fig. 11. Detail of the blastopore of a stage-12 embryo (x 250), in sagittal section.
The archenteron cavity is less obvious because the section is not quite in the median
plane. Note the different appearances of the mesodermal invagination (M) in dorsal
and ventral regions.
Fig. 12. Coronal section: stage-12 embryo, dorsal aspect (x 250). The cells in the
dorsal ectoderm are tightly packed and the underlying mesoderm forms a continuous
layer across the midline.
Histology of neural induction in Xenopus
553
The dorsal ectodermal layer was thickest approximately half way between the
edge of the lip and the blastocoele cavity (Fig. 10). Here there were three tiers
of cells, the inner one of which was clearly columnar. Proceeding towards the
blastopore, although the ectoderm became thinner, the cells remained closely
grouped. Towards the blastocoele, however, the ectoderm reverted to a twotiered arrangement in which the cells were more loosely packed. In some specimens it was noted that the ectodermal thickening and rearrangement apparently
13
Fig. 13. Sagittal section: stage-13 embryo (x!20), survey picture. Note that the
archenteron cavity (A) has expanded. The arrow heads indicate the gradual decrease
in dorsal mesodermal thickness between the blastopore (Q) and the cephalic end.
extended further rostrally than the position reached by the invaginating mesodermal cells. This might have been an illusion due to the difficulty in distinguishing mesodermal cells from endodermal ones at the apex of the advance.
Alternatively, it might have indicated that the inductive effect can be exerted at
a distance of ten cell diameters or more.
Mid-coronal sections (Fig. 12) confirmed that the dorsal ectoderm was in
554
14
D. TARIN
100//m
Histology of neural induction in Xenopus
555
some places three-tiered with an inner layer of columnar cells. The dorsal
mesoderm contained rounded cells which were more closely packed in the
midline than elsewhere and the archenteron cavity was clearly lined with a
continuous layer of endoderm.
Summary of features of phase I
While mesodermal invagination is well under way in the dorsal area at stage
10} (i.e. before archenteron formation begins) it is quite evident that the first
ectodermal responses to inductive stimuli are not seen until stage 11} or 12.
These consist of increase in the total thickness and number of layers of the
ectoderm and the appearance of the columnar cells in the inner layer. Some
features of mesodermal invagination differed in dorsal and ventral regions.
Phase II. Stages 13-17
In this phase the neural plate appeared and then began to fold in the production of the neural tube. Internal changes included a rapid expansion in the
size of the archenteron cavity to form the primitive gut and the segregation of
the notochord from the two lateral sheets of mesoderm.
Stage 13
Externally the blastopore was small and slit-shaped indicating imminent
closure and the dorsal ectoderm was less pigmented than other regions.
Sagittal sections showed an enormously expanded archenteron and a correspondingly decreased blastocoele (Fig. 13). As in the stage-12 embryos the
archenteron had a complete and continuous endodermal lining.
The closing blastopore formed the caudal end of the developing embryo and
the dorsal and ventral components of the continuing mesodermal invagination
in this region were very different. Dorsally, it extended the whole length of the
embryo and gradually declined in thickness from caudal to cephalic end (Fig. 13).
Starting in the dorsal lip of the blastopore the cells were closely grouped together
(Fig. 14) and slightly more rostrally became arranged in columnar array to
form a mesodermal sheet 5-6 columnar cells thick. This then narrowed until in
the head region a single layer of flattened cells (the prechordal plate) lay between
the brain and the endodermal lining of the archenteron (Fig. 15).
In contrast the mesodermal cells in the ventral lip of the blastopore were rounder
and more irregularly arranged with large intercellular spaces (Fig. 14). The ventral
FIGURES 14, 15
Fig. .14. Detail of caudal end of stage-13 embryo, sagittal section (x400). Same
embryo as Fig. 13. The dorsal mesodermal layer is thicker, has more closely grouped
cells and extends further rostrally than its ventral counterpart.
Fig. 15. Detail of rostral end of stage-13 embryo, sagittal section (x400). Same
embryo as Figs. 13 and 14. The brain (B) and spinal cord (/) portions of the neural
plate can now be distinguished. The single layer of endodermal cells lining the roof
of the archenteron is also easily seen.
556
17/
18
Fig. 16. Coronal section stage-13 embryo (x220). The dorsal mesoderm has
segregated into a notochord between two lateral sheets (K). Note the continuous
endodermal lining of the archenteron cavity. The dorsal ectoderm temporarily reverts
to its original thickness about this time but the cells in the presumptive neural plate
remain closely packed.
Fig. 17. Coronal section stage-13 embryo (x 1450). Detail of the mid-dorsal region
showing that the notochord (N) is closely applied to the neural plate (/), the paraxial
mesoderm (^T)and the endoderm (H). Clumps of metachromatic material (R) are also
present in the spaces between these tissues.
Fig. 18. Ventral ectoderm stage-13 embryo (x 1450). The thickness is similar to that
of the dorsal ectoderm (Fig. 17) but in contrast the inner tiers of cells are loosely
arranged with large intercellular spaces.
Histology of neural induction in Xenopus
557
part of the invaginating mesodermal sleeve did not extend for more than one-third
of the length of the embryo.
The ectoderm also differed in dorsal and ventral regions of the stage 13
embryos. Although of approximately similar thickness (except at the rostral
end where the brain was forming) the dorsal ectodermal cells were closely
packed together and the inner tier was low columnar in shape. The ventral
ectoderm consisted of two tiers of cuboidal cells with small intercellular spaces
in the inner one (compare Figs. 17 and 18). It will be remembered that the
dorsal ectoderm thickened at stage 12 and this transient reversion to a thinner
structure in which the cells remained columnar and closely attached was of
some interest. It was seen only in the trunk region and for a brief period.
At the head end the dorsal ectoderm was four to six columnar cells thick and
this constituted the developing brain plate (Fig. 15). It had a prominent rostral
margin and tapered caudally to merge with the spinal cord. In this region the
inner ectodermal cells were sometimes not as closely packed as in Fig. 15. The
outermost, as in other regions, were not columnar and were always tightly
joined together.
The ectomesodermal junction was well defined in both dorsal and ventral
regions but had no special features in either. Once again the degree of separation
between the two germ layers varied slightly. Metachromatic material was
prominent in the extracellular spaces of both dorsal and ventral lips of the
blastopore. More rostrally, in the trunk region, there was none extracellularly
but some cytoplasmic metachromasia was observed. At the head end similar
material was once again present in quantity in the extracellular spaces of the
brain plate and underlying mesoderm and at the ectomesodermal junction.
Coronal sections showed that the dorsal mesoderm had segregated into three
components consisting of the notochord and two lateral sheets. There was little
evidence as yet of subdivision of the lateral masses to form somites (paraxial
mesoderm) and lateral plate mesoderm. The notochord was in contact dorsally
with the neural plate and ventrally with the single layer of endodermal cells
forming the roof of the archenteron cavity (Figs. 16, 17).
Stage 14
External features. Early neural plate formation could be distinguished by a
slight prominence of the dorsal surface. The caudal part was most evident and
featured three faint grooves radiating rostrally from the blastopore. The brain
plate was, however, not visible.
His to logically these embryos were similar to those of the previous stage and
the main changes affected the dorsal ectoderm, which was now clearly thicker
than elsewhere. This thickening constituted the neural plate which displayed a
well-marked median (neural) groove, and a prominent rostral edge. The timelapse cinematographic study mentioned earlier showed that at this period of
development a distinct ridge moved across the dorsal surface of the embryo
558
20
D. TARIN
100 //m
Histology of neural induction in Xenopus
559
and in the wake of this the neural plate could be distinguished. Examination of
histological sections of embryos from stages 13-16 showed that it was the progressive rostral extension of this structure which was responsible for the moving
ridge phenomenon.
In most places the neural plate was composed of three tiers of closely packed
cells, the inner one of which was columnar. Laterally the neural plate merged
with the two-tiered non-neural ectoderm. The boundary between these was not
distinct but corresponded roughly with the junction of somitic and lateral plate
components of the underlying mesoderm. A further point of difference between
the two types of ectoderm was that intracellular metachromasia was present in
the neural plate, whereas in the lateral ectoderm similar material was in the
extracellular space. (The cephalo-caudal distribution of metachromasia was the
same as at stage 13.)
As with the previous stage the notochord was very close to the neural plate,
endoderm and lateral sheets of mesoderm. The latter was now, however, further
subdivided into paraxial and lateral plate sections. The paraxial mesoderm is the
forerunner of the somites and coronal sections showed that the cells were
elongated and rearranged in radial fashion giving a rosette-like appearance (see
figures of later stages).
Stage 15
External features. The complete outline of the neural plate was clearly seen
on the surface of the embryo. Most embryos were still round at this stage.
Once again histological sections showed only few changes since the previous
stage. Sagittal sections showed that the brain plate was thicker and longer.
Metachromatic material was still present although reduced. Most was intracellular although some was still observed at the ectomesodermal junction in the
trunk region. There was little in the vicinity of the brain plate.
Coronal sections showed that the neural plate was much thicker than the
FIGURES
19-21
Fig. 19. Coronal section stage 15 (x400), dorsal aspect. The neural groove, black
asterisk, is evident and the neural plate, white asterisk, contains columnar cells which
slope inwards at the edges. The paraxial mesoderm (K) has segregated from the
lateral plate mesoderm (T) and is now thicker than the notochord. Metachromatic
granules are present in the archenteron cavity and also in the space between the
embryo and the vitelline membrane.
Fig. 20. Coronal section stage 18 (x 400), dorsal aspect. The neural plate is much
thicker and the neural groove deeper than in the previous figure. The paraxial
mesoderm is also thicker and contains an eccentrically placed myocoele cavity, arrow.
Note the rosette-like arrangement of its columnar cells.
Fig. 21. Coronal section stage 19 (x400), dorsal aspect showing further dorsoventral enlargement of the somites, deepening of the neural groove and thickening
of the neural plate. Notice how the boundary between the somites and the neural
plate is more oblique than in previous stages.
560
D. TARIN
lateral ectoderm and the transition was moderately sharp (Fig. 19). The deeper
part of the neural plate contained columnar cells which at its edges sloped
towards the neural groove. It was also noted that clumps of metachromatic
material were present at the four corners of the notochord and that its shape
had become rounder. Accompanying this change was an apparent reduction in
its dorso-ventral diameter relative to the somites which now projected above it.
In fact the measurements recorded in Table 1 show that the notochord stayed
Table 1. Dimensions of the notochord and somites between stages 1.3 and 19
A
Stage
No. of
specimens
13
2
14
2
15
2
16
2
17
18
1
2
19
2
Vertical
measurement
Transverse
measurement
Om)
111
126
132
126
120
126
126
120
117
120
117
111
126
78
96
96
96
90
90
120
90
90
87
84
81
102
Somites.
Vertical dimension
(jim) only. Figure
indicates mean of
each pair
108
126
177
180
162
156
195
171
192
OO OO
ON O
Notochord
207
204
The general trend is for the notochord to stay constant in size while the somites
gradually expand with particularly large increments between stages 13 and 14 and
stages 18 and 19.
almost constant in size and that the somites enlarged vertically. Thus the neural
groove dipped into a longitudinal trough formed between the somites and
remained closely applied to the notochord which formed the floor of this
depression.
Stages 16 and 17
During these stages the neural plate started to fold along a cephalo-caudal
axis to form the neural tube. Histological sections showed no dramatic internal
changes; only a gradual thickening of the neural plate coupled with a deepening
of the neural groove so that by the end of stage 17 the two sides of the neural
plate were approximating to one another. Under each half lay the masses of
paraxial mesoderm forming the presumptive somites and these gradually
increased in size relative to the notochord which remained attached to the
Histology of neural induction in Xenopus
561
ectodermal cells in the base of the neural groove. At the same time the embryo
started to elongate and the archenteron cavity increased in size.
Summary of Phase II
Between stages 12 and 13 there were considerably changes in the structure of
the embryo, particularly in the segregation of the neural plate into brain and
spinal regions and of the mesoderm into notochord and paraxial masses. Sub-
Fig. 22. Sagittal section, stage 18 (x 100) survey picture. The archenteron is larger
and the blastocoele has correspondingly decreased in size. The mesoderm is almost
uniform in thickness between the blastopore and mid-trunk level. More anteriorly
it rapidly becomes thinner as the brain is reached. The rectangle indicates the area
shown in Fig. 23.
sequently, however, there was a period of relative quiescence in which the
neural plate gradually thickened, although at a greater pace in the brain than
elsewhere, and eventually (stage 16) started folding about a longitudinal axis.
Phase III. Stages 18 and 19
The neural tube was almost completed in this phase and a trend towards
regionalization of the brain was noted.
562
23'
D. TARIN
Histology of neural induction in Xenopus
563
Stage 18
In coronal sections the neural groove was narrow and deep (Fig. 20). The
neural plate consisted of a deeper part, two to three tiers of columnar cells
thick and a superficial one composed of a single tier of tightly apposed rounded
cells. In the neural groove the cells of the superficial tier became columnar and
occupied most of the thickness of the neural plate as described by Schroeder
(1970). The edges of the neural plate were now sharply defined and lay over the
middle of the paraxial mesoderm. In contrast to the underlying mesoderm the
neural plate contained numerous mitotic figures.
The notochord and the paraxial mesoderm looked much the same as previously
with the exception that a myocoele cavity could now be distinguished in the
latter. This contained metachromatic material and was eccentrically placed in
the presumptive somite mass so that the cells medial and inferior to it were
columnar and those superior and lateral were small and rounded (Fig. 20).
The first evidence of subdivision of the brain plate into three parts, separated
by grooves on the ventral aspect of the brain plate was observed in sagittal
sections at this stage (Figs. 22, 23). The anterior, middle and posterior segments
thus created represent the prosencephalon, mesencephalon and rhombencephalon respectively. In contrast to these developments the ventral ectoderm and
mesoderm were substantially unchanged in thickness, cellular arrangement and
distribution since stage 13 or 14.
Deep to the brain lay the very thin portion of the mesodermal sheet known as
the prechordal plate (Fig. 23). Under the prosencephalon this consisted of a
single layer of flattened irregularly arranged cells. This increased to approximately three layers of loosely packed cells in the mesencephalic region. More
caudally the mesoderm was much thicker and the columnar cells were closely
packed. Moreover, the trunk and caudal sections of the dorsal mesoderm were
now of roughly uniform thickness (Fig. 22) thus differing from the situation in
earlier stages (see stage 13).
The ectomesodermal junction was very tight in the posterior part of the
embryo but more rostrally small spaces were occasionally present.
Metachromatic material, both extra- and intracellular, was still present at this
stage but was considerably reduced. In the ectoderm only minute traces remained
FIGURES 23, 24
Fig. 23. Detail of brain of stage-18 embryo (x 400). Same embryo as Fig. 22. The
brain is divided into three regions (U, V, W) by ventral grooves. The rhombencephalon (WO merges imperceptibly with the spinal cord (/) and the prosencephalon
(£/) has a sharp boundary with the ectoderm of the stomadeal membrane (Z). The
prosencephalon (U) and mesencephalon (K) are underlain by a very thin sheet of
mesoderm - the prechordal plate (X).
Fig. 24. Detail of brain of stage-19 embryo (x 400), sagittal section. The subdivision of
the brain is more marked than at stage 18 and the rostral groove is much deeper.
36
EMB 26
564
D. TARIN
but in the mesoderm moderate quantities of extracellular metachromasia were
still visible in the prechordal plate and at the caudal end of the embryo. Coronal
sections revealed that similar material was still present at the corners of the
notochord and at infrequent intervals around the somite blocks. A small
blastocoele cavity was still evident and this contained metachromatic granules
as did the capacious archenteron.
Stage 19
Externally the neural folds were in apposition along the length of the embryo.
The protuberant spinal cord and flattened but laterally expanded brain were
easily recognizable. Fusion of the neural folds was imminent.
Sagittal sections revealed that the regionalization of the brain was more
evident with a deep ventral groove separating the prosencephalon from the
mesencephalon (Fig. 24). Once again there were several mitoses in the ectoderm
but apparently none in the mesoderm.
The thin prechordal plate was present only under the prosencephalon and the
anterior part of the mesencephalon. The hind-brain overlay a thicker sheet of
mesoderm, the cells of which were not arranged so regularly as under the spinal
cord.
Coronal sections (Fig. 21) showed a very deep neural groove, the neck of
which was constricted by the apposing neural folds in some parts of the embryo.
The deeper portion, which is known to form the central canal of the spinal cord,
was very small in comparison with the mass of nervous tissue around it and the
floor of the neural groove was lined with microvilli.
The somites were by this stage very thick in a dorsoventral plane and the
notochord and lateral mesoderm were relatively small. The notochord was almost
round in cross-section and there were only traces of extracellular metachromatic
material associated with it. The archenteron cavity still contained abundant
metachromatic granules. Elsewhere in the embryo there was virtually no extracellular metachromatic material left. The intracellular variety was, however,
still widely present.
DISCUSSION
The results of the grafting experiments described in the first part of this paper
are similar to those obtained in chicks (Waddington & Schmidt, 1933), fish
(Oppenheimer, 1936) and other amphibia (Spemann & Mangold, 1924). They
show that induction of the nervous system by mesoderm does occur in Xenopus
laevis and that, as in other amphibian species (Spemann, 1938) the portion of
the mesoderm responsible for this invaginates through the dorsal lip of the
blastopore.
The histological observations reveal a fairly clear-cut sequence of events in
neural induction. Initial thickening occurs in the mid-dorsal ectoderm at about
stage 1H and spreads over the rest of the dorsal surface by stage 12-12^. This is
Histology of neural induction in Xenopus
565
rapidly followed by segregation of brain and spinal regions, clearly recognizable
by stage 13.
There is next a period of relative quiescence during which the neural plate
continues to thicken and mechanical changes bring about folding around a
longitudinal axis. Then, between stages 17 and 19 regionalization of the brain
begins with the segregation of its substance into three main masses separated
from each other by deep constrictions.
These observations show that the morphological events comprising neural
induction are complex and gradual. Thus the nervous system does not appear ab
initio as a preformed unit with inherent structural differences in various regions
but, instead, as a diffuse thickening of the dorsal ectoderm (interpreted as
'activation') which subsequently becomes regionally modified to form the
different parts of the C.N.S. (interpreted as 'transformation'). One therefore
expects that the inductive processes responsible for these changes will prove to
be similarly complex and sequential rather than dependant on a single rapid event.
This conclusion is similar to those reached by other investigators using grafting
and other experimental manipulations (Nieuwkoop et al. 1952; Takaya, 1955;
Eyal-Giladi, 1954), and supports in principle the activation-transformation
hypothesis advanced by Nieuwkoop et al. (1952) and the similar scheme proposed
by Sala (1955).
Although the present work has established when the thickening of the dorsal
ectoderm and its regionalization take place in Xenopus neural induction, the
timing of the stimuli which trigger these responses remains unknown. However,
the work of Spemann (1938) and his associates shows that this is not of particular importance because inductive stimuli are present for long after the
neural tube is formed and it is the ability of the ectoderm to respond which
decides when induction begins. Thus, we should consider (a) whether there are
any features which persist for long periods during induction and might represent
the agents responsible, and {b) whether there are any changes in the ectoderm
prior to induction which indicate when the cells become competent to respond
to the inductive stimulus.
Consider first item (a): in a histological study it is of course only possible to
identify visible features, and the only one of these which persisted for long enough
to represent a possible inductive agent was the extracellular metachromatic
material. The exact chemical nature of this is at present uncertain, although the
types of compounds possessing this staining property, which could be present at
this stage of development are quite limited. The most likely alternatives are either
the nucleic acids or some complex polysaccharide such as glycogen. Early
investigators claimed both ribonucleic acid (Brachet, 1941, 1947) and glycogen
(Woerdeman, 1933; Raven, 1933; see also review by Spemann, 1938) to be
present in quantity in the dorsal lip of the blastopore but these reports did not
specifically discuss the extracellular distribution of such material. More recent
workers using the electron microscope also differ in their claims, for while Van
36-2
566
D. TARIN
Gansen & Schramm (1969) found only glycogen in the intercellular spaces of the
gastrula of Xenopus laevis, Kelly (1970) contended that both RNA and glycogen
are present in this site. The chemical nature of the intercellular material is
therefore still not settled and its identification will require further work.
Any assessment of the role of the extracellular metachromatic material in
development must of course take into account its distribution in the various
parts of the embryo and the regional pattern of its disappearance. As it is
present in both dorsal and ventral regions of the embryo from stage 11 onwards
(see above) it seems unlikely that it is specifically involved in neural induction.
(The counter-argument, that the ventral ectoderm might be simply unresponsive
to the inductive stimulus, is refuted by the production of secondary nervous
systems on the ventral aspect by transplantation of the dorsal lip of the blastopore - see above). On the other hand, it has to be conceded that while the substances in dorsal and ventral lips of the blastopore are histochemically similar
(Tarin, 1971a), these tests are relatively crude. For instance, they will not
distinguish one family of RNA or glycogen from another. Thus, in different
regions, there may be minute chemical differences in the molecular structure of
the metachromatic material which are of great biological significance.
Consideration of the regional pattern of disappearance of this material is
also of some value in attempting to assess its function. It was noted above that it
persisted for longest in the dorsal and ventral lips of the blastopore and in the
region of the developing brain. This might indicate a role in cellular movement
or in other activities involving high energy consumption. Further studies on the
chemical composition and significance of this material are at present in progress
and will be reported separately.
With regard to item (b) this light microscopical study did not result in the discovery of any changes which might assist in assessing when the ectoderm acquires
the competence to respond to primary embryonic induction. Further work using
histochemical and electron microscopical methods (Tarin, 1971 a, b), has also
failed to reveal any obvious features of value for this purpose.
It seems therefore that a proper assessment of when reactivity to the activating
and regionalizing stimuli is acquired and lost will depend on grafting procedures.
Yet these, too, have their technical limitations as evidenced by the conflicting
results of comparable experiments. Some of these discrepancies are probably
due to the difficulty of separating the ectodermal and mesodermal layers at
their natural interface. As noted in the results the relationship between these
two sheets of cells is usually very close although it is somewhat variable. Thus,
unless future experiments are controlled by the histological assessment of
portions of the grafted material or the present methods are refined so that
either the ectoderm or the mesoderm is specifically marked in some way
(Tarin, 1971 c) prior to the cutting of the graft, these discrepancies will
continue.
The slight variability of the closeness of apposition of the ectodermal and
Histology of neural induction in Xenopus
567
mesodermal layers in different embryos and in different parts of the same embryo
is compatible with the view that the inducing agent is a diffusible substance for
it is known that neural induction can occur even across a Millipore filter providing it is less than 25 pan. thick (Gallera, Nicolet & Bauman 1968), (i.e. the interacting tissues do not have to be in contact). Even in the most extreme cases the
separation did not exceed 10 /im in the specimens described above.
It seems established by the work of several investigators (Spemann, 1931;
Holtfreter, 1933; Alderman, 1935; Deuchar, 1953; Takaya, 1955; see also
review by Saxen & Toivonen, 1962) that the segregation of the neural plate into
different regions (regionalization) is effected by the underlying mesoderm. What
is not agreed, however, is whether the mesoderm itself is a mosaic of separate
areas each responsible for the evocation of a different part of the central nervous
system or whether regionalization is the result of variation in the length of
exposure to cumulative influences exerted by the mesoderm as it slides under the
ectoderm. As might be expected in such a situation there are various items of
experimental evidence which are considered to support or oppose each of these
interpretations. For instance, Waddington & Deuchar (1952) substituted the
neural plate of late gastrulae with vitally stained ectoderm from early ones and
found that, in the embryos which survived, a whole normal neural axis was
formed from the marked transplant. This suggests that regionalization is not
produced by differential effects exerted during mesodermal imagination and
tends to favour the alternative possibility of regional differences in the mesoderm. On the other hand, Eyal-Giladi (1954) removed portions of ectoderm
from the dorsal surface at different stages during gastrulation and transplanted
them to the ventral aspect of the embryos. She noted the development of progressively more caudal neural structures in the grafts taken at successively later
stages of mesodermal invagination, and attributed this sequence to differences
in the length of exposure to cumulative effects exerted by the mesoderm sliding
underneath the dorsal surface.
The histological results presented above do not permit a choice between these
alternatives. However, the pertinence of the present paper to the problem is
that the further grafting experiments which will be needed to understand how
regionalization occurs can be performed at suitable stages selected on the basis
of the histological changes.
The mechanism of folding of the neural plate to form a neural tube is a
problem related to this study of neural induction. In contrast to the newt, where
the neural plate is only one cell thick (Burnside & Jacobson, 1968), in Xenopus
it is composed of several cell layers which all participate in the folding process.
Thus it seems unlikely that the deformation in the shape of the surface ectodermal cells described by some investigators (Waddington & Perry, 1966;
Baker & Schroeder, 1967; see also Wren & Wessels, 1969) is alone an adequate
causal factor in this species because it is difficult to see how this could achieve
more than the production of a surface dimple or wrinkle. Although such a
568
D. TARIN
mechanism might initiate the formation of a neural groove it could be expected
to have little effect on the deeper layers of the ectoderm. It is therefore imagined
that to accomplish folding of the full thickness of the neural plate other factors
must be in operation. In fact, it seems highly likely that the formation of a
longitudinal gutter, by the sinking of the notochord and the relatively fast dorsal
expansion of the somite masses between stages 13 and 19 makes a significant
contribution to this process. These histological observations confirm those of
Schroeder (1970) who drew similar conclusions and argued that this mechanism
augmented the effects of cellular rearrangement and alteration of cell shape.
The continuity of the endodermal lining of the archenteron cavity throughout
gastrulation in Xenopus contrasts markedly with the mode of formation of the
gastro-intestinal tube in urodele amphibians. In such embryos the roof of the
archenteron cavity is initially formed by mesoderm which must segregate from
the more laterally placed endoderm before the latter can grow medially to meet
its neighbour under the mesoderm and thus fill the defect in the endodermal
tube. The importance of this observation for future work on Xenopus is that it
implies that the fate map of this animal differs considerably from others in
having no mesoderm represented on the surface immediately prior to gastrulation. In turn, this suggests that the mesoderm is derived from deeper layers of
the lining of the blastula. These deductions are supported by the results of
preliminary surface marking experiments using vital dyes (D. Tarin, unpublished
observations), and exactly concur with the views of Nieuwkoop & Florschutz
(1950).
A further point of agreement between this work and theirs is that, in this
animal, mesodermal invagination occurs well before the invagination of the
archenteric cavity. These two studies are also complementary in many respects
where they do not overlap and the special contributions of the present work are
to provide a detailed account of the relationships between ectoderm and mesoderm during neural induction, the distribution and fate of metachromatic
material and the changes in the ectoderm during individuation of the nervous
system. The analysis of these particular problems was facilitated by the use of
1 [im thick sections of material embedded in Araldite, which can be recommended as providing clearer visualization of histological features than paraffin
wax sections.
I am very grateful to Mrs E. K. Jones for the outstanding quality of the technical assistance
she has provided throughout this work. I also wish to thank Professor R. L. Holmes for
reading and criticizing the manuscript and the Nunield Foundation for providing financial
support for this research programme.
Histology of neural induction in Xenopus
569
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