/ . Embryol. exp. Morph. Vol. 36, 3, pp. 513-522, 1976
Printed in Great Britain
513
Scanning electron microscopy of lithium-induced
exogastrulae of Xenopus laevis
By J. L. SMITH, J. C. OSBORN AND M. STANISSTREET 1
From the Department of Zoology, University of Liverpool
SUMMARY
Lithium-induced exogastrulae are abnormal embryos which fail to complete gastrulation
and do not form normal neural structures. Scanning electron microscopy has been used to
compare the surface structure of the ectoderm cells of exogastrulae with that of the ectoderm
cells of normal embryos and has shown that the appearance of ciliated cells is delayed in
exogastrulae. In addition, the surface structure of endoderm cells, which remain exposed in
these embryos, has been studied.
INTRODUCTION
Recently the scanning electron microscope has been used to great advantage
to study the morphological changes which occur during normal early embryogenesis. Tarin (197J) has confirmed that cells at the blastopore groove of amphibian embryos change shape during gastrulation, and has demonstrated the
presence of microvilli at the blastopore groove of the gastrula and on the floor
of the neural groove of the neurula of Xenopus laevis. More recently, Monroy,
Baccetti & Denis-Donini (1976) have used scanning electron microscopy to
show that cells lining the blastocoel of Xenopus blastulae form microvilli just
before gastrulation, whereas the invaginated chordamesoderm cells form filopodia. Two groups of workers, Billett & Courtney (1973) and Kessel, Beams &
Shih (1974), have studied the appearance of ciliated cells in differentiating
amphibian ectoderm, using Amblystoma maculatum and Rana pipiens respectively. Their results are comparable - ciliated cells first appear at the neurula
stage and increase in number up to the tail-bud stage. Grunz, Multier-Lajous,
Herbst & Arkenberg (1976) have used scanning electron microscopy to
show differences in the surface structure of ectoderm isolated from Triturus
alpestris gastrulae and cultured with and without a vegetalizing inducer.
It has been known for many years (Backstrdm, 1954) that treatment of amphibian embryos with lithium chloride produces abnormalities which are to
some extent characteristic of the stage at which the embryos are treated. Such
embryos are 'vegetalized' since lithium treatment causes overproduction of
structures derived from 'vegetal' endoderm and underproduction of structures
1
Authors' address: Department of Zoology, P.O. Box 147 University of Liverpool, 6938X,
U.K.
33
EMB 36
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J. L. SMITH, J. C. OSBORN AND M. STANISSTREET
derived from 'animal' ectoderm. The most extreme example of vegetalization is
exogastrulation when embryos fail to gastrulate normally and therefore do not
form normal neural structures. Such ' vegetalized' embryos may be produced
with agents other than lithium chloride such as amines (Stanisstreet, 1974) or
theophylline (Pays-de-Schutter, Kram, Hubert & Brachet, 1976) (see Stanisstreet & Osborn, 1976).
In this paper we have studied the exogastrula of Xenopus laevis produced by
lithium treatment. As well as the overall morphology of the abnormal embryos,
two aspects have been studied in detail: the time of appearance of ciliated cells
on exogastrula ectoderm; and the structure of endoderm cells, which are exposed
in exogastrulae and can thus be studied without recourse to fracturing the
embryos.
MATERIALS AND METHODS
Production of exogastrulae
Embryos were obtained by injecting pairs of adult Xenopus laevis with
chorionic gonadotrophin ('Pregnyl', Intervet Ltd.) and were staged according
to Nieuwkoop & Faber (1956). When the embryos had reached stage 5 (16-cell
stage) the jelly coats were removed chemically (Dawid, 1965), and the embryos
were washed and subsequently cultured in 10 % Steinberg saline, pH 7-3 (Steinberg, 1957). Some of the embryos within one batch were treated with (MM
lithium chloride in 10 % Steinberg saline for 3 h to produce a high percentage of
exogastrulae (Stanisstreet, 1974). After treatment, embryos were washed and
kept in 10 % Steinberg saline. Lithium-treated and control embryos were observed and fixed at various times after lithium treatment (Table 1). Throughout
the experiment the temperature was controlled at 21 ±% °C.
Preparation of embryos for electron microscopy
Immediately prior to fixation, the vitelline membrane was removed with
watchmakers' forceps. The embryos were fixed for 6 h in 2-5 % glutaraldehyde
plus 2 % paraformaldehyde with 2-5 mM calcium chloride in 0-1 M cacodylate
buffer, pH 7-2 (modified from Karnovsky, 1965) and then washed in changes of
cacodylate buffer containing 2-5 mM calcium chloride over 2 h. The embryos
were dehydrated in a graded series of alcohols, and the ethanol was then gradually replaced with amyl acetate: occasionally embryos were left at this stage.
Embryos were dried using the critical-point method, transferred to the microscope stubs with a fine hair brush as recommended by Tarin (1971), and affixed
with adhesive. Finally the embryos were coated with gold-palladium and observed and photographed with a Cambridge 'Stereoscan' scanning electron
microscope.
Stereoscan of abnormal Xenopus embryos
515
Table 1. Appearance of cilia on ectoderm of normal embryos and lithiuminduced exogastrulae of Xenopus laevis
Figures in brackets indicate number of embryos studied.
Time after start of lithium treatment (h)
Control embryos
Stage
Presence of cilia
Lithium-treated embryos
Stage
Presence of cilia
12
25
31
35
51
Gastrula
Neurula
Tail-bud
—
24-25
32-33
1H
19-20
Late
neurula
22-23
(11)
(8)
(6)
(5)
(9)
—
+
+++
—
+++
—
+++
—
El
(7)
—
E2
(6)
—
E3
(5)
E4
(8)
Early exogastrula
EO
(5)
—
Exogastrula
++
+++
RESULTS
The surface structure of normal embryos of Xenopus laevis has been comprehensively described by Tarin (1971) and since our observations confirmed his,
the control embryos will be described only for comparison with the abnormal
embryos. For convenience, abnormal embryos were classified as early exogastrulae (EO), exogastrulae (El) and late exogastrulae (E2, E3, E4). As can be
seen from Table 1, these stages correspond to certain normal stages of Nieuwkoop & Faber (1956). At each stage little variability in the presence or absence
of cilia was noted, but it is possible that some variability in the time at which
cilia appeared was masked by the time intervals used.
Twelve hours
At 12 h the control embryos had reached stage 11-^ (late gastrula). The lip of
the blastopore formed a complete circle with a diameter equal to approximately
half that of the whole embryo, within which yolky cells were visible (Fig. 1). As
reported by Tarin (1971) the cells of the blastopore groove were covered with
many microvilli (Fig. 2).
In the lithium-treated embryos (EO), the blastopore formed a complete but
much larger circle, so that more yolky cells were visible (Fig. 3). Such embryos
were more susceptible to fracturing at the yolk plug during critical-point drying,
possibly indicating a change in the degree of adhesiveness of the cells in this
region. Like the normal gastrulae, the cells of the 'blastopore lip' of these
embryos showed microvilli (Fig. 4).
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J. L. SMITH, J. C. OSBORN AND M. STANISSTREET
Fig. 1. Xenopus gastrula. (x 53)
Fig. 2. Microvilli on blastopore lip of Xenopus gastrula. (x 2570)
Fig. 3. Early exogastrula (E0). (x 57)
Fig. 4. Microvilli on blastopore lip of early exogastrula. (x 3300)
Fig. 5. Xenopus neurula. (x 67)
Fig. 6. Flank ectoderm of Xenopus neurula. (x 1425)
Stereoscan of abnormal Xenopus embryos
517
Twenty-five hours
By 25 h the control embryos had reached stage 19-20 (neurulae). The neural
folds were closing and the embryo was covered with ectoderm (Fig. 5). Higher
power examination showed that the ectoderm cells had discrete borders and
small marginal pits (Fig. 6). Some differentiation of the ectoderm was apparent as reported by Tarin (1971) the floor of the neural groove showed microvilli
(Fig. 7) and in the lateral ectoderm occasional sunken cells with a few short cilia
were apparent (Fig. 8).
The lithium-treated embryos had reached the exogastrula stage (El). Exogastrulae were dumbell-shaped and consisted of a sphere covered with pigmented ectoderm from which protruded a sphere of yolky endoderm cells
(Fig. 9). Examination of the ectoderm of exogastrulae showed a similar general
picture to neurula ectoderm (Fig. 10), but ciliated cells were not present and
there appeared to be more marginal pits. Examination of endoderm cells showed
a different cell surface structure: endoderm cells were large and 'raspberry-like'
and often showed a central depression (Fig. 11). It is possible that this 'raspberry ' appearance was due to the collapse of the cell membrane onto the underlying large yolk platelets. Under higher power examination some cells showed
microvilli (Fig. 12).
Thirty-one hours
At 31 h the control embryos had reached the late neurula stage (stage 22-23).
The most noticeable change in the structure of the ectoderm was the increase in
the occurrence of cilia. Ciliated cells were more frequent, forming an approximately regular pattern (Fig. 13) and each ciliated cell had more and longer cilia.
The appearance of the endoderm cells of late exogastrulae (E2) was similar to
their appearance in the previous stage (El), although in a few areas tube-like
structures were visible (Fig. 14). In the ectoderm, marginal pits were still
frequent, and cilia were not apparent.
Thirty-five hours
By this time the control embryos were at stage 24-25. Ciliated cells formed a
more regular array, and cilia were longer (15). The ectoderm of exogastrulae
(E3) now showed the presence of some ciliated cells (Fig. 16), and thus appeared
to differentiate some 10 h later than normal ectoderm.
Fifty-one hours
At 51 h the control embryos had reached stage 32-33, and were highly ciliated.
The cell borders were raised and discrete, and very few marginal pits were
present (Fig. 17). The ectoderm of later exogastrulae (E4) presented a picture
similar to that of stage 32-33 embryos. More ciliated cells were present than in the
previous stage, and the cilia were longer (Fig. 18). Thus it appears that the differentiation of exogastrula ectoderm is delayed rather than inhibited completely.
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J. L. SMITH, J. C. OSBORN AND M. STANISSTREET
11
Fig. 7. Floor of neural groove of Xenopus neurula showing microvilli. (xl700)
Fig. 8. Flank ectoderm of Xenopus neurula showing ciliated cell, (x 3145)
Fig. 9. Lithium-induced exogastrula (El). (x55)
Fig. 10. Ectoderm of lithium-induced exogastrula (El), (x 1285)
Fig. 11. Endoderm of lithium-induced exogastrula (El). (x230)
Fig. 12. Endoderm of lithium-induced exogastrula (El), (x 880)
Stereoscan of abnormal Xenopus embryos
Fig. 13. Flank ectoderm of stage 22-23 embryo, (x 620)
Fig. 14. Endoderm of late exogastrula (El). (x4140)
Fig. 15. Ciliated cell of stage 24-25 embryo. (x2520)
Fig. 16. Ectoderm of later exogastrula (E3). (x 735)
Fig. 17. Ectoderm of stage 32-33 embryo, (x 1140)
Fig. 18. Ectoderm of E4 exogastrula. (x995)
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J. L. SMITH, J. C. OSBORN AND M. STANISSTREET
DISCUSSION
In the preparation of delicate material for scanning electron microscopy the
possibilities of the obliteration of surface detail and generation of artifactual
structures must be considered. Whilst the fixation of material appears to present
few problems, the method of drying the specimens can alter the final appearance
(Billett & Courtney, 1973). Tarin (1971), who first used the scanning electron
microscope to observe amphibian embryos, used air drying from fluoracil.
Billett & Courtney (1973) compared two methods of drying specimens, air
drying from acetone and freeze substitution, and found the latter to be a better
method. We have used the more recent method of critical-point drying, which
appears to give good preservation of surface structure.
Our observations on normal embryos of Xenopus laevis support those of
previous workers: both in the appearance of microvilli at gastrulation and
neurulation, and in the progressive appearance of ciliated cells in the ectoderm
from neurulation onwards. Light microscopical and stereoscan observations of
lithium-treated embryos suggest that the onset of gastrulation is not delayed,
although gastrulation is abnormal. We have shown (Osborn & Stanisstreet,
in preparation) that exogastrulae (El in Table 1) have about half as many
cells as control neurulae, and that by the onset of gastrulation, lithium-treated
embryos have significantly fewer cells than gastrulae. Thus it is possible that the
abnormal gastrulation of lithium-treated embryos could be due to the presence
of fewer but larger cells in these embryos. However, the results of a series of
experiments by Cooke (1972, 1973 a, 1973£) suggest that inhibition of cell
division by Mitomycin-C does not prevent differentiation or post-gastrula
morphogenesis.
The present results suggest that cell differentiation of ectoderm, judged by the
appearance and growth of cilia, is delayed by some hours in lithium-induced
exogastrulae. This finding is confirmed in principle by transmission electron
microscope studies (Osborn, Smith & Stanisstreet, in preparation) which suggest
that the formation of mucous-secreting cells, a characteristic of differentiated
ectoderm (Billett & Gould, 1971), is delayed in exogastrulae. The reason for this
delay in cell differentiation is not clear.
Ectoderm isolated from gastrulae forms ciliated cells (Grunz 1973, Grunz
et ah 1976) and recently it has been shown that the animal blastomeres of the
8-cell stage of Triturus alpestris are determined to form ciliated epidermis (Grunz
et ah 1976). Thus the differentiation of ciliated epidermis does not appear to rely
on inductive interactions which could be altered by lithium treatment.
It could be that ciliated cells appear later in cultured isolated ectoderm than in
ectoderm in situ: ectoderm taken from gastrulae of Xenopus laevis and cultured
at 15 °C for 15 h did not show cilia (Billett, 1968). If the differentiation of
isolated ectoderm is delayed, lithium treatment might mimick the isolation effect
in some way. However, no absolute comparisons of the time of appearance of
Stereoscan of abnormal Xenopus embryos
521
cilia and mucous vesicles in isolated ectoderm and whole embryos using the
same species at the same temperature are available.
Treatment of isolated ectoderm with a vegetalizing agent prevents the formation of ciliated cells (Grunz et al. 1976). However, presumably in this system
ectoderm cells are ' vegetalized' into endoderm and mesoderm cells which do not
form cilia. It is conceivable that lithium, which is also a 'vegetalizing' agent,
could act in a similar but less extreme manner to delay cilia formation in
exogastrulae produced by a short pulse of lithium treatment. The mechanism of
the vegetalizing action of the lithium ion is not known, but biochemical comparisons between lithium-treated and control embryos have shown several gross
differences at the transcriptional and translational level (reviewed by Stanisstreet & Osborn, 1976), and it is likely that the delay in cell differentiation of
ectoderm in lithium-induced exogastrulae is due to some lesion in the mechanisms
of gene expression.
We wish to thank Mr C. J. Veltkamp for his expert help with the scanning electron microscopy, and Dr C. F. H. Vickers of the Department of Dermatology who allowed us to use the
critical-point drying apparatus. J. C. O. thanks the Wellcome Trust for a Research Scholarship.
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{Received 17 May 1976, revised 5 July 1976)