/ . Embryol. exp. Morph. Vol. 35, 1, pp. 139-148, 1976
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Printed in Great Britain
The formation of the gonadal ridge in
Xenopus laevis
II. A scanning electron microscope study
By C. C. WYL1E,1 M. BANCROFT 1 AND J. HEASMAN 1
From the Department of Anatomy, University College London
SUMMARY
This paper studies the surface morphology of the developing gonadal ridge in X. laevis
between stages 44 and 49 (Nieuwkoop & Faber, 1956). During this period the primordial
germ cells (PGCs) move laterally from the dorsal mesentery of the gut to the position of the
presumptive gonadal ridge. As they do so the coelomic lining cells lateral to the mesentery
differentiate into a specialized, longitudinally orientated band, stretching nearly the full
length of the dorsal mesentery on each side. The PGCs migrate beneath this band of cells,
which thus becomes the germinal epithelium of the gonadal ridge. We have demonstrated
by irradiation experiments that this specialized band of cells can differentiate independently,
in the absence of the PGCs.
INTRODUCTION
The early events of gonadal ridge formation in Xenopus, as in other anurans,
are known only in outline (Witschi, 1929; Kalt & Gall, 1974; Wylie & Heasman,
1975; Whitington & Dixon, 1975). It is thought, but by no means certain,
that the primordial germ cells (PGCs) migrate from the gut along the dorsal
mesentery to the posterior body wall. They then migrate laterally to the site
of formation of the paired gonadal ridges. The evidence for this sequence of
events is largely drawn from work in other species, e.g. the chick (Dubois,
1968) and the mouse (Peters, 1970).
In a previous study (Wylie & Heasman, 1975) the fine structure of PGCs,
and the cells with which they associate to form the gonadal ridge, has been
described. In this paper the surface morphology of gonadal ridge formation
is studied.
MATERIALS AND METHODS
(1) Growth of embryos. Eggs were artificially fertilized to obtain synchronously developing batches of embryos. Embryos were allowed to develop until
hatching in Gurdon-modified Barth's saline (Gurdon, 1968) at 10 x dilution,
and thereafter in degassed tapwater at 21 °C.
1
Authors' address: Department of Anatomy, University College London, Gower Street,
London WC1E6BT, U.K.
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C. C. WYLIE, M. BANCROFT AND J. HEASMAN
(2) Preparation for scanning electron microscopy (SEM). At the desired
morphological stage (Nieuwkoop & Faber, 1956) tadpoles were first anaesthetized with low concentrations of MS 222. The abdominal cavity was then carefully
opened and, using a hypodermic syringe, the abdominal cavity was washed out
with fixative. This prevents the precipitation of coelomic fluid constituents on to
the posterior abdominal wall. Two fixatives were found to give good results:
Karnovsky's fluid (Karnovsky, 1965) and 4 % glutaraldehyde in 0-2 M phosphate
buffer, pH 7-4 (Lofberg, 1974). After fixing overnight the tadpoles were dissected
by removing the front and sides of the abdominal cavity, the abdominal contents
and the head and tail of the tadpole. A photomicrograph of such dissection is
published elsewhere (Wylie & Roos, 1975). The tadpoles were then postfixed in
1 % osmium tetroxide, dehydrated through an ethanol series, followed by an
ethanol/Freon series, before being critical-point-dried in a Polaron criticalpoint drying apparatus from liquid CO2. The specimens were mounted on
aluminium stubs with UHU glue, coated with gold in a sputter coating unit, and
examined in a Cambridge Stereoscan S4-10 scanning electron microscope
operated at 10 kV.
(3) Irradiation of fertilized eggs. These were chemically dejellied, using 0-2 %
papain in 2 % cysteine hydrochloride, and placed on a quartz glass strip in a
drop of saline over an ultraviolet lamp. The lamp delivers a dose of 518-4 ergs/
mm2/min. A dose of 29000 ergs/mm2 was found to eliminate PGCs from the
ensuing embryos. Doses less than this reduced, but did not entirely eliminate,
the PGCs.
RESULTS
(1) The median gonadal ridge stage
PGCs were found in the dorsal mesentery, and clumped at the root of the
mesentery, between stages 43-45. At this stage the PGCs bulge outwards from
the dorsal mesentery, at or near its junction with the posterior body wall (Wylie
& Heasman, 1975).
Figure 1 shows a montage of this region on the left-hand side of a tadpole at
stage 44. The following observations can be made:
(a) There are numerous bulges in the dorsal mesentery. These represent
PGCs covered over by a thin layer of cytoplasm of the mesentery cells.
(b) The detailed surface morphology of the cells is difficult to see, due to the
presence of many small, round projections from the surface of most of the cells.
These projections are due to the presence of large yolk granules, which have
not been used at this stage, pushing outwards against the surface membranes
of the cells. These have been seen many times in material sectioned for light and
electron microscopy (Wylie & Heasman, 1975).
(c) The junction of the mesentery and posterior body wall is almost featureless. There is no sign of an indifferent gonadal ridge to which the PGCs are
moving, or indeed any organized tissue at all in this region.
SEM of the early gonadal ridge in X. laevis
FIGURES .1 AND 2
Fig. 1. Scanning electron micrograph montage of stage-44 tadpole showing the
junction of the posterior body wall (B) and gut mesentery (M). The PGCs are seen
as large bulges (arrowed) beneath the surface epithelium.
Fig. 2. Stage-47/8 tadpole. The free edge of the mesentery (M), where the gut has been
removed, is visible. The germ cells are now visible as bulges beneath the newly
differentiated germinal epithelium.
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C. C. WYLIE, M. BANCROFT AND J. HEASMAN
(2) The early-paired-gonadal-ridge stage
Figure 2 shows a montage of scanning photomicrographs of the full length of
the right side of the dorsal abdominal wall of a tadpole at stage 47-48. The yolk
granules have disappeared from the cells in this region now, so that their surfaces
can be more easily studied.
The PGCs can now be seen as bulges lateral to the root of the mesentery, in
an irregular order down nearly the full length of the abdominal cavity.
The most striking observation is that there is now a highly organized band of
somatic cells which covers over the PGCs at the site of the gonadal ridge. These
cells are elongated in a cranio-caudal axis and are interdigitated with each other.
It has been shown before (Wylie & Heasman, 1975) that the PGCs are inevitably
covered at this stage by a layer of somatic cells. However, in conventional crosssections there is no hint of the complexity and organization of these cells. The
somatic cells are presumably the precursors of the germinal epithelium of the
gonad at later stages.
Figure 3 shows a rather more detailed view of the gonadal ridge at this stage.
Figures 4-6 show PGCs, covered with somatic cells, from progressively more
caudal areas of the gonadal ridge shown in Fig. 3. There are distinct craniocaudal differences in the ridge, with the more cranial regions seemingly more
highly organized than those more caudal. We have observed this in all embryos
(about 20) of this stage studied.
Figure 4 is a stereo pair of scanning micrographs, and shows a typical cranial
bulge in the gonadal ridge. Here, many somatic cells are involved in covering
the PGCs. They are more obviously spindle-shaped and stand out further from
the posterior body wall. At the caudal end (Figs. 5, 6) the somatic cells become
progressively more flattened and plate-like. They overlap each other, showing
small surface projections resembling microvilli at their boundaries. The caudal
end of the ridge does not stand out as far from the posterior body wall.
Three-dimensional inspection of these bulges reveals cilia-like processes on
their surfaces. These are shown to greater advantage in a micrograph taken
from another tadpole of the same stage (Fig. 7). Each somatic cell of this bulge
has one cilium, more or less centrally placed.
The distribution of cells of the gonadal ridge at this stage (Figs. 3-6) suggests
that the cranial end is beginning to proliferate more rapidly than the caudal end.
This phenomenon is emphasized by studying later stages in ridge formation
(Fig. 8). Here the cranial end is seen to be growing much more rapidly than the
caudal end. Indeed, the most cranial part seems to be almost an independent
body at this stage. This more rapid growth of the cranial end is paradoxical in
the light of previous studies (Wylie & Heasman, 1975), which show that the
PGCs move laterally to form the gonadal ridge later at the cranial end.
These observations raise two important questions concerning the specialized
band of somatic cells:
SEM of the early gonadal ridge in X. laevis
ICraniall
Fig. 3. Montage of stage-47 gonadal ridge to show the surface appearance of the
early gonadal ridge cells.
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C. C. WYLIE, M. BANCROFT AND J. HEASMAN
FIGURES
4-7
Figs. 4-6. These are higher-power micrographs of areas A, B and C in Fig. 3, to
show the cranio-caudal differences in the gonadal ridge at this stage. Fig. 4 is a
stereo pair to show the detailed surface morphology of the differentiated gonadal
ridge cells.
Fig. 7. Area of gonadal ridge from stage-48 tadpole to show the single cilium in the
centre of each coelomic lining cell of the gonadal ridge.
SEM of the early gonadal ridge in X. laevis
145
(a) their origin,
(b) whether the PGCs have a role in the organization or differentiation of these
cells to form the gonadal ridge.
With respect to the first question, we think it most likely that the somatic
cells are present before the PGCs arrive, as squamous epithelial cells of the
coelomic lining. Light and electron microscopy of cross-sections demonstrate
the presence of coelomic epithelial cells at the junction of the mesentery and
posterior body wall. We think that these become organized concomitantly with
the arrival of the PGCs, to form the gonadal ridge.
We were able to test the second question by seeing whether the somatic cells
were capable of differentiating and organizing themselves in the absence of
PGCs. Newly fertilized eggs were irradiated at the vegetal poles with 29000 ergs/
mm2 of u.v. light. The embryos were allowed to develop and examined for the
presence of PGCs. This is a well-established method for causing sterility in some
anurans by preventing the early differentiation of the PGCs (Bounoure, Aubry &
Auck, 1954; Smith, 1966). Figure 9 shows an embryo so treated, examined at
stage 48. The specialized strip of somatic cells has formed normally, but there
are none of the bulges characteristic of PGCs beneath it. The bulge at the cranial
end of the ridge was found, on re-embedding and sectioning, to contain no
PGCs. The strip of specialized somatic cells is therefore capable of differentiating
without any influence from the PGCs.
All the irradiated specimens examined (20) showed a disorganized mass of
somatic cells at the cranial end of the sterile gonadal ridge (Fig. 9). On careful
examination these were also found in unirradiated controls (Fig. 2). In histological sections of controls, these cranial bulges were usually found to be devoid
of PGCs (Fig. 10A). In sections immediately caudal to these bulges (Fig. 10B)
PGCs reappear. These bulges stick out for some distance into the coelomic
cavity and often appear to be appendages to the main part of the gonadal ridge
(Fig. 11). The significance of this rapidly growing cranial part of the gonadal
ridge, which is usually sterile in control embryos, is uncertain. It may be the
homologue of Bidder's organ in the toad (Witschi, 1933).
CONCLUSIONS
We have shown in this study that the PGCs move laterally from the root of
the mesentery to form the paired gonadal ridges by stage 47-48 under our
conditions. Concomitant with this movement, a population of somatic cells,
possibly arising from the coelomic lining in this area, differentiate to form a
highly organized band of tissue, which completely covers the PGCs. These are
probably the precursor cells of the germinal epithelium of the developing gonad.
The somatic cells can differentiate by themselves without any influence from the
PGCs, which come to lie beneath them.
The appearance of these somatic cells raises the question of whether they
IO
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146
C. C. WYLIE, M. BANCROFT AND J. HEASMAN
•
[cranial
SEM of the early gonadal ridge in X. leavis
147
Fig. 11. SEM stereo pair of the cranial end of the gonadal ridge from a stage-49
tadpole. This stereo pair shows the difference in surface morphology between the
cranial bulge of cells and the main part of the gonadal ridge.
play any role in the organization of the gonadal ridge. Two obvious possibilities
present themselves:
(a) the PGCs are moved passively from the median gonadal ridge to their
final, more lateral position, by the morphogenetic movements undergone by
the sheet of coelomic lining cells, as they condense to form a narrow strip of
gonadal ridge cells.
FIGURES
8-10
Fig. 8. Low-power SEM to show gonadal ridge of stage-49/50 tadpole. Both ridges
are visible since the micrograph was taken from directly above the posterior body
wall.
Fig. 9. Stage-48 tadpole, to show the differentiation of the gonadal ridge cells in the
absence of PGCs. Note the characteristic rounded mass of cells at the cranial end
of the ridge.
Fig. 10. (A) Light micrograph of haematoxylin and eosin-stained 7 /*m wax section
from stage-49 tadpole. Notice the bulge of somatic cells, devoid of germ cells, at
cranial end of the gonadal ridge. (B) Twenty-one /*m further caudally, the cranial
bulge of somatic cells is being replaced by the main part of the gonadal ridge,
containing typical PGCs.
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C. C. WYLIE, M. BANCROFT AND J. HEASMAN
(b) the somatic cells exert a chemotactic effect on the PGCs, which actively
migrate laterally towards them.
The latter hypothesis is most likely, given the evidence from mouse (Blandau,
White & Rumery, 1963; Peters, 1970) and chick (Dubois, 1968) embryos. It
remains to be seen, however, whether the large PGCs of Xenopus embryos are
capable of independent locomotion, and if so, whether the cells of the coelomic
lining destined to form the germinal epithelium play any role in attracting them.
We are indebted to Mrs M. Reynolds and Mrs P. Beveridge for technical assistance, and
to Mr R. F. Moss and Miss D. Bailey for photographic assistance.
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{Received 17 July 1975)