/. Embryol. exp. Morph. Vol. 35, 1, pp. 125-138, 1976
125
Printed in Great Britain
The formation of the gonadal ridge in
Xenopus laevis
I. A light and transmission electron microscope study
By C. C. WYLIE 1 AND J. HEASMAN 1
From the Department of Anatomy, University College London
SUMMARY
In Xenopus laevis tadpoles, between stages 44 and 49 (Nieuwkoop & Faber, 1956), the
primordial germ cells (PGCs) migrate from the dorsal mesentery of the gut to the site of the
presumptive gonadal ridge. This paper describes the process at the light- and electronmicroscope levels. The PGCs in the mesentery, which at first are very large and yolk-laden,
seem to lie entirely within the cellular matrix of the mesentery, although this is not obvious in
light micrographs. Where the PGCs bulge out into the coelomic cavity, they stretch the
somatic cell covering to a thin, cytoplasmic layer. The somatic cells of the mesentery are held
together around them at this stage by well-differentiated desmosomes. At this, and subsequent
stages, the PGCs have cytoplasmic processes, roughly the size of microvilli, which are irregularly distributed over their surfaces, and which are inserted between surrounding somatic
cells. Whether these processes play any role in locomotion or exploration of the substrate is
uncertain.
As the PGCs move laterally from the root of the mesentery to the presumptive gonadal
ridge, the coelomic lining cells which cover them, initially with a very thin squamous layer,
differentiate to form the cuboidal cells of the germinal epithelium. Several interesting ultrastructural features of these cells, and the PGCs, are described, particularly in the light of their
surface interaction.
In the light of the morphological data presented here, particularly of the cell surfaces
involved, we conclude that both active locomotion by the PGCs and passive movement by
the morphogenetic movements of the cells around them contribute to the establishment of
the early gonadal ridge.
INTRODUCTION
The primordial germ cells (PGCs) of certain anuran amphibians are formed
very early in embryogenesis (Bounoure, 1954; Bladder, 1960; Smith, 1966).
They are already determined in the endoderm of the embryonic blastulae. They
become incorporated by the morphogenetic movements of gastrulation into the
mass of yolky cells which form the gut; and from here move to the top of the
dorsal mesentery of the gut, and eventually form the paired undifferentiated
gonadal ridges on the posterior abdominal wall (Witschi, 1929).
The mechanism by which the PGCs move from the primitive gut to the site
of the presumptive gonad is unclear, as is the origin of the somatic cells with
1
Authors' address: Department of Anatomy, University College London, Gower Street,
London WC1E 6BT, U.K.
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C. C. WYLIE AND J. HEASMAN
which they associate and form the gonadal ridge. In the chick embryo (Dubois,
1968) it has been demonstrated that the PGCs leave their early position in the
'germinal crescent', migrate actively into the bloodstream, and leave it in the
capillaries of the gonadal ridge. They migrate actively in organ culture (Dubois,
1964) and seem to be under a chemotactic influence from the germinal epithelium
of the gonadal ridge (Dubois, 1968). In the mouse embryo the PGCs, demonstrated by their content of alkaline phosphatase, can be seen in the dorsal
mesentery of the gut 'migrating' to the gonadal ridge (Mintz, 1957). In vitro
they have been shown to exhibit amoeboid movements (Blandau, White &
Rumery, 1963).
The position in Xenopus laevis is complicated by two factors:
(1) Their enormous size, due to large quantities of yolk, which calls into
question their capacity for active movement.
(2) The absence of any obvious gonadal ridge to which they migrate.
The mechanism by which the PGCs arrive at the position of the presumptive
gonad is unclear therefore. This paper seeks to elucidate the early events of
gonadal ridge formation, using light microscopy and transmission electron
microscopy.
MATERIALS AND METHODS
Fertilized Xenopus laevis eggs were obtained by injecting chorionic gonadotrophin into the dorsal lymph sac to induce ovulation and mating (600 units
female, 400 units male). The embryos were sterilized by rapid washing in 70 %
alcohol. Healthy fertilized eggs were sorted and their outer jelly coats manually
removed with forceps. The embryos developed in 1/10N Holtfreters saline.
Tadpoles were harvested at stages 42-49 (Nieuwkoop & Faber, 1956).
For LM studies tadpoles were fixed in Bouin's fixative, dehydrated, embedded
in paraffin wax and cut in 7 /im sections. Sections were stained with haematoxylin and eosin.
For electron microscope studies tadpoles were placed in Karnovsky fixative
(Karnovsky, 1965). As the tadpoles were found to be very resistant to infiltration
by the embedding medium, the anterior body wall and part of the posterior
body wall were dissected during fixation. After 2 h fixation the material was
post-fixed with 1% OsO4 (1 h), dehydrated and embedded in Araldite resin.
2 /tm and ultrathin sections were cut using a Reichert ultramicrotome. Thin
sections were stained with lead acetate and viewed with a Philips 300 electron
microscope.
RESULTS AND DISCUSSION
Light microscopy
At stage 42 the cellular details and cell boundaries of all the cell types present
are obscured by large quantities of yolk. The identification of PGCs at
this stage is difficult, although in occasional sections the large pale-staining,
Morphology of developing gonadal ridge in Xenopus laevis
127
lobulated nuclei, which are characteristic of PGCs at later stages, can be seen.
Fig. 1 shows such a cell, attached to the posterior body wall, lateral to the
broad mesentery.
By stage 45/46 the dorsal mesentery has become relatively narrow and the
yolk platelets have disappeared from the endodermal and splanchnic mesoderm
cells. The PGCs, however, retain their yolk longer than other cells, which makes
them easy to recognize in section (Figs. 2, 3). They are also characterized by
their enormous size, and large multi-lobed nuclei, with one or two nucleoli. The
position of the PGCs is variable at this stage. They are frequently seen attached
to the dorsal mesentery of the gut (Fig. 2). In this position they often appear to
be only attached along one of their surfaces, with the rest of the cell free in the
coelomic cavity. At this stage there are definite cranio-caudal differences in the
number and location of the PGCs. At the cranial end of the root of the mesentery
(Fig. 3 A) there are a greater number of them, arranged as a clump across the
midline. This forms the 'median germ ridge' described in earlier work (Witschi,
1929). Further caudally, there are fewer PGCs, and they tend to be found more
lateral to the root of the mesentery (Fig. 3B, C). These figures also show that the
PGCs at the root of the mesentery, and on the posterior body wall, often appear
to be devoid of a cellular covering. On several occasions, PGCs were found in
the lumen of a blood vessel (Fig. 4). Whilst chick PGCs are known to use blood
vessels as a pathway for migration, they have not been reported previously in
the blood vessels of Xenopus. Occasionally a mitotic PGC is seen (Fig. 5),
although these are rare at this stage.
At stage 47 the position of the PGCs is similar to that at stages 45/46. They
gradually diminish in size, due mainly to the loss of yolk platelets. Fig. 6 shows
two PGCs on the cranial end of the gut mesentery and one at its root. Some
PGCs, both on the mesentery and on the posterior body wall, have an obvious
squamous covering of somatic cells, presumably those of the coelomic lining
(Fig. 7). Others still appear to have surfaces free in the coelomic cavity.
During subsequent development the PGCs move, or are moved, laterally,
and by stage 49/50 typical gonadal ridges lateral to the root of the mesentery are
seen (Fig. 8). These consist of a thick squamous, or even cuboidal surface
covering, the germinal epithelium, which completely surrounds the PGCs.
There is no strict sequence in the above events of gonadal ridge formation.
In some clutches of embryos the yolk platelets are lost from the PGC cytoplasm
as early as stage 45, whereas in other clutches yolk is still present at stage 48.
Similarly, PGCs are found attached to the dorsal mesentery at all stages between
42 and 49, albeit in fewer numbers at the later stages.
From these observations several interesting questions arise.
(1) How do the PGCs arrive at the posterior body wall? There appear to be
two possibilities. The first is that the endoderm containing germ cells is separated
from the bulk of the gut endoderm by the formation of the dorsal mesentery
from splanchnic mesoderm. Germ cells subsequently found in the dorsal
C. C. WYLIE AND J. HEASMAN
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Morphology of developing gonadal ridge in Xenopus laevis
129
mesentery could be explained as ectopic cells which eventually degenerate.
Alternatively the germ cells seen in the mesentery could be the last arrivals of
an active migration from the gut endoderm up the dorsal mesentery and laterally
to the gonadal ridges. PGCs found in the bloodstream could be either ectopic
cells or using the blood vessels to orientate themselves along their migratory path.
(2) If the germ cells are actively migratory, how do such large cells move and
over what substrate? It is impossible to demonstrate by light microscopy
whether they move within the cellular matrix of the gut mesentery and posterior
body wall, or have surfaces free in the coelom. The origin and role of the somatic
cells associated with germinal ridge formation is also obscure.
For elucidation of these problems the material was examined with the electron
microscope.
Ultrastructural features of germ cells
The outstanding features of PGCs in the stages examined (stages 44-49) are:
(a) Large size relative to the surrounding cells (Fig. 9). Stage-45 PGCs have
much more cytoplasm than the somatic cells, containing many large yolk
platelets and grey, amorphous, lipid droplets. In contrast, stage-48 PGCs are
relatively much smaller, with very few yolk platelets, and the prominent feature
is now a mass of mitochondria (Figs. 10, 12). Stage-49 PGCs are more rounded
than previously and they protrude further into the abdominal cavity, forming
the definitive gonadal ridge (Fig. 11)
(b) The PGC nucleus has two striking features: firstly it is multi-lobed with
FIGURES
1-8
Fig. 1. The developing gut (G) and dorsal mesentery (/«) at stage 42. Note the
presence of a PGC at this early stage (arrowed) already attached to the posterior
abdominal wall.
Fig. 2. Stage 45, the dorsal mesentery is now narrowed to one or two cells in width.
Three PGCs are attached to one side of the mesentery (arrowed).
Fig. 3. A, B, C, Sections from the cranial end, the middle and the caudal end, of
the same dorsal mesentery. Note the cranio-caudal differences in length of mesentery,
and distribution of PGCs.
Fig. 4. Stage-46 tadpole to show PGCs in various positions on the cranial end of
the dorsal mesentery. One PGC (arrowed) is in the lumen of the posterior vena cava.
Fig. 5. Section from the middle of the dorsal mesentery of stage-46 tadpole, to
show a mitotic PGC.
Fig. 6. Section through cranial end of the dorsal mesentery at stage 47. Two PGCs
are visible attached to the mesentery, and one at its root. Note decrease in size of
the PGCs and loss of their yolk compared to stage 46.
Fig. 7. Section from the middle of the dorsal mesentery at stage 47, to show squamous
cell covering of the PGCs.
Fig. 8. Stage-49/50 tadpole, to show further development of the gonadal ridge.
Note the proliferation of cells covering the PGCs to form a cuboidal epithelium.
In regions where there are not PGCs (arrowed), the germinal epithelium forms a
solid ridge of cells.
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C. C. WYLIE AND J. HEASMAN
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Morphology of developing gonadal ridge in Xenopus laevis
131
a very large surface area, and secondly it contains a prominent electron-dense
nucleolus (Figs. 9, 11).
(c) Throughout the stages studied the germ cells contain many characteristic
mitochondria. These are typically larger and more electron-dense than those of
the somatic cells around them and contain many cristae and dense granules
(Fig. 14A, B).
(d) Fig. 12 shows part of a germ cell of a stage-48 tadpole. At this stage
electron-dense bodies can be seen attached to mitochondria. These bodies are
scattered throughout the cytoplasm and have been seen infrequently attached
to the nuclear membrane. They are 130-160 nm in diameter, uniform in density
and not enclosed in membrane. Such structures have not been seen in germ cells
before stage 48 in this study. Similar aggregates have been reported in stage-42
PGCs (Kalt, 1973). However, in the above-mentioned study, the gonadal ridges
are far more advanced than those conventionally seen in stage-42 Xenopus
embryos (Nieuwkoop & Faber, 1956). It has been suggested that similar electrondense bodies reported in Rana and Xenopus fertilized eggs, and in subsequent
stages in PGC differentiation, may represent the germ-cell determinants. This
can only be established by demonstrating that some factor within these electrondense granules does, in fact, influence differentiation.
(e) In most germ cells a relatively mitochondrion and yolk-platelet-free
cytoplasmic area can be distinguished near the nucleus (Fig. 14 A, C). It consists
of a collection of membranous vesicles and hollow tubules resembling an extensive Golgi complex. These structures have not previously been reported in
Xenopus PGCs and their role in germ cell metabolism is unclear.
Ultrastructural features of the somatic cells of the mesentery and gonadal ridge
At stage 44 the cells which constitute the dorsal mesentery are continuous
laterally at its root with a single layer of coelomic lining cells on the posterior
body wall. These cells are morphologically similar, and are probably all of
FIGURES 9—13
Fig. 9. Stage 45. The posterior abdominal wall, lateral to the dorsal mesentery. The
PGC protrudes into the coelomic cavity (C) and is surrounded by a thin layer of
coelomic lining cells. Note the convoluted nucleus of the PGC and the pigment
cells (P) on the posterior body wall, yp = yolk platelets, L = lipid droplet.
Fig. 10. PGC at stage 48 on the posterior wall lateral to the dorsal mesentery. Note
the coelomic lining cells (cl) covering both dorsal and ventral aspects of the PGC.
Fig. 11. PGC at stage 49 in the gonadal ridge. Note the three distinct structures (a,
b and c) in the PGC nucleus.
Fig. 12. PGC at stage 48. Note the electron-dense bodies (arrowed) attached to the
mitochondria. See text for details.
Fig. 13. Surface of PGC at root of dorsal mesentery, stage 45. The PGC has cytoplasmic processes (cp) seen in T.S. and L.S. Note subcortical filaments (arrowed).
9-2
C. C. WYLIE AND J. HEASMAN
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Morphology of developing gonadal ridge in Xenopus laevis
133
splanchnic mesodermal origin. The nuclei are large, more densely staining than
the PGC nuclei and contain condensations of chromatin particularly at the
nuclear membrane (Fig. 15). Their cytoplasm is rich in granular endoplasmic
reticulum and Golgi apparatus. At stage 49 the flattened somatic cells have
rounded up and have the more characteristic appearance of the adult germinal
epithelium (Fig. 16).
The relationship between germ cells and somatic cells in the
mesentery and gonadal ridge
The light microscope studies described above demonstrate repeatedly germ
cells apparently devoid of a somatic cell covering (Figs. 3, 4). Such cells appear
to be attached to the posterior body wall only along one surface while the body
of the germ cell protrudes freely into the coelom. However, we have demonstrated by ultrastructural examination that all the PGCs on the mesentery (Fig.
17) and posterior body wall (Fig. 9) are completely covered by somatic cells.
Where PGCs protrude into the coelom, cytoplasmic extensions from adjacent
coelomic lining cells form a thin layer over their surface (Figs. 9-11, 17). On
one occasion, however, it was noticed that the surface covering was not continuous, although some surface material was present (Fig. 13). At this point
there is a large number of surface projections from the PGCs whose possible
role is discussed later.
In all stages examined, coelomic lining cells of the mesentery and posterior
body wall are firmly attached to each other at points of contact by desmosomes.
Such membrane specializations can frequently be seen where two coelomic lining
cells overlap on the surface of a germ cell (Fig. 18). In this situation, both
somatic cells tend to have parallel protrusions outwards from the general surface
(Fig. 14B). These protrusions can be correlated with microvillous-like processes
seen in scanning electron micrographs of the gonadal ridge (Wylie, Bancroft &
Heasman, 1975).
Although there is an abundance of specialized junctions between somatic
cells (Figs. 9, 14B, 17C, 24), no junctions have been seen between germ cells
and coelomic lining cells in the mesentery. However, occasional junctions have
FIGURES
14—16
Fig. 14. (A) PGC at stage 45 on the posterior abdominal wall. Note the extensive
Golgi complex (G). (B) High-power view of region A in Fig. 14 A to show contact
of two coelomic lining cells over surface of PGC. Note parallel protrusions of these
somatic cells and cell junction types. Compare the mitochondria of the PGC and
somatic cells. (C) High-power view of region B of Fig. 14 A to show vesicles and
tubules of Golgi complex (C), and mitochondrion between two lipid droplets.
Fig. 15. Coelomic lining cell on posterior body wall of stage-45 tadpole. Note the
heterochromatin in the nucleus and the granular endoplasmic reticulum (arrowed).
Fig. 16. Germinal epithelial cells covering a PGC in the gonadal ridge of a stage-49
tadpole. Note cuboidal shape of these cells compared to Fig. 15.
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C. C. WYLIE AND J HEASMAN
17C
o-5 fim
Fig. 17. (A) PGC in the dorsal mesentery of stage-44 tadpole. The cytoplasm is full
of yolk platelets (Y) and lipid droplets (L). Note that the PGC is completely covered
by a thin layer of cytoplasm of the mesentery cells. (B) High-power view of region
A of Fig. 17A to show a cytoplasmic process from the dorsal end of the PGC.
(C) High-power view of region B of Fig. 17A to show overlapping somatic cells,
held together by a desmosome.
Morphology of developing gonadal ridge in Xenopus laevis
135
been identified between germ cells and somatic cells on the posterior body wall
during gonadal ridge formation. Two types of junction have been seen: occasional
desmosomes (Fig. 19) and less specialized regions where the intercellular space
and cell membranes are obscured by electron-dense material (Fig. 20). These
findings suggest a. close structural and possibly functional association between
the two types of cell from as early as stage 45 when the gonadal ridge is first
developing.
Germ cells are found together in clusters on two occasions in their development, first at stage 45, in a single median ridge at the root of the mesentery, and
later in the paired gonadal ridges. Only on one occasion was a specific junction
seen between germ cells in the median cluster (Fig. 21). This consists of electrondense material obscuring the intercellular space for 300 nm.
A characteristic feature of the germ-cell surface in stages 44-49 is the presence
of small cytoplasmic processes. Fig. 17 B shows the dorsal end of a germ cell in
the mesentery at stage 44-45, with a short pseudopodium extending forwards.
Fig. 22 indicates a similar cytoplasmic process of a PGC at stage 47 between
two somatic cells. This protrusion is similar to those reported in endodermal
PGCs of mouse embryos (Spiegelman & Bennett, 1973). Cytoplasmic processes
are seen projecting from the surface of PGCs in areas where they are not closely
opposed to the somatic cells (Figs. 13, 23). The processes vary considerably in
length, but the width is fairly constant, being 87 nm on average. The microvilli
of Xenopus gut are uniformally 96 nm in diameter and of constant length. Therefore the processes seen here are most likely to be microvilli on account of their
size. Scanning electron microscopy of isolated PGCs in vitro (Wylie, unpublished
observations) shows that these processes are evenly distributed on the PGC
surface. This difference in distribution in vivo and in vitro suggests they are labile.
The microvilli themselves seem to possess some substructure and in places the
microfilaments present in the cortical cytoplasm seem to extend into the microfilamentous skeleton. This, together with their locality, suggests their role may
not be purely absorptive, but that they may be used for exploration of the
substrate, or movement over it.
Fig. 24 shows an interesting feature of germ-cell/somatic-cell association.
Here two somatic cells surround a PGC. The coelomic lining cell nearest the
germ cell contains vesicles which in three cases are continuous with the cell
surface and with the intercellular space. Such vesicles are frequently found in
somatic cell cytoplasm, especially over the germ cell surface. Their function is
obscure, but they may be secretory or pynocytotic vesicles, and possibly sites of
communication between PGCs and somatic cells. An interesting correlate of
such a system of communication has been demonstrated in chick (Cuminge &
Dubois, 1974). Using pulse-chase techniques, glycoproteins synthesized in
germinal epithelium cells have been demonstrated to be taken up by the germ cells.
It has been postulated that such a phenomenon may form the basis of a
characteristic guidance mechanism for the migrating PGCs.
C. C. WYLIE AND J. HEASMAN
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Morphology of developing gonadal ridge in Xenopus laevis 137
CONCLUSIONS
From these observations the following conclusions can be drawn:
(1) Germ cells in the mesentery are ensheathed in somatic cell cytoplasm,
have cytoplasmic processes between coelomic lining cells and are not associated
by junctions with them. This evidence is suggestive of a picture of germ cells
moving up the mesentery, not static and degenerating in it. A similar conclusion
has been drawn from a study of the fine structure of mouse PGCs (Spiegelman,
1973). Germ cells found in the dorsal mesentery of mouse embryos have small
cytoplasmic processes containing micron"laments and do not exhibit junctions
with other cells. The relatively small and sparse nature of these cytoplasmic
processes in both Xenopus and mouse embryos is not consistent with an amoeboid
type of locomotion. The mechanism of migration remains to be determined.
(2) Germ cells on the posterior body wall are also ensheathed in somatic
cells, which at stage 48 become cuboidal and form the germinal epithelium.
These cells are strongly held together by desmosomes. Germ cells possess
occasional pseudopodial extensions between coelomic lining cells, which suggests
continued germ-cell movement. However, some junctions are present between
PGCs and somatic cells from stage 45 onwards, suggesting the development of
a more permanent association between the two cell types.
(3) The conclusion from this study is that both the passive and active theories
of gonadal ridge formation may be correct (Witschi, 1929; Whitington &
Dixon, 1975). Some of the germ cells present in the gut endoderm are transported
passively to the posterior body wall and are surrounded by splanchnic mesodermal cells by the formation of the dorsal mesentery. Other PGCs may be
trapped within the mesentery on its formation, or may still lie in the dorsal
endoderm. These cells then actively migrate up the mesentery, forcing their
way between the coelomic lining cells, always enclosed in a cellular sheath. At
FIGURES 18-24
Fig. 18. High-power electron micrograph to show a desmosome linking two coelomic
lining cells of the posterior abdominal wall at stage 45.
Fig. 19. High-power view of a desmosome linking a PGC at stage 46, with a coelomic
lining cell. Note the cytoplasmic process of the PGC (arrowed).
Fig. 20. High-power view of a junction between a PGC and a coelomic lining cell
at stage 48.
Fig. 21. A specialized cell junction between two PGCs at the root of the mesentery
in a stage-45 tadpole.
Fig. 22. A cytoplasmic process from a PGC at stage 47, extending between two
coelomic lining cells on the posterior body wall.
Fig 23. Cytoplasmic processes from the surface of a PGC at stage 49.
Fig. 24. Vesicles (arrowed) in a coelomic lining cell adjacent to a PGC on the
posterior abdominal wall (stage 45). Note that some of these vesicles open into the
intercellular space. A desmosome links the two coelomic lining cells.
138
C. C. WYLIE AND J. HEASMAN
the root of the mesentery PGCs are surrounded by somatic cells of splanchnic
mesodermal origin. These latter cells proliferate and differentiate into the germinal epithelium, enclosing the PGCs. The final position of the germ cells in
the lateral gonadal ridge may be the result of both active migration and passive
movement due to germinal epithelial cell proliferation and narrowing of the
dorsal mesentery. In order to prove that the PGCs of Xenopus laevis do migrate
actively it is necessary to demonstrate that they are capable of locomotion.
Evidence for this is presented elsewhere.
Our grateful thanks are due to Mrs M. Reynolds and Mrs P. Beveridge for excellent
technical assistance, and to Mr R. F. Moss and Miss D. Bailey for help with the photography.
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(Received 17 July 1975)