/ . Embryol. exp. Morph. Vol. 42, pp. 149-161, 1977
Printed in Great Britain © Company of Biologists Limited 1977
149
Studies on the locomotion of primordial germ cells
from Xenopus laevis in vitro
By JANET HEASMAN, 1 TIM MOHUN, 1 AND C C. WYLIE
From the Department of Structural Biology,
St George's Hospital Medical School, London
SUMMARY
The mechanism of embryonic cell movement is poorly understood. Primordial germ cells
(PGCs) of the anuran amphibian Xenopus laevis migrate individually from their site of
determination in the embryonic endoderm to their site of differentiation, in the developing
gonad. PGCs have been isolated during their migratory phase from tadpoles, and their movement studied in vitro on a variety of natural and artificial substrates.
On all artificial substrates used, including acid-washed glass, tissue-culture plastics, polyL-Iysine-coated glass, and collagen, the PGCs move by amoeboid extrusion of hemispherical
lobopodia. Several considerations make it unlikely that this is the mechanism employed
in vivo.
On living cellular substrates, e.g. monolayers of Xenopus laevis embryonic cells, adult
kidney cells, and adult mesentery cells, PGCs become firmly attached and undergo phases
of elongation and contraction. They move by elongation, coupled with the extrusion of
filopodia, followed by waves of contraction, and ultimately by retraction of the trailing end
of the cell. Evidence is presented that this is the mode of locomotion normally employed
by PGCs in vivo.
INTRODUCTION
Morphogenetic movements are characteristic of the cells of early embryos
of all animals. The mechanism of embryonic cell movement, and the factors by
which they recognize their correct location, are poorly understood.
In vertebrate embryos, two cell types in particular, neural crest and primordial germ cells (PGCs), migrate individually (rather than in sheets) over quite
long distances from their sites of determination to their sites of differentiation.
We have studied the migration of the PGCs of Xenopus laevis (Wylie &
Heasman, 1976; Wylie, Bancroft & Heasman, 1976). These cells arise in the
vegetal pole of the earliest embryonic stages (Bounoure, 1954; Blackler, 1960;
Smith, 1966) and, after gastrulation, migrate from the embryonic gut to the
root of its dorsal mesentery, and thence laterally across the dorsal abdominal
wall, to the site of formation of the gonadal ridge.
Although several light and electron microscope studies have been made of
1
Author's address: Department of Structural Biology, St George's Hospital Medical
School, Cranmer Terrace, London SW17 ORE, U.K.
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J. HEASMAN, T. MOHUN AND C. C. WYLIE
this process (Witschi, 1929; Kalt, 1973; Whitington & Dixon, 1975; Kamimura, Ikenishi, Kotani & Matsuno, 1976), we still do not know the mechanism
of PGC movement, nor the factors which guide them to their destination.
This paper describes further studies on PGC movement in vitro, using a variety
of natural and artificial substrates. We have found that on living amphibian
cell monolayers, Xenopus PGCs move by a mechanism consistent with their
appearance in fixed, normal material.
MATERIAL AND METHODS
(1) Isolation ofPGCs. Embryos were obtained using chorionic gonadotrophin
to induce mating in pairs of toads. Embryos were allowed to develop to stages
42-43 (Nieuwkoop & Faber, 1956), and left overnight in distilled water containing 1 mg/ml sulphadiazine, to reduce the fauna and flora of the gut. The
tadpoles were dissected as previously described (Wylie & Roos, 1976), and
transferred to Ca2+- and Mg2+-free Steinberg's saline, pH 7-6, containing
0-1% trypsin and 0-05% collagenase. Disaggregated PGCs were collected
from the mesentery and dorsal body wall, and placed in Petri dishes containing
60% Liebovitz medium, 30% distilled water, 10% foetal calf serum, and a
variety of antibiotics (see later).
(2) Preparation of substrates. Glass slides were coated with collagen (prepared by the method of Ehrmann & Gey, 1956), or with 5 /^g/ml of poly-Llysine solution.
Monolayers of amphibian cells were obtained from X. laevis tadpoles of
stage 50 or older. These were left in distilled water, containing 1 mg/ml sulphadiazine, overnight and then sterilized externally by washing briefly in 70%
alcohol. The tadpoles were macerated and placed in disaggregating saline, as
above. After three serial trypsin treatments, cell pellets were resuspended in
60 % Liebovitz medium. We found that X. laevis tadpoles are the hosts of a
wide variety of bacteria and fungi. These were controlled in primary cultures by
gentamycin (50/^g/ml) and fungizone (2-5/*g/ml). A line of adult X. laevis
kidney cells (Harris Biological Ltd.) was maintained in 70% Liebovitz
medium, containing 20% distilled water, 10% foetal calf serum, and antibiotics.
Monolayers of adult mesentery cells from X. laevis were prepared by explanting pieces of adult mesentery, and stretching them beneath glass rings on plastic.
The outgrowing cells form a monolayer, which can be contact-inhibited when
it reaches the required diameter, by seeding adult kidney cells around it.
RESULTS
Serial sections of tadpoles of stage 42-45 (Nieuwkoop & Faber, 1956) show
PGCs at all stages along their migratory route (Fig. 1). They move up the
The locomotion of Xenopus germ cells in vitro
151
Fig. 1. Phase contrast photomicrograph of 2/tm thick araldite section, through the
posterior body wall and mesentery of stage-44 tadpole, showing three large PGCs
on the dorsal mesentery (A, B, and C).
Fig. 2. Micrograph of PGC on acid-washed glass (Nomarski optics) showing large,
hemispherical lobopodia.
Fig. 3. Sequence of micrographs (Nomarski optics) to show the adhesion of a
fibroblast from Xenopus laevis tadpole to plastic. L = lobopodium; arrow indicates
lobopodium which has adhered to the substrate.
Fig. 4. Micrograph (Nomarski optics) showing Rana sylvatica PGC attached to a
monolayer of Xenopus cells The yolk platelets are over-exposed in order to correctly
expose the filopodium.
dorsal mesentery to its root, and then laterally across the dorsal abdominal
wall, to the site at which the gonadal ridge will form upon their arrival (Wylie
& Heasman, 1976). We have isolated the PGCs from these stages, in order to
study their activity in vitro, on a variety of substrates:
(1) Acellular substrates: acid-washed glass, tissue-culture plastic, poIy-L-lysine
coated glass, and collagen-coated glass
On all these surfaces, PGCs are active, showing similar behaviour. They
extend large, clear, hemispherical lobopodia, into which they push the yolky
cytoplasm (Fig. 2, see also Wylie & Roos, 1976). However, several considera-
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J. HEASMAN, T. MOHUN AND C. C. WYLIE
Fig. 5. Phase contrast micrograph to show Xenopus PGC on a monolayer of adult
Xenopus kidney cells. It is elongated, and possesses a filopodium. The culture
has been fixed, and stained with OsO4.
Fig. 6. Phase contrast micrograph to show an isolated PGC on adult kidney cells.
It has a slight transverse constriction. The yolk platelets and lipids are stained
black with OsO4.
Fig. 7. Living PGC on adult mesentery cells. The transverse constriction almost
divides the cell in two.
tions make it unlikely that this process is similar to PGC movement in
vivo.
(a) When time-lapse films are made from the side, PGCs are seen to extend
lobopodia at all surfaces, in all planes of space. When sufficient cytoplasm has
been pushed into a lobopodium to alter the centre of gravity of the cell, it
topples over. When filmed from above, the PGCs tumble about in this manner
quite randomly, without becoming firmly attached to the substrate.
(b) No large, hemispherical lobopodia have been seen in either light or
electron microscope studies of Xenopus PGCs in vivo.
(c) Non-confluent X. laevis somatic cells in culture put out exactly the same
types of processes when adhering to the substrate. Figure 3 shows one such
somatic cell in the process of sticking down to a plastic culture dish. At first,
large hemispherical lobopodia are extended (Fig. 3d). Some of these flatten
out, after adhering to the substrate (Fig. 3 b). Finally, the cell becomes flattened
and stretched by its attached processes, and comes to resemble the other cells
in the dish (Fig. 3c).
It seems, therefore, that the lobopodia seen on PGCs on these substrates
represent attempts by the cell to adhere, by a mechanism common to all cell
types. The PGCs evidently cannot attach themselves properly to these substrates.
(2) Cellular substrates: cultured cell monolayers
When seeded onto monolayers of amphibian cells, PGCs exhibit behaviour
totally different from that observed on artificial substrates. For 2-3 days they
The locomotion o/Xenopus germ cells in vitro
153
do not stick to the underlying cells, but extend lobopodia, and change shape
in an apparently random manner. They then adhere firmly to the substrate,
and cannot be removed by washing. This strong attachment is not speciesspecific, since Rana syhatica and Rana pipiens PGCs will stick to X. laevis
monolayers, and vice versa. However, PGCs from none of these species will
stick to mammalian cells in culture, so some specificity is involved.
After adhering to the substrate, the PGCs remain obviously active, and exhibit a number of cellular phenomena which we have divided, rather arbitrarily,
into the following categories:
Filopodia formation. As on glass, PGCs put out yolk-free processes, but in
this case they are long thin filopodia, of variable length. Figures 4 and 5 show
such processes. As far as we can establish, filopodia are not extended in all
planes of space, but are confined to one pole of the elongated cell.
Elongation. Figures 4 and 5 also show that, in contrast to their behaviour on
artificial substrates, PGCs are able to elongate to a considerable degree on
the surfaces of the cells beneath them, achieving lengths of over 70 jum in the
process. The same PGC, viewed over a period, alternately elongates and rounds
up again.
Constrictions. Elongated PGCs often show transverse constrictions. These
vary in extent from those which produce only a slight 'waist' to the cell (Fig. 6)
to those which almost divide it in two (Fig. 7).
During the appearance of these phenomena, PGCs on cellular substrata
remain obviously motile when observed over long periods. We therefore
turned to time-lapse cinemicrography to see whether the cellular phenomena
observed play any identifiable role in the movement of these cells. It became
obvious from such time-lapse studies that PGCs move actively over the underlying cells, and that they do so only when performing sequences of elongation
and contraction. Rounded-up PGCs only move passively, by movement of
the underlying substrate. Figure 8 shows a sequence of frames from afilm,lasting
approximately 4 h. The cell is seen to show a distinct series of changes, during
which it becomes displaced by about 52 ju,m, over a cell diameter from its
original position. The PGC first extends a filopodium, some part of which presumably sticks to the substrate. At first this process is yolk-free (Fig. $b), but
soon the yolky cytoplasm is pushed forwards into the filopodium from behind
(Fig. 8c). The first part of the cell to enter the filopodium is the nucleus, though
this is not obvious from the print. Gradually, over the next hour (Figs. 8 c-f)
the yolky cytoplasm is pushed forward into the leading end of the cell. At the
point which marks the original base of the filopodium, the cortex fails to expand
and a well-marked constriction develops. Yolk granules are clearly seen to
accelerate through this constriction, suggesting that it may aid in generating
the propulsive force of these cells, but since not all motile PGCs show such
constrictions, they do not seem to be vital to the movement process. Following
the forward movement of the cytoplasm the trailing end of the cell is withdrawn,
and the PGC rounds up into its new position (Fig. 8g).
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J. HEASMAN, T. MOHUN AND C. C. WYLIE
The locomotion o/Xenopus germ cells in vitro
155
Figure 9 shows a sequence of frames from a different film, illustrating another
phenomenon frequently seen in PGC movement. The cell is already elongated
at the start of this sequence. The cytoplasm is pushed forwards into the leading
end of the cell by a series of peristalsis-like waves of contraction. These pass
forwards from the rear of the cell, giving it an undulating appearance in the
film. As more yolk passes forwards, the trailing end becomes narrower, giving
the cell a pear-shaped appearance. The trailing end is then withdrawn (Fig.
9c-d). Following this, the PGC pushes forwards again (Fig. 9e), the same
process is repeated, and the trailing end withdrawn (Fig. 9/). This sequence of
frames lasted for 7 h, and resulted in displacement of the PGC by about 81 jum,
from leading edge to leading edge.
The question next arises as to whether the mode of locomotion demonstrated
by PGCs in vitro bears any relation to their normal movement in vivo. Evidence
for such a supposition must include the demonstration, in fixed normal material,
of the following:
(1) PGCs both rounded up, and elongated.
(2) Transverse constrictions, and/or evidence of contraction of the hind end.
(3) Filopodia, at the leading end of the cell, i.e. nearest the site of gonadal
ridge formation.
These facts are not easy to establish in fixed material. At the early stages in
mesentery formation, there are only about 15 PGCs per tadpole; of these,
only a small proportion may be motile at any one time. However, careful
study of thick and thin sections shows a striking similarity between PGCs in
FIGURE 8
Fig. 8. Series of frames from a time-lapse film, showing movement of PGC over a
cellular substrate.
(a) Time 0: There are two PGCs in the field of view. One is rounded up (arrowed)
and one is elongated.
(b) 115 min: One of the PGCs has now extended a filopodium (arrowed).
(c) .122 min: The filopodium is now longer. Material from the body of the cell has
begun to enter the filopodium. The area at its base is, in fact, the nucleus, although
this is not clear from the print.
(d) 143 min; (e) 148 min: Progressively more of the yolky cytoplasm has been
propelled forwards,fillingthefilopodium.A constriction (C) is developing at a point
which corresponds to the original base of the filopodium.
(/) 183 min: Active streaming forwards of the cytoplasm has continued. The
constriction is now more marked. Anterior to the constriction is cytoplasm which
has been propelled forwards.
(g) 239 min: During this period the field of view has drifted out of focus. However,
it can still be seen that the PGC has withdrawn its trailing end, and rounded up
again.
(h) During this sequence of events, the PGC moves by over a cell diameter. This
tracing shows the first and last frames superimposed.
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J. HEASMAN, T. MOHUN AND C. C. WYLIE
The locomotion o/Xenopus germ cells in vitro
157
vitro and in vivo. Figure 10 shows a cluster of PGCs at the root of the mesentery
in a Rana sylvatica tadpole. Two are rounded up, and one is clearly elongated.
Furthermore, when examined with high-power Nomarski optics (Fig. 10&), the
leading end of the elongated cell is seen to possess a filopodium and, towards
the rear of the cell, a transverse constriction is present. This PGC may be undergoing the sequence of activities seen in vitro in Fig. 8. One difference is that
in vivo the PGC is surrounded on all sides by cells, not just at one surface, as
in vitro. Given that difference, the PGCs in Figs. 8 and 106 are strikingly similar.
We have also, on two occasions, found filopodia extending from the leading
ends of PGCs in electron micrographs of fixed, normal embryos (Fig. 11).
The scarcity of these is not necessarily evidence of their insignificance, for
relatively few PGCs in a tadpole may have extended filopodia at the time of
fixation. We know that filopodia remain extended after fixation, from their
appearance in fixed cultures (Fig. 5).
Many PGCs in vivo, along the normal course of their migration, show constrictions similar to those found in vitro. Figure 12 shows such a cell, towards the
root of the mesentery. It bears a distinct resemblance to the PGC which is
demonstrably motile in Fig. 9. We also find that the degree of constriction
varies in vivo. Figure 13 shows a thick section (400 nm) viewed under the electron
microscope, exhibiting the 'waist' often seen in vitro (compare with Figs. 6
and 8 c).
DISCUSSION
The results presented here show that PGCs isolated from X. laevis embryos,
at the stage when they are normally migrating to the gonadal ridge, exhibit
motility when placed on cultured monolayers of Xenopus cells. The evidence
suggests that the PGCs move by a mechanism consistent with their appearance
in vivo. This mechanism seems to include:
(1) The extrusion of filopodia at their leading ends. This establishes the
polarity of the cell. The exact role of the filopodium is uncertain; it may be
involved in direction finding, or in forming a new attachment to the substrate.
FIGURE 9
Fig. 9. Series of frames from a time-lapse film.
(a) Time 0: The PGC is already elongated.
(b) 35 min; (c) 91 min: The trailing end of the cell is becoming thinner, owing to
waves of contraction passing along it, which push the cytoplasm forwards.
(d) 175 min: The trailing end is now being retracted, with net displacement of the
PGC on the underlying cells, which do not move.
(e) 301 min: The leading pole has now pushed forwards again.
(/) 441 min: Following more contractions, the trailing end is withdrawn again.
(g) Tracings of the positions of the PGC in the first and last frames, to show the
displacement of the cell during this process.
II
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J. HEASMAN, T. MOHUN AND C. C. WYLIE
The locomotion o/Xenopus germ cells in vitro
159
Filopodia have been reported to be involved in the movement of several cell
types, e.g. fibroblasts of neural crest origin (Bard & Hay, 1976), sea-urchin
coelomocytes (Edds, 1977), and ascidian tunic cells (Izzard, 1974). The latter
two cell types show multiple filopodia per cell.
(2) Contractions of the cell surface. These seem to be of two types: single
constrictions, and peristaltic waves. The latter are seen in time-lapse films to
push the yolky cytoplasm forwards. The yolk granules are an excellent marker
for this process. Constrictions are seen frequently, both in vitro and in vivo.
However, we do not know their role in locomotion. One possibility is that they
are the site of anterior attachment of the cell, and thus represent a fixed point
past which the cell contents can be pushed. The fact that they seem to arise at
the point of origin of the filopodia seems to support this hypothesis.
(3) Retraction of the trailing edge of the cell. The PGCs are stuck down very
firmly to the underlying monolayer, and cannot be removed by vigorous washing.
Adhesive contacts must be made and broken during cell movement, but the
nature of these remains unknown.
It is unusual for cells to adhere to living monolayers in vitro. DiPasquale &
Bell (1974) have shown that a variety of epithelial cells and fibroblasts fail to
stick to or move over one another's upper surfaces in culture. PGCs appear
to be an exception to this rule. We have shown in a previous study (Wylie &
Heasman, 1976) that the substrate for normal migration of PGCs is the inner
surface of the coelomic lining cells which form the dorsal mesentery of the gut
(see also Fig. 13). A cellular substrate is therefore normal for them.
The fact that 2 or 3 days elapse before PGCs adhere to monolayers suggests
that, following trypsin treatment, they need to resynthesize surface components
before they can continue with their normal functions.
The rates of movement described here are very low. We have recorded displacements of 50-100 /tm, in sequences lasting from 2 to 24 h. These rates are
misleading, because PGCs spend variable lengths of time rounded up between
FIGURES
10-12
Fig. 10. Photomicrograph (Nomarski optics) of an unstained 2/*m section through
the root of the mesentery of a Rana sylvatica tadpole. The fixation has fortuitously
caused cell contraction, and thus the boundaries of the PGCs can be seen. Several
PGCs can be seen, two of which are rounded up (A and B), and the other elongated
(C). The elongated PGC possessed a filopodium (F) at its leading end, and a
constriction (C), shown in high magnification in Fig. 10(6). N = Nucleus of coelomic
lining cell.
Fig. 11. Electron micrograph of a PGC from stage-44 Xenopus tadpole. The cell
situated at the root of the mesentery, possesses a filopodium, from its leading end,
which separates two somatic cells (SC).
Fig. 12. Micrograph (Nomarski optics) of an unstained araldite section (2/«n).
A large PGC is seen arriving at the root of the mesentery. It shows a transverse
constriction. Note its resemblance to the PGC filmed in vitro (Fig. 9).
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J. HEASMAN, T. MOHUN AND C. C. WYLIE
Fig. 13. Low power electron micrograph of a thick section (400 nm) from the
mesentery of a stage-44 tadpole. The constriction (C) is shown for comparison
with the one seen in vitro in Fig. 6.
active phases. The rates of movement are, however, entirely consistent with the
observed distances of migration in vivo. The distance between gut tube and
site of gonadal ridge formation is about 1-2 mm (see Fig. 1) at the time of
germ-cell migration, and the time taken for migration is days, rather than hours.
We are still faced with many problems with regard to PGC locomotion. We
need to study their ultrastructure carefully, to establish whether or not the
cellular specializations observed possess the structural and contractile elements
necessary for the postulated mechanism of locomotion. We do not know how
the germ cells attach themselves to the substrate. This obviously plays a crucial
role in their movement both in vivo and in vitro. Lastly, we do not know the
factors which guide the PGCs to their destination. They move in random directions in vitro; however, they are always polarized, with one pole leading.
Despite these many unknowns, PGCs in vitro constitute a potentially exciting
model system, with which we can study the cellular and molecular basis of germ
cell locomotion and guidance.
Our grateful thanks are due to Mrs M. Reynolds and Mr R. F. Moss for excellent technical
assistance; and to Liz Adam, for recent help with the electron microscopy of thick sections.
Our thanks are also due to Professor R. D. Allen, in whose laboratory at Dartmouth
College part of this work was undertaken (supported by grant no. GM-22356 from the
National Institute of Health).
One of us (J.H.) is a Teaching and Research Fellow, supported by the Wellcome Trust,
whose help is gratefully acknowledged.
Tim Mohun is supported by the Cancer Research Campaign, whose generous financial
support for this work is gratefully acknowledged.
The locomotion o/Xenopus germ cells in vitro
161
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{Received 25 March 1977, revised 8 July 1977)