/. Embryo., exp. Morph. Vol. 44, pp. 71-80, 1978
Printed in Great Britain © Company of Biologists Limited 1978
71
Cinemato graphical study of cell migration in the
opened gastrula of Ambystoma mexicanum
By H. Y. KUBOTA 1 AND A. J. DURSTON 2
From the Hubrecht Laboratory, Utrecht, Netherlands
SUMMARY
The migration of inner marginal cells was studied in the Ambystoma gastrula, using
scanning electron micrography and time-lapse cinemicrography.
Scanning electron micrographs of gastrulae which were fixed while intact revealed that
the migrating cells have flattened lamellipodia at their anterior end and a rounded cell body,
which can sometimes be seen to be attached to a neighbouring cell by a slender posterior
process. Films of opened gastrulae showed actively moving cells, with the same features
described above. Details of their movements are reported and discussed in relation to the
mechanism of gastrulation.
INTRODUCTION
The morphogenetic movements that occur during amphibian gastrulation
probably depend on many factors. These may include: extension and imagination of flask cells (Rhumbler, 1902; Ruffini, 1925; Holtfreter, 1943tf, 1944;
Baker, 1965); extension of mesoderm and endoderm (Vogt, 1929; Schechtman,
1942); epibolic spreading of ectoderm (Schechtman, 1942; Holtfreter, 1943&,
1944); differential adhesiveness of the germ layers (Trinkaus, 1969); and active
migration of invaginating cells (Nakatsuji, 1974; \915a,b, 1976). The importance of the various factors, their relations to each other, and the ways in which
cell behaviour is coordinated in the gastrula are not well understood.
The analysis of gastrulation would be facilitated if we could observe details
of the cell behaviour associated with this process in vivo. This has been achieved
for morphogenetic movements in the transparent embryos of sea urchins and
fishes, by using time-lapse cinemicrography (Dan & Okazaki, 1956; Gustafson
& Kinnander, 1956; Gustafson & Wolpert, 1963; Trinkaus & Lentz, 1967;
Trinkaus, 1973). It has provided important insights into gastrulation and epiboly
in these animals. In amphibia, the inner cell movements associated with gastrulation have never been observed because the germ layers are thick and opaque.
In the present study, we have attempted to overcome this technical difficulty by
1
Author's address: Laboratory of Developmental Biology, Department of Zoology,
Faculty of Science, Kyoto University, Kyoto, 606 Japan.
"Author's address: Hubrecht Laboratory, Uppsalalaan 8, Universiteitscentrum 'De
Uithof', Utrecht, The Netherlands.
72
H. Y. K U B O T A A N D A. J. D U R S T O N
(b)
(«)
Fig. 1. Schematic representation of the operation, (a) The ventral and lateral
marginal zone of the initial gastrula (stage 10) is cut at the place indicated using
sharp tungsten needles, (b) The animal cap is opened on the collodion-coated coverslip. En, Inner endodermal cell mass; A, animal cap; B, blastopore; D , dorsal
marginal zone.
opening axolotl gastrulae and filming the inside of the gastrula. We report some
qualitative details of the movement of migrating inner marginal cells and discuss
the mechanism of gastrulation.
MATERIALS AND METHODS
Eggs of the axolotl, Ambystoma mexicanum, were used throughout. The eggs
were manually de-jellied with sharpened watchmaker's forceps and stored in
sterile tap water.
Operation and cinematography
Operations were carried out in Steinberg's solution, buffered to pH 7-6-8-0
by 3 mM HEPES-NaOH instead of Tris-HCl, and containing Ca 2+ and Mg 2+ at
one-third strength. The vitelline membrane was removed just before operation.
The operations were performed, using a hair loop and a tungsten needle, on
a layer of 1 % agar in a glass dish. The ventral and lateral marginal zone of the
initial gastrula (usually stage 10 of Harrison) (Harrison, 1969) was cut with
special care so as not to damage the endodermal mass, and the animal cap was
opened on a collodion-coated coverslip in a glass dish (Fig. la, b). The margin
of the opened animal cap was weighted by glass bridges made of a fragment of
coverslip, to prevent it from curling up. Time-lapse cinematographs were made
from above, using incident light, and a frame interval of 15 sec to 4 min.
Fixation and processing for scanning electron microscopy
Embryos were fixed for 15 h in a mixture of 2-5 % glutaraldehyde and 1 %
acrolein in 0-05 M cacodylate buffer at pH 7-6. In order to expose the inside of
the embryos to fixative, they were cut in various planes during prefixation, using
sharp tungsten needles and a razor blade. They were then washed for at least
10 h in three changes of 0-05 M cacodylate buffer, and post-fixed for 15 h in 1 %
osmium tetroxide in phosphate buffer at pH 7-6. They were dehydrated in
ascending concentrations of acetone, and critical point dried, using liquid CO2,
Cell migration in gastrula of Ambystoma
Fig. 2. A scanning electron micrograph of the inner endodermal mass of the initial
gastrula (stage 10) from which the animal cap was removed. En, Inner endodermal
mass; D, dorsal marginal zone; m, inner marginal cells.
Fig. 3. Inner endodermal cells of the initial gastrula (stage 10) are connected to each
other by many fine cell processes.
Fig. 4. Scanning electron micrograph of the early gastrula (stage 10£), cut in the midsagittal plane. Inner marginal cells are seen on the inner surface of the blastocoel
wall.
Fig. 5. Inner marginal cells moving across the inner surface of the blastocoel wall.
Leading cells have very thin lamellipodia at their front end. Pseudopodia from
following cells may overlap the leading cells. Leading cells may be attached to
following cells by a thin process. Bl, Inner surface of the blastocoel wall; /, lamellipodium; pp, posterior process.
73
74
H. Y. KUBOTA AND A. J. DURSTON
0-5 mm
Fig. 6. Frames (separated by 156 min intervals) from a film showing the movement
of inner marginal cells. Note that leading cells form flattened pseudopodia from
their advancing end. En, Inner endodermal cells; m, inner marginal cells; Bl, blastocoel wall.
while mounted on aluminium stubs covered with conducting silver paint. They
were coated with gold and examined in a Cambridge S4 'stereoscan' scanning
electron microscope (SEM).
RESULTS
1. Scanning electron microscopy
As a control for any abnormalities in opened gastrulae and to aid interpretation of the time-lapse films, we made scanning electron micrographs of
inner marginal cells in gastrulae that were fixed while still intact. Fig. 2. shows
an upper view of the endodermal mass of an initial gastrula (Harrison stage 10),
from which the animal cap was removed after fixation. At this stage the blastopore has formed along the edge of the dorsal marginal zone, but the inner
marginal cells remain quiescent. The figure shows some small cells (m) between
the dorsal marginal cells (D) and the endoderm (En). These are inner marginal
cells. The endoderm cells are connected to each other by fine processes, but no
Cell migration in gastrula of Ambystoma
75
125/<m
Fig. 7. High power view of migrating cells. Fan-shaped cells are each connected to
posterior tissue by a posterior process. Rounded cells have probably lost contact with
the ectoderm./, Fan shaped cell; r, rounded cell.
cells have lamellipodia at this stage (Fig. 3). Fig. 4 shows the inside view of
a later gastrula (stage lO-}), cut through the mid-sagittal plane. Inner marginal
cells are now migrating across the inner surface of the ectodermal layer. Most
migrating cells adhere loosely to each other, but some are well separated. Each
has thin lamellipodia (/) at its leading edge and, typically, a rounded rear end.
Following cells may cover the backs of leading cells or leading cells may be
connected to following tissue by a single slender posterior process (Fig. 5).
2. Time-lapse
cinematography
Embryos were opened at various stages and (usually) filmed till after the
inner marginal cells finished migration. The following observations are from
20 successful films.
Inner cell migration begins by about 1 h after stage 10£ (embryos opened at
blastula stages do not show premature migration). At this stage the blastoporal
groove has just formed, as a depression of the surface of the inner marginal
zone and most or all of the inner marginal cells are hidden under the endodermal
cell mass. They now emerge and migrate away from the endoderm, across the
surface of the ectodermal layer (Fig. 6). The active movements of these cells are
directional (forward and approximately perpendicular to the local boundary of
the inner marginal tissue; never towards, over or under posterior neighbours).
They are often, but not invariably, intermittent. Individual leading cells advance
till their cell body is 25-50 /*m ahead of neighbouring cell bodies, then either
stop until following cells catch up with them or are pulled back (see below).
They then advance again.
76
H. Y. KUBOTA AND A. J. DURSTON
In clear films one can see that all migrating cells have anterior lamellipodia,
as seen in the SEM (compare Fig. 5 with Fig. 6C, D, E; Fig. 7) and that all are
connected to following tissue. In many cases the rear end of a leading cell extends
into a slender process, which is connected to a following cell (as in the SEM:
Fig. 5). These cells migrate as far ahead of neighbours as permitted by the restraining process. They thus become elongated and fan-shaped (Fig. 7). Other
leading cells are overlapped by posterior neighbours (as in the SEM: Fig. 5), but
are probably also restrained by posterior processes. Particular cells can be seen,
at different times, to be connected by a posterior process and to be overlapped
by neighbours. Intermittently moving cells become fan shaped as they move ahead
and remain so when they stop. If they are pulled back, they round up and
retract their lamellipodia. Presumably, they lose contact with the ectodermal
substrate and are pulled back by contraction of the posterior process.
At the beginning of some film sequences, one can see isolated cells that are
well ahead of the migrating cell mass (> 100 /*m) and evidently not connected
to it. We suspect that these, as well as isolated cells seen in SEM, are separated
when the embryo is processed (opened or fixed, respectively). Isolated cells seen
in films move at a reduced rate, or may even fail to move, suggesting that
interactions among migrating cells are required for the normal directed movement. To test this idea, we transferred small groups of middle gastrula mesoderm
cells (stage 11) onto the ectodermal sheet of an early gastrula (stage 10£). These
cells move apart, but do not move in any preferred direction relative to the
orientation of the ectoderm. This result will be discussed in detail elsewhere
(Kubota, in preparation). It supports the idea that interactions among migrating
cells, rather than interactions between migrating cells and ectoderm lead to
directed migration.
Posterior (endoderm) cells follow inner marginal cells across the ectoderm
surface. They never overtake them. This fact may also be relevant for understanding gastrulation (see Discussion).
The trajectory of migration in an opened gastrula differs from that in an
intact gastrula. The leading cells may migrate across the ectodermal region
corresponding to the animal pole (their final destination in the intact embryo).
They may continue till they reach the glass bridge placed on the cut edge of the
ectoderm. This corresponds to the ventral marginal zone in the intact embryo.
The velocity of migration at the leading edge is about 60/mi/h. This is slow
compared with the velocity in normal gastrulation (Ignat'eva, 1963). No mitoses
were observed among the migrating cells.
More than half of the embryos filmed showed intermittent pulsations, with
a mean interval of about 30 min. These varied in position, timing and type.
They occur most often in the ectoderm or in the ectoderm/mesoderm bilayer
(but sometimes also in the endoderm). They commonly entail localized bulging
of the cell sheet (causing local separation of the apices of endoderm or mesoderm
cells), followed by relaxation. In most of the successful films these pulsations
Cell migration in gastrula of Ambystoma
77
either had no clear relation to migration (the cell front moved more or less
smoothly) or else the relationship could not be assessed. In two cases, the inner
marginal cells were seen to make synchronized intermittent movements. In the
clearer case, they went through synchronous activity cycles similar to those
described for individual cells above. These were synchronized with contractions
of the underlying ectoderm.
DISCUSSION
The migration of inner amphibian gastrula cells has been investigated using
vital staining, transmission electron microscopy and scanning electron microscopy. It has never previously been observed directly. In this study we made
time-lapse films of inner cells in gastrulae opened under conditions intended to
disturb gastrulation as little as possible. We used a culture medium having a low
Ca 2+ level (0-005 g/J.) and high pH (8-0), similar to blastocoel fluid in the
early gastrula (Stableford, 1949, 1967). In this medium, inner marginal cells are
not excessively adhesive; they migrate freely; and they resemble inner cells in
intact gastrulae in their shape and general mode of migration. We believe that
inferences made about these opened gastrulae will be useful for understanding
normal gastrulation. We note that opening the gastrula has consequences for
the migration process. One important effect is that the trajectory of migration
is altered, as described above. This leads to conclusions about the mechanism
of gastrulation (see below).
From both the time-lapse films and the scanning electron micrographs, it was
evident that inner marginal cells begin to make lamellipodia and to migrate soon
after stage 10£. Satoh, Kageyama & Sirakami (1976) have observed that isolated
amphibian cells show a sharp increase in the incidence of circus movement at
the beginning of gastrulation. This may reflect the onset of competence to make
lamellipodia in the intact embryo. Our films make it clear that inner marginal
cells migrate actively over the ectodermal substrate. Nakatsuji (19756, 1976) had
previously suggested that this was so and, further, that mesodermal and
pharyngeal endoderm cells provide the main motive force for gastrulation, and
tow the (passive) vegetal endoderm cells into the embryo . Our observations from
films are compatible with his second suggestion (since endoderm cells keep
their (posterior) position relative to the inner marginal cells). They do not
prove it.
Our observations provide general clues as to the way in which cell migration
is controlled in the gastrula. The fact that rather normal migration occurs in
opened gastrulae rules out models for directed migration that depend on the
integrity of the gastrula (e.g. migration up an electrical potential gradient set
up by active transport of Na + into the blastocoel (Cohen & Morrill, 1969)). The
fact that, in opened gastrulae, leading cells overshoot their normal end point and
proceed till an obstacle (the glass bridge) prevents further progress also rules
out models that depend on a fixed attractive point in the ectoderm (e.g. chemo6
EMB
44
78
H. Y. KUBOTA AND A. J. DURSTON
taxis to a fixed source of chemotactant or contact guidance along a system of
fibrils leading to a fixed destination). It is also clear that cell migration is not
limited by contact with a fixed ectodermal template (see Gustafson & Wolpert,
1963). The geometry of migration does not rule out certain types of model that
depend on an ectodermal guidance system. For example: a chemotactant
maximum might be defined by peaks in concentrations of the reactants in
a Turing-type system (Turing, 1952). If a cut ectodermal edge transiently
becomes a leaky boundary for these reactants, this could result in formation of
attractant maxima at the cut edges of the ectoderm, with results for cell
migration as observed. However, previous evidence already suggests that
migrating cells are not guided by the ectoderm (Cooke, 1972; Spemann, 1967),
and our observations of migration by isolated cells support this view (see
Results, p. 6).
Our observations of the behaviour of inner marginal cells lead us to suggest
that migration is controlled by interactions between these cells and endoderm
cells and probably also by interactions among the migrating cells themselves.
The most important features of migration are directional (forward) movements,
by connected cells, moving at the limits of their posterior processes and low
amplitude movements without predictable orientation by isolated cells and cells
in small groups. These features would result if cells are stimulated to move
directionally because they are repelled by their posterior neighbours. Initially,
these neighbours will be endoderm cells. Later, the effective neighbours could
either be vegetal endoderm (now distant) or following migrating cells, or
both. This interpretation is consistent with the general geometry of gastrulation
(movement away from the endoderm, to the edge of the ectoderm sheet in
opened gastrulae; movement away from the endodermal mass till the leading
(pharyngeal endoderm) cells meet similar (ventral endoderm) cells in the intact
gastrula). The specific possibilities are: that migrating cells show negative
chemotaxis (to a substance made by endoderm cells or by themselves and
endoderm cells) or that there is an interaction based on cell contact. We note
that negative chemotaxis has already been demonstrated in some amphibian
cells (Twitty & Niu, 1954). We are investigating both possibilities. Any specific
model for the control of cell migration must take into account the details of
gastrulation; for example, that the leading (prechordal and presumptive
notochord) cells are eventually overtaken by posterior (pharyngeal endoderm)
cells in the intact blastula, but not in opened gastrulae. We do not exclude the
possibility that fluid exchange between the archenteron and the blastocoel may
also play a role in regulating tissue movements during later gastrulation.
We were interested in the pulsations that occur in opened embryos because
it seems probable that gastrulae contain a signalling system or systems that
co-ordinate the behaviour of the many cells involved in the process. The
discussion above concerns possibilities for the control of one facet of gastrulation (inner cell migration). The pulsations show that numbers of ectoderm,
Cell migration in gastrula of Ambystoma
79
mesoderm and endoderm cells can be triggered to move or contract synchronously and also that movements of the ectoderm and of migrating cells can
be synchronized with each other. They may reflect the existence of a signalling
system (or systems) that regulates normal gastrulation and also cell migration,
via the interactions proposed above. However, from the variability of the
pulsations and the fact that high-amplitude pulsations like them are seldom
detectable at the surface of intact gastrulae, we deduce that they reflect a derangement of the normal system produced by wounding.
We are grateful to Professor P. D. Nieuwkoop and Dr J. Faber for discussion and
criticism and to Dorothy Parsons for the typing. We thankDr W. Berendsen (State University,
Utrecht) for taking the SEM photographs and Dr J. Bluemink and W. v. Maurik, for help
with the SEM. H. Y. Kubota received a U.N.E.S.C.O. travel fellowship.
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{Received 27 June 1977, revised 27 October 1977)