1 Division of Mammalian Development, National Institute for Medical Research,
The Ridgeway, Mill Hill, London NW7 1AA, UK
2 Division of Developmental Biology, National Institute for Medical Research,
The Ridgeway, Mill Hill, London NW7 1AA, UK
3 Wellcome Trust/Cancer Research UK Institute and Department of Zoology,
University of Cambridge, Tennis Court Road, Cambridge CB2 1QR, UK
Author for correspondence (e-mail:
shankar{at}srinivas.org)
Accepted 25 November 2003
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SUMMARY |
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Key words: Mouse embryo, Anterior visceral endoderm, Patterning, Morphogenesis, Migration, Embryo culture, Time lapse imaging
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Introduction |
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DiI-labelling experiments show that the AVE moves unilaterally from its
initial position at the distal tip of the egg cylinder to the future anterior
of the embryo, thereby converting a proximodistal axis to an anteroposterior
axis (Thomas et al., 1998).
Embryos mutant for cripto or Otx2 fail to effect this movement
(Ding et al., 1998
;
Kimura et al., 2000
;
Perea-Gomez et al., 2001
), and
the epiblast becomes mispatterned such that the distal region adopts an
anterior character and the proximal region a posterior character.
The central cells of the blastocoel floor of the Xenopus embryo
(Jones et al., 1999) and the
hypoblast of the chick embryo (Foley et
al., 2000
) are thought to correspond to the mouse AVE. The fates
of these regions are similar to that of the AVE and, like the AVE, they
express genes such as Hex. The regions also show functional
similarities, capable of imposing anterior character on Xenopus
ectoderm (Jones et al., 1999
)
and restricting the formation of multiple primitive streaks in chick
(Bertocchini and Stern, 2002
).
Significantly, the anterior movement of AVE cells is conserved in the
equivalent tissues in both species (Jones
et al., 1999
), again highlighting the significance of this
process.
Although the unilateral movement of AVE cells is crucial for anterior
patterning of the embryo, very little is known about how it takes place. One
suggestion is that it involves different rates of proliferation of anterior
and posterior cells of the visceral endoderm or the polarised division of
these cells (Lawson and Pedersen,
1987; Thomas et al.,
1998
). Another is that the AVE might be carried unilaterally as
part of a global movement of the entire visceral endoderm
(Weber et al., 1999
). In
contrast to these passive means of movement, it is also possible that AVE
cells actively migrate to their anterior position, implying that they respond
to, or are directed by, environmental cues. It is impossible to distinguish
between these possibilities by studying fixed specimens, and we have therefore
developed a system to observe AVE movement in real time.
Our results show that AVE cells actively migrate from the distal tip of the egg cylinder to presumptive anterior regions. Time-lapse imaging reveals that this migration comes to an abrupt halt at the junction of the epiblast with the extra-embryonic ectoderm. Cell tracking reveals that once they reach this border, cells spread laterally in both directions, with highly convoluted paths. Confocal microscopy shows that migrating AVE cells retain direct contact with the epiblast at all times. Together, our results show that the anterior movement of AVE cells is the result of active cell migration, perhaps in response to cues from the epiblast or the extracellular matrix surrounding it.
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Materials and methods |
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Time-lapse imaging of embryos
Phase contrast and epifluorescence digital time-lapse images were acquired
using the Deltavision system from Applied Precision. Embryos were cultured
directly on the stage of an Olympus IX70 inverted microscope and imaged using
an Olympus 20x objective with a numerical aperture of 0.4. EGFP was
excited using a standard 100 W mercury vapour lamp (Osram), with 490/20
excitation and 528/38 emission filters from Chroma. Exposure times were
between 0.5 and 1.0 seconds per fluorescent image. Five images from different
focal planes were usually captured at each time point. Fluorescent images from
multiple focal planes were de-convolved and an extended-focus image was
projected for each time point. When cultured embryos drifted in the field of
view, projected images from different time points were manually set in
register using Adobe Photoshop. QuickTime movies were compiled from individual
still images using the Graphic Converter programme.
Quantitation of filopodial orientation
For the purpose of this study, filopodia were defined as cellular processes
that were transient, were less than a fourth of a cell diameter in width at
their base, and had at least one side that formed an angle of less than
120° with respect to the tangent that passed through the base of the
filopodium. The Volocity program (Improvision) was used to outline 23
migrating AVE cells from seven embryos and then to calculate the centroids of
each cell. The base and the tip of each filopodium on each cell were also
marked, and their coordinates were determined. The length of each filopodium
was calculated as the square root of
[(x2-x1)2+(y2-y1)2],
where (x1, y1) and (x2, y2) are
the co-ordinates of the base and the tip of the filopodium. The `radius' of
the cell was computed using the same formula, except that (x1,
y1) and (x2, y2) were the coordinates of the
base of the filopodium and the centroid of the cell. The length of the
filopodium was expressed as a fraction of the `radius' of the cell. The angle
of the filopodium with respect to the proximodistal axis of the embryo was
determined as the arc tangent of
[(y2-y1)/(x2-x1)], where
(x1, y1) and (x2, y2) are the
coordinates of the base and tip of the filopodium, respectively. The programme
CricketGraph was used to plot the data as a polar graph.
Cell tracking
Cell tracking was performed using the tracking module of the Volocity
programme (Improvision). Eight cells were manually outlined using a graphics
tablet and their centroids calculated at each time point. The path taken by
cells was generated using Volocity. Data on the positions of the cells were
imported into Microsoft Excel, where all further calculations were performed.
The ratio of the movement of each cell was calculated as the absolute value of
(y2-y1)/(x2-x1) where
(x1, y1) and (x2, y2) are the
coordinates of the centroids of a cell at two consecutive time points. The
ratios of all eight cells tracked were then averaged for each time point. The
distance separating two cells was calculated as the square root of
[(x2-x1)2+(y2-y1)2]
where (x1, y1) and (x2, y2) are
the co-ordinates of the centroids of cells 1 and 2, respectively. The distance
covered by a cell in each time interval was computed using the same formula,
except that (x1, y1) and (x2, y2)
were the coordinates of the same cell at two consecutive time points.
Phalloidin staining and confocal imaging of embryos
Embryos were fixed for 1 hour at 4°C in a solution of 4%
paraformaldehyde in PBS, rinsed once at room temperature in PBT (0.1%
Triton-100 in PBS) and then stained for 2 hours at 4°C in 0.5 µg/ml
TRITC-Phalloidin (Sigma) in PBT. They were then washed once at room
temperature in PBT and mounted on a slide using DAPI-Vectashield mounting
medium (Vector Laboratories). Confocal images of the embryo were captured on a
Leica TCS-SP upright microscope and de-convolved using the Hugyens programme
from Scientific Volume Imaging. Confocal stacks were rendered as 3D volumes
using Volocity (Improvision).
Whole-mount in situ hybridisation
Embryos were dissected at roughly 5.75 dpc, after the AVE was likely to
have moved anteriorly. Embryos were fixed in a solution of 4% paraformaldehyde
in PBS and in situ hybridisation was carried out following standard procedures
(Wilkinson, 1992).
Hex (Thomas et al.,
1998
), and Cer1
(Thomas et al., 1997
) probes
were as described.
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Results |
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Embryos were dissected and set up in culture at approximately 5.5 dpc (early egg cylinder stage), when Hex is expressed at the distal tip of the embryo. Phase-contrast and EGFP fluorescence images were captured every 10-15 minutes over a period of 8-10 hours. Of 32 embryos cultured under these conditions, 27 developed normally, as indicated by morphological criteria (expansion of pro-amniotic cavity into the extra-embryonic ectoderm and overall growth) and by the unilateral shift of the AVE from the distal tip of the egg cylinder to the prospective anterior (Fig. 1, and see Movie 1 at http://dev.biologists.org/supplemental/).
AVE cells migrate up to a proximal boundary
In 12 of the 27 embryos that developed normally, we were able to discern
individual cells of the AVE in great detail as they moved to the prospective
anterior of the embryo. AVE cells at the distal tip of the embryo are
columnar, tightly clumped together and inactive (Figs
2,
5). As soon as movement is
initiated, however, they undergo a dramatic change in morphology. In
particular, they become squamous and motile, and they project filopodial
processes, primarily in the direction of motion
(Fig. 2,
Fig. 3A-D,G, and Movies 3 and 5
at
http://dev.biologists.org/supplemental/).
Filopodia are frequently greater than one cell radius in length
(Fig. 3G) and they often make
contact with surrounding cells (Fig.
3A-D), allowing for the possibility of intercellular
communication. AVE cells are occasionally seen to divide
(Fig. 3E,F), but not in any
consistent orientation. They show the hallmarks of migration, in that they
translocate and project filopodia, predominantly in the direction of their
motion.
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Tracking AVE cells
To compare the trajectories of individual AVE cells during migration, we
computed the positions over time of AVE cells in representative embryos.
Fig. 4 shows the tracks of
eight cells in the embryo shown in Fig.
2 (see Movies 2 and 3 at
http://dev.biologists.org/supplemental/).
The tracks of AVE cells in two other embryos (Movies 4, 5, 6 and 7) are shown
in supplementary Fig. S1. The tracks of the eight cells in
Fig. 4 show that not all reach
the extra-embryonic ectoderm. The leading cells (cells 1, 2 and 3 in
Fig. 4A) do reach the
extra-embryonic ectoderm and start to spread laterally. The cells behind them,
however (cells 4, 5, 6, 7 and 8 in Fig.
4A,B), stop migrating proximally and start spreading laterally
before reaching the extra-embryonic ectoderm, presumably because they are
obstructed by the leading cells. Cells 5 and 6, which are sisters, share a
common track before they divide. They have separate tracks after division, but
remain in contact with each other throughout their further migration
(Fig. 4B). The same is observed
for cells 4 and 7, which are also sister cells
(Fig. 4A,B).
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The tracks of all cells in Fig. 4 move to the left at the boundary. This is a result of the embryo `rolling' during culture (see Movie 2 at 7 hours of culture). To correct for this rolling, and to compare the lateral directions in which cells move at the boundary, we calculated the distances between cells over time, which should be less affected by the rolling. The separation between two representative pairs of cells is shown in Fig. 4D. Cells 1 and 3 move away from each other after reaching the boundary, whereas cells 2 and 4 move towards one another. This indicates that on reaching the boundary, the lateral movement of AVE cells does not occur in a coordinated manner, but that cells move independently of one another in either direction.
AVE cells migrate in direct contact with the epiblast
Our observations show that AVE cells migrate from the distal region of the
embryo to the junction between the epiblast and the extra-embryonic ectoderm.
Once AVE cells have migrated some distance proximally, EGFP-expressing cells
can be seen intermingled with non-expressing cells (Figs
2,
3). To investigate the
significance of this apparent mixing and to determine whether AVE cells
migrate on top of more proximal visceral endoderm cells, we examined
phalloidin-stained prestreak Hex-GFP transgenic embryos by confocal
microscopy.
The AVE is often discernible as a thickening of the visceral endoderm
(Fig. 5A). Kimura and
colleagues have suggested that the AVE is thicker than surrounding visceral
endoderm because it consists of two cell layers that form a stratified
cuboidal epithelium (Kimura et al.,
2000). However, detailed confocal imaging of six embryos shows
that the AVE comprises a single layer of approximately 10-15 cells, their
columnar nature accounting for the observed thickening
(Fig. 5). The columnar cells of
the AVE are polarised, their nuclei displaced towards the epiblast and showing
stronger actin staining on the surface away from the epiblast
(Fig. 5C).
As AVE cells migrate proximally, they lose their columnar nature and become squamous. In confocal sections through nine embryos, migrating AVE cells are never seen on top of other visceral endoderm cells but are apposed to cells of the epiblast (Fig. 5D). This suggests that anterior migration occurs directly on the epiblast rather than on other cells of the visceral endoderm.
Significantly, we observe that even prior to migration, when AVE cells are
at the distal tip of the embryo, they comprise a population of both
Hex-GFP expressing and non-expressing cells
(Fig. 5C), indicating that the
salt-and-pepper appearance of migrating AVE cells is likely to be a
consequence of the initial heterogeneity within the AVE. Although some
intermingling with non-AVE cells may occur during migration, this is likely to
be limited, because little cell mixing occurs in the visceral endoderm
(Gardner and Cockroft, 1998). A
similar salt-and-pepper pattern of expression is seen with endogenous
Hex transcripts as well as other markers of the AVE, such as
Cerberus-like (Fig. 6),
suggesting that expression of the Hex-GFP transgene is a true
reflection of Hex transcription and not a result of position effect
variegation.
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Discussion |
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Migration of AVE cells
As they begin to move proximally, cells of the AVE become squamous and
their behaviour becomes highly dynamic; they change their shapes continuously
and project filopodia in the direction in which they are moving. Our results
suggest that most of the movement of AVE cells can be accounted for by
migration. The increase in the surface area occupied by AVE cells as a result
of their becoming squamous might contribute to their translocation to some
extent, but is unlikely to be the primary motive force. For example, cells
occasionally switch positions with respect to one another (cells 3 and 4 in
Fig. 4A and Movie 3; cells 2
and 3 in supplementary Fig. S1, panel B, and Movie 7). This would not occur if
cell movement were the result of passive expansion due to changes in cell
shape. Cell division is also unlikely to contribute significantly to their
movement because cleavage does not occur with a consistent orientation and
because the movement is completed in four to five hours, during which time
only one or two AVE cells are usually observed to divide. The sister cells in
Fig. 4B, for example, have
migrated a substantial distance proximally before they divide. Global movement
of the surrounding visceral endoderm is also unlikely to contribute
significantly to AVE movement. Although most AVE cells move proximally, some
invariably remain at the distal tip of the embryo (Figs
1,
2). This would not occur if the
entire visceral endoderm were moving.
A barrier to migration
Our data show that AVE cells migrate proximally, to the boundary of the
epiblast and extra-embryonic ectoderm, and then abruptly begin to move
laterally. This boundary provides an endpoint to the proximal migration of AVE
cells and positions them such that they can undertake their subsequent role of
patterning the underlying epiblast. We note that not all migrating AVE cells
reach the boundary; rather, those that arrive first appear to prevent cells
behind them from moving further proximally. It is possible that cells at the
boundary become `squashed' by later-arriving cells, causing them to become
elongated along an axis parallel to the junction between the epiblast and
extra-embryonic ectoderm.
In retrospect, the existence of this boundary can be inferred from previous
cell lineage analyses. When pre-streak embryos are labelled in the region we
now recognise as the AVE, and then cultured to early streak stages, labelled
cells are observed to have moved proximally, but never beyond the epiblast
(Lawson and Pedersen,
1987).
What prevents leading AVE cells from migrating beyond the epiblast? One
possibility is that migration is controlled by the underlying epiblast.
Confocal images show that AVE cells migrate in direct contact with the
epiblast. The functions of genes such as cripto and ß-catenin, which are
necessary for AVE migration, are required in the epiblast and not the visceral
endoderm (Ding et al., 1998;
Huelsken et al., 2000
). If AVE
cells require contact with the epiblast in order to migrate, this would
explain why they do not migrate onto the extra-embryonic ectoderm.
Alternatively, signals from the extra-embryonic ectoderm (or the visceral
endoderm overlying the extra-embryonic ectoderm) might actively repel AVE
cells, preventing them from migrating beyond the epiblast. We note that later
in development AVE cells do move beyond the epiblast; during gastrulation they
are displaced onto the forming yolk sac by the anterior definitive endoderm
(Lawson and Pedersen, 1987;
Thomas and Beddington, 1996
).
This might occur because AVE cells have themselves changed, as suggested by
the fact that they downregulate markers such as Hex
(Thomas et al., 1998
). Another
possibility is that by these stages the extra-embryonic ectoderm has been
displaced by the forming yolk sac, which does not repel AVE cells.
Interestingly, in embryos mutant for angiomotin, the AVE is not displaced onto
the extra-embryonic region during gastrulation
(Shimono and Behringer, 2003
).
Analysis of these mutant embryos should help resolve this issue. We hope to
address these, and other questions, by continuing to observe mouse embryos in
real time as they develop in culture.
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ACKNOWLEDGMENTS |
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Footnotes |
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These authors contributed equally to this work
* Present address: Molecular Embryology Group, MRC Clinical Sciences Centre,
Imperial College London, Hammersmith Hospital Campus, Du Cane Road, London W12
ONN, UK
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