Department of Biology, University of North Carolina, Chapel Hill, NC 27599, USA
Accepted 17 October 2002
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SUMMARY |
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Key words: Cell migration, In vitro system, Gastrulation, Morphogenesis, Polarity, Actomyosin cytoskeleton, C. elegans
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INTRODUCTION |
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One of the key cell rearrangements during development is gastrulation, the
process by which metazoan embryos internalize their future endoderm, resulting
in proper positioning of the three germ layers the endoderm, the
mesoderm and the ectoderm. The mechanisms of gastrulation differ between
animals, ranging from the simple to the complex. For example, in the nematode
Caenorhabditis elegans, embryos internalize their endoderm by the
ingression of only two cells (Sulston et
al., 1983), whereas, in chick embryos, the process is complex,
requiring the coordinated movement of entire tissues immediately followed by
neurulation (Schoenwolf,
1991
). Owing to its simplicity, the gastrulation process in C.
elegans is particularly well suited to studying individual cell movement
and positioning during development.
C. elegans development follows a precise program of cell divisions
(Sulston et al., 1983). The
one-cell embryo divides into the anterior cell AB, which gives rise to most of
the nervous system and epidermis, while the posterior cell, P1,
produces the endoderm, germline and most of the mesoderm
(Fig. 1A). Among the
descendents of P1 are several important players in gastrulation:
the endoderm precursors Ea and Ep, and their neighbors, the germline founder
cell P4, and the granddaughters of MS, which produce primarily
mesoderm (we use `MSxx' to indicate any one granddaughter of MS). Gastrulation
begins with the ingression of Ea and Ep towards the center of the embryo,
leaving a space between the cells and the eggshell known as the ventral cleft.
Meanwhile, MSxx and P4 move towards each other and fill the ventral
cleft (Fig. 1B,C)
(Sulston et al., 1983
). In the
experiments described in this paper, we analyzed the displacement of the Ea
and Ep cells relative to their immediate neighbors. For convenience, we refer
to these movements alone as gastrulation, although these initial movements are
followed by internalization of almost half of the cells in the developing
embryo to complete the entire process of gastrulation
(Sulston et al., 1983
;
Nance and Priess, 2002
).
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A major advantage of using C. elegans embryos to study cell
rearrangement is the potential to combine the powerful tools of direct cell
manipulation, cell biology and genetics. An in vitro culture system exists
whereby the eggshell and vitelline envelope can be removed (which we refer to
as devitellinization), and the resulting naked embryos can be cultured in
embryonic growth medium (Edgar,
1995). This in vitro system was previously used to dissect
cell-cell signaling and cell fate determination
(Goldstein, 1992
;
Shelton and Bowerman, 1996
;
Lin et al., 1998
;
Berkowitz and Strome, 2000
),
and is thus well suited to study many aspects of embryogenesis. Use of in
vitro models in other animal systems, such as Keller explants of
Xenopus embryos, has allowed detailed examination of morphogenetic
movements at the cellular level (Wilson
and Keller, 1991
).
Despite the numerous advantages of using C. elegans to study
morphogenesis, the mechanisms that underlie C. elegans gastrulation
are unknown. The success or failure of gastrulation is often noted in
characterizing mutants, but few studies have actually addressed the
mechanistic requirements for gastrulation. Proper endoderm fate appears to be
required for gastrulation, as mutations that prevent endoderm specification
also result in a gastrulation-defective phenotype (reviewed by
Maduro and Rothman, 2002). In
addition, proper cell cycle length may also be essential, as early Ea/Ep
division appears to prevent gastrulation
(Schierenberg et al., 1980
;
Knight and Wood, 1998
). Other
factors, such as the formation of small blastocoel-like spaces, an
apical-basal polarized distribution of PAR proteins, and an accumulation of
myosin at the ventral sides of Ea and Ep might play roles in gastrulation
(Nance and Priess, 2002
), but
their functional requirements have not been tested. Therefore, it is currently
unclear what mechanisms control this important event.
In this study, we first asked if we could extend the C. elegans in vitro system to study morphogenetic movements during gastrulation. We also addressed the following questions. What are the mechanisms of cell movement? Is chemotaxis involved? What is the role of the cytoskeleton? What determines the direction of cell movement?
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MATERIALS AND METHODS |
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Devitellinization and blastomere culture/isolation
Devitellinization and blastomere culture techniques were performed
essentially as described (Edgar,
1995; Goldstein,
1992
), with the following exceptions: 10% hypochlorite (Sigma) was
used; the enzyme solution consisted of 3 units/ml chitinase (Sigma), 6.6 mg/ml
chymotrypsin (Sigma) and 0.1% penicillin-streptomycin (Gibco); and the Edgar's
Growth Medium (EGM, also called Embryonic Growth Medium)
(Edgar, 1995
) did not contain
heavy metals. In cases where a mouth pipette and needle were insufficient for
blastomere separation, a micropipette was pulled by hand over a flame and the
uncut tip was laid down between blastomeres to separate them. In most
experiments, embryos were checked the next day for the presence of
birefringent gut granules, an indicator of proper cell fate specification
(Babu, 1974
; Laufer, 1980). A
small percentage of embryos did not make gut granules (
3% of all
experiments), and these were not included in data analyses.
4-D videomicroscopy
Embryos with eggshells were placed in egg buffer on a 0.1% poly-L-lysine
(Sigma) coated coverslip, inverted over a 3% agar pad, and sealed with
petroleum jelly. Devitellinized embryos and isolated blastomeres were placed
in 15 µl EGM on a coverslip with clay feet at its corners and sealed with
petroleum jelly. Images were acquired on a C2400-07 Hamamatsu Newvicon video
camera (Hamamatsu Photonics) mounted on a Nikon Eclipse 800 microscope (Nikon
Instrument Group). Time-lapse images were acquired at 1 µm sections every
30 seconds using 4D Grabber, and subsequently analyzed with 4D Viewer
(Integrated Microscopy Resource, University of Wisconsin, Madison).
Quantification of cell movements
For timing measurements, the beginning of gastrulation movement was chosen
by the frame in each movie where P4 and MSxx began to move towards
each other. Because there is a slight variation in cell cycle length from
embryo to embryo, cell cycle lengths were normalized: the beginning of
movement was expressed as a fraction of the length of each embryo's total
Ea/Ep cell cycle. For angle quantification measurements, two different
approaches were used because of a difference between wild-type and mutant
embryos (see Results). In all cases, the angles were measured using a
protractor, with the line joining the centers of the Ea and Ep cell nuclei as
the (0°) reference point. A measurement was taken at the indicated initial
time point, and that value was subtracted from the measurement at the final
time point, to give the total movement. Means and standard deviations were
calculated as described for circular distributions
(Zar, 1999). Watson-Williams
tests revealed significant differences between the four embryo types for the
three approaches (see Table 1).
Multiple comparisons were made using pair-wise Watson-Williams tests. This
procedure is analogous to using an ANOVA to determine the main effect of
embryo type followed by pair-wise t-tests for multiple means
comparisons between the embryo types. (This type of procedure produces a real
risk of type 1 errors but could not be avoided because post hoc means
comparison tests for circular data have not been developed.) It is likely that
our angle measurements of P1 isolates are underestimates, as we
measured movements in one plane of a multiplane movie, and cells sometimes
moved partially along the z-axis (in or out of the x, y
plane observed). For measurements of apical surface/length, lengths from the
apical border of Ea/MSx(x) to Ep/P4 were measured from time-lapse
images using Metamorph software, where the measuring tool was calibrated to
micrometers. Two timepoints were taken: the first was at P4 birth
and the second was taken 20 minutes later. Three independent measurements were
taken from each embryo at each time point, and were then averaged together to
obtain a representative number for each embryo. Six embryos from each
treatment were analyzed.
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Scanning electron microscopy
Blastomeres were cultured and allowed to develop to gastrulation stages.
The cells were washed twice with simplified culture medium (SCM)
(Goldstein, 1995). The embryos
were then fixed, post-fixed and washed as previously described
(Priess and Hirsh, 1986
).
Cells were placed on a 0.1% poly-L-lysine coated coverslip, and subsequently
dehydrated through a series of graded ethanol washes (30%, 50%, 70%, 100%,
100%). Critical-point drying was performed using CO2 as the
transition solvent, and the coverslip was mounted on an aluminum stub and
sputter coated to a thickness of 15 nm with 60:40 Au:Pd alloy on a Hummer X
Sputtering System (Anatech). Samples were observed at an accelerating voltage
of 20 kV and photographed using a Cambridge S200 SEM. Images were acquired
using NIH Image software and processed with Adobe Photoshop software.
Immunofluorescence
Blastomeres were cultured and allowed to develop to gastrulation stages.
They were then washed twice with SCM, fixed with 4% paraformaldehyde (Electron
Microscopy Sciences), and washed three times with PBT (phosphate-buffered
saline with 1% Tween-20). For phalloidin, embryos were extracted with
sonicated 0.5% Triton-X-100 for 5 minutes immediately following fixation. The
following concentrations were used: 1:50 Alexa Fluor 488 Phalloidin (F-actin,
Molecular Probes) and 1:500 mouse -phosphotyrosine (P-Tyr-102, Cell
Signaling Technology). Alexa Fluor 488-conjugated goat anti-mouse secondary
antibody was used at 1:1000 (Molecular Probes). Confocal images were obtained
as described above. Epifluorescence imaging was attained with a Hamamatsu Orca
II (model C4742-98) charge-coupled device camera mounted on a Nikon Eclipse
E600FN. Adobe Photoshop software was used to process the images for
publication.
Fluorescent microsphere experiments
For each experiment, 0.2 µm yellow-green fluorescent,
carboxylate-modified latex microspheres (Molecular Probes) were freshly
diluted 1:4 in SCM, sonicated for 1 minute and added to P1 isolates
in EGM. The microspheres and P1 isolates were quickly mixed
together using a mouth pipette for three minutes, after which the
P1 isolates were transferred through two washes of EGM. The
microspheres attach non-specifically to protein surfaces
(Wang et al., 1994). The
isolates were placed in 2 µl of EGM in one well of an eight-well slide
(ICN), with the three surrounding wells also containing 2 µl of EGM. A
coverslip was gently placed over the four wells, and sealed with valap (1:1:1
vaseline, lanolin and paraffin). Simultaneous DIC and GFP time-lapse images
were acquired every 30 seconds at three planes, 1-1.5 µm apart, using a
Hamamatsu Orca II (model C4742-98) charge-coupled device camera mounted on a
Nikon Eclipse E600FN. The Metamorph software package (Universal Imaging) was
used for microscope automation, image acquisition and image analysis. DIC and
GFP images were combined into one image for analysis. Microsphere movements
were traced by hand onto transparencies, from the beginning to the end of
gastrulation movements, or until the microspheres went out of plane. As both
the microspheres and the cells were moving at this time, the tracing template
position was adjusted at each time interval along with cell movement, to make
it possible to discern microspheres that were moving faster relative to cell
boundaries from those remaining stationary on a moving cell. Kymographs of
bead movement on Ea and Ep were made using Metamorph software to confirm
tracing data. Tracings were scanned into the computer as TIFF images and then
re-drawn using Canvas software. For vector addition, each cell was divided up
into quadrants, and vectors were added if quadrants contained more than three
vectors. Microspheres along cell boundaries or those that did not move were
not included in the calculations. All calculations were performed by
decomposing the vectors into x and y components. To obtain the average
velocities for the set of microspheres from each quadrant, we first took the
average of the x and y components separately, then extrapolated the total
distance the microsphere would be expected to move by multiplying the distance
from the added vector by a time ratio (total time/observed time) to obtain the
total, average distance for all vectors in that quadrant. Then, we converted
the relative distances into µm and divided this total, extrapolated
distance by the total gastrulation time (25.2 minutes) to obtain the average
rate of movement. Angle/direction of movement was calculated by calculating
the arctangent of (y/x).
Blastomere recombination experiments
P1 cells were isolated and allowed to develop in EGM. To rotate
partial isolates relative to each other, P1 isolates were first
split at one of three cell boundaries. For Ea and Ep recombinations, Ea and Ep
were separated 5 minutes after division and recombined. For MSxx and
P4 recombinations, the MS or P3 cell was separated from
the rest of the isolate and recombined one cell division later, when E had
divided into Ea and Ep. Although the partial P1 isolates were not
deliberately rotated, we refer to them as rotated because they were placed
back in contact without regard for their original rotational orientations;
examining centrosome positions has demonstrated that this procedure can
randomize rotational orientations
(Goldstein, 1995
). The
recombined P1 isolates were mounted on coverslips and filmed as
described above. Analysis of the direction of movement depended on whether
MSxx and P4 moved in the same plane or not. In all cases, MSxx and
P4 always moved towards the Ea/Ep boundary. If both cells moved in
the same plane, the movement was either 0° (towards each other) or
180° (away from each other). If a cell moved in or out of plane, a
reference cell was chosen (see Fig.
8) and the non-reference cell was estimated to move 45°,
90° or 135° off the axis of movement of the reference cell.
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RESULTS |
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The C. elegans embryo develops inside a vitelline envelope, which
is surrounded by a chitinous eggshell
(Wood, 1988). It has
previously been reported that gastrulation does not occur in devitellinized
embryos, suggesting that the vitelline envelope might produce a
micro-environment required for gastrulation
(Schierenberg and Junkersdorf,
1992
). However, half of the embryos cultured in that study did not
make rhabditin/gut granules, a marker of endoderm differentiation in C.
elegans embryos, suggesting that the embryonic culture medium used might
not have supported development as well as EGM (see Materials and Methods).
To determine if gastrulation can occur in vitro, we devitellinized embryos, filmed their development in EGM, and compared them with intact embryos (Fig. 2A-H). Gastrulation movements occurred consistently in devitellinized embryos (Fig. 2E-H, six out of six embryos), and these movements occurred at the same time as they do in intact embryos (see below). Therefore, the eggshell and the vitelline envelope do not serve as required surfaces for gastrulation forces to act against or as sources for signals that are necessary for gastrulation to occur.
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Gastrulation does not require AB descendants
Gastrulation movements in the descendents of isolated P1 cells
were previously noted in some partial embryos
(Laufer et al., 1980;
Edgar, 1995
). We wanted to
extend what was observed by asking if these movements occur consistently in
vitro, and to what extent the movements were similar to those seen in intact
embryos. First, we tested whether gastrulation requires AB descendants, either
as substrates for cell crawling or as a source of essential signals.
Time-lapse imaging of P1 isolates showed that gastrulation movements occur consistently in vitro without AB descendants (Fig. 2I-P, 12/12 embryos). Without the eggshell or the AB descendants as reference points, we could not assess whether or not the E cells in the P1 isolates were moving into what was once the center of the embryo. However, we always observed P4 and MSxx moving towards each other, as they do in intact embryos (12/12 embryos), although not to the same extent (see below). The descendants of P1 exhibited variable division patterns, producing partial embryos that ranged from a dumbbell shape, in which the neighbors of the E cells made multiple cell-cell contacts with the E cells (Fig. 2I-L), to a linear orientation, in which there were single cell-cell contacts between the E cells and their neighbors (Fig. 2M-P). Partial embryos of all geometries underwent gastrulation movements (Fig. 2I-P). As we observed that both the leading and trailing edges of the neighboring cells were displaced in the direction of movement (data not shown), we infer that these are directed movements rather than cells simply spreading over the surfaces of Ea and Ep. Our results suggest that single cell-cell contacts between the E cells and their immediate neighbors, P4 and MSxx, are sufficient for at least some gastrulation movements to occur.
Because P1 isolates would enable us to perform cell manipulations that are not feasible in intact, or even devitellinized, embryos, we were interested in determining to what extent gastrulation movements occurred normally in descendents of P1 isolates. Thus, we next asked to what degree the movements we observed were similar to those in intact embryos in both the timing as well as the extent (distance) of the movements. In intact embryos, gastrulation always began after Ea and Ep were born, but before they divided (Fig. 3A, 10/10 embryos). Similarly, the onset of movements in both devitellinized embryos (six out of six embryos) and in P1 isolates (12/12 embryos) always occurred before the end of the Ea/Ep cell cycle (Fig. 3A).
Next, we quantified the extent of movement by measuring the angle of
movement of P4 and MSxx relative to a reference axis defined by the
positions of Ea and Ep nuclei (see Materials and Methods). As a negative
control, we chose mom-2 mutants, as gastrulation fails in embryos
produced by mom-2 mutant mothers
(Rocheleau et al., 1997;
Thorpe et al., 1997
).
mom-2 mutants, like all gastrulation-defective mutants identified to
date in C. elegans (Knight and
Wood, 1998
; Shi and Mello,
1998
; Zhu et al.,
1997
; Tabara et al.,
1999
), have shortened Ea and Ep cell cycles, with Ea and Ep
dividing about 15 minutes earlier than wild type. Therefore, as a time
reference, we used the cell cycles of neighboring cells because those are not
different in wild-type and mom-2 embryos.
In wild-type, intact embryos, gastrulation movements can begin as early as P4 birth (Fig. 3A). Because our measurements are affected by cell divisions, and because MSxx is born 12 minutes after P4, we first examined movement of P4 during this 12 minute interval before MSxx birth. The amount of P4 cell movement from all three wild-type groups (intact embryos, devitellinized embryos and P1 isolates) was significantly greater than that of P4 movement in mom-2 mutant embryos (Fig. 3B, Table 1).
Next, we examined the angle of movement by both P4 and MSxx
during the 30 minute period from when MSxx is born until it divides. MSxx
moved significantly more in each of the wild-type groups than in P1
isolates from mom-2 mutant embryos
(Fig. 3C,
Table 1). Additionally, MSxx
movement in another gastrulation-defective mutant, gad-1
(Knight and Wood, 1998), was
much less than in wild-type groups (data not shown). We were surprised to find
that P4 cells, during this time period, moved to a similar extent
in both wild-type P1 isolates and P1 isolates derived
from mom-2 embryos. This result is not specific to mom-2, as
analysis of gad-1 produced a similar result (data not shown). The
data suggests that the P4 cells in these mutants may retain the
ability to move or be translocated during this time period, or that the extent
of P4 movement in wild-type P1 isolates is not as robust
as that of MSxx movement.
We conclude that devitellinized embryos and P1 isolates exhibit several aspects of gastrulation movements seen in intact embryos. First, the onset of movements occurs at the same time in intact embryos, devitellinized embryos, and in P1 isolates. Second, the movements of P4 and MSxx are always in the same direction as in intact embryos. Additionally, we found that in most cases, the extent of cell movement in wild-type P1 isolates was significantly greater than that of gastrulation-defective mutant P1 isolates. We note, however, that the extent of some cell movements in intact embryos is significantly greater than that in devitellinized embryos and in P1 isolates (Table 1), suggesting that the eggshell, vitelline envelope and/or AB cells may be required for the full extent of gastrulation movements.
MSxx does not chemotax towards P4
It is possible that P4 and MSxx send chemotactic signals that
could be used as cues to move towards each other. We tested this hypothesis by
removing the potential sources of chemoattractants. Either MS or P2
(the grandparents of MSxx and P4) were removed from P1
isolates, and the truncated isolates were subsequently filmed to document cell
movements in the manipulated embryos. When we removed P2, MSxx
appeared to move as it would in unmanipulated P1 isolates
(Fig. 4D-F). Analysis with
angle measurements confirmed this observation, as the average extent of
movement of MSxx in truncated isolates was similar to that found in normal
P1 isolates (Fig.
4H) and was significantly greater than the movement seen in
mom-2 P1 isolates
(Table 1). Although we also
observed movement by P4 after removing MS, the movement was not
significantly greater than that in gastrulation-defective mutants, or
significantly less than that in wild-type P1 isolates
(Table 1). Therefore, we cannot
make a conclusion for P4 on the basis of this experiment alone. We
conclude that MSxx does not rely on a chemotactic signal from P4
during gastrulation.
Intact microfilaments are required for gastrulation
As the cytoskeleton plays a central role in cell motility, we asked whether
microtubules and/or microfilaments were required for gastrulation. To test
this, we permeabilized intact, gastrulating embryos by laser ablating holes in
the eggshell and then exposed the embryos to various cytoskeletal inhibitors.
Exposing the embryos to taxol, which suppresses microtubule dynamics in living
cells (Yvon et al., 1999), or
to the microtubule-destabilizing drug nocodazole, had the expected effects on
microtubule distribution in gastrulating embryos, but neither drug prevented
gastrulation (Fig. 5A-C). We
conclude that microtubules do not play an essential role specifically during
C. elegans gastrulation in producing movements.
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By contrast, all embryos subjected to the microfilament assembly inhibitors cytochalasin D or latrunculin B did not gastrulate (Fig. 5E,F), suggesting that intact microfilaments are required for gastrulation. Examination of the actin distribution following drug treatment confirmed that actin microfilaments were no longer cortically enriched following treatment (Fig. 5E,F). To determine whether microfilaments are required only for gastrulation to initiate, or also for cells to continue moving, we exposed embryos to cytochalasin D during the middle of gastrulation, when MSxx and P4 were about halfway towards meeting each other. Gastrulation halted immediately upon exposure to the drug (six out of six embryos). To ensure that cytochalasin D was not simply killing the embryos, we performed washout experiments to test the reversibility of the drug. Upon washout of cytochalasin D, gastrulation consistently resumed, after an average of 11±6 minutes (15/15 embryos). In addition, when we examined the rate of movement, we confirmed that during cytochalasin D treatment, the E cells did not ingress (Table 2). After washout, the rate of Ea/Ep movement was roughly equivalent to untreated control embryos (Table 2). Therefore, intact microfilaments are required throughout C. elegans gastrulation.
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Cells do not appear to move via a classical crawling mechanism
Actin-dependent mechanisms of motility mediated by cell protrusions are
well documented in other motility systems, such as growth cones, keratocytes
and fibroblasts (Mitchison and Cramer,
1996). We were interested in whether gastrulating cells in C.
elegans also move by a similar mechanism. Crawling cells typically send
out actin-rich protrusions, in the form of filopodia or lamellipodia, towards
the direction of movement. Actin polymerization at the leading edge of the
cell, in combination with traction at the cell base, results in forward
displacement of the cell body. Simultaneously, the cortex and cell surface
move rearward relative to the direction of movement. Finally, the very rear of
the cell must de-adhere from the substrate so that there is a total forward
motion of the cell (Mitchison and Cramer,
1996
). In the case of gastrulation, Ea and Ep could be actively
migrating towards the center of the embryo, or the neighboring cells could be
crawling towards each other, using Ea and Ep as a substrate.
We used several independent methods to test if cells were indeed crawling
via actin-mediated protrusions. Our initial approach was to look for
extensions or cell shape changes in gastrulating P1 isolates.
Tracings of the cell shapes from time-lapse images during the time of
gastrulation showed that P4 and MSxx exhibited subtle cell shape
changes at the leading edge towards the direction of movement, while Ea and Ep
did not make any such observable shape changes (data not shown). This
suggested that P4 and MSxx might be making extensions and crawling
towards each other, using the E cells as substrates. To test this, we used
scanning electron microscopy (SEM) to investigate whether or not the cells
produced extensions. Examination of both devitellinized embryos and
P1 isolates by SEM showed no consistent evidence of filopodia or
lamellipodia (intact, n=8, data not shown; P1 isolates,
n=9, Fig. 6A). In some
cases (four out of eight intact embryos, two out of nine P1
isolates), we saw flattened protrusions at cell boundaries reminiscent of
those reported previously in devitellinized embryos
(Nance and Priess, 2002).
However, there was never more than one cell per embryo exhibiting a protrusion
and the cell that exhibited the protrusion varied from embryo to embryo. In
another approach, we reasoned that if the cells had actin-rich extensions,
then we should see an increased intensity of filamentous actin (F-actin) at
the leading edge of the motile cells. Staining P1 isolates with
fluorescent phalloidin, which marks F-actin, did not reveal an enrichment of
actin at any specific cell-cell boundary (n=8,
Fig. 6B). Another marker of
active protrusions is phosphotyrosine, which has been shown to be enriched at
the leading edge of crawling cells including nematode sperm and
Aplysia growth cones (Italiano et
al., 1996
; Wu and Goldberg,
1993
). We observed no polarized enrichment of phosphotyrosine at
any specific cell-cell leading edge (n=12,
Fig. 6C, and legend). We also
used PAR-2::GFP as a live cortical marker in P4
(Boyd et al., 1996
) and found
that the cortex of the P4 cell did not make protrusions as it moved
over the ventral surface of Ep (data not shown). Finally, tracking the
surfaces of cells with fluorescent markers showed that the surface of MSxx
does not move rearwards relative to the movement of the cell body, as would be
expected from protrusion based crawling (see below). Together, these
approaches suggest that cells do not form lamellipodia or filopodia and are
unlikely to move via protrusions during C. elegans gastrulation.
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Myosin is required for cell movement
If cells do not move via protrusion-based cell crawling, then they must
either move through another microfilament-dependent process, or microfilaments
must play an indirect role in movement. Recent evidence showed an accumulation
of NMY-2, a non-muscle myosin type II, at the ventral cortex of Ea and Ep
during gastrulation (Nance and Priess,
2002), but whether or not this accumulation was required for
gastrulation was not tested. Therefore, we asked if myosin activity was
required for gastrulation.
As loss of NMY-2 results in severe defects in establishment of polarity and
in execution of cytokinesis very early in embryogenesis, before gastrulation
(Guo and Kemphues, 1996), we
tested the role of myosin activity using ML-7, a potent and specific inhibitor
of myosin light chain kinase (Saitoh et
al., 1987
). As ML-7 has not been used previously in C.
elegans embryos, we performed dose response experiments to test the
specificity of the effect of ML-7 and two related compounds, ML-9 and H-7, on
gastrulation. ML-9 is also a MLCK inhibitor, but has ten-times less affinity
for MLCK compared with the affinity of ML-7, whereas H-7 is a specific
inhibitor of PKA, but exhibits a very weak affinity for myosin light chain
kinase (Saitoh et al., 1987
;
Mabuchi and Takano-Ohmuro, 1990). We found that while 250 µM ML-7 was
sufficient to completely inhibit gastrulation, 750 µM ML-9 and 4 mM H-7
were required to inhibit gastrulation (Fig.
7). We also found that exposure of embryos to 250 µM ML-7
immediately halted cell movement at the beginning of gastrulation (six out of
six embryos), as well as in the middle of gastrulation (seven out of seven
embryos). 750 µM H-7 was insufficient to inhibit gastrulation, but these
embryos arrested early, before morphogenesis, suggesting other, potentially
PKA-dependent defects (seven out of seven embryos). These experiments suggest
that ML-7 specifically inhibits MLCK in C. elegans embryos and that
myosin activity is required for cell movements throughout C. elegans
gastrulation.
Cell surface tracking shows evidence of cell contraction
Our experiments with pharmacological agents suggest that actin and myosin
play an essential role in C. elegans gastrulation, but the mechanism
of movement is still unclear. One possible mechanism is that MSxx and
P4 could be actively moving via a rotating mechanism, where the
cells would roll like wheels towards each other onto the ventral sides of Ea
and Ep, possibly mediated by local gradients in cell adhesion. An alternative
model is that a ventral, actomyosin-based contraction toward the Ea and Ep
border drives the neighboring cells closer together. One prediction of this
model is that the apical (ventral) surfaces of Ea and Ep should decrease in
size during gastrulation, while embryos treated with the myosin inhibitor
would not. We therefore measured the distance along the apical surface between
the Ea/MSx(x) border and the Ep/P4 border (see Materials and
Methods). We found that untreated, control embryos underwent an average apical
reduction of 7.7 (±2.3) µm during gastrulation, whereas the apical
surfaces of embryos treated with ML-7 was reduced by 1.1 (±2.3) µm
during the same time frame. Therefore, the apical surface areas of Ea and Ep
decrease during gastrulation, while myosin-inhibited embryos do not undergo
significant changes.
Although these measurements are consistent with the contraction model, they
are also consistent with models in which cells roll and also with models where
the apical surfaces decrease as a result of basolateral expansion. To assess
directly how cell surfaces behave, we tracked the surfaces of cells with
fluorescent microspheres and filmed them during gastrulation. Fluorescent
microspheres are useful markers for tracing the movement at the cell surface
because they do not become engulfed by cells and are relatively photostable
(Wang et al., 1994). Thus, if
Ea and Ep were undergoing actomyosin contraction, then the microspheres on the
ventral cortex of Ea and Ep would converge toward the Ea/Ep boundary. However,
if the neighboring cells were rotating, then the microspheres on the
neighboring cell surfaces would be displaced in the direction of movement.
Finally, if Ea and Ep were basolaterally expanding, then the beads on the
apical surface of Ea and Ep should either remain stationary or move slightly
away from the Ea/Ep boundary.
Partial embryos were incubated with microspheres, and a large number of microspheres were observed on the ventral surfaces of Ea, Ep and MSxx; whether or not this distribution is nonrandom and reflective of an increased degree of adhesivity at these surfaces is unclear. As these surfaces had a sufficiently large number of microspheres, we limited our analysis to these surfaces. We found that microspheres on the ventral sides of Ea and Ep generally moved toward the Ea-Ep boundary (Fig. 8). We also observed microspheres on the ventral side of MSxx moving along the cell surface towards Ea (Fig. 8). These results are consistent with the model that the ventral sides of Ea and Ep contract, and that MSxx might additionally contribute to movement by rotating towards Ea during gastrulation.
Polarity in Ea and Ep dictates the direction of movement of
neighboring cells
The observations that P4 and MSxx move toward each other in
P1 isolates, that a non-muscle myosin is enriched in the ventral
side of Ea and Ep (Nance and Priess,
2002), that myosin activity is required for gastrulation and that
the ventral sides of Ea and Ep contract during gastrulation suggest that Ea
and Ep may be ingressing via a myosin-based contraction of their ventral
surfaces. One prediction of this hypothesis is that Ea and Ep should be
capable of dictating the direction of movement of their neighbors, regardless
of the orientation of the neighbors. To test this, we rotated cells relative
to each other by bisecting a P1 isolate at various cell boundaries,
recombining the isolated halves, and filming the partial embryos to document
the ensuing direction of movement (see Materials and Methods,
Fig. 9). We tested three
variations of rotation experiments, in which we separated and recombined
P1 isolates at the Ea and Ep, Ea and MSxx, or Ep and P4
boundaries.
|
We found that when we separated and recombined Ea and Ep, MSxx and P4 did not move towards each other in the same axis as they did in unmanipulated P1 isolates. Instead, the cells moved toward the Ea/Ep boundary in a range of directions relative to each other (Fig. 9A). Thus, when Ea/Ep orientation was randomized, so was the direction of movement by MSxx and P4. In addition, because the neighbors move in different directions, this result further confirms that MSxx and P4 are not chemotaxing towards each other.
In isolates where MSxx and Ea were separated and recombined, MSxx always moved in the same direction as P4 (10/10 embryos; Fig. 9B). Similarly, in most cases, P4 moved in the same direction as MSxx where P4 had been recombined with Ep (seven out of ten embryos; Fig. 9C). Curiously, in a small number of Ep/P4 recombination cases, the neighboring cells moved in the same plane toward each other, but in the opposite direction (three out of ten embryos; Fig. 9C). It is possible that P4 may influence the orientation by which polarity is established in Ep, although this has not been tested. We conclude that Ea and Ep are polarized in a way that can generally dictate the direction of movement of their neighbors.
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DISCUSSION |
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|
Additional mechanisms may contribute additively or redundantly to ventral contraction of Ea and Ep. There are indications that some neighboring cells may make an active contribution to movement. For example, in P1 isolates, MSxx sometimes moves not only to the Ea/Ep boundary but also beyond this point (data not shown), and beads on the ventral surface of MSxx move in the direction of cell movement (Fig. 8), suggesting that MSxx might be rolling in this direction, perhaps driven by cell-cell adhesion. This does not appear to occur in P4, as P4 never passes the Ea/Ep boundary in P1 isolates (data not shown).
Although many mechanisms may contribute to C. elegans gastrulation, other potentially redundant mechanisms can be excluded by our results. First, our cell manipulation experiments suggest that neighboring cells do not chemotax towards each other. Second, the cells most likely do not move by protrusion-based cell crawling, as no protrusive structures were observed during gastrulation, and surfaces do not move in the directions expected in protrusion-based cell crawling. Thus, C. elegans gastrulation could be used as a model to examine cell motility that is not dependent on cell protrusions.
Additionally, because the descendants of P1 isolates can gastrulate in vitro, we conclude that at least three classes of mechanisms for cell positioning are not essential for gastrulation movements, although the possibility that they make redundant or additive contributions to movements cannot be discounted. First, buckling forces or spatial restriction of cell divisions requiring physical constraints, such as those provided by the eggshell and vitelline envelope, cannot be necessary for gastrulation because gastrulation can occur in devitellinized embryos. For example, the P3 division in intact embryos is aligned with the eggshell, so that when P4 is born, it might push Ep towards the center of the embryo. However, oriented P3 cell division constrained by the eggshell cannot be required to initiate gastrulation movements because gastrulation can occur in vitro.
Second, we can rule out the necessity of a neighbor annealing mechanism in which, as the neighboring cells (P4, MSpp, MSpa, MSap, ABplpa and ABplpp) converge on the ventral side of Ea and Ep (Fig. 1C), their adhesiveness to each other drives their convergence, sealing up the ventral cleft. As the AB cells are not required for gastrulation movements, and as movements can occur in P1 isolate orientations in which the only neighboring cells, P4 and MSxx, do not contact each other until the end of gastrulation movements (Fig. 2M-P), we conclude that neighbor annealing is not required for gastrulation.
Third, a model whereby movement is driven exclusively by differential
adhesion between cells is unlikely. The differential adhesion hypothesis
(Steinberg, 1963) proposes
that cell rearrangements can be directed by differences in the adhesive
strengths of cells. Cells that show some degree of motility tend to maximize
adhesive contacts, resulting in the most adhesive cells ending up in the
center of a group of cells, with the less adhesive cells surrounding them
(Steinberg and Takeichi,
1994
). For example, it is possible that Ea and Ep could be more
adhesive than their neighbors, and this differential adhesion could drive
their ingression as they become surrounded by their less adhesive neighbors.
However, differential adhesion alone cannot be a sufficient mechanism as Ea
and Ep in linear P1 isolates exhibit gastrulation movements without
being surrounded by neighboring cells. In addition, there is, to date, no
evidence for a role for adhesion molecules in C. elegans
gastrulation, as molecules such as catenins, cadherins and extracellular
matrix components do not show upregulation in the E cells at the time of
gastrulation, and embryos carrying mutations in genes that encode adhesion
proteins do not arrest until late embryogenesis, during the events of ventral
closure and elongation (reviewed by
Michaux et al., 2001
).
However, large-scale knockouts of adhesion molecules have not been performed,
and functional redundancy may have prevented a gastrulation phenotype from
being detected thus far.
Cell contraction in morphogenesis
Our model of ventral contraction (Fig.
10) is reminiscent of apical constriction, a long-proposed
mechanism for certain morphogenetic events. Cells that contract at their
apical sides basolaterally expand, which in a cell sheet can cause bending or
invagination of the sheet at the site of the apically constricting cells
(Lewis, 1947;
Odell et al., 1981
). Because
of their shape, these cells are often described as flask or bottle cells
(Rhumbler, 1899
;
Rhumbler, 1902
;
Ruffini, 1925
). Apical
constriction has been suggested to play a role in morphogenetic movements in a
wide range of organisms, including gastrulation in shrimp
(Hertzler and Clark, 1992
),
fly (Young et al., 1991
),
jellyfish (Byrum, 2001
), sea
urchin (Kimberly and Hardin,
1998
), white sturgeon (Bolker,
1993
), rabbit (Viebahn et al.,
1995
) and frog (Keller,
1981
; Hardin and Keller,
1988
), as well as in primitive streak formation in chick and rat
embryos (Solursh and Revel,
1978
), and neurulation in frogs
(Jacobson et al., 1986
).
Whether apical constriction actually drives shape changes in cell sheets has
been directly tested in Xenopus, sea urchin and Drosophila
gastrulation. In Xenopus and sea urchin embryos, removal or ablation
of bottle cells showed that the initial invagination of the epithelial sheet
required the bottle cells (Keller,
1981
; Hardin and Keller,
1988
; Kimberly and Hardin,
1998
). The cellular mechanism by which bottle cells invaginate is
unclear. In Drosophila embryos, myosin and actin are enriched at the
apical cortex of gastrulating cells (Young
et al., 1991
). In addition, disrupting a pathway that regulates
actin through RhoA prevents concerted apical constriction, halting
gastrulation (Barrett et al.,
1997
; Hacker and Perrimon,
1998
). However, some clusters of cells still undergo apical
constriction, suggesting that this pathway is required for concerted cell
shape changes, but not for apical constriction.
The apical constriction model predicts a local actomyosin-driven cell
contraction (Odell et al.,
1981). Our results demonstrate that C. elegans
gastrulation is an example of apical constriction. We define the apical and
basal sides of cells based on findings by Nance and Priess
(Nance and Priess, 2002
) that
cell cortices facing the outside of the embryo, or apical side, have an
enrichment of the protein PAR-3. NMY-2 also accumulates at the apical side
(Nance and Priess, 2002
) and
we show, through pharmacological studies, that both actin and myosin activity
are required for gastrulation. Furthermore, the apical surfaces of Ea and Ep
contract, and the polarity of Ea and Ep is important for the direction of
movement by their neighbors. As adherens junctions and tight junctions are not
found this early in embryogenesis (Krieg
et al., 1978
), we conclude that apical constriction can function
to position blastomeres in early embryos, even before such cell-cell junctions
form. The availability of both genetic tools and direct manipulation of cells
should contribute to the usefulness of C. elegans as a system for
understanding the mechanism of apical constriction in early embryos.
![]() |
ACKNOWLEDGMENTS |
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Footnotes |
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