Department of Cell Biology, Harvard Medical School, Boston, MA 02115, USA
* Author for correspondence (e-mail: marc{at}hms.harvard.edu)
Accepted 23 January 2003
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SUMMARY |
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Key words: Xenopus, Gastrulation, Xbra, Cell migration, Convergent extension, Wnt
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INTRODUCTION |
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Active cell migration of the prechordal mesoderm is characterized by the
ability of single cells to spread and crawl upon a fibronectin substrate.
During gastrulation, the cells generate bipolar, actin-rich lamellipodia and
actively migrate upon the extracellular matrix secreted by the blastocoel
roof. Interaction with a fibronectin substrate is necessary, as blockade of
cell surface interactions with fibronectin, using either peptides that mimic
fibronectin (GRGDSP) or fibronectin-blocking antibodies, abolishes migration
(Winklbauer, 1990). Prechordal
mesoderm cells migrate in vitro at the same rate as they do in the embryo,
about 2.5 µm/minute (Wacker et al.,
1998
; Winklbauer,
1990
). Migrating cells come to rest at the future anterior end of
the embryo, where they play a crucial role in patterning the anterior
neurectoderm (Sive et al.,
1989
). Inhibition of active cell migration of the prechordal
mesoderm, as caused by fibronectin-blocking antibodies, can lead to a variety
of head defects (Marsden and DeSimone,
2001
).
In contrast to prechordal mesoderm cell migration, convergent extension is
not so much a migrating process as a cell sorting process. Groups of cells
rearrange and intercalate to change the overall shape of a tissue. During
gastrulation, chordamesoderm cells rearrange from a largely isotropic
organization, intercalate and generate a rod-like structure that will
differentiate into notochord. Cell-cell adhesion, rather than cell-substrate
adhesion involving fibronectin, is crucial for this process. Unlike the case
of active cell migration, peptides that mimic fibronectin (GRGDSP) have no
effect on the convergence and extension movements of isolated explants
(Ramos and DeSimone, 1996;
Ramos et al., 1996
;
Winklbauer and Keller, 1996
).
Cell-cell adhesion molecules such as cadherins and protocadherins are
important for convergent extension. Because cell-cell contact is required for
convergent extension, this behavior cannot be observed in isolated single
cells, rather, it is analyzed in the context of an intact explant. Defects in
this process result in shortened trunks with a fully formed head
(Wallingford and Harland,
2001
).
These two behaviors are discrete and non-overlapping. Prechordal mesoderm
exhibits active cell migration, but not convergent extension; conversely,
chordamesoderm undergoes convergent extension, while only a few cells
dissociated from the chordamesoderm actively migrate on fibronectin
(Wacker et al., 1998).
Gastrulation in Xenopus laevis probably presents the most accessible
system to study control of these fundamental processes of cell and tissue
morphogenesis. Though many growth factors, transcription factors and signaling
molecules have been identified in the process of cytodifferentiation, the
control of cell behavior and cell motility is not well understood.
To understand better each cell behavior from both a developmental and cell
biological standpoint, we have examined embryonic tissues in explants. The
animal cap, a tissue explant whose unperturbed fate is ventral ectoderm, can
be induced to express prechordal or chordamesoderm markers via treatment with
the TGFß factor activin (Symes et
al., 1994; Gurdon et al.,
1996
; Gurdon et al.,
1999
). Depending on the conditions of the treatment, either
migration or convergent extension can be induced. We have used this system to
study factors that are important in the induction or regulation of either
behavior.
VegT, a transcription factor of the T-box family,is necessary for both cell behaviors. By contrast, Xbra, another T-box transcription factor, although required for convergent extension, inhibits cell migration. This cell migration block can be partially rescued by inhibiting convergent extension downstream of Xbra. In addition, Xbra appears to inhibit cell migration by specifically inhibiting adhesion to fibronectin. We propose that Xbra functions as a fundamental switch to keep cell migration and convergent extension as mutually exclusive behaviors in adjacent domains during gastrulation.
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MATERIALS AND METHODS |
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Prechordal and chordamesoderm explant preparation: at stage 10, the
vitelline was removed and incisions made on either side of the dorsal
blastopore lip. A third incision was made at the blastopore lip, leaving an
explant of involuted prechordal mesoderm, chordamesoderm (preinvolution) and
neural tissue. Involuted prechordal mesoderm was lifted off of the explant as
an intact piece by inserting an eyebrow knife into Brachet's cleft. The
remaining explant was trimmed to remove neural tissue, leaving a
chordamesoderm explant. For the mesendoderm extension assay, explants were
prepared as described (Davidson et al.,
2002).
Activin-induced convergent extension and cell migration assays
Sibling animal caps were dissected at stage 9. Half of the caps were left
intact and treated with activin protein for one hour (1 U/ml in 1xMMR).
They were then transferred from activin solution into 1xMMR and allowed
to heal and grow until stage 19, when they were scored for convergent
extension. The rest of the caps were dissociated in CMFM (Ca2+ and
Mg2+ free medium) and then treated with activin protein (1 U/ml in
1xCMFM) for 1 hour. The dissociated cells were subsequently plated in
Modified Barth's Solution (MBS) into fibronectin-coated chambered coverslips
(VWR). Coverslips were coated with 0.1 mg/ml fibronectin (Sigma, diluted to
the appropriate concentration with MBS) for 2 hours at room temperature, and
then blocked with bovine serum albumin (BSA; 50 mg/ml in MBS).
For the migration assay, cells were analyzed in the following manner: a field of cells was randomly chosen, and images were captured at 30 second intervals for at least 20 minutes (15 minutes for control samples: uninjected/untreated cells and uninjected/Activin-treated cells). Images were assembled into timelapse form (using Openlab software) and played back at a speed of either 10 or 20 frames per second. A cell was scored as positive for migration if it both translocated across the substrate and exhibited active protrusions.
Image analysis
Timelapse analysis of dissociated cells was performed using a Hamamatsu
C2400 CCD Camera attached to a Zeiss Axiovert 135. Images were acquired and
timelapse files assembled using Openlab software (Improvision). Fluorescence
images were acquired using a Princeton Instruments cooled CCD Camera. Color
images were acquired with a Sony 3CCD Color Camera mounted onto a Zeiss Stemi
SV11 Stereoscope.
RNAs/DNAs
Plasmid DNAs were linearized overnight and purified using the Qiagen PCR
Purification Kit. All RNAs were synthesized using the mMessage mMachine kit
(Ambion) for capped RNA, purified using the Qiagen RNeasy Mini Kit, and
subsequently precipitated in ethanol and resuspended in RNAse-free water.
XbraDNABD is a delection in amino acids 206-229
(Kispert and Hermann, 1993
),
Xdsh mutants are all as in
(Rothbacher et al., 2000
).
VegT-EnR was constructed as described previously
(Horb and Thomsen, 1997
).
Xbra-EnR was constructed as described previously
(Conlon et al., 1996
). dn Wnt11
was constructed as previously (Tada and
Smith, 2000
).
All constructs are in a pCS2 backbone
(Rupp et al., 1994;
Turner and Weintraub, 1994
),
except for Xenopus Activin, which is in pSP64T.
Activin protein
Xenopus oocytes were harvested by manual defolliculation, and
injected with 30 ng of capped RNA encoding full-length Xenopus
activin. Oocytes were cultured in OR2 solution for oocyte storage (+ 0.5 mg/ml
BSA) in 96-well plates for 2 days at 18°C (five oocytes per well in 200
µl culture medium). The supernatant was collected, filtered, aliquotted and
stored at -80°C. Activity of each batch was tested, and was consistently
found to be 20 U/ml [units as defined by Green et al.
(Green et al., 1992)].
Rhodamine-phallodin staining
Cells were fixed for 20 minutes in MBS + 3.7% formaldehyde. Samples were
permeabilized with MBS + 0.1% Triton X-100 for 5 minutes, and then blocked in
MBS + 0.1% Triton X-100 + 2% BSA for 10 minutes. Cells were stained for 20
minutes using 1 µg/ml rhodamine-phalloidin (Sigma) in blocking solution.
All incubations were done at room temperature.
Cell spreading assay
Cells dissociated in CMFM (as described above) were plated into
fibronectin- or poly-L-lysine-coated chambered coverslips (VWR). Fibronectin
substrates containing a `high' concentration of fibronectin were prepared:
coverslips were coated with 0.2 mg/ml fibronectin (Sigma, diluted with MBS)
for 3 hours at room temperature, and then blocked with BSA (50 mg/ml in MBS)
for 30 minutes at room temperature. Poly-L-lysine-coated coverslips were
prepared: coverslips were coated with a solution of 1 mg/ml poly-L-lysine
(Sigma) in water for 30 minutes at room temperature. The coverslips were then
rinsed 10 times with MilliQ H2O, and finally blocked with BSA (50
mg/ml in MBS) for 30 minutes at room temperature.
For the dose response experiment, coverslips were coated as described here with the following concentrations of fibronectin: low (0.1 mg/ml), medium (0.15 mg/ml) and high (0.2 mg/ml).
After plating, cells were allowed to recover and spread on the substrate for 2 hours, at which time they were fixed and processed for rhodamine-phalloidin staining. A cell was scored as spread if it took on a spread morphology: flattened with irregular borders and exhibiting actin-rich protrusions extending along the substrate.
Mesendoderm extension assay
The mesendoderm extension assay was performed as described
(Davidson et al., 2002).
Coverslips were coated with 0.1 mg/ml fibronectin (Sigma, diluted with MBS)
overnight at 4°C. Coverslips and plastic petri dishes were blocked with
Danilchik's For Amy [DFA, containing 1 mg/ml BSA, as described previously
(Davidson et al., 2002
)] for at
least 1 hour before explants were mounted. Timelapse analysis was performed
using a Hamamatsu C2400 CCD Camera attached to a Zeiss Stemi SV11 Stereoscope.
Images were captured and assembled into a timelapse file using Openlab
software (Improvision).
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RESULTS |
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VegT is necessary for both activin-induced cell migration
and convergent extension
VegT, also known as Xombi, Brat and Antipodean,
is a member of the T-box family of transcription factors, and is a direct
downstream target of activin signaling
(Horb and Thomsen, 1997;
Lustig et al., 1996
;
Stennard et al., 1996
;
Zhang and King, 1996
). Ectopic
expression of VegT can induce both endoderm and mesoderm. In
addition, oocyte depletion (Zhang et al.,
1998
) shows that maternal VegT RNA is required for both
endoderm and mesoderm development. We tested whether VegT is required
for cell migration and convergent extension. A dominant inhibitory form of
VegT (VegT-EnR) was constructed by fusing its DNA-binding
domain to the repressor domain of the Drosophila engrailed gene; this
form of VegT will repress its normal downstream targets. Previously,
VegT-EnR has been demonstrated to inhibit expression of mesodermal
markers (Gsc, Xbra and Xlim-1) and mesodermal patterning;
VegT-EnR also inhibited blastopore formation
(Horb and Thomsen, 1997
). As
might be expected, expression of VegT-EnR blocks both cell migration
and convergent extension (Fig.
2). Cells injected with an inhibitory dose of VegT-EnR
look like untreated animal cap cells. These data suggest that VegT
activity is necessary for both activin-induced cell migration and convergent
extension.
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Xbra inhibits cell shape changes induced by dominant active
Rac or dominant active Cdc42
As the control of cell migration is largely unexplored in embryos, the
downstream targets through which Xbra inhibits cell migration remain
largely unknown. Downstream transcriptional targets could act on any number of
processes in order to inhibit cell migration, for example, extracellular
signaling, intracellular signaling to the cytoskeleton, actin polymerization
or cellular adhesion. To define better the mechanisms by which Xbra
inhibits cell migration, we examined the response of the cells to small
GTPases involved in the cytoskeleton and adhesion.
The Rho family of small GTPases has been shown in cultured cell lines to
have specific effects on cell morphology and the actin cytoskeleton. Activated
Cdc42 injected into cells induces filopodia, while activated Rac induces
lamellipodia (Nobes and Hall,
1995). Xenopus animal cap cells respond similarly to
these activated GTPases. To avoid early embryonic phenotypes, V12 Cdc42 or V12
Rac were injected as expression plasmids into both blastomeres at the two-cell
stage, thereby limiting expression to post mid-blastula transition. Under
these conditions, V12 Cdc42 induces filopodia and V12 Rac induces elaborate
lamellipodia (Fig. 6A). When
wild-type Xbra is co-injected with the activated form of either
GTPase, the cell shape changes are inhibited
(Fig. 6B); the cells have the
same morphology as those that have not been injected. By contrast,
co-injection of Goosecoid (Gsc), a transcription factor
expressed in the prospective prechordal mesoderm, has negligible effects (data
not shown).
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Xbra inhibits cell spreading on fibronectin, but not
poly-L-lysine
To distinguish between these two possibilities, cell spreading assays were
performed. When a twofold higher concentration of fibronectin is used to coat
coverglasses (see Materials and Methods), dissociated, uninjected animal cap
cells (not treated with activin) spread upon the substrate. The cells are
flattened, exhibiting an irregular border and few small spiky protrusions
(Fig. 6C). By comparison, cells
plated on a poly-L-lysine substrate also spread, exhibiting an irregular
border and, often, large flat lamellae
(Fig. 6C). Because the
injection of activated GTPases is not necessary for the cells to exhibit these
morphological behaviors, this represents a simple assay for adhesion and
spreading upon a substrate. Injection of Xbra inhibits cell spreading
upon fibronectin, such that the cells assume a round, unspread, unadherent
morphology (Fig.
6C,6D).
However, expression of Xbra has no effect on spreading upon
poly-L-lysine. Fig. 6E shows
dose response to Xbra in the cell spreading assay upon fibronectin.
Three fibronectin concentrations were tested: low, medium and high. Low is
equal to the amount used in previous migration assays, and high is equal to
the amount used in Fig. 6D for
the previous cell spreading assay (see Materials and Methods for details).
These data suggest that in a simple assay to test adhesion and cell spreading,
Xbra can inhibit adhesion specifically to fibronectin. Thus, the
effects mediated by Xbra are less likely to be general effects upon
the cytoskeleton and contractility, and may be specific to the process of
adhesion to fibronectin.
|
In addition to cell spreading assays, visual actin polymerization assays
were performed to test the ability of small GTPases to induce actin
polymerization in extracts (Ma et al.,
1998). Small scale extracts were made from animal caps from either
uninjected embryos or embryos injected animally with Xbra (500 pg
RNA) at the two-cell stage. GTP
S-loaded Cdc42 and GTP
S-loaded
Rac induced actin polymerization equally well in extract from uninjected
animal caps as in extract from Xbra-injected animal caps (S. Eden,
K.M.K. and M.W.K., unpublished). Taken together, these data suggest that
Xbra inhibits adhesion, not actin polymerization, and, more
specifically, adhesion of cells to a fibronectin substrate.
The effects of Xbra are recapitulated in the endogenous
populations of cells that undergo either cell migration or convergent
extension, in the marginal zone
We have used activin-induced animal caps to recreate, as best as possible,
conditions in the marginal zone, where endogenous populations of cells undergo
either migration or convergent extension. We therefore wished to test whether
Xbra has the same effects in the marginal zone cells themselves. For
this, we injected Xbra into the marginal zone of the two dorsal cells
of a four-cell embryo. At stage 10, head mesoderm was dissected from the
resulting gastrulae, and the dissociated cells assayed. As shown in
Fig. 7A, expression of
Xbra inhibits the ability of the prechordal mesoderm cells to
migrate, in a dose-dependent manner. Sibling embryos were cultured until early
tailbud stages, and, as shown, overexpression of Xbra in the dorsal
marginal zone causes anterior truncations
(Fig. 7B). Therefore, even
though migration is inhibited in only 50% of prechordal mesoderm cells, this
is clearly enough to cause severe defects in head development. Another assay
was used to test prechordal mesoderm migration. Davidson et al. have developed
an assay to test the rate and extent of mesendoderm extension, in which the
head mesoderm migrates upon a fibronectin substrate as an intact mantle
(Davidson et al., 2002).
Timelapse analysis was performed to compare mesendoderm extension in dorsal
marginal zone explants (see Materials and Methods) from
Xbra-injected, uninjected and Xbra-EnR-injected embryos
(explants numbered 1, 2, and 3, respectively in
Fig. 7C). As shown, the
Xbra-injected explant initially extends at a similar rate as the
other explants. However, by about 7.5 hours
(Fig. 7C), the
Xbra-injected explant has reached its maximum extension, while the
uninjected and Xbra-EnR-injected explants continue to extend for
another 2.5 hours (Fig. 7C).
Thus, even as an intact mantle, inhibition of migration by 50% causes a clear
decrease in the extent of mesendoderm migration. Meanwhile, migration of the
Xbra-EnR-injected explant was indistinguishable from that of the
uninjected explant, not surprisingly, as expression of Xbra-EnR has
no effect in the in vitro cell migration assay.
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The effects of Xbra on the behavior of chordamesoderm cells
can be recapitulated by mutants of Xdsh that inhibit the Wnt PCP
pathway
We wondered to what extent the effects of Xbra inhibition in the
marginal zone could be recapitulated by inhibition of the Wnt signaling
pathway, specifically the planar cell polarity pathway which has been shown to
be necessary for convergent extension
(Wallingford and Harland,
2001). To determine whether the effect of Xbra upon cell
behavior can be largely attributed to downstream effects on cell fate or a
more direct effect upon cell motility, different Dishevelled mutants
were tested within the chordamesoderm for effects on cell migration.
Dishevelled (Dsh) is an intracellular mediator of Wnt
pathway signal transduction. It is a modular protein with multiple functions:
the DIX domain mediates cell fate decisions via the Wg/Wnt pathway, while the
PDZ domain mediates effects on cell behavior via the planar cell polarity
(PCP) pathway (Rothbacher et al.,
2000
; Wallingford et al.,
2000
). We tested whether the effects of Xbra-EnR on
chordamesoderm cell behavior could be recapitulated with mutants of
Dsh that specifically block the planar cell polarity pathway
downstream of a Wnt signal, in this case specifically Wnt11.
Xdsh
DIX (deletion of the DIX domain), Xdsh
PDZ
(deletion of the PDZ domain) and Xdd1 (deletion of most of the PDZ domain and
part of the following region) were tested for effects on both convergent
extension and cell migration within the chordamesoderm.
As reported previously, chordamesoderm explants expressing
XdshPDZ and Xdd1 fail to elongate; explants expressing
Xdsh
DIX are also partially inhibited from undergoing
convergent extension (Fig.
9A). As shown in previous figures, explants expressing either dn
Wnt11 or Xbra-EnR are also inhibited in convergent extension
movements. However, these same chordamesoderm explants, when assayed for cell
migration, reveal that overexpression of specific Xdsh mutants (as
well as dn Wnt11), causes an increased fraction of cells to undergo cell
spreading and migration on a fibronectin substrate
(Fig. 9B). It is notable that
the mutants that inhibit the Wnt PCP pathway do so without altering cell fate
in the dorsal mesoderm, as measured by staining for differentiated muscle and
notochord (using the antibodies Tor70, MZ15 and 12/101), as well as Northern
blot analysis of the genes Xlim1, Xnr3, Gsc, Xotx2, Chordin and
Xbra (Sokol, 1996
;
Wallingford and Harland,
2001
). These data suggest that specific inhibition of the Wnt PCP
pathway, which does not alter cell fate, is sufficient to partially switch
dorsal mesoderm cells from convergent extension behavior to cell migration. In
addition, cell migration may be a default state within the dorsal mesoderm,
which requires active signals to inhibit components of the migration
machinery, such as adhesion to fibronectin, and promotes convergent
extension.
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DISCUSSION |
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Xbra is a transcription factor that appears early in the trunk compartment of the organizer, where it has long been known to have a role in trunk mesoderm development. We demonstrate here that Xbra actively inhibits cell migration, both in activin-induced animal cap cells, and in the endogenous prechordal mesoderm. This inhibition of cell migration can be rescued by inhibiting convergent extension. These experiments suggest that in the embryo, Xbra acts as a morphogenetic switch not only promoting convergent extension, but also actively repressing cell migration. As expected for such a switch, blocking Xbra activity in the dorsal mesoderm via dominant-negative Xbra (Xbra-EnR) increases the number of cells in the chordamesoderm that spread on fibronectin and actively migrate. Under these conditions, the default behavior of the tissue appears to be cell migration. Therefore, one unappreciated role of endogenous Xbra is to inhibit cell migration within the chordamesoderm, while fostering convergent extension movements. Xbra, which has a role in the eventual cytodifferentiation of the chordamesoderm, also has an immediate dual role in morphogenetic movements.
Wnt11 is a direct downstream target of Xbra which is necessary for
convergent extension (Tada and Smith,
2000). Although Wnt11 signaling is not required for cell migration
(Fig. 5A), expression of
dominant-negative Wnt11 reverses the inhibitory effects of Xbra on
activin-induced cell migration. These same effects can be produced in the
dorsal mesoderm, with dn Wnt11 as well as deletion mutants of
Dishevelled, which inhibit the Wnt planar cell polarity pathway
(Dsh
PDZ, Xdd1). Notably, these Dsh mutants have no
effect on cell fate (Sokol,
1996
; Wallingford and Harland,
2001
).
Taken together, these data suggest that there exist within the dorsal mesoderm two cell behaviors, with cell migration as a default state. Expression of Xbra within the prospective chordamesoderm produces a domain where cell migration is inhibited. The ground state may be restored by inhibition of Xbra itself or the downstream Wnt planar cell polarity pathway. In the embryo, Xbra is excluded from the prechordal mesoderm, allowing these cells to exhibit active cell migration. Xbra acts as a cell behavior switch that is crucial for the proper anteroposterior development of the embryo. Inhibition of Xbra activity in the chordamesoderm results in embryos with a shortened trunk, a consequence of a failure of convergent extension; these cells now actively migrate. Misexpression of Xbra in the prechordal mesoderm results in embryos with head truncations, as a consequence of a failure of cell migration.
It has been reported that Gsc and Mix.1, two
transcription factors expressed in the dorsal marginal zone of gastrula stage
embryos, synergize to repress the expression of Xbra
(Latinkic and Smith, 1999).
Expression of dominant inhibitory forms of either transcription factor results
in inappropriate expression of Xbra in the prechordal mesoderm, and
anterior truncation of the embryos. From the results presented here, it is
likely in those experiments that derepression of Xbra in the
prechordal mesoderm inhibited prechordal mesoderm migration, producing
anterior truncations.
Niehrs and colleagues have reported on the gene anti-dorsalizing
morphogenetic protein (ADMP), expressed in the chordamesoderm, which also
seems to be part of a network that maintains the subdivision between
prechordal and chordamesoderm (Dosch and
Niehrs, 2000). It will be interesting to explore the relationship
between ADMP and Xbra. It has already been shown that ADMP can induce
the expression of Xbra (Moos et
al., 1995
), but whether Xbra also affects the expression
of ADMP is unclear. Similarly, the effects of ADMP specifically on cell
behavior have yet to be determined. Although ADMP is expressed solely in the
chordamesoderm, Xbra is expressed throughout the mesoderm in a
circumblastoporal ring and is specifically excluded from the prechordal
mesoderm. Xbra may be expressed beyond the chordamesoderm to control
various degrees of convergent extension required throughout the mesoderm to
ensure proper blastopore closure via radiolateral convergent extension
movements.
The fact that Xbra expression is maintained in chordamesoderm but not paraxial mesoderm may be important for establishing an axis and polarity for mediolateral convergent extension. In terms of inducing convergent extension in paraxial mesoderm, prolonged Xbra expression may not be required. There may be other signals, specifically from the chordamesoderm, that propagate mediolateral convergent extension behavior, and there may be other genes induced within the paraxial mesoderm itself that play a role in the behavior of its cells.
Just how does Xbra inhibit cell migration? Ectopic expression of Xbra has several effects: inhibition of cell migration, inhibition of the morphological effects elicited by activated Rac and Cdc42 in dissociated animal cap cells, and inhibition of adhesion to fibronectin in a simple cell spreading assay. The inhibition of cell migration depends on the ability of Xbra to bind DNA and activate transcription, which suggests that these effects are mediated by one or more downstream transcriptional targets of Xbra. Wnt11, although required for convergent extension, is not sufficient to inhibit cell migration. Therefore, Xbra must induce the expression of other factors which can, at the very least, inhibit adhesion to fibronectin, either alone or in conjunction with Wnt11. It will be interesting to learn the downstream target genes of Xbra that may be involved in cell adhesion and movement.
In both the activin-induced animal cap system and the endogenous
chordamesoderm, expression of Xbra or Xbra-EnR alters
expression level of many markers as early as stage 10, including Xbra
itself, Mix.1, Xwnt-8, Gsc, Pintallavis, Xnot, Chordin, Noggin and
Wnt11 (Conlon and Smith, 1999;
Tada and Smith, 2000
).
Although changes in any of these genes may represent a change in cell fate, it
is notable that some of the effects described here can be recapitulated using
mutants of Dsh that do not alter cell fate in the dorsal
mesoderm.
In summary, vertebrate embryos possess two distinct cell behaviors that pattern the dorsal side of the embryo. Convergent extension, which can be assayed only in populations of cells, leads to notochord formation and patterns the trunk. Cell migration, which can be studied in individual cells, leads to different cell fates and inductive effects, which are confined to the anterior neural plate. Distinct as they are, in Xenopus, these two behaviors arise from adjacent cell populations; both can be produced downstream of the same signaling protein, activin. The distinction in cell behavior depends on the domain of brachyury expression. Brachyury actively inhibits cell migration while inducing the tissue to undergo convergent extension via the Wnt planar cell polarity pathway. However, Wnt11 is not sufficient to inhibit cell migration. Therefore, the downstream targets of Xbra that inhibit cell migration are unknown, but are capable of inhibiting cell shape changes induced by small GTPases and adhesion to fibronectin in a simple cell spreading assay. These findings raise two further questions: what is the mechanism by which Xbra regulates convergent extension and cell migration, and what establishes the domains of cell behavior? The key to both of these is the regulation of Xbra expression and its downstream targets.
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ACKNOWLEDGMENTS |
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