1 Department of Anatomy and Developmental Biology, University College London,
Gower Street, London WC1E 6BT, UK
2 Millennium Nucleus in Developmental Biology, Facultad de Ciencias, Universidad
de Chile, Casilla 653, Santiago, Chile
3 Fundacion Ciencia para la Vida, Zanartu 1482, Santiago, Chile
* Author for correspondence (e-mail: r.mayor{at}ucl.ac.uk)
Accepted 8 April 2005
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SUMMARY |
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Key words: Neural crest, Cell migration, Wnt, Wnt11, Fz7, Non-canonical, PCP
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Introduction |
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Once the neural crest is induced at the border of the neural plate, its
cells delaminate and move along specific routes to their destination in the
embryo. A number of molecules are known to participate in neural crest
delamination and migration, such as cadherins, Rho GTPases, Noggin and several
extracellular matrix molecules (Borchers
et al., 2001; Bronner-Fraser
et al., 1992
; Henderson et
al., 2000
; Hoffmann and
Balling, 1995
; Kimura et al.,
1995
; Liu and Jessell,
1998
; Nakagawa and Takeichi,
1995
; Nakagawa and Takeichi,
1998
; Perris and Perissinotto,
2000
; Pla et al.,
2001
; Sela-Donenfeld and
Kalcheim, 1999
; Sela-Donenfeld
and Kalcheim, 2000
; Takeichi
et al., 2000
; Vallin et al.,
1998
; Van de Putte et al.,
2003
; Yagi and Takeichi,
2000
). However, the mechanisms by which extracellular signals are
integrated with cell adhesion and cytoskeletal modification to orchestrate the
cell movements underlying delamination and movement of the neural crest are
still unclear.
Mesoderm is another tissue that undergoes extensive cell movement. In
recent years, evidence has accumulated from studies in zebrafish and
Xenopus embryos that supports the notion that the migration of
mesodermal cells during gastrulation is dependent on factors similar to those
involved in planar cell polarity (PCP) in Drosophila, which are
activated by non-canonical Wnt signalling (for reviews, see
Keller, 2002;
Mlodzik, 2002
;
Myers et al., 2002
;
Ueno and Greene, 2003
;
Veeman et al., 2003b
;
Wallingford et al., 2002
).
Non-canonical Wnt signalling (Planar Cell Polarity or Wnt-Ca2+)
affects convergent extension movements through a pathway similar to the
Drosophila PCP pathway. One element in this pathway is the protein
Dishevelled (Dsh); a domain of this protein is required for PCP and for
convergent extension in vertebrates
(Axelrod et al., 1998;
Boutros et al., 1998
;
Heisenberg et al., 2000
;
Tada and Smith, 2000
).
Perturbation of non-canonical Wnt signalling disrupts the mediolateral
elongation and alignment of dorsal mesodermal cells, and the mediolateral
stabilization of cell protrusions
(Wallingford et al., 2000
). In
addition, interference with the non-canonical Wnt signalling pathway of
zebrafish, Xenopus or mouse embryos, either genetically or by the use
of morpholinos, produces defects in convergent extension of the mesoderm and
failures in neural tube closure
(Carreira-Barbosa et al.,
2003
; Curtin et al.,
2003
; Darken et al.,
2002
; Goto and Keller,
2002
; Heisenberg et al.,
2000
; Jessen et al.,
2002
; Kibar et al.,
2001
; Kilian et al.,
2003
; Park and Moon,
2002
; Rauch et al.,
1997
; Takeuchi et al.,
2003
; Veeman et al.,
2003b
).
Here, we present evidence that the non-canonical Wnt pathway regulates neural crest migration. We conclude from the effect of expressing different mutants of the Dsh proteins in Xenopus embryos that canonical (ß-catenin-mediated) Wnt signalling participates in neural crest induction, whereas the non-canonical (PCP or Wnt-Ca2+) pathway controls neural crest migration. Grafts of cells containing fluorescent markers and expressing specific Dsh mutants show that non-canonical Wnt signalling is essential for neural crest migration. We show that Wnt11 is expressed in the ectoderm of Xenopus embryos in a region adjacent to the neural crest cells that expresses the Wnt receptor Fz7. Loss- and gain-of-function experiments of Wnt11 indicate that this ligand is required for neural crest migration in vivo. In addition, localized overexpression of Wnt11 in Xenopus embryos provokes an abnormal migration of the neural crest cells towards the region of high Wnt expression. Finally, by performing time-lapse analysis, we show that the non-canonical Wnt signal controls neural crest migration on a fibronectin substrate by stabilizing the protrusions of the migrating neural crest cells.
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Materials and methods |
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In vitro RNA synthesis and microinjection of mRNAs
All cDNA was linearized and transcribed, as described by Harland and
Weintraub (Harland and Weintraub,
1985) (New England Biolabs). For injections and lineage tracing,
mRNA was resuspended in DEPC-water, and co-injected into two- or
eight-cell-stage embryos with fluorescein dextran or rhodamine dextran (FDX,
RDX; Molecular Probes) using 8-12 nl needles as described by Aybar et al.
(Aybar et al., 2003
). The
constructs used were Slug (Mayor
et al., 1995
); Wnt11
(Ku and Melton, 1993
);
Fz7 (Medina et al.,
2000
); dd1 and dd2 (Sokol,
1996
); and Dsh-
N, Dsh-DEP+ and dnWnt11
(Tada and Smith, 2000
).
In vitro culture, time lapse and immunostaining of neural crest cells
In vitro culture of neural crest cells was performed as described by
Alfandari et al. (Alfandari et al.,
2003). For time-lapse recordings of migrating neural crest, images
were collected on a Nikon Eclipse E1000 Microscope using a Jenoptik/Jena cam.
Images were collected every 2 minutes and time-lapse stacks were assembled and
viewed in OpenLab software. Protrusive activity was quantified by counting new
protrusions extending, existing protrusions withdrawn, or stable protrusions
(present in both the first frame and the last frame of the movie).
Phalloidin-rhodamine and microtubule staining was performed by incubating with
phalloidin-rhodamine (Sigma-Aldrich), or with a monoclonal antibody against
-tubulin (Sigma-Aldrich) for 1 hour; the secondary antibody used was
IgG-FITC (Sigma-Aldrich). Lamellipodia were counted as large when they
occupied more than one-third of the cell border, and as normal when they were
smaller than one-third of the cell border.
Scanning electron microscopy (SEM)
Embryos were microinjected and their neural crest cultured in vitro as
described above. They were then fixed in 0.2 M cacodylate buffer and 1.5%
glutaraldehyde, and rinsed in 0.1 M cacodylate buffer, as described previously
(Sadaghiani and Thiebaud,
1987). Critical point drying was performed by using ethanol and
liquid nitrogen.
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Results |
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Based on these results, we decided to use Dsh-DEP+ and Dsh-N to study
neural crest migration. Embryos were injected as described, but fixed at
stages when neural crest migration is taking place (stages 24-25). Injection
of either construct produced a dramatic effect on neural crest migration as
visualized by Slug expression
(Fig. 2A,B). On the injected
side, Slug-expressing cells were seen in a group on the surface of
the embryo with no indication of cell migration (white arrowheads in
Fig. 2A,B), whereas the
uninjected side (the control) showed the normal streams of cephalic neural
crest cell migration (red arrowheads in
Fig. 2A,B). To inhibit the
PCP/Wnt-Ca2+ pathway specifically in neural crest cells, the
following experiment was performed (Fig.
2C). FDX was injected at the one-cell stage, either alone or
together with mRNA encoding Dsh-DEP+. Then, at the early neurula stage, the
prospective neural crest was grafted into a normal embryo. Host embryos were
then cultured to stage 26, when the distribution of the fluorescent neural
crest cells was examined. Grafts of control neural crest cells show a normal
distribution, with typical streams of migrating cephalic neural crest cells
(red arrowheads in Fig. 2D,F).
However, grafts of cells expressing Dsh-DEP+ showed complete inhibition of
migration of the neural crest (white arrowhead in
Fig. 2E,G), consistent with the
phenotype shown in Fig. 2B.
|
|
The expression of Wnt11 was compared with that of the neural crest marker Slug at different times during development (Fig. 3). Our results show that just before migration of the neural crest (stage 17) Wnt11 is expressed adjacent to the prospective migrating cells (Fig. 3A-C,E-G). The prospective neural crest, defined by expression of Slug (Fig. 3A,E), is adjacent to a continuous band of Wnt11-expressing cells flanking the prospective pathway of migration (Fig. 3B,F). Double in situ hybridization for Slug and Wnt11 shows Wnt11 expression at the most lateral side of Slug expression (Fig. 3C,G). The continuous band of Wnt11 that borders the cephalic neural crest is not uniform; there are regions where Wnt11 seems to be expressed more strongly or in a larger population of cells (compare black and white arrows in Fig. 3C). Once neural crest cells start to migrate (Fig. 3I), the Wnt11-expressing cells do not move, instead they remain on the dorsal aspect of the neural tube (Fig. 3J) while the neural crest cells move underneath them (Fig. 3K).
Although no specific Wnt11 receptor has been identified, there is some
evidence that suggests that PCP Wnt signalling involves Fz7
(Carreira-Barbosa et al.,
2003; Djiane et al.,
2000
; Medina et al.,
2000
; Sumanas and Ekker,
2001
; Winklbauer et al.,
2001
). We examined the distribution of the Fz7 receptor in neural
crest cells. Our results show expression of Fz7 in different regions
of the neural ectoderm, as has been described previously
(Djiane et al., 2000
;
Wheeler and Hoppler, 1999
),
including in the pre-migratory neural crest
(Fig. 3D,H) and the migrating
crest cells (Fig. 3L). A
comparison of Fz7 and Slug expression indicates that
Fz7 is expressed in a subpopulation of neural crest cells located
adjacent to the Wnt11-expressing cells in the ectoderm
(Fig. 3E,G,H). Interestingly,
these cells are probably the first cells to delaminate. In summary, early in
neural crest migration, Wnt11 is present in cells adjacent to the
first migrating cells, which also express the receptor Fz7
(Fig. 3M). Once the neural tube
closes, the early migrating crest cells move away and beneath the
Wnt11-expressing cells, so that later migrating cells come into
contact with the source of Wnt11 signalling
(Fig. 3N).
|
|
Finally, we analyzed whether the inhibition of neural crest migration by
dnWnt11 could be rescued by intracellular activation of the
PCP/Wnt-Ca2+ pathway in the neural crest cells
(Fig. 6). The normal migration
of the neural crest (Fig. 6B)
was inhibited by the expression of dnWnt11
(Fig. 6C); however, when these
embryos received a graft of neural crest taken from an embryo injected with
Dsh-N, an activator of the non-canonical pathway
(Tada and Smith, 2000
),
complete rescue of neural crest migration was observed
(Fig. 6D,E).
Non-canonical Wnt signalling controls cell protrusion in the migrating neural crest cells
The non-canonical Wnt pathway controls convergent extension of mesoderm
during gastrulation movements by instigating a directional activity of the
lamellipodia that favours movement in one direction
(Carreira-Barbosa et al.,
2003; Wallingford et al.,
2000
). Although no similar directionality of individual migratory
neural crest cells has been described, we decided to investigate whether we
could identify similar cell behaviour, controlled by non-canonical Wnt
signalling. We cultured neural crest cells in vitro and analyzed their
behaviour (Fig. 7A). In control
explants, neural crest cells migrated normally
(Fig. 7B), as described
previously (Alfandari et al.,
2003
); however, in explants taken from embryos injected with 1 ng
of Dsh-DEP+ mRNA or 2 ng of dnWnt11, migration of cells was
strongly inhibited (Fig. 7C-E).
To examine the effect at a cellular level, we performed a time-lapse analysis
of the migrating neural crest cells. The number and shape of cell protrusions
was counted in control and Dsh-DEP+ expressing cells in frames from time-lapse
video movies (Fig. 7F-I). Our
results indicate that in explants from Dsh-DEP+ expressing embryos, there were
less cell protrusions than in control cells. The frequency of crest cells
withdrawing rather than extending cell processes is greater in the Dsh-DEP+
cells than in the control cells (Fig.
7J). To extend these observations, we visualised actin
microfilaments with phalloidin-rhodamine, and microtubules by immunostaining,
and then analyzed the size and types of lamellipodia
(Fig. 7K-P). In control cells,
lamellipodia were larger and more polarized than in the Dsh-DEP+ expressing
cells, whereas the Dsh-DEP+ expressing cells exhibited more filopodia than the
control neural crest cells (Fig.
7Q). A typical control cell is shown in
Fig. 7L,M (more than 50% of
cells), while typical Dsh-DEP+ expressing cells are shown in
Fig. 7O,P (more than 90% of
cells, although most of the cells were found forming groups and very few were
isolated). We also analyzed the morphology of the migrating neural crest by
SEM (Fig. 7R-T). Control
migrating cells exhibited large lamellipodia at the front of migration (yellow
arrows in Fig. 7R,R'),
while cells injected with Dsh-DEP+
(Fig. 7S,S') or
dnWnt11 (Fig.
7T,T') exhibited long filopodia that frequently were
connecting the more packed cells (red arrows).
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Discussion |
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Several observations suggest that the localized expression of Wnt11 adjacent to the premigratory neural crest is essential for its function. First, overexpression of Wnt11 in one half of the embryo completely blocks normal neural crest migration (Fig. 4) and suggests a requirement for localized expression for this molecule. Second, when Wnt11 was expressed in a localized manner opposite to its normal expression, the neural crest cells migration was blocked (Fig. 5). Third, when Wnt11 was expressed in the normal route of crest migration the cells migrated actively under the Wnt11-expressing cells (Fig. 5). Taken together, these results show that localized expression of Wnt11 is required to activate the PCP/Wnt-Ca2+ pathway and to control neural crest migration. We do not know the molecular function of this localized Wnt expression in the neural crest cells, but the situation in Drosophila PCP suggests that a gradient of Wnt11 could determine the asymmetric expression of PCP molecules that direct crest migration. No asymmetrical localization of PCP proteins has been observed in any vertebrate system.
In addition to the evidence suggesting that Wnt11 is required for neural
crest migration, our data based on Dsh mutants show that non-canonical Wnt
signalling participates in neural crest migration. Dsh-DEP+, a
dominant-negative form of Dsh that contains the DEP domain and lacks the DIX
and PDZ domains, has been an incisive reagent for analysing the role of the
non-canonical pathway in neural crest migration. This blocks the
PCP/Wnt-Ca2+ pathway without affecting canonical signalling
(Tada and Smith, 2000). It
produces a strong inhibition of neural crest migration in vivo
(Fig. 2) and in vitro
(Fig. 7), either when injected
into one side of the embryo or when specifically expressed in neural crest
cells. Analysis of early neural crest markers shows no effect of Dsh-DEP+ on
neural crest induction (Fig.
1), indicating that non-canonical signalling does not participate
in neural crest induction. Another Dsh mutant, dd1 is also able to
block neural crest migration (data not shown), but it also interferes with the
canonical Wnt pathway (Tada and Smith,
2000
), and as a consequence reduces neural crest induction
(Bastidas et al., 2004
).
Overexpression of Wnt11 dramatically affected neural crest migration, but also
affected neural crest induction in some cases. This small effect could be
explained either by an indirect effect on mesoderm or by inhibition of the
canonical signal through the non-canonical Wnts
(Torres et al., 1996
;
Prieve and Moon, 2003
;
Maye et al., 2004
). Inhibition
of canonical Wnt signalling by a dominant-negative form of Tcf3 does not
inhibit neural crest migration (F. Romero and R.M., unpublished). There is
convincing experimental evidence that shows that canonical Wnt signalling is
involved in neural crest induction and cell differentiation
(Dorsky et al., 1998
;
Garcia-Castro et al., 2002
;
LaBonne and Bronner-Fraser,
1998
; Lee et al.,
2004
; Lewis et al.,
2004
; de Melker et al.,
2004
; Tan et al.,
2001
; Villanueva et al.,
2002
). Wnt signalling evidently plays a crucial role in neural
crest development, in the canonical pathway in induction and in the
non-canonical pathway in neural crest migration, as we have shown here.
|
The participation of PCP on cell movements during gastrulation has been
very well characterized, although how Wnt controls cell movement remains
unknown. Migration of the neural crest cells requires an
epithelial-mesenchymal transition (EMT), an elaborate process that occurs in
many steps. There is an initial delamination step that is essential for the
second step of neural crest migration. Our results using Dsh and Wnt11
mutants, which show inhibition of cell movement both in vivo and in vitro, are
compatible with an inhibition of delamination or posterior cell movement of
the neural crest cells. Localized expression of Wnt11, by a graft of
Wnt11-expressing ectoderm, shows an effect on crest migration that is
dependent on the position of the graft. We propose that Wnt11 can trigger a
cellular activity required for cell movement during delamination and/or cell
migration, and that the crest cells require additional cues to translate this
into an effective cell migration. The possibility that neural crest was
induced in the graft is ruled out, as competence for neural crest induction is
lost at the stage at which the tissue was transplanted
(Mancilla and Mayor, 1996;
Bastidas et al., 2004
). It is
still possible that Wnt11 promotes cell proliferation
(Ouko et al., 2004
), although
this seems unlikely as cell numbers in our in vitro cultures did not increase
after stimulating Wnt11 signalling. We, therefore, favour an effect of Wnt11
on neural crest migration, instead of cell proliferation. A recent report
shows that PCP signalling controls the orientation of cell division during
gastrulation (Gong et al.,
2004
). No analysis of cell division orientation during neural
crest migration has been reported, but inhibition of the cell cycle blocks
neural crest migration (Burstyn-Cohen and
Kalcheim, 2002
; Saka and
Smith, 2001
). Thus, it is possible that Wnt11 signalling controls
cell migration by controlling cell divisions.
Neural crest migration in vitro and in vivo is blocked by Dsh and Wnt11
mutants, to a similar extent (Fig.
7). The ability to block cell migration in vitro suggests that the
neural crest cells have already responded to Wnt11 signalling at the time of
the dissection. This is possible, as the Wnt11-expressing cells are
adjacent to the neural crest and it would be difficult to exclude them from an
in vitro culture. Analysis of cell protrusions in migrating crest cells in
vitro shows that non-canonical Wnt signalling is required to stabilize the
lamellipodia. Inhibition of the PCP pathway increases the number of cells with
filopodia with a less-polarized phenotype than the control neural crest cells.
Similar functions for the PCP pathway and Wnt11 have been described during
gastrulation in Xenopus and zebrafish embryos
(Ulrich et al., 2003;
Wallingford et al., 2000
). We
propose that Wnt11 controls cytoskeletal behaviour or cell adhesion properties
in neural crest migration, and that it is required to generate the cell
protrusions necessary for locomotion.
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ACKNOWLEDGMENTS |
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