1 Laboratoire de Génétique et de Physiologie du
Développement (LGPD). Developmental Biology Institute of Marseille
(IBDM), CNRS UMR 6545. Université de la Méditerranée,
Campus de Luminy, case 907, 13288 Marseille, Cedex 09, France
2 Department of Biology, San Francisco State University, 1600 Holloway Ave., San
Francisco, CA 94132, USA
3 School of Life Sciences, Arizona State University, Tempe, AZ 85287, USA
Authors for correspondence (e-mail:
c.linker{at}ucl.ac.uk;
marcelle{at}ibdm.univ-mrs.fr)
Accepted 30 June 2005
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SUMMARY |
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Key words: Chick, Somite, Wnt, Paraxis, ß-Catenin, Epithelial-mesenchymal transition
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Introduction |
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The chick somite represents an attractive experimental model where EMT processes can be studied in vivo. The possibility of expressing molecules that can either activate or inhibit specific signalling pathways in the somites, by means of the electroporation technique, together with the analysis of gene expression and of cell morphology, represent powerful tools with which to analyse the regulation of EMT in a three-dimensional system.
Newly formed somites comprise an outer epithelium, the cortex, surrounding
a central cavity, the somitocoel. Cortex cells are bottle-shaped and polarised
(apical side facing the somitocoel), and they express, at the adherens
junctions located at their apical end, the epithelial markers N-cadherin,
ß-catenin and F-actin (Gros et al.,
2005). As somites differentiate, cortex cells undergo multiple EMT
processes to give rise to their derivatives: the ventral portion of the somite
first dissociates into a mesenchyme, the sclerotome, which becomes organised
around the notochord and neural tube and differentiates into axial cartilage
and bones. The remaining dorsal epithelium, the dermomyotome, is the source of
all skeletal muscles of the body and the limbs, as well as precursors for the
dermis of the back (Christ and Ordahl,
1995
). Skeletal muscles form in two stages: first, cells located
at the epithelial borders of the dermomyotome delaminate into the nascent
primary myotome to generate postmitotic myocytes
(Gros et al., 2004
); the
second stage is characterised by the massive entry of embryonic muscle
progenitors within the myotome, triggered by the EMT of the dermomyotome
(Gros et al., 2005
;
Relaix et al., 2005
;
Kassar-Duchossay et al., 2005). Concomitantly, cells located in the medial
domain of the dermomyotome dissociate and migrate as single cells towards the
ectoderm, where they condense to form the dermis of the back
(Brill et al., 1995
;
Olivera-Martinez et al., 2002
;
Gros et al., 2005
; Ben Yair et
al., 2005). Finally, limb, tongue and diaphragm muscle precursors dissociate
from the epithelial border of the lateral dermomyotome and migrate as single
cells to later differentiate into their cognate muscles
(Bladt et al., 1995
;
Brand-Saberi et al., 1996
;
Dietrich et al., 1999
;
Heymann et al., 1996
).
The analysis of paraxis (Tcf15) mutant mice has shown that the
epithelial organisation of somites is crucial for the later morphogenesis of
trunk muscles. These mutants display somites that are unable to organise
themselves into an epithelium, and although the specification of skeletal
muscles takes place correctly, the spatial organisation of their muscles is
grossly altered (Burgess et al.,
1996; Wilson-Rawls et al.,
1999
). These data indicate that muscle progenitors of the body use
the epithelial sheet of the dermomyotome as a scaffold on which they organise.
Thus, an important step towards a comprehension of the organisation of the
trunk skeletal muscles lies in an understanding of the tissue interactions and
the molecular networks that regulate the epithelial organisation of
somites.
Embryonic manipulations have shown that both the ectoderm and the dorsal
neural tube can promote the induction or the maintenance of dermomyotome
structure (Borycki and Emerson,
2000; Burgess et al.,
1995
; Correia and Conlon,
2000
; Dietrich et al.,
1998
; Dietrich et al.,
1997
; Fan and Tessier-Lavigne,
1994
; Goulding et al.,
1991
; Maroto et al.,
1997
; Reshef et al.,
1998
; Spence et al.,
1996
). In vivo and in vitro experiments support a model where Wnt
proteins mediate this dorsalising activity of the neural tube and ectoderm
(Capdevila et al., 1998
;
Fan et al., 1997
;
Maroto et al., 1997
;
Wagner et al., 2000
;
Münsterberg et al., 1995
;
Stern et al., 1995
;
Galli et al., 2004
). Whether
or not these tissues and molecules regulate the epithelial organisation of
somites has not been systematically addressed in these experiments.
Importantly, Pax3, which has been generally used as a dorsal somitic marker,
cannot be considered as an adequate epithelium marker, because it is expressed
in paraxis-null mice, where the epithelial structure is not present
(Burgess et al., 1996
;
Wilson-Rawls et al., 1999
).
Moreover in the Pax3 knockout mice, the epithelial structure of the
somite is initially normal, indicating that Pax3 does not play an active role
in this process (Daston et al.,
1996
). This is not due to a functional redundancy with Pax7, which
is expressed in somites in a similar fashion as Pax3, because in
Pax3/Pax7 double knockout mice, epithelial somites initially
form (Frédéric Relaix and Margaret Buckingham, personal
communication).
While the phenotype of paraxis-null mice clearly implicates this molecule
in the epithelialisation of the somite, the mechanisms that regulate its
expression are poorly understood. Studies in chick and mouse indicate that
paraxis expression is initiated independently of surrounding structures in a
mesoderm autonomous way (Correia and
Conlon, 2000; Linker et al.,
2003
; Palmeirim et al.,
1998
). Sosic and colleagues
(Sosic et al., 1997
) have
shown that ectoderm provides signals that maintains paraxis expression and the
epithelial organisation of somites, but they have not addressed the role of
Wnt in this process. Wnt6 is the only Wnt known to be expressed in the
ectoderm overlying the anterior segmental plate and early somites of the chick
embryo (Cauthen et al., 2001
;
Marcelle et al., 2002
;
Schubert et al., 2002
;
Rodriguez-Niedenführ et al.,
2003
), which makes it a likely candidate to play a role in this
process. Schmidt and collaborators
(Schmidt et al., 2004
) have
recently shown that Wnt6 promotes the epithelial organisation of the segmental
plate mesenchyme. However, the effect of this molecule in later events of
somite differentiation, such as dermomyotome formation and dermis
specification, was not addressed.
In this study, we have analysed the tissue and molecular interactions that
regulate the epithelial organisation of somites. Through a systematic removal
of tissues surrounding the somites, followed by a time-course analysis, we
show that the ectoderm is not responsible for the formation, but for the
maintenance of the epithelial structure of the somite. Our experiments confirm
that Wnt6 is the molecule from the ectoderm responsible for this effect. In
addition, we show evidence that this action is specific to Wnt6, while Wnt1
exerts a distinct function in the medial compartment of the dermomyotome. We
then characterised the intracellular signalling pathway activated by Wnt6 in
the somites. Three Frizzled receptors, Fz1, Fz2 and Fz7 are
expressed in epithelial somites and in the dermomyotome
(Linker et al., 2003).
Although the expression of all three is lost upon removal of the ectoderm,
Wnt6 maintains Fz7, but not Fz1 or Fz2 expression,
suggesting a specific role for Fz7 in mediating Wnt6 activity. Blocking the
activity of ß-catenin or Lef1 in the dermomyotome leads to a loss of
somite cell polarisation, and a de-epithelialisation of the dermomyotome,
indicating that Wnt6 activity is mediated by a ß-catenin-dependent
pathway. Finally, we demonstrate that paraxis is a target of the
ß-catenin signal and that its prolonged expression is sufficient to
counter the de-epithelialisation of the dermomyotome that normally takes place
during late embryonic development. These results establish that a
ß-catenin activity, initiated by Wnt6 through Fz7 and mediated by
paraxis, is required for the maintenance of the epithelial
organisation of somites.
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Materials and methods |
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Antibodies, immunohistochemistry, confocal analysis on cryosections
Phalloidin Alexa 456 (Molecular Probes) was used to detect F-actin. The
following antibodies were used: a mouse monoclonal against N-cadherin (Sigma
C-8538) and a polyclonal antibody against GFP (Torrey Pines Biolabs). Sections
were examined with a confocal microscope Zeiss LSM 510 Meta.
Injection of Wnt6-expressing cells and electroporation
Electroporation was performed as described
(Scaal et al., 2004). For the
observation of electroporated somites, embryos were fixed in 4% formaldehyde,
cleared in 90% glycerol/H2O and examined with a confocal
microscope. Image stacks (50-100 µm thick) were treated with Metamorph
(Universal Imaging) and Imaris (Bitplane) image analysis software for image
visualisation and 3D reconstruction. The full-size mouse paraxis cDNA
clone (provided by A. Rawls), a dominant-negative form of Xenopus
ß-catenin (kindly provided by P. McCrea)
(Montross et al., 2000
), and a
dominant-negative form of chick Lef1 [kind gift of J. C. Ispizua-Belmonte
(Kengaku et al., 1998
)] were
cloned into the pCLGFPA electroporation vector
(Scaal et al., 2004
), which
drives the gene of interest under the control of a CMV/chick ß-actin
enhancer/promoter. The pCLGFPA vector also contains the coding sequence for
the reporter gene e-GFP under the control of a SV40 promoter/enhancer. Cell
counting was statistically analysed. Results were different from each other
with a 95% confidence; P<0.005 were evaluated by a Student's
t-test. COS cells were transfected using the Lipofectamine agent
(Gibco-BRL) with 1 µg of the pHYK vector containing the full-length mouse
Wnt6 cDNA (obtained from A. McMahon and L. Burrus)
(Burrus and McMahon, 1995
).
Forty-eight hours after transfection, cells were collected by trypsinisation,
labelled with DiI (DM-DiI, Molecular Probes) and pressure-injected into
somites of stage 12HH developing embryos using a Picospritzer (General Valve
Corporation), as described previously
(Linker et al., 2003
).
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Results |
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The ectoderm maintains the epithelial structure of the dermomyotome
We have investigated the role of the tissues surrounding the paraxial
mesoderm on somite epithelialisation. This was carried out over an extended
period of time to follow the progression of the phenotypes obtained after
these manipulations. We have analysed the expression of paraxis and
of the Wnt receptors Frizzled 1, Frizzled 2 and Frizzled 7.
In these experimental conditions, our results demonstrate that the surgical separation of the rostral segmental plate mesoderm from axial (neural tube and notochord, Fig. 1A) or lateral (lateral plate mesoderm, Fig. 1B) structures had no visible effects on the formation of epithelial somites (Fig. 1C,D) or on the expression of paraxis (10/11, Fig. 1F), Fz1 (8/8, not shown), Fz2 (11/11, not shown) and Fz7 (9/9, Fig. 1C-E,G).
We then tested whether the ectoderm overlying the segmental plate and somites plays a role in the formation, and/or the maintenance, of an epithelial dermomyotome. Ectoderm was separated from the paraxial mesoderm by the positioning of a tantalum foil between these tissues (Fig. 2A). Embryos were then allowed to develop for varying times, and their morphology and gene expression were examined. After 3 hours incubation, two somites had formed under the membrane (Fig. 2B), their epithelial structure was normal in section (not shown) and they expressed the three Fz proteins normally (Fz1 9/9, Fz2 6/6 not shown and Fz7 7/7, Fig. 2B). The same result was obtained if embryos were re-incubated for 6 or 8 hours (not shown, 13/13). Twelve hours of separation from the ectoderm did not affect the morphology of somites (Fig. 2I). However, the dorsal epithelium, did not express paraxis (9/9, Fig. 2G), pax3 (8/8, Fig. 2E) or any of the three Frizzled (Fz1 10/10, Fz2 8/8 not shown, Fz7 10/10; Fig. 2C,I). Finally, when embryos were allowed to develop for 24 hours after the manipulation, the dermomyotome was no longer recognisable. Instead, the entire somite presented a mesenchyme-like structure (Fig. 2J) and the expression of all the markers was completely abolished (Fz1 18/18, Fz2 16/20 not shown, and Fz7 17/17, Fig. 2D,J; paraxis 13/13, Fig. 2H; pax3 8/8, Fig. 2F).
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|
Overlapping and unique properties of Wnt6 and Wnt1 on somites
Wnt6 is the only member of the Wnt family known to be expressed in the
ectoderm overlying the segmental plate and newly formed somites, in the chick
embryo (Marcelle et al., 2002;
Schubert et al., 2002
;
Rodriguez-Niedenfüihr, 2003). For this reason, Wnt6 was the best
candidate to be playing a role in the maintenance of the epithelial structure
of the dermomyotome.
To test this hypothesis, Wnt6-expressing cells (DiI-labelled) were injected in the anterior segmental plate, the ectoderm above the injected region was separated from segmental plate by an impermeable membrane as described before, and embryos were allowed to develop for 24 hours (Fig. 3A). Under these conditions, paraxis (26/31, Fig. 3H) and pax3 (11/18, Fig. 3G) expression was rescued in dermomyotomal cells close to the injection site. Interestingly, Wnt-6 injected cells were able to rescue Fz7 expression (9/12, Fig. 3B,F), but failed to rescue Fz1 (15/15, Fig. 3D) or Fz2 (14/21, Fig. 3E) expression. In transverse sections, we could also clearly observe an epithelial dermomyotme-like structure around the Wnt6-expressing cells, at the place where paraxis and Fz7 expression was rescued (Fig. 3B). Control, untransfected cells did not rescue the expression of any of these genes (Fz7 9/11, paraxis 11/12; pax3 8/8, not shown), or the epithelial structure of the dermomyotome.
A number of studies have implicated Wnt molecules from the neural tube in
the patterning of the somites, as well as the control of their
epithelialisation. To test whether the observed effects were a specific
response to Wnt6, we performed the same set of experiments using
Wnt1-expressing cells. Under these conditions, we observed that Wnt1 is not
able to rescue Fz1 (11/13, Fig.
3J) or Fz7 expression (17/18,
Fig. 3L). By contrast,
paraxis (12/13, Fig. 3N)
and pax3 (18/19, Fig.
3M) expression was rescued around the injected cells in most
embryos, whereas Fz2 expression was distinctively, albeit faintly, activated
in 60% of the analyzed embryos (8/13, Fig.
3K). On sections, we could observe that an epithelial,
dermomyotome-like structure was present around the Wnt1 cells
(Fig. 3C). Further confirmation
that Wnt1 and Wnt6 display distinct function in somite differentiation came
from the comparison of their role in the patterning of the dermomyotome. As
Wnt1 regulates Wnt11 expression in the dorsomedial lip of
the dermomyotome (Marcelle et al.,
1997), we compared the induction of Wnt11 by Wnt1 with that of
Wnt6. Whereas Wnt1-expressing cells were able to rescue, and ectopically
induce, Wnt11 expression (5/5,
Fig. 3O), Wnt6-expressing cells
were never able to do so under the same experimental conditions (6/6,
Fig. 3I).
Together, these data indicate that Wnt1 and Wnt6 activate overlapping and
distinct cellular responses in the paraxial mesoderm. Although it is tempting
to postulate from our data that Wnt6 and Wnt1 mediate their activity through
Fz7 and Fz2, respectively, further analysis of this process will be needed to
determine whether this is indeed the case. We observed that Wnt1 and Wnt6 are
both able to rescue the epithelial organisation of somites. However, as the
epithelial organisation of somites was never perturbed in the absence of
neural tube (the source of Wnt1), this indicates that Wnt6 is the endogenous
molecule regulating the epithelial organisation of somites in the embryo.
Thus, it is likely that under our experimental conditions, Wnt1 mimics the
activity of Wnt6 on the epithelial organisation of somites. Alternatively,
Wnt1 (or a Wnt1-like molecule in the neural tube) and Wnt6 cooperate for the
epithelialisation of the somite: Wnt from the neural tube might provide
additional epithelialisation function, resulting in a
`hyper-epithelialisation' in the medial dermomyotome. Indeed, the medial
border of the dermomyotome (the DML) displays a stronger expression of
adherens junction molecules (ß-catenin, N-cadherin and actin) than the
rest of the dermomyotome (Gros et al.,
2005). This should result in an increased cell adhesion in the
DML, which might be functionally relevant during myotome formation. At
present, we cannot exclude any of the two possibilities.
|
This was performed by the electroporation of dominant-negative forms of
ß-catenin or Lef1 molecules in newly formed somites
(Kengaku et al., 1998;
Montross et al., 2000
).
Constructs containing either of the factors, together with the GFP reporter,
were electroporated in the dorsal region of newly formed epithelial somites
(somites I to IV). An expression plasmid containing only GFP served as
control. After 24 hours of incubation, embryos were analysed by confocal
microscopy. Control dermomyotomal cells exhibited a typical pseudo-epithelial
morphology, with bottle-shaped cells extending long cytoplasmic filopodia
towards the ectoderm, and expressing at their apical end the adherens junction
markers N-cadherin and actin (Fig.
4A-D). By contrast, somites electroporated with DN ß-catenin
(Fig. 4E) or DN Lef1 constructs
(Fig. 4F-H) displayed a
disorganised dermomyotome composed of round cells, which have lost the
majority or their cytoplasmic extensions. In those cells, N-cadherin was
redistributed evenly along the cell membrane
(Fig. 4G,H). In embryos
electroporated with DN Lef1, we did not observe any significant increase in
activated caspase 3 staining, indicating that round cells were not dying (not
shown).
These data show that blockage of the Wnt ß-catenin canonical pathway promotes the de-epithelialisation of dermomyotomal cells, implying that Wnt6, which is secreted by the ectoderm, initiates a ß-catenin-dependent cascade that is required for the maintenance of the epithelial structure of the dermomyotome.
paraxis acts downstream of ß-catenin to maintain the epithelialisation of the dermomyotome
The phenotype of paraxis-null mice clearly implicates this molecule in
somite epithelialisation (Burgess et al.,
1996). Thus, an attractive possibility was that Wnt6 secreted by
the ectoderm interacts with Fz7 activating the ß-catenin-dependent
cascade responsible for regulation of the expression of paraxis,
which controls the epithelial morphology of the dermomyotome.
The first requirement for such hypothesis is that Fz7 expression should precede paraxis expression in the presomitic mesoderm during development. To test this, we have analysed the expression of Fz7 and paraxis in two halves of the same embryo. By doing this, we were able to directly compare the expression of each gene in embryonic tissues at the exact same age and axial level (Fig. 5A). This analysis clearly showed that Fz7 expression precedes paraxis expression in the presomitic mesoderm (Fig. 5D, 5/5). A second requirement is that the expression of Fz7 should be downregulated before the expression of paraxis when the ectoderm is removed. To test how the expression of these two genes is lost in the absence of ectoderm, we placed two membranes, one at each side of the embryo. Embryos were then reincubated and one half was tested for paraxis and the other for Fz7 expression (Fig. 5B). As expression of both genes is lost after 12 hours (Fig. 2), we decided to decrease the time of incubation. After 10 hours of incubation, Fz7 expression was greatly reduced, or absent, while in the same embryos exhibits a robust expression of paraxis (Fig. 5E, 11/11). These results support the hypothesis that paraxis could be a downstream target of Fz7 activation. To test this directly, we have analysed paraxis expression in dermomyotomal cells where either ß-catenin or Lef1 activity was inhibited. Following the electroporation of somites with DN-ß-catenin (Fig. 5C, black arrowheads) or DN-Lef1 (Fig. 5F-G), we observed that paraxis expression was downregulated in the electroporated cells, whereas in control cells (Fig. 5C, white arrowheads) or in GFP electroporated cells, paraxis was expressed normally (not shown). These results strongly suggest that Wnt signalling mediated by ß-catenin is required for maintenance of paraxis expression in the dermomyotome.
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|
Wnt6 or paraxis expression are sufficient to counter the de-epithelialisation of the dermomyotome
Between day 3 and day 6 of development, the dermomyotome de-epithelialise
to give rise to the dermis of the back
(Brill et al., 1995;
Christ and Ordahl, 1995
;
Couly and Le Douarin, 1988
;
Olivera-Martinez et al., 2000
;
Olivera-Martinez et al., 2002
;
Gros et al., 2005
). The
de-epithelialisation of the dermomyotome is progressive, first observed in the
cervical region of an E3 embryo (stage 18 HH), in a central domain of the
dermomyotome. Then it advances posteriorly along the embryonic axis and
towards the borders of the dermomyotome; the medial and lateral borders of the
dermomyotome are the last to dissociate between E5 and E6
(Marcelle et al., 2002
;
Scaal et al., 2004
;
Gros et al., 2005
).
Interestingly, Wnt6 expression, which is homogeneous in the ectoderm located
above newly formed somites, is progressively downregulated in all but the
ectoderm located above the somitic borders
(Marcelle et al., 2002
). This
is followed in the dermomyotome by a similar loss in the expression of
paraxis, which is initially homogeneous and becomes progressively
restricted to the epithelial borders of the dermomyotome
(Fig. 6A,B). Although the
reason for the Wnt6 progressive spatial restriction in the ectoderm is
unknown, an interesting hypothesis is that it could account for the
de-epithelialisation of the dermomyotome. To analyze this possibility, we have
injected Wnt6-expressing cells under the medial region of the dermomyotome at
E2.5, and the embryos were left to develop for 48 hours. In this way, we
presented a continuous source of Wnt6 to a central region of the dermomyotome
that should otherwise have undergone de-epithelialisation. Under these
conditions, high level of paraxis expression was maintained around
the source of Wnt6 (8/8, Fig.
6A-C), while de-epithelialisation did not take place
(Fig. 6D,E). These observations
indicate that the dissociation of the dermomyotome observed between E3 and E6
may be due to the disappearance of the epithelium-maintaining signal, Wnt6.
Evidently, this observation does not exclude the possibility that
dissociation-promoting signals may also participate in this process.
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Discussion |
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Finally, our observation that genes implicated in cell epithelialisation
(e.g. paraxis) and cell differentiation (e.g. pax3) are
regulated in a similar manner by the presence or absence of the ectoderm (and
by Wnt6) underlines the close interconnection of both processes during
mesoderm maturation. Although this link has been demonstrated in many
developmental and pathological EMTs, it remains largely unexplained at a
molecular level. Recent discoveries on the role of GSK-3ß provide
important insights into this crucial question: in addition to its role in
mediating the canonical ß-catenin dependent Wnt signal, GSK3ß acts
as a molecular sensor for a number of signalling pathways (MAPK-, PI(3)K/Akt-,
SHH/Gli-dependent pathways) to regulate the activity of the zinc-finger
transcription factor snail, which is a major player during
epithelial-mesenchymal transitions (Zhou
et al., 2004). It will be important to determine whether such
molecular networks play a role in both the differentiation process and the EMT
of the dermomyotome.
Wnt6-Fz7: a specific pathway regulating the epithelial structure of the dermomyotome?
Our experiments indicate that Wnt6 secreted by the ectoderm is responsible
for the maintenance of the epithelial structure of the dermomyotome.
Wnt6-expressing cells are able to rescue the epithelial structure of the
somites when ectoderm is removed. Under these conditions, dermomyotome cells
maintain the expression of Fz7, whereas the expression of
Fz1 and Fz2 is not maintained. As Fz1, Fz2 and
Fz7 are the only Frizzled receptors identified by PCR in early
somites (Linker et al., 2003),
this observation suggests that Fz7 is the receptor that mediates the
signal transduction of Wnt6 in somites. The Wnt6 signal also maintains the
expression of paraxis and pax3. These data confirm those
recently obtained by Schmidt et al.
(Schmidt et al., 2004
).
However, we significantly extend their findings by showing that the effect
that we observed after Wnt6 ectopic expression is specific to this molecule,
showing that Wnt1 has a distinct action on somites. Wnt1 is able to maintain
Fz2 expression (instead of Fz7 as Wnt6) and to induce the
formation of a dorsomedial, Wnt11-positive compartment in the
dermomyotome, whereas Wnt6 is unable to do so.
These data show that in vertebrates, Wnts can control the activity of their
receptors through a modulation of their transcription. A similar regulatory
mechanism has been observed in the Drosophila wing imaginal disc, where
Wingless inhibits the transcription of Drosophila Fz2, contributing
to the generation of a gradient of its activity
(Cadigan et al., 1998;
Lecourtois et al., 2001
).
Later during wing development, Wingless is capable of activating
Drosophila Fz3 transcription
(Sato et al., 1999
;
Sivasankaran et al., 2000
).
The fact that different Wnts maintain the transcription of different Fz
receptors argues for the specificity of the ligand-receptor interaction. Our
data suggest that Wnt6 mediates its response through Fz7, whereas Wnt1 binds
Fz2.
Paraxis is a downstream target of the ß-catenin signalling activated by Wnt6
Intracellularly, Wnt molecules can signal through multiple pathways: the
canonical pathway, which leads to the stabilisation of ß-catenin in the
cytoplasm that in turn translocates to the nucleus and binds TCF/LEF proteins
to regulate transcription (Huelsken and
Birchmeier, 2001); and the non-canonical pathways, the signal
transduction of which is very diverse and includes Ca2+ flux, JNK,
and both small and heterotrimeric G proteins
(Veeman et al., 2003
).
Although the activation of the non-canonical pathway has been generally linked
to the control of cell architecture and movement in the embryo, in this work
we show that this is not general to all tissues. In the somites, Wnt6 signals
through the ß-catenin pathway, to activate paraxis
transcription, that in turns control the maintenance of its epithelial
structure. The molecular mechanism by which paraxis controls the
architecture of the dermomyotome is unclear. Recent work from Nakaya et al.
(Nakaya et al., 2004
), has
implicated the small GTPases Cdc42 and Rac1 in paraxial mesoderm
epithelialisation. Correct levels of the activity of both proteins are
necessary for PSM cells to incorporate into the epithelium
(Nakaya et al., 2004
).
Interestingly, they also show that the epithelial morphology induced by
paraxis requires the activity of Rac1, and they suggest that paraxis
indirectly controls the function of Rac1 and/or Cdc42. Together with our own
data, it is tempting to propose that the ß-catenin-dependent signal
regulates the epithelialisation of somites at least in part via the activation
of paraxis expression, that in turns control the expression of cytoskeletal
modulators of the Rac1/Cdc42/RhoA family. It is noteworthy that some of these
modulators [Pebble protein in Drosophila
(Smallhorn et al., 2004
), and
the Cap1 and Quattro proteins in zebrafish
(Daggett et al., 2004
)] have
been specifically involved in the migration and delamination of mesodermic
cells. Further analyses will be needed to establish the participation of these
molecules during somite formation and differentiation.
|
Epithelialisation: a crucial step of mesoderm morphogenesis
As mesoderm cells mature, they progress rostrally in the PSM and become
competent to respond to both intrinsic and extrinsic differentiation signals
(Dubrulle et al., 2001;
Fan and Tessier-Lavigne, 1994
;
Linker et al., 2003
;
Stern et al., 1995
;
Stern and Hauschka, 1995
;
Tajbakhsh et al., 1998
;
Zhang et al., 2001
). The
description of the paraxis-null mutant mouse strongly suggests that somite
epithelialisation is not an essential step in the differentiation of the
somite derivatives. In these mice, myogenic differentiation is initiated
correctly, but throughout the entire somite rather than only at its medial
border. As a consequence, myocytes form and elongate, but in a disorganised
fashion (Wilson-Rawls et al.,
1999
). This observation suggests that the epithelial structure of
the dermomyotome provides a scaffold on which myocytes orient themselves in
space. Alternatively, epithelialisation might be required for the correct
expression of positional signals that are required for the orientation of the
myocytes.
Together, our data allow us to propose an integrated model for Wnts
function during somite epithelialisation and differentiation
(Fig. 7). First, at the
posterior segmental plate level, a signal from axial structures is required
for the epithelialisation of somites. That signal can be mimicked by
Wnt6-expressing cells (Schmidt et al.,
2004); however, Wnt6 cannot be the endogenous Wnt molecule acting
at this level, as it is only expressed in the ectoderm. Wnt3a, which is
strongly expressed in the caudal PSM and neural tube
(Marcelle et al., 1997
), could
represent this factor. Second, the Wnt6 signal, which is secreted by the
ectoderm, probably binds the Fz7 receptor in segmental plate cells to control
paraxis expression through the activation of the canonical,
ß-catenin pathway. The permanent source of Wnt6 from the ectoderm allows
the maintenance of the epithelial structure and paraxis expression in
dorsal cells, while ventral cells (that receive Hedgehog signals) lose
paraxis expression, undergo an EMT and differentiate into sclerotome.
Finally, cells located in the medial-most region of the dermomyotome receive a
Wnt1 signal from the dorsal neural tube, which, maybe through Fz2 receptors,
imposes a medial character on these cells through the induction of
Wnt11 expression. As somites differentiate, cells from the four
borders of the dermomyotome generate myocytes. The decrease of Wnt6 and
paraxis expression in the central region of the dermomyotome probably triggers
its de-epithelialisation, allowing dorsally the migration of dermis precursors
and ventrally that of muscle progenitors. The addition of a permanent source
of Wnt6 or paraxis during this period maintains the central dermomyotomal
cells in an epithelial state, impeding their migration and further
differentiation.
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ACKNOWLEDGMENTS |
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Footnotes |
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Present address: University College London, Department of Anatomy and
Developmental Biology, Gower Street, London WC1E 6BT, UK
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