1 Division of Developmental Biology, National Institute for Medical Research,
The Ridgeway, Mill Hill, London NW7 1AA, UK
2 Wellcome Trust/Cancer Research UK Institute and Department of Zoology,
University of Cambridge, Tennis Court Road, Cambridge CB2 1QR, UK
3 Department of Molecular Genetics, University of Illinois at Chicago, Chicago,
Illinois 60607-7170, USA
* Author for correspondence (e-mail: jim{at}welc.cam.ac.uk)
Accepted 20 February 2003
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SUMMARY |
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Key words: Xenopus, Cyr61, CCN family, Wnt signalling, Cell adhesion, Gastrulation
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INTRODUCTION |
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In addition, the roles of cell adhesion molecules, such as laminin
(Nakatsuji, 1986) and
fibronectin (Marsden and DeSimone,
2001
; Reintsch and Hausen,
2001
; Winklbauer and Keller,
1996
), in the regulation of gastrulation are becoming clearer, as
are the functions of their receptors, including the integrins
(Davidson et al., 2002
;
Ramos et al., 1996
;
Whittaker and DeSimone, 1993
).
The successful prosecution of gastrulation and cell fate specification
requires the coordination and integration of all these activities. In an
attempt to shed light on this issue, we have focused on the cysteine-rich
secreted protein Cyr61, a member of the CCN (Cyr61, CTGF, Nov) family. Members
of the CCN family are versatile proteins, exhibiting properties that might
well be expected of key regulators of gastrulation: they associate with the
extracellular matrix; they can mediate cell adhesion, cell migration and
chemotaxis; and they can augment the activity of peptide growth factors
(Lau and Lam, 1999
).
Significantly, Cyr61 can also induce signalling events, such as activation of
ERK and Rac, and the induction of gene expression in fibroblasts
(Chen et al., 2001a
;
Chen et al., 2001b
).
Members of the CCN family have four characteristic domains, each encoded by
a separate exon, and each of which is defined by similarities to other
families of secreted proteins (Fig.
1A) (Bork, 1993).
The first domain, which follows the secretory sequence, is similar to the
IGF-binding domain of insulin-like growth factor binding proteins (IGFBPs).
The second region, usually referred to as the von Willebrand factor type C
(VWC) domain, resembles the cysteine-rich domains of chordin, a BMP antagonist
(Sasai et al., 1994
). The
third domain has homology to the thrombospondin (TSP) type I repeat. The
fourth, or C-terminal (CT), region contains cystine knot domains
(Vitt et al., 2001
) and is
characterised by its similarity to slit proteins
(Bork, 1993
), which are
involved in axonal pathfinding (Brose and
Tessier-Lavigne, 2000
;
Rothberg et al., 1988
). A
region of variable sequence and length is positioned between the VWC and TSP
domains.
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MATERIALS AND METHODS |
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cDNA isolation and plasmid construction
Sequence encoding Xenopus Cyr61 (Xcyr61) was isolated by
the polymerase chain reaction (PCR) using degenerate primers based on the
conserved cysteines, in domains 2 and 3 of mammalian Cyr61 proteins, that
flank the central variable region. Primers used were:
The resulting fragment was used as a probe to isolate a near-full-length
cDNA from a tadpole cDNA library (GenBank Accession Number AF320592). This
fragment was also used as a probe in RNAase protection assays. Primer
extension analysis suggested that this cDNA lacked 100 nucleotides from
the 5' end (data not shown; full details are available on request).
The Xcyr61 open reading frame was cloned into the vectors pSP64T and pcDNA3. Deletion constructs comprising domains 1, 2 and 3, domains 1 and 2, and domain 1 alone were created by PCR using standard techniques; they were then inserted into pSP64T or pcDNA3. A construct comprising the CT domain alone (domain 4) was created by two rounds of PCR. This construct consisted of sequence preceding domain 1, including the secretory signal, fused in frame to domain 4.
Other expression plasmids were as follows: Xwnt8
(Sokol et al., 1991), Dsh
(Sokol et al., 1995
), dd1
(Sokol, 1996
) and
ß-catenin (Domingos et al.,
2001
).
Adhesion/cell spreading assay
Lab-Tek chamber slides (Nalge Nunc International) were coated overnight at
4°C, or for 3-4 hours at room temperature, with 50 µg/ml fibronectin
(Sigma), 10 µg/ml mouse Cyr61 (Kireeva
et al., 1996) or 10 µg/ml human Cyr61
CT
(Grzeszkiewicz et al., 2001
)
dissolved in NAM containing 7.5% of the normal divalent cation concentration
(LCMM) and 0.1% bovine serum albumin (BSA). Adhesive surfaces were blocked
with LCMM containing 0.1% BSA for 1 hour at room temperature. Animal pole
explants were dissociated in Ca2+- and Mg2+-free medium
(CMFM) containing 0.1% BSA, and the cells were plated at a density of
approximately two animal pole equivalents per chamber in LCMM/0.1% BSA
containing 10 U/ml activin (Cooke et al.,
1987
). Heparin (Sigma) was used at a final concentration of 10
µg/ml. Cell spreading was documented by photomicrography. Cells were
subsequently fixed in MEMFA and stained with phalloidin/FITC (Sigma) for
observation by confocal microscopy.
Antisense morpholino oligonucleotides
These were purchased from GeneTools, LLC (Oregon, USA). Sequences were:
Whole-mount in situ hybridisation and immunostaining
In situ hybridisation and immunohistochemistry were carried out essentially
as described (Harland, 1991),
using a hydrolysed Xcyr61 cDNA (see above), cardiac actin
(Mohun et al., 1984
) or XAG-1
(Sive et al., 1989
) cDNAs, or
anti-mouse Cyr61 (Kireeva et al.,
1997
) or anti-muscle 12/101
(Kintner and Brockes, 1984
)
antibodies.
Scanning electron and confocal microscopy
Scanning electron microscopy of Xenopus embryos was carried out as
described (Howard et al.,
1992). Confocal microscopy was carried out using a Leica scanning
laser microscope.
RNAase protection assays
RNA was prepared using the acid guanidinium thiocyanate-phenol-chloroform
method (Chomczynski and Sacchi,
1987). RNAase protection analysis was carried out essentially as
described by Jones and colleagues (Jones
et al., 1995
), using RNAase T1 alone for all probes. An
Xcyr61 probe was made using the original Xcyr61 PCR fragment, which
was cloned into pBluescript KS. This plasmid was linearised with
EcoRI and transcribed with T7 RNA polymerase. Probes for
siamois (Lemaire et al.,
1995
) and ornithine decarboxylase
(Isaacs et al., 1992
) were as
described.
Luciferase assays
Luciferase assays were performed using the Promega Dual-Luciferase assay
kit. Embryos were injected with 10 pg TOPFLASH
(van de Wetering et al.,
1997), 10 pg pRL-SV40/TK as a reference plasmid, and an
appropriate amount of RNA encoding components of the canonical Wnt signalling
pathway. Animals caps were dissected at stage 8.5 and cultured in 75% NAM for
3-4 hours. They were then suspended in 10 µl of 1x Passive Lysis
Buffer per cap and, after centrifugation, 5 µl was taken for assay. All
values were expressed as Relative Luciferase Units (Firefly luciferase
activity/Renilla luciferase activity), with the value for the DNA alone sample
being set at unity. Each experiment shown was carried out at least three
times.
Western blotting
Western blots were carried out as described
(Tada et al., 1997), using
antibodies raised against fibronectin
(Marsden and DeSimone, 2001
)
or against Hsp70 (Sigma).
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RESULTS |
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RNAase protection analysis using a probe directed against Xcyr61a revealed that Xcyr61 is expressed during oogenesis and that transcripts are detectable at least until early blastula stage 6 (Fig. 1C). Zygotic expression begins during neurula stages (Fig. 1C), and in situ hybridisation (which is likely to detect both Xcyr61a and Xcyr61b) showed that transcripts were present in somites, heart, notochord and blood vessels during tailbud and tadpole stages (Fig. 1D-F).
Our attempts to visualise Xcyr61 protein by immunocytochemistry using two
anti-peptide antibodies did not meet with success, although in some
experiments reactive material was observed in western blots of embryos at the
early gastrula stage (data not shown). In an effort to overcome this problem
and to examine the localisation of Cyr61 protein, we injected RNA encoding
mouse Cyr61 into Xenopus embryos at the one-cell stage and detected
the resulting protein using a well characterised antibody directed against
mouse Cyr61 (Kireeva et al.,
1997). Surprisingly, despite the widespread distribution of the
injected RNA (data not shown), Cyr61 protein was detectable only in the roof
of the blastocoel at late blastula and early gastrula stages, in a pattern
resembling that of fibronectin (Fig.
1G-I). This suggests that the localisation of Xcyr61 is regulated
in some way.
Overexpression of Xcyr61 interferes with gastrulation movements
Overexpression of Xcyr61 by RNA injection into the one-cell stage embryo
caused gastrulation defects (Fig.
2A,B) In particular, there was a severe delay of blastopore
closure, tissue appeared to accumulate around the blastopore, and embryos did
not elongate fully along the anteroposterior axis. As discussed below, these
defects may result from disruption of epiboly and of convergent extension
movements.
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These effects were investigated in more detail by studying isolated dorsal
marginal zone tissue. Dissected dorsal marginal zone explants undergo
gastrulation movements, including epiboly and convergent extension, in a
manner that resembles their behaviour in the embryo
(Keller and Danilchik, 1988).
By contrast, dorsal marginal zone regions derived from embryos injected with
Xcyr61 mRNA gastrulated abnormally. In particular, epiboly was
severely disrupted because the pigmented ectodermal cells that normally cover
the yolky mesendodermal tissue (Fig.
2G) failed to do so (Fig.
2H).
Antisense oligonucleotides directed against Xcyr61 also cause
gastrulation defects
To assess the role of Xcyr61 in early development in more detail,
we designed and tested three antisense morpholino oligonucleotides
(Fig. 3A). Antisense morpholino
oligonucleotides have been shown to block translation of their target RNAs
efficiently in both Xenopus and zebrafish embryos
(Heasman et al., 2000;
Nasevicius and Ekker, 2000
).
The first morpholino oligonucleotide (MO1) was directed against
Xcyr61a and the second (MO2) against Xcyr61b. The two
morpholino sequences differed by only one base and it seemed likely that each
would interfere with the translation of both Xcyr61 alleles. Indeed,
as we describe below, the two oligonucleotides gave identical phenotypes
following injection into Xenopus embryos. However, to confirm the
specificity of the two oligonucleotides we also designed MO3, which matches
both Xcyr61a and Xcyr61b and does not overlap with MO1 and
MO2. All three antisense morpholino oligonucleotides inhibited in vitro
translation of Xcyr61a in a specific manner
(Fig. 3B).
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Bisection of gastrula-stage embryos injected with Xcyr61 antisense morpholino oligonucleotides revealed changes in the structure of both the marginal zone and the animal pole region. The superficial and deep layers of the marginal zone, which are usually tightly adherent, became separated and their constituent cells were more loosely packed (Fig. 4I-K). The animal pole regions of injected embryos were thicker than those of controls (Fig. 4I-K). Failure of the animal pole region to undergo thinning during gastrulation suggested that epiboly and radial intercalation was disrupted in such embryos. Cells in the animal pole regions of these embryos appeared rounder, less adherent and, as in the marginal zone, more loosely packed (Fig. 4I-K). Scanning electron microscopy confirmed these impressions (Fig. 4L,M), and also indicated that migration of large flat mesendodermal cells, visible at the bottom of Fig. 4L, was impaired in MO1-injected embryos (Fig. 4M). Similar results have been obtained with morpholino oligonucleotide MO2. These changes in the animal pole blastomeres are likely to be caused by a decrease in cell adhesion rather than by apoptosis: we observed no significant increase in TUNEL-staining cells in embryos injected with either MO2 or MO3 (data not shown).
The effects of the morpholino oligonucleotides on the structure of the
animal cap are consistent with the idea that the downregulation of
Xbra observed in Fig.
4D was not the primary cause of the gastrulation defect
illustrated in Fig. 4, because
Xbra is not expressed in the animal hemisphere. Rather, it seems
likely that gastrulation was disrupted. at least in part. because Xcyr61
protein did not accumulate in the roof of the blastocoel. This may in turn
disturb the distribution of other components of the extracellular matrix. For
example, in control embryos, fibronectin forms an elaborate fibrillar network
in the blastocoel roof (Fig.
4N) and this acts as a substrate for the adhesion and migration of
involuted mesoderm cells (Marsden and
DeSimone, 2001). By contrast, in MO2-injected embryos, there was a
dramatic reduction in the amount of fibronectin in the extracellular matrix of
the blastocoel roof, and the remaining fibrils appeared disorganised
(Fig. 4O; data not shown). The
apparent reduction in levels of extracellular fibronectin is likely to be
caused by a defect in fibronectin fibril assembly rather than by a decrease in
overall levels: western blotting experiments indicated that levels of
fibronectin were similar in control and morpholino-injected embryos
(Fig. 4P).
The similar phenotypes produced by the three antisense morpholino oligonucleotides, two of which are non-overlapping, suggests that their effects are specific. The specificity of MOs 1-3 was examined further by performing `rescue' experiments. Such experiments are difficult to interpret when, as here, overexpression of a gene product produces a phenotype similar to that observed following inhibition of its function; an observation that, in our experiments, suggests that normal gastrulation requires both precisely controlled levels and localisation of Xcyr61. Nevertheless, we observed that whereas 91% of MO2-injected embryos exhibited either the more severe `small eyes' or `gastrulation defect' phenotype, co-injection of 200 pg Xcyr61 RNA reduced this figure to 50% (Fig. 5). These observations also suggest that the effects of the antisense morpholino oligonucleotides are specific.
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We first used activin-treated animal pole cells
(Smith et al., 1990) to show
that purified mouse Cyr61, like fibronectin, can support cell adhesion during
gastrulation (Fig. 6).
Significantly, cells adherent to Cyr61 proved to have a different shape from
those adherent to fibronectin (Fig.
6A,B). The latter were usually polarised, with prominent filopodia
and fewer lamellipodia (Fig.
6A), whereas cells adherent to Cyr61 were characterised by large
lamellipodia frequently distributed in a near-symmetrical fashion around the
cell (Fig. 6B). Because cell
migration requires the dynamic formation and disappearance of lamellipodia and
associated focal adhesions, it is possible that cells adherent to Cyr61 are
not as motile as those adherent to fibronectin. Defects in gastrulation might
therefore arise from either depletion or overexpression of Xcyr61.
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We noted that cells of the blastocoel roof and marginal zone of embryos
injected with Xcyr61 antisense morpholino oligonucleotides were loosely packed
and apparently less adherent (Fig.
4I-M). To determine whether this was associated with a decrease in
cell adhesion, cells from the blastocoel roofs of control embryos or of
embryos injected with antisense morpholino oligonucleotides were dissociated
by culture in Ca2+ and Mg2+-free medium and then
reaggregated by addition of Ca2+
(Torres et al., 1996). Control
cells formed large aggregates within minutes
(Fig. 6I), whereas those
derived from embryos injected with antisense morpholino oligonucleotides
formed only small cell groups (Fig.
6J), which suggests that their capacity to form
Ca2+-dependent contacts was compromised.
Cyr61 can induce secondary axes in the Xenopus embryo
The results described above suggest that Xcyr61 regulates
gastrulation through its influence on cell-cell and cell-matrix adhesion, but
it is also possible that it influences cell signalling, perhaps through
cooperation with growth factors. We note, for example, that depletion of
Xcyr61 caused downregulation of Xbra expression
(Fig. 4C,D), and mammalian
Cyr61 has been shown to cooperate with several growth factors in vitro and to
induce changes in gene expression and cell morphology
(Chen et al., 2001a;
Chen et al., 2001b
).
To examine the ability of Xcyr61 to influence early embryonic signalling, we first injected Xcyr61 mRNA into ventral or dorsal blastomeres of Xenopus embryos at the four- to eight-cell stage. Dorsal injections resulted in the formation of embryos with enlarged heads, which suggests that Xcyr61 has dorsalising activity (data not shown), and, consistent with this, ventral injections caused secondary axis formation (Fig. 7A). In a few cases (1-5%, depending on the egg batch) complete secondary axes were induced (Fig. 7B), but more frequent were partial secondary axes (5-30%; Fig. 7C,D) or the induction of ectopic muscle (30-80%; Fig. 7E,F). In addition, most injected embryos had blastopore closure defects, perhaps reflecting the ability of Xcyr61 to regulate gastrulation movements.
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Xcyr61 also induced the TOPFLASH synthetic reporter, which
responds directly to Wnt/ß-catenin signalling, in ventral marginal zone
tissue (Fig. 7L). The level of
TOPFLASH induction by Xcyr61 was modest compared with that obtained
with Xwnt8 (not shown) or Dishevelled
(Fig. 7L), consistent with the
observation that Xcyr61 usually induces incomplete secondary axes. A
Xcyr61 construct comprising just the IGFBP domain also activated the
TOPFLASH reporter, and with higher activity than the full-length protein.
Xcyr61 is likely to activate TOPFLASH by acting through the canonical
Wnt signal transduction pathway involving Dishevelled and Gsk3; the ability of
the IGFBP domain to activate the reporter is inhibited by the
dominant-negative Dishevelled construct dd1 (data not shown)
(Sokol, 1996).
The above experiments address Wnt function during cleavage stages of development. At later stages, during gastrulation, Wnt signalling through the canonical pathway promotes ventrolateral fates. We found that dorsal injection of a plasmid expressing Xcyr61 caused a reduction in head and eye formation, suggesting that Xcyr61 can also activate Wnt signalling during gastrulation (data not shown). Thus, in two independent contexts, our results are consistent with the idea that Xcyr61 causes stimulation of Wnt signalling.
Cyr61 can also antagonise Wnt/ß-catenin signalling
The results described above show that Xcyr61 has weak axis-inducing
activity that is likely to occur through the Wnt signalling pathway. In an
effort to elucidate the molecular basis of this phenomenon, we investigated
whether Xcyr61 could act synergistically with Xwnt8 in such
an assay. Surprisingly, instead of observing synergism between Xcyr61
and Xwnt8, we observed antagonism; Xcyr61, which alone
induces partial secondary axis formation
(Fig. 8C) inhibited secondary
axis induction by Xwnt8 (Fig.
8A,B; Table 1). This inhibitory effect requires the TSP and CT
domains (domains 3 and 4), since deletions that lacked these regions
(construct 1,2) could not inhibit secondary axis formation
(Fig. 8D), although they
retained the ability to induce secondary axes
(Fig. 8E). It is likely that
the inhibitory activity resides in the CT domain because this alone (provided
with the secretory signal) proved to be sufficient to block
Xwnt8-induced secondary axis formation
(Fig. 8F), although it was
unable to induce secondary axes (Fig.
8G). Xcyr61 can therefore both induce and inhibit
secondary axis formation.
It is possible that Xcyr61 interferes with secondary axis formation by Xwnt8 by some indirect means, perhaps through its effects on cell adhesion. However, we find that Xcyr61 inhibits Xwnt8-induced activation of the TOPFLASH reporter in animal caps, which suggests that it interferes with Wnt/ß-catenin signalling directly (Fig. 8H). This inhibitory activity of Xcyr61 requires the CT domain, as does its ability to block secondary axis formation (Fig. 8E,H). Xcyr61 interfered only slightly with the ability of Dishevelled or ß-catenin to induce the TOPFLASH reporter (Fig. 8H,I), which suggests that it acts upstream of Dishevelled, perhaps at the level of the cell membrane.
Members of the Wnt family also signal through the so-called planar polarity
pathway, which in vertebrate embryos is involved in the control of
gastrulation. We investigated whether Xcyr61 can regulate the planar
polarity pathway by asking whether it can prevent activin-induced elongation
of Xenopus animal pole regions
(Symes and Smith, 1987;
Tada and Smith, 2000
).
Injection of full-length Xcyr61 mRNA inhibited such elongation
(Fig. 9A-C), an effect that
requires the CT domain (Fig.
9D). Although elongation of animal caps was inhibited in these
experiments, the induction of mesodermal cell types, such as muscle-specific
actin, was only slightly reduced (Fig.
9E-J). These results suggest that Xcyr61 also interferes with
convergent extension, perhaps by reducing Wnt signalling through the planar
polarity pathway, and that this inhibition requires the CT domain.
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Xcyr61 can inhibit BMP signalling as well as modulate the Wnt
pathway
The four modules of Cyr61 protein include the cysteine-rich (CR) VWC domain
(domain 2; Fig. 1A). CR repeats
are also present in other CCN family members, such as CTGF
(Abreu et al., 2002), and also
in chordin (Sasai et al.,
1994
) and short gastrulation
(Francois and Bier, 1995
). In
these molecules the CR repeats bind to, and inhibit the action of, members of
the BMP family (Abreu et al.,
2002
). We have tested Xcyr61 for anti-BMP activity by
overexpression in animal pole explants. The doses of Xcyr61 RNA used
in whole-embryo assays (100 pg to 1 ng) had little effect on animal caps but 4
ng Xcyr61 RNA induced cement gland formation and caused weak
activation of NCAM (Fig. 10;
data not shown), which suggests that Xcyr61 can, at least to some extent,
inhibit BMP signalling. In further experiments, we found that co-expression of
Xcyr61 and the truncated BMP receptor tBR induces additional
heads, which is not observed with either construct alone
(Fig. 10C-E). Because head
induction requires the simultaneous inhibition of Wnt and BMP signalling, this
suggests that the main function of Xcyr61 is to modulate Wnt
signalling, rather than to inhibit the BMP pathway.
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DISCUSSION |
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Adhesive properties of Xcyr61
Although we have been unable to detect endogenous Xcyr61 mRNA
during gastrulation, it is likely that Xcyr61 protein accumulates during
cleavage stages. Results obtained in tissue culture indicate that once
incorporated into the extracellular matrix, Cyr61 is very stable, with a
half-life exceeding 24 hours (Yang and
Lau, 1991). Experiments involving misexpression of the mouse gene
suggest that the Xenopus gene product accumulates in the blastocoel
roof (Fig. 1G,H), and in this
respect Xcyr61 would resemble fibronectin, another gene product that is
expressed throughout the early embryo (Lee
et al., 1984
). Like fibronectin, Cyr61, through its CT domain,
supports the adhesion of Xenopus blastomeres
(Fig. 6B). The morphology of
cells plated on Cyr61 differs from that of cells adherent to fibronectin, and
it seems likely that the behaviour of blastomeres adherent to the two
substrates would differ, with those attached to fibronectin being more motile
than those attached to Cyr61. Migratory behaviour during gastrulation might
therefore depend on the levels of the two molecules in the extracellular
matrix of the blastocoel roof.
However, it is likely that the influence of Xcyr61 on the extracellular
matrix and on gastrulation is more profound than this because interference
with Xcyr61 synthesis disrupts fibronectin accumulation in the blastocoel
roof, no doubt exacerbating significantly the effects of merely losing Xcyr61
from the extracellular matrix; we note that embryos depleted of fibronectin
have a similar phenotype to those lacking Xcyr61
(Marsden and DeSimone,
2001).
Interference with cell-matrix adhesion is one way in which depletion or overexpression of Xcyr61 might disrupt gastrulation. Another is through interference with cell-cell adhesion. A re-aggregation assay (Fig. 6I,J) shows that depletion of Xcyr61 from the embryo compromises Ca2+-induced cell adhesion. Together, these data suggest that Xcyr61 plays a role in gastrulation through its own ability to support cell-matrix adhesion, through its role in the assembly of the extracellular matrix and through its influence on cell-cell adhesion.
Xcyr61 can both activate and inhibit Wnt signalling
Another way in which Xcyr61 might affect gastrulation is through modulation
of the Wnt signalling pathway. Direct evidence that Xcyr61 affects the
canonical Wnt signalling pathway is provided by experiments in which
overexpression of Xcyr61 causes the formation of (usually partial) secondary
axes in Xenopus embryos (Fig.
7A-F). It also induces the expression of Siamois
(Fig. 7G) and cardiac actin
(Fig. 7H-K) in isolated ventral
marginal zone tissue, and activates expression of the TOPFLASH reporter in
these cells (Fig. 7L). This
ability of Xcyr61 to activate the Wnt pathway, which is weak compared with
that of Xwnt8 (Fig. 7G) or
Dishevelled (Fig. 7L), is
likely to be mediated by the IGFBP domain (domain 1)
(Fig. 7L).
However, to our surprise Xcyr61 also proved to be capable of inhibiting Wnt
signalling. Thus, Xcyr61 prevented the formation of secondary axes in response
to Xwnt8 (Fig. 8A-G), and, not
only did it fail to activate the TOPFLASH reporter in animal caps, it
inhibited its activation by Xwnt8 (Fig.
8H,I). Our experiments suggest that this inhibition is mediated by
the CT domain (domain 4): this region of the protein is capable, alone, of
preventing the formation of secondary axes
(Fig. 8F) and it is required
for inhibition of TOPFLASH activation (Fig.
8H). The ability of Xcyr61 to both activate and inhibit the Wnt
pathway is discussed below. Together, these experiments are consistent with
the suggestion that Xcyr61 inhibits the elongation of activin-treated animal
caps by interfering with Wnt signalling. It is possible that the decrease in
Xbra expression in embryos injected with Xcyr61 antisense morpholino
oligonucleotides (Fig. 4D) is
caused, in part, by the downregulation of Wnt signalling
(Arnold et al., 2000;
Yamaguchi et al., 1999
), and
this may also contribute to the disruption of gastrulation
(Beddington et al., 1992
;
Conlon and Smith, 1999
).
Xcyr61: a versatile modular molecule
Together, our results indicate that Xcyr61 is a versatile molecule that
probably plays several roles in early Xenopus development. It is
involved in the assembly of the extracellular matrix, in cell-matrix and
cell-cell adhesion, in the upregulation and inhibition of Wnt signalling, and
(albeit weakly) in the inhibition of BMP signalling. Some of these activities
can be ascribed to particular domains of the protein. Adhesion of blastomeres
to Cyr61 requires the CT domain, for example, as does the inhibition of Wnt
signalling, where this domain is sufficient to inhibit secondary axis
induction by Xwnt8 (Fig. 8F).
By contrast, stimulation of Wnt signalling appears to be mediated by the IGFBP
domain (domain 1).
The abilities of the CT domain to regulate cell adhesion and to inhibit Wnt
signalling may be related. This domain is required for the adhesion of
fibroblasts (Grzeszkiewicz et al.,
2001) and of Xenopus blastomeres
(Fig. 6D) to Cyr61, and it also
mediates the interaction of Cyr61 with heparan sulphate proteoglycans (HSPGs)
(Chen et al., 2000
). Exogenous
heparin blocks the adhesion of Xenopus cells to Cyr61
(Fig. 6H) and this is likely to
occur as a result of competition with cell-associated HSPGs for sites on
Cyr61. HSPGs have also been implicated in the regulation of Wnt signalling
(Topczewski et al., 2001
;
Tsuda et al., 1999
), and it is
possible that the ability of Xcyr61 to bind HSPGs is related to its ability to
inhibit Wnt signalling. Another potential link between cell adhesion and the
modulation of Wnt signalling by Xcyr61 might be provided by the integrins,
which are the only known receptors for Cyr61
(Bökel and Brown, 2002
;
Grzeszkiewicz et al., 2001
;
Lau and Lam, 1999
).
Integrin-mediated adhesion of cells to the extracellular matrix recruits
Dishevelled to the plasma membrane
(Marsden and DeSimone, 2001
),
and this may enhance the ability of cells to respond to a Wnt signal.
Activation of Wnt signalling by Xcyr61 can occur through the IGFBP domain
(domain 1). We do not know if the Wnt-stimulating activity of domain 1 of
Xcyr61 is related to its putative IGF binding activity, although we note that
activation of the IGF receptor in Xenopus embryos inhibits the Wnt
pathway (Pera et al., 2001;
Richard-Parpaillon et al.,
2002
). It is possible that domain 1 of Xcyr61 binds endogenous
members of the insulin-like growth factor family and relieves this
inhibition.
It is intriguing that Xcyr61 can function both as an activator and as an inhibitor of Wnt signalling. These two activities can be observed in similar cellular contexts (the equatorial and vegetal regions of the Xenopus embryo) and so it is unlikely that the different activities are caused by the presence or absence of specific co-factors. Rather, our experiments suggest that Xcyr61 can act to elevate Wnt signalling when it is at a low level and inhibit it when the level is high. It might behave, in effect, as a Wnt `buffer'. This may explain why, in many embryos, depletion of Xcyr61 has little effect on axis formation.
Conclusion: the role of Xcyr61 in the Xenopus embryo
Our results suggest that Xcyr61 is a multifunctional molecule that plays a
key role in modulating and integrating many pathways and types of cell
behaviour during Xenopus development. These include cell-cell and cell-matrix
adhesion, the stimulation and repression of Wnt signalling, and the inhibition
of BMP signalling. In view of this wide range of activities, it is not
surprising that for gastrulation to proceed normally, the level of Xcyr61
needs to be precisely controlled. In the future, we plan to investigate each
of these activities separately, by investigating in more detail the functions
of different domains of the molecule, and by using antisense morpholino
oligonucleotides to inhibit splicing of individual domains of the endogenous
protein. We also plan to define the mechanism by which Xcyr61 modulates Wnt
signalling.
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ACKNOWLEDGMENTS |
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REFERENCES |
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