1 Division of Developmental Neurobiology, National Institute for Medical
Research, The Ridgeway, Mill Hill, London NW7 1AA, UK
2 Division of Developmental Biology, National Institute for Medical Research,
The Ridgeway, Mill Hill, London NW7 1AA, UK
* Authors for correspondence (e-mail: nitasak{at}nimr.mrc.ac.uk and rek{at}stowers-institute.org)
Accepted 3 June 2003
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SUMMARY |
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Key words: Wise, Wnt signalling, Xenopus
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INTRODUCTION |
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Analysis of neural patterning is complicated by the tissue interactions and
dynamic morphogenetic movements that occur during gastrulation.
Xenopus animal caps provide a simplified system for studying
patterning events separate from morphogenetic movements. Animal caps alone
form epidermis in culture, but when treated with antagonists of BMP signalling
they can be induced to adopt an anterior neural fate
(Hemmati-Brivanlou and Melton,
1997). This anterior neural tissue is capable of altering its
positional identity to a more posterior character under the influence of
signals from tissues surrounding the neural tube or by ectopic application of
posteriorising factors, such as RA, FGF and Wnt family members
(Baker et al., 1999
;
Blumberg et al., 1997
;
Domingos et al., 2001
;
Itoh and Sokol, 1997
;
Kolm et al., 1997
;
Lamb and Harland, 1995
;
McGrew et al., 1997
;
McGrew et al., 1995
;
Pownall et al., 1996
).
Experiments in Xenopus have shown that planar signals within the
neuroectoderm and vertical signals from the underlying mesoderm work in
concert to control regional identity of the nervous system
(Doniach, 1993). Although
early AP specification of the nervous system occurs during gastrulation, the
neural cells are not irreversibly committed to a particular identity. Grafting
experiments in several species have revealed that plasticity in regional
character is retained after gastrulation
(Cox and Hemmati-Brivanlou
,
1995; Gould et al., 1998
;
Grapin-Botton et al., 1997
;
Itasaki et al., 1996
;
Muhr et al., 1997
;
Trainor and Krumlauf, 2000
;
Woo and Fraser, 1997
),
suggesting that neural cells are actively receiving signals and communicating
with surrounding tissues at later stages.
In this study, we performed a functional screen to search for novel factors derived from tissues surrounding the neural tube with the potential to alter the AP character of neuralised Xenopus animal caps. We have identified a novel gene, Wise, expressed in the surface ectoderm. Wise encodes a secreted protein that is capable of inducing posterior neural markers, and modulates the Wnt signalling pathway in a context-dependent manner. Our results provide a novel mechanism for modulating the Wnt pathway and support a role for Wnt signalling in the neural patterning process.
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MATERIALS AND METHODS |
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DNA constructs
The dominant-negative Dishevelled construct DIX, specific to the canonical
Wnt pathway (Axelrod et al.,
1998) was made by creating a stop codon after amino acid 170
(Glutamine) by PCR and sub-cloned into pCS2+. Tagged Wise constructs were
generated by PCR, and their activity was confirmed by injection into
Xenopus embryos with noggin and assayed for induction of
en2 in animal caps.
E1-2 IgG (lacking EGF repeat 1 and 2) and
E3-4 IgG (lacking EGF repeat 3 and 4) of human LRP6 were generated by
fusing the extracellular domains E3-4 and E1-2 of LRP6
(Mao et al., 2001
) to the IgG
Fc domain (Tamai et al.,
2000
). A FLAG tag was attached to chick Frizzled 1 extracellular
domain (amino acids 1-199) by PCR. Other constructs were as previously
published.
RNA and morpholino injection
The relevant amounts of RNA injected per embryo were as follows. Initial
screening: noggin (500 pg) and RNA from pools (12 ng).
Fig. 1B: noggin (500
pg) and Wise (150, 300, 600, 1200 pg).
Fig. 1C: Wise (30 ng).
Fig. 1D,E: noggin (500
pg), Wise (600 pg) and lacZ (100 pg).
Fig. 3: Wise (300-500
pg) and lacZ (50 pg); Wise morpholino (30 ng).
Fig. 4A: Wise (chick,
Xenopus) (500 pg). Fig.
4B-K: control or Wise morpholino (33 ng).
Fig. 5A: noggin (500
pg), Wise (600 pg); Wnt8 (200 pg),
LRP6 (1 ng);
Dsh(dd1) (1.2 ng), GSK3
(500 pg) and LEF
N (200 pg).
Fig. 5B: noggin (500
pg), Wnt8 (50 pg), Wise (600 pg) and
Fz8 (2
ng). Fig. 5C: noggin
(500 pg), Wnt8 (600 pg), Wise (800 pg),
Wnt8
(800 pg). Fig. 5E-G:
Tcf3 (300 pg), Wnt8 (25 pg), Wise (300 pg).
Fig. 6A-C: Wnt8 (5
pg), Wise (200 pg) and
Dsh (DIX) (1 ng).
Fig. 6D: Wise (1 ng),
Wnt8 (100 pg), Dsh (1 ng) and ß-catenin (200
pg). Fig. 6E,F: tBR
(900 pg) and Wise (50 pg). Fig.
7C: Wise (1 ng). Fig.
7D: Wise (0.5 ng, 1 ng).
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Protein analysis
To test the secretion of Wise protein, 15 oocytes were injected with RNA
encoding HA-tagged Wise and incubated in a 96-well dish with 150 µl of OR2
medium (Wallace et al., 1973)
+ 0.01% BSA for 2 days. Oocytes and the conditioned medium were collected
separately and used for western blotting with an anti-HA antibody (Roche).
For immunoprecipitation of conditioned medium, 293 cells were transfected
with DNA and the conditioned medium (opti-MEM, Gibco) was concentrated 20- to
40-fold by ultrafiltration. Wnt8-myc medium was collected from S2 cells as
described (Hsieh et al.,
1999). The medium was mixed and incubated overnight at 4°C.
Immunoprecipitation was performed using protein A beads (Amersham) and
wash-buffer 150 mM NaCl, 50 mM Tris-HCl (pH 7.5) and 0.1% Triton X-100 as
previously described (Hsieh et al.,
1999
; Tamai et al.,
2000
).
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RESULTS |
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Structural and functional properties of Wise
The predicted Wise protein consists of 206 amino acids and contains a
cysteine knot-like domain found in a number of growth factors, as well as in
Slit, mucin and CCN (Cef10/Cyr61, CTGF and Nov) family members
(Bork, 1993)
(Fig. 2A). Among these, the
C-terminal domain of the CCN family members showed the highest homology to
Wise, but other motifs conserved within the CCN family were absent in Wise
(Fig. 2B). Hence, Wise is
related to but not a member of the CCN family. A homology search revealed that
Wise showed the highest amino acid identity (38%) to Sclerostin (SOST),
identified by positional cloning of the gene mutated in sclerosteosis
(Brunkow et al., 2001
). There
are a number of EST sequences homologous to Wise in zebrafish, mouse and human
databases (Fig. 2A), but none
was found in the Drosophila or C. elegans genomes.
|
Wise expression
In situ hybridisation analyses revealed that Wise is highly
expressed in the surface ectoderm of the embryo in a dynamic pattern. In
chick, expression was first detectable broadly at stage 9, and then localised
in the posterior surface ectoderm overlying the presomitic mesoderm by stage
10-11 (Fig. 1F,G). This
expression resolved into a small posterior domain in the tail bud by stage 13
(data not shown). At later stages, Wise was expressed in other
tissues such as branchial arches, limbs and feather buds (data not shown). By
cloning the Xenopus homologue, we found that transcripts were first
detected at the gastrula stage (Fig.
1H), and in comparison to chick more extensively along the AP axis
in the surface ectoderm at the neurula stage
(Fig. 1I). Other localised
expression at neurula and tailbud stages was seen in the stomodeal-hypophyseal
anlage (Fig. 1J-L), the ventral
diencephalon (Fig. 1N) and
presumably cranial and lateral line placodes
(Fig. 1J,M,N). Some of the
corresponding patterns were seen in chick embryos as well (data not
shown).
Functional analysis of Wise in Xenopus embryos
The function of Wise was first studied in Xenopus embryos using
overexpression approaches. Injection of Wise RNA in amounts >200
pg into the whole embryo lead to gastrulation defects and loss of eyes (data
not shown). At lower amounts (<100 pg), gastrulation proceeded normally,
but neural tube closure was abnormal and the neural plate appeared thicker and
shorter than controls (data not shown). To evaluate further the effects of
Wise on development of the neural tube, RNA or DNA was injected into
specific blastomeres at 4-16 cell stages. When Wise RNA injections
were targeted to presumptive neural regions, lateral expansion of the neural
plate on the injected side was observed
(Fig. 3A,B). AP specific
markers (en2 and Krox20) were generally displaced laterally
and posteriorly (Fig.
3D,E,G,H). When Wise injection was targeted to the
forebrain region, ectopic expression of Krox20 and slug was
observed (Fig. 3H,K). This
indicates that forebrain cells acquired a more posterior character in response
to Wise. Other defects included a failure in eye and cement gland formation
(Fig. 3M,N, and data not
shown). Conversely, when Wise RNA was injected ventrally, ectopic
cement glands were induced (Fig.
3P,Q). Identical results were obtained using DNA constructs for
injection, where Wise expression commenced at mid-blastula stages
under the control of a cytoskeletal actin promoter (data not shown). Thus,
ectopic expression of Wise altered aspects of AP patterning in
embryos, as well as in animal caps.
To analyse the role of Wise in normal Xenopus development, we injected antisense morpholino oligonucleotides that specifically interfere with translation of Wise (Fig. 4A). By injection of morpholino oligonucleotides into both blastomeres at the two-cell stage, embryos became cyclopic (Fig. 4B-E). Localised injection of the morpholino into one of the dorsal animal blastomeres at four to eight cell stages resulted in a reduction of neural tissue (Fig. 3C), decreased amount of slug-positive neural crest cells (Fig. 3L), and the formation of a smaller eye (Fig. 3O) on the injected side. AP neural markers such as en2 and Krox20 and a cement gland marker XCG were not obviously affected (Fig. 3F,I,R). In the forebrain, Wise morpholino oligonucleotides caused loss of the olfactory placode revealed by Emx2 staining (Fig. 4F-I). Histological sections at the trunk level of neurula stage embryos showed that Wise morpholino injection caused thicker surface ectoderm (Fig. 4J,K). These results indicated that Wise has endogenous roles in controlling eye, olfactory placode and surface ectoderm formation. The strong expression of Wise in cranial placodes at the tailbud stage (Fig. 1M,N) further suggests a key role for Wise in placode formation. Wise is likely to play a permissive role in the eye development, as the eye tissue shows only a small domain of expression at the tailbud stage (Fig. 1M), and depletion of Wise by morpholino injection affects only growth of the eye and not the initial formation of the eye primordium, as marked by Xrx1 (Fig. 3O).
The fact that ectopic expression of Wise RNA and depletion of
endogenous translation by the morpholino oligonucleotides both result in
similar defects in eye formation (Fig.
3N,O; Fig. 4L)
suggests that this process requires a precise level of signalling mediated by
Wise. Defects in eye formation are also observed following injection of
molecules that activate Wnt signalling (Wnt8 DNA, Frizzled3)
(Christian and Moon, 1993;
Rasmussen et al., 2001
) as
well as molecules that inhibit the pathway (Frzb1, Crescent)
(Pera and De Robertis, 2000
).
Other aspects of the phenotypes observed by altering Wise expression
are also reminiscent of those seen when the Wnt signalling pathway is
perturbed. For example, ectopic induction of cement gland is observed
following injection of GSK3ß (Itoh et
al., 1995
), and proper gastrulation and convergent extension
movements require Wnt11 and Dishevelled
(Heisenberg et al., 2000
;
Tada and Smith, 2000
;
Wallingford et al., 2000
).
Wnts/ß-catenin function as posteriorising factors in animal cap assays
(Domingos et al., 2001
;
McGrew et al., 1995
), and also
regulate induction of neural crest cells
(Saint-Jeannet et al., 1997
;
LaBonne and Bronner-Fraser,
1998
; Garcia-Castro et al.,
2002
). These similarities prompted us to test whether
Wise acts through or modulates the Wnt signalling pathway.
Wise activates the canonical Wnt pathway in animal caps
As Wnts and Wise both induce en2 expression in
noggin-injected animal caps, we investigated whether the ability of
Wise to induce en2 requires Wnt signalling. To test this,
Wise RNA was co-injected with blocking reagents of the Wnt pathway
such as wild type GSK3ß (Dominguez et
al., 1995) and dominant-negative (dn) versions of Wnt8
(Hoppler et al., 1996
),
Frizzled8 (Itoh and Sokol,
1999
), LRP6 (Tamai et al.,
2000
), Dishevelled (Sokol,
1996
) or Lef1 (Vleminckx et
al., 1999
). These reagents either eliminated or attenuated the
ability of Wise to induce en2 in neuralised animal caps
(Fig. 5A,B). With respect to
the intracellular component Dishevelled, only dominant-negative constructs
affecting the canonical Wnt pathway abolished en2 induction (data not
shown). Wnt8 and Wise showed an additive effect in induction of en2
(Fig. 5C). To confirm the
activation of the canonical Wnt pathway by Wise, we examined its effects in
other assays. First, in animal cap explants in the absence of noggin,
Wise showed a weak dorsalising activity by inducing siamois and
Xnr3, two direct targets of the Wnt signalling pathway
(Brannon et al., 1997
)
(Fig. 5D). Second, Injection of
Wise RNA increased nuclear accumulation of ß-catenin in animal caps, in a
manner similar to that of Wnt8. This phenomenon was enhanced by co-injection
of Tcf3, a co-factor of ß-catenin for transcriptional activation
(Fig. 5E-G). As animal caps
were assayed before mid-blastula transition, we believe the observed effect is
not due to secondarily induced transcription. These data suggest that Wise
activates Wnt signalling and requires components of the canonical pathway to
induce the signal in animal caps.
It is important to note that, although Wise and Wnts both activate the
canonical pathway, there are distinct differences in their outputs.
Wnt8 RNA (50 pg) is sufficient to induce both en2 and
Krox20 (Domingos et al.,
2001), and a higher amount (600 pg) induces only Krox20
(Fig. 5C). By comparison, it
takes much higher amounts of Wise RNA (300-600 pg) just to induce
en2, and 1.2 ng of Wise RNA is only sufficient to weakly induce
Krox20 (Fig. 1B).
Similarly, Wnt8 robustly induces siamois and Xnr3 at a low
amount of RNA (100 pg), although Wise only induces these genes in a relatively
weak manner, even at the highest levels of RNA (100-1000 pg)
(Fig. 5D). These results show
that Wise has weaker posteriorising and dorsalising activities in comparison
with Wnt8, raising the possibility that there are both quantitative and
qualitative differences in the outputs of the Wnt pathway when activated by
these two proteins. The fact that Wnt8 induces dorsal mesoderm in animal caps
in the presence of noggin
(Domingos et al., 2001
)
although Wise does not (Fig.
1B), further supports their qualitative difference.
Wise can interfere with Wnt signals
Although induction of en2 can be explained by activation of Wnt
signalling, the effects of injected Wise RNA on cement gland
induction (Fig. 3Q) resemble
those observed when the Wnt pathway is inhibited
(Itoh et al., 1995). Thus, it
is possible that in some contexts Wise blocks Wnt signalling. When
Wnt8 RNA is injected into a ventral vegetal blastomere at the four to
eight cell stage, it induces an ectopic secondary axis
(Smith and Harland, 1991
;
Sokol et al., 1991
)
(Fig. 6A). Based on the ability
of Wise to induce siamois and Xnr3, we expected that Wise on
its own might induce a secondary axis or work in synergy with Wnt8 in this
process. However, injection of Wise RNA did not exhibit secondary
axis formation (data not shown). Rather, co-injection of Wise RNA
completely blocked Wnt8-induced secondary axis formation
(Fig. 6B), as did a dominant
negative form of Dishevelled (Fig.
6C). This inhibitory activity was confirmed at the molecular level
in ventral marginal zone explants by demonstrating that the Wnt-dependent
induction of siamois and Xnr3 is greatly reduced by
co-injection of Wise (Fig.
6D). These results suggest that in the presence of both Wnt8 and
Wise, Wise interferes with the level of activity of Wnt8. Wise had no
effect on the ability of injected intracellular components such as Dishevelled
and ß-catenin to induce Xnr3 and siamois
(Fig.6D), suggesting that Wise
functions extracellularly to interfere with the canonical Wnt pathway. The
inhibitory effect of Wise on Wnt signalling was further examined by assaying
secondary head induction, which can be induced by simultaneous inhibition of
both BMP and Wnt signalling (Glinka et
al., 1997
) (Fig.
6E). Co-injection of Wise and a dominant-negative BMP receptor
(Suzuki et al., 1994
) induced
a complete secondary axis with eyes and cement gland
(Fig. 6F), demonstrating that
Wise functions as a Wnt inhibitor in this context. This contrasts with our
analysis in animal caps, which reveals that Wise does not interfere with the
action of Wnt in the induction of Krox20
(Fig. 5C). Therefore,
modulation of the Wnt pathway by Wise (activation or inhibition) varies with
respect to both target genes and cellular contexts.
Wise might affect the planar cell polarity pathway of Wnt
signalling
Although the activating and inhibiting properties of Wise in animal caps
and embryos described above are dependent upon the canonical Wnt pathway, it
is possible that Wise also influences the planar cell polarity (PCP) pathway
that branches at Dishevelled. Wnt11 is required for proper convergent
extension movements of mesoderm during gastrulation in Xenopus and
Zebrafish, and this has been shown to be dependent upon the PCP pathway of Wnt
signalling (Heisenberg et al.,
2000; Tada and Smith,
2000
; Wallingford et al.,
2000
). Animal caps cultured in the presence of activin form
mesoderm, and undergo convergent extension movements that can be blocked by
reagents that either elevate or decrease Wnt signalling
(Tada and Smith, 2000
). This
implies that precise levels of Wnt signalling through the PCP pathway are
essential for coordinated cell movements.
In this animal cap assay, injection of Wise RNA blocked cell movements preventing elongation of animal caps, but had no affect on activin-induced mesoderm formation (Fig. 7). This suggests that Wise might influence the Wnt-dependent PCP pathway, but whether this is mediated by its ability to activate or inhibit the pathway cannot be distinguished. This effect on cell behaviour in animal caps is consistent with and may explain the phenotypic effects observed in Wise-injected whole embryos. Wise perturbed the morphogenesis of the neural tube, which failed to close. It was thickened and shortened, and there was a lateral expansion, broadening or posterior-shift of AP markers. Many of these defects appear to relate to abnormal convergent extension movements during gastrulation. However, the fact that morpholino antisense oligonucleotides do not interfere with gastrulation (Fig. 4D) and neural AP patterns (Fig. 3F,I), and that Wise is not predominantly expressed at gastrula stage (Fig. 1H), both suggest that endogenous Wise is unlikely to be involved in normal gastrulation movements. This assay suggests that Wise has a potential to interfere with Wnt-mediated PCP as well as the canonical pathway.
Wise interacts with Wnt co-receptor LRP6
Wise encodes a secreted protein and interacts with the Wnt pathway
extracellularly (Fig. 5A,B;
Fig. 6D). Therefore, to begin
to approach the mechanisms of action, we investigated potential physical
interactions of Wise with Wnt family members or their putative co-receptors
Frizzled8 (Hsieh et al., 1999)
and LRP6 (Tamai et al., 2000
)
(Fig. 8). We mixed conditioned
medium of 293 or S2 cells containing a secreted form of LRP6
(Tamai et al., 2000
) or
Frizzled8 (Hsieh et al., 1999
)
with Wise conditioned medium, and assayed for interactions by
immunoprecipitation (IP). In this assay, Wise bound to LRP6 but not to
Frizzled8 (Fig. 8A) or Wnt8
(Fig. 8B). Recent studies have
shown that individual members of the Dickkopf (Dkk) family of secreted
proteins can either antagonise or stimulate Wnt signalling through interaction
with LRP6 (Brott and Sokol,
2002
; Mao et al.,
2001
; Wu et al.,
2000
). Therefore we performed IP experiments to determine if Wise
shares common binding sites with Dkk1 or Wnt on LRP6. The extracellular domain
of LRP6 contains four EGF repeats and Dkk1 interacts with repeats 3-4, while
Wnt interactions seem to involve mainly repeats 1-2
(Mao et al., 2001
). We found
that Wise binds to LRP6 and a variant where EGF repeats 3 and 4 are deleted
(
E3-4), but not to one in which EGF repeats 1 and 2 are removed
(
E1-2) (Fig. 8A).
Conversely, Dkk1 binds to LRP6 and
E1-2, but not to
E3-4
(Fig. 8A). These results show
that Wise shares the domain on LRP6 essential for interaction with Wnts and
that Wise and Dkk1 modulate LRP6 activity by interacting through different
domains. We also tested whether Wise and Wnt8 can bind to LRP6 at the same
time or whether they compete for binding. As shown in
Fig. 8C, Wise interferes with
the binding of Wnt8 to LRP6. This suggests a mechanism whereby Wise inhibits
Wnt signalling by competing with Wnt8 for binding to LRP6
(Fig. 8D).
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DISCUSSION |
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Role for Wise in surface ectoderm
The ability of Wise to interact with the Wnt pathway and the fact that it
is normally expressed in a transient manner in the non-neural surface
ectoderm, suggest that it might have a role in modulating Wnt signalling in
this tissue. Morpholino antisense oligonucleotide against Wise caused thick
surface ectoderm (Fig. 4K), and
overexpression of Wise by RNA injection caused expanded neural plate
(Fig. 3B). These results
suggest that endogenous expression of Wise at the edge of the neural
plate might regulate the balance of neural and non-neural ectoderm transition.
It is known that a balance between Wnt and BMP signalling in the surface
ectoderm and dorsal neural tube is important in modulating dorsal fates and
the generation of neural crest cells
(Dickinson et al., 1995;
Garcia-Castro et al., 2002
;
Liem et al., 1995
;
Trainor and Krumlauf, 2002
).
Furthermore, Wnts in the surface ectoderm influence patterning of the
underlying somites and their derivatives
(Capdevila et al., 1998
;
Munsterberg et al., 1995
). The
distribution and timing of Wise expression in the surface ectoderm together
with the result of morpholino experiments suggest that it promotes precise
levels of Wnt signalling to control some of these interactions.
Wise, Wnts and patterning
Wise was isolated on the basis of its ability to alter AP neural
patterning, an activity consistent with its interaction with the Wnt cascade.
Recent studies provide us with evidence on involvement of Wnt pathway in AP
patterning (Davidson et al.,
2002; Domingos et al.,
2001
; Kiecker and Niehrs,
2001
). Furthermore, an increasing number of extracellular and
intracellular inhibitors of Wnt signalling have been found, highlighting the
considerable complexity in the nature of regulating this cascade. The roles
for Wise in Wnt signalling raises the possibility that other members of this
class of cysteine-knot proteins may also exert some of their functions by
modulating Wnt activity. Indeed, the CCN family member Cyr61 is also capable
of regulating Wnt signalling, although its mode of action is unknown
(Latinkic et al., 2003
). Wise
is distinguished from other Wnt modulators as it seems to have multiple roles
in modulating and integrating the readout of Wnt signalling depending upon the
local context.
Even though Wise requires the canonical Wnt pathway to posteriorise
noggin-treated animal caps, there are differences in the patterns of induction
compared with stimulation by Wnt ligands such as Wnt8. Wise induces
en2 at low levels of injected RNA, and en2 plus
Krox20 only at high levels (Fig.
1B). By contrast, en2 and Krox20 are
simultaneously induced by Wnt8, even with very low amounts of RNA, and Wnt8
can induce more posterior Hox genes such as Hoxb9
(Domingos et al., 2001).
Another difference is that although Wnts or ß-catenin downregulate
forebrain markers (Otx2, BF1) at the same time as inducing posterior
genes (McGrew et al., 1997
;
McGrew et al., 1995
)
Wise does not (Fig.
1B). The basis of these differences is not clear. It has been
suggested that en2 is under the direct regulation of Tcf3
(McGrew et al., 1999
), which
seems to reflect processes in the isthmic region, where Wnt1 is required for
expression of en2 (Danielian and
McMahon, 1996
; McMahon et al.,
1992
). However, the activation of other downstream targets, such
as Krox20 and Hoxb9, could be indirect and involve multiple
steps downstream of the direct action of ß-catenin and Lef/Tcf. For
example, Wise does not induce mesoderm in the presence or absence of
noggin, whereas Wnts or ß-catenin do
(Sokol, 1993
). As mesoderm can
influence AP patterning, the differences associated with induction by Wnt
might in part be mediated indirectly through mesoderm. Hence, even though Wise
and Wnts stimulate the same pathway, there are differences in the nature of
their outputs and Wise appears to be a much weaker inducer of posterior
genes.
Dual roles for Wise: a context dependent agonist and antagonist
Recent studies on secreted proteins that affect Wnt signalling suggest
complex mechanisms modulating the canonical Wnt pathway. Different
Frizzled-related protein and Dkk family members exhibit opposite effects in a
variety of in vivo and in vitro assays
(Bradley et al., 2000;
Brott and Sokol, 2002
;
Li et al., 2002
;
Mao and Niehrs, 2003
;
Wu et al., 2000
). The
activation of the Wnt pathway by Wise is either weaker or different than that
seen using Wnts because it takes higher concentrations of Wise to induce
en2 and Krox20 and the relative levels of induction of
siamois and Xnr3 are much lower. With respect to inhibition,
in the presence of both Wnt and Wise, Wise competes with Wnts for the binding
to LRP6. This could result in either a less efficient activation of the
receptors, which masks Wnt dependent activity, or a complete block of receptor
activity (Fig. 8D). It is also
possible that Wise could affect Wnt signalling through additional mechanisms.
The interaction of Wise with LRP6 may also interfere with the function of
Dkks, which could result in either activation or inhibition of the Wnt pathway
depending upon which Dkk family member is present. There remains the
possibility that Wise interacts with other receptors or modulators that work
through intracellular Wnt signalling components. We observed that Wise
interferes with cell movements in activin-treated animal caps
(Fig. 7), consistent with the
gastrulation defects observed in Wise-injected whole embryos. As the
pathway involved in cell movements does not appear to require LRP6
(Semenov et al., 2001
), this
result implies that Wise could interact with other proteins for its function.
The studies presented here reveal new mechanisms through which a fine balance
in Wnt signalling is regulated in various developmental processes.
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ACKNOWLEDGMENTS |
---|
![]() |
Footnotes |
---|
Present address: Box 252, The Rockefeller University, 1230 York Avenue, New
York, NY 10021, USA
Present address: Wellcome Trust/Cancer Research UK Institute and Department
of Zoology, University of Cambridge, Tennis Court Road, Cambridge CB2 1QR,
UK
¶ Present address: Stowers Institute for Medical Research, 1000 East 50th
Street, Kansas City, MO 64110, USA
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