Department of Microbiology and Molecular Genetics, Harvard Medical School, and Department of Medicine, Beth Israel Deaconess Medical Center, 330 Brookline Avenue, Boston, MA 02215, USA
Author for correspondence (e-mail:
sergei.sokol{at}mssm.edu)
Accepted 14 July 2004
![]() |
SUMMARY |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: Frodo, Wnt, TCF, ß-catenin, Neural, Xenopus
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Recent studies have led to the realization that the Wnt pathway involves
multiple branches, including signaling through ß-catenin, activation of
Jun N-terminal kinases, Rho GTPases and Ca2+ ion signaling
(Boutros et al., 1998;
Habas et al., 2001
;
Kinoshita et al., 2003
;
Yamanaka et al., 2002
).
Despite the existence of many protein targets, it is commonly accepted that
the activation of Wnt target genes involves ß-catenin and transcriptional
factors of the T cell factor (TCF) family. The TCF proteins in complex with
Groucho family members repress the transcription of their targets
(Cavallo et al., 1998
;
Roose et al., 1998
), but may
be activated or derepressed by an associated co-factor such as ß-catenin.
TCFs have been reported to function as transcriptional repressors for
anteroposterior axis specification in C. elegans embryos
(Meneghini et al., 1999
) and
during vertebrate head development
(Houston et al., 2002
;
Kim et al., 2000
). The
predominant model of canonical Wnt signaling assumes that the upstream
components of the pathway, such as Dishevelled (Dsh), stabilize ß-catenin
and promote its association with TCF, thereby converting TCFs into
transcriptional activators (Nusse,
1999
).
Dishevelled appears to be essential for all branches of the Wnt pathway
(Boutros and Mlodzik, 1999;
Sheldahl et al., 2003
;
Sokol, 2000
). In the canonical
Wnt pathway, Dsh is proposed to inhibit the activity of GSK3, a
serine-threonine protein kinase that targets ß-catenin for degradation
(Cook et al., 1996
;
Itoh et al., 1998
;
Siegfried et al., 1992
;
Yost et al., 1998
). In
addition, Dsh associates with and inhibit the function of Axin, a component of
the ß-catenin destruction complex
(Cliffe et al., 2003
;
Itoh et al., 2000
;
Kishida et al., 1999
;
Li et al., 1999
;
Smalley et al., 1999
;
Zeng et al., 1997
). In an
attempt to gain insight into Dsh function, many Dsh-associated proteins have
been identified (Wharton,
2003
). Among those are Frodo
(Gloy et al., 2002
;
Gillhouse et al., 2004
) and
Dapper (Cheyette et al., 2002
),
two closely related proteins that contain a highly conserved N-terminal
leucine zipper domain and a C-terminal PDZ-binding domain. Whereas Frodo and
Dapper are 90% similar in primary amino acid sequence, they are expressed in
different patterns and reveal different activities in functional assays
(Cheyette et al., 2002
;
Gloy et al., 2002
). Both Frodo
and Dapper have been implicated in mesoderm and neural tissue development, but
their specific roles and molecular mechanism of action remain to be
elucidated. This study investigates the function of these proteins using the
morpholino-mediated loss-of-function approach. Our data suggest that Frodo and
Dapper are involved in more than one step of the signaling cascade and may
function in a pathway that is parallel to ß-catenin.
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Xenopus embryos and microinjections
In vitro fertilization and embryo culture in 0.1xMarc's modified
Ringer's solution (MMR) were carried out as described
(Peng, 1991). Staging was
according to Nieuwkoop and Faber
(Nieuwkoop and Faber, 1967
).
For microinjection, embryos were transferred to 3% Ficoll 400 (Pharmacia) in
0.5xMMR and injected at the 4- to 16-cell stages with 5 nl of mRNA or
morpholino solution. For rescue experiments and luciferase reporter assays,
the same blastomere was injected with a morpholino, followed by mRNA or DNA
injection 15-20 minutes later. For lineage tracing, RNA encoding nuclear
ß-galactosidase (nßgal) was injected together with morpholinos at 20
pg/embryo, and ß-galactosidase activity was visualized with the Red-Gal
substrate (Research Organics). FrdMO and DprMO have been characterized
previously (Cheyette et al.,
2002
; Gloy et al.,
2002
). Control morpholino (CoMO) had the following sequence:
5'-AGAGACTTGATACAGATTCGAGAAT-3'. For mRNA synthesis,
pCTX-HA-Frodo, pCTX-HA-Frd337, pCTX-HA-Frd186, pCS2-Flag-Frd337,
pCS2-ß-catenin (S>A) (Liu et al.,
1999
), pCS2-TCF3VP16 (Vonica
et al., 2000
), pCS2-nßgal
(Turner and Weintraub, 1994
)
and pT7TS-HA-TCF3 (Molenaar et al.,
1996
) were linearized with NotI or XbaI. Capped
synthetic RNAs were generated by in vitro transcription with SP6 or T7 RNA
polymerase using the mMessage mMachine kit (Ambion).
Whole-mount in situ hybridization
Whole-mount in situ hybridization was carried out according to Harland
(Harland, 1991) with slight
modifications as described previously
(Hikasa et al., 2002
). For
Frodo in situ hybridization, embryos were rehydrated in 1xPBS, 0.1%
Tween 20 and bisected with a razor blade before hybridization.
Digoxigenin-labeled antisense RNA probes were synthesized from pBS59-chd
(chordin) (Sasai et al.,
1994
), pDor3 (Xnr3)
(Smith et al., 1995
), pGsc
(Cho et al., 1991
), pBSSK-Xsox2
(Green et al., 1997
),
pBSKS-nrp1 (Richter et al.,
1990
), pBSSK-Frodo (Gloy et
al., 2002
) and pBSSK-myoD
(Hopwood et al., 1989
) using
the DIG labeling mixture (Boehringer Mannheim). 5-Bromo-4-chloro-3-indolyl
phosphate (Sigma) and Nitro blue tetrazolium (Sigma) were used for chromogenic
reactions.
RNA isolation and RT-PCR
Total embryo RNA for RT-PCR was extracted from stage 10 or stage 11 embryos
injected with morpholinos by proteinase K-phenol extraction as described
(Itoh and Sokol, 1997). cDNA
was made from DNase-treated RNA using Superscript first strand synthesis
system (Invitrogen). RT-PCR was carried out as previously described
(Itoh and Sokol, 1997
).
Primers for RT-PCR were: cerberus,
5'-GCTTGCAAAACCTTGCCCTT-3' and
5'-CTGATGGAACAGAGATCTTG-3'; chordin,
5'-AACTGCCAGGACTGGATGGT-3' and
5'-GGCAGGATTTAGAGTTGCTTC-3'; Xnr3,
5'-CGAGTGCAAGAAGGTGGACA-3' and
5'-ATCTTCATGGGGACACAGGA-3'; siamois,
5'-CTCCAGCCACCAGTACCAGATC-3' and
5'-GGGGAGAGTGGAAAGTGGTTG-3'; gsc,
5'-TTCACCGATGAACAACTGGA-3' and
5'-TTCCACTTTTGGGCATTTTC-3'; vent1,
5'-GCATCTCCTTGGCATATTTGG-3' and
5'-TTCCCTTCAGCATGGTTCACC-3'; EF1a,
5'-CAGATTGGTGCTGGATATGC-3' and
5'-ACTGCCTTGATGACTCCTAG-3'.
Transfections, GST pull-down assays, immunoprecipitation and western analysis
COS7 cells were cultured in Dulbecco's modified Eagle's medium (Gibco)
supplemented with 10% fetal calf serum and 50 µg/ml of gentamicin (Sigma).
For GST pull-down assays, cells were transiently transfected using the Fugene
6 transfection reagent (Roche) with the following plasmids: pEBG (0.1 µg),
pEBG-XTCF3 (10 µg), pEBG-NTCF (12 µg), pCTX-HA-Frodo (10 µg),
pCTX-HA-Frd337 (1 µg) and pCTX-HA-Frd186 (1 µg). After 30 hours in
culture, transfected cells were lysed in 500 µl of lysis buffer [1% Triton
X-100, 50 mM Tris-HCl (pH. 7.5), 50 mM NaCl, 1 mM EDTA, 0.1 mM
phenylmethylsulfonylfluoride, 10 mM NaF, 1 mM Na3VO4].
Supernatants were cleared at 12,000 g for 5 minutes and
incubated with glutathione-agarose beads (Sigma) for 2 hours at room
temperature. The beads were washed three times with lysis buffer and boiled in
the SDS-PAGE sample buffer. Immunoprecipitation and western analysis were
performed as described (Gloy et al.,
2002
). Monoclonal 12CA5 and M2 antibodies (Sigma) were used for
detection of HA- and FLAG-tagged proteins, respectively. Antibodies to
non-phosphorylated ß-catenin were from Upstate Biotechnology.
Luciferase reporter assays
Four-cell stage embryos were injected into a single ventral animal
blastomere with 30 pg of pSia-Luc reporter DNA
(Fan et al., 1998) together
with the indicated RNAs. At early gastrula stage (stage 10+), embryos were
homogenized in 50 mM Tris-HCl (pH 7.5). Supernatants were cleared by
centrifugation at 12,000 g for 3 minutes and assayed for
luciferase activity as previously described
(Fan et al., 1998
). Every
experimental group included four samples, each comprising five embryos. All
transcriptional assays were repeated at least three times.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Injections of either FrdMO or DprMO resulted in shortened embryos with mild
anterior abnormalities, whereas a control morpholino with a similar base
composition did not significantly alter normal development
(Fig. 1A-C). The simultaneous
injection of FrdMO and DprMO resulted in dorsally bent embryos lacking head
structures even at half the dose (Fig.
1D; Table 1),
suggesting that Frodo and Dapper synergize during early development. These
developmental abnormalities were suppressed by full length Frodo mRNA lacking
the morpholino target sequence, indicating that Frodo can functionally
substitute for Dapper in this assay (Fig.
1E; Table 1). These
findings confirm the specificity of morpholino effects. Interestingly, mRNA
for stabilized ß-catenin (Liu et al.,
1999) also rescued head structures, including cement gland and
eyes, but failed to restore proper morphogenetic movements
(Fig. 1F;
Table 1). These observations
indicate that Frodo and Dapper are necessary for both head development and
morphogenetic movements accompanying body axis elongation.
|
|
|
|
|
|
To test whether nrp1 is a target of ß-catenin signaling, we
analyzed nrp1 levels in embryos injected with ß-catenin
morpholino (ßcatMO). In these embryos, overall levels of nrp1
were not significantly affected. Whereas the nrp1 expression domain
narrowed down at the posterior neural tube, it expanded anteriorly
(Fig. 3J, left), indicating the
anterior shift of cell fates. These embryos had enlarged forebrain, midbrain
and the cement gland (Fig. 3L,
right), consistent with previously published data
(Heasman et al., 2000). Thus,
the effect of ßcatMO on nrp1 is clearly different from the
effects of FrdMO and DprMO, suggesting that the zygotic ß-catenin
function is not required for nrp1 expression.
Selective downregulation of organizer markers in embryos depleted of Frodo and Dapper
The expression of Frodo and Dapper in the dorsal mesoderm
(Cheyette et al., 2002;
Gloy et al., 2002
) suggests
that they play a role in the formation and function of the Spemann organizer,
a dorsal signaling center conserved in all vertebrates
(Harland and Gerhart, 1997
).
As the neuroectoderm is adjacent to the organizer in the early embryo
according to fate maps (Moody,
1987
; Vodicka and Gerhart,
1995
), deficient head development in FrdMO and DprMO-injected
(FDM) embryos (Fig. 1) may be
caused by either abnormal neuroectoderm development or impaired organizer. To
discriminate between the two possibilities, we studied the organizer markers
chordin, Xnr3 and goosecoid (gsc) by in situ
hybridization in embryos dorsally injected with FrdMO or/and DprMO
(Fig. 4A;
Table 4). Either morpholino
significantly reduced chordin and Xnr3 expression, whereas
co-injection of both MOs resulted in a much stronger effect on marker
expression, suggesting that head defect in FDM embryos is caused by impaired
organizer. We note that chordin appears to be the marker that is most
sensitive to the loss of Frodo and Dapper. Moreover both Frodo and
ß-catenin RNAs recovered chordin and Xnr3 expression
(Fig. 4) and suppressed head
defects (Fig. 1) in FDM
embryos, supporting the conclusion that Frodo and Dapper are essential
activators of chordin and Xnr3. Surprisingly, gsc
which is another organizer-specific gene and a target of the ß-catenin
pathway (Watabe et al., 1995
),
was not affected by FrdMO and DprMO (Fig.
4A). In fact, we occasionally observed a slight expansion of
gsc in FDM embryos. By contrast, ßcatMO strongly reduced the
expression of all three organizer markers
(Fig. 4A). These results show
that the effect of FrdMO and DprMO on organizer genes is gene specific.
|
|
Frodo associates with TCF3 and is required for TCF-dependent, but not ß-catenin-dependent, reporter activation
As organizer formation is thought to depend on early ß-catenin
function, we next examined the requirement of Frodo and Dapper in canonical
Wnt/ß-catenin signaling. Despite high sequence similarity, Frodo and
Dapper have been shown to oppositely modulate Wnt signal transduction
(Cheyette et al., 2002;
Gloy et al., 2002
). Thus, we
directly compared the effects of Frodo and Dapper on a Wnt-dependent
luciferase reporter (Fan et al.,
1998
). Consistent with our previous finding
(Gloy et al., 2002
), we
observed that injections of either Frodo or Dapper RNA enhance Dsh-dependent
activation of the reporter at 0.5 ng and 2 ng, whereas reporter activity is
suppressed at 6 ng of both RNAs (Fig.
5A). Thus, both Frodo and Dapper synergize with Dsh at low and
medium levels, but can act as inhibitors of Dsh signaling at high levels.
|
Whereas Frodo associates with Dsh through its C-terminal region
(Gloy et al., 2002), we
noticed that both the C-terminal and the N-terminal domains of Frodo can act
as Dsh antagonists. This suggests that these two domains associate with
different components of Wnt signaling machinery. We then evaluated the
possible association of HA-tagged Frodo and TCF3 fused with
glutathione-S-transferase (GST-TCF3) in transfected mammalian COS7 cells. This
GST pull-down assay demonstrated that Frodo specifically binds GST-TCF3, but
not GST (Fig. 5C,D). Further
analysis using Frodo deletion constructs revealed that GST-TCF3 binds the
large N-terminal fragment of Frodo (Frd337), but not the smaller fragment
Frd186 (Fig. 5C,D), implying
that the conserved region of Frodo located between amino acids 186 and 337 is
necessary for the association of Frodo and TCF3. The binding of the N-terminal
region of Frodo and TCF3 has been also confirmed in Xenopus embryos
using immunoprecipitation analysis (Fig.
5E). These observations indicate that Frodo interacts with both
Dsh and TCF and implicate Frodo in Wnt signal transduction downstream of
Dsh.
Based on the properties of the C-terminal Dsh-binding domain that acted in
a dominant-negative manner, we previously concluded that Frodo acts at the
level of Dsh (Gloy et al.,
2002). As the N-terminal Frd337 fragment binds TCF, we tested
whether this region of Frodo would interfere with signaling by TCF3-VP16, a
construct in which TCF3 lacking the ß-catenin binding region is fused to
the transcriptional activator VP16 (Vonica
et al., 2000
). This construct is predicted to activate Wnt target
genes independently of ß-catenin. The N-terminal Frd337 fragment, but not
Frd186, inhibited the ability of TCF3-VP16 to stimulate the pSia-luc reporter
in a dose-dependent manner (Fig.
5F), suggesting that Frodo acts in the Wnt pathway at the level of
TCF.
To determine the role for endogenous Frodo/Dapper in TCF signaling, we evaluated the effect of FrdMO and DprMO on the activity of TCF3-VP16 in the pSiaLuc reporter assay. Our results show that the activity of TCF3-VP16 was dramatically downregulated in FDM embryos, but it was not significantly affected by the control morpholino (Fig. 6A). This finding suggests that Frodo and Dapper are required for TCF-mediated transcription. We next tested if overexpression of Frodo influences the binding of ß-catenin to TCF3. The amount of ß-catenin precipitated with GST-TCF3 in GST pull-down assays was not significantly affected by Frodo (Fig. 6B), indicating that the TCF3-ß-catenin and TCF3-Frodo complexes may be independently regulated.
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The requirement for Frodo and Dapper in neuroectoderm may reflect the early
predisposition of dorsal ectoderm to neural and mesodermal fates observed by
others (Sharpe et al., 1989;
Sokol and Melton, 1991
). As
Frodo and Dapper morpholinos inhibit the expression of chordin, a
gene implicated in neural induction
(Wessely et al., 2001
), it is
possible the effect of FrdMO on neural development is due to the early
suppression of chordin. However, this explanation is not very likely
as the depletion of chordin results in the reduced sox2
expression domain according to a previous study
(Oelgeschlager et al., 2003
),
whereas sox2 is virtually eliminated in embryos depleted of Frodo or
Dapper (Fig. 3). Dorsal animal
injection of a dominant interfering TCF construct was reported to reduce
nrp1 expression but did not have an effect on muscle actin
(Baker et al., 1999
). Taken
together, these observations raise a possibility that Frodo/Dapper and TCF
cooperate in neural tissue development.
Several observations argue that the suppression of panneural markers by the morpholinos is not a consequence of decreased organizer activity. First, the requirement of Frodo for neural marker expression can be observed already at early/midgastrula stages. Second, to avoid morpholino effects on the organizer the injections have been performed at the 8- to 16-cell stage into a single dorsal animal blastomere that predominantly contributes to ectodermal tissues. Lineage tracing experiments demonstrate that only the cells injected with the specific morpholinos were affected. If the morpholinos inhibited the organizer, non cell-autonomous effects would be expected. Third, despite profound neural marker defects, myoD expression did not change, indicating that mesodermal specification remains largely unaffected (Fig. 3G,H; Table 3). In these experiments, injection of ßcatMO at the same location did not have a detectable influence on organizer markers (data not shown), further arguing that the injections were restricted to the responding ectoderm, as ßcatMO efficiently inhibited organizer genes when supplied to the dorsal margin (Fig. 4). These observations suggest that Frodo and Dapper are required for early neural development in the responding ectoderm.
Our results also argue that Frodo and Dapper are also needed for the proper
function of the organizer, because dorsal marginal injection of Frd/Dpr
morpholinos (FDM) significantly reduced organizer markers, such as
chordin and cerberus, Wnt responsive genes
(Sasai et al., 1994;
Wessely et al., 2001
), and
Xnr3, a direct Wnt target
(McKendry et al., 1997
;
Smith et al., 1995
). In
contrast to these genes, other targets of Wnt signaling, such as
siamois and gsc (Cho et
al., 1991
; Watabe et al.,
1995
), were not affected in FDM embryos, suggesting that Frodo and
Dapper are involved only in some aspects of Wnt signaling. The latter
observation reinforces the idea of the heterogeneity of the organizer
(Zoltewicz and Gerhart, 1997
)
and the conclusion that different organizer-specific genes are regulated by
different molecular mechanisms (Hamilton
et al., 2001
).
The available evidence is consistent with the view that Frodo and Dapper
are structurally and functionally related and play redundant roles during
development. The effects of Frodo and Dapper morpholinos are very similar in
the assays that we have conducted. Simultaneous injection of both morpholinos
revealed significant synergy of Frodo and Dapper. The functional differences
between Frodo and Dapper proposed in the early reports
(Cheyette et al., 2002;
Gloy et al., 2002
) may be due
to the doses or specific assays used. As Frodo is likely to play a scaffolding
role in signal transduction, its effect on signaling is predicted to be
dose-sensitive. In fact, we observed that at low doses Frodo and Dapper act
synergistically with Dsh in both axis induction and reporter assays, whereas
at higher doses they behave as antagonists
(Fig. 5A)
(Gloy et al., 2002
). Moreover,
our transcriptional assays (Fig.
6) reveal opposing roles for Frodo/Dapper at different levels of
the signaling cascade. Although these proteins appear to be required for
TCF-mediated transcriptional activation, they function as negative regulators
for Wnt8-dependent responses. Detailed analysis of the molecular mechanisms
involved warrants further studies.
Our data reveal significant functional differences between Frodo/Dapper and ß-catenin. First, whereas Frodo and Dapper are required for sox2 and nrp1 expression, ß-catenin does not seem to be necessary for pan-neural marker expression, although our data support its role in anteroposterior patterning of the neural tissue. Second, expression of organizer markers, including chordin, Xnr3 and gsc, is reduced in ß-catenin-depleted embryos, whereas only chordin and Xnr3, but not gsc, are affected in FDM embryos. Third, Frodo RNA, but not ß-catenin RNA, can restore normal morphogenetic movements during gastrulation and neurulation. Finally, Frodo and Dapper morpholinos strongly suppress TCF-dependent stimulation of the pSiaLuc reporter, but not ß-catenin-dependent stimulation of the reporter. These results allow us to propose a model in which Frodo transduces Wnt signals to target genes in a pathway parallel to that of ß-catenin. Consistent with this model, we find that Frodo associates with both Dsh and TCF. The physical interaction of Frodo and TCF may provide an additional, ß-catenin-independent control over TCF function. As Frodo is predominantly found in cell nuclei (data not shown), it may be involved in the direct activation of TCF-dependent transcription or derepression of TCF3. Future studies will be aimed at the elucidation of the molecular mechanism used by Frodo to upregulate TCF3 activity.
![]() |
ACKNOWLEDGMENTS |
---|
![]() |
Footnotes |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Baker, J. C., Beddington, R. S. and Harland, R. M.
(1999). Wnt signaling in Xenopus embryos inhibits bmp4 expression
and activates neural development. Genes Dev.
13,3149
-3159.
Boutros, M. and Mlodzik, M. (1999). Dishevelled: at the crossroads of divergent intracellular signaling pathways. Mech. Dev. 83,27 -37.[CrossRef][Medline]
Boutros, M., Paricio, N., Strutt, D. I. and Mlodzik, M. (1998). Dishevelled activates JNK and discriminates between JNK pathways in planar polarity and wingless signaling. Cell 94,109 -118.[Medline]
Cadigan, K. M. and Nusse, R. (1997). Wnt
signaling: a common theme in animal development. Genes
Dev. 11,3286
-3305.
Cavallo, R. A., Cox, R. T., Moline, M. M., Roose, J., Polevoy, G. A., Clevers, H., Peifer, M. and Bejsovec, A. (1998). Drosophila Tcf and Groucho interact to repress Wingless signalling activity. Nature 395,604 -608.[CrossRef][Medline]
Cheyette, B. N., Waxman, J. S., Miller, J. R., Takemaru, K., Sheldahl, L. C., Khlebtsova, N., Fox, E. P., Earnest, T. and Moon, R. T. (2002). Dapper, a Dishevelled-associated antagonist of beta-catenin and JNK signaling, is required for notochord formation. Dev. Cell 2,449 -461.[Medline]
Cho, K. W., Blumberg, B., Steinbeisser, H. and de Robertis, E. M. (1991). Molecular nature of Spemann's organizer: the role of the Xenopus homeobox gene goosecoid. Cell 67,1111 -1120.[Medline]
Cliffe, A., Hamada, F. and Bienz, M. (2003). A role of Dishevelled in relocating Axin to the plasma membrane during wingless signaling. Curr. Biol. 13,960 -966.[CrossRef][Medline]
Cook, D., Fry, M. J., Hughes, K., Sumathipala, R., Woodgett, J. R. and Dale, T. C. (1996). Wingless inactivates glycogen synthase kinase-3 via an intracellular signalling pathway which involves a protein kinase C. EMBO J. 15,4526 -4536.[Abstract]
Fan, M. J., Gruning, W., Walz, G. and Sokol, S. Y.
(1998). Wnt signaling and transcriptional control of Siamois in
Xenopus embryos. Proc. Natl. Acad. Sci. USA
95,5626
-5631.
Gillhouse, M., Wagner Nyholm, M., Hikasa, H., Sokol, S. Y. and Grinblat, Y. (2004). Two Frodo/Dapper homologs are expressed in the developing brain and mesoderm of zebrafish. Dev. Dyn. 230,403 -409.[CrossRef][Medline]
Gloy, J., Hikasa, H. and Sokol, S. Y. (2002). Frodo interacts with Dishevelled to transduce Wnt signals. Nat. Cell Biol. 4,351 -357.[Medline]
Green, J. B., Cook, T. L., Smith, J. C. and Grainger, R. M.
(1997). Anteroposterior neural tissue specification by
activin-induced mesoderm. Proc. Natl. Acad. Sci. USA
94,8596
-8601.
Habas, R., Kato, Y. and He, X. (2001). Wnt/Frizzled activation of Rho regulates vertebrate gastrulation and requires a novel Formin homology protein Daam1. Cell 107,843 -854.[CrossRef][Medline]
Hamilton, F. S., Wheeler, G. N. and Hoppler, S.
(2001). Difference in XTcf-3 dependency accounts for change in
response to beta-catenin-mediated Wnt signalling in Xenopus blastula.
Development 128,2063
-2073.
Harland, R. M. (1991). In situ hybridization: an improved whole-mount method for Xenopus embryos. In Methods in Cell Biology, Vol. 36 (ed. B. K. Kay and H. B. Peng), pp. 685-695. San Diego, CA: Academic Press.[Medline]
Harland, R. and Gerhart, J. (1997). Formation and function of Spemann's organizer. Annu. Rev. Cell Dev. Biol. 13,611 -667.[CrossRef][Medline]
Heasman, J., Kofron, M. and Wylie, C. (2000). Beta-catenin signaling activity dissected in the early Xenopus embryo: a novel antisense approach. Dev. Biol. 222,124 -134.[CrossRef][Medline]
Hikasa, H., Shibata, M., Hiratani, I. and Taira, M. (2002). The Xenopus receptor tyrosine kinase Xror2 modulates morphogenetic movements of the axial mesoderm and neuroectoderm via Wnt signaling. Development 129,5227 -5239.[Medline]
Hopwood, N. D., Pluck, A. and Gurdon, J. B. (1989). MyoD expression in the forming somites is an early response to mesoderm induction in Xenopus embryos. EMBO J. 8,3409 -3417.[Abstract]
Houston, D. W., Kofron, M., Resnik, E., Langland, R., Destree,
O., Wylie, C. and Heasman, J. (2002). Repression of
organizer genes in dorsal and ventral Xenopus cells mediated by maternal
XTcf3. Development 129,4015
-4025.
Itoh, K. and Sokol, S. Y. (1997). Graded amounts of Xenopus dishevelled specify discrete anteroposterior cell fates in prospective ectoderm. Mech. Dev. 61,113 -125.[CrossRef][Medline]
Itoh, K., Krupnik, V. E. and Sokol, S. Y. (1998). Axis determination in Xenopus involves biochemical interactions of axin, glycogen synthase kinase 3 and beta-catenin. Curr. Biol. 8,591 -594.[Medline]
Itoh, K., Antipova, A., Ratcliffe, M. J. and Sokol, S.
(2000). Interaction of dishevelled and Xenopus axin-related
protein is required for wnt signal transduction. Mol. Cell.
Biol. 20,2228
-2238.
Kim, C. H., Oda, T., Itoh, M., Jiang, D., Artinger, K. B., Chandrasekharappa, S. C., Driever, W. and Chitnis, A. B. (2000). Repressor activity of Headless/Tcf3 is essential for vertebrate head formation. Nature 407,913 -916.[CrossRef][Medline]
Kinoshita, N., Iioka, H., Miyakoshi, A. and Ueno, N.
(2003). PKC delta is essential for Dishevelled function in a
noncanonical Wnt pathway that regulates Xenopus convergent extension
movements. Genes Dev.
17,1663
-1676.
Kishida, S., Yamamoto, H., Hino, S., Ikeda, S., Kishida, M. and
Kikuchi, A. (1999). DIX domains of Dvl and axin are necessary
for protein interactions and their ability to regulate beta-catenin stability.
Mol. Cell. Biol. 19,4414
-4422.
Knecht, A. K., Good, P. J., Dawid, I. B. and Harland, R. M.
(1995). Dorsalventral patterning and differentiation of
noggin-induced neural tissue in the absence of mesoderm.
Development 121,1927
-1935.
Li, L., Yuan, H., Weaver, C. D., Mao, J., Farr, G. H., 3rd,
Sussman, D. J., Jonkers, J., Kimelman, D. and Wu, D.
(1999). Axin and Frat1 interact with dvl and GSK, bridging Dvl to
GSK in Wnt-mediated regulation of LEF-1. EMBO J.
18,4233
-4240.
Liu, C., Kato, Y., Zhang, Z., Do, V. M., Yankner, B. A. and He,
X. (1999). beta-Trcp couples beta-catenin
phosphorylation-degradation and regulates Xenopus axis formation.
Proc. Natl. Acad. Sci. USA
96,6273
-6278.
Makarova, O., Kamberov, E. and Margolis, B. (2000). Generation of deletion and point mutations with one primer in a single cloning step. Biotechniques 29,970 -972.[Medline]
McKendry, R., Hsu, S. C., Harland, R. M. and Grosschedl, R. (1997). LEF-1/TCF proteins mediate wnt-inducible transcription from the Xenopus nodal-related 3 promoter. Dev. Biol. 192,420 -431.[CrossRef][Medline]
Meneghini, M. D., Ishitani, T., Carter, J. C., Hisamoto, N., Ninomiya-Tsuji, J., Thorpe, C. J., Hamill, D. R., Matsumoto, K. and Bowerman, B. (1999). MAP kinase and Wnt pathways converge to downregulate an HMG-domain repressor in Caenorhabditis elegans. Nature 399,793 -797.[CrossRef][Medline]
Mizuseki, K., Kishi, M., Matsui, M., Nakanishi, S. and Sasai,
Y. (1998). Xenopus Zic-related-1 and Sox-2, two factors
induced by chordin, have distinct activities in the initiation of neural
induction. Development
125,579
-587.
Molenaar, M., van de Wetering, M., Oosterwegel, M., Peterson-Maduro, J., Godsave, S., Korinek, V., Roose, J., Destree, O. and Clevers, H. (1996). XTcf-3 transcription factor mediates beta-catenin-induced axis formation in Xenopus embryos. Cell 86,391 -399.[Medline]
Moody, S. A. (1987). Fates of the blastomeres of the 32-cell-stage Xenopus embryo. Dev. Biol. 122,300 -319.[Medline]
Nieuwkoop, P. D. and Faber, J. (1967).Normal Table of Xenopus laevis (Daudin) . Amsterdam, The Netherlands: North Holland.
Nusse, R. (1999). WNT targets. Repression and activation. Trends Genet. 15, 1-3.[CrossRef][Medline]
Oelgeschlager, M., Kuroda, H., Reversade, B. and de Robertis, E. M. (2003). Chordin is required for the Spemann organizer transplantation phenomenon in Xenopus embryos. Dev. Cell 4,219 -230.[Medline]
Peng, H. B. (1991). Xenopus laevis: practical uses in cell and molecular biology. Solutions and protocols. Methods Cell. Biol. 36,657 -662.[Medline]
Richter, K., Good, P. J. and Dawid, I. B. (1990). A developmentally regulated, nervous system-specific gene in Xenopus encodes a putative RNA-binding protein. New Biol. 2,556 -565.[Medline]
Roose, J., Molenaar, M., Peterson, J., Hurenkamp, J., Brantjes, H., Moerer, P., van de Wetering, M., Destree, O. and Clevers, H. (1998). The Xenopus Wnt effector XTcf-3 interacts with Groucho-related transcriptional repressors. Nature 395,608 -612.[CrossRef][Medline]
Sanchez, I., Hughes, R. T., Mayer, B. J., Yee, K., Woodgett, J. R., Avruch, J., Kyriakis, J. M. and Zon, L. I. (1994). Role of SAPK/ERK kinase-1 in the stress-activated pathway regulating transcription factor c-Jun. Nature 372,794 -798.[Medline]
Sasai, Y., Lu, B., Steinbeisser, H., Geissert, D., Gont, L. K. and de Robertis, E. M. (1994). Xenopus chordin: a novel dorsalizing factor activated by organizer-specific homeobox genes. Cell 79,779 -790.[Medline]
Sharpe, C. R., Pluck, A. and Gurdon, J. B. (1989). XIF3, a Xenopus peripherin gene, requires an inductive signal for enhanced expression in anterior neural tissue. Development 107,701 -714.[Abstract]
Sheldahl, L. C., Slusarski, D. C., Pandur, P., Miller, J. R.,
Kuhl, M. and Moon, R. T. (2003). Dishevelled activates
Ca2+ flux, PKC, and CamKII in vertebrate embryos. J. Cell
Biol. 161,769
-777.
Siegfried, E., Chou, T. B. and Perrimon, N. (1992). wingless signaling acts through zeste-white 3, the Drosophila homolog of glycogen synthase kinase-3, to regulate engrailed and establish cell fate. Cell 71,1167 -1179.[Medline]
Smalley, M. J., Sara, E., Paterson, H., Naylor, S., Cook, D.,
Jayatilake, H., Fryer, L. G., Hutchinson, L., Fry, M. J. and Dale, T.
C. (1999). Interaction of axin and Dvl-2 proteins regulates
Dvl-2-stimulated TCF-dependent transcription. EMBO J.
18,2823
-2835.
Smith, W. C., McKendry, R., Ribisi, S., Jr and Harland, R. M. (1995). A nodal-related gene defines a physical and functional domain within the Spemann organizer. Cell 82, 37-46.[Medline]
Sokol, S. Y. (1996). Analysis of Dishevelled signalling pathways during Xenopus development. Curr. Biol. 6,1456 -1467.[Medline]
Sokol, S. Y. (1999). Wnt signaling and dorso-ventral axis specification in vertebrates. Curr. Opin. Genet. Dev. 9,405 -410.[CrossRef][Medline]
Sokol, S. (2000). A role for Wnts in morpho-genesis and tissue polarity. Nat. Cell Biol. 2,E124 -E125.[CrossRef][Medline]
Sokol, S. and Melton, D. A. (1991). Pre-existent pattern in Xenopus animal pole cells revealed by induction with activin. Nature 351,409 -411.[CrossRef][Medline]
Sokol, S. Y., Klingensmith, J., Perrimon, N. and Itoh, K. (1995). Dorsalizing and neuralizing properties of Xdsh, a maternally expressed Xenopus homolog of dishevelled. Development 121,3487 .[Medline]
Tada, M. and Smith, J. C. (2000). Xwnt11 is a
target of Xenopus Brachyury: regulation of gastrulation movements via
Dishevelled, but not through the canonical Wnt pathway.
Development 127,2227
-2238.
Turner, D. L. and Weintraub, H. (1994). Expression of achaete-scute homolog 3 in Xenopus embryos converts ectodermal cells to a neural fate. Genes Dev. 8,1434 -1447.[Abstract]
Vodicka, M. A. and Gerhart, J. C. (1995).
Blastomere derivation and domains of gene expression in the Spemann Organizer
of Xenopus laevis. Development
121,3505
-3518.
Vonica, A., Weng, W., Gumbiner, B. M. and Venuti, J. M. (2000). TCF is the nuclear effector of the beta-catenin signal that patterns the sea urchin animal-vegetal axis. Dev. Biol. 217,230 -243.[CrossRef][Medline]
Wallingford, J. B., Rowning, B. A., Vogeli, K. M., Rothbacher, U., Fraser, S. E. and Harland, R. M. (2000). Dishevelled controls cell polarity during Xenopus gastrulation. Nature 405,81 -85.[CrossRef][Medline]
Watabe, T., Kim, S., Candia, A., Rothbacher, U., Hashimoto, C., Inoue, K. and Cho, K. W. (1995). Molecular mechanisms of Spemann's organizer formation: conserved growth factor synergy between Xenopus and mouse. Genes Dev. 9,3038 -3050.[Abstract]
Wessely, O., Agius, E., Oelgeschlager, M., Pera, E. M. and de Robertis, E. M. (2001). Neural induction in the absence of mesoderm: beta-catenin-dependent expression of secreted BMP antagonists at the blastula stage in Xenopus. Dev. Biol. 234,161 -173.[CrossRef][Medline]
Wharton, K. A., Jr (2003). Runnin' with the Dvl: proteins that associate with Dsh/Dvl and their significance to Wnt signal transduction. Dev. Biol. 253, 1-17.[CrossRef][Medline]
Yamanaka, H., Moriguchi, T., Masuyama, N., Kusakabe, M.,
Hanafusa, H., Takada, R., Takada, S. and Nishida, E.
(2002). JNK functions in the non-canonical Wnt pathway to
regulate convergent extension movements in vertebrates. EMBO
Rep. 3,69
-75.
Yost, C., Farr, G. H., 3rd, Pierce, S. B., Ferkey, D. M., Chen, M. M. and Kimelman, D. (1998). GBP, an inhibitor of GSK-3, is implicated in Xenopus development and oncogenesis. Cell 93,1031 -1041.[Medline]
Zeng, L., Fagotto, F., Zhang, T., Hsu, W., Vasicek, T. J., Perry, W. L., 3rd, Lee, J. J., Tilghman, S. M., Gumbiner, B. M. and Costantini, F. (1997). The mouse Fused locus encodes Axin, an inhibitor of the Wnt signaling pathway that regulates embryonic axis formation. Cell 90,181 -192.[Medline]
Zoltewicz, J. S. and Gerhart, J. C. (1997). The Spemann organizer of Xenopus is patterned along its anteroposterior axis at the earliest gastrula stage. Dev. Biol. 192,482 -491.[CrossRef][Medline]