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First published online 25 October 2006
doi: 10.1242/dev.02657
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Laboratory for Vertebrate Body Plan, RIKEN Center for Developmental Biology, 2-2-3 Minatojima Minami, Chuo-ku, Kobe 650-0047, Japan.
* Author for correspondence (e-mail: saizawa{at}cdb.riken.jp)
Accepted 22 September 2006
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SUMMARY |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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Key words: Shisa, Somitogenesis, Wnt, FGF, Xenopus
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INTRODUCTION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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;
Dubrulle and Pourquie, 2004a;
Saga and Takeda, 2001). This
transition point, termed a `wavefront', `differentiation wavefront' or
`determination front' (Cooke and Zeeman,
1976; Dubrulle and Pourquie,
2004a), has been thought to be located at the border between the
rostral and caudal PSM and marked by changes in the expression of two basic
helix-loop-helix (bHLH) transcription factors. Mesogenin1
(Mes1; Xenopus ortholog of Mespo) marks the caudal
PSM (Joseph and Cassetta,
1999; Yoon et al.,
2000; Yoon and Wold,
2000); Thylacine1 (Thy1; Xenopus
ortholog of Mesp) is expressed in two or three bilateral stripes that
correspond to the anterior half of the somitomeres I-III
(Fig. 3G) in the rostral PSM.
The most posterior Thy1 stripe in S-III is thought to represent PSM
cells that have just passed the wavefront
(Buchberger et al., 1998;
Saga et al., 1997;
Sawada et al., 2000;
Sparrow et al., 1998). As
somitogenesis proceeds, the wavefront moves posteriorly; this movement is
tightly coupled with axial elongation.
A well-known signal constituting the gradient is FGF. The increase in the
FGF/MAPK signaling suppresses the maturation of PSM cells and delays the
transition to the segmental fate, consequently generating smaller somites. By
contrast, the inhibition of FGF/MAPK signaling promotes the maturation of PSM
cells and the generation of larger somites
(Delfini et al., 2005;
Dubrulle et al., 2001;
Dubrulle and Pourquie, 2004b;
Sawada et al., 2001).
The Wnt signal emitted from the tailbud has been implicated in the
mechanism of the segmentation clock by which periodicity of segmentation is
generated. In the mutant mouse harboring the Wnt3a hypomorphic allele
vestigial tail (vt), expressions of several segmental clock
genes are severely reduced or disrupted, including Axin2, Lunatic
fringe, Nkd1 and Snail1 (Snai1 - Mouse Genome
Informatics) (Aulehla et al.,
2003; Dale et al.,
2006; Ishikawa et al.,
2004). In the segmentation, increases and decreases in
Wnt/ß-catenin signaling generate smaller and larger somites,
respectively, suggesting that Wnt signaling also functions as an inhibitor of
the PSM maturation as FGF signaling does
(Aulehla et al., 2003). It has
thus been suggested that the wavefront is settled at the position where the
level of FGF and Wnt signaling goes below a certain threshold
(Aulehla and Herrmann, 2004;
Dubrulle and Pourquie, 2004a).
However, as the FGF8 expression is strongly reduced in vt/vt
mice, Wnt might set up the wavefront indirectly by regulating the FGF
signaling (Aulehla and Herrmann,
2004). Thus it remains elusive whether individual regulation of
both FGF and Wnt signals are required for the positioning of the
wavefront.
The mechanisms establishing the PSM gradient have been explained in two
ways. One is the axial elongation by which somitic precursors progressively
move away from the tailbud, where cells actively transcribe and translate
FGF8 and Wnt3a genes. The decay and diffusion of the ligand
protein regulate the gradient activity. Furthermore, as a result of a slow
decay of FGF8 transcripts, cells establish an FGF8 mRNA
gradient in the PSM; this mRNA gradient has been suggested to generate a
shallow FGF8 protein gradient and to regulate the maturation of PSM
(Dubrulle and Pourquie,
2004b). The other mechanism is the antagonistic relationship
between retinoic acid (RA) and the FGF gradient. The RA signal constitutes a
gradient that is rostrocaudally decreasing and has been suggested to promote
the maturation of PSM. The increase and decrease in RA activity causes a
posterior and anterior shift of the wavefront, respectively, through the
indirect regulation of FGF signaling (Diez
del Corral et al., 2003;
Moreno and Kintner, 2004). By
contrast to the gradient in extracellular ligands, however, it is largely
unknown whether, and how, cells regulate their responsiveness to FGF and Wnt
signaling for setting up the morphogenetic gradient in the PSM.
Wnts bind to the Frizzled (Fz) family of the seven-pass transmembrane
receptor and activates a downstream target, Dishevelled. In the
Wnt/ß-catenin (canonical) pathway, this suppresses the activity of a
protein kinase GSK3 (a negative regulator of this signaling), stabilizes
ß-catenin and activates transcriptional targets
(Nusse, 2005). Wnt-Fz also
initiates at least two other signaling cascades, planer cell polarity (PCP)
and Wnt/Ca2+ pathway
(Wallingford and Habas, 2005).
In the early Xenopus embryos, integrations of this signaling govern
numerous biological processes, including axis formation, convergent-extension
cell movements, mesodermal differentiation and cell adhesion. FGF binds to the
receptor tyrosine kinase FGF receptor family (FGFR1-4), and induces its
dimerization and transphosphorylation. Subsequently, the small GTPase Ras
transmits the FGFR signal and activates the protein kinase cascade
Raf-MEK1/2-ERK1/2, which phosphorylates and activates various transcription
factors (Goldfarb, 2001). FGF
functions in the mesoderm and neural induction and their differentiation
(De Robertis and Kuroda, 2004;
Slack et al., 1996). We have
recently reported isolation and functional characterization of Shisa1
(previously called Shisa) (Yamamoto et
al., 2005), a cell-autonomous inhibitor for both Wnt and FGF
signaling, involved in Xenopus head formation. Shisa physically
interacts with an immature form of Fz and FGFR in the endoplasmic reticulum
(ER) and inhibits their protein maturation and cell surface transportation,
thereby suppressing events being initiated by ligand-receptor interactions of
Wnt and FGF signaling.
Here we have identified Shisa-related genes in Xenopus, Shisa2 and Shisa3, which inhibit both Wnt and FGF signals through the retention of their receptors in the ER as Shisa1 does. Knockdown study of Shisa2 suggests that it plays an essential role in the maturation of PSM cells by individual attenuation of both FGF and Wnt signaling.
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MATERIALS AND METHODS |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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).
Morpholino antisense oligomers were obtained from Gene Tools: 5mis
Shisa2MO1, 5'-GAGGCGTGCAACCACATCACTGGC-3';
Shisa2MO1, 5'-GAGCCCTCCAACCACATGACTGGG-3';
Shisa2MO2, 5'-ACTCCTCTCACGGGCAGCAAAAGTC-3'; 5mis
Shisa2MO3, 5'-ACATGCCATTTATTAGCTCCTCTAG-3';
Shisa2MO3, 5'-AGATCCCATTTATTACCTGCTGTAG-3'.
Shisa1MO was described previously
(Yamamoto et al., 2005).
Cloning and construction of Xenopus Shisa2 and Shisa3
The expressed sequence tag (EST) clone (Accession number CF286494: IMAGE
5516153) encoded a partial Xenopus Shisa2 coding sequence (CDS),
lacking 227 nt from the 3' end of the CDS. To determine the full-length
Shisa2 cDNA sequence, 3' RACE of the tadpole stage total RNA
was carried out with a SMART RACE cDNA Amplification Kit (CLONTECH). The two
forward primers 5'-GGTGGCAATTTGCTGTTGCAGATGT-3' and
5'-AGTGCGAGCTGCGCTACTGCTGTT-3' were used in a nested way. By the
sequences of five independent clones, a full-length sequence of
Shisa2 cDNA was determined. The EST sequence of the BI449671 clone
contained the full-length of Shisa3 CDS. The CDS of Shisa2
and 3 were amplified by RT-PCR and subcloned into pCS2
(Shisa2/pCS2 and Shisa3/pCS2).
Shisa2-HA, Shisa2-Flag, Shisa3-HA and
Shisa3-Flag were also generated by PCR. Other constructs were
described previously (Yamamoto et al.,
2005).
RT-PCR
RT reaction was carried out with MLTV (Invitrogen) using 500 ng of total
RNA, isolated with RNA-STAT-60 (TEL-TEST Inc.) from embryos, animal caps or
tailbud explants. PCR amplification was carried out for 28 cycles with the
following thermal cycle profile: denaturation at 94°C for 30 seconds,
annealing at 55°C for 45 seconds and extension at 68°C for 45 seconds,
followed by a final extension at 72°C for 5 minutes. The primers used
were: Shisa2, forward 5'-ACGATTCGACCATCTGCTG-3' and
reverse 5'-CAGTTGGTTTGGGATCGAGT-3'; Mes1, forward
5'-GAGACAACGGAGCTCTCACC-3' and reverse
5'-AATCCAGCCTGGTGTTTCAG-3'; FGF8, forward
5'-ACCTCCATCCTGGGCTATCT-3' and reverse
5'-GCCCCTTCCATTAGTCTTCC-3'; Wnt3a, forward
5'-GCGATTTTTGGACCAGTGTT-3' and reverse
5'-TTCTGCCTGCTTCATTGTTG-3'; Hes6, forward
5'-GGCTGCTGATCTTCTGAACC-3' and reverse
5'-CCTTCTCCCCTTCAGATTCC-3'; Shisa2 F,
5'-ACGATTCGACCATCTGCTG-3'; Shisa2 R1,
5'-GAAATTCCATCATCCCAACC-3'; Shisa2 R2,
5'-CAGTTGGTTTGGGATCGAGT-3'. Other primer sequences and conditions
for PCR reaction were carried out as described previously
(Yamamoto et al., 2005).
In situ hybridization, whole-mount immunostaining and western blotting
Whole-mount in situ hybridization was performed according to described
procedure (Sive et al., 2000).
Signals were developed with BM Purple (Roche) or BCIP (Roche). The probes used
for in situ hybridization were transcribed from Mes1, XL322e02ex
(NIBB); Thy1, XL220g19 (NIBB); ESR9, XL224g01ex (NIBB);
Arp-A, XL146e16 (NIBB); Cyp26, XL322k18ex (NIBB);
Rarg, XL275p11ex (NIBB); Raldh2, XL191i17 (NIBB);
Shisa2, EXL1051-5991502 (Open Biosystems). Xbra,
MyoD, Papc and Wnt3a were transcribed as described
(Kim et al., 1998;
Rupp and Weintraub, 1991;
Smith et al., 1991;
Wolda et al., 1993).
FGF8 cDNA was isolated by RT-PCR and cloned into the pGEMT-E vector
(Promega). Western blotting was performed as described previously
(Yamamoto et al., 2005).
Antibodies against phospho-p44/42 MAP kinase (dp-ERK)(Cell Signaling), p44/42
MAP kinase (Cell Signaling) and HA (Covance) were used at 1:1000 dilution.
Whole-mount immunostaining was performed according to described procedure
(Kuroda et al., 2005).
Immunofluorescent staining, luciferase assay and coimmunoprecipitation assay
Immunofluorescent staining, luciferase assay and co-immunoprecipitation
assay were carried out as described previously
(Yamamoto et al., 2005). Cell
transfection into COS cells was performed with Lipofectamine 2000
(Invitrogen), according to the manufacturer's instructions.
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RESULTS |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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). A
search of a Xenopus EST database with the amino acid sequence of
Shisa1 allowed us to identify two genes that encode Shisa family proteins
harboring the two conserved CRDs (Fig.
1A,B); they are referred to as Shisa2 and Shisa3
(Accession number CF286494 for Shisa2 and BI449671 for
Shisa3). Among Shisa1, 2 and 3, the amino acid sequences are well
conserved in the amino-terminal half, including the CRDs, but not in the
carboxy-terminal half (Fig.
1C).
In the Xenopus animal cap assay, ectopic expression of Wnt8 or
treatment with bFGF (FGF2) protein induces the expression of Xnr3 and
Xbra, respectively (Brannon et
al., 1997; McKendry et al.,
1997; Pownall et al.,
1996). Shisa2 and 3 inhibited this induction in a dose-dependent
manner, as Shisa1 does (Fig.
1D,E) (Yamamoto et al.,
2005). Shisa2 and 3 neither induced neuroectoderm in animal caps
or a secondary axis on the ventral side, nor did they inhibit
Nodal/Activin-dependent Mix2 expression (data not shown). These
results indicate that Shisa2 and 3 uniquely inhibit Wnt and FGF signaling, but
not Nodal/activin or BMP signaling.
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Shisa2 and Shisa3 antagonize Wnt and FGF signaling by the retention of their receptors in the ER
Shisa2 and Shisa3 do not have a known ER retention signal, as is also true
of Shisa1. In Hek293T cells, however, HA-tagged Shisa2 and Shisa3 specifically
localized in the ER (Fig.
2A-B''). We examined the mode of action of Shisa2 and 3 in
Wnt signaling in Hek293T cells. Ligand cells expressing Wnt3a and receptor
cells expressing Fz8 and Lrp6 together with TOPFLASH reporter
(Korinek et al., 1997) were
prepared independently and mixed for stimulation. The non-cell-autonomous
action of Wnt3a elevated the reporter activity (threefold;
Fig. 2C, lane 2). When either
Shisa 2 or 3 was coexpressed with Wnt-ligand, reporter activity was not
affected (Fig. 2C, lanes 5 and
6); however, expression of Shisa2 or 3 in the receptor cells suppressed the
reporter activity below the basal level
(Fig. 2C, lanes 3 and 4),
demonstrating that Shisa2 and 3 cell-autonomously inhibit Wnt signaling in
these cells.
To analyze whether Shisa2 and 3 physically interact with Fz and FGFR,
receptors tagged with HA were coexpressed together with Shisa2- or
Shisa3-tagged Flag, and immunoprecipitated with anti-Flag mAb. We found the
low molecular weight form of Fz8 and FGFR1 could be immature glycosylated Fz
and FGFR (Yamamoto et al.,
2005) in the precipitates of the Shisa2 and 3, indicating that
Shisa2 and 3 physically interact with immature forms of Fz and FGFR
(Fig. 2D,E). Furthermore,
Shisa2 and 3 retain the Fz8 and FGFR1 in the ER
(Fig. 2F-I'' for Shisa2;
data not shown for Shisa3). We also examined whether Shisa2 retains other Fz
homologs in the ER. In Xenopus, Fz2 and 7 are expressed in the PSM:
they share 78% identities in the amino acid level
(Deardorff and Klein, 1999;
Sumanas et al., 2000). We
found that Shisa2 retained Fz7 in the ER (n=20/20; data not shown).
Altogether these results indicate that Shisa2 and 3 inhibit both Wnt and FGF
signaling through the regulation of protein maturation and cell surface
transportation of their receptors within the ER as Shisa1 does.
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). We
found that injection of Shisa2 RNA rescued this phenotype, suggesting
that Shisa1 and 2 are functionally exchangeable in vivo at a molecular level
(Fig. 2J-J'').
Expression of Shisa2 in the Xenopus embryo
RT-PCR analysis showed that the Shisa2 expression was weak
throughout early embryogenesis and increased after the tailbud stage
(Fig. 3A). Whole-mount in situ
hybridization showed maternal and/or zygotic expression of Shisa2 in
the entire animal hemisphere by blastula stage (data not shown).
Shisa2 expression in the PSM was first detected at the beginning of
neurulation (Fig. 3B). As
somitogenesis proceeded, the Shisa2 expression moved posteriorly
(Fig. 3C), covering all the
somitomeres (S-I, -II, -III) that have committed to segmentation and are
visualized by Thy1 expression
(Fig. 3E,E',G). The
Thy1 expression in the S-I and -II extended into the lateral plate
mesoderm; however, Shisa2 expression in this region was below the
detectable level. In the caudal PSM, Shisa2 showed a graded
expression: high anteriorly and low posteriorly, extended laterally and partly
overlapping with Mes1 expression, a marker for the caudal PSM and
tailbud (Fig. 3F,F',G).
At the tadpole stage, Shisa2 expression was detected in the middle of
each somite, precursors of the ventral body wall muscle, the otic and optic
vesicles, head mesenchyme and brachial arches
(Fig. 3D). We also found unique
Shisa3 expressions in ventral forebrain and ventral hindbrain at the
tailbud stage (data not shown). Shisa3 might play a role in these tissues;
however, this study focused on the role of Shisa2 in segmental patterning.
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-tubulin
(Fig. 4B). In these studies,
the MOs were unilaterally injected, the uninjected side serving as an internal
control. During gastrulation, these MOs had no effect on the expression of
Xbra (a panmesodermal marker) or MyoD (a myogenic marker),
suggesting that Shisa2 has no role in mesoderm induction, or in the
dorsoventral and anteroposterior patterning
(Fig. 4C-D''). At the
early neurula stage, the paraxial mesodermal region stained by MyoD
expression was symmetrical in the MO-injected and uninjected side, suggesting
that the early allocation of somitic precursors was not affected by the MO
injection (Fig.
4E-E'').
In the early segmentation period (stage 18; in Xenopus, the first
somite buds off at stage 16-17) (Hamilton,
1969), unilateral injection of MO1 and MO2 elicited the anterior
shift of the expression of Thy1 and Papc
(Kim et al., 1998) for a
distance of one to two segments; the level and the mediolateral width of their
expression was not affected (Fig.
4F-G''). The anterior borders of Mes1, Xbra, FGF8
and Wnt3a expressions in the caudal PSM and the tailbud were also
expanded anteriorly in the Shisa2MO-injected side
(Fig. 4H-K''). These
results suggest that knockdown of Shisa2 causes delay of the maturation of PSM
cells.
Although we tried to rescue the Shisa2 morphant phenotypes by co-injection of carefully titrated Shisa2 RNAs, the morphogenetic defects of gastrulae caused by ectopic Shisa2 expression severely disturbed early allocation of somitic precursors (as shown in Fig. 1F-I'): this made it difficult for us to come to any conclusion on this issue. To further confirm the specificity of the Shisa2 morphant phenotype, we generated a third Shisa2MO (MO3; Fig. 4A'), which inhibits splicing of endogenous Shisa2 mRNA (Fig. 4B'). We found that unilateral injection of MO3 also resulted in the anterior shift of the expression of Thy1 and Mes1 (Fig. 4L-M'). These results further support the specificity of the Shisa2 morphant phenotypes.
Next we examined whether knockdown of Shisa2 caused anterior extension of
Wnt and FGF signaling activities. In mice, Axin2 has been thought to
be a direct downstream target of Wnt3a in the PSM
(Aulehla et al., 2003). In
Xenopus, expression of the Axin-related gene (Arp-A)
(Itoh et al., 2000) was also
under the control of Wnt signaling but not FGF (see
Fig. 8C-C''). In the
Shisa2 morphant, the expression of Arp-A and phospho-ERK (dp-ERK)
staining were extended anteriorly (Fig.
4N-O'), indicating that knockdown of Shisa2 caused anterior
extension of both Wnt and FGF signaling activities. We also found the anterior
extension of retinoic acid hydroxylase Cyp26
(Hollemann et al., 1998)
expression and RA receptor gamma Rarg
(Pfeffer and De Robertis,
1994), but not that of a dehydrogenase of RA Raldh2
(Chen et al., 2001) in the
Shisa2 morphant (Fig. 4P,Q;
data not shown for Raldh2).
Next we examined whether the depletion of Shisa2 affects the cyclic
expression of the segmental clock gene. In Xenopus, three distinct
phases of cyclic expression are observed for ESR9
(Hairy/Enhancer-of-split related 9), a possible component in the
Notch signaling cascade (Fig.
5A-C) (Li et al.,
2003). By the Shisa2 depletion, the anterior border of the
ESR9 expression was expanded anteriorly; however, the phase of the
expression was not affected (Fig.
5D-F for MO1; data not shown for MO2).
Histological analysis of the longitudinal horizontal section demonstrated that Shisa2 depletion elicited anterior displacement of the most newly formed somite for a distance of one to two somites, accompanying the anterior expansion of PSM. The serial sections showed that the position of the first somite was symmetrically at the same level in the MO-injected and uninjected sides, and the segment number was reduced by the knockdown of Shisa2 (Fig. 5G-L). Our data suggest that the altered morphogenetic gradient by the Shisa2 deficiency affected the generation of somites for a few segments and consequently reduced their number.
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), and then
stained for Mes1 and Thy1 expressions. These treatments
affect the maturation of the caudal PSM and the most newly formed segment in
the S-III, but not older segments (e.g. S-I). In the uninjected side, both RA
and SU5402 treatments abolished Mes1 expression
(Fig. 6A-C) and induced ectopic
Thy1 expression in the caudal PSM
(Fig. 6D-F; ectopic
Thy1 expression is indicated by red arrowheads)
(Moreno and Kintner, 2004).
The treatments did not affect the endogenous Shisa2 expression
(Fig. 6J-J''). In the
Shisa2-depleted side, both the reduction of Mes1 expression and the
ectopic induction of Thy1 in the caudal PSM were significantly
inhibited (Fig. 6A-F,G-I).
|
). RA treatment in wild-type embryos directly activated
ubiquitous expression of Cyp26
(Loudig et al., 2000)
(Fig. 6K'). The knockdown
of Shisa2 had no effect on the RA-induced Cyp26 expression
(Fig. 6K''). These results
suggest that the inhibition of the effects of RA on the wavefront by the
Shisa2 knockdown would be indirect. In the tailbud explants, the depletion of Shisa2 also inhibited the reduction of Mes1 expression by SU5402 treatment, the expression of FGF8 and Wnt3a being unchanged (Fig. 6L). This Shisa2 knockdown effect was not caused by the reactivation of FGF signaling, as the SU5402 treatment strongly suppressed the activation of ERK/MAPK in both control and Shisa2-depleted embryos (Fig. 6M). These results strongly suggest that Shisa2 functions in the maturation of the PSM cells by regulating signals other than RA and FGF signaling, or by both FGF and Wnt signaling together.
Shisa2 promotes maturation of PSM cells through the inhibition of both Wnt and FGF signaling
As Shisa2 inhibits Wnt and FGF signaling, it is possible that inhibition of
both these types of signaling canceled the anterior shift of the
S-III/Thy1 stripe in the Shisa2 morphants. To inhibit Wnt signaling,
we injected a small amount of Gsk3 RNA, an inhibitor of the canonical
Wnt pathway but not of Wnt/PCP: this had no or little effect on the early
allocation of PSM precursors at gastrulation (data not shown). Embryos
radially receiving Gsk3 RNA were subsequently injected with
Shisa2MO1 unilaterally and were treated with SU5402 at the early
neurula stage for 1.5 hours. This manipulation generated a larger posterior
shift of the S-III/Thy1 stripe in the Shisa2-depleted side and
positioned the stripes in a symmetrical manner
(Fig. 7A-A''').
Next we further examined this issue using dorsal explants that were generated at the early neurula stage (stage 15) (Fig. 7B). The explants were treated for 1.5 hours with SU5402 and/or WIF1 protein, and subsequently analyzed for Mes1 and Thy1 expression. The knockdown of Shisa2 expanded Mes1 expression anteriorly, as seen in the whole embryo (Fig. 7C). The inhibition of either Wnt or FGF signaling alone reduced Mes1 expression in the uninjected side, whereas it was insufficient to abolish this expression in the Shisa2-depleted side (Fig. 7C',C''). Inhibition of both types of signaling together, however, efficiently abolished Mes1 expression in the caudal PSM of the Shisa2 morphants (Fig. 7C''',F).
We then examined whether the inhibition of Wnt signaling by the WIF1 or conditioned media containing FzCRD (a soluble form of Fz8), together with SU5402 treatment, abolishes the anterior shifted S-III/Thy1 stripe in the Shisa2 morphants. As in the case of Mes1 expression, the inhibition of either signaling alone was insufficient to cancel the anterior shift of the stripe (Fig. 7D',D'',E,G); however, inhibition of both types of signaling together positioned the stripe symmetrically (Fig. 7E',E'',G). Altogether, the present results strongly suggest that Shisa2 promotes maturation of the PSM cells by the individual inhibition of both Wnt and FGF signaling.
Positioning of the wavefront by Wnt and FGF signaling in the normal condition
The dorsal explants from wild-type embryos were treated with WIF1 or SU5402
for 1.5 or 3 hours. In the 3 hour treatment, but not in the 1.5 hour
treatment, WIF1 and SU5402 suppressed MAPK phosphorylation and Arp-A
expression, respectively (Fig.
8A-C''). These results show that the inhibition of each
signaling for a longer period induces mutual regulation of their signaling
activities.
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DISCUSSION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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).
Shisa1 uniquely inhibits Wnt and FGF signaling by suppressing the protein
maturation of their receptors in the ER. It remains uncertain, however,
whether this regulatory mechanism functions in other Wnt- and FGF-related
events during vertebrate embryogenesis. To address this issue, we isolated and
characterized Xenopus Shisarelated genes, Shisa2 and
3. Our data indicate that Shisa-related molecules constitute a
functionally conserved new gene family. Furthermore, we show that
Shisa2-mediated regulation of FGF and Wnt signaling plays an essential role in
segmental patterning during somitogenesis.
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; Yoon and Wold,
2000). It seems likely that the termination of Mes1
expression is a prerequisite to activate the expression of another bHLH
transcription factor, Thy1, an essential factor for specifying a
position of the segment boundary and the anteroposterior polarity of somites
(Morimoto et al., 2005;
Nomura-Kitabayashi et al.,
2002; Saga et al.,
1997; Sawada et al.,
2000; Sparrow et al.,
1998). It has recently been reported that Mesp2 (mouse
ortholog of Thy1) arrests the oscillation of Notch activity and
initiates the segmentation program in the rostral PSM
(Morimoto et al., 2005;
Takahashi et al., 2000). Thus,
the transition of the expression of these two transcription factors seems to
be coincident with the wavefront. We found that both Wnt and FGF signaling are
required in the initiation and/or maintenance of Mes1 expression, and
that the Shisa2-mediated inhibition of these two types of signaling is
required for the proper termination of Mes1 expression
(Fig. 7). FGFR1, Fz7 and Fz2
are expressed in the PSM (Deardorff and
Klein, 1999; Golub et al.,
2000; Sumanas et al.,
2000); Shisa2 would play an essential role in the regulation of
the morphogenetic gradient by controlling protein maturation of these
receptors. These present results provide an additional context, segmentation
in the PSM, in which Shisa-mediated signaling regulation controls
cell-autonomous competence to respond to Wnt and FGF signaling for the
establishment of vertebrate body patterning.
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). We show, however, that in the absence of Shisa2 function,
the regulation of FGF and Wnt ligands is not sufficient to generate the
morphogenetic gradient for proper segmental patterning. Shisa2-mediated
suppression of FGF and Wnt signaling in the PSM is required for setting up the
gradient. Shisa2 is expressed strongly in the region covering the
wavefront, but its expression is low in the posteriormost PSM
(Fig. 3). As somitogenesis
proceeds, Shisa2 expression moves posteriorly, coupled with axial
elongation. Shisa2 inhibits surface expression of Fz and FGFR by retaining
them in the ER. Thus, it is likely that Fz and FGFR are expressed at a high
level in the posteriormost PSM and at a lower level in the anterior PSM. In
this context, cells in the posterior PSM can respond to FGFs and Wnts better
than those in the anterior PSM. In cooperating with the gradients of the
ligand proteins, the gradients in the receptor expression may play a crucial
role in setting up the morphogenetic gradient. As the knockdown of Shisa2
delays the maturation of PSM cells through the anterior extension of FGF and
Wnt signaling activities, this mechanism would contribute to the formation of
the morphogenetic gradient in the PSM.
In Xenopus, RA signaling is reported to inhibit FGF signaling by
upregulating the MAPK phosphatase MKP3. A negative regulation of FGF signaling
by RA is also reported in chick embryos
(Diez del Corral et al., 2003;
Moreno and Kintner, 2004).
These reports suggest that RA signaling modifies the morphogenetic gradient at
least in part by suppressing FGF signaling. Although the RA treatment or
inhibition of FGFR caused a posterior shift of the wavefront
(Moreno and Kintner, 2004)
(Fig. 6), neither of them could
suppress the phenotypes of the Shisa2 knockdown. The data indicate that
inhibition of FGF by Shisa2 alone cannot explain the loss-of-function
phenotype. The inhibition of both FGF and Wnt signals, however, strongly
suppressed the phenotype and shifted the wavefront posteriorly
(Fig. 7). Furthermore, in the
normal condition we found that the inhibition of these two types of signaling
independently repositioned the third Thy1 stripes posteriorly
(Fig. 8). Altogether these
results strongly suggest that Shisa2-mediated individual inhibition of Wnt and
FGF signaling is required for the proper positioning of the wavefront.
The segmental clock in Shisa2-knockdown
The components of the Notch signaling cascade are involved in the segmental
clock (Bessho and Kageyama,
2003; Giudicelli and Lewis,
2004; Pourquie,
2003; Rida et al.,
2004). In Xenopus, the expression of ESR9 and
the closely related ESR10 are oscillated in the caudal PSM
(Li et al., 2003). We have
found that the loss of function of Shisa2 expanded the anterior border of
ESR9 expression but did not affect its oscillation
(Fig. 5). It has been suggested
that FGF and Wnt signaling not only controls the maturation of the PSM and the
position of the wavefront but also the segmental clock system. In
vt/vt mutants, the expression of the cyclic genes is affected
(Aulehla et al., 2003;
Dale et al., 2006;
Ishikawa et al., 2004).
LEF/TCF factors, a component of the Wnt signaling cascade, directly control
the expression of Delta-like1, thereby controlling the segmental
clock (Galceran et al., 2004;
Hofmann et al., 2004). In
zebrafish, Her13.2 (Hes6-related hairy/Enhancer split-related), which
functions downstream of FGF signaling, controls the expression of cyclic genes
such as Her1 and Her7
(Kawamura et al., 2005). By
contrast to these previous reports, Shisa2-mediated inhibition of FGF and Wnt
signals did not affect the phase of expression of the ESR9. It is
tempting to speculate that the high level of FGF and Wnt signals in the
tailbud is the source of generation of the cyclic expression of the genes.
Shisa2 controls the FGF and Wnt signals in the region more anterior to the
tailbud; thus it controls the wavefront but not the segmental clock.
In summary, Shisa2 plays an essential role in the maturation of PSM and establishment of proper segmental patterning by the individual inhibition of Wnt and FGF signaling.
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ACKNOWLEDGMENTS |
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REFERENCES |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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