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First published online 30 November 2005
doi: 10.1242/dev.02178
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1 Division of Developmental Neurobiology, Graduate School of Medical Sciences,
Kumamoto University, Kumamoto 860-8556, Japan.
2 The 21st Century COE program "Cell Fate Regulation Research and
Education Unit", Kumamoto University, Kumamoto 860-0811, Japan.
3 Department of Anatomy, University of Cambridge, Downing Street, Cambridge CB2
3DY, UK
4 PRESTO, JST, 4-1-8 Honcho, Kawaguchi, Saitama 332-0012, Japan.
5 Department of Oncology, The Hutchison/MRC Research Centre, University of
Cambridge, Hills Road, Cambridge CB2 2XZ, UK.
* Author for correspondence (e-mail: ohta9203{at}gpo.kumamoto-u.ac.jp)
Accepted 25 October 2005
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SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: Ectoderm, Neural crest, Neural plate, Epidermis, X-TSK, BMP, Notch, Xenopus
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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;
Le Douarin and Kalcheim,
1999). Several factors secreted from the neural plate, non-neural
ectoderm or mesoderm show neural crest inducing activity in ectodermal
explants, when applied alone or in combination. Yet, how the neural crest is
specified at the junction between the neural plate and the epidermis remains
unclear.
It has been proposed that bone morphogenetic protein (BMP) signaling plays
an important role in specification of cell fates at the neural
plate/ectodermal border in mouse, chick and frog embryos
(Kanzler et al., 2000;
Liem et al., 1995;
Marchant et al., 1998). During
neural development in Xenopus, BMPs are first expressed in the whole
ectoderm, but they are then gradually downregulated in the presumptive neural
plate, while BMP antagonists such as Noggin, Chordin and
Follistatin are secreted from the dorsal axial mesendoderm (reviewed
by Sasai and De Robertis,
1997). Several in vitro experiments suggest that a gradient of BMP
activity in the ectoderm is established by interactions between BMPs expressed
in the ectoderm and BMP inhibitors secreted from the axial mesendoderm,
resulting in the specification of the neural crest at a specific threshold of
BMP signaling levels (LaBonne and
Bronner-Fraser, 1998; Marchant
et al., 1998; Nguyen et al.,
1998). However, neural crest formation is specifically restricted
to the posterior border of the neural plate, excluding the anterior neural
plate (Hopwood et al., 1989;
Mayor et al., 1995). This
cannot be explained by the BMP gradient alone, and posteriorizing factors such
as Wnts, fibroblast growth factors (FGF) and retinoic acid (RA), are required
for this anteroposterior localization
(Villanueva et al., 2002).
Activation of Wnt signaling in the Xenopus embryo or in neuralized
animal cap induces neural crest specification, while Wnt inhibition by either
Wnt8 knockdown or overexpression of a dominant-negative Tcf3
abrogates neural crest specification in zebrafish
(LaBonne and Bronner-Fraser,
1998; Lewis et al.,
2004; Mayor et al.,
1995; Tan et al.,
2001). These results led to the two-signal model, suggesting that
the neural crest is specified in the posterior neural plate border by the
intersection of canonical Wnt signaling with moderate levels of BMP signaling
(LaBonne and Bronner-Fraser,
1998; Marchant et al.,
1998; Villanueva et al.,
2002). Recently, the Notch signaling pathway has also been shown
to play an important role in neural crest specification, by regulating the
expression of BMP4 and the BMP target gene Msx1 at the
lateral edge of the neural plate (Glavic
et al., 2004).
Here, we show that the Xenopus Tsukushi gene (X-TSK) is strongly expressed at the border of the neural plate at the time of neural crest specification, and that its expression in the ectoderm is regulated by BMP signaling. By biochemical analysis and overexpression assays in Xenopus, we show that: (1) X-TSK works as a BMP antagonist by direct binding to BMPs; (2) X-TSK modulates activation of Notch signaling by directly binding to X-delta-1 extracellular region. Furthermore, X-TSK can regulate BMP4 transcription indirectly via modulation of the Notch signaling pathway. Both gain- and loss-of-function assays demonstrate that X-TSK plays a crucial role in patterning of the ectoderm and especially in neural crest specification. We argue that X-TSK functions in the crosstalk of BMP and Notch signaling pathways at the boundary between the neural and non-neural ectoderm to determine the correct specification of the neural crest progenitors.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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For RT-PCR, total RNA was isolated by RNeasy Kit (QIAGEN) and TRIZOL
purification system (Invitrogen). The obtained total RNA was treated with
DNase (Invitrogen) for removing genome DNA contamination, and first-strand
cDNA was synthesized using oligo d(T)12-18 primer and Superscript
III reverse transcriptase (Invitrogen). PCR was performed with the Expand Long
template PCR system 3 (Roche), with the following primers: Xslug-U
5'-CAATGCAAGAACTGTTCC-3'; Xslug-D
5'-TCTAGGCAAGAATTGCTC-3'; XAG-1
(Sive and Bradley, 1996);
XBF-1U, 5'-TCAACAGCCTAATGCCTGAAGC-3'; XBF-1D,
5'-GCCGTCCACTTTCTTATCGTCG-3'; Xotx2
(Blitz and Cho, 1995);
Sox2 (De Robertis et al.,
1997); Msx1
(Tríbulo et al., 2003);
BMP4 (Dale et al.,
1992); XK81 (LaBonne
and Bronner-Fraser, 1998); ODC
(Agius et al., 2000);
Cardiac actin (Stutz et al., 1986); Sox9 and Zic5
(Monsoro-Burq et al., 2003);
XESR-1 (Wittenberger et al.,
1999); X-notch-1U, 5'-TCCTGATTTATATTGCTTATCCGAGT-3';
and X-notch-1D, 5'-TTACAGAAGTGTTAACAGCAACAACA-3'.
Embryological methods, in situ hybridization and immunostaining
Xenopus embryos were obtained as previously described
(Newport and Kirschner, 1982)
and staged according to Nieuwkoop and Faber
(Nieuwkoop and Faber, 1967).
For animal cap assays, mRNA was injected into the animal pole of two- or
four-cell stage embryos. Animal caps (ACs) were dissected from stage 8-9
embryos in 1x MBS and cultured in 0.5x MBS until early neurula
(stage 14), mid neurula (stage 17) or early tailbud (stage 21/22) stages,
depending on the experiment. For dissections of ventral marginal zone (VMZ) or
dorsal marginal zone (DMZ) explants, embryos were marginally injected into
ventral or dorsal blastomeres at the four-cell stage with X-TSK, BMP4
or BMP4 +X-TSK mRNAs. After culturing to the early gastrula
stage (stage 10-10+), VMZ or DMZ explants (comprising about 60°
of the VMZ or DMZ) were dissected in 1x MBS and cultured in 0.5x
MBS until stage 24-26. Dorsal lateral marginal zone (DLMZ)-AC conjugates were
prepared as described by Bonstein et al.
(Bonstein et al., 1998).
Whole-mount in situ hybridization was performed as previously described
(Harland, 1991;
Shain and Zuber, 1996).
Pigmented embryos and explants were bleached after the color reaction
(Mayor et al., 1995). The
following probes were used: Xdlx3
(Woda et al., 2003);
ADAM13 (Alfandari et al.,
1997); XAG-1 (Bradley
et al., 1996); XK81
(Jonas et al., 1985);
Sox2 (Mizuseki et al.,
1998); Xrx1 (Casarosa
et al., 1997); N-tubulin
(Richter et al., 1988);
cardiac actin (Mohun et al.,
1984); Xotx2 (Pannese et al.,
1995); Xotx5b (Vignali et al.,
2000); X-ESR-1
(Wettstein et al., 1997).
X-delta-1 was cloned independently
(Kiyota et al., 2001).
Xbmp4 cDNA was subcloned into pBSSK (-) from a BMP4
expression vector. Xslug, Sox9, Zic5, Msx-1, Hairy2A and
Trp-2 cDNAs were isolated by RT-PCR.
We raised polyclonal antibody against X-TSK in rabbits (QIAGEN). The specificity of this antibody was checked by SDS-PAGE and western blot. Myc-His tagged X-TSK and X-TSK protein in the lysate of embryo can be detected in blot (data not shown). For double or triple staining, the hybridized embryo was sectioned by cryostat, and detected by anti-X-TSK antibody with Cy3-conjugated secondary antibody (Jackson Immuno Research).
Microinjection of mRNA, short inhibitory double strand RNA (siRNA) or morpholino oligonucleotides
Capped mRNAs were synthesized from linearized plasmid templates with the
mMessage Machine kit (Ambion). Embryos were injected with 500-4000 pg
mRNA/embryo at the two- or four-cell stage in 0.1x MMR with 4% Ficoll
and kept in it for a few hours. They were then transferred into 0.1x MMR
for subsequent culturing. The following RNAs were made as previously described
(Kuriyama and Kinoshita,
2001): X-TSK (pCS2+-X-TSK), truncated BMP
receptor (tBR) (Graff et al.,
1994), Chordin (Sasai
et al., 1994), BMP4
(Fainsod et al., 1994), Xnr1
(Jones et al., 1995),
Notch Intracellular domain, X-delta-1 and dominant-negative
form of Delta (Chitnis et al.,
1995), Suppressor of hairless DNA binding mutant, and
Su(H)/Notch ankyrin repeats (Ank) fusion construct
(Wettstein et al., 1997).
We used the computer program provided on Dr Gregory Hannon's website
(http://www.cshl.edu/gradschool/hannon.html)
to predict the short-hairpin-siRNA sequence for gene silencing of
X-TSK. Myc-His tagged X-TSK was transfected into COS-7 cells with
either a control or X-TSK siRNA-expressing vector and downregulation of X-TSK
protein synthesis was checked by western blotting. After selecting the
effective sequences, we used a X-TSK siRNA designed for the following
sequence: 5'-GATACCTCGTATCTGGATCTC-3'. C-TSK siRNA, which degrades
C-TSK mRNA effectively, was used as control siRNA
(Ohta et al., 2004). C-TSK
mRNA was used for a rescue experiment of X-TSK siRNA. The applicable region of
the C-TSK sequence was 5'-GATACCTCCTACTTGGATTTG-3'. In
another loss-of-function experiment, we used a morpholino antisense oligo (MO)
against X-TSK, designed to target the following sequence:
5'-TCTAACAATGGCTCTTTCTTCTTGG-3' (Gene Tools). To distinguish the
effect of MO on lateral expression of X-TSK from MO injection to deplete the
organizer expression (Ohta et al.,
2004), we injected it into ventral border of animal dorsal
blastomere at the eight-cell stage corresponding to future D121 and D122 in
the cell fate map of 32-cell stage embryo
(Moody, 2000).
Western blotting and immunoprecipitation
Myc-His tagged X-TSK, Myc-His tagged C-TSK, a Flag tagged BMP4 plasmids or
a Flag tagged X-delta-1 extracellular domain plasmids were transfected into
COS-7 cells with LipofectAMINE 2000 (Invitrogen) and the supernatants were
harvested after 96 hours culture in serum-free Opti-MEM (Invitrogen). The
immunoprecipitation experiment was performed as previously described
(Ohta et al., 2004).
Myc-tagged and Flag-tagged proteins were detected after blotting using an
anti-Myc antibody 9E10 or anti-Flag antibody M2 (Sigma), respectively.
Xenopus mRNA electroporation
mRNA electroporation was performed as described by Sasagawa et al.
(Sasagawa et al., 2002). mRNA
(200 nl) of X-TSK (2 µg/µl) and GFP (1 µg/µl), or only GFP (3
µg/µl) were injected into the space under the vitelline membrane, and
electroporated at stage 11.5. The position of electroporation was at the
border between the lightly pigmented dorsal ectoderm and darkly pigmented
lateral ectoderm. Electroporated embryos were selected under the fluorescent
microscope for GFP expression at stage 14, fixed at the stage 15/16, then
analyzed by whole-mount in situ hybridization.
Staining of the branchial arch cartilages
The same procedure was used as previously described
(Berry et al., 1998). Samples
were fixed overnight in 4% paraformaldehyde/PBS, dehydrated in 95% ethanol and
stained with Alcian Blue solution [95% ethanol:acetic acid:0.3% Alcian Blue in
70% ethanol (8 ml:2 ml:1 ml)]. After the staining, samples were left in 95%
ethanol for 2 days and then stained with Alizarin Red solution [0.1% Alizarin
Red in 95% ethanol:1% potassium hydroxide (0.25 ml:10 ml)] for 2 days. After 6
hours incubation in 1% potassium hydroxide, samples were transferred into 100%
glycerol solution for image acquisition.
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). A
Xenopus tailbud cDNA library was screened using C-TSK as a
probe, obtaining a cDNA fragment encoding for a predicted protein of 338 amino
acid residues, which corresponds to the Xenopus TSK gene
(X-TSK). We have previously reported about other Tsukushi homologues
in zebrafish, mouse and human (Ohta et
al., 2004), all of which have similar characteristics of
structure. At the amino acid level, X-TSK shares 59% identity with C-TSK, and
48-49% identity with zebrafish, mouse and human TSK.
Maternal X-TSK expression is observed in the animal hemisphere
from unfertilized eggs to early gastrula embryos
(Ohta et al., 2004). However,
at the early neurula stage (stage 13), X-TSK expression is hardly
detectable in the presumptive neural plate region, and restricted to the
non-neural ectoderm (Fig. 1A),
where its levels increase by stage 14, especially in the presumptive anterior
neural fold (Fig. 1C). At these
stages, the spatiotemporal expression pattern of X-TSK closely
resembles that of Xbmp4 (Fig.
1D) (Fainsod et al.,
1994; Hemmati-Brivanlou and
Thomsen, 1995; Schmidt et al.,
1995) and BMP target genes, such as Xdlx3
(Fig. 1B)
(Woda et al., 2003).
In addition, X-TSK is expressed in the prospective cranial neural
crest, in a similar domain to that of Xslug
(Fig. 1E,F)
(Mayor et al., 1995). Sections
of hybridized embryos at these stages revealed that X-TSK is
expressed in a broad area overlapping with the neural fold, but it is hardly
observed inside the neural plate or in the underlying mesoderm
(Fig. 1I), while Xslug
expression is restricted to the neural crest region
(Fig. 1J). We immunostained
Xslug hybridized embryos with an anti-X-TSK antibody on sections
(Fig. 1K,L). X-TSK protein is
expressed in the superficial layer of the epidermis and the neural crest
region, and also in the proximal edge of the neural crest region in the same
layer of the Xslug expression domain
(Fig. 1L, arrowhead). At the
early tailbud stage (stage 23), X-TSK expression is observed in
cranial neural crest cells, the dorsal retina and the lens placode
(Fig. 1G). Comparison with
Xbmp4 showed that X-TSK has a more distinct expression
pattern at this stage (Fig.
1H). In the migrating cranial neural crest, X-TSK
expression is observed strongly in the mandibular crest segment, weakly in the
distal tip of the hyoid crest segment, and in the anterior and posterior
branchial crest segments (Fig.
1M). This expression is similar to that of Sox9
(Fig. 1N)
(Spokony et al., 2002) and
ADAM13 (Fig. 1O)
(Alfandari et al., 1997), while
Xslug is downregulated in neural crest cells after they leave the
neural tube (Fig. 1P).
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X-TSK antagonizes BMP4 activity and directly binds to BMP proteins in vitro
In a previous study, we showed that C-TSK has dorsalizing activity
when overexpressed in Xenopus embryos, which is due to its ability to
bind to BMPs directly and antagonize BMP signaling in the extracellular space
(Ohta et al., 2004). In
addition, C-TSK expression partially overlaps with BMP expression only at the
posterior marginal zone and the posterior primitive streak during chick
gastrulation. However, the expression of X-TSK largely overlaps with
BMP4 in the ectoderm of Xenopus neurula embryos, and is controlled by
BMP4. Thus, to confirm whether these dorsalizing and anti-BMP activities are
conserved in X-TSK, we performed overexpression experiments of
X-TSK mRNA in Xenopus embryos, and found that X-TSK
can dorsalize ventral mesoderm both in ventral marginal zone explants and in
animal caps injected with low doses of Xnr1 mRNA, as detected by
explant elongation and induction of cardiac actin expression (see Fig. S1 in
the supplementary material). In addition, in co-injection experiments,
X-TSK can antagonize the ventralizing effects of BMP4 overexpression
in dorsal marginal zone explants (see Fig. S1 in the supplementary material).
To examine whether X-TSK can bind to BMP4 directly, we carried out an
immunoprecipitation assay between BMP4 and X-TSK or C-TSK proteins
(Fig. 3A). When Myc-tagged
X-TSK or C-TSK was reacted with FLAG-tagged BMP4, immunoprecipitation of both
proteins with nickel chelating resins pulled down BMP4. These data indicate
the direct binding of X-TSK to BMP4. Thus, the basic molecular characteristics
of Tsukushi are also conserved in the Xenopus homologue.
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;
Wilson et al., 1997). To
further test the ability of X-TSK as a BMP antagonist, we performed animal cap
assays and analyzed the induction of the dorsoanterior ectodermal markers.
Using whole-mount in situ hybridization, we could detect strong induction of
cement gland markers, but weak induction of neural markers, in
X-TSK-injected caps (see Fig. S2 in the supplementary material). We
then analyzed X-TSK-injected caps by semi-quantitative RT-PCR, which
provides more sensitivity than in situ hybridization
(Fig. 3B). In these
experiments, X-TSK induced the cement gland marker XAG-1,
the telencephalic marker XBF-1, the forebrain and cement gland marker
Xotx2, and the pan-neural marker Sox2 in a dose-dependent
manner (Fig. 3B, lanes 3-5).
Interestingly, X-TSK overexpression had only a weak, if any, effect
on the expressions of BMP4 and its target gene Msx1
(Fig. 3B). Comparison with the
effects of another BMP antagonist, Noggin, showed that, even at high
doses of injected mRNA (4 ng), the effects of X-TSK mRNA on the
induction of cement gland/neural markers and on the repression of
BMP4 and Msx1 were clearly weaker than those of
Noggin mRNA. Co-overexpression of X-TSK and Noggin
also slightly reinforced the effects of Noggin
(Fig. 3B, lanes 1,2). These
effects were direct and not due to the induction of dorsal mesoderm, as no
activation of cardiac actin was detected in the injected caps. Altogether,
these data indicate that, while BMP antagonists such as Noggin can
strongly inhibit BMP activity and strongly neuralize ectodermal explants,
X-TSK overexpression causes a moderate repression of BMP signaling.
Thus, X-TSK may induce peripheral neural plate character in the
ectoderm via direct induction of intermediate levels of BMP signaling.
Loss of X-TSK function inhibits neural crest formation in vivo
Several studies in Xenopus have suggested that intermediate levels
of BMP signaling are necessary for neural crest specification at the neural
plate border (LaBonne and Bronner-Fraser,
1998; Marchant et al.,
1998). Thus, it was interesting to see, as described above, that
X-TSK is a BMP antagonist strongly expressed in the presumptive
cranial neural crest, which can promote intermediate levels of BMP signaling
in ectodermal explants. To determine the role of X-TSK, we performed
loss-of-function experiments using siRNA
(Zhou et al., 2002). The
inhibitory effect of X-TSK siRNA (X-TSK-si) sequence was confirmed by the
inhibition of X-TSK-Myc protein production after co-transfection in COS cells
(data not shown). X-TSK-si was also effective in reducing the levels of
endogenous X-TSK mRNA in Xenopus embryos, as shown by RT-PCR
(Fig. 4C). Although the
endogenous expression of X-TSK increased from stage 13 in uninjected
embryos, this increase was completely prevented in embryos injected with
X-TSK-si. Injection of X-TSK-si into one animal blastomere at the four-cell
stage (0.3-0.5 pmol/cell) caused abnormal neural fold formation in the neural
plate and increased pigmentation at the site where the neural fold is normally
formed (Fig. 4A,B).
We then analyzed the effects on X-TSK depletion on ectodermal patterning
using molecular markers after co-injection of X-TSK-si with ß-gal mRNA
(see Table 1). The expression
of the neural crest markers Sox9, Zic5 and Xslug was
inhibited in the ß-gal positive area
(Fig. 4E,G,L). No effects were
detected after injection of C-TSK siRNA (C-TSK-si)
(Fig. 4D,F). In addition, while
the expression of the neural plate markers Sox2 and Xrx1
were not affected by X-TSK-si injection
(Fig. 4H,J), the expression of
the epidermal marker XK81 was enhanced
(Fig. 4I) to the same extent of
the increased pigmentation as shown in Fig.
4B. In these embryos, no changes were detectable in the expression
of Xbmp4 (Fig. 4K).
These effects were rescued by co-injection of both 500 pg C-TSK mRNA
(Fig. 4L,M) or 250 pg
X-TSK mRNA (Fig.
4N,O), indicating that these phenotypes are specifically caused by
the depletion of the TSK protein (Table
2). Previously, we performed morpholino oligonucleotide (MO)
injections into the prospective dorsal midline of the embryo to determine the
effects of X-TSK depletion on the Spemann organizer and neural induction. In
those experiments, we showed that X-TSK MO impairs anterior neural plate
specification, as shown by a reduction in the expression domain of
Sox2 and Xrx1 (Ohta et
al., 2004). To confirm siRNA effects and to avoid indirect effects
due to abnormal organizer formation, we performed MO injections into the
prospective lateral ectoderm, as described in the Materials and methods
section. In these conditions, both Sox9 and Xslug were
diminished by X-TSK MO injection (Fig.
4P,Q). These effects were rescued by the injection of N-cad-X-TSK
mRNA, where the N-terminal region of X-TSK, including the initiation
codon and the signal peptides, were replaced by a myc-tagged N-cadherin signal
peptide (Fig. 4R,S; see also
Table 2). We also examined the
effects of X-TSK depletion on the expression of Hairy2A and
Msx1, which identify a pre-neural crest region before neural crest
specification (Glavic et al.,
2004; Tríbulo et al.,
2003). Both Hairy2A and Msx1 were downregulated
by X-TSK-si (Fig. 4U,W), but
not C-TSK-si (Fig. 4T,V). To
confirm the relationship between X-TSK protein reduction and the observed
phenotypes, we sectioned the embryos hybridized with neural crest markers and
immunostained them with an anti-TSK antibody
(Fig. 4X). In the embryos
showing downregulation of Sox9 and upregulation of XK81
after X-TSK-si injection, X-TSK protein expression in the neural crest was
reduced on the injected side, while X-TSK endogenous expression was detectable
in the proximal edge of the neural crest region on the control side
(Fig. 4X, arrowheads).
Morphological and molecular analysis at later stages of development confirmed
that, in agreement with the reduction of early neural crest markers at neurula
stages, inhibition of X-TSK function strongly repressed formation of neural
crest derivatives such as melanocytes and the branchial arch cartilages (see
Fig. S3 in the supplementary material). Altogether, these results suggest that
X-TSK is required at an early step of neural crest specification
upstream of Hairy2A and Msx1, and that in the absence of TSK
function in the ectoderm the presumptive neural crest region is at least
partially specified as epidermis.
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).
Sox9 induction was much reduced in X-TSK-si-injected caps conjugates
with DLMZ explants (Fig. 5B;
15.9%, n=44), but not in uninjected caps conjugated with
X-TSK-si-injected DLMZ (Fig.
5C; 75%, n=28). Expressions of the neural crest marker,
Zic5 and Xslug were also decreased in a dose-dependent
manner in X-TSK-si-injected caps after conjugation with DLMZ, as detected by
RT-PCR analysis (Fig. 5G). By
contrast, the expression of Sox9 was strongly increased in conjugates
of DLMZ with animal caps overexpressing X-TSK mRNA
(Fig. 5A,D; 100%,
n=15), while it was decreased in uninjected caps conjugated with
X-TSK-overexpressing DLMZ (Fig.
5A,E; 41.6%, n=24). These results indicate that
X-TSK expression in the ectoderm enhances the response to neural
crest inducing signals derived from DLMZ, while X-TSK overexpression
in the dorsolateral mesoderm has the opposite effect. As X-TSK overexpression
in the mesoderm has a dorsalizing effect (see Fig. S1 in the supplementary
material), this opposite effect may be explained by dorsalization of the
DLMZ.
In Xenopus embryos, neural crest specification requires the action
of posteriorizing signals such as Wnts, FGFs and retinoic acid acting on cells
with intermediate levels of BMP signaling at the border of the neural plate
(Villanueva et al., 2002).
These signals may be at least partially produced by the DLMZ
(Bang et al., 1999;
Monsoro-Burq et al., 2003;
Monsoro-Burq et al., 2005;
Wu et al., 2003).
XWnt-8 overexpression induces ectopic neural crest markers in vivo
(LaBonne and Bronner-Fraser,
1998), and it can induce neural crest specification in animal caps
in cooperation with BMP antagonists (Chordin or Noggin)
(Christian et al., 1991;
LaBonne and Bronner-Fraser,
1998). Therefore, we tested whether X-TSK could induce
neural crest specification in cooperation with XWnt-8. X-TSK-injected
caps induced the cement gland marker XAG-1
(Fig. 3B,
Fig. 5I), but did not show any
significant expression of the neural crest marker Xslug
(Fig. 5L), while
XWnt-8-injected caps showed weak expression of both XAG-1
and Xslug (Fig. 5J,M).
By contrast, when X-TSK and XWnt-8 were co-overexpressed,
Xslug was strongly induced, while XAG-1 expression was
reduced compared with X-TSK-injected caps
(Fig. 5H,K). RT-PCR analysis
confirmed that co-injection of X-TSK and XWnt-8
significantly enhanced expression of the neural crest markers Sox9
and Zic5, compared with single X-TSK or XWnt-8
injection (Fig. 5N). Therefore,
the BMP antagonistic activity of X-TSK is sufficient for neural crest
specification in cooperation with XWnt-8.
|
). Thus, BMP antagonistic activity is required for neural
crest formation after gastrulation. To study X-TSK function in vivo
at these later stages, we performed mRNA electroporation in stage 11.5
Xenopus embryos (Fig.
5O-R). In these conditions, overexpression of X-TSK in
the anterior neural fold extended the expression domain of Sox9
(Fig. 5P, arrowhead), and
induced ectopic expression in the epidermal region
(Fig. 5Q, arrowhead), while GFP
mRNA electroporation had no effect (Fig.
5O, arrowhead). Xslug expression was also enhanced in the
electroporated side (Fig. 5R).
The electroporation of X-TSK mRNA after early neurula stage 13 caused
a slight enhancement of the cranial-facial neural crest at stage 21 (data not
shown). These data strongly suggest that X-TSK upregulation in the
lateral ectoderm after gastrulation during normal development
(Fig. 1C,M) is involved in
cranial neural crest specification.
Overexpression experiments suggest that X-TSK can modulate BMP4 transcription and regulate activation of Notch signaling
In this study, gain-of-function and loss-of-function analyses clearly
suggest the importance of X-TSK-mediated BMP antagonism in ectodermal
patterning and neural crest specification. To shed light on X-TSK activity
during ectodermal development, we performed additional overexpression
experiments in Xenopus embryos. When X-TSK mRNA was injected
into one dorsoanimal blastomere of four-cell stage embryos (1 ng/cell;
co-injected with 200 pg of ß-gal mRNA as a lineage tracer), the
expression of the neural crest marker Xslug was suppressed on the
injected side, while ß-gal alone did not cause any specific phenotype
(Fig. 6A,B). Sox9
expression was also suppressed by X-TSK, and even more strongly by a
membrane-bound form of X-TSK (X-TSK-CD2), obtained after
fusion with the CD2 transmembrane domain
(Chang et al., 2001)
(Fig. 6C,D). The fact that the
effects of both the secreted and membrane-bound forms of X-TSK were very
similar, and that the secreted form of X-TSK affected only the area
immediately around the ß-gal-stained region, suggest that X-TSK acts as a
short-range factor and does not disperse far away. Unilateral injection of
X-TSK also caused the expansion of the neural plate marker
Sox2 (Fig. 6E), the
inhibition of the epidermal marker XK81
(Fig. 6F), and a downregulation
of Hairy2A and Msx-1 in the presumptive neural crest
territory (Fig. 6K)
(Table 3). ß-Gal mRNA did
not change the expression of Hairy2A and Msx-1 (data not
shown) (Table 3). These effects
were dose dependent. Similar to X-TSK overexpression, injection of
Chordin mRNA, another BMP antagonist, also caused an expansion of the
neural plate (Fig. 6G), and the
inhibition of neural crest formation (Fig.
6H). These data confirm that X-TSK acts as a BMP antagonist in
vivo, although less efficiently than Chordin. However, Chordin was
not effective in restoring neural crest gene expression in X-TSK-depleted
embryos (Fig. 6I,J), suggesting
that the function of X-TSK in neural crest formation may not be simply
explained by its anti-BMP activity.
|
). In
fact, similar to the results described by Glavic et al.
(Glavic et al., 2004),
overexpression of Notch-ICD mRNA into early embryos repressed
Xslug expression (Fig.
6P), while overexpression of X-delta-1Stu, a
dominant-negative ligand of Notch, caused upregulation of Xbmp4
expression (Fig. 6R). As these
effects are similar to those of X-TSK overexpression, we then
examined in more detail whether the effects of TSK overexpression are
mediated by modulation of the Notch signaling pathway. We first checked the
effects of TSK overexpression on primary neurogenesis, which is known
to be controlled by Notch signaling
(Chitnis et al., 1995).
Analogous with what has previously been shown for Notch overexpression
(Chitnis et al., 1995;
Chitnis and Kintner, 1996),
TSK overexpression repressed the expression of N-tubulin, a
marker of differentiated neurons, and that of X-delta-1
(Fig. 6S,T). Moreover,
X-TSK overexpression upregulated the Notch-target gene,
X-ESR-1 (Fig. 6V).
Finally, we found that the repression of neural crest specification by
TSK overexpression could be overcome by co-injection with
Su(H)DBM mRNA, which acts as an antagonist of Notch
signaling (Wettstein et al.,
1997) (Fig. 6W)
(Table 3), while
Su(H)DBM mRNA alone very slightly enhanced Sox9
expression (data not shown) (Table
3). By contrast, co-overexpression of X-TSK with
Su(H)/Ank, which works as an agonist of Notch signaling as described
by Wettstein et al. (Wettstein et al.,
1997), strongly inhibited Xslug and Sox9
(Fig. 6Z, data not shown)
(Table 3). Su(H)/Ank
alone also inhibited the expression of neural crest markers, though more
weakly than Notch ICD (Fig.
6Y) (Table 3).
However, Xbmp4 upregulation by X-TSK overexpression in the
lateral ectoderm could not be rescued by co-injection of
Su(H)DBM mRNA (Fig.
6X) (Table 3),
consistent with the fact that the same effect is obtained after overexpression
of X-delta-1Stu (Fig.
6R). By contrast, the upregulation of Xbmp4 expression by
X-TSK overexpression could be partially prevented by co-injection of
Su(H)/Ank mRNA, though some patches of ectopic Xbmp4
expression in the lateral area of ß-gal-positive cells were still
detectable (Fig. 6BB, arrows).
Su(H)/Ank mRNA alone inhibited Xbmp4 expression very weakly
(Fig. 6AA, arrowhead).
Altogether, these results suggest that TSK overexpression has
complex, bimodal effects on the regulation of Notch signaling. Specifically,
the repression of neural crest markers and primary neurogenesis appear to be
consistent with enhanced Notch signaling, while the activation of
Xbmp4 transcription seems to be related to diminished Notch
signaling.
|
To clarify whether TSK works as an activator or an inhibitor of Notch
signaling, we performed animal cap experiments by using the Notch-target gene
XESR-1 expression as a readout for the activation of the Notch
signaling pathway (Fig. 7B). As
the expression of X-delta-1 in early gastrula stages is localized to
the marginal zone, uninjected animal caps express Notch, but not
X-delta-1 (Wittenberger et al.,
1999). Thus, Notch activation does not normally occur in isolated
animal caps (Kiyota and Kinoshita,
2002). We then activated Notch signaling in animal caps by
injection of X-delta-1 mRNA, and assessed the effects of X-TSK gain-
or loss-of-function on this activation. Synthetic mRNAs and/or X-TSK MO were
injected into four-cell stage embryos, animal caps were excised from stage 8.5
embryos, and analyzed by RT-PCR at stage 9.5. As expected, both Notch
ICD mRNA and X-delta-1 mRNA caused induction of XESR-1
(Fig. 7B, lanes 3, 4). By
contrast, co-injection of X-TSK with X-delta-1 caused a much
lower induction of XESR-1 compared with X-delta-1 alone,
while X-TSK alone could not induce X-ESR1
(Fig. 7B, lanes 4-6).
Remarkably, co-injection of X-TSK-MO together with X-delta-1 also strongly
blocked X-ESR1 activation by X-delta-1
(Fig. 7B, lanes 7, 8). Taken
together, these data suggest that the endogenous levels of X-TSK present in
uninjected animal caps (Fig.
2D,J) are required for activation of Notch signaling by X-delta-1.
Conversely, higher levels of TSK, as those resulting from X-TSK
overexpression, can cause inhibition of X-delta-1 activity, suggesting that
X-TSK may differentially modulate X-delta-1 activity in a dose-dependent
manner in vivo.
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DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Signaling activities of X-TSK
X-TSK encodes for a secreted protein belonging to the SLRP family.
Although some members of this family, such as decorin, have
previously been shown to bind TGF-ß and modulate its activity
(Yamaguchi et al., 1990),
their role during embryonic development was not clear. We recently described
the chick TSK homolog and showed that it works as a BMP antagonist during
chick gastrulation (Ohta et al.,
2004), while another SLRP member, Biglycan, has also been shown to
modulate BMP activity during Xenopus early development
(Moreno et al., 2005). Similar
to its chick counterpart, X-TSK works as a BMP antagonist, as indicated by
overexpression experiments in Xenopus embryos and in vitro assays. In
fact, X-TSK overexpression can dorsalize ventral mesoderm (see Fig.
S1 in the supplementary material), and it can induce cement gland and neural
tissue, but not the dorsal mesodermal marker cardiac actin in animal caps
(Fig. 3, see Fig. S2 in the
supplementary material), thus mimicking the effects of other known BMP
inhibitors, such as Chordin, Noggin or a dominant-negative BMP
receptor (Sasai et al., 1995;
Sive and Bradley, 1996). In
whole embryos, X-TSK overexpression can enhance dorsoanterior fates
and repress ventrolateral fates, and these effects are similar to those
produced by Chordin overexpression
(Fig. 6G). The opposite effect,
namely a reduction of dorsal and an expansion of ventral structures, is
observed after X-TSK depletion (Fig.
4) (Ohta et al.,
2004). Finally, X-TSK can antagonize the ventralizing
activity of BMP4 in mesodermal explants, and it can bind BMP proteins
in vitro. Altogether, these data indicate that X-TSK is endowed with a clear
BMP antagonistic activity.
|
) and the
expression of the Notch-target gene X-ESR-1
(Wettstein et al., 1997).
Finally, suppression of neural crest formation by X-TSK
overexpression was efficiently rescued by a dominant-negative Su(H),
which inhibits Notch signaling, but not by an active form of Su(H)
(Fig. 6). Together, these data
clearly suggest that X-TSK may also work, at least in part, via activation of
Notch signaling. However, the effect of X-TSK on BMP4 expression in
the anterior peripheral region of the neural plate is similar to that of a
dominant-negative Notch ligand, X-delta-1Stu
(Glavic et al., 2004), and
this X-TSK effect can be prevented, at least in part, by an active form of
Su(H), but not by a dominant-negative Su(H)
(Fig. 6). This suggests that
X-TSK overexpression may also cause inhibition of Notch signaling at
the neural/epidermal border. To gain more insight into the interaction between
X-TSK and X-delta-1, we performed gain- and loss-of-function experiments of
X-TSK in isolated animal caps where Notch signaling was simultaneously
activated by X-delta-1 overexpression
(Fig. 7B). Surprisingly, we
found that while the endogenous levels of X-TSK in isolated animal caps are
necessary for X-delta-1 activity, higher X-TSK levels resulting from
X-TSK overexpression inhibits X-delta-1 function. Although further
work will be needed to dissect the biochemical mechanism by which X-TSK exerts
these dual effects on X-delta-1 activity, these results identify X-TSK as a
new modulator of the Notch signaling pathway, which works by directly binding
X-delta-1 in the extracellular space.
X-TSK is required for ectodermal patterning and neural crest specification
The work described in this paper uncovers a novel function for TSK family
members, i.e. the control of neural crest specification. During gastrulation
and neurulation, X-TSK expression is downregulated in the middle of
the neural plate, while it remains in the non-neural ectoderm and it
accumulates to strong levels at the neural plate border
(Fig. 1). This is the region
where the neural crest is specified, and therefore it was reasonable to see
that both X-TSK gain and loss of function affected neural crest
formation. In particular, neural crest specification was inhibited in
X-TSK-depleted embryos, which also showed an expansion of the
epidermal ectoderm and, depending on the approach, a reduction of the neural
plate (Fig. 4)
(Ohta et al., 2004). In these
experiments, the observation that the neural plate was reduced in embryos
injected with a X-TSK-targeted morpholino
(Ohta et al., 2004), but not
siRNA, may be explained with the fact that siRNA did not apparently affect
X-TSK levels during gastrulation, when X-TSK is expressed in
the dorsal ectoderm and mesoderm, while the morpholino may be already
effective at these stages (Ohta et al.,
2004). By contrast, X-TSK overexpression after
gastrulation caused an expansion of the neural crest
(Fig. 6). In addition, though
X-TSK on its own was not able to induce neural crest markers in
animal caps, it could strongly cooperate in this process with the dorsolateral
mesoderm or XWnt-8, both of which provide a posteriorizing signal
required for neural crest specification
(Fig. 5)
(Villanueva et al., 2002).
These experiments also showed that, consistent with it expression pattern at
the neural plate border (Fig.
1), X-TSK function is required in the ectoderm for neural crest
specification.
How does X-TSK control neural crest specification? Clearly, its BMP
antagonistic activity is likely to play a role. In fact, other BMP inhibitors,
such as Chordin or Noggin, can mimic X-TSK ability to induce
neural crest markers in cooperation with XWnt-8
(LaBonne and Bronner-Fraser,
1998; Mayor et al.,
1995). In addition, both gain- and loss-of-function analysis
suggest that X-TSK has a more general role in the mediolateral patterning of
the ectoderm, which is best explained with its anti-BMP function. A specific
gradient of BMP signaling is well-known to be required to specify the neural
plate, neural crest and epidermal domains in the Xenopus ectoderm
(Marchant et al., 1998).
Therefore, one possibility is that the BMP-inhibitory activity of X-TSK,
localized at the neural plate border and in the non-neural ectoderm, is
essential for the proper shaping of the BMP gradient required for ectodermal
patterning, and that the action of BMP antagonists secreted from the dorsal
midline, such as Chordin and Noggin, is not sufficient in this respect. This
would be in agreement with previous observations that removal of the dorsal
marginal zone does not prevent neural crest specification, though it strongly
affects neural plate induction (Marchant
et al., 1998).
The fact that another BMP antagonist, namely Chordin, could not
efficiently rescue the neural crest reduction in X-TSK-depleted embryos
(Fig. 6I,J) suggested the
interesting possibility that X-TSK may also control neural crest specification
via alternative mechanisms distinct from direct BMP antagonism through
protein-protein interaction. First, different from other BMP antagonists,
X-TSK can upregulate BMP4 expression in the non-neural ectoderm,
while BMP signaling can positively regulate X-TSK expression. In
addition, as described above, we found clear indications that X-TSK may also
work in neural crest specification via modulation of the Notch signaling
pathway. Remarkably, it has been already shown that Notch signaling plays a
role in neural crest specification and that it can regulate expression of
BMP4 and genes associated with BMP signaling in the presumptive
neural crest region (Glavic et al.,
2004). Moreover, during Xenopus development, it has been
described that Notch and its target gene Hairy2A are expressed in the
neural crest territory, while the Notch ligands, Delta and Serrate, are
expressed in the cells surrounding the prospective crest cells
(Glavic et al., 2004). An
attractive hypothesis is that, by directly interacting with both BMP4 and
X-delta-1 in the extracellular space at the neural/epidermal border, and by
indirectly regulating BMP4 transcription in this region, X-TSK may work as a
crucial molecular intersection between BMP and Notch signaling in the
territory where the neural crest is specified.
|
), the
expression pattern of Notch and Delta can only partially account for the
specific localization of the neural crest region. Furthermore, although
caudalizing signals such as Wnt, FGF and RA are thought to be responsible for
the localized induction of the neural crest at the lateral, but not the
anterior, neural border, some FGFs and RA-producing enzymes, such as FGF8 and
Raldh2, are expressed in the anterior neural border from early neurula stages
(Lupo et al., 2005).
Therefore, it is tempting to speculate that the presence of higher levels of
X-TSK in the anterior neural border and in the proximal edge of the neural
crest in the lateral neural border, compared with the neural crest itself,
might be an additional mechanism to restrict the boundaries of the neural
crest domain
A model of consecutive steps neural crest specification
Previously, two-signal models of neural crest specification were proposed
(LaBonne and Bronner-Fraser,
1998; Villanueva et al.,
2002), which suggested the requirement of intermediate levels of
BMP signaling and posteriorization factors. Based on the results presented in
this paper, we propose a model of consecutive steps in neural crest
specification that contemplates the dynamic expression pattern of
X-TSK and sequential molecular interaction during ectodermal
development.
First, during gastrulation, X-TSK is expressed in the whole animal
ectoderm and in the dorsal mesoderm (Ohta
et al., 2004). Dorsal midline signals, such as Chordin,
Noggin and X-TSK, bind to BMPs and directly inhibit their
activity in the dorsal ectoderm (Sasai et
al., 1994) (Fig.
8A, phase I), leading to the specification of the neural plate at
these stages (reviewed by Sasai and De
Robertis, 1997).
Subsequently, X-TSK expression is downregulated in the presumptive neural plate, while it is maintained and enhanced in the ectoderm flanking the neural plate (phase II). At these stages, in the presumptive neural crest regi
