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First published online 30 August 2006
doi: 10.1242/dev.02579
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1 Department of Developmental Neurobiology, Graduate School of Medical Sciences,
Kumamoto University, Kumamoto 860-8556, Japan.
2 PRESTO, JST, 4-1-8 Honcho Kawaguchi, Saitama, Japan.
3 21st Century COE, Kumamoto University, Kumamoto 860-8556, Japan.
4 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 4 August 2006
 |
SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: Chick, Tsukushi, BMP antagonist, VG1, Primitive streak, Hensen's node
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). The anterior tip of the primitive streak is known both as
Hensen's node and the organizer because of its ability to dorsalize the
mesoderm and to induce and pattern the nervous system
(Streit and Stern, 1999).
Embryological studies have shown that formation of the primitive streak and
the organizer is regulated by two key signaling centers that work at different
stages during gastrulation. First, during pre-streak stage, the posterior
marginal zone (PMZ), a small posterior domain within the marginal zone, has a
crucial role in inducing adjacent epiblast cells to form the primitive streak.
When this domain is transplanted to an ectopic site of the marginal zone of a
host embryo, it induces a secondary axis including the organizer
(Eyal-Giladi and Khaner, 1989;
Khaner and Eyal-Giladi, 1989).
Later in gastrulation, during primitive streak stages, a second signaling
center located in the middle primitive streak (MPS) is involved in controlling
induction of the organizer at the anterior tip of the primitive streak.
Transplantation experiments have shown that this tissue, known as the `node
inducing center', is sufficient to induce adjacent cells to become organizer
(Joubin and Stern, 1999).
Remarkably, the inducing activities of both the PMZ and the MPS have been
shown to be mediated by the same classes of signaling molecules, namely VG1, a
TGFß superfamily member, and WNT proteins
(Joubin and Stern, 1999).
Grafts of VG1- and WNT-expressing cells in pre-streak embryos can reproduce
the activity of the PMZ to induce primitive streak
(Joubin and Stern, 1999),
while VG1 and WNT1 misexpression during gastrulation can mimic the ability of
the MPS to induce an ectopic organizer
(Joubin and Stern, 1999).
However, the action of VG1 and WNT signals needs to be complemented at both
stages by mechanisms that locally lower the levels of BMP signaling, as
members of this different class of TGFß molecules have been shown to have
a very strong inhibitory activity towards primitive streak and Hensen's node
formation (Streit et al.,
1998).
We have previously described the isolation of chick Tsukushi (TSK), a
soluble molecule belonging to the small leucine-rich proteoglycan (SLRP)
family (Ohta et al., 2004). We
showed that TSK is expressed in the primitive streak and in Hensen's node
during chick gastrulation, where it works as a BMP antagonist. Previous gain-
and loss-of-function experiments have indicated that TSK function is both
sufficient and necessary to provide a BMP antagonistic activity required for
induction of Hensen's node by the MPS
(Ohta et al., 2004). Here, we
report on novel observations that provide evidence that TSK controls formation
of the chick embryonic axis by also regulating the activity of VG1. We show
that alternative splicing of the chick Tsukushi gene generates two isoforms
that differ in their C-terminal region. Remarkably, these two isoforms have
different expression patterns during chick gastrulation and different
biochemical activities. The TSKA isoform, which corresponds to the
originally described TSK (Ohta et al.,
2004), is a strong BMP antagonist, the expression of which becomes
progressively restricted to Hensen's node and to the anterior primitive streak
during gastrulation. By contrast, the newly identified TSKB isoform
is a significantly weaker BMP antagonist than TSKA, which becomes
enriched in the MPS. Both TSKA and TSKB directly bind to VG1 in vitro and show
clear functional interactions with VG1 in facilitating embryonic axis
induction both in Xenopus and chick embryos. Finally,
loss-of-function experiments demonstrate that TSKB, but not TSKA, function is
required in the MPS for induction of Hensen's node. Taken together, these
experimental evidences provide considerable advance on our understanding of
the molecular mechanism that controls embryonic axis formation in chick, by
identifying TSK isoforms as crucial modulators of different branches of
TGFß signaling during gastrulation.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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;
Hamburger and Hamilton, 1951).
Embryos were explanted in EC culture
(Chapman et al., 2001). The
expression vectors for TSKA (Ohta et al.,
2004), TSKB, VG1 (Shah et al.,
1997), Nodal (Bertocchini and
Stern, 2002), FGF8B (Sato and
Nakamura, 2004) or chordin
(Streit et al., 1998) were
transfected into COS-7 cells using LipofectAmine 2000 (Gibco-BRL). Forty-eight
hours later, cell pellets containing 1000-2000 cells were generated by hanging
drop cultures (Ohta et al.,
1999). Rat B1-fibroblast cells secreting WNT1 were used as
described (Shimizu et al.,
1997) (a gift from J. Kitajewski). Cell aggregates were grafted to
the indicated regions in the embryo using a micropipette. Embryos were
incubated for different periods, fixed and processed for in situ
hybridization. The experiments described here, like those of previous works in
this field, rely on misexpression of soluble factors delivered by grafts of
transfected cells.
Cloning and RT-PCR
The developing primitive streak was dissected from chick embryos at stage
2. Explants were taken from chick embryos at stage 3 and subdivided into the
anterior primitive streak (PS-A) and the posterior primitive streak (PS-P).
Explants from chick embryos at stage 4 were also taken and subdivided into the
Hensen's node (HN), the PS-A, the middle primitive streak (PS-M) and the PS-P.
Total RNA from each fraction was extracted with TRIZOL reagent (Invitrogen)
and first-strand cDNA was synthesized using oligo d(T)12-18 primer
and Superscript II reverse transcriptase (Invitrogen). PCR was performed with
the following primers: TSK-S
(5'-ATGAATTCACGATGCAGTTCCTAGCCTGGTTCAAC-3') and TSK-AS
(5'-ATCTCGAGCGTGGGGTTTTGGACGGTTGAGGT-3'). PCR amplification (30
cycles) yielded a TSKB cDNA band of 1.5 kb. Excision of a gel slice at
1.1 kb DNA size, followed by DNA elution and further PCR amplification
(20 cycles) using the same primer set, produced a TSKA cDNA band of 1.1 kb.
After subcloning and sequencing of the amplified cDNA fragments, two types of
Tsukushi cDNAs were identified. We designated the original TSK as TSKA
(Ohta et al., 2004) and the
other as TSKB (GenBank Accession Number AB195969).
For RT-PCR of chordin and GAPDH, the following primers were used: Chordin-S (5'-GGCACCACGGGTGAAGTGCACTGCG-3'); Chordin-AS (5'-GCGGCTCCATGCCTCTGCTGT-3'); GAPDH-S (5'-GGCTGCTAAGGCTGTGGGGA-3'); GAPDH-AS (5'-TATCAGCCTCTCCCACCTCC-3').
In situ hybridization
In situ hybridization was performed as described
(Ohta et al., 2004).
Digoxigenin (DIG)-labeled antisense and sense RNA probes of TSK, brachyury,
goosecoid and chordin were produced from their constructs subcloned into
pBluescript II (Stratagene).
Immunoprecipitation
An immunoprecipitation (IP) assay was carried out as previously described
(Ohta et al., 2004). Briefly,
COS-7 cells were transfected with the following constructs: Myc-His tagged
TSKA (Ohta et al., 2004),
Myc-His tagged TSKB, Myc-tagged VG1
(Shah et al., 1997),
Flag-tagged BMP7 (Ohta et al.,
2004) and Flag-tagged BMP4. The conditioned medium
containing the tagged proteins was incubated for 12 hours at 4°C. After
incubation with ProBond resins (Invitrogen), the beads were washed with IP
buffer without BSA or the same buffer with high salt (450 mM), 0.5%
Triton-X-100 and 0.5% CHAPS. The bound proteins were subjected to SDS-PAGE and
blotted onto a nylon membrane. Myc-tagged and Flag-tagged proteins were
immunodetected using the antibodies 9E10 (DSHB) and M2 (Sigma),
respectively.
Xenopus secondary axis formation
Xenopus laevis embryos were staged according to Nieuwkoop and
Faber (Nieuwkoop and Faber,
1967). For the secondary axis induction experiment, mRNA was
injected into the ventro-vegetal midline at eight-cell stage embryo. The
judgment of complete or incomplete secondary axis is as same as described by
Lemaire et al., (Lemaire et al.,
1995).
Electroporation of siRNA into chick embryos
The `TSKs' siRNA (5'-CTCTCTGGAAGTCCTTCCA-3') was used to
silence the function of both TSKA and TSKB. TSKA siRNA
(5'-GTCTGCAGGTGCAGAGATA-3') which silences the TSKA specifically
(Ohta et al., 2004) and
control1 siRNA; 5'-CAAGATCCAGAAGGTCGGT-3'. COS-7 cells were
transfected with Myc-tagged TSKA or TSKB and with 2 µg of control1,
TSKA or TSKs siRNA, and the amount of TSKA and TSKB proteins were
analyzed as described above. For electroporation, 0.2 µl siRNA solution
(0.5 µg/µl), including 0.05% Fast Green, was microinjected into the
space between the vitelline membrane and the epiblast. The MPS was transfected
by electroporation with two successive square pulses of 7 V and 50 ms using
platinum square electrodes (2 mm x 2 mm) 3.5 mm apart. The middle of the
primitive streak, roughly 200 µm x 600 µm, was excised from
transfected donor embryos and implanted into the lateral position to Hensen's
node of a host embryo. The embryos were incubated for 6 hours at 38.5°C
and in situ hybridization was performed with TSK, chordin or goosecoid probe.
To confirm the ability of TSKs siRNA in the chick, after electroporation, the
node was excised and cultured for 6 hours at 38.5°C, and then the
transfected MPSs were excited for RT-PCR using the following primers: TSK-S
and TSK-AS1 (5'-ATACATGTTCACAAAACGTAAGGCGC-3'); GAPDH-S
and GAPDH-AS, VG1-S
(5'-TAGAATTCAAGTGCTTACAACGTCCCCGTC-3'); VG1-AS
(5'-ATCTCGAGTCTGCAGCCACACTCATCCAC-3').
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). Sequence
alignment revealed extensive conservation of TSK sequences across different
organisms, with the exception of TSK C-terminal region that appears to be more
divergent from that of other TSK homologs (data not shown). We speculated that
the TSK gene produces another isoform with higher conservation of the
C-terminal region by alternative splicing. To test this possibility, we
performed RT-PCR on RNA extracted from stage 4 chick embryos and found two
types of TSK cDNAs (Fig. 1A).
One of these cDNA corresponds to the previously isolated TSK cDNA
(Ohta et al., 2004) and we
re-designated it as TSKA. However, the second cDNA encodes for a new
isoform of TSK, that we named TSKB, the C-terminal sequence of which
is conserved with respect to those of other TSK orthologs. A chicken genome
database search (Ensembl Chicken Genome Browser) indicated that the Tsukushi
gene is located on chromosome 1. Fig.
1B shows the alignment of TSKB cDNA and amino acid
sequences at the C-terminal region. TSKB cDNA sequence is identical
to that of the Tsukushi genomic DNA. In the TSKA isoform, nucleotides
990-1309 (red) are deleted by alternative 5' splicing.
Fig. 1C schematize the
alternative 5' splicing of TSK pre-RNA, resulting in the conversion of
TSKB to TSKA.
|
), was
predominantly expressed in the anterior fragment
(Fig. 2B). At stage 4, when the
primitive streak has reached its full extension, we divided the embryo axial
structures into four parts, namely Hensen's node (HN), and the anterior,
middle and posterior parts of the primitive streak (PS-A, PS-M and PS-P).
TSKA expression was observed exclusively in the anterior part (HN and
PS-A) of the embryonic axis, while TSKB was expressed throughout the
axial structures with a peak of expression at the MPS. At this stage, chordin
was specifically detected in Hensen's node
(Lawson et al., 2001;
Skromne and Stern, 2002)
(Fig. 2C). At all stages
examined, TSKB was expressed at higher levels than TSKA,
because with the same set of primers the TSKB isoform was detectable
after a lower number of PCR cycles than was the TSKA isoform.
TSKB is a significantly weaker BMP antagonist than TSKA
We have previously shown that TSKA binds to BMP4 directly and inhibits its
activity in vitro and in vivo (Ohta et
al., 2004). To investigate whether TSKB can also work as a BMP
antagonist, we initially performed overexpression assays in Xenopus
embryos. Injection of 600 pg of TSKB mRNA into a ventral vegetal
blastomere of eight-cell stage embryos
(Fig. 3B) did not induce
ectopic axial structures, although 400 pg of TSKA mRNA were very
efficient at inducing a secondary axis
(Fig. 3A)
(Ohta et al., 2004). We next
performed co-immunoprecipitation assays. When Myc-His-tagged TSKA or TSKB was
reacted with Flag-tagged BMP4, immunoprecipitation with nickel-chelating
resins pulled down BMP4 (Fig.
3C,D). BMP4 was not detected by immunoblotting when the TSKB and
BMP4 complex was washed under high stringency conditions, although binding of
BMP4 to TSKA was still detectable (Fig.
3D). Although both TSKA and TSKB can pull down BMP4 in a
dose-dependent manner, the amount of BMP4 precipitated by TSKA was higher than
that pulled down by TSKB (Fig.
3E). The band intensity of lane 2 is three times stronger than
that of lane 6, as measured by NIH Image software (data not shown). These data
suggest that TSKA can bind BMP4 more strongly than TSKB. As shown in
Fig. 3F, similar to TSKA, TSKB
can also bind to BMP7 (Ohta et al.,
2004).
We then examined whether TSKB exerts a similar BMP antagonistic activity to
TSKA (Ohta et al., 2004). It
has been shown that the MPS acts as a Hensen's node-inducing center in chick
embryos, but its activity is inhibited by BMP signals secreted from the
periphery of the embryo (Joubin and Stern,
1999). Therefore, aggregates of COS-7 cells expressing TSKA or
TSKB were grafted with the MPS into the lateral edge of a host embryo at stage
3+ and cultured for 6 hours. On the other side of the embryo,
control COS-7 cells and the MPS were implanted
(Fig. 3G). In the presence of
TSKA, the MPS induced the ectopic expression of chordin in 12/20 (60%) embryos
(Fig. 3H), whereas only 3/15
(20%) embryos were positive on the TSKB implanted side
(Fig. 3I), which was similar to
the effect of grafting the MPS together with control cells [6/35 (17%)].
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|
). As
WNT1 and WNT8C have been shown to belong to the same functional subclass of
WNT proteins by several different assays
(Joubin and Stern, 1999), we
used a stable fibroblast cell line secreting WNT1
(Shimizu et al., 1997). When
VG1+WNT1 cells were grafted together at the periphery of the area pellucida at
stage 3+, they showed a weak node-inducing activity [1/8 (13%)]
(data not shown). We then grafted VG1+WNT1 cells along with three aggregates
of TSKA or TSKB-secreting cells (Fig.
3J). In the presence of TSKA, VG1+WNT1 induced ectopic expression
of chordin in 6/15 (40%) of embryos (Fig.
3K), while no induction was obtained with TSKB-expressing cells
[0/16 (0%)] (Fig. 3L), which
was similar what obtained by grafting VG1+WNT1 cells together with control
cells [1/31 (3%)]. In conclusion, our experimental results indicate that,
although TSKB retains some weaker BMP4-binding ability in vitro, its
biological activity is much weaker than that of TSKA.
TSK binds VG1 directly in vitro and functionally interacts with VG1 during axis formation
The PMZ of the chick embryo works as an inducing center responsible for
initiating primitive streak formation in the adjacent epiblast
(Bachvarova et al., 1998). The
molecular synergism between VG1 and WNT activities, whose expression overlaps
in the PMZ, is required and sufficient for initiating primitive streak
formation (Skromne and Stern,
2001). At later stages, during gastrulation, VG1 and WNT signals
are also co-expressed and define a second inducing center in the MPS, where
they play a role in the induction of Hensen's node
(Joubin and Stern, 1999). The
fact that TSKB expression specifically peaks at the MPS
(Fig. 2C), suggested to us that
TSK also interacts with VG1 in addition to BMPs. To further investigate on
this possibility, we compared the expression of TSK, VG1 and WNT8C by at
pre-streak stage and during gastrulation. At stage XII, TSK is detected in the
area opaca, the PMZ and Koller's sickle
(Fig. 4A). WNT8C is expressed
all around the area opaca and the marginal zone, most strongly in PMZ, while
VG1 is specifically expressed in the PMZ
(Fig. 4B,C). At primitive
streak stages, both VG1 and WNT8C are expressed in the MPS
(Fig. 4E,F)
(Joubin and Stern, 1999), as
well as TSK (Fig. 4D). Thus,
TSK and VG1 expression domains partially overlap both in the PMZ and in the
MPS.
|
). The
binding between both TSKs and VG1 was inhibited by BMP4
(Fig. 4H)
(Ohta et al., 2004). As TSK
interacts with VG1 in vitro, we subsequently examined whether these molecules
can also functionally interact in vivo. To this aim, we initially performed
co-overexpression experiments in Xenopus embryos. When 100 pg of
VG1 mRNA were injected into a ventral vegetal blastomere of the
eight-cell stage embryo, a weak secondary axis was induced lacking any
anterior structures [10/21 (47%); Fig.
4I] (Seleiro et al.,
1996), while 400 pg of VG1 mRNA resulted in gastrulation
defects (data not shown). However, when 100 pg of VG1 mRNA were
co-injected with 400 pg of TSKB mRNA, a well-defined secondary axis
was induced in 54% of the cases (26/48). Furthermore, in 17% (8/48) of the
embryos a nearly complete secondary axis was induced that also contained
anterior structures (Fig. 4J).
The TSKA isoform was not suitable for this assay because it can induce a
secondary axis by itself (Ohta et al.,
2004). These observations suggest that TSK interacts with VG1
during chick early development.
To gain more insight into this interaction, we transplanted TSKA- or
TSKB-expressing cells in combination with VG1-producing cells in the marginal
zone or in the area pellucida at stage XII and examined the expression of
brachyury, a specific marker of the primitive streak mesoderm
(Lawson et al., 2001).
Fig. 4K shows the schematic
diagram of the transplantation experiments. As previously described
(Skromne and Stern, 2001),
misexpression of VG1 in the area pellucida induced an ectopic primitive streak
in only 6/28 (21%) of the grafted embryos, whereas the combination of VG1+WNT1
caused a much stronger effect [9/14 (64%)]
(Table 1). When TSKA or TSKB
was misexpressed alone in the area pellucida, neither of them could induce
ectopic expression of brachyury [TSKA 0/10 (0%), TSKB 0/8 (0%)]
(Table 1). By contrast, when
TSKA or TSKB were misexpressed in the area pellucida together with VG1, an
ectopic primitive streak developed in a significantly higher number of cases
compared with VG1 misexpression alone [TSKA+VG1 18/30 (60%), TSKB+VG1 23/46
(50%) (Fig. 4L,
Table 1)]. Notably, TSK could
also strengthen the effect of VG1+WNT misexpression, because, in the presence
of TSKA or TSKB, induction of ectopic expression of brachyury raised up to
26/30 (87%) and 25/29 (86%) of embryos, respectively
(Table 1). In conclusion, our
data suggest that TSK interacts with VG1 during axis formation in the chick
embryo.
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|
We first verified the effectiveness of these siRNAs by immunoblotting and
RT-PCR. COS-7 cells were transfected with control1, TSKA or TSKs
siRNA, plus plasmid DNA for Myc-His-tagged TSKA or TSKB. Immunoblotting of
cell culture supernatants showed that TSKA and TSKB protein products were
nearly fully depleted in the presence of TSKs siRNA, whereas TSKA
siRNA specifically interfered with TSKA, but not TSKB protein expression. TSKA
and TSKB expression was not affected in the presence of control1 siRNA (see
Fig. S1 in the supplementary material). We then checked whether TSKs siRNA is
able to downregulate the endogenous expression of TSK. As we have not yet
succeeded in electroporating chick embryos at pre-streak stage, we have not
been able to use siRNA to interfere with TSK expression in the early-forming
primitive streak. As shown in Fig. S2C (see supplementary material), the
expression of TSK was even more strongly upregulated in the MPS, which acts to
promote node regeneration after surgical removal of the node
(Joubin and Stern, 1999). We
then analyzed induction of TSK expression after ablation of the node and
anterior primitive streak in embryos electroporated with TSKs, TSKA
or control1 siRNAs. Although TSK expression was upregulated after surgery in
control embryos or after electroporation of control1 or TSKA siRNA
(see Fig. S2C-E in the supplementary material), we hardly detected any
expression of TSK mRNA in the MPS of embryos electroporated with TSK siRNA,
although some ectopic induction of TSK expression was detectable in both
lateral sides of the operated area (see Fig. S2F in the supplementary
material). We also examined the effects of TSK depletion on the expression of
chordin and goosecoid, which have been used as node markers
(Joubin and Stern, 1999),
without noticing any evident effects on their expression (see Fig. S2G-N in
the supplementary material).
We showed that TSKB RNA accumulates in the MPS during gastrulation
(Fig. 2C) and TSK expression is
strongly upregulated in the MPS during node regeneration (see Fig. S2C in the
supplementary material). As the MPS works as a node-inducing center that
specifically expresses VG1 (Joubin and
Stern, 1999), we speculated that TSK may play an important role in
the MPS by regulating induction of Hensen's node in cooperation with VG1. To
test this hypothesis, we carried out an implantation experiments using MPS
from either control embryos or from embryos electroporated with TSKs,
TSKA or control siRNA, and compared their ability to induce an
ectopic Hensen's node. Grafts of MPS were implanted into the lateral side of
the node into stage 3+ host embryos as schematized in
Fig. 5A and the expression of
organizer markers was analyzed 6 hours later. As previously described
(Joubin and Stern, 1999),
untransfected primitive streak induced ectopic expression of chordin in 20/50
(40%) embryos (Fig. 5C), which
is similar to what was obtained after grafting MPS electroporated with either
control1 or TSKA siRNA [17/44 (39%) and 12/32 (38%)] embryos with
ectopic chordin expression, respectively
(Fig. 5D,E). By contrast,
transplantation of TSKs siRNA-electroporated MPS resulted in only 8/40 (20%)
embryos showing ectopic chordin expression
(Fig. 5F). We also used
goosecoid as a more specific marker of Hensen's node because chordin is
expressed in the notochord and in early organizer precursors as well as in the
mature organizer itself (Chapman et al.,
2002), and we obtained similar results. In particular, grafts of
untransfected, control1 siRNA- and TSKA siRNA-electroporated MPS
induced ectopic expression of goosecoid in 12/40 (30%), 12/42 (29%) and 12/44
(27%) embryos, respectively (Fig.
5G-I). By contrast, grafts of TSKs siRNA-electroporated MPS
induced ectopic goosecoid expression in only 7/53 (13%) embryos
(Fig. 5J). Taken together,
these results indicate a requirement of TSK activity for Hensen's node
induction by the MPS.
|
).
This inhibitor acts downstream of VG1 but upstream of Nodal and Chordin,
because VG1+chordin or VG1+Nodal, but not VG1 alone, could efficiently bypass
the inhibition (Bertocchini et al.,
2004). Does TSK also work downstream of VG1 and this unknown
inhibitor? To test this possibility, we first examined whether TSK expression
is induced by the misexpression of VG1 in the anterior part of pre-streak
embryos. In this assay, VG1 has been shown to induce a cascade of gene
activation, including Nodal expression after 6 hours, followed by chordin
after 12 hours (Skromne and Stern,
2002). We found that TSK is induced by VG1 after 12 hours (see
Fig. S3 in the supplementary material), suggesting that TSK also works
downstream of VG1. We then performed further experiments to determine whether
TSK works downstream of the VG1-induced inhibitor. We first grafted a pellet
of VG1-expressing cells on the lateral margin of stage XII embryos, followed 6
hours later by implantation of another pellet of cells expressing TSKA, TSKB
or other molecules (VG1, chordin, Nodal and FGF8B), which are known to be
involved in primitive streak formation
(Fig. 6A)
(Bertocchini et al., 2004).
Weak, if any, ectopic induction of primitive streak took place with any of
these molecules (Fig. 6B-D; see
Table 2). We then grafted a
cell aggregate expressing VG1 on one side, followed 6 hours later by cells
expressing VG1 together with one of the above-described molecules on the
opposite side (Fig. 6E-H; see
Table 2). If TSK works upstream
of the VG1-induced inhibitor, co-expression of VG1 and TSK should not be
sufficient to induce an ectopic axis. If TSK works downstream of the
inhibitor, misexpression of VG1 and TSK could be enough to bypass the
inhibitory step. Grafting a second VG1 pellet together with either TSKA, TSKB,
Nodal or chordin-expressing cells induced ectopic primitive streak formation
[21/42 (50%) embryos with VG1+TSKA; 37/76 (49%) embryos with VG1+TSKB; 17/43
(40%) embryos with VG1+Nodal; 22/44 (50%) embryos with VG1+chordin]
(Fig. 6E-H; see
Table 2), suggesting that the
inhibitor works downstream of VG1 but upstream of TSKs, Nodal and chordin.
However, Nodal or chordin alone is not able to induce an ectopic primitive
streak in this assay (Bertocchini et al.,
2004). Similarly, weak, if any, ectopic induction was obtained by
misexpressing chordin or Nodal together with TSKA or TSKB; 7/48 (15%) embryos
with chordin+TSKA; 6/44 (14%) embryos with chordin+TSKB; 0/44 (0%) embryos
with TSKA+Nodal; 2/53 (4%) embryos with TSKB+Nodal
(Table 2). These weak effects
are similar to those obtained after transplantation of chordin or Nodal
pellets alone [chordin 6/54 (11%); Nodal 1/44 (2%)]
(Table 2). Chordin, a strong
BMP antagonist (Sasai et al.,
1994), has also been shown to act downstream of the VG1-induced
inhibitor, since chordin+VG1 could bypass the inhibitory step and induce
ectopic primitive streak formation
(Bertocchini et al., 2004).
However, we found that chordin+VG1 induced an ectopic primitive streak more
efficiently when misexpressed together with TSKA or TSKB [39/51 (76%) and
25/32 (78%) embryos, respectively, showing an ectopic primitive streak,
compared with 22/44 (50%) embryos] (Fig.
6I,J; see Table 2),
suggesting that TSK is not simply working as a BMP antagonist in this assay.
Taken together, these results indicate that TSK acts downstream of VG1 and a
VG1-induced inhibitor during primitive streak formation.
|
|
DISCUSSION |
|---|
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
). Genomic sequence analysis of the TSK gene indicates that
these two isoforms are predicted to be generated by alternative 5'
splicing of TSK pre-mRNA, a process that is well known to modulate RNA
splicing in a developmental and/or cell-type-specific fashion
(Cartegni et al., 2002). In the
process of alternative 5' splicing, it is believed that cis-acting
elements are present in the coding sequences of spliced genes, allowing the
splicing machinery to distinguish between genuine and pseudoexons and to
modulate the selection of alternative splice sites. The mechanisms that are
responsible for the recognition and function of these exonic signals are now
starting to be elucidated (Cartegni et al.,
2002). However, the possible cis-acting elements that may control
alternative splicing of the TSK gene are presently unknown. We have identified
TSK orthologs in zebrafish, Xenopus, mouse and human, all of which
have a B-type structure in their C-terminal region (data not shown). Analysis
of nucleotide databases did not allow the identification of Tsukushi type A
isoforms in these organisms. However, recent progresses in the study of
pre-mRNA splicing may open the way to discover type A-related isoforms in
other animal species.
TSKA and TSKB isoforms are differentially regulated and possess distinct biochemical activities
Expression analysis of TSKA and TSKB revealed that they
are differentially regulated in chick early development. During initial
formation and extension of the primitive streak, both isoforms were
co-expressed throughout the developing primitive streak. However, when the
primitive streak has reached its full extension, TSKA expression is
specifically restricted to Hensen's node and the anterior part of primitive
streak. By contrast, TSKB is expressed throughout the primitive
streak, but it is enriched in the MPS. Hensen's node releases BMP-antagonizing
signals to pattern the mesoderm and ectoderm tissues
(Joubin and Stern, 1999). The
MPS instead functions as a node-inducing center because of the specific
localization of VG1 and WNT signals
(Joubin and Stern, 1999).
Therefore, the differential regulation of TSKA and TSKB expression in Hensen's
node and the primitive streak suggests that these TSK isoforms participate in
different signaling events during gastrulation.
Indeed, our experimental analysis highlighted a clear difference in the
anti-BMP activities of TSKA and TSKB. We have previously shown that TSKA can
bind to BMP directly and has a strong anti-BMP activity in vivo
(Ohta et al., 2004). This was
demonstrated by its ability to induce a secondary axis in Xenopus
embryos through mesoderm dorsalization and induction of neural tissue,
activities that are well known to depend on BMP antagonism in amphibians. In
chick embryos, TSKA was sufficient and necessary to provide an anti-BMP signal
required for Hensen's node induction by the MPS
(Ohta et al., 2004;
Joubin and Stern, 1999). In
contrast to TSKA, TSKB binds to BMP much more weakly in vitro and shows a much
weaker BMP inhibition in vivo (Fig.
3). Taken together, the differential expression patterns and
biochemical properties of TSKA and TSKB suggest that, although TSKA is
involved in protecting the developing chick axial structures from the
inhibitory influence of BMP signals (Ohta
et al., 2004), TSKB is involved in different signaling activities
from BMP antagonism.
TSK isoforms interact with VG1 in the induction of the primitive streak and Hensen's node
The enrichment of TSKB expression at the MPS, where VG1 expression is also
localized, suggested to us that TSKB could interact with VG1 in addition to
BMPs. This hypothesis was confirmed by the observation that both TSKA and TSKB
can bind VG1 in vitro. Binding of TSKs to VG1 was competed by BMP4, suggesting
that TSKs binds to VG1 and BMPs through at least partially overlapping binding
sites. Biological assays performed in Xenopus and chick embryos
revealed that the interaction between TSKs and VG1 is of a synergistic rather
than antagonistic kind. These results suggested that TSKs functions as an
activator of VG1 and modulates the node-inducing activity of the MPS by
regulating VG1 function in chick development. We tackled this question
directly by loss-of-function experiments using TSK-targeted siRNA
(Fig. 5). The fact that
TSKA-targeted siRNA was not effective in this assay, and that TSKA RNA
expression was hardly detectable in the MPS, suggest that TSKB, but not TSKA,
works in the node-inducing center. However, as we were not able to produce an
effective TSKB-specific siRNA, this hypothesis could not be formally
demonstrated.
|
;
Skromne and Stern, 2001).
Thus, it seems likely that TSK also interacts with VG1 at the level of the PMZ
to control the primitive streak formation. This idea is strongly supported by
overexpression experiments (Fig.
4). In Xenopus embryos, co-overexpression of TSKB
together with VG1 caused a much stronger induction of a secondary axis than
VG1 alone. In chick embryos, VG1 alone is a poor inducer of primitive streak
in the area pellucida, but VG1 and TSKs can induce an ectopic primitive streak
efficiently. Thus, the current results support the idea that TSKs regulates
VG1 activity and primitive streak induction in the PMZ, although further work
will be needed to prove the requirement of TSKs in this process.
The inducing activities of the PMZ and the MPS are both mediated by VG1 and
WNT signals (Joubin and Stern,
1999), and the lowered levels of BMP signaling are required in
these induction events, because increased BMP levels inhibit both primitive
streak and Hensen's node formation (Streit
et al., 1998). Could our results be explained with TSK simply
working as a BMP antagonist? This seems to be unlikely for the following
reasons. First, TSKA and TSKB induce the primitive streak together with VG1
with similar efficiencies. This is in striking contrast to the fact that TSKB
is a much weaker BMP antagonist than TSKA. Second, loss-of-function
experiments indicate that, in the induction of an ectopic organizer by the
MPS, TSKA and TSKB functions are specifically required in Hensen's node and
the MPS, respectively [(Ohta et al.,
2004), this work, Fig.
5)]. The most likely explanation is that induction of an ectopic
organizer by the MPS requires two distinct signaling activities: the anti-BMP
activity of TSKA secreted from the endogenous node, and a signaling activity
of TSKB secreted from the MPS, which is different from BMP antagonism.
Finally, when TSKB- or chordin-secreting cells were grafted together with
VG1-producing cells on the opposite side of an embryo that was previously
grafted with a pellet of VG1-expressing cells into the lateral margin at
pre-streak stage, both TSKB and chordin could bypass the inhibitory effects
induced by the first VG1 pellet. In this experiment, TSKB could still
significantly enhance the primitive streak formation, even when misexpressed
in the presence of both VG1 and chordin. In conclusion, our observations
indicate that the anti-BMP activity of TSK is not sufficient to explain all of
its biological effects, and that an additional signaling activity of TSK, most
probably modulation of VG1 function, is involved.
In Fig. 7, we summarize the
possible functions of TSK isoforms during chick gastrulation. At pre-streak
stage (Fig. 7A), TSK expression
is detectable in the area opaca, the PMZ and Koller's sickle. The primitive
streak is induced by VG1 and WNT signals secreted from the PMZ, and it
requires local lowering of BMP signaling in the posterior epiblast, possibly
owing to the action of chordin released from Koller's sickle
(Streit et al., 1998). TSK
isoforms play a dual role in the process of primitive streak formation. On the
one hand, TSKA collaborates with Chordin to decrease BMP activity in the
posterior epiblast, in accordance with our previous findings that TSKA and
chordin cooperate in blocking BMP signaling
(Ohta et al., 2004).
Furthermore, the BMP activity positively regulates TSK expression
(Kuriyama et al., 2006). On
the other hand, both TSKs could positively regulate VG1 activity in the PMZ,
thus facilitating induction of the streak. At primitive streak stages
(Fig. 7B), both TSKs are
initially expressed throughout the primitive streak. However, as the primitive
streak reaches its full extension, the expression of TSKA becomes restricted
to the anterior part of the streak and Hensen's node, while TSKB expression
becomes enriched in the MPS. VG1 and WNT signals secreted from the MPS control
the induction of the node at the anterior tip of the streak
(Joubin and Stern, 1999).
Organizer induction also requires inhibition of BMP2/4 and BMP7 signals,
released from the periphery of the epiblast and the posterior streak,
respectively (Joubin and Stern,
1999). TSKA is involved in counteracting BMP2/4 signals spreading
from the periphery in collaboration with chordin
(Ohta et al., 2004), and it
may also transiently antagonize BMP7 signals from the posterior streak, thus
creating permissive conditions for organizer induction by the MPS. However,
TSKA expression is not maintained in the middle streak. By contrast, sustained
and increased expression of TSKB in this inductive center may promote
node-inducing activity by positively regulating VG1 activity, as highlighted
by the present work.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/133/19/3777/DC1
|
ACKNOWLEDGMENTS |
|---|
|
Footnotes |
|---|
Present address: Department of Anatomy and Developmental biology,
University College London, Gower Street, London, WC1E 6BT, UK 
Present address: Smurfit Institute of Genetics, Trinity College Dublin,
Dublin 2, Ireland
|
REFERENCES |
|---|
|
TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Bachvarova, R. F., Skromne, I. and Stern, C. D. (1998). Induction of primitive streak and Hensen's node by the posterior marginal zone in the early chick embryo. Development 125,3521 -3534.[Abstract]
Bellaris, R. (1986). The primitive streak. Anat. Embryol. 174,1 -14.[Medline]
Bertocchini, F. and Stern, C. D. (2002). The hypoblast of the chick embryo positions the primitive streak by antagonizing nodal signaling. Dev. Cell 3, 735-744.[CrossRef][Medline]
Bertocchini, F., Skromne, I., Wolpert, L. and Stern, C. D.
(2004). Determination of embryonic polarity in a regulative
system: evidence for endogenous inhibitors acting sequentially during
primitive streak formation in the chick embryo.
Development 131,3381
-3390.
Cartegni, L., Chew, S. L. and Krainer, A. R. (2002). Listening to silence and understanding nonsense: exonic mutations that affect splicing. Nat. Rev. Genet. 3, 285-298.[CrossRef][Medline]
Chapman, S. C., Collignon, J., Schoenwolf, G. C. and Lumsden, A. (2001). Improved method for chick whole-embryo culture using a filter paper carrier. Dev. Dyn. 220,284 -289.[CrossRef][Medline]
Chapman, S. C., Schubert, F. R., Schoenwolf, G. C. and Lumsden, A. (2002). Analysis of spatial and temporal gene expression patterns in blastula and gastrula stage chick embryos. Dev. Biol. 245,187 -199.[CrossRef][Medline]
Eyal-Giladi, H. and Kochav, S. (1976). From cleavage to primitive streak formation: a complementary normal table and a new look at the first stages of the development of the chick. I. General morphology. Dev. Biol. 49,321 -337.[CrossRef][Medline]
Eyal-Giladi, H. and Khaner, O. (1989). The chick's marginal zone and primitive streak formation. II. Quantification of the marginal zone's potencies-temporal and spatial aspects. Dev. Biol. 134,215 -221.[CrossRef][Medline]
Hamburger, V. and Hamilton, H. L. (1951). A series of normal stages in the development of the chick. J. Morphol. 88,49 -92.[CrossRef]
Joubin, K. and Stern, C. D. (1999). Molecular interactions continuously define the organizer during the cell movements of gastrulation. Cell 98,559 -571.[CrossRef][Medline]
Khaner, O. and Eyal-Giladi, H. (1989). The chick's marginal zone and primitive streak formation. I. Coordinative effect of induction and inhibition. Dev. Biol. 134,206 -214.[CrossRef][Medline]
Kuriyama, S., Lupo, G., Ohta, K., Ohnuma, S., Harris, W. A. and
Tanaka, H. (2006). Tsukushi controls ectodermal
patterning and neural crest specification in Xenopus by direct
regulation of BMP4 and X-delta-1 activity. Development
133, 75-88.
Lawson, A., Colas, J. F. and Schoenwolf, G. C. (2001). Classification scheme for genes expressed during formation and progression of the avian primitive streak. Anat. Rec. 262,221 -226.[CrossRef][Medline]
Lemaire, P., Garrett, N. and Gurdon, J. B. (1995). Expression cloning of Siamois, a Xenopus homeobox gene expressed in dorsal-vegetal cells of blastulae and able to induce a complete secondary axis. Cell 81, 85-94.[CrossRef][Medline]
Nieuwkoop, P. D. and Faber, J. (1967). Normal Table of Xenopus laevis (Daudin). Amsterdam: North Holland.
Ohta, K., Tannahill, D., Yoshida, K., Johnson, A. R., Cook, G. M. and Keynes, R. J. (1999). Embryonic lens repels retinal ganglion cell axons. Dev. Biol. 211,124 -132.[CrossRef][Medline]
Ohta, K., Lupo, G., Kuriyama, S., Keynes, R., Holt, C. E., Harris, W. A., Tanaka, H. and Ohnuma, S. (2004). Tsukushi functions as an organizer inducer by inhibition of BMP activity in cooperation with Chordin. Dev. Cell 7, 347-358.[CrossRef][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.[CrossRef][Medline]
Sato, T. and Nakamura, H. (2004). The Fgf8
signal causes cerebellar differentiation by activating the Ras-ERK signaling
pathway. Development
131,4275
-4285.
Seleiro, E. A., Connolly, D. J. and Cooke, J. (1996). Early developmental expression and experimental axis determination by the chicken Vg1 gene. Curr. Biol. 6,1476 -1486.[CrossRef][Medline]
Shah, S. B., Skromne, I., Hume, C. R., Kessler, D. S., Lee, K. J., Stern, C. D. and Dodd, J. (1997). Misexpression of chick Vg1 in the marginal zone induces primitive streak formation. Development 124,5127 -5138.[Abstract]
Shimizu, H., Julius, M. A., Giarre, M., Zheng, Z., Brown, A. M. and Kitajewski, J. (1997). Transformation by Wnt family proteins correlates with regulation of beta-catenin. Cell Growth Differ. 8,1349 -1358.[Abstract]
Skromne, I. and Stern, C. D. (2001).
Interactions between Wnt and Vg1 signalling pathways initiate primitive streak
formation in the chick embryo. Development
128,2915
-2927.
Skromne, I. and Stern, C. D. (2002). A hierarchy of gene expression accompanying induction of the primitive streak by Vg1 in the chick embryo. Mech. Dev. 114,115 -118.[CrossRef][Medline]
Streit, A. and Stern, C. D. (1999). Mesoderm patterning and somite formation during node regression: differential effects of chordin and noggin. Mech. Dev. 85, 85-96.[CrossRef][Medline]
Streit, A., Lee, K. J., Woo, I., Roberts, C., Jessell, T. M. and Stern, C. D. (1998). Chordin regulates primitive streak development and the stability of induced neural cells, but is not sufficient for neural induction in the chick embryo. Development 125,507 -519.[Abstract]
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