1 Department of Developmental and Cell Biology, and the Developmental Biology
Center, 4213 McGaugh Hall, University of California, Irvine, CA 92697-2300,
USA
2 Department of Cell Biology, 360 McCallum Building, 1530 3rd Avenue South,
University of Alabama, Birmingham, AL 35294-0005, USA
Authors for correspondence (e-mail:
kwcho{at}uci.edu
and
cchang{at}uab.edu)
Accepted 7 July 2003
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SUMMARY |
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Key words: Twisted gastrulation, Bone morphogenetic proteins, Chordin, BMP1, BMP4, Tolloid, BMP signaling, Xenopus, CCN family
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Introduction |
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BMPs bind to heteromeric transmembrane receptor complexes that function to
directly phosphorylate the intracellular signal transducers Smads 1, 5 and 8
(reviewed by Itoh et al.,
2000). These Smad proteins translocate to the nucleus upon
activation where they function to regulate transcription of BMP target genes.
The availability of BMP ligands is regulated in the extracellular environment
by a variety of positive and negative factors. BMPs, like other members of the
TGFß superfamily, signal as obligate disulfide-linked dimers that are
synthesized and proteolytically processed from precursor pre-proproteins. BMP
dimers are secreted into the extracellular environment where they interact
with cell surface proteoglycans and other secreted factors that act to
modulate their behavior. Most secreted BMP modulators are inhibitors of ligand
activity that act by sequestering BMP ligands from their cell surface
receptors. These inhibitors include noggin, chordin, follistatin and members
of the DAN family of proteins (Smith and
Harland, 1992
; Sasai et al.,
1994
; Hemmati-Brivanlou et
al., 1994
; Bouwmeester et al.,
1996
; Fainsod et al.,
1997
; Hsu et al.,
1998
). Among these, the regulation of BMP signaling by chordin
appears the most complex.
Chordin is specifically expressed in the dorsal marginal zone (Spemann's
organizer) of the Xenopus gastrula stage embryo and its ectopic
expression in ventral mesoderm results in the development of secondary dorsal
axes (Sasai et al, 1994).
Chordin functions to oppose the action of BMPs, which are expressed broadly in
the early gastrula and are ventralizing factors
(Dale et al., 1992
;
Jones et al., 1992
). BMPs
induce the expression of ventral-specific transcription factors and chordin
blocks ventralization by BMPs to maintain dorsal cell fates
(Sasai et al., 1994
;
Piccolo et al., 1996
).
Chordin's activity, like its Drosophila counterpart encoded by
short gastrulation (sog)
(Zusman et al., 1988
;
Francois et al., 1994
), is in
turn opposed by secreted tolloid-related metalloproteases including BMP1 and
Xolloid (Marques et al., 1997
;
Piccolo et al., 1997
;
Scott et al., 1999
;
Wardle et al., 1999
;
Blitz et al., 2000
). These
metalloproteases act by endoproteolytically cleaving chordin at several
specific sites (Scott et al.,
1999
). Cleavage of chordin results in the separation of its two
BMP binding domains onto separate cleavage products that appear to have
10-fold lower affinities for BMP ligands
(Larrain et al., 2000
).
Whether or not the cleavage products have reduced half-lives in vivo is
currently unclear, however, cleavage appears to function in the early embryo
to either limit chordin's range of action or to alter the shape of a chordin
protein gradient.
Recently it has become clear that the modulation of BMP signaling by
chordin is more complex. We, and others, have identified the secreted protein
twisted gastrulation (TSG) as a BMP modulatory protein
(Oelgeschlager et al., 2000;
Chang et al., 2001
;
Ross et al., 2001
;
Scott et al., 2001
). TSG
physically interacts with both BMP4 ligand and chordin. This interaction may
either lead to stronger repression of BMP signaling through BMP sequestration
(Chang et al., 2001
;
Ross et al., 2001
;
Scott et al., 2001
), or
possibly to more readily release BMP4 ligand from chordin cleavage products
after endoproteolysis by the metalloproteases (Oelgelschlager et al., 2000;
Larrain et al., 2001
).
Finally, TSG also enhances the proteolysis of chordin at an underutilized site
(Scott et al., 2001
) and
therefore may function to enhance chordin inactivation and hence up-regulate
(de-repress) BMP signaling (Scott et al.,
2001
; Larrain et al.,
2001
). However, while the in vivo functional significance of this
latter cleavage remains to be determined, it may also result in the production
of a hyperinhibitory version of chordin, as has been suggested in the case of
Drosophila SOG [`super-sog' (Yu
et al., 2000
)]. These observations have led to a complicated
picture. One model suggests that TSG may function as both a BMP activator and
also as a BMP inhibitor, and its mode of behavior depends on its coexpression
with other factors (e.g. metalloproteases).
Superficially, some of these proposed mechanisms appear at odds with
others. Drosophila genetics initially provided evidence consistent
with the idea that TSG functions in the dorsal midline of the fly embryo
(Zusman and Weischaus, 1985; Mason et al.,
1994) as a BMP activator to enhance signaling by the
Drosophila BMP encoded by decapentaplegic (dpp).
The suggestion that Drosophila TSG functions as a BMP (DPP) activator
was based on correlative evidence. TSG is required for proper amnioserosal
development in the dorsal midline of the fly embryo (Zusman and Weischaus,
1985; Mason et al., 1994
) and
slight reductions in the level of DPP resulted in loss of amnioserosa
(Arora and Nusslein-Volhard,
1992
; Ferguson and Anderson,
1992
). These data supported the notion that TSG might function to
boost DPP signaling to higher levels in the dorsal midline. However,
paradoxically, the Drosophila chordin homolog SOG, a known BMP
inhibitor, is also required for amnioserosal development
(Marques et al., 1997
). To
bring together these different observations, it has been proposed that the
function of TSG as a BMP inhibitor (together with SOG) might facilitate higher
level BMP signaling in the dorsal midline by `transporting' BMPs to this
region where they are then released by metalloprotease cleavage of SOG-TSG-BMP
complexes (Ross et al., 2001
).
While it is clear that TSG is required both for BMP inhibition in dorsolateral
regions and for high level BMP signaling in the Drosophila dorsal
midline (Ross et al., 2001
),
this transport mechanism remains hypothetical. Finally, a recent study on
mouse TSG, using a knockout strategy, demonstrated that TSG-deficient mice
have thymocyte and bone defects (Nosaka et
al., 2003
). While the behavior of Smad1 phosphorylation in
thymocytes is consistent with TSG functioning as a BMP inhibitor, the bone
defects observed can be more easily explained, with current knowledge, if TSG
is an activator (Nosaka et al.,
2003
). Therefore, the possibility that TSG can function directly
to both antagonize and activate BMP signaling remains a viable model.
While the biochemistry has supported all of the multiple scenarios postulated, in vivo support for many of these conclusions have been primarily provided by forced overexpression studies. Therefore, we wished to examine the in vivo function of TSG using a loss-of-function approach in Xenopus embryos, the vertebrate system where the role of BMP signaling is most thoroughly studied, to attempt to provide evidence for a role for TSG as either a positive or a negative regulator of the BMP pathway. This would focus our attention toward certain models for further testing. Toward this end we have used morpholino antisense oligonucleotides (MOs) to inhibit the translation of endogenous TSG (and chordin) and have examined the phenotypes of Xenopus embryos during early development. We report that MO `knockdown' of TSG expression on the dorsal side of the embryo results in a reduction of anterior head structures, and this phenotype can be rescued by either restoring TSG expression or by elevating chordin expression. Furthermore, we have found that loss of TSG function does not appear to significantly reduce dorsal marker gene expression in early gastrulae, but at late gastrula to early neurula stages markers of dorsal cell fates are reduced at the expense of expanded ventral marker gene expression, implying a role for TSG as a BMP antagonist. Using MOs to both TSG and chordin together we also show that embryos have stronger ventralization phenotypes consistent with previous suggestions that these molecules coordinately act to inhibit BMP signaling. Finally, we show that TSG is required for an efficient response of embryos or explanted animal caps to ectopically expressed chordin. These in vivo data support a model whereby TSG primarily functions to assist chordin as a BMP inhibitor to establish or maintain dorsal cell fates during gastrulation.
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Materials and methods |
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Whole-mount in situ hybridizations
Whole-mount in situ hybridizations were performed according to the method
of Harland (Harland, 1991)
using BM purple (Boehringer-Mannheim) as substrate. In situ probes were made
as follows. Goosecoid probe was prepared by linearizing pBSSKXgscH43
with EcoRI and RNA was transcribed using T7 RNA polymerase
(Cho et al., 1991
). Otx2 and
sizzled probes were prepared as previously described
(Blitz and Cho, 1995
;
Blitz et al., 2000
). MyoD and
Sox2 probes were prepared from pBSSKII+ based clones from an arrayed library,
and Krox20 was prepared from pGEM4-Krox20. All of these were digested with
EcoRI and T7 RNA polymerase was used. HoxB9 probe was a kind gift
from Bruce Blumberg.
Plasmid templates for in vitro transcription
Xenopus and human TSG mRNAs
(Scott et al., 2001;
Chang et al., 2001
) were
prepared from plasmid templates after cloning the XTSG coding region into
pCS2+. To generate a Xenopus TSG rescue construct, PCR product was
generated using the oligonucleotides
5'-CCCGCTAGCATGCAATAAGGCTCTCTGTGCTA-3' and
5'-CCCCTCGAGTTAAACCATACAGTTCACGCACTT-3' which was directionally
cloned between the NheI and XhoI sites of pCS2+mTSG
replacing the mouse TSG sequences (Scott
et al., 2001
). The resulting plasmid, pCS2+BM40-XTSG encodes the
Xenopus TSG protein as a fusion to the signal peptide from the
extracellular matrix protein BM40. Chd-MO rescue experiments utilized chordin
mRNA synthesized from pCS2+Xchordin (kindly provided by Eddy De Robertis),
which lacks its 5' UTR and therefore lacks Chd-MO target sequences.
pCS2+ template DNAs representing the two different chordin genomic copies, and
containing Chd-MO complementary sequences, were reconstructed by substituting
the 5' EcoRI-BglII fragment of pCS2+Xchordin with DNA
fragments produced by RT-PCR that contain its `native' 5' UTR or the
5' UTR of the redundant pseudotetraploid copy (GenBank accession number
AW460332). To generate these PCR products, the respective 5' oligos
5'-GGGGAATTCTACGAGACAGAACGTTTGGAACCAC-3' and
5'-GGGGAATTCAGCTTGGTTCGGGACAACCACAAA-3' were used in combination
with the 3' oligo 5'-GTGCATAACTCCGAATGGTTC-3'. The integrity
of all constructs was verified by sequencing. Noggin RNA was synthesized with
SP6 polymerase using EcoRI-linearized pSP64T-Noggin template. All
chordin and TSG mRNAs were prepared using SP6 polymerase from pCS2+ based
plasmids linearized with NotI.
RT-PCR
The protocol for RT-PCR was as previously described
(Blitz and Cho, 1995) except
that in some cases we used ethidium bromide-stained agarose gels to analyze
the reaction products. Primers used to amplify otx2 and histone H4 were as
previously described (Blitz and Cho,
1995
). Msx1 primers: 5'-GATTCGTTGATAGGATCGCACT-3' and
5'-GGTCTCTCCCAGGTTTCCTA-3'. Vent2 primers:
5'-AGGCCATTTGTTAGATATTAATC-3' and
5'-GTATTTTTCATAGAATATACACGC-3'. Otx2, histone H4, vent2 and msx1
amplification reactions used 27, 24, 30 and 30 cycles respectively for agarose
gels, and 21 or 25 cycles for 5% PAGE with radioactive PCR products. All
RT-PCR reactions were done three times, with somewhat varying extents of shift
in the response of the animal caps to chordin or noggin in the presence of
TSG-MO (e.g. 2- to 5-fold shift in the dose requirement for noggin RNA to
induce otx2 in the animal caps). Representative experiments are shown.
Morpholinos and in vitro translations
Capped mRNAs were synthesized using Ambion mMessage Machine T3 and SP6 in
vitro transcription kits. Inhibition of translation of TSG and chordin mRNAs
was performed as previously described
(Taylor et al., 1996).
Briefly, 1 µg of each capped mRNA was translated using the Promega Rabbit
Reticulocyte System and [35S]methionine in the absence or presence
of 2.5 µM of the specified morpholino antisense oligonucleotide. Reactions
were RNAse A treated, subjected to SDS-PAGE, and labeled proteins were
detected by autoradiography. Two different morpholino oligos were tested for
their efficacy in inhibiting TSG translation (see
Fig. 1A). One TSG-MO
(5'-AGGAAAGAGGGCTTCATACTTGGCC-3') hybridizes to the region around
the translational start site of the published TSG sequences (GenBank accession
numbers AF279246 and AF245221), and the other was designed against an
unpublished allele of Xenopus TSG, which contains the sequence
5'-GCCAATTATGAAGCCCTCTTTCCTT-3' at the translation start
(the polymorphic base is in bold and the ATG is underlined). The two TSG-MOs
differ in their sequences by one nucleotide in the middle. Both TSG-MOs
efficiently inhibited the synthesis of TSG protein containing either
translational start sequences, and both efficiently induced similar phenotypes
in vivo. We therefore refer to both morpholino oligos as TSG-MO and do not
distinguish between the versions of these MOs used in the text. Two chordin
MOs, designated ChdA-MO and ChdB-MO, were designed against the published
chordin allele (Sasai et al.,
1994
) and a sequence for a chordin EST (GenBank accession number
AW460332). These are 5'-CAGCATTTTTGTGGTTGTCCCGAAC-3' and
5'-GGGACACTGCATTTTTGTGGTTCCA-3', respectively. Throughout the text
we designate an equimolar mixture of these simply as Chd-MO. Luciferase
control RNA was provided with the Promega Rabbit Reticulocyte. `Control
morpholino' refers to Genetools `Standard' MO. We find that injection of
control MO even at concentrations as high as 60 ng/embryo has no observable
effect on Xenopus development. The sequences of GDF6-MO and BMP7-MO
used for control for blastopore closure are: GDF6-MO
5'-GCAGAGGGCTCCTGTATGTATCCAT-3' (GenBank accession number
AF155125) and BMP7-MO 5'-CTGTCAAAGCATTCATTTTGTCAAA-3' (GenBank
accession number U38559).
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Results |
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As we also wished to examine the in vivo functional relationship between
TSG and chordin, we designed MOs to inhibit chordin translation. Since it was
not possible to easily make one chordin MO complementary to both
pseudotetraploid isoforms, we designed two different Chd-MOs (ChdA-MO and
ChdB-MO; Fig. 1A) each directed
against one of the chordin isoforms and tested them either alone or in
combination. In similar fashion to the TSG-MO, we first tested the efficacy of
our chordin MOs in in vitro translations with various chordin mRNA templates.
Both of the chordin MOs most efficiently inhibited translation of their
respective target mRNAs (`ChdA' and `ChdB' mRNAs) to which they were designed
(Fig. 1C, lanes 4-11).
Furthermore, each of the Chd-MOs also reduced the translation of the other
chordin mRNAs. Finally, neither of the Chd-MOs inhibited the translation of
luciferase (Fig. 1C, lanes
1-3), TSG (as discussed above; Fig.
1B, lane 6 and 7), or a chordin mRNA containing a 5' UTR
lacking MO recognition sequences (5'-Chd;
Fig. 1C, lanes 12-15). In
conclusion, TSG and chordin protein translation in vitro is specifically
inhibited by TSG-MO and Chd-MOs respectively.
TSG-MO and Chd-MOs block TSG and chordin function in vivo
To further examine the specificity of the TSG-MO, we sought to inhibit TSG
overexpression phenotypes by coinjecting the TSG-MO and TSG mRNA to which the
TSG-MO was designed. Overexpression of TSG in Xenopus embryos results
in a complex set of malformations in late tailbud and early tadpole stages
including most prominently a reduction of head structures and defective
ventral tail fin development (Chang et al.,
2001; Scott et al.,
2001
; Oelgeshlager et al., 2000;
Larrain et al., 2001
). Head
reduction and abnormal posterior morphogenesis first becomes visible during
neurula stages (data not shown) and can readily be seen in `pre-tailbud' stage
24 embryos (Fig. 2B). However,
coinjection of the TSG-MO together with XTSG mRNA results in rescue of embryo
morphology (Fig. 2C).
Therefore, taking the in vitro translation data together with these in vivo
observations, we suggest that the TSG-MO specifically inhibits XTSG function
in vivo.
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Embryonic TSG loss-of-function phenotypes
To determine how TSG regulates BMP signaling in vivo, we initially examined
the TSG loss-of-function phenotype in TSG-MO microinjected embryos (`TSG-MO
embryos'). As the consequences of overexpression (hyperventralization) and
inhibition (hyperdorsalization) of BMPs in early Xenopus embryos have
been well characterized, we wished to establish whether knockdown of TSG
expression would result in either of the typical BMP overexpression or BMP
inhibition phenotypes. Microinjection of TSG-MO into each blastomere at the
four-cell stage resulted in defects in both head and tail development in frog
tadpoles. The embryos show reductions in head size, a curved body axis and
minor tail defects (Fig. 3B;
for more detailed description see also below). To further dissect the function
of TSG in dorsal (low BMP signals) and ventral (high BMP signals) regions, we
also injected the TSG-MO into the dorsal or the ventral blastomeres only.
Microinjection of TSG-MO into the ventral blastomeres resulted in a
ventralward bending of the tail and abnormal tail fin development in 71% of
the embryos (n=80; Fig.
3E), suggesting that TSG may be required on the ventral side in
early frog development. Since TSG is expressed in the ventral region at
gastrula stages and the ventral-posterior region of the trunk and the tail at
tailbud stages (Oelgeschlager et al.,
2000), the ventral injection phenotype may be the result of either
an early or a late effect on TSG function in the ventral marginal zone derived
tissues. Microinjection of the TSG-MO into each of the dorsal blastomeres at
the four-cell stage resulted in reduction of head structures with many embryos
displaying defective or even absent eyes (78%, n=181;
Fig. 3D). This result suggests
that TSG may play an important role(s) in dorsoanterior specification. In
addition, a significant number of embryos (89%, n=181) show a
sideways bending of the body axis and have smaller eyes
(Fig. 3D). This reduction in
anterior development suggests that the TSG-MO induces a `moderate' elevation
of BMP signaling. A similar result has also been recently reported when
embryos are depleted of chordin
(Oelgeschlager et al., 2003
)
and, therefore, these results imply that endogenous TSG, like chordin, may
function to inhibit BMP signaling on the dorsal side. We thus further
characterized the phenotype of TSG-MO embryos in more detail.
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|
To determine whether other BMP antagonists can also rescue the TSG-MO phenotype, we coinjected the TSG-MO with increasing doses of noggin RNA into the dorsal blastomeres of four-cell stage embryos. We found that 5 pg to 10 pg of noggin RNA is sufficient to rescue the phenotype of TSG-MO embryos (Fig. 4D). As the TSG-MO phenotype can be rescued by coinjection with either chordin or noggin mRNAs, our results suggest that TSG may function as a BMP inhibitor in Xenopus. Loss of TSG expression may lead to elevated BMP signaling in the embryo which can be suppressed by BMP inhibitors to restore normal development of early embryos.
The TSG-MO and Chd-MOs cooperate to inhibit dorsal specification
In zebrafish, TSG and chordin cooperate with each other to regulate dorsal
development (Ross et al.,
2001), however it is presently unclear whether TSG plays a similar
role in dorsal specification in Xenopus. The rescue of the TSG-MO
embryos with chordin mRNA (discussed above) suggested that TSG and chordin may
cooperate to specify dorsal tissues in Xenopus as well. To further
explore this possibility, we coinjected the TSG-MO and Chd-MOs and examined
the morphology of the injected embryos.
We first examined the embryos at gastrula stages. While embryos injected
with the TSG-MO into each four-cell stage blastomere had the normal onset of
gastrulation, as judged by the timing of the appearance of the dorsal lip,
reduction of TSG expression resulted in slowing of the progression of
blastopore closure (data not shown). Interestingly, microinjection of the
Chd-MOs has a similar effect in delaying blastopore closure (data not shown),
however injection of the `control' MO, or MOs to Xenopus GDF6 or
BMP7, had little to no effect on the timing of blastopore closure (data not
shown) demonstrating the specificity of the TSG-MO and Chd-MOs for this
effect. Furthermore, we could partially rescue the blastopore closure defect
induced by TSG-MO by coninjection of a wild-type TSG mRNA that cannot be
inhibited by the MO (data not shown). The delay in blastopore closure mimics
the effect of overexpression of low doses (20 pg mRNA/blastomere) of BMP2
(data not shown) suggesting that both TSG and chordin function to inhibit BMP
signaling. Importantly, similar to what has been shown in the case of BMP
overexpression (Jones et al.,
1992), the blastopore does eventually close. At neurula stages,
the embryos injected with the TSG-MO had recovered to a large extent, though
they showed a shortened neural axis (data not shown). Taken together, these
data suggest that the TSG-MO and Chd-MOs may induce embryonic defects after
the onset of gastrulation (see also below).
We next sought to determine whether coinjection of the Chd-MOs together with the TSG-MO would result in an enhancement of the TSG-MO phenotypes. We first examined the phenotype of embryos coinjected dorsally with both TSG-MO and the Chd-MOs at tadpole stages. When injected into the dorsal side at 10 ng/blastomere, the TSG-MO and Chd-MOs each induced mild to moderate reductions in head (dorsoanterior) development (Fig. 5B,C); however when the two MOs were coinjected into the dorsal blastomeres, the resulting embryos had severely reduced or even missing heads and shortened anterior-posterior body axes (Fig. 5D; 84%, n=55, of co-injected embryos had barely discernible head structures). We also examined embryos that were injected with the MOs in all blastomeres. While dorsal injection of this `high' dose of TSG-MO (10 ng/blastomere) causes head defects that are rescuable with TSG mRNA (Fig. 4A), injection of this dose into all blastomeres of four-cell stage embryos results in death of the embryos during late neurula stages (data not shown). We have not attempted to analyze the mechanisms responsible for this embryonic death. At lower doses of MO (5 ng MO into each four-cell stage blastomere), both TSG-MO and Chd-MOs each induced a `weak' phenotype (Fig. 5F,G), while coinjection of these MOs together led to a stronger reduction of dorsoanterior structures concomitant with an apparent enlargement of the ventroposterior region (Fig. 5H). Finally, the combined MO phenotype we observed does not appear to be caused by a mere increase in the total amount of injected MOs, because (1) increasing the total dose of MO by coinjecting the TSG-MO together with a control MO did not result in more severe changes in embryo morphology than that of TSG-MO embryos (data not shown); (2) when the total amount of MO per blastomere was kept constant, the phenotype induced by the combination of low doses (5 ng of each MO/dorsal blastomere) of TSG-MO and Chd-MOs was always more severe than that when each individual MO was used at a higher dose (10 ng/blastomere; data not shown). Our results thus suggest that TSG and chordin cooperate in vivo to regulate dorsal development.
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|
TSG is required for efficient inhibition of BMP signaling by chordin
in animal caps and secondary axis induction
We, and others, previously suggested that TSG functions as a BMP inhibitor
(Scott et al., 2001;
Chang et al., 2001
;
Ross et al., 2001
). These
studies also provided in vitro and in vivo overexpression data supporting the
notion that the inhibitory activity of TSG on BMP signaling might occur
through its ability to enhance the interaction between chordin and BMP
ligands. One prediction of this model is that reduction in the level of TSG
expression would result in a decreased response to ectopic chordin expression.
To test this hypothesis, we assayed the ability of chordin to induce secondary
axes when overexpressed in the ventral marginal zone. We overexpressed chordin
in the ventral marginal zone of embryos in the presence or absence of the
TSG-MO and assayed for development of secondary axes at early tailbud stage.
We found that, while chordin efficiently induced secondary axes
(Fig. 7B; 95%, n=60),
the TSG-MO completely inhibited chordin-mediated secondary axis formation
(Fig. 7C; 0%, n=57).
Furthermore, a control MO had no effect on chordin's activity in this assay
(98%, n=58). To further rule out the possibility that this inhibition
of chordin function might instead be due to more general `non-specific'
effects on secondary axis formation we sought to examine the effects of the
TSG-MO on secondary axes induced by other secreted signaling molecules. Early
Wnt signaling acts via a canonical ß-catenin-mediated pathway to induce
expression of siamois, twin, and Xnr3 resulting in the
formation of secondary axes. Furthermore, expression of these genes is not
affected by elevating BMP signaling
(Laurent et al., 1997
).
Therefore, we microinjected Xwnt8 mRNA into the ventral marginal zone
at the four-cell stage in the presence
(Fig. 7E) or absence
(Fig. 7D) of coinjected TSG-MO
and examined the effects of TSG depletion on Xwnt8-induced secondary axis
formation. We found that the frequency of formation of secondary axes by Xwnt8
was unaffected by reduction of TSG expression. Furthermore, we tested the
effect of TSG-MO on secondary axis induction by noggin, another secreted BMP
inhibitor. As shown in Fig.
7F-H, the ability of noggin to induce a secondary axis was not
blocked by the TSG-MO either. These data suggest that the effect of TSG-MO on
secondary axis induction by chordin are the result of chordin's BMP inhibitory
function and that TSG is required for efficient BMP inhibition by chordin in
vivo.
|
This inability of chordin to induce Xotx2 or to repress
Msx1 or Vent2 may explain why low doses of chordin could not
rescue the phenotype induced by the dorsally injected TSG-MO
(Fig. 4C). We did, however,
observe suppression of the TSG-MO phenotype when chordin was injected at high
doses, suggesting that chordin does not absolutely require TSG function. To
test whether this is the case, we further analyzed the BMP inhibitory activity
of chordin over a wider range of RNA doses and with a more sensitive detection
method using radioactive nucleotides in the PCR reaction. As shown in
Fig. 7J, we observed a dramatic
shift in the caps' response to chordin in the absence as compared to the
presence of TSG-MO. While 10 pg chordin mRNA alone induced high levels of
Xotx2 and repressed Msx1 efficiently, it appears that
approximately 20- to 50-fold higher doses of chordin were required to achieve
a similar level of induction/repression when TSG-MO was present
(Fig. 7J, compare lane 4 to
lanes 16 and 17). To address whether the TSG-MO blocks BMP antagonists other
than chordin in animal caps, we performed a similar assay using the unrelated
BMP inhibitor noggin. While the TSG-MO had an effect on induction of
Xotx2 and repression of Msx1 and XVent2 by noggin,
this reduction in noggin's BMP inhibitory behavior appears to be weaker,
perhaps as little as two- to fourfold (Fig.
7K). From these experiments, we conclude that, while the TSG-MO
can reduce the BMP inhibitory behavior of noggin in these overexpression
experiments, its ability to alter chordin function appears to be significantly
stronger. Finally, while it has previously been shown that expression of
chordin itself can be induced by overexpression of BMP inhibitors
(Blitz et al., 2000)
(suggesting that TSG might indirectly influence BMP inhibition by noggin
through noggin's induction of chordin), significant levels of chordin were not
induced at these doses of noggin (data not shown). Therefore, we conclude that
endogenous TSG is important for determining the strength of the inhibitory
activity of chordin towards BMP, but TSG may also function indirectly, in a
chordin-independent manner, to establish appropriate levels of BMP signaling
in vivo.
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Discussion |
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High-level TSG overexpression results in head truncations
(Oelgeschlager et al., 2000;
Chang et al., 2001
;
Ross et al., 2001
;
Scott et al., 2001
) consistent
with models that it might promote BMP signaling. Furthermore, when chordin is
coexpressed with high levels of TSG, secondary axis formation is inhibited
(Oelgeschlager et al., 2000
;
Ross et al., 2001
) (our
unpublished data), consistent with the notion that TSG can inhibit chordin
function. These observations suggest that `high-level expression' of TSG
promotes BMP signaling. However, other experiments lead to opposite
conclusions. For example, when low doses of ectopic chordin are expressed
ventrally, in amounts incapable of inducing secondary axes, the addition of
low doses of overexpressed TSG together with chordin resulted in secondary
axis induction (Scott et al.,
2001
). These and other results of TSG overexpression
(Chang et al., 2001
;
Ross et al., 2001
) provided in
vivo evidence that TSG can also function as a BMP inhibitor. Finally,
curiously certain TSG loss-of-function phenotypes in zebrafish embryos show
similarities to some of the phenotypes achieved by TSG overexpression in
Xenopus (Oelgeschlager et al.,
2000
; Chang et al.,
2001
; Ross et al.,
2001
; Scott et al.,
2001
). This raises the possibility that TSG may function
differently in Xenopus and zebrafish embryos. As it remains unclear
whether endogenous TSG indeed has dual activities in regulating BMP signaling,
we have undertaken a loss-of-function approach in Xenopus embryos to
examine the role of in vivo levels of TSG to determine whether TSG might
function as a BMP inhibitor or activator, or both.
In the present study, loss of TSG function, by inhibition of its
translation using morpholino oligonucleotides, resulted in a reduction of head
structures. The eyes are often defective or even missing in TSG knockdown
embryos. Since head formation requires inhibition of the BMP pathway
(Glinka et al., 1997;
Piccolo et al., 1999
;
Bachiller et al., 2000
), this
observation is consistent with the notion that BMP signals may be elevated
when levels of TSG expression are reduced. This hypothesis is supported by our
analysis of marker expression in TSG-MO embryos. At neurula and tailbud
stages, expression of several dorsal and anterior markers are moderately
reduced, while the expression of the ventral marker sizzled is
expanded. Additionally, phenotypes of head reduction and expansion of the
sizzled domain were found when chordin expression is reduced by
chordin antisense MOs (Fig. 6)
(Oelgeschlager et al., 2003
).
Furthermore, we observe that chordin or noggin, like wild-type TSG, rescues
the head defect induced by the TSG-MO. These data suggest that TSG functions
in vivo to inhibit BMP signaling.
TSG inhibits BMP signalling during early to midgastrulation to
maintain dorsal cell fates
TSG loss-of-function embryos do not show any obvious defects at early
gastrula stages. Both dorsal and ventral markers are expressed at apparently
normal levels and in the correct spatial patterns, and initiation of dorsal
blastopore lip formation is normal. The effects of reduced TSG expression
arise at midgastrula stages, when changes in some markers of dorsal-ventral
patterning become apparent. Later in gastrulation we observed a slowing in the
progression of blastopore closure in TSG-MO embryos. The lack of an early
effect by the TSG-MO can be explained in two ways. Either maternally deposited
TSG protein, which is not affected by the antisense oligonucleotides, may be
present to function at blastula to early gastrula stages and the residual low
level of TSG may therefore be sufficient for early embryogenesis; or loss of
TSG function may only affect embryo development after the onset of
gastrulation. We favor the latter hypothesis as we have found that knockdown
of chordin expression with antisense morpholino oligos similarly induces
defects in frog embryos after the initiation of gastrulation, but not earlier.
The fact that both chordin and TSG loss-of-function embryos show a delayed
effect on marker expression and embryo morphology suggests that reduced BMP
inhibition at early gastrula stages may reveal its effects only during later
development of Xenopus embryos. This observation is also consistent
with a previous report that ectopic BMP overexpression alters the embryo
development after the onset of gastrulation
(Jones et al., 1996). It has
been shown that, when overexpressed at high doses, BMPs can arrest
gastrulation without influencing the initial formation of the dorsal lip or
marker expression (Dale et al.,
1992
). At low to intermediate doses of BMP, which mimic TSG-MO or
Chd-MOs injections, formation of the dorsal lip is still normal and the
blastopore eventually closes (Jones et
al., 1992
; Jones et al.,
1996
), but the closure is also delayed (our unpublished
observations). The slow progression through gastrulation in both TSG-MO and
Chd-MO embryos may therefore be due to enhanced BMP signaling as well. These
observations are also reminiscent of a recent report of BMP regulating cell
movements during gastrulation in the zebrafish embryo
(Myers et al., 2002
).
Therefore, consistent with these previous observations, it is perhaps not
surprising that altering the levels of expression of BMP regulators with MOs
(as we have performed in the present study) might also not reveal phenotypes
until midgastrulation.
TSG and chordin cooperate to inhibit BMP signaling in dorsal cell
fate specification
We also examined the relationship between TSG and chordin in dorsal
specification by coinjecting the TSG-MO together with Chd-MOs. While
inhibition of translation of each of these genes individually resulted in
relatively weak reductions in the extent of dorsal development, simultaneous
inhibition of both TSG and chordin resulted in moderate-to-strong
ventralization. These data further support the notion that both TSG and
chordin act in the same direction; they both inhibit BMP signaling. TSG on its
own is a weak BMP inhibitor as it is incapable of inducing secondary axes
(Oelgeschlager et al., 2000;
Ross et al., 2001
;
Scott et al., 2001
) unless it
is expressed at high doses as a membrane-tethered protein
(Chang et al., 2001
). These
observations are consistent with the finding that TSG has a ten-fold lower
binding affinity for BMP ligands than chordin
(Oelgeschlager et al., 2000
).
These observations are also consistent with the previous biochemical evidence
that chordin and TSG physically interact with one another. TSG can promote
chordin's BMP binding and BMP inhibitory activities
(Chang et al., 2001
;
Ross et al., 2001
;
Scott et al., 2001
) and
therefore cooperative effects of BMP inhibition by both TSG and chordin on
dorsal cell fate specification in the present study are in good agreement with
the suggestion that these proteins function together to form an inhibitory
complex with BMP4. Finally, a loss-of-function study in zebrafish has also
suggested that TSG acts in partnership with chordin to inhibit the activities
of BMPs (Hammerschmidt et al.,
1996
; Ross et al.,
2001
). An antisense TSG MO induces a ventralized phenotype that is
similar to moderate chordin loss-of-function mutants, and chordin and TSG MOs
synergistically enhance the expansion of blood islands, a ventral tissue type.
Our results thus bolster the idea that the function of vertebrate TSGs may be
conserved in that TSG cooperates with chordin to inhibit BMP signaling.
Are there two distinct modes of chordin-mediated BMP inhibition?
An unexpected result from our studies suggests that TSG not only assists
chordin in inhibition of BMP signaling, but may be required for efficient BMP
inhibitory activity of chordin. This result is surprising since purified
chordin can directly bind to BMPs in vitro to prevent their association with
their cognate receptors, thus inhibiting BMP signaling
(Piccolo et al., 1996). As the
chordin-BMP interaction is enhanced by TSG, we and others suggested that TSG
enhances chordin's BMP binding activity to increase BMP inhibition
(Chang et al., 2001
;
Ross et al., 2001
;
Scott et al., 2001
). Our
morpholino coinjection experiments with chordin
(Fig. 7) are consistent with
this idea. Furthermore, the expression patterns of TSG and chordin suggest
that the embryo might utilize TSG as a cofactor to enhance the activity
chordin only when higher level BMP inhibition cannot be achieved by chordin
alone.
In the early stages, the expression domain of chordin overlaps with two
regulators of chordin, TSG and BMP1/Xolloid
(Goodman et al., 1998;
Scott et al., 2001
), and their
expression segregates into different regions as development proceeds. TSG is
ubiquitously distributed during early gastrulation
(Scott et al., 2001
) and
overlaps chordin in Spemann's organizer during late blastula and early
gastrula stages. The presence of the negative chordin regulator BMP1/Xolloid
in all cells during early Xenopus development, and the early
expression of BMPs 2, 4 and 7 in the organizer itself may necessitate a
requirement for TSG to enhance chordin's BMP inhibitory activity in order to
permit dorsal gene expression and establishment of an `appropriately sized'
organizer (Stewart and Gerhart,
1990
). The intricate balance between the levels of TSG,
BMP1/Xolloid, BMP ligands and chordin (and other BMP inhibitors) at these
stages may thus determine the level of BMP inhibition by chordin in early
gastrulae.
By late gastrulation, TSG expression is excluded from the dorsal side of
the embryo (Oelgeschlager et al.,
2000; Scott et al.,
2001
), and Xolloid is detected only in posterior ectodermal
patches (Goodman et al.,
1998
). Chordin at these stages is expressed in the dorsal mesoderm
underlying the neural plate (Sasai et al.,
1994
), a TSG-free zone. It is thus likely that at these later
stages, conditions are such that the embryo no longer requires a reliance of
chordin on TSG to enhance its ability to bind and inhibit BMPs. At these later
stages, chordin may therefore block BMPs appropriately even in the absence of
TSG in these developmental contexts. It is thus conceivable that there may be
two distinct modes of chordin-mediated BMP inhibition, TSG-dependent and
TSG-independent.
It is also interesting to speculate that TSG may interact with other proteins in vivo to regulate early frog development, as ventrally injected TSG-MO induces ventroposterior defects, even though chordin expression is absent from the ventral region and Chd-MOs have no effect on early frog embryogenesis when expressed ventrally (data not shown). Further studies are required to understand all the partners of TSG and how they regulate early Xenopus development.
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ACKNOWLEDGMENTS |
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