1 Howard Hughes Medical Institute and Department of Biological Chemistry,
University of California, Los Angeles, CA 90095-1662, USA
2 University of Pennsylvania, Department of Cell and Developmental Biology, 421
Curie Boulevard, Philadelphia, PA 19104-6058, USA
Author for correspondence (e-mail:
derobert{at}hhmi.ucla.edu)
Accepted 20 May 2003
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SUMMARY |
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Key words: Twisted-gastrulation, BMP, Chordin, Tolloid, Crossveinless, TGFß, Cell-cell signaling, Xenopus, Zebrafish, CR modules
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INTRODUCTION |
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Chordin is a large secreted protein abundantly expressed in Spemann's
organizer, reaching extracellular concentrations in the 6-12 nanomolar range
(Piccolo et al., 1996).
Chordin binds directly to BMP and prevents BMP binding to its cognate
receptor, causing dorsalization of the embryo in overexpression studies
(Sasai et al., 1995
;
Piccolo et al., 1996
). The
binding of Chordin to BMP is mediated by four cysteine-rich domains (CRs) of
about 70 amino acids each (Larraín
et al., 2000
). A large number of additional extracellular proteins
containing CR domains have now been identified, and interactions with BMP or
TGFß have been documented for many of them (reviewed in
García-Abreu et al.,
2002
).
In Drosophila, the chordin homolog short
gastrulation (sog) is expressed in ventral neuroectoderm and is
required for the formation of neural tissue
(François et al.,
1994). In addition, Sog is necessary for the formation of the
dorsal-most tissue of the fly embryo, the amnioserosa, which requires maximal
BMP activity (Ross et al.,
2001
; Eldar et al.,
2002
).
In zebrafish, the ventralized chordino phenotype is caused by a
loss-of-function mutation in the zebrafish chordin gene
(Schulte-Merker et al., 1997;
Hammerschmidt and Mullins,
2002
). The neural plate and dorsal mesoderm are reduced, and
ventral mesoderm is expanded in chordino mutants
(Hammerschmidt et al., 1996a
;
Gonzalez et al., 2000
). The
opposite phenotype, dorsalization, is observed in bmp2b/swirl,
bmp7/snailhouse, Smad5/somitabun and Tolloid/mini-fin mutants
(Mullins et al., 1996
;
Hammerschmidt and Mullins,
2002
). In addition, chordino;swirl double
mutants display the swirl phenotype, confirming that Chordin
functions genetically as an anti-BMP
(Hammerschmidt et al., 1996b
).
Recent studies on swirl/chordino genetic interactions indicate that
in zebrafish, Chordin also functions in the formation of ventral tail fin
tissue that requires maximal BMP signaling
(Wagner and Mullins, 2002
).
This suggests that in vertebrates Chordin, like Sog in the amnioserosa of the
fly, may promote BMP signaling as well.
In mouse, the targeted inactivation of chordin results in a
ventralized gastrulation phenotype only in a low percentage of the homozygous
mutant embryos. Most mutants lack anterior notochord and pharyngeal endoderm,
causing a phenotype similar to human DiGeorge syndrome, but do not have
gastrulation phenotypes (Bachiller et al.,
2003). However, mice homozygous for mutations in chordin
and noggin lack the forebrain, indicating that Noggin can partially
compensate for the loss of Chordin
(Bachiller et al., 2000
).
In Xenopus, inhibition of Chordin production by morpholino
oligonucleotides causes a phenotype similar to that of zebrafish
chordino, with embryos developing with smaller heads and enlarged
ventroposterior structures
(Oelgeschläger et al.,
2003). This relatively weak ventralization contrasts with the
strong requirement for Chordin observed when the embryos are experimentally
manipulated. Indeed, dorsalization of embryos by LiCl
(Kao and Elinson, 1988
),
dorsal mesoderm induction by Activin
(Green et al., 1992
) and CNS
induction by dorsal lip grafts, all show a complete dependence on the presence
of Chordin (Oelgeschläger et al.,
2003
).
Tsg is a co-factor of Chordin that can bind both to BMP and to Chordin,
generating trimolecular complexes that antagonize BMP signaling
(Oelgeschläger et al.,
2000; Chang et al.,
2001
; Scott et al.,
2001
; Larraín et al.,
2001
). The cleavage of Chordin by the Xolloid (Xld)/Tolloid (Tld)
zinc-metalloproteinase generates protein fragments that include intact
BMP-binding cysteine-rich modules (CRs) and retain anti-BMP activity
(Larraín et al., 2000
).
Tsg facilitates the cleavage of Chordin by Tolloid
(Scott et al., 2001
;
Larraín et al., 2001
)
and antagonizes the residual anti-BMP activity of the proteolytic cleavage
products of Chordin (Oelgeschläger et
al., 2000
; Larraín et
al., 2001
). Thus, in this second aspect of its activity Tsg
behaves as a pro-BMP. The cleavage of Chordin by Xld/Tld constitutes the
molecular switch that controls the anti-BMP and pro-BMP activities of Tsg
protein in this biochemical pathway
(Larraín et al.,
2001
).
The multifunctional properties of Tsg hamper the analysis of its function
in embryonic patterning. For example, in Xenopus embryos
overexpression of xTsg has a pro-BMP effect, resulting in ventralization
(Oelgeschläger et al.,
2000). In zebrafish, overexpression of zTsg causes an anti-BMP,
dorsalized phenotype (Ross et al.,
2001
). The difference in phenotype may be attributed to a lower
endogenous activity of Tolloid during early zebrafish development
(Connors et al., 1999
;
Larraín et al., 2001
).
Indeed, the ventralizing activity of xTsg in Xenopus could be
reversed by overexpression of a dominant-negative Xolloid
(Larraín et al.,
2001
).
In the present study, we dissected the anti-BMP and pro-BMP activities of
xTsg by generating a series of point mutations that affected specifically one
or the other activity of xTsg. The Tsg protein contains two evolutionary
conserved cysteine-rich domains. The C-terminal cysteine-rich region does not
have any significant homology to known protein motifs and its role in the
biochemical activities of Tsg is unknown. The N-terminal cysteine-rich domain
of Tsg has some homology to the BMP-binding modules of Chordin (CRs) and is
necessary and sufficient for the direct interaction of xTsg with BMP
(Oelgeschläger et al.,
2000). As shown here, mutations in the N-terminal domain generated
mutant xTsg proteins that no longer bound BMP and had greatly enhanced
ventralizing activities, preventing the formation of CNS and dorsal mesoderm
in Xenopus embryos. This hyperventralizing activity of xTsg mutants
required an intact C-terminal domain, and phenotypes were much stronger than
those caused by Chordin loss-of-function, both in Xenopus and
zebrafish embryos. Hyperventralizing xTsg mutations specifically antagonized
proteins containing BMP-binding modules of the Chordin type. Our data indicate
that xTsg inactivates CR-modules through its C-terminal conserved domain and
that xTsg may interact, in addition to Chordin, with other CR-containing
proteins required for the regulation of early dorsoventral patterning.
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MATERIALS AND METHODS |
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A combination of two anti-chordin morpholinos was used as
described (Oelgeschläger et al.,
2003).
Embryo manipulations and chordino genotyping
Microinjections, in situ hybridization and RNA synthesis were performed as
described (Piccolo et al.,
1997; Oelgeschläger et
al., 2000
; Sive et al.,
2000
). For LiCl rescue experiments, embryos were microinjected at
the two to four cell stage four times with a total of 2 ng TsgW67G
or 8 ng of a 1:1 mixture of the two Chordin morpholinos. Embryos at the 32-64
cell stage were treated for 29 minutes with 120 mM LiCl in 0.1xMBS
saline (Larraín et al.,
2000
; Sive et al.,
2000
). The dorsoanterior index (DAI) was determined at stage 30
(Kao and Elinson, 1988
).
Treatment of animal cap explants with human recombinant Activin protein
(R&D Systems) was as described
(Piccolo et al., 1999
). RNA
was isolated at stage 25. RTPCR conditions and PCR primers used have been
described elsewhere (Sasai et al.,
1995
)
(http://www.hhmi.ucla.edu/derobertis/index.html).
To genotype chordinott250 mutant embryos two primers were
used for PCR: chd-2 GCA GAA ACG TCT ACG TTT CC and chd-3 CGT TTT AGT TGG TGC
TCT TGA CG. Following digestion with MspI, the wild-type allele was
digested whereas the mutant allele was not.
Protein biochemistry
Xenopus Tsg and the mutant Tsg proteins were affinity purified
using the anti-FLAG M2 affinity gel column (Sigma). The anti-FLAG column (0.5
ml) was washed once with 5 ml 0.1 M glycine, pH 3.5 and three times with 5 ml
aliquots of TBS (50 mM Tris HCl, 150 mM NaCl, pH 7.4). Conditioned medium from
human 293 cells transfected with pCS2-ChdN-xTsg or Tsg point mutants was
harvested and loaded directly onto the column. After washing three times with
10 ml TBS, the bound protein was eluted with five aliquots of 1 ml TBS
containing 100 µg/ml FLAG peptide (Sigma). Protein concentrations were
estimated by comparison with BSA standards after Coomassie Blue staining.
Analyses of protein secreted by animal cap explants were performed as
described (Oelgeschläger et al.,
2000). The supernatant was used for western blots probed with an
antibody specific for the inter-repeat region of Chordin (
-I-Chd)
(Piccolo et al., 1997
) that
had been blot-affinity purified
(Larraín et al., 2001
).
For the expression of proteins in co-cultures, 293T cells were independently
transfected with mouse Chordin, Bmp4, Tsg and TsgW66A expression
plasmids using FuGENE (Roche), and chemical crosslinking and
immunoprecipitation experiments carried out as described
(Oelgeschläger et al.,
2000
; Larraín et al.,
2001
).
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RESULTS |
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|
The C-terminal cysteine-rich domain of Tsg did not show any significant homology to known protein motifs in the databases but is conserved across Tsgs from different species. Mutations in several of its conserved cysteine residues were tested, two of which (xTsgC180A, xTsgC198A) had a phenotypic effect causing dorsalization, with reduced trunk and enlarged head structures (Fig. 1I and data not shown).
When W67G, the strongest ventralizing (pro-BMP) mutation, was combined with one of the dorsalizing mutations (C198A), the resulting double mutant (xTsgW67G+C198A) had almost no ventralizing activity in overexpression experiments (Fig. 1J, compare with 1H). However, in a subset of embryos injected with this double mutant weak phenotypes, including reduced head structures and defects in tail development could be observed. Mutation of a potential glycosylation site (xTsgS54A) resulted in a typical xTsg phenotype, with reduced head structures and a posteriorized anus, although the phenotype was stronger than its wild-type counterpart (compare Fig. 1D,F). S54 seems to be a site of glycosylation in vivo, as the mutant protein had faster electrophoretic mobility (Fig. 1K, compare lanes 1 and 3). Perhaps the enhanced activity of this glycosylation site mutant is due to increased diffusibility in the embryo.
We conclude from these data that mutations in the N-terminal cysteine-rich domain enhanced the ventralizing (pro-BMP) activity of Tsg, whereas those in the C-terminal domain caused the opposite effect, dorsalization. The phenotype of the double mutant further demonstrated that the hyperventralizing activity of xTsg mutants requires the function of the C-terminal cysteine-rich domain.
The hyperventralizing xTsgW67G mutant
To compare the ventralizing effects of wild-type xTsg and
TsgW67G on CNS patterning, we analyzed the neural plate marker
Sox2 by in situ hybridization. At early neurula stages,
microinjection of 1 ng of xTsg mRNA led to reduction of the neural
plate and the formation of a ring of Sox2 positive cells surrounding
the slit-shaped blastopore (Fig.
2A,B). This ring may indicate a posteriorization of the embryo,
and correlated with the posteriorized anus phenotype at tadpole stages. The
mass of posteriorized and detached endoderm expressed Sox2, which
marks the proctodeum (Fig.
2K,L) (Chalmers et al.,
2000). At higher concentrations (4 ng), xTsg mRNA reduced
the neural plate further, indicating that the wild-type protein has pro-BMP
effects (Fig. 2C)
(Oelgeschläger et al.,
2000
). Results were different when the hyperventralizing mutant
TsgW67G was microinjected: the reduction of the neural
plate was much more severe and the ring of Sox2-positive cells as
well as the posteriorized anus were not seen
(Fig. 2D,I).
|
In histological sections, embryos overexpressing wild-type xTsg showed a
mild reduction of the spinal cord and somites
(Fig. 2M,N) and in some cases a
hypoplastic notochord (data not shown)
(Oelgeschläger et al.,
2003). By contrast, embryos injected with TsgW67G
showed considerable reduction of the brain, spinal cord, notochord and somites
(Fig. 2O) (data not shown).
We conclude that xTsg mRNA inhibited development of the anterior neural plate and posteriorized the embryo. In the hyperventralizing mutant TsgW67G, the pro-BMP activities of wild-type xTsg were exacerbated.
Tsg and TsgW67G antagonize dorsalization of mesoderm by
Activin
In Xenopus, formation of dorsal mesoderm is mediated by a
horizontal signal secreted by Spemann's organizer
(Dale and Slack, 1987). This
signal can be mimicked by treatment of ectodermal explants with Activin
protein (Green et al., 1992
;
Dyson and Gurdon, 1998
).
Activin induces the expression of chordin in ectodermal explants, and
we have recently shown that this expression of endogenous Chordin is required
for dorsal mesoderm induction
(Oelgeschläger et al.,
2003
). Treatment of ectodermal explants with 2 ng/ml Activin
triggered the convergence-extension movements that accompany dorsal mesoderm
formation (Fig. 3B). In
explants microinjected with xTsg or TsgW67G,
animal cap elongation was blocked (Fig.
3C,D). RT-PCR analysis confirmed that the induction by Activin of
the dorsal mesodermal markers MyoD and
-Actin and of
the pan-neural marker NCAM was inhibited by wild-type xTsg
or TsgW67G mRNAs (Fig.
3F). Overexpressed xTsg is known to induce degradation of Chordin
protein secreted by Spemann's organizer
(Larraín et al., 2001
).
As expected, microinjection of xTsg mRNA led to a decrease of
full-length Chordin protein secreted by Activin-treated animal caps
(Fig. 3E, lanes 2, 3). By
contrast, TsgW67G increased the amount of Chordin protein secreted
by Activin-treated animal caps (Fig.
3E, lane 4). Thus, although full-length Chordin protein was
abundantly produced in animal caps in the presence of TsgW67G
(Fig. 3E, lane 4), this Chordin
protein was inactive in mesoderm dorsalization
(Fig. 3F, lane 5).
|
Hyperventralizing Tsg proteins do not bind BMP
To investigate the molecular mechanisms that underlie the ventralizing
activities of xTsg and TsgW67G, we carried out a series of
functional and biochemical experiments. As reported previously, xTsg RNA
induced expression of the cement gland marker XAG-1 in animal cap
explants (Fig. 4A, lane 3)
(Chang et al., 2001;
Larraín et al., 2001
).
However, this anti-BMP effect was not sufficient to induce the expression of
the pan-neural marker NCAM, which requires even lower levels of BMP
signaling (Fig. 4A). We tested
the various xTsg point mutants in animal cap explants and found that, like
wild-type xTsg, the dorsalizing mutant xTsgC198A induced
XAG-1 but not NCAM expression
(Fig. 4A, lane 5). The
hyperventralizing mutations xTsgS36A, xTsgC59A and
xTsgW67G did not induce XAG-1 expression in animal cap
explants (Fig. 4A, lane 4 and
data not shown). One possible explanation for this difference was that the
N-terminal mutations might prevent binding of xTsg to BMP.
|
TsgW67G binds to Chordin
To test whether binding to Chordin was affected by the mutations,
affinity-purified mutant xTsg proteins were incubated with full-length
Xenopus recombinant Chordin protein, and complexes chemically
crosslinked with disuccinimidyl suberate (DSS). Wild-type xTsg protein formed
a high molecular weight complex in the presence of Chordin, corresponding to a
dimer of xTsg bound to Chordin (Fig.
4C, lane 2). A similar complex was observed in the presence of
TsgW67G and Chordin protein
(Fig. 4C, lane 4). By contrast,
Chordin-Tsg complexes were not detectable using the C-terminal mutant
TsgC198A (Fig. 4C, lane 6).
We conclude from these biochemical experiments that the hyperventralizing mutant TsgW67G is not affected in its ability to bind to Chordin, and that dorsalizing mutations in the C-terminal domain prevent binding of xTsg to Chordin.
TsgW67G does not form ternary complexes with Chordin and
BMP
We next asked whether the lack of BMP binding activity in hyperventralizing
xTsg mutants affected the formation of ternary complexes with BMP and Chordin.
Affinity-purified xTsg proteins (at a concentration of 20 nM) were incubated
with 5 nM BMP4 and 2 nM Chordin protein, chemically crosslinked with DSS, and
BMP4 visualized with a monoclonal antibody specific for BMP4
(Masuhara et al., 1995).
Incubation of BMP4 and Chordin protein led to the formation of a high
molecular weight complex with a molecular mass corresponding to a BMP4 dimer
bound to a Chordin monomer (Fig.
4D, lane 2). Incubation of wild-type Tsg protein with Chordin and
BMP4 generated a ternary complex corresponding to a Tsg dimer bound to a BMP4
dimer and Chordin (Fig. 4D,
lane 3).
By contrast, when TsgW67G protein was used, only the Chordin-BMP4 complex was detectable (Fig. 4D, lane 4). When the same blot was incubated with a Flag-Tsg specific antibody, complexes of Chordin and TsgW67G were detectable (Fig. 4E, lane 4). As ternary complexes were not formed, this indicates that the full-length Chordin protein bound to TsgW67G lost its BMP4 binding ability. This result was surprising, as Chordin is a large protein containing four BMP-binding domains of the CR type. Tsg is a rather small molecule, five times smaller than Chordin, with only one BMP interaction domain. These results raise the possibility that TsgW67G might inactivate the BMP-binding activity of Chordin in an active way (see below).
The inability of TsgW67G to form ternary complexes with Chordin and BMP was confirmed using a co-culture system of human 293T kidney cells. Cell cultures separately transfected with expression constructs for mouse Chordin, BMP4, mTsg or mTsgW66G were mixed, cultured together for 24 hours, and proteins secreted into the culture medium analyzed after chemical crosslinking. These conditions greatly increased the avidity of the monoclonal antibody for BMP4 in trimolecular complexes. (Fig. 4F, lanes 2-4 and data not shown). Even under these conditions, which greatly facilitate the detection of trimolecular Tsg-Chd-BMP complexes, the TsgW66G mutation precluded the binding of BMP4 to Chordin complexes (Fig. 4F, lane 6).
We conclude from these biochemical experiments that the hyperventralizing mutations prevent binding of Tsg to BMP4 but allow binding to full-length Chordin. Binding of xTsgW67G to Chordin prevents binding of BMP to either protein, and this inhibition correlates with the enhanced ventralizing activity of this Tsg mutant.
TsgW67G inhibits CR-modules
When Chordin is digested by the Tolloid/Xolloid metalloprotease the
molecule is cleaved twice, releasing intact CR modules that can still bind and
inhibit BMP. Unlike full-length Chordin, individual CR modules do not form
stable complexes with xTsg. However, wild-type xTsg has the ability to
antagonize the residual anti-BMP activity of the proteolytic fragments
(Oelgeschläger et al.,
2000; Larraín et al.,
2001
). We tested the effects of TsgW67G on
Xenopus CR1, a protein that mimics the N-terminal cleavage product.
Microinjection of mRNA encoding xCR1 induced the expression of the anterior
neural marker genes Rx2a and Six3, the pan-neural marker
gene NCAM, and the cement gland marker XAG-I in animal cap
explants (Fig. 5A, lane 3).
Co-injection of wild-type xTsg mRNA inhibited the expression of
anterior neural marker genes but expression of XAG-1 was still
observed (Fig. 5A, lane 4).
Co-injection of TsgW67G mRNA completely blocked the
induction of XAG1 and of neural markers by xCR1
(Fig. 5A, lane 5). In addition,
expression of the epidermal BMP target gene Msx1, which was inhibited
by xCR1, was restored by TsgW67G mRNA
(Fig. 5A).
|
TsgW67G is specific for CR-containing proteins
We next tested the effect of TsgW67G on other inhibitors of BMP
signaling that lack CR-modules. Ventral injection of tBR mRNA
resulted in the formation of partial secondary axes that were not affected by
co-injection of TsgW67G mRNA
(Fig. 5D,E). This shows that
TsgW67G acts on the BMP signaling pathway, upstream of the BMP
receptor. Microinjection of noggin mRNA induced secondary axes that
were not affected by co-injection of TsgW67G mRNA
(Fig. 5F,G). By contrast, axes
induced by injection of xCR1 mRNA were abolished by co-injection of
TsgW67G (Fig.
5B,C). As TsgW67G does not affect dorsalization by tBR
or Noggin, we conclude that the effects of TsgW67G in the embryo
are specific for CR modules.
Hyperventralizing Tsgs compared with Chordin loss of function
Chordin downregulation after microinjection of morpholino oligonucleotides
results in a moderate ventralization of Xenopus embryos comparable
with that of chordino mutant zebrafish
(Oelgeschläger et al.,
2003). The phenotype of the hyperventralizing mutants
xTsgW67G and xTsgC59A is much more severe, causing
extensive loss of the CNS, particularly in the anterior
(Fig. 1G,H). Striking pro-BMP
effects of xTsgW67G were observed in LiCl-treated
Xenopus embryos. Embryos treated at the 32-cell stage with 120 nM
LiCl expressed organizer genes such as Chordin throughout the
marginal zone at gastrula and cause the development of embryos with radial
head structures lacking trunk-tail structures
(Fig. 6A,B; Dorsoanterior
index, DAI=9.25, n=16). The effect of LiCl can be blocked by
microinjection of Chordin morpholinos
(Oelgeschläger et al.,
2003
). Even in the presence of LiCl, Chordin morpholinos produced
a chordino-like weakly ventralized phenotype
(Fig. 6D). Overexpression of
TsgW67G had a strong effect on LiCl treated embryos. Microinjection
of TsgW67G mRNA into each of the four blastomeres resulted
in embryos that were strongly ventralized and lacked CNS and axial structures,
despite having been treated with LiCl (Fig.
6C, DAI=1.1, n=11). Thus, the ventralizing effect of
TsgW67G is much stronger than that of Chordin loss of function, and
suppressed most dorsal development even in what should have been dorsalized
LiCl-treated embryos.
|
|
Whereas wild-type xTsg overexpression may cause both dorsalization and
ventralization in zebrafish, hyperventralizing xTsgW67G
and xTsgC59A constructs had only pro-BMP effects. As seen
in Fig. 7E-G, the anterior CNS
was hypoplastic, lacking brain and eyes in the most severe cases, and trunk
somites were defective or absent. The degree of ventralization caused by
xTsgW67G and xTsgC59A was more severe
(Fig. 7) than the
ventralization caused by complete loss-of-function of Chordin in zebrafish
(Hammerschmidt et al., 1996a;
Ross et al., 2001
;
Wagner and Mullins, 2002
) or
by knock-down using Chordin morpholinos in Xenopus
(Oelgeschläger et al.,
2003
). When 800 pg xTsg W67G mRNA was injected
into embryos resulting from din/+ x din/+ crosses
(n=147), we recovered 14 embryos that had a din/din genotype
by PCR but a ventralized phenotype stronger than that in chordino
mutants. We used the tt250 allele of chordino, which results
in a null mutation (Hammerschmidt et al.,
1996a
; Schulte-Merker et al.,
1997
; Wagner and Mullins,
2002
). Therefore, these experiments demonstrate that
xTsgW67G can exert ventralizing effects even in the absence of
Chordin protein.
As the hyperventralizing Tsg activities are specific for proteins containing CR modules (Fig. 5), these data indicate that other CR-containing proteins, in addition to Chordin, must be required for the establishment of dorsal cell fate in Xenopus and zebrafish development.
![]() |
DISCUSSION |
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Mutations in conserved cysteines of the C-terminal domain at positions 180 and 198 resulted in the opposite phenotype, a mild dorsalization, with enlarged head structures and shortened trunks. This phenotype indicated anti-BMP activity, which could be explained by their maintaining BMP-binding ability in combination with a loss of the ventralizing activity contained in the C-terminal domain. When the strongest ventralizing and dorsalizing mutations were combined in the same molecule, the resulting molecule had almost no activity in overexpression assays. Thus, the two conserved domains of Tsg appear to have opposing biological activities.
The hyperventralizing Tsg mutants
Biochemical studies showed that xTsgC59A and xTsgW67G
mutants were unable to bind BMP4 but were still able to bind to full-length
Chordin. The C-terminal mutations, however, were unable to bind Chordin,
suggesting that their dorsalizing activity is caused exclusively by BMP
binding. The strongest ventralizing mutant, xTsgW67G, bound to
full-length Chordin and prevented the recruitment of BMP into a ternary
complex (Fig. 4D,F).
The hyperventralizing mutants of xTsg described here are not a neomorphic
activity resulting from the point mutations. This is because wild-type xTsg
also has ventralizing effects in Xenopus embryos
(Fig. 2B,C)
(Oelgeschläger et al.,
2000). Wild-type xTsg mRNA has very potent ventralizing
effects (inhibition of CNS, notochord or muscle) in Xenopus embryos
in which the levels of Chordin have been reduced by microinjection of
chordin antisense morpholino oligos
(Oelgeschläger et al.,
2003
).
xTsgW67G was not only able to antagonize the activity of
full-length Chordin, but also that of an isolated CR module
(Fig. 5). We had previously
shown that CR1 was able to bind and antagonize BMP and that this binding could
be competed by increasing amounts of xTsg protein without formation of ternary
complexes (Larraín et al.,
2001). It was assumed that xTsg acted via competition for BMP
binding. However, as xTsgW67G did not bind BMP, the inactivation of
the anti-BMP activity of this CR module must be achieved through a different
molecular mechanism. This raises the possibility that the C-terminal domain of
Tsg contains a biochemical activity able to inactivate CR domains. Although
the C-terminal domain of Tsg presented some homology to bacterial nitrate
reductases (narH) in blast searches, these similarities were weak and the
catalytic residues not conserved. It will be important to compare this domain
of Tsg to other enzymes once its three-dimensional structure is available.
The N-terminal domain could have an autoinhibitory effect on the
biochemical activity of the C terminus. Autoinhibitory mechanisms by
intramolecular interactions have been described for a variety of enzymes
including the Src tyrosine kinase, GSK3ß and ubiquitin ligases
(Nguyen and Lim, 1997;
Harwood, 2001
;
Du et al., 2002
), and are
commonly described as the jack-knife model. A requirement of the N-terminal
domain for autoinhibition of Tsg might explain why multiple mutations in this
domain have similar phenotypic effects on an intriguing activity that resides
in the C terminus of the molecule.
xTsg must regulate multiple CR-containing proteins
The ventralizing (pro-BMP) activity of xTsgW67G was specific for
proteins containing CR modules, as it was able to block Chordin and an
isolated CR-domain, but did not inhibit the structurally unrelated BMP
antagonist Noggin or a dominant-negative BMP receptor (tBR). Biochemical
analyses have so far failed to detect stable binding of xTsg or
xTsgW67G to isolated CR domains
(Oelgeschläger et al.,
2000; Larraín et al.,
2001
) (M.O. and E.M.D.R., unpublished). The ventralizing activity
of xTsgW67G mRNA is very potent, for it can almost completely
eliminate formation of CNS and dorsal mesoderm when overexpressed in
Xenopus or zebrafish embryos. In addition, xTsgW67G
completely blocked the dorsalizing effects of LiCl and Activin protein.
The effects of xTsgW67G cannot be solely due to inactivation of
endogenous Chordin protein. In zebrafish embryos, overexpression of
hyperventralizing Tsg mutants (Fig.
7) resulted in embryos that were much more ventralized than a
chordino null allele
(Hammerschmidt et al., 1996a;
Schulte-Merker et al., 1997
;
Wagner and Mullins, 2002
).
Experiments using chordino mutant zebrafish embryos confirmed that
xTsgW67G can exert ventralizing effects that are independent of the
presence of Chordin. Taken together, these results indicate that
hyperventralizing xTsg mutants must act on other CR-containing proteins in
addition to Chordin.
Chordin is probably only the tip of the iceberg. CR-containing proteins are
part of a growing family of secreted proteins
(Garcia-Abreu et al., 2002),
many of which have been shown to bind BMP. For example, CTGF and isoforms of
procollagens contain single CR modules and have been shown to bind both BMPs
and TGFßs (Abreu et al.,
2002
; Zhu et al.,
1999
; Larraín et al.,
2000
). Kielin, CRIM1 (cysteine-rich motor neuron 1), Crossveinless
2, Neuralin 1/Ventroptin and Neuralin 2 contain multiple CR repeats and have
been implicated in the regulation of BMP and TGFßs
(Matsui et al., 2000
;
Kolle et al., 2000
;
Larraín et al., 2000
;
Coffinier et al., 2001
;
Nakayama et al., 2001
;
Sakuta et al., 2001
;
Coffinier et al., 2002
). Any of
these CR-containing proteins, or as yet undiscovered ones, may contribute to
dorsoventral patterning in the course of normal development.
![]() |
ACKNOWLEDGMENTS |
---|
![]() |
Footnotes |
---|
Present address: Departamento de Biologia Celular y Molecular, Centro de
regulación Celular y Patologia, P. Universidad Católica de
Chile, Alameda 340, Santiago, Chile
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