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First published online 26 January 2006
doi: 10.1242/dev.02250
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1 Max-Planck-Institute of Immunobiology, Stuebeweg 51,79108 Freiburg,
Germany.
2 Department of Physiological Chemistry II, Biocenter, University of Wuerzburg,
Am Hubland, 97074 Wuerzburg, Germany.
Author for correspondence (e-mail:
hammerschmid{at}immunbio.mpg.de)
Accepted 14 December 2005
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SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: Zebrafish, Crossveinless 2, Bmp
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). Bmps are members of the transforming growth factor ß
(Tgfß) superfamily of proteins, which signal through transmembrane
receptor complexes consisting of type I and type II serine/threonine kinases.
Activation of the receptors leads to phosphorylation of type I by type II
receptors, followed by phosphorylation of intracellular R-Smad proteins by
type I receptor kinases. Upon their phosphorylation, R-Smads dissociate from
the receptors, form complexes with Co-Smads, like Smad4, and translocate to
the nucleus to participate in the transcriptional regulation of target genes
(Baker and Harland, 1997;
Heldin et al., 1997).
Given the high sensitivity of developing cells to Bmp signals, the activity
of Bmps is under tight spatial and temporal regulation. One important level of
regulation occurs in the extracellular space, mediated by various secreted
Bmp-binding proteins (Balemans and Van Hul,
2002). Some of these have rather complex functions. Chordin for
example binds to Bmps and prevents the interaction of Bmps with their
receptors, accounting for Bmp inhibition
(Piccolo et al., 1996). In
addition, Chordin can have subtle long-range pro-Bmp effects, as demonstrated
for the Drosophila Chordin and Bmp homologues Sog and Dpp: Sog/Dpp
complexes seem to diffuse within the extracellular space, with Sog carrying
Dpp away from the Sog source (Ashe and
Levine, 1999; Decotto and
Ferguson, 2001). By cleaving Bmp/Dpp-bound Chordin/Sog, Tolloid
metalloproteases can then release biologically active Bmps from the complex
(Piccolo et al., 1997),
resulting in increased Bmp/Dpp levels at the sites of Tolloid activity
(Ashe and Levine, 1999;
Blader et al., 1997;
Decotto and Ferguson, 2001).
Another factor with a similar dual effect on Bmp signaling is Twisted
gastrulation, which promotes binding of Chordin to Bmps (anti-Bmp effect), but
also the cleavage of Chordin by Tolloid (pro-Bmp effect)
(Chang et al., 2001;
Larrain et al., 2001;
Oelgeschlager et al., 2000;
Ross et al., 2001;
Scott et al., 2001;
Shimmi and O'Connor, 2003).
These examples show that Bmp-binding proteins cannot be generally categorized
as anti- or pro-Bmp factors, but rather can have differential functions
depending on the molecular context.
In the early zebrafish and Xenopus embryo, ventrally expressed
Bmps and dorsally expressed Bmp inhibitors establish a ventral-to-dorsal
gradient of Bmp activity, which is required for patterning of the dorsoventral
axis (De Robertis et al.,
2000; Hammerschmidt and
Mullins, 2002; De Robertis and
Kuroda, 2004; Schier and
Talbot, 2005). Accordingly, the zebrafish mutants bmp2b/swirl,
bmp7/snailhouse, alk8/lost-a-fin (a type I Bmp receptor),
smad5/somitabun and tolloid/minifin display dorsalized
phenotypes (Bauer et al., 2001;
Connors et al., 1999;
Dick et al., 2000;
Hild et al., 1999;
Kishimoto et al., 1997;
Mintzer et al., 2001;
Nguyen et al., 1998;
Schmid et al., 2000). By
contrast, the chordin/dino mutant is ventralized
(Fisher et al., 1997;
Schulte-Merker et al., 1997),
with additional subtle dorsalized traits in ventral-most derivatives,
reflecting the dual anti- and pro-Bmp function of Chordin described above
(Hammerschmidt and Mullins,
2002; Wagner and Mullins,
2002). The effects of loss of Tsg function on dorsoventral
patterning of the zebrafish embryo are less clear. Although according to one
report, Tsg is a Bmp antagonist (Ross et
al., 2001), other studies come to the opposite conclusion,
indicating a pro-Bmp effect of Tsg (Little
and Mullins, 2004; Xie and
Fisher, 2005).
The Bmp binding of Chordin is mediated by CR repeats that are characterized
by ten cysteine residues with a conserved spacing pattern
(Larrain et al., 2000). CR
domains are also present in Crossveinless 2 (Cvl2 or Cv-2). Although in
vertebrates, the role of Cvl2 had not been analyzed via loss-of-function
studies as yet, analyses of Drosophila mutants indicate that it is
required to enhance Bmp signaling during wing vein development
(Conley et al., 2000).
cvl2 gain-of-function experiments in vertebrate cell culture systems
and Xenopus embryos led to conflicting results, showing either pro-
or anti-Bmp effects (Binnerts et al.,
2004; Kamimura et al.,
2004; Moser et al.,
2003; Coles et al.,
2004).
Here, we describe the first loss-of-function study for a vertebrate crossveinless 2 homologue. We show that zebrafish cvl2 acts as a Bmp signaling-promoting factor during early embryonic development and that this activity has Chordin-dependent and -independent aspects. We also show that proteolytic cleavage can convert Cvl2 from an anti- to a pro-Bmp factor and modifies its interaction with the extracellular matrix. These findings might help to explain the controversies about the role of Cvl2 reported in previous gain-of-function studies.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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)
dinott250; ogontm305
(Hammerschmidt et al., 1996a)
and ogonm60
(Solnica-Krezel et al., 1996).
The generation of hsp70::bmp2b and hsp70::noggin3 transgenic
fish lines will be described elsewhere (F.R. and M.H., unpublished).
Isolation of zebrafish crossveinless2, RT-PCR and generation of expression constructs
Conditions and PCR primers used for cloning of zebrafish cvl2, for
the generation of expression constructs, for temporal RT-PCR analyses and for
genomic mapping of cvl2 are available from the authors upon request.
cvl2-N contains amino acids 1-355, in cvl2-CM residues
350-355 (VFGDPH) are replaced by LVPRGS.
Morpholino oligonucleotides
The sequences of the used cvl2 antisense morpholino
oligonucleotides are: TTA CTG GAG GAG ACA GAC ACA GCA T (ATG-MO=MO1)
corresponding to nucleotides +1 to +25 of the cDNA; CTA AAT TCG CTC CAG ACG
CAC GGG (UTR-MO=MO2) corresponding to nucleotides -25 to -2 in the
5'UTR. Sequences of the respective mismatch control MOs were: ACa GGA
cGA GAC AGA CAg AGC tTC C (-2 to +23); CTA AAT TCc CTg CAc ACG gAC cGG. Unless
stated otherwise, 1 nl containing 2.4 ng MO was injected per embryo.
In situ hybridization and immunohistochemistry
In situ hybridization and whole-mount immunostaining was performed as
described previously (Hammerschmidt et
al., 1996a). Staining for phosphorylated Smads was done using an
anti-pSmad1/5/8 antibody (Cell Signaling Technology) at a concentration of
1:200 and a Cy3-coupled secondary antibody (Jackson ImmunoResearch Labs).
Graphical illustrations of staining intensities were generated from Photoshop
images of vegetal views, using ImageJ software
(http://rsb.info.nih.gov/ij/).
For Western blot analyses, dechorionated midgastrula embryos were
dissociated in Ca-free Ringer's solution; cells were separated from yolk via
centrifugation, and protein extracts were prepared by standard procedures
(Westerfield, 1994). Extracts
were separated on 8% polyacrylamide gels. Myc-tagged fusion proteins were
detected with anti c-myc antibody 9E10 (Santa Cruz Biotechnology),
anti-pSmad1/5/8 antibody (Cell Signaling Technology) was used at a
concentration of 1:1000.
Biacore analyses
A detailed description of the expression and purification of recombinant
proteins, microsequencing of the Cvl2 fragments and interaction assays is
available from the authors upon request. Recombinant Chd protein was obtained
from R&D systems (Minneapolis, MN).
Cell culture and heparin binding experiments
Plasmids pCS2-cvl2-CM, pCS2-cvl2-WT and
pCS2-cvl2-N were transfected into HEK293 cells with the
calciumphosphat method, efficiency was controlled by co-transfection of
pCS2-gfp. Transfected cells at 
60% confluence were incubated
with serum-free medium for 48 hours. Separation of supernatant, cells and ECM
was carried out as described previously
(Novikova et al., 2000).
Briefly, the supernatant was collected and the cells were washed off with cold
PBS. The ECM attached to the culture dish was washed off with 95°C sample
buffer (50 mM Tris, pH 6.8, 1% SDS, 4% glycerol).
Heparin binding assays were carried out as described by Jasuja et al.
(Jasuja et al., 2004), using
proteins from cell extracts obtained in the transfection experiments.
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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1 hour before the onset of
gastrulation, and continued during all later developmental stages investigated
(Fig. 1B).
Whole-mount in situ hybridization revealed that during blastula stages,
cvl2 was broadly expressed at rather low levels (not shown). During
gastrula stages (shield, 60% epiboly, 80% epiboly), cvl2 transcripts
were predominantly found on the ventral side of the embryo, with from ventral
to dorsal decreasing mRNA levels (Fig.
1C,D,I). Additionally, transcripts became detectable around nuclei
of the yolk syncytial layer (YSL, Fig.
1E,K) and in the prechordal plate (arrow in
Fig. 1D). At the tailbud stage
(end of gastrulation), cvl2 was most prominently expressed in the
lateral mesoderm along the entire anteroposterior length of the embryo
(Fig. 1E,F). During
segmentation stages, this domain became restricted to the anterior half of the
embryo (Fig. 1G,H) and to the
tailbud mesoderm (see Fig. S2A in the supplementary material). Interestingly,
all these expression domains correspond to areas of bmp expression:
bmp2b, bmp4 and bmp7 are expressed ventrally, bmp2b
in the YSL, and bmp4 in the prechordal plate
(Dick et al., 2000;
Nikaido et al., 1997;
Schmid et al., 2000).
cvl2 expression depends on Bmp signaling
Analysis of cvl2 expression in mutants with altered Bmp activity
revealed a positive regulation of cvl2 by Bmp signaling. In
chordin/dino mutants, ventral expression of cvl2 was
expanded to the dorsal side (Fig.
1I,J). By contrast, in bmp2b/swirl mutant embryos,
ventral expression was absent (Fig.
1K). These data suggest that cvl2 expression is under the
positive control of Bmp signaling. The same appears to be true for later
stages of zebrafish development, when cvl2 was expressed in a variety
of different tissues, including the otic vesicles and the pharyngeal pouches
(Fig. 1L and Fig. S2 in the
supplementary material). Using transgenic zebrafish lines expressing
bmp2b or the Bmp inhibitor noggin3 under the control of a
heatshock-inducible promoter (Halloran et
al., 2000) (F.R. and M.H., unpublished), we obtained complete loss
of this late cvl2 expression upon blockage of Bmp signaling
(Fig. 1N). However,
bmp2b overexpression strongly increased cvl2 transcript
levels without inducing cvl2 expression at ectopic sites
(Fig. 1M).
Cvl2 is required to promote Bmp signaling during dorsoventral patterning of the zebrafish embryo
To study whether Cvl2 is required for early zebrafish development, we
carried out loss-of-function experiments, using antisense morpholino
oligonucleotides (MO) (Nasevicius and
Ekker, 2000). Used MOs efficiently inhibited translation of
cvl2 transcripts, as revealed via western blotting of protein
extracts from embryos co-injected with cvl2 MOs and mRNA encoding a Cvl2-Myc
fusion protein (Fig. 2A).
Phenotypically, such MO-mediated loss of Cvl2 function led to reduced Bmp
signaling and moderate dorsalization of the embryo.
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)
(Fig. 2G,H), cvl2
itself (Fig. 2I,J),
gata2 (ventral ectoderm) (Detrich
et al., 1995) (Fig.
2K,L) and tfap2a (ventral ectoderm)
(Knight et al., 2003) (see
Fig. S3E,F in the supplementary material) at midgastrula stages, and a reduced
number of ventral cell types such as blood precursors (marked by expression of
gata1) (Detrich et al.,
1995) (Fig. 2O,P)
at early segmentation stages. By contrast, expression of tbx6, a
marker for the entire non-axial mesoderm largely under the control of Wnt8
(Szeto and Kimelman, 2004;
Ramel et al., 2005), was only
moderately affected in midgastrula cvl2 morphants (Fig. S3C,D), while
the expression domains of dorsal markers like otx2
(Li et al., 1994)
(Fig. 2M,N), which mark
anterior neuroectoderm, were ventrally expanded. Accordingly, at segmentation
stages, cvl2 morphants displayed an expansion of dorsal and paraxial
cell types, as apparent by a broadening of hindbrain rhombomeres 3 and 5,
revealed by krox20 expression
(Fig. 2Q,R), and by a
broadening of the anterior somites, revealed by myoD expression (see
Fig. S3G,H in the supplementary material). These data indicate that Cvl2 is
required for ventral specification both in the mesoderm and the ectoderm.
Similar results were obtained with two different cvl2 MOs, but not
with the corresponding mismatch control MOs. The effect of used MOs was
specific, as shown by the rescue of the morphant phenotype to near wild-type
condition upon co-injection of low doses of mRNA encoding zebrafish Cvl2 (for
the UTR-MO) or Drosophila Cvl2 (for both the UTR-MO and the ATG-MO;
Fig. 2S,T; and data not shown).
The rescue with Drosophila cvl2 mRNA further indicates that the
function of Cvl2 proteins has been conserved across invertebrate and
vertebrate species.
Partial loss of Cvl2 synergistically enhances dorsalization of Bmp pathway mutants
To further analyze the functional interaction of cvl2 with the Bmp
pathway, we injected suboptimal amounts of cvl2 MO into mutants with
weakly reduced Bmp activity, such as tolloid/minifin. minifin
embryos, as well as embryos injected with low doses of cvl2 MO (0.8
ng per embryo), display a very subtle expansion of the neuroectoderm, as shown
by krox20 staining (Fig.
2U-W). Injection of the same low doses of cvl2 MO into
minifin embryos, however, resulted in strongly enhanced dorsalization
(Fig. 2X). The same enhancement
was obtained upon co-injection of low doses of cvl2 MO with low doses
of chordin, noggin or twisted gastrulation RNA, or upon
injection into weakly dorsalized alk8/lost-a-fintm110
mutants (confirmed by genotyping; data not shown). Together, these results
strengthen the notion that during early dorsoventral patterning, Cvl2 has an
essential ventralizing function and functionally interacts with Bmp
signaling.
Loss of Cvl2 function has differential and moderate effects in chordin mutants
We next analyzed the functional interaction of cvl2 with Chordin,
performing epistasis analyses by injecting cvl2 MOs into homozygous
chordin mutants to generate double-deficient embryos.
chordin/dino mutants display elevated Bmp signaling and are
ventralized, as morphologically indicated by a reduction of the brain, a
derivative of the dorsal ectoderm, and an enlargement of the blood islands, a
derivative of the ventral mesoderm
(Hammerschmidt et al., 1996a;
Hammerschmidt et al., 1996b).
The phenotype in the ventral tail fin, a derivative of the ventral ectoderm,
is more complex. Although other ventralized mutants such as
sizzled/ogon (Martyn and
Schulte-Merker, 2003; Wagner
and Mullins, 2002; Yabe et
al., 2003) display a duplication of the ventral tail fin over its
entire length (see Fig. S3J in the supplementary material), a variable
percentage of chordin mutants show only duplications in
posterior-most regions of the ventral tail fin, whereas more anteriorly,
ventral tail fin tissue is missing (Fig.
3A-D). This feature reflects the additional subtle pro-Bmp
activity of Chordin in regions of the ventral ectoderm that require peak Bmp
levels (see Introduction).
To quantify the effects of cvl2 MOs in the ventral tail fin, we classified chordin mutants according to the length of the fin gap (class I: no gap, Fig. 3B; class II: partial gap, Fig. 3C; class III: complete gap, Fig. 3D), reflecting the degree of reduction of Bmp activity in this tissue. Injection of low doses of cvl2 MO (0.8 ng/embryo), which had no effect in wild-type siblings, significantly enhanced the loss of ventral tail fin tissue of chordin mutants (see numbers in Fig. 3B-D). However, ventralized traits at other sites of chordin mutants appeared less sensitive to Cvl2 levels. Thus, the blood islands of mutants even injected with highest doses of cvl2 MOs (3 ng/embryo) remained their increased size, as also confirmed by gata1 staining at the 10-somite and 24 hpf stage (data not shown). Similarly, injection of highest doses of cvl2 MOs into dino mutants or chordin morphants only led to a very subtle alleviation of the reduced krox20 expression (Fig. 3E). The unresponsiveness of these phenotypic traits of chordin mutants to loss of Cvl2 indicates that here, Chordin is epistatic to Cvl2, suggesting that Cvl2 fulfills part of its pro-Bmp function via an inhibitory effect on Chordin. However, the enlarged gap in the ventral tail fin of chordin mutants injected with cvl2 MO suggests that Cvl2 in addition promotes Bmp signaling independently of a putative Chordin-antagonizing function.
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45 kDa
including the Myc-tag) in addition to full-length Cvl2 protein (90 kDa
including the Myc-tag) (Fig.
4A, left lane). The smaller fragment was obtained only under
reducing electrophoresis conditions, whereas under non-reducing conditions,
only one band corresponding to full-length protein was visible
(Fig. 4A, right lane). The size
of full length Cvl2 is slightly larger than the calculated mass (86 kDa
including the Myc-tag), presumably owing to glycosylation of the N-terminal
part (see Kamimura et al.,
2004). Together, these data indicate that a large region of
zebrafish Cvl2 is cleaved into two fragments that remain associated via
disulfide bonds, similar to the situation recently reported for human Cvl2
(Binnerts et al., 2004).
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Proteolytic cleavage converts Cvl2 from a dorsalizing to a ventralizing factor
In order to analyze the impact of this proteolytic cleavage on the
biological activity and the molecular properties of Cvl2, we generated two
mutant constructs of Cvl2: a full-length construct in which the cleavage site
is destroyed (Cvl2-CM) and a construct representing only the N-terminal half
of Cvl2 containing the five CR domains (Cvl2-N, amino acids 1-355)
(Fig. 4B). These constructs,
when injected into zebrafish embryos or transfected into SF9 cells, led to
protein bands of the expected sizes (Fig.
4C, Fig. 6A). In
contrast to wild-type Cvl2-WT, which can both ventralize and dorsalize, these
two opposite effects were completely separated in the two mutant versions of
Cvl2. Although the mRNA encoding cleavage-resistant Cvl2-CM caused strong
dorsalization (Fig. 4F,J,N and
Table 1), injection of
cvl2-N mRNA led to ventralized phenotypes only
(Fig. 4G,K,O;
Table 1). Co-injection of
cvl2-CM and cvl2-N mRNA caused intermediate phenotypes,
similar to the effect of cvl2-wt
(Table 1). Together, these
results indicate that uncleaved Cvl2 acts as an inhibitor of Bmp signaling,
which upon proteolytic processing can be converted to a Bmp
signaling-promoting factor, the apparent in vivo role of Cvl2 according to the
loss-of-function studies described above.
The ventralizing effect of Cvl2-N strictly depends on Bmps and occurs in competition with the dorsalizing effect of Chordin
Our observation that cvl2 morphants display reduced levels of
phosphorylated Smad1/5 proteins suggests that Cvl2 acts by modulating Bmp
signaling. To study whether this is also true under gain-of-function
conditions, we injected cvl2-N mRNA into bmp2b morphant
embryos. Indeed, while sibling embryos injected with cvl2-N mRNA
alone were ventralized, embryos co-injected with cvl2-N mRNA and
bmp2b MO were as strongly dorsalized as control embryos injected with
bmp2b MO only (Fig.
5A-D; Table 1).
This indicates that Cvl2 requires Bmps to fulfill its ventralizing activity,
suggesting that it is a Bmp signaling-promoting factor that acts at the level
or upstream of Bmp proteins. By contrast, embryos co-injected with
chordin and cvl2-N mRNAs displayed a strong reduction of
dorsalization compared with sibling embryos injected with chordin
mRNA alone (Fig. 5E,F;
Table 1). This shows that Cvl2
can rescue the blockage of Bmp signaling by Chordin, consistent with the
results of the Cvl2-Chordin epistasis analyses described above
(Fig. 3).
The ventralizing effect of Cvl2-N is partly independent of Chordin
Comparing the ventralized traits of chordin mutants in the absence
or presence of Cvl2, we had concluded that Cvl2 has both Chordin-dependent and
Chordin-independent pro-Bmp effects (Fig.
3). To examine the Chordin-independent effect under
gain-of-function conditions, cvl2-N mRNA was co-injected with maximal
amounts of chordin MOs. Nevertheless, chordin morphants
co-injected with cvl2-N mRNA displayed an enhancement of all
ventralized traits, including a further expansion of the blood island and a
further reduction of the size of the head, as well as a loss of the gap in the
ventral tail fin (Fig. 5G,H;
Table 1). Although we cannot
rule out that some residual Chordin protein is present in morphants, this
result suggests that Cvl2 has an additional, Chordin-independent pro-Bmp
function.
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90% purity
(Fig. 6A). Comparing
cleavage-resistant Cvl2-CM with the CR repeats-containing Cvl2-N fragment, and
with wild-type Cvl2 (Cvl2-WT, 80% cleaved/associated;
Fig. 6A, lane 4), all three
immobilized versions were found to bind Bmp2 with nearly identical high
affinity (apparent KD 1.2 nM)
(Table 2 and
Fig. 6B). Similar affinities
were determined for Bmp4 and Bmp7, while binding to the more distantly related
growth and differentiation factor 5 (Gdf5) was about 25-fold weaker
(Table 2). The affinities
between Cvl2 and Bmp members are similar to the affinity between Bmp2 and the
type I receptor BRIA (Keller et al.,
2004). Together, these data indicate that Cvl2 strongly binds to
Bmps, that this binding occurs via its N-terminal CR repeats, and that binding
is not affected by the proteolytic cleavage of Cvl2.
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25 nM;
Fig. 6B;
Table 3). We found that
saturation of Bmp2 with Cvl2-WT or Cvl2-N blocked binding of Chordin to Bmp2
(Fig. 6D), although Chordin
readily bound to immobilized Bmp2 alone (KD 12 nM;
Fig. 6C,
Table 3), consistent with
previous reports (Larrain et al.,
2000). This shows that Cvl2 and Chordin bind to overlapping
epitopes of Bmp2, and that direct binding of Cvl2 and Chordin to Bmps is
mutually exclusive.
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;
Paine-Saunders et al., 2002;
Jasuja et al., 2004), we
wanted to examine whether differential affinity to the ECM might account for
the opposite effects of uncleaved and cleaved Cvl2. For this purpose, we transfected plasmids encoding Myc-tagged Cvl2-WT or Cvl2-N into HEK293 cells, cultured cells for 48 hours and isolated proteins from cells, supernatants and ECM. Upon transfection with Cvl2-WT, which gives rise to both cleaved/associated and uncleaved Cvl2 in cell extracts (data not shown), the two forms distributed differently in the extracellular domain. Whereas uncleaved Cvl2 was found in the ECM fraction only, cleaved Cvl2 was in the supernatant (Fig. 6E, lanes 3-5). Similarly, Cvl2-N, which lacks the C-terminal half, was present only in the supernatant, but not in the ECM fraction (Fig. 6E, lanes 6,7). This shows that the C-terminus containing the vWFd domain is required for binding of uncleaved Cvl2 to the ECM, while cleavage of Cvl2 strongly reduces ECM binding, although the N- and C-terminal fragments remain associated.
As association of Bmps, Chordin and Noggin with the ECM mainly occurs via
heparan sulfate proteoglycans (HSPGs)
(Ruppert et al., 1996;
Paine-Saunders et al., 2002;
Jasuja et al., 2004), we next
tested whether Cvl2 can bind to heparin-coated sepharose beads. After
incubation of Myc-tagged Cvl2-N and Cvl2-CM with the beads, proteins were
eluted with increasing concentrations of NaCl. The elution profiles showed
that Cvl2-CM binds to heparin with much higher affinity than Cvl2-N (elution
maximum for Cvl2-CM at 1.25-1.5M NaCl, for Cvl2-N at 0-0.5M NaCl,
Fig. 6G), suggesting that
differential binding to HSPGs might account for the difference in binding to
the ECM between cleaved and uncleaved Cvl2.
To study the functional relevance of heparin binding, we sought to
interfere with the heparin and ECM binding of Cvl2-CM. Cvl2 contains 67 basic
amino acids (Arg and Lys) as potential binding sites for HSPGs, making it
difficult to completely abolish heparin binding. We focused on one site that
conforms to a consensus heparin binding site (see
Hileman et al., 1998), which
is located at amino acids 393-396 (RRTR), 40 residues C-terminal of the
cleavage site. Removal of these four residues from Cvl2-CM reduced the
affinity for heparin beads (elution maximum at 0.75-1M NaCl;
Fig. 6F) and led to the release
of a significant fraction of the protein into the supernatant after
transfection into HEK293 cells (Fig.
6G). Importantly, injection of mRNA for this construct
(cvl2-CM
393-396) into zebrafish embryos had a
strongly reduced dorsalizing activity compared with cvl2-CM, although
both proteins were synthesized at equivalent levels
(Table 1;
Fig. 6H). These data suggest
that association of uncleaved Cvl2 with HSPGs of the ECM significantly
contributes to its anti-Bmp activity, possibly by tethering Bmps at the ECM,
making them inaccessible for their transmembrane receptors.
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DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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During gastrula stages, Bmps are expressed in a graded fashion with highest
levels at the ventral pole. According to the morphogen concept, this Bmp
gradient defines positional values and differential cell fates along the
dorsoventral axis. Cvl2 promotes Bmp signaling at all positions of the
gradient: in the presence of the Cvl2-dependent positive feedback loop, this
gradient reaches higher maximum levels at the ventral pole and is steeper, as
revealed by pSmad1/5 immunostaining (Fig.
2B). Clearly, an increased slope should yield a higher spatial
resolution of positional values along the dorsoventral axis, thereby
facilitating the translation of the gradient into differential cell fate. In
addition, the Cvl2-dependent positive feedback should ensure higher gradient
stability over time. A similar role of Cvl2 to stabilize territories of Bmp
signaling might also occur during later processes of vertebrate development,
when cvl2 displays a highly dynamic, but restricted expression at
various sites that are supposed to be under the control of Bmps
(Fig. 1; see Fig. S2 in the
supplementary material) (Coffinier et al.,
2002; Coles et al.,
2004; Kamimura et al.,
2004). Such a role of zebrafish Cvl2 to locally elevate Bmp
signaling levels is consistent with the function of its counterpart
crossveinless2 during wing development in Drosophila
(Conley et al., 2000). Here,
the developing crossveins of the pupal wing show increased Bmp signaling
activity, revealed by increased Mad (the Drosophila Smad1/5
homologue) phosphorylation, although neither the Drosophila bmp
homologs decapentaplegic and glass bottom boat, nor their
type I receptor thick veins show elevated expression in this region.
However, cvl2 expression is upregulated in this territory, and in
cvl2 mutants, the elevated Mad phosphorylation is lost
(Conley et al., 2000).
Chordin-dependent and -independent pro-Bmp effect of Cvl2
Our embryological epistasis analyses revealed a significant dependence of
Cvl2 activity on Chordin function. Thus, we found that although loss of Cvl2
in wild-type embryos leads to reduced Bmp signaling and a dorsalization of the
embryo, loss of Cvl2 in chordin mutants had only very moderate
dorsalizing effects and failed to rescue particular ventralized traits of the
mutants, as revealed by the persistent enlargement of the blood islands and
the persistent reduction of the neuroectoderm
(Fig. 3). This indicates that
in the absence of Chordin, Cvl2 cannot display its full pro-Bmp effect,
suggesting that it normally promotes Bmp signaling by antagonizing Chordin.
This notion of Cvl2-Chordin competition is further supported by our
overexpression studies (Fig.
5), showing that application of Cvl2-N can rescue the
dorsalization caused by Chordin, whereas it cannot compensate for the loss of
Bmp signaling caused by knockdown of Bmps.
In addition, we could detect Chordin-independent pro-Bmp effects of Cvl2
both in gain- and loss-of-function experiments. In the loss-of-function
scenario, this effect was most apparent in the ventral tail fins, the tissue
supposed to derive from the ventral-most ectoderm. In chordin
mutants, it is reduced, pointing to a pro-Bmp effect of Chordin in ventral
tail fin tissue. Recent experiments with temporally controlled Bmp
inactivation indicate that Bmp function driving ventral tail fin formation is
required during post-gastrulation stages
(Pyati et al., 2005), and the
same might be true for Chordin. In addition, Chordin might act during gastrula
stages, transporting Bmps against their gradient into ventral-most regions to
reach peak Bmp levels in concert with Tolloid function
(Hammerschmidt and Mullins,
2002; Wagner and Mullins,
2002). In any case, our findings that both in chordin and
in tolloid mutants, dorsalized traits of the ventral tail fin tissue
are further enhanced by loss of Cvl2 suggests that here, Cvl2 has a pro-Bmp
effect independently of the Chordin-Tolloid system (Figs
2,
3 and data not shown).
Consistent results pointing to a Chordin-independent pro-Bmp effect of Cvl2
were obtained in our gain-of-function experiments, showing that Cvl2-N can
further ventralize chordin morphant embryos
(Fig. 5).
Molecular mechanisms of Cvl2 function: Bmp receptors and Tsg
Cvl2 contains five CR domains, which were initially characterized in
Chordin, but are also present in other Bmp-regulating secreted proteins
(Garcia Abreu et al., 2002).
Accordingly, we found that both cleaved and uncleaved full length Cvl2, as
well as its N-terminal fragment containing the CR domains, readily and
specifically bind Bmps (Fig.
6). Binding affinities to immobilized Bmp2 were similar to that
determined for Chordin under identical conditions (KD 25 nM
for Cvl2, 12 nM for Chordin; Table
3). We further found that direct binding of Chordin or Cvl2 to
Bmp2 is mutually exclusive (Fig.
6), consistent with the opposing roles of Cvl2 and Chordin during
zebrafish development. In this respect, it appears that the Chordin-dependent
pro-Bmp effect found in the zebrafish embryo is due to an `anti-Chordin'
function, with Cvl2 competing with Chordin for free and possibly also for
Chordin-prebound Bmps. Along the same lines, the Chordin-independent
Bmp-promoting effect of Cvl2 could be due to competition with other Bmp
inhibitors or Bmp-binding proteins.
We also searched for differences in the molecular properties of Cvl2-Bmp
versus Chd-Bmp complexes that could account for the opposite effects of Cvl2
and Chd on Bmp signaling. One possibility could be that, in contrast to Chd
(Larrain et al., 2000),
binding of Cvl2 to Bmp does not block the interaction between Bmps and their
transmembrane receptors, similar to how it has been previously reported for
the anti-Bmp factor Follistatin (Iemura et
al., 1998) and the CR domain-containing pro-Bmp protein Kcp
(Lin et al., 2005). To test
this notion, we performed competitive Biacore analyses between Chd or Cvl2,
Bmp2 and type I (BmpRIA) or type II (BmpRII) receptors. However, obtained
results were inconclusive. Thus, depending on which of the components were
immobilized, each of the tested proteins (Cvl-N, Cvl-CM or Chd) either blocked
Bmp-BmpR interaction, or was permissive, whereas we failed to detect crucial
differences between Cvl-N on one side, and Cvl-CM and Chd on the other (J.Z.
and W.S., unpublished).
Alternatively, Cvl2 could act at the level of other components of
Bmp-containing complexes, such as Twisted gastrulation (Tsg). In
gain-of-function experiments, Tsg can display both pro- and anti-Bmp effects,
as has been described for Cvl2. As Cvl2, Tsg binds Bmps, mediated by a partial
CR-like domain (Oelgeschlager et al.,
2003). Tsg also binds Chordin, thereby facilitating binding of
Chordin to Bmps, accounting for its anti-Bmp effect
(Chang et al., 2001;
Oelgeschlager et al., 2000).
In addition, Tsg promotes cleavage of Chordin by the metalloprotease Tolloid,
resulting in a pro-Bmp effect
(Oelgeschlager et al., 2000;
Scott et al., 2001;
Larrain et al., 2001;
Shimmi and O'Conner, 2003;
Little and Mullins, 2004;
Xie and Fisher, 2005). In view
of this, we tested whether Cvl2 might act via Tsg, but Biacore analyses
indicated that Cvl2 and Tsg do not physically interact (J.Z. and W.S.,
unpublished). However, we cannot rule out that Cvl2 binds to higher order
complexes containing Chd and/or Tsg.
Molecular mechanisms of Cvl2 function: HSPGs and the ECM
Recently, it has been reported that the Bmp-antagonizing function of
Chordin depends on the binding of Chordin to heparan sulfate proteoglycans
(HSPGs) (Jasuja et al., 2004).
HSPGs are either components of the extracellular matrix (ECM) or remain
attached to the cell membrane, usually via GPI anchors. In
Drosophila, the membrane-bound Glypican protein Dally has been shown
to promote signaling by the Bmp homologue Dpp, acting as some kind of
co-receptor (Fujise et al.,
2003). In this light, both the Chordin-dependent and the
Chordin-independent pro-Bmp effects could be explained by a role of Cvl2 to
regulate the distribution of Bmps within the extracellular space, or the
recruitment of Bmps to their cell-surface receptors. Here, we found that
uncleaved Cvl2 binds to heparin with an affinity very similar to that reported
for Chordin, and also to the ECM of transfected cells. Removal of a putative
heparin binding site from uncleaved Cvl2 results in reduced affinity for
heparin, reduced binding to the ECM and decreased anti-Bmp activity in
embryos. Moreover, co-injection of exostosin1 MOs, targeting a key
enzyme of HSPG synthesis, strongly reduced the dorsalizing activity of
cvl2-CM mRNA (F.R. and M.H., unpublished). This suggests that similar
to Chordin, uncleaved Cvl2 requires binding to HSPGs to exert its anti-Bmp
activity.
By contrast, cleavage of Cvl2 into two fragments that remain associated via disulfide bonds apparently leads to a conformational change that abolishes Cvl2 binding to the ECM (Fig. 6). As Bmps usually display strong affinity to the ECM (Reddi, 2002) and as cvl2 is co-expressed with bmps, binding of Bmps to cleaved Cvl2 could prevent sequestration of Bmps at the ECM, thereby helping to increase the availability of Bmps for receptor activation on target cells. Alternatively, or in addition, cleaved Cvl2 might facilitate the interaction between Bmps and their HSPG co-receptors.
In summary, our biochemical analyses indicate that Cvl2 binds to Bmps, that
Cvl2 and Chordin compete for Bmp binding, and that cleaved Cvl2, in contrast
to uncleaved Cvl2 and Chordin, lacks binding to the ECM. However, how this
leads to the pro-Bmp effect of Cvl2 observed in vivo, remains largely unclear.
In any case, our results highlight that binding of CR proteins to Bmps does
not necessarily lead to an inhibition of Bmps, as in the case of Chordin
(Picollo et al., 1996), but can, by contrast, be required to promote Bmp
signaling, consistent with recent data obtained for the Kielin protein Kcp
during renal regeneration in mouse (Lin et
al., 2005).
Proteolytic processing of Cvl2 as a possible mechanism underlying its contrary effects in different contexts
Cvl2 gain-of-function studies in cell culture systems and in
Xenopus embryos have yielded controversial results, revealing either
pro- or anti-Bmp effects. Our findings that Cvl2 can be proteolytically
cleaved, and that cleaved and uncleaved Cvl2 display similar affinities to
Bmps, but different affinities to the ECM, might provide a biochemical
explanation for this phenomenon. Our overexpression studies further indicate
that Cvl2 must be present in the cleaved state to elicit its pro-Bmp effect,
while the contrary phenotype obtained in our loss-of-function studies suggests
that it is this cleaved version which is predominantly present during early
zebrafish development to mediate the essential pro-Bmp effect of Cvl2.
However, this might be different during other processes and/or in other
organisms, where the uncleaved version might be present in excess, leading to
a net anti-Bmp function of Cvl2. Such differences in the ratio of uncleaved
versus cleaved protein could depend on the synthesis and turnover rate of
Cvl2, or the abundance of Cvl2 proteases. According to our data, proteolytic
activity is present in human (Fig.
6E) and insect (Fig.
6A) cell cultures, as well as in zebrafish embryos
(Fig. 4A). The nature of the
protease, if existent, remains unclear. We could not detect altered ratios of
Cvl2 cleavage in tolloid gain-or loss-of-function experiments (data
not shown), ruling out this apparent candidate. In any case, a spatial and
temporal control of Cvl2 cleavage would offer an efficient and flexible system
for the fine-tuning of Bmp activity, allowing context-dependent switching of a
potent Bmp inhibitor to a Bmp agonist.
|
ACKNOWLEDGMENTS |
|---|
|
Footnotes |
|---|
Supplementary material for this article is available at http://dev.biologists.org/cgi/content/full/133/5/801/DC1
* These authors contributed equally to this work 
Sars Centre for Marine Molecular Biology, Thormøhlensgt 55, N-5008,
Bergen, Norway
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REFERENCES |
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Knigh
-pSmad1/5 immunoreactivity. (E,F) p-Smad1/5 intensity along
dorsoventral axis of cvl2 morphant (gray in F) and control sibling
(black in F) at 80% epiboly stage. Signal intensities were measured from
vegetal views, along a yellow line indicated in E for one of the investigated
controls (n=7). (G-X) Whole-mount in situ hybridization with probes
indicated in lower left corners. (G-N) Eighty percent epiboly stage.
(G,H,K-N) Lateral views; (I,J) vegetal views, dorsal rightwards. Ratios of
affected embryos were: eve1, 33/39; cvl2, 66/71;
gata2, 31/32; otx2, 45/53. (J) Only the ventral expression
of cvl2 is reduced in the morphant, while expression in the
prechordal plate is normal. (O,P) Ten-somite stage, view of tailbud,
anterior leftwards; expression domain of the blood precursor marker
gata1 is reduced in cvl2 morphant (29/37 embryos).
(Q-X) Five-somite stage, anterior leftwards; the krox20
expression domains are ventrally expanded in cvl2 morphant (R, 53/68
embryos), but rescued to near wild-type condition in embryo co-injected with
cvl2 MO and Drosophila cvl2 mRNA (T, 46/47 embryos). (U-X)
Low doses of cvl2 MO1 enhance the mild dorsalization of
tolloid/minifin mutants (40/43 in cross of two mfn
homozygotes, 22/97 in cross of two mfn heterozygotes), while having
no or a much weaker effect in wild-type embryos injected in parallel (0/33).
Control embryos are injected with a mismatch cvl2 morpholino.
Abbreviations: Coom, Coomassie Blue staining of gel loaded with same amounts
of protein; PonRed, Ponceau Red staining of blots after immunoreaction.
