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First published online May 16, 2007
doi: 10.1242/10.1242/dev.000901
1 Instituto Gulbenkian de Ciência, 2781-901 Oeiras, Portugal.
2 Institute for Biotechnology and Bioengineering, Centro de Biomedicina
Molecular e Estrutural, Universidade do Algarve, 8005-139 Faro,
Portugal.
* Authors for correspondence (e-mails: atavares{at}igc.gulbenkian.pt; jbelo{at}ualg.pt)
Accepted 27 March 2007
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SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: Cerberus, FoxH1, Nodal signaling, Left-right asymmetry, Transcriptional regulation, Chick, Mouse
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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; Yokouchi et al.,
1999; Zhu et al.,
1999). Cerberus-related proteins have been identified in other
vertebrate species: the founding member Xenopus Cerberus (XCer)
(Bouwmeester et al., 1996),
zebrafish Charon (Hashimoto et al.,
2004), mouse Cerberus-like (hereafter denominated Cerl-1)
(Belo et al., 1997;
Biben et al., 1998;
Shawlot et al., 1998) and
mouse Cerberus-like-2 (Cerl-2; also known as Dand5 or Dante)
(Marques et al., 2004).
Xenopus XCer, mouse Cerl-1 and chick Cer genes are
syntenic
(www.metazome.net)
and, at early stages, are expressed in equivalent embryonic structures, such
as the anterior endomesoderm, anterior visceral endoderm and hypoblast,
respectively (Bouwmeester et al.,
1996; Belo et al.,
1997; Foley et al.,
2000). Mouse Cerl-1 and chick Cer transcripts
are also detected in the anterior definitive mesendoderm
(Belo et al., 1997;
Rodriguez Esteban et al.,
1999). However, at later stages, Cerberus-related genes
have very distinct patterns: XCer expression is no longer detected,
mouse Cerl-1 transcripts are found in nascent somites and presomitic
mesoderm, zebrafish charon and mouse Cerl-2 are expressed
around the node region (Cerl-2 expression levels are higher on the
right side), and chick Cer is expressed in the left paraxial and
lateral plate mesoderm (Bouwmeester et al.,
1996; Belo et al.,
1997; Rodriguez Esteban et
al., 1999; Marques et al.,
2004; Hashimoto et al.,
2004). The understanding of how these different patterns of
expression are generated may bring some insights into the evolution of
Cerberus-related genes and their functions in the different
vertebrate species.
A conserved regulator of vertebrate left-right patterning is
Nodal, a member of the transforming growth factor-ß (TGF-ß)
family of signaling molecules that is expressed in the node region and left
lateral plate mesoderm (reviewed by Hamada
et al., 2002; Schier,
2003). In the mouse embryo, Nodal activity is restricted to the
left side by Cerl-2 (Marques et al.,
2004), by the midline barrier and by Lefty2, a Nodal
antagonist also expressed in the left lateral plate mesoderm (reviewed by
Juan and Hamada, 2001). The
left-side expression of Nodal and Lefty2 is directly
regulated by Nodal itself. Our present findings demonstrate that cCer
asymmetric expression is also directly activated by Nodal signaling and
suggest that the cis-regulatory sequences of Cerberus-related genes
have diverged among vertebrates.
Zebrafish charon, mouse Cerl-2 and chick Cer
have all been implicated in the determination of the left-right axis, but
their functions seem to differ: zebrafish charon and mouse
Cerl-2 have a role in preventing Nodal signals from crossing to the
right side (Hashimoto et al.,
2004; Marques et al.,
2004), whereas chick Cer was reported to have a role in
transferring the positional information from the node to the left lateral
plate mesoderm (Rodriguez Esteban et al.,
1999; Yokouchi et al.,
1999). At the molecular level, Cerberus-related proteins behave as
antagonists of members of the TGF-ß family
(Hsu et al., 1998;
Rodriguez Esteban et al.,
1999; Piccolo et al.,
1999; Belo et al.,
2000). During left-right patterning, zebrafish Charon and mouse
Cerl-2 proteins were shown to act as Nodal antagonists
(Hashimoto et al., 2004;
Marques et al., 2004), whereas
cCer has been proposed to act as a bone morphogenetic protein (BMP) antagonist
(Rodriguez Esteban et al.,
1999; Yokouchi et al.,
1999). Chick Cer would allow the expression of Nodal in
the left lateral plate mesoderm by inhibiting the repressive activity of BMPs
on Nodal transcription. However, more recent reports have shown that
BMP signaling is indeed essential for the activation of Nodal expression in
the left lateral plate (Piedra and Ros,
2002; Schlange et al.,
2002), leaving the role of cCer in left-right patterning
unexplained. Our results from overexpression and knockdown experiments
demonstrate that cCer acts as a negative regulator of Nodal expression and
prevents Nodal signaling from crossing to the right side. In conclusion, we
propose that chick Cer, zebrafish Charon and mouse Cerl-2 evolved different
regulatory mechanisms but retained a similar role in restricting Nodal
activity to the left side.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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To recognize possible binding sites for known transcription factors,
cCer 5' genomic sequences were analyzed using MatInspector
Professional release 7.4 (Quandt et al.,
1995).
To identify the transcription initiation site(s), 5' rapid amplification of cDNA ends was performed using total RNA from HH3-9 chick embryos and the RLM-RACE kit (Ambion). PCR products were size-fractionated by agarose gel electrophoresis, purified using a gel extraction kit (Qiagen), cloned into the pGEM-T Easy vector (Promega) and sequenced.
DNA constructs and morpholinos
cCer 5' genomic sequences were subcloned into an enhanced
green fluorescence protein (EGFP) reporter vector containing the EGFP
coding sequence and the SV40 early mRNA polyadenylation signals from pEGFP-N3
(Clontech). Deletions or point mutations of FoxH1- and SMAD-binding elements
were designed according to the literature
(Zhou et al., 1998;
Mostert et al., 2001) and
introduced into the Cer0.36-EGFP construct by PCR-based site-directed
mutagenesis.
For the enhancer assays, cCer genomic sequences were either
amplified by PCR (PCR1-5) or synthesized as complementary oligonucleotides,
and subcloned into the p1229-EGFP enhancer-less vector. This vector
carries the human ß-globin minimal promoter and was generated by
replacing the lacZ gene in the ß-globinlacZ BGZA or p1229 vector
(Yee and Rigby, 1993) with the
EGFP coding sequence (Clontech).
Chick expression plasmids were based on a modified pCAGGS-MCS vector (gift
from D. Henrique, Instituto de Medicina Molecular, Lisbon, Portugal)
(Niwa et al., 1991). The
coding sequence of Xenopus Cerberus-short (XCerS) was
amplified by PCR from a pCS2-XCerS vector (gift from S. Piccolo)
(Piccolo et al., 1999). The
cCer coding sequence (cCerCDS) was isolated by reverse transcriptase
(RT)-PCR according to the published sequence (GenBank accession no. AF179484)
(Rodriguez Esteban et al.,
1999) and subcloned into the XhoI and NotI sites
of pCAGGS-MCS.
The pCAGGS-RFP vector (gift from D. Henrique), carrying the cDNA
of monomeric red fluorescent protein (RFP; Clontech)
(Campbell et al., 2002) under
the control of the CAGGS promoter, was used to control the extent and
efficiency of electroporation.
To generate the luciferase (luc) reporter constructs, cCer regulatory sequences were amplified by PCR (using Cer-EGFP plasmids as template) and subcloned into the pGL2-Basic vector (Promega).
Fluorescein-tagged antisense morpholino oligonucleotides (cCer MO:
5'-CATGGTCCTGCTGATGCTGTAGATC-3'; cCer CoMO:
5'-CATcGTCgTGCTcATGaTGTAcATC-3', mismatches in lowercase) were
designed and produced by Gene Tools. The efficacy of cCer morpholinos to
inhibit the translation of Cer-Luc reporter constructs was tested in
a cell-free transcription/translation system (see Fig. S3 in the supplementary
material) (Summerton et al.,
1997).
Bead implantation and whole-mount in situ hybridization
Fertilized chicken eggs (Quinta da Freiria) were incubated at 37.5°C
for the appropriate period. Embryos were staged according to Hamburger and
Hamilton (HH) (Hamburger and Hamilton,
1951), explanted at HH stage 4-7 (HH4-7) together with the
vitelline membrane and anchored to a metacrilate ring according to the
protocol of New (New, 1955).
Affigel-blue beads (Bio-Rad) were soaked in Shh protein [1 mg/ml in 0.1%
bovine serum albumin (BSA)/phosphate-buffered saline (PBS); R&D Systems];
heparin acrylic beads (Sigma) were soaked in recombinant Nodal protein (0.5
mg/ml; R&D Systems); and AG1-X2 anion-exchange beads (Bio-Rad) were soaked
either in SU5402 [3 mM in dimethylsulfoxide (DMSO); Calbiochem]
(Mohammadi et al., 1997) or in
SB-431542 (10 mM in DMSO; Tocris) (Inman
et al., 2002). Treated embryos were cultured at 37.5°C in a
humid chamber, fixed in 4% paraformaldehyde and processed for whole-mount in
situ hybridization.
Whole-mount in situ hybridization on chicken and mouse embryos was
performed as described by Liguori et al.
(Liguori et al., 2003).
Detailed descriptions of the RNA probes used are available from the authors on
request. Embryos were developed with BM purple (Roche) for purple color and
with INT/BCIP (Roche) for orange.
Embryo electroporation
Embryos were processed for New culture
(New, 1955) at HH3-5 and
transferred into a silicon rubber pool containing a 2 mm-square cathode
(CY700-1Y electrode; Nepa Gene). The ring was then covered with warmed Hank's
buffer (GibcoBRL) and the embryo was injected with a DNA solution (0.5-3
mg/ml; 0.1% Fast Green; Sigma) using a pulled glass capillary and an IM-300
microinjector (Narishige). Electroporation was performed by placing a 2
mm-square anode (CY700-2 electrode; Nepa Gene) over the embryo and applying
five pulses (10 V for 50 ms at 350 ms intervals) using a square wave
electroporator (ECM830; BTX). The embryo was then placed on a 30 mm Petri dish
with albumen (New, 1995), incubated for the appropriate period of time
(7-48h), and observed under a fluorescence stereomicroscope (Leica
MZ16FA).
Luciferase reporter assay
Capped sense mouse Nodal mRNA was synthesized using the mMessage
mMachine kit (Ambion). Eggs were obtained from Xenopus laevis
females, cultured and microinjected as previously described
(Medina et al., 2000).
Embryonic stages were determined according to Nieuwkoop and Faber
(Nieuwkoop and Faber, 1967).
Xenopus embryos were injected in each animal blastomere of the
eight-cell stage with a total of 200 pg of reporter plasmid, with or without
Nodal mRNA (50 pg), and 25 pg of pTK-Renilla luciferase. Animal caps
were isolated from the blastula stage, cultured until sibling embryos reached
stage 12 and lysed in 20 µL of passive lysis buffer per cap. Firefly and
Renilla luciferase values were obtained by analyzing 20 µL lysate by the
standard protocol provided in the Dual Luciferase Assay kit (Promega) in a
luminometer (MicroLumatPlus, Berthold Technologies). Each assay was performed
in triplicate and repeated independently at least twice.
Generation of transgenic mouse embryos
The transgenic mouse line Cer2.5-EGFP was generated by
microinjection of linearized reporter construct DNA into the pronuclei of
fertilized eggs from FVB mice, as described
(Nagy et al., 2003). F1
embryos were collected from embryonic day (E)7.5 to E10.5, observed under a
fluorescence stereomicroscope (Leica MZ16FA), fixed in 4% paraformaldehyde and
processed for whole-mount in situ hybridization. For histological analyses,
embryos were embedded in gelatin, cryosectioned and photographed under a
fluorescence microscope (Leica DMRA2). In some slides, cell nuclei were
labeled with DAPI (Molecular Probes).
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). In turn, Nodal was shown to induce the expression of
XCer (Osada et al.,
2000) and mouse Cerl-1
(Waldrip et al., 1998;
Brennan et al., 2001).
Therefore, the induction of cCer expression by Shh
(Rodriguez Esteban et al.,
1999; Yokouchi et al.,
1999; Zhu et al.,
1999) might be mediated by Nodal. To test this hypothesis, beads
soaked in recombinant mouse Nodal protein were implanted on the right side of
HH6 chick embryos and cCer expression was examined by whole-mount in
situ hybridization. Indeed, ectopic expression of cCer was observed
in the right-side mesoderm of these embryos (81%, n=33,
Fig. 1A-F). cCer was
also induced in embryos electroporated with a Dorsalin-Nodal
expression construct (pCAGGS-DcNodal)
(Bertocchini and Stern, 2002),
but with less efficiency (35%, n=23, data not shown).
|
). In the mouse embryo, Nodal expression in the left
lateral plate mesoderm is directly activated by Nodal protein produced in the
node (Saijoh et al., 2003;
Yamamoto et al., 2003).
Accordingly, we observed that exogenous Nodal protein is able to activate
Nodal expression in the right lateral plate mesoderm of chick embryos
(68%, n=19, Fig.
1C-F). Nodal protein induced ectopic cCer expression in
less than 1 hour (73%, n=11, Fig.
1B and data not shown) and Nodal expression in
approximately 2 hours (71%, n=7,
Fig. 1D), whereas beads soaked
in Shh protein started to activate cCer and Nodal
transcription no sooner than 4-5 hours after implantation (66%, n=6,
Fig. 1H; 0% 2 hours after
implantation, n=5, data not shown). Taken together, these
observations suggest that the transcription of cCer and
Nodal is directly regulated by Nodal.
To determine whether endogenous Nodal signaling is necessary for
cCer expression, chick embryos were electroporated with a pCAGGS
expression vector containing the Nodal-specific antagonist Xenopus
Cerberus-short (XCerS)
(Piccolo et al., 1999;
Bertocchini and Stern, 2002).
Embryos were co-electroporated with pCAGGS-red fluorescent protein (RFP) and
initially scored for the co-localization of RFP fluorescence and
XCerS mRNA (data not shown). As expected, the inhibition of Nodal by
XCerS resulted in the downregulation of cCer (89%, n=9,
Fig. 1J) and Nodal
(85%, n=13, data not shown), whereas control electroporations had no
effect (n=10, Fig.
1I). Similarly, cCer expression was also repressed by
SB-431542, an inhibitor of Nodal receptors (88%, n=16, see Fig. S1 in
the supplementary material). Therefore, we conclude that endogenous Nodal
signaling is required for normal activation of cCer and
Nodal expression in the left lateral plate mesoderm. In addition,
ectopic induction of cCer by Shh protein was inhibited by XCerS (86%,
n=7, Fig. 1L), which
demonstrates that Nodal signaling is required for the activation of
cCer expression by Shh.
Identification of the cCer left-side enhancer
To investigate further whether cCer is a direct target of Nodal
signaling, we analyzed the regulatory sequences responsible for cCer
transcription in the left-side mesoderm. For this, cCer 5'
genomic sequences of different lengths were subcloned into an enhanced green
fluorescence protein (EGFP) reporter vector (Cer-EGFP constructs) and
introduced into chick embryos by microinjection and electroporation in New
culture (New, 1955). A
representation of these constructs and their electroporation results are
summarized in Fig. 2A.
|
; Yokouchi et al.,
1999; Zhu et al.,
1999). Fluorescent cells are later detected in the foregut and
heart (data not shown), which are derivatives of the chick anterior
mesendoderm. The subsequent analysis of EGFP expression driven by
shorter fragments (Cer0.9, Cer0.4 and Cer0.36) revealed a similar pattern
(Fig. 3A,C, and data not
shown). However, left-side expression was specifically disrupted in embryos
electroporated with Cer0.34-EGFP and shorter constructs (data not
shown). These observations indicate that the cCer left-side enhancer
is located in the 360 bp 5' region and that the -360 to -340 sequence
contains an essential regulatory element.
FoxH1 and SMAD elements are essential for cCer enhancer activity in the left-side mesoderm
To confirm that cCer left-side expression is directly activated by
Nodal, we first analyzed the cCer left-side enhancer sequence and
looked for the presence of FoxH1- and SMAD-binding sites. These transcription
factors are nuclear effectors of the Nodal signaling pathway (reviewed in
Schier and Shen, 2000) and
were shown to directly regulate the asymmetric expression of the Nodal,
Lefty2 and Pitx2 genes
(Saijoh et al., 2000;
Osada et al., 2000;
Yashiro et al., 2000;
Shiratori et al., 2001).
Sequence analysis of the cCer 360 bp 5' region (Cer0.36) using
MatInspector software (Professional release 7.4) revealed the presence of a
possible TATA box at -60, and consensus binding sites for several putative
regulators of cCer transcription, including two FoxH1 and one SMAD
element (Fig. 2B).
|
;
Mostert et al., 2001)
(indicated in Fig. 2B). Each of
the F1del, F1mut, F2del, F2mut, Sdel and Smut constructs was electroporated
into chick embryos, and EGFP fluorescence was analyzed both in the anterior
mesendoderm and in the left-side mesoderm (results are summarized in
Fig. 2C). All constructs were
able to drive EGFP expression in the anterior mesendoderm
(Fig. 3B and data not shown),
but left-side expression was specifically abolished
(Fig. 3D and data not shown).
These observations demonstrate that the FoxH1 and SMAD sites in
Cer0.36 are essential for the induction or maintenance of left-side
transcription. In addition, the functions of the FoxH1 and SMAD elements in the cCer left-side enhancer were quantified in luciferase reporter assays with Xenopus animal caps. The Cer0.36 reporter construct was clearly activated in the presence of Nodal (Fig. 3I). However, luciferase activity was reduced with the introduction of mutations in one of the FoxH1 or SMAD elements (F1mut, Smut and F2mut constructs; Fig. 3I). Taken together, our results indicate that the cCer left-side enhancer is directly activated by the Nodal-FoxH1/SMAD signaling pathway.
FoxH1 and SMAD elements are sufficient to activate the cCer left-side enhancer
We next investigated whether the FoxH1 and SMAD elements in the
cCer left-side enhancer are sufficient to induce left-side
expression. For this, potential regulatory sequences were subcloned into
enhancerless vectors that contain the human beta-globin minimal promoter
upstream of either the EGFP or the luciferase reporter gene. The
potential enhancer sequences tested were either shorter fragments of
Cer0.36 (PCR1-5) or combinations of individual FoxH1 (F) and SMAD (S)
elements (FSF, FF and FS; results are summarized in
Fig. 2D). Embryos
electroporated with PCR1, PCR2, PCR4, FF or FS did not display EGFP
expression in the left-side mesoderm (Fig.
3F,H, and data not shown). By contrast, asymmetric expression was
detected in embryos electroporated with the PCR3, PCR5 or FSF constructs
(Fig. 3E,G, and data not
shown). Accordingly, luciferase activities of the reporter constructs that
lack one of the FoxH1 or SMAD elements (PCR1, PCR2, FS and FF) were severely
reduced when compared with those of PCR3 or FSF
(Fig. 3J). These observations
indicate that the FSF module in the cCer left-side enhancer is
sufficient to activate asymmetric expression.
Regulation of the cCer left-side enhancer by Nodal signaling
Asymmetric expression of cCer is induced by Shh on the left side
and repressed by fibroblast growth factor 8 (FGF8) on the right side
(Rodriguez Esteban et al.,
1999; Yokouchi et al.,
1999; Zhu et al.,
1999). To investigate whether the cCer enhancer region is
also regulated by these signaling molecules, chick embryos were electroporated
with Cer0.4-EGFP and grafted on the right side of the node with beads
soaked either in Shh protein or in the FGF receptor-1 inhibitor SU5402. In
addition to the expected left-side pattern, EGFP expression was
activated on the right side both by the Shh protein (100%, n=10,
Fig. 4C) and by SU5402 (62%,
n=13, Fig. 4D). These
observations demonstrate that the cCer left-side enhancer is
regulated by Shh and FGF signaling in the same way as cCer
expression.
To confirm that cCer enhancer activity is regulated by Nodal, embryos were electroporated with the Cer0.4-EGFP reporter construct and grafted with beads soaked in Nodal protein. As expected, Nodal was able to ectopically induce EGFP expression in the right-side mesoderm (100%, n=11, Fig. 4B; compare with control, n=5, Fig. 4A). Conversely, when embryos were co-electroporated with the PCR5-EGFP reporter construct and the Nodal antagonist XCerS (pCAGGS-XCerS), cCer enhancer activity was specifically repressed in the left-side mesoderm (64%, n=11, Fig. 4F; compare with control, n=4, Fig. 4E). XCerS did not have an effect on anterior mesendoderm expression (n=4, Fig. 4G,H). These observations indicate that Nodal signaling is required for the regulation of cCer transcription in the left-side mesoderm.
|
; Belo et al.,
1997). However, at later stages, chick Cer is expressed
in the left-side mesoderm (Rodriguez
Esteban et al., 1999; Yokouchi
et al., 1999; Zhu et al.,
1999) (see also Fig.
1A), whereas mouse Cerl-1 expression is found in the
rostral domain of the nascent somites and presomitic mesoderm
(Belo et al., 1997) and mouse
Cerl-2 is expressed in the node region
(Marques et al., 2004). In
order to determine whether the upstream regulators of cCer expression
are conserved in mouse, we generated a transgenic line carrying the
cCer regulatory region (Cer2.5-EGFP) and analyzed reporter
gene expression in mouse embryos at different stages. At E7.5, EGFP
fluorescence was detected in the anterior mesendoderm (data not shown), an
expression domain common to chick Cer and mouse Cerl-1
genes. However, at E8.5, EGFP was expressed in the left lateral plate
mesoderm (Fig. 5A,B), which is
a cCer-specific pattern. As in Cer-EGFP-electroporated chick
embryos, fluorescent cells were also found in the foregut and heart of E8.5
transgenic embryos (Fig.
5Aa,B). These results indicate that the upstream regulators of
cCer expression are present not only in tissues that express both
cCer and mouse Cerl-1 (i.e. anterior mesendoderm), but also
in the mouse left-side mesoderm, a region that expresses cCer but not
the mouse Cerl genes.
In the mouse embryo, the asymmetric expression of both Nodal and
Lefty2 is directly regulated by Nodal signaling
(Saijoh et al., 2000;
Saijoh et al., 2003;
Yamamoto et al., 2003). In
Cer2.5-EGFP mouse embryos, EGFP mRNA expression is
exclusively detected in the left lateral plate mesoderm at E8.25, and
coincides with the expression patterns of Nodal
(Fig. 5C,C') and
Lefty2 (Fig.
5D,D'), which reinforces the hypothesis that cCer
regulatory sequences are directly regulated by Nodal.
Nodal signaling is negatively regulated by cCer
In the chick embryo, Lefty is expressed in the midline (as is
mouse Lefty1) and in a small posterior domain of the left lateral
plate mesoderm at late stages, whereas the cCer expression pattern is
much more similar to that of mouse Lefty2 in the left-side mesoderm
(Rodriguez Esteban et al.,
1999; Ishimaru et al.,
2000). Like Lefty proteins, Cerberus-related molecules were shown
to act as Nodal antagonists in zebrafish
(Hashimoto et al., 2004),
Xenopus (Hsu et al.,
1998; Piccolo et al.,
1999), chick (Bertocchini and
Stern, 2002) and mouse (Belo et
al., 2000; Marques et al.,
2004) embryos. Therefore, we proposed that cCer has taken the role
of mouse Lefty2 in the left-side mesoderm, and acts to restrict the range of
Nodal signaling. To test this hypothesis, we have performed cCer
overexpression and knockdown experiments in chick embryos. Because
Nodal transcription is autoregulated, Nodal expression was
analyzed as a readout of Nodal signaling.
Chick embryos electroporated on the left side with a pCAGGS expression vector containing the cCer coding sequence (pCAGGS-cCerCDS) showed a dramatic reduction or absence of Nodal expression in the left lateral plate mesoderm, but not in the node region (95%, n=20, Fig. 6B; compare with control, n=4, Fig. 6A). On the other hand, when cCer was misexpressed on the right side, Nodal was never ectopically induced (n=18, Fig. 6C) and was downregulated only in one embryo (6%, n=18, data not shown). In this embryo, it is possible that the cCer protein had traveled from the right to the left side, where it inhibited the Nodal signal. In addition, the expression of the Nodal target gene Pitx2 was downregulated by cCer on the left side (56%, n=16, data not shown), whereas it was never induced on the right side (n=11, data not shown). At older stages, chick embryos showed reversed heart looping when cCer was overexpressed on the left side (47%, n=15, data not shown), but not when cCer was misexpressed on the right side (n=7) nor in control electroporations (n=4). Taken together, these observations suggest that cCer may act as a negative regulator of Nodal signaling.
To investigate the effect of cCer downregulation on Nodal
expression, fluorescein-tagged morpholino oligonucleotides against
cCer (MO) or against a related sequence (five mismatches; CoMO; see
Fig. 2B) were electroporated
into the future left-side mesoderm of HH4-6 chick embryos. At HH8-11,
Nodal transcription was ectopically induced on the right side of cCer
morphant embryos (MO: 51%, n=37,
Fig. 6E,F versus CoMO: 7%,
n=27, Fig. 6D). In
nine HH10-11 morphant embryos, Nodal expression on the right was
higher than on the left side (Fig.
6F). This observation can be explained by a right-side-biased
amplification of Nodal signaling, as predicted by the self-enhancement and
lateral inhibition (SELI) model in the absence of Nodal inhibitors
(Nakaguchi et al., 2006). The
Lefty midline expression domain was normal in cCer MO-treated embryos
(n=26, see Fig. S2 in the supplementary material), suggesting that
the midline barrier was not affected. At older stages, cCer knockdown
resulted in the inversion of heart looping (43%, n=7 versus CoMO: 0%,
n=5, data not shown). These results indicate that the main function
of cCer in the left-side mesoderm is to prevent Nodal signaling from crossing
to the right side.
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|
DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
; Yokouchi et al.,
1999; Zhu et al.,
1999). The inconsistency between these and our results may be a
consequence of the usage of different Nodal overexpression methods: we grafted
beads soaked in active Nodal protein (mature form; R&D Systems), whereas
others used retroviral vectors carrying a Bmp4-Nodal fusion protein that were
introduced either by direct injection
(Rodriguez Esteban et al.,
1999) or by implantation of expressing cell pellets
(Yokouchi et al., 1999;
Zhu et al., 1999). In fact,
induction of ectopic cCer expression was much less efficient when we
used a Dorsalin-Nodal fusion construct (34%, versus 81% with Nodal protein
beads) and was never detected when we overexpressed chick Nodal complete cDNA
(n=10, data not shown). Therefore, it is possible that the proprotein
convertase required for Nodal maturation is present at low levels in the right
side of the chick embryo (Constam and
Robertson, 2000), or that the Nodal protein encoded by these
constructs is less stable than the recombinant protein
(Le Good et al., 2005).
|
; Osada et al.,
2000; Saijoh et al.,
2003; Yamamoto et al.,
2003). Accordingly, our results indicate that Nodal signaling
positively regulates Nodal expression in the left lateral plate in
the chick embryo (Fig. 1). This
observation supports the hypothesis that Nodal itself is the intermediary
signal that transfers the asymmetric information from the node to the lateral
plate (Pagán-Westphal and Tabin,
1998).
cCer left-side enhancer is regulated by Nodal signaling via FoxH1 and SMAD elements
Here we have provided evidence that the FoxH1 and SMAD elements present in
the cCer left-side enhancer are essential and sufficient for the
activation of asymmetric expression (Figs
2,
3). FoxH1 and SMAD
transcription factors are nuclear mediators of Nodal signaling (reviewed by
Schier and Shen, 2000). As
expected, Nodal is necessary and sufficient to activate the cCer
left-side enhancer (Fig. 4).
These observations, together with the evidence that cCer asymmetric
expression is activated by Nodal (Fig.
1), indicate that Nodal signaling directly regulates cCer
transcription in the left-side mesoderm via the activity of FoxH1 and SMAD
factors. In the future, the isolation of chick FoxH1 and the investigation of
its direct binding and activation of the FoxH1 elements present in the
cCer left-side enhancer may bring additional support to these
results.
cCer restricts the range of Nodal signaling to the left side
Previous reports have proposed that cCer is able to induce ectopic
Nodal expression on the right side by antagonizing the repressive
activity of BMPs on Nodal transcription
(Rodriguez Esteban et al.,
1999; Yokouchi et al.,
1999). However, BMPs can have opposite effects on Nodal
expression: Bmp4 is a negative regulator of Nodal in the right side of the
chick node at early stages (HH5-6)
(Monsoro-Burq and Le Douarin,
2001), whereas Bmp2 positively regulates Nodal signaling in the
lateral plate mesoderm at later stages (HH7-8)
(Piedra and Ros, 2002;
Schlange et al., 2002).
Because cCer was introduced at early stages (HH6) on the right side of the
node (Rodriguez Esteban et al.,
1999; Yokouchi et al.,
1999), it might be inducing Nodal expression by blocking
the inhibitory function of Bmp4 in the node region. In fact, when
cCer-expressing cells were implanted at later stages (HH7-8) in the
lateral plate, Nodal expression was not affected
(Zhu et al., 1999).
In our hands, cCer misexpression in the node region (n=7,
data not shown) or right lateral plate
(Fig. 6C) was never able to
induce Nodal, whereas cCer overexpression on the left side
actually repressed Nodal (Fig.
6B). Conversely, Nodal was ectopically expressed on the right side
of cCer-knockdown embryos (Fig.
6E,F). These findings revealed that cCer acts as a
negative regulator of Nodal signaling. Similar results have been obtained with
the Nodal antagonist Lefty: Nodal activity was repressed by the ectopic
expression of chick Lefty (Rodriguez
Esteban et al., 1999) or mouse Lefty1 or Lefty2
(Yoshioka et al., 1998) in the
chick embryo, whereas it was upregulated on the right side of Lefty2 mutant
mice (Meno et al., 2001).
Given the similarities between the expression patterns and functions of chick
Cer and mouse Lefty2 (reviewed by
Juan and Hamada, 2001), we
propose that cCer has taken the role of mouse Lefty2 in left-right patterning,
and acts in addition to the midline barrier to confine Nodal signaling to the
left side.
Feedback model of cCer and Nodal regulation
Taken together, our findings suggest a feedback mechanism by which Nodal
signaling is restricted in the left lateral plate mesoderm of the chick embryo
(Fig. 7). During the
establishment of the left-right axis, Nodal expression is first
activated in the left perinodal region by the Notch and Shh signaling pathways
(reviewed by Raya and Izpisua-Belmonte,
2004). This initial Nodal signal directly induces cCer
expression via the activation of the cCer left-side enhancer by FoxH1
and SMAD transcription factors. We hypothesize that SMAD2 and/or SMAD3
regulate cCer transcription, because they are thought to transduce
Nodal signal in the left-side mesoderm (reviewed by
Schier, 2003) and
phospho-SMAD2 has been detected in the chick lateral plate mesoderm
(Faure et al., 2002).
Additionally, we propose that, as in the mouse embryo (reviewed in
Hamada et al., 2002), Nodal
also activates its own transcription, leading to the amplification of
Nodal and cCer expression throughout the left lateral plate.
The partial overlap of the Nodal and cCer expression domains
is possibly determined by functional differences in their regulatory regions
and/or in Nodal and cCer diffusion rates, as proposed for the mouse Nodal and
Lefty2 proteins (Nakaguchi et al.,
2006). Together with the midline barrier, cCer has a key role in
preventing the Nodal signal from crossing to the right side. Ultimately, the
negative-feedback regulation of Nodal signaling by cCer results in the
downregulation of Nodal and cCer expression in the left
lateral plate mesoderm. Further support for this model may come from the
analysis of chick Nodal transcriptional regulation as well as from
the investigation of the diffusion rates and stability of Nodal and cCer
proteins.
Evolution of Cerberus-related genes: divergence of gene regulation but conservation of function in left-right patterning
Unlike other known Cerberus-related genes, cCer is
expressed on the left side of the paraxial and lateral plate mesoderm.
Variations in the expression patterns of orthologous genes may arise either
from the presence of particular cis-regulatory elements in their genomic
sequence, or from the existence of differences in the localization and
activation status of their upstream regulators, or both. Cross-species studies
of cis-regulatory sequences are likely to help distinguish between these two
hypotheses. In our study, the analysis of Cer-EGFP transgenic mouse
embryos revealed that the upstream regulators of the cCer left-side
enhancer (i.e. Nodal) are present in the mouse left lateral plate mesoderm,
and suggested that the regulatory regions of Cerberus-related genes
have diverged in chick and mouse. In agreement with this, the comparison
between the cCer left-side enhancer and non-coding sequences of
human, mouse, Xenopus and Fugu Cerberus-related genes using
ConSite and VISTA programs was unable to detect any conserved FoxH1-binding
sites or other common regulatory elements. FoxH1- and SMAD-binding sites are
indeed present in the asymmetric enhancers of several left-side-specific
genes, such as the ascidian, Xenopus, mouse and human Nodal
genes, mouse and human Lefty2 genes, and mouse and Xenopus
Pitx2 genes (Saijoh et al.,
2000; Osada et al.,
2000; Yashiro et al.,
2000; Shiratori et al.,
2001). Our findings add cCer to this list, and underscore
the essential role of evolutionarily conserved FoxH1-SMAD modules in the
transcriptional regulation of asymmetric gene expression
(Osada et al., 2000).
Although chick Cer, zebrafish charon and mouse
Cerl-2 have different expression patterns, the Cerberus-related
proteins encoded by these genes seem to have a conserved function in
left-right development, which is to restrict Nodal signaling to the left side
of the embryo. Nodal expression in the node region also differs among
vertebrate species: it is bilateral in zebrafish and in early mouse embryos,
whereas it is restricted to the left side in the chick embryo (reviewed in
Raya and Izpisua-Belmonte,
2004). This difference may justify the need for a Nodal antagonist
in the node of zebrafish and mouse embryos, which is not required in the chick
node.
The divergence in gene regulation between chick, Xenopus and mouse Cerberus homologues, here demonstrated by the presence of a FoxH1-SMAD module in the cCer regulatory region, has conveyed a novel scenery for the activity of cCer and enabled it to take the role of Lefty2 as a negative-feedback regulator of Nodal signaling in the left lateral plate. In the future, the analysis of novel Cerberus-related molecules involved in the left-right development of other vertebrate species (such as Xenopus and rabbit) should provide further insight into the evolution of Cerberus gene regulation and function.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/134/11/2051/DC1
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ACKNOWLEDGMENTS |
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