1 Max Delbrueck Center for Molecular Medicine, Robert-Roessle-Strasse 10, 13125
Berlin, Germany
2 Department of Molecular Genetics, The University of Texas, M. D. Anderson
Cancer Center, Houston, TX 77030, USA
3 Center for Advanced Biotechnology and Medicine and Dept. of Pediatrics,
UMDNJ-Robert Wood Johnson Medical School, 679 Hoes Lane, Piscataway, NJ 08854,
USA
4 Department of Immunology, University Hospital Utrecht, NL-3584 CX Utrecht, The
Netherlands
5 Department of Pharmacology, Kyoto University Graduate School of Medicine,
Yoshida-Konoe-cho, Sakyo-ku, Kyoto 606-8501, Japan
* Author for correspondence (e-mail: wbirch{at}mdc-berlin.de)
Accepted 10 September 2003
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SUMMARY |
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Key words: Microarray, Anteroposterior axis, Gastrulation, Signalling pathways, Tdgf1, Nanog
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Introduction |
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The formation of the primary body axes during early embryogenesis is
controlled in many organisms by canonical Wnt/ß-catenin signal
transduction (reviewed by Sokol,
1999; De Robertis et al.,
2000
). The expression of Wnt target genes is regulated by nuclear
ß-catenin that is bound to transcription factors of the Lef/Tcf family
(Behrens et al., 1996
;
Molenaar et al., 1996
;
He et al., 1998
;
Tetsu and McCormick, 1999
). In
Xenopus, depletion of ß-catenin mRNA interferes with the
formation of the dorsoanterior axis, while injection of Wnt mRNA promotes axis
formation (Smith and Harland,
1991
; Sokol et al.,
1991
; Heasman et al.,
1994
). In zebrafish, misexpression of Wnt receptors, or mutations
in Axin or Ichabod, which encode a ß-catenin
scaffolding protein and a regulator of ß-catenin localisation, modulate
the function of the dorsal organiser and impair the formation of the
anteroposterior axis (Nasevicius et al.,
1998
; Kelly et al.,
2000
; Heisenberg et al.,
2001
). In the mouse, ablation of ß-catenin results in failure
to orient the anteroposterior axis
(Huelsken et al., 2000
), i.e.
the movement of an anterior signalling centre from the distal to the anterior
visceral endoderm does not occur (Thomas
and Beddington, 1996
). Studies employing chimeras between mutant
and wild-type embryos indicate that ß-catenin acts in the embryonic
ectoderm at this developmental step.
Signals provided by the TGF-ß/Nodal pathway are also essential for the
formation of the primary body axes (reviewed by
De Robertis et al., 2000;
Whitman, 2001
). Nodal signals
are transduced via Smads, which accumulate in the nucleus, interact with other
transcription factors, and regulate gene expression
(Chen et al., 1996
;
Liu et al., 1996
;
Zhang et al., 1996
;
Watanabe and Whitman, 1999
).
Cripto, an essential co-receptor of Nodal, acts together with activin
receptors to transmit the Nodal signal via Smad2 and Smad3
(Gritsman et al., 1999
;
Yeo and Whitman, 2001
;
Kumar et al., 2001
;
Yan et al., 2002
). Previous
studies have shown a requirement for Nodal signalling in anteroposterior axis
formation in both zebrafish and mice. In particular, zebrafish that carry
double mutations in the cyclops and squint genes, which
encode Nodal-related ligands, display a mispositioned anteroposterior axis
(Feldman et al., 1998
). In the
mouse, Nodal mutants lack a proximodistal axis as indicated by the
absence of the signalling centres in extra-embryonic ectoderm and visceral
endoderm, and as a consequence, do not form an anteroposterior axis
(Conlon et al., 1994
;
Varlet et al., 1997
;
Brennan et al., 2001
). Mutants
of Cripto establish a proximodistal axis, but fail to position the
nascent anteroposterior axis in the correct orientation
(Ding et al., 1998
). Thus, in
early mouse embryogenesis, Nodal signals are required for the formation of the
anteroposterior axis, but its co-receptor Cripto is required only for the
proper positioning of the anteroposterior axis. Whether ß-catenin and
Nodal/Cripto act in the same or in parallel pathways during the formation and
positioning of the anteroposterior axis has been unclear.
Subsequently, Wnt/ß-catenin and TGF-ß signals regulate the
formation of the vertebrate mesoderm. In Xenopus, ectodermal
expression of factors of the TGFß family induces mesoderm (reviewed by
De Robertis et al., 2000;
Kimelman and Griffin, 2000
),
and TGFß and Wnt signals cooperate to induce expression of brachyury that
specifies mesoderm (Vonica and Gumbiner,
2002
). In the mouse, Wnt3 mutants lack a primitive streak
and mesoderm, but form the anteroposterior axis
(Liu et al., 1999
). In
cultured epithelial cells, Wnt3 signals are transmitted via ß-catenin,
suggesting that Wnt3 uses the canonical Wnt/ß-catenin pathway
(Shimizu et al., 1997
).
Brachyury is expressed in the mesoderm, and is a direct transcriptional target
for canonical Wnt/ß-catenin signals
(Yamaguchi et al., 1999
;
Arnold et al., 2000
;
Galceran et al., 2001
).
Nodal mutant mice lack a primitive streak and only rarely form
patches of mesoderm (Conlon et al.,
1994
), and experiments employing chimeric embryos indicate that
these essential Nodal signals are generated in the epiblast
(Varlet et al., 1997
). By
contrast, Cripto mutants do form mesoderm that arises at an ectopic
position (Ding et al., 1998
).
Thus, the comparison of Wnt3 and Cripto mutants indicates
that formation of the anteroposterior axis and of mesoderm can occur
independently of each other. However, ß-catenin is required in both
processes.
In the adult, deregulation of both Wnt/ß-catenin and TGF-ß
signals play important roles in the establishment and progression of cancer.
Mutations in Apc (Groden et al.,
1991; Kinzler et al.,
1991
), which encodes a regulator of ß-catenin stability, or
in ß-catenin (Morin et al.,
1997
; Rubinfeld et al.,
1997
) are found in the majority of human colorectal cancers
(reviewed by Polakis, 2000
).
Mutations in Axin and conductin (now known as Axin2), which
encode scaffolding proteins of the ß-catenin degradation complex, have
been identified in medulloblastomas and colorectal carcinomas
(Liu et al., 2000
;
Satoh et al., 2000
;
Dahmen et al., 2001
). Genes
that encode proteins of the TGFß signalling pathway act as tumour
suppressor genes: Smad2 and Smad4 in human colorectal cancer
(Eppert et al., 1996
;
Thiagalingam et al., 1996
),
and Tgfbr2 in colon tumours with microsatellite instability
(Markowitz et al., 1995
). The
Nodal co-receptor Cripto is overexpressed in many colon carcinomas and can
transform fibroblasts and epithelial cells
(Niemeyer et al., 1998
;
Salomon et al., 2000
).
Target genes of ß-catenin during formation of the anteroposterior axis and the mesoderm have not been analyzed in a systematic and genome-wide manner. Using microarray technology, we identify Cripto, which encodes a Nodal co-receptor, as a primary target of ß-catenin. This defines the basis of a novel molecular interaction between Nodal and ß-catenin signalling pathways in early embryogenesis. Interestingly, Cripto expression in the early embryo and in tumours depends on ß-catenin signals. Comparison of the genome-wide expression profiles of ß-catenin, Cripto and Wnt3 mutant embryos results in the identification of novel genes that are active during formation of the anteroposterior axis and of the mesoderm.
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Materials and methods |
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Microarray analysis
RNA and genomic DNA were isolated from single mouse embryos, using Trizol
(GibcoBRL), according to the manufacturer's instructions. RNA was reverse
transcribed using a T7-promoter tagged polyT primer, and antisense RNA was
produced using T7 RNA polymerase. Antisense RNA was examined by gel
electrophoresis, and equivalent amounts of RNA from three to five age-matched
embryos of the same genotype were pooled to generate a microarray probe.
Probes were biotin-labeled in a second round of T7 amplification, as
previously described (Luo et al.,
1999). Profiles of ß-catenin-, Cripto- and
Wnt3-deficient mice were compared with profiles of wild-type
littermates of the same genetic background. Microarray profiling was performed
in triplicate from independent preparations of embryonic RNA using Affymetrix
Mu11k GeneChips (11.000 sequences). Isolated tissue domains were profiled in
duplicate using MG-U74A GeneChips (8.000 sequences).
GeneChip image analysis, data normalisation and comparative analysis were
performed using the statistical algorithm of Affymetrix Microarray Suite (MAS)
5.0 software following the manufacturer's guidelines. Ranking and filtering of
genes was performed using Microsoft Excel. ß-Catenin-responsive genes
were defined as genes that changed expression more than twofold, with a Change
p-Value limit of 0.1 in cross-wise comparative analyses. Genes commonly
deregulated in ß-catenin and either Cripto or Wnt3
mutants were defined as genes that changed expression greater than twofold, or
with a Change p-Value limit of 0.1. GeneChip data of isolated tissues were
normalised and visualised using Silicon Genetics GeneSpring software. The
significance of the overlap between genes deregulated in both ß-catenin
and Cripto or Wnt3 mutants was calculated using the
hypergeometric distribution function
(Feller, 1968). The microarray
data set is available on our website
http://www.mdcberlin.de/~zelldiff
Tissue culture
Ls174T cells that express tetracycline-inducible, dominant-negative Tcf4
have been described before (van de
Wetering et al., 2002). For northern blot analysis, RNA was
isolated using Trizol (GibcoBRL) before and 12 hours after induction with 1
µg/ml doxycyclin. For ß-catenin-Tcf4-dependent reporter assays, 293T
cells were transfected by calcium phosphate/DNA co-precipitation with 1 µg
reporter constructs, 0.1 or 0.4 µg Tcf4-ß-catenin-fusion-construct, 1
µg pSV-ß-Gal-Plasmid, and empty pCMVneo to 4 µg DNA per well.
Transfections and measurements were performed in triplicate.
Plasmid construction
Cripto enhancer sequences were amplified from a cosmid clone using
the primer sequences 5'-AAT CAC TTT GCC ACC CTG TC-3' and
5'-ACT GCG CAG AAG CTG ATG G-3' and cloned into the
SalI-sites of FOPflash (Molenaar
et al., 1996) to replace the FOP sites. Putative Lef/Tcf-binding
sites were mutated using PCR mutagenesis and the primer sequences 5'-GGG
CGA TAA ATC AAC TGC GTT TGT GTC CTC TTC TGG-3', 5'-GTC
CTC CGG AAT CCT GCG ATT CCT TCG AGA GGA C-3', 5'-GTC
CTC TCC ATG TGC TGC GAT GGC TGG CTA GAT-3' (mutated
nucleotides are underlined). Cripto 5' flanking sequences were
amplified using the primer sequences 5'-GCC AGT GTG GAC AAG TCC
TG-3' and 5'-CTT CGA CGG CTC GTA AAA AC-3' and cloned into
the SalI site of pBL-luc.
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Results |
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We found that 106 and 60 genes are deregulated in
ß-catenin-/- embryos at E6.0 and E6.5, respectively,
corresponding to the stages at which the anteroposterior axis and the mesoderm
are formed (for selected genes see Table
1, for a complete list see Table S1 at
http://dev.biologists.org/supplemental/).
The profiling data do not provide information whether these genes are
modulated directly or indirectly by ß-catenin. Many genes that are
modulated in ß-catenin-/- embryos encode signalling molecules
or transcription factors. Remarkably, Cripto is the most prominently
downregulated gene at E6.0, and we provide evidence below that Cripto
may be a direct transcriptional target of ß-catenin. We also find
deregulated expression of Msx1, Tm7Sf1, Tssc3 (Phlda2-Mouse
Genome Informatics) Afp, Sfrp1, Fas1 (Tnfsf6-Mouse Genome
Informatics), Sim2, Neurod1, Frz7 and other genes at this stage.
Among these, Msx1 and Frz7 have previously been identified
to be regulated by ß-catenin, but it is not known whether this regulation
is direct or indirect (Willert et al.,
2002; Kielman et al.,
2002
).
|
In control studies, we also examined transcriptional changes that occur between E6.0 and E6.5 in wild-type embryos during normal development. We found that 130 genes are downregulated at E6.5 in wild type, and of these, 127 were also downregulated in ß-catenin mutants (Fig. 1A). In addition, 673 genes were upregulated in wild-type embryos at E6.5, and of these, 591 were also upregulated, and only 82 were downregulated in ß-catenin mutants (green and red in Fig. 1A, respectively). Thus, despite the severe phenotypic defects in ß-catenin mutant embryos, the general program of gene expression is overall highly similar to wild type at these developmental stages.
|
|
Comparison of expression profiles of ß-catenin, Cripto and Wnt3
mutant embryos
To define groups of genes regulated by signals that direct the formation of
the anteroposterior axis and the mesoderm, we determined the expression
profiles of Cripto and Wnt3 mutant embryos at E6.0 and E6.5
and compared them with those of ß-catenin-/- embryos. We found
30 (out of 106) genes commonly deregulated in ß-catenin and
Cripto mutants at E6.0 (see Table S1 at
http://dev.biologists.org/supplemental/).
The number of these commonly deregulated genes is high, and indeed statistical
analysis showed that it is larger than expected to occur randomly
(P<0.01, as determined by the hypergeometric distribution
function). We find downregulated expression of Msx1, Ppp2ca, Ctsz and
other genes in both ß-catenin and Cripto mutants, which are
expressed in the embryonic ectoderm, but also in other tissue domains
(Fig. 3A, upper panel). We also
find upregulated expression of Neurod1, Sim2 and other genes in the
embryonic ectoderm, that in wild-type embryos are typically expressed in
developing neural tissues (Fig.
3A, lower panel). General neural markers like Sox1/2 are
not upregulated. The commonly deregulated genes may be positioned genetically
downstream of Cripto, and thus may not be directly controlled by
ß-catenin. These results, in conjunction with our finding that
Cripto expression depends on ß-catenin in the mouse embryo at
E6.0, suggest that ß-catenin regulates specific gene expression programs
via Cripto, which is required for anteroposterior axis
positioning.
|
A gain-of-function mutation of ß-catenin promotes Cripto
expression and disturbs anteroposterior axis formation
As loss of ß-catenin led to a loss of Cripto, we asked
whether increased ß-catenin signals may promote Cripto
expression. Constitutively active ß-catenin can be produced in mice by a
gain-of-function mutation in the ß-catenin gene by using the
Cre-loxP technology (Harada et
al., 1999; Soshnikova et al.,
2003
). We introduced this conditional mutation in the early embryo
by employing a mouse strain that expresses Cre recombinase early and
ubiquitously under the control of the CMV promoter
(Nagy et al., 1998
). Using
this technique, the floxed exon 3 of ß-catenin is removed, which leads to
constitutively active ß-catenin that cannot be phosphorylated at the N
terminus and cannot be degraded. We examined the expression of essential genes
in embryos at E6.5 by immunohistochemisty and in situ staining of transversal
sections. In wild-type embryos, ß-catenin is produced asymmetrically,
i.e. enriched in the cytoplasm on the posterior side, as is expression of
conductin, which is a direct target of ß-catenin signals
(Lustig et al., 2002
;
Jho et al., 2002
). In the
gain-of-function mutants, ß-catenin and conductin were highly expressed,
but now symmetrically in the entire epiblast
(Fig. 4A-D). Remarkably,
Cripto expression that is also increased on the posterior side in the
wild type, was as well highly expressed symmetrically
(Fig. 4E,F). These data
indicate that the asymmetric expression of Cripto is a result of
asymmetric ß-catenin function, strongly suggesting that Cripto
is a ß-catenin target. Moreover, in ß-catenin gain-of-function
mutants, we also found loss of asymmetry of the expression of Nanog,
brachyury and Cerberus, which mark the posterior ectoderm, posterior
mesoderm and anterior visceral endoderm, respectively, and we found
downregulation of the ectodermally expressed gene Pou5f1/Oct4
(Fig. 4G-N).
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Discussion |
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We have demonstrated an unexpected mechanism underlying the genetic
interaction between ß-catenin and Nodal/Cripto signalling during early
mouse embryogenesis. Previous studies in Xenopus have demonstrated an
interaction between the ß-catenin and Nodal pathways through the
coordinate transcriptional regulation of downstream targets such as
Twin, a homeobox gene, which is essential for the formation of the
Spemann organiser (Nishita et al.,
2000; Labbe et al.,
2000
). We show that ß-catenin signalling in the mouse embryo
impinges upon the Nodal pathway through regulation of the Nodal co-receptor
Cripto. Cripto expression is absent in ß-catenin-deficient
embryos, while it is enforced in ß-catenin gain-of-function mutants.
Nodal-/- mice do not express Cripto
(Brennan et al., 2001
), and
thus the regulation of Cripto is not solely dependent on
ß-catenin, but requires Nodal activity as well. We find that
Nodal is expressed appropriately in ß-catenin mutants,
indicating that loss of Cripto is not due to the absence of Nodal.
We have identified genes that are commonly downregulated in both
Cripto- and ß-catenin-deficient embryos at E6.0, e.g. Sox11,
Ppp2ca and Plk. These latter genes are expressed in the
embryonic ectoderm and may be targets of Cripto. Alternatively, Cripto may
signal to associated tissue domains, e.g. the visceral endoderm and the
extra-embryonic ectoderm during this developmental stage. It is known that
developmental signalling pathways interact across tissue borders
(Struhl and Basler, 1993;
Brennan et al., 2001
). We have
also identified genes that are commonly upregulated in both Cripto
and ß-catenin-deficient embryos at E6.0, e.g. Sim2, Neurod1 and
Aqp4. Interestingly, these genes are often expressed in neural
tissues (Kimura et al., 2001
).
We also found loss of expression of the ectodermally expressed gene
Pou5f1/Oct4 in ß-catenin gain-of-function mutants. Consistent
with these findings, recent studies indicate that ß-catenin-mediated
signals can block ectodermal differentiation of embryonic stem cells, whereas
the suppression of such signals induces neuroectodermal differentiation
(Kielman et al., 2002
;
Aubert et al., 2002
). Moreover,
precocious expression of the neurally expressed genes Hesx and
Six3 has previously been noted in Cripto mutants
(Ding et al., 1998
). The
increased expression of neurally expressed genes in ß-catenin and
Cripto mutants may be indicative of premature neuroectodermal
differentiation, and may imply that these genes act downstream of Cripto.
The comparison of genes deregulated in ß-catenin and Wnt3
mutants at E6.5 and E6.0 allowed us to dissect the sequence of events that
leads to primitive streak and mesoderm formation. In control embryos, genes
like brachyury (Wilkinson et al.,
1990), Nanog and Eomes
(Russ et al., 2000
) are
expressed early in the embryonic ectoderm before the primitive streak and
mesoderm are formed, and these genes are deregulated at both E6.0 and E6.5 in
ß-catenin and Wnt3 mutants (early genes). Subsequently, genes
like Fgf8 (Sun et al.,
1999
) and Evx1 (Dush
and Martin, 1992
) are expressed in the developing mesoderm of
control embryos, and these are deregulated at E6.5, but not at E6.0 in
ß-catenin and Wnt3 mutants, owing to absence of mesoderm (late
genes). Among the set of early genes, we have identified the homeobox gene
Nanog, which is required for maintenance of pluripotency of epiblast
cells prior to implantation (Chambers et
al., 2003
; Mitsui et al.,
2003
). At E6.0, Nanog is expressed in the proximal
epiblast, and this expression domain shifts to more distal and posterior
positions in the E6.5 embryo. Nanog expression is thus similar to the
expression pattern of Wnt3 (Liu
et al., 1999
), suggesting that Nanog may be regulated by
Wnt3/ß-catenin signals during gastrulation. Our analysis therefore
indicates that Nanog may have additional functions in the post-implantation
mouse embryo.
Gene expression profiling revealed that the vast majority of genes that
change expression between E6.0 and E6.5 in wild-type embryos are regulated in
a similar manner in ß-catenin mutants, i.e. they are either commonly up-
or commonly downregulated. This finding suggests that the loss of
ß-catenin affects specific, and not general developmental programs.
Moreover, most genes that are deregulated in ß-catenin mutant embryos are
expressed in specific domains of the wild-type embryo, suggesting
tissue-specific functions. Surprisingly, a large group of ß-catenin
modulated genes are expressed in extra-embryonic tissues of control embryos,
and only a fraction of these are also deregulated in Cripto mutants.
It is thus possible that additional ß-catenin-dependent developmental
programs regulate gene expression in visceral endoderm and extra-embryonic
ectoderm. In accordance with this, a loss of regionalisation of the visceral
endoderm in ß-catenin mutants was previously observed by electron
microscopy (Huelsken et al.,
2000). A recent study using conditional inactivation of
ß-catenin has indicated a requirement of ß-catenin in the visceral
endoderm (Lickert et al.,
2002
). Together with our previous results, i.e. that
ß-catenin function is required in the embryonic ectoderm
(Huelsken et al., 2000
), these
data may indicate that ß-catenin has multiple essential roles in early
embryogenesis.
We also observed regulation of Cripto expression by ß-catenin
during tumourigenesis. Cripto expression is regulated by Lef/Tcf in
colon cancer cells, and by ß-catenin in colon adenomas of Min mice.
Cripto was previously found to be overexpressed in a wide range of
epithelial tumours, including colon, gastric and endometrial carcinomas, which
also frequently harbour activating mutations in the Wnt/ß-catenin
signalling pathway (Salomon et al.,
2000). Tissue culture studies have implicated Cripto in
cell survival, proliferation control, and oncogenic transformation
(Ciardiello et al., 1991
;
Niemeyer et al., 1998
),
indicating that Cripto expression contributes to malignancy. Our work
suggests that activation of ß-catenin signalling in tumours may be
responsible for Cripto overexpression. Cripto may thus be a
direct and critical target gene of Lef/Tcf transcription factors in
tumorigenesis and could potentially represent a suitable extracellular target
for future tumour therapy.
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ACKNOWLEDGMENTS |
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Footnotes |
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