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First published online May 30, 2007
doi: 10.1242/10.1242/dev.001206
1 Clinical Research Institute of Montreal, Montreal, Quebec, Canada.
2 Department of Cellular and Molecular Medicine, University of Ottawa, Ottawa,
K1H 8M5, Canada.
Author for correspondence (e-mail:
dlohnes{at}uottawa.ca)
Accepted 29 March 2007
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SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: Cdx1, Wnt, Lef/Tcf, Transcription, Anteroposterior patterning, Somitogenesis
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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It is well established that the Hox gene products are crucial mediators of
AP vertebral patterning. The 39 murine Hox genes are distributed in four
clusters, Hoxa to Hoxd, which are likely to have arisen from
duplication of an ancestral complex related to the Drosophila
melanogaster homeotic gene complex (HOM-C)
(Duboule, 1998
;
Duboule and Dollé,
1989; Ferrier and Holland,
2001; Lemons and McGinnis,
2006). In the mouse, Hox transcripts are first detected at
embryonic day 7.5 (E7.5) in the primitive streak, with expression subsequently
expanding to a fixed rostral limit in the neurectoderm and paraxial mesoderm
(Kmita and Duboule, 2003;
Deschamps and van Nes, 2005).
Both the onset and rostral limit of expression are generally related to the
chromosomal location of a given Hox gene within a cluster, with 3'
members expressed earlier and reaching a more anterior limit than their
5' counterparts. This results in staggered domains of Hox gene
expression along the AP axis, which is believed to comprise a `Hox code' for
vertebral patterning (Burke et al.,
1995; Gaunt, 1994;
Kessel and Gruss, 1991).
Grafting experiments in the chick have demonstrated that the cues controlling
both vertebral AP patterning and somitic Hox gene expression are acquired
early and are fixed in the anterior presomitic mesoderm prior to overt somite
segmentation (Dubrulle and
Pourquié, 2004; Christ
et al., 1974; Christ and
Ordahl, 1995; Kieny et al.,
1972; Nowicki and Burke,
2000).
Understanding the molecular mechanisms involved in establishing Hox gene
expression has been the focus of considerable research. A number of
transcription factors that impact directly on Hox gene expression have been
documented, including the vertebrate Cdx (caudal) gene family of homeodomain
transcription factors, Cdx1, Cdx2 and Cdx4
(Beck et al., 1995;
Gamer and Wright, 1993;
Meyer and Gruss, 1993). In the
murine embryo proper, Cdx1 and Cdx2 expression initiates in
the primitive streak region at E7.5, followed by Cdx4 at E8.5
(Epstein et al., 1997;
Lohnes, 2003). This results in
a nested, caudal-high pattern of Cdx expression in both ectodermal
and mesodermal compartments, with Cdx1 exhibiting a rostral-most
limit of expression, followed by Cdx2 and Cdx4. This pattern
of transcript distribution is maintained until extinction of expression in the
tail bud, with loss first of Cdx1, followed by Cdx4 and
Cdx2. These dynamic patterns of expression, together with the results
of gain-of-function and transgenic reporter experiments, have been proposed to
be indicative of a functional Cdx gradient that regulates the spatial
expression of target genes along the major body axis
(Marom et al., 1997;
Charité et al., 1998;
Gaunt et al., 2004).
Numerous studies have clearly demonstrated key roles for Cdx gene products
in vertebral patterning, and this is believed to occur, at least in part,
through direct regulation of Hox gene expression. Consistent with this,
Cdx1-null and Cdx2 heterozygotes, as well as
Cdx1;Cdx2+/-, Cdx1;Cdx4 and Cdx2;Cdx4
compound mutants, display vertebral homeosis of the cervical and anterior
thoracic regions, which correlates with posterior shifts in the rostral
mesodermal limit of several Hox genes
(Chawengsaksophak et al.,
1997; Subramanian et al.,
1995; van Den et al.,
2002, van Nes et al.,
2006). Cdx-binding sites have also been identified in the promoter
regions of numerous Hox genes (Subramanian
et al., 1995), some of which have been shown to direct spatial
expression in vivo (Charité et al.,
1998; Gaunt et al.,
2004). Gain- and loss-of-function studies in chick and
Xenopus embryos are also consistent with roles for Cdx members in AP
patterning via regulation of Hox gene expression
(Bel-Vialar et al., 2002;
Isaacs et al., 1998).
A number of signaling pathways impact on axial patterning, including
retinoic acid (RA), Wnt and fibroblast growth factor (Fgf). However, the
precise mechanisms by which these signaling molecules influence Hox gene
expression as relates to vertebral patterning are not fully understood. Prior
work has shown that Cdx1 is a target of both RA and Wnt signaling,
suggesting that Cdx1 might serve to relay information from Wnt and retinoid
pathways to mesodermal Hox gene expression
(Lohnes, 2003).
RA signals through binding to the RA receptors (RAR
, ß and
) which, together with a retinoid X receptor (RXR, ß or
) heterodimeric partner, induces transcription of target genes through
cis-acting RA-response elements (RAREs)
(Altucci and Gronemeyer, 2001;
Bastien and Rochette-Egly, 2001; Blomhoff
and Blomhoff, 2006; Mark et
al., 2006; Mic et al.,
2003). Cdx1 expression is attenuated in certain RAR-null
mutant backgrounds, and is induced by RA treatment in vivo. Consistent with a
direct relationship, the Cdx1 promoter harbors an atypical RARE that
is essential for a subset of Cdx1 expression and function in vivo
(Houle et al., 2000;
Houle et al., 2003).
In the canonical Wnt pathway, Wnt occupation of frizzled receptor leads to
stabilization of cytoplasmic ß-catenin, which translocates to the nucleus
and associates with members of the Lef/Tcf family of transcription factors
(Lef1, Tcf1, Tcf3 and Tcf4) to activate transcription of target genes
(Moon et al., 2002;
Logan and Nusse, 2004).
Wnt3a hypomorph vestigial tail (vt) mutants
(Greco et al., 1996) exhibit
vertebral defects associated with reduced Cdx1 expression and
posteriorized Hox gene expression (Ikeya
and Takada, 2001; Prinos et
al., 2001). Two Lef/Tcf-response elements (LREs) have been
identified in the proximal Cdx1 promoter that are candidates for
response to Wnt (Lickert et al.,
2000; Lickert and Kemler,
2002; Prinos et al.,
2001).
Analysis of mice mutated for the Cdx1 RARE has clearly
demonstrated the in vivo relevance of retinoid signaling as regards
Cdx1 expression and function. To more definitely determine the
importance of Wnt signaling alone or with RA in the regulation of
Cdx1, we have derived mice in which the LRE, or the LRE plus the
RARE, has been functionally inactivated by gene targeting in embryonic stem
cells. Both the LRE and the LRE+RARE-null mutants exhibit near complete
extinction of Cdx1 expression as well as skeletal defects and
posterior shifts in the expression of several Hox genes, which closely
resembles the defects seen in Cdx1-null mutants. These data support a
crucial role for Wnt signaling, through these specific LRE motifs, as a key
regulator of Cdx1 expression in vivo. Moreover, in agreement with
prior work indicating a strong synergy between RA and Wnt signaling on the
Cdx1 promoter (Prinos et al.,
2001), LRE-null mutant embryos were markedly refractory to
induction of Cdx1 by RA. Finally, recent data support a role for Wnt
signaling in the regulation of Notch-dependent somitogenesis. A high incidence
of vertebral fusions was observed in both LRE and LRE-RARE-null mutant
offspring, suggesting that Cdx1 might play a role in this process.
However, we did not observe any overt changes in the expression of several
members of the Notch signaling pathway in these backgrounds.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). The targeting vectors were generated from a 5.8 kb
KpnI-HindIII genomic fragment, subcloned into pBluescript
KS+ (Stratagene), that encompassed the proximal Cdx1 promoter, the
first exon and part of the first intron
(Fig. 1). The LRE sequences
were mutated and SfuI sites incorporated, as described previously
(Prinos et al., 2001). For the
LRE+RARE targeting construct, part of the RARE in the LRE mutant targeting
vector was converted to a SacI site (GAAGGGGAGCTCCCCCT;
mutation underlined) using the Quick Change Site-Directed Mutagenesis Kit
according to the manufacturer's (Stratagene) instruction. A bifunctional
floxed thymidine kinase/neomycin resistance cassette
(loxPGK-TK-Neolox) (Iulianella
and Lohnes, 2002) was then subcloned into the unique XhoI
site present in the intron of either construct. R1 embryonic stem cells were cultured on murine embryonic fibroblasts under standard conditions. Cells were electroporated with 40 µg of linearized targeting vector and selected with G418 (200 µg/ml) for 10 days. Surviving clones were isolated, expanded and assessed for homologous recombination by genomic Southern blot analysis using SacI digestion and hybridization with a probe 5' to the sequences used to generate the targeting construct (probe A in Fig. 1). Positive clones were confirmed for the fidelity of recombination by Southern blot analysis using additional restriction enzymes and hybridization with an internal probe (probe B in Fig. 1). Incorporation of the mutated RARE in targeted clones was determined by genomic Southern blot analysis by virtue of the novel SacI site introduced in this motif (Fig. 1), whereas integration of the mutated LRE was assessed by SfuI digestion of a PCR product generated by primers flanking these sequences (Fig. 1).
Germline chimeras were derived by injection of targeted ES clones into
C57BL/6 blastocysts according to standard procedures
(Hogan et al., 1994). F1
animals from chimera-C57BL/6 outcrosses bearing the targeted allele were bred
with homozygous CMV-Cre mice
(Dupé et al., 1997) and
offspring assessed for excision of the floxed Tk-Neo selection
cassette by genomic Southern blot analysis as depicted in
Fig. 1. Subsequent genotyping
of established mutant lines was performed by PCR using primers flanking the
XhoI site of the first intron (forward,
5'-ATCCTGGCGCAGTCCCTC-3'; reverse,
5'-AGGACAAGAGTGGTCGTGG-3'); the PCR product of the mutant allele
exhibited an increased size owing to the presence of residual loxP
sequences (Fig. 1).
Analysis of mutant offspring
Mice were mated overnight and noon of the day of vaginal plug detection was
considered as E0.5. In situ hybridization and skeletal preparations were
performed as previously described (Allan et
al., 2001; Houle et al.,
2003), using either wild-type littermates or CD1 offspring as
controls. Embryos to be compared were stage-matched according to established
criteria and processed in parallel. Probes for in situ hybridization were
generated from previously described plasmids: Cdx1
(Houle et al., 2000),
Hoxa3 (Manley and Capecchi,
1995), Hoxd3 (Condie
and Capecchi, 1993), Hoxb4
(Folberg et al., 1999),
Hoxd4 (Featherstone et al.,
1988) and Lfng
(Mustonen et al., 2002).
Plasmids for probes for Notch1 and Hes5 were a kind gift
from C. C. Hui (The Hospital for Sick Children, Toronto, Canada) and the
Delta1 (Dll1) probe was kindly provided by O.
Pourquié (Stowers Institute for Medical Research, Kansas City, MO).
Embryo culture, Wnt3a treatment and oral RA gavage were carried out as
described previously (Prinos et al.,
2001; Houle et al.,
2000).
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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;
Prinos et al., 2001), and data
from transgenic models is consistent with a role for these motifs in
regulating spatial-temporal expression of Cdx1 in vivo
(Lickert and Kemler, 2002). A
crucial role for the Cdx1 RARE has been clearly established by gene
targeting, although ablation of this element in vivo does not fully
recapitulate the findings from transgenic analysis
(Houle et al., 2003). RA and
Wnt have also been shown to synergistically activate the Cdx1
promoter in tissue culture models (Prinos
et al., 2001). However, the precise in vivo role of the LRE, alone
or in concert with the RARE, has not been thoroughly investigated. To evaluate the importance of the LRE and the interplay between retinoid and Wnt signaling in the control of Cdx1 expression in vivo, we derived mice mutated for the LRE or the LRE+RARE. Targeting constructs containing a mutated LRE or mutated LRE+RARE were used to generate recombinant ES clones (Fig. 1). Chimeras generated from targeted cells for either mutation gave germline transmission. Subsequent excision of the selection cassette was effected by crossing F1 offspring with CMV-Cre mice (Fig. 1B). All of the resulting heterozygotes passed the mutant allele to their offspring at the predicted mendelian ratio.
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).
However, novel point mutations were introduced to inactivate the RARE. To
determine if this element was devoid of function, the mutated RARE was
amplified from LRE-RARE homozygote mutant offspring and subcloned into a
previously described Cdx1 reporter vector
(Houle et al., 2000),
replacing the cognate wild-type element. This mutant RARE reporter failed to
respond to exogenous RA in transfection assays in F9 embryocarcinoma cells
(data not shown), demonstrating that this element was functionally
inactive.
LRE and LRE+RARE mutations phenocopy loss of Cdx1 function
The murine axial skeleton is normally composed of occipital bones, derived
from condensation of the four rostral-most somites, and a vertebral column
composed of seven cervical (C1-C7), 13 thoracic (T1-T13), six lumbar (L1-L6),
three or four sacral (S1-S3/S4) and 31 caudal vertebrae. Many vertebrae
exhibit specific morphological characteristics related to their position along
the AP axis. The first cervical vertebra (C1, or atlas) exhibits thick neural
arches and possesses a ventrally located tubercle, the anterior arch of the
atlas (AAA; see Fig. 2A, for
example). The neural arches of C2 are intermediate to those of C1 and
more-caudal cervical vertebrae. C2 also possesses a second vertebral body, the
dens axis, which articulates with C1. C3, C4 and C5 are morphologically
similar, whereas C6 is distinguished by ventrally protruding anterior
tuberculi. The thoracic vertebrae are characterized by the presence of ribs,
the first seven of which (T1-T7) are attached to the sternum.
RARE+/- offspring do not exhibit any overt vertebral anomalies
(Table 1,
Fig. 2A)
(Houle et al., 2003). By
contrast, approximately 40% of LRE+/- and LRE+RARE+/-
offspring exhibited a C2 to C1 transformation, indicated by a broader neural
arch on C2. This transformation was, however, less expressive in
LRE+/- offspring as an ectopic AAA was never observed in this
background (Table 1,
Fig. 2B,C). By contrast, the
penetrance and expressivity of C2 to C1 anterior homeotic transformation
(including a C2 AAA) in the LRE+RARE+/- mutants was comparable to
that observed in Cdx1 heterozygote offspring
(Table 1).
|
). In
contrast to RARE loss-of-function, ablation of the LRE or the LRE+RARE
resulted in anterior homeotic transformations and vertebral fusions that
largely recapitulated the defects seen in Cdx1-null offspring
(Table 1,
Fig. 2F-H). C1 displayed a
reduced neural arch and loss of the AAA and was fused to, or in close
apposition with, the basioccipital bone. C2 to C1, C3 to C2, C6 to C5 and C7
to C6 transformations were also observed, as evidenced by the morphological
criteria described above. However, both the LRE and the LRE+RARE-null mutants
were distinguished from Cdx1-null offspring by a high incidence of
fusions between neural arches of the first two or three cervical vertebrae
(Table 1,
Fig. 2I,J). Moreover, relative
to Cdx1 mutants, fusion between C1 and the basioccipital bone was
less expressive in the LRE and LRE-RARE-null backgrounds. Together, with
expression analysis (below), these data suggest that some residual Cdx1
function exists in these backgrounds.
|
), Cdx1 transcripts were first detected in the late
primitive streak region in wild-type embryos at E7.5
(Fig. 3A). Expression peaked
around E8.0 in the tail bud and then declined until E9.5, by which time
transcripts in the caudal embryo were barely detectable
(Fig. 3B,C). Relative to
controls, Cdx1 expression in the primitive streak was barely
detectable at earlier stages, with a weak signal in some LRE-null and
LRE+RARE-null mutants at the early head-fold stage (E7.75;
Fig. 3D,G). As observed in
controls, expression peaked around E8.0 in both LRE-null and LRE+RARE-null
mutants, although transcript levels remained greatly reduced and were no
longer detected after the 4- to 5-somite stage
(Fig. 3E,F,H,I; note that LRE
and LRE+RARE mutants were stained four times longer than controls, to reveal
residual expression).
Altered Hox gene expression in LRE and LRE+RARE mutants
Cdx1-null mice exhibit vertebral defects that reflect those of
certain Hox group 3- and group 4-null mutants
(Subramanian et al., 1995).
Consistent with this relationship, the rostral mesodermal limit of expression
of a number of these Hox genes is posteriorized in Cdx1-null mutants
(Allan et al., 2001;
Houle et al., 2003). By
contrast, only Hox group 4 genes are affected in RARE mutant offspring, in
agreement with the restricted effects on C2 morphogenesis seen in this
background.
To determine the relationship between Hox gene expression and the vertebral
defects in LRE and LRE+RARE mutants, we assessed the expression patterns of
Hoxa3, Hoxd3, Hoxb4 and Hoxd4. In wild-type embryos at E9.5,
the mesodermal limit of expression of Hoxa3 and Hoxd3 is at
the fifth somite (Fig. 4A,D)
(Condie and Capecchi, 1993;
Gaunt, 1988;
Sham et al., 1992). As for
Cdx1 (but not RARE) mutants, expression of these genes was
posteriorized by one somite in both LRE and LRE+RARE-null mutants
(Fig. 4B,C,E,F). The normal
rostral limit of Hoxb4 and Hoxd4 in the mesoderm is at
somite 6 (Fig. 4G,J)
(Featherstone et al., 1988;
Gaunt et al., 1989). Again,
expression was posteriorized by one somite in both LRE and LRE+RARE-null
mutants (Fig. 4H,I,K,L); an
identical posteriorization is also seen in both Cdx1 and RARE-null
mutants (Houle et al., 2003;
van Den et al., 2002). These
results are in agreement with skeletal analyses
(Fig. 2), and underscore a
crucial role for Wnt signaling in vertebral patterning through regulation of
Cdx1.
Response of LRE-null and LRE+RARE-null mutants to exogenous Wnt and RA
To verify if the LRE is required for regulation by Wnt signaling ex vivo,
embryos were cultured in the absence or presence of Wnt3a-conditioned medium,
and Cdx1 expression assessed. As described previously
(Prinos et al., 2001),
Cdx1 expression was modestly but reproducibly induced by
Wnt3a-conditioned medium in wild-type embryos at all stages assessed
(Fig. 5A,B and data not shown).
By contrast, induction was markedly attenuated in LRE and LRE+RARE mutants at
E7.5-8.5 (Fig. 5C-F and data
not shown; note that mutants were stained four times longer than controls to
reveal residual expression).
Targeted mutation of the RARE blocks the response of a Cdx1
reporter to RA in tissue culture, but does not completely prevent induction in
vivo, perhaps owing to the presence of alternate RAREs
(Houle et al., 2000;
Houle et al., 2003;
Gaunt et al., 2003). Prior
work has also shown a strong synergistic interaction between RA and Wnt3a on
the Cdx1 promoter in embryocarcinoma cells
(Prinos et al., 2001). To
determine the potential relevance of this interaction in vivo, we assessed
Cdx1 expression in LRE-null embryos 4 hours after RA treatment in
utero. Whereas treatment clearly induced Cdx1 expression at E7.5 and
E8.5 in wild-type controls (Fig.
6A-D), this effect was markedly reduced in LRE-null embryos at
E7.5 (Fig. 6E,F) and E8.5
(Fig. 6G,H). Given that Wnt
signaling is active in the caudal embryo at these stages, these results are
consistent with previous findings in tissue culture, and underscore a role for
RA and Wnt signaling through these specific motifs in coordinately regulating
Cdx1 transcription.
Notch signaling and somitogenesis in LRE+RARE mutants
Fusions between one or more of the first three cervical vertebrae were
frequently observed in LRE and LRE+RARE mutants, suggesting a defect in
somitogenesis. Both Wnt and RA signaling have been implicated in this process,
the former operating upstream of the Notch pathway and impacting on the somite
`clock', whereas RA has been proposed to antagonize Fgf8-dependent events
related to the wavefront (Aulehla and
Herrmann, 2004; Iulianella et
al., 2003; Dubrulle et al.,
2001; Dubrulle and
Pourquié, 2004). Moreover, RA, Wnt, Fgf and Notch pathways
have all been linked to regulation of Hox gene expression and vertebral
patterning (Cordes et al.,
2004; Dubrulle et al.,
2001; Yamaguchi et al.,
1994; Zakany et al.,
2001).
As Cdx1 is a target of both Wnt and retinoid signaling pathways,
it could conceivably serve as an intermediary in somitogenic events upstream
of Notch. To this end, we compared expression of the Notch pathway components
Dll1, Lfng, Notch1, Mesp2 and Hes5 between wild-type and
LRE+RARE mutants. The vertebral fusions seen in LRE and LRE+RARE mutants
involved the first three cervical vertebrae, which are derived from somites
5-7. As the somite determination front is located in a region corresponding to
four prospective somites in the unsegmented paraxial mesoderm
(Dubrulle et al., 2001),
expression patterns were compared using embryos of zero to five somites. No
overt alterations in transcript levels or distribution were observed for these
particular markers in these mutant backgrounds (see Fig. S1 in the
supplementary material and data not shown).
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DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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; Lickert and
Kemler, 2002; Prinos et al.,
2001). Cdx1 is also regulated by RA through an atypical
RARE, and mutagenesis studies clearly demonstrate a role for retinoid
signaling in directing a subset of Cdx1 expression. Moreover, strong
synergistic interaction between RA and Wnt signaling on transcription from the
Cdx1 promoter has been described in embryocarcinoma cells, and this
interaction is dependent on the LRE and RARE
(Prinos et al., 2001). To more
precisely address the role of Wnt and RA signaling in the regulation of
Cdx1 expression, we derived and analyzed murine lines lacking the LRE
or LRE+RARE.
|
|
). These sequences are conserved in the mouse, but are
approximately 1 kb 3' of the residual loxP sequences. It would
therefore appear unlikely that these exogenous lox sequences would
impact on Cdx1 expression through interference with these enhancer
sequences, although this has not been formally tested.
Retinoid signaling has been implicated in early stages of Cdx1
expression (Houle et al.,
2000; Houle et al.,
2003), whereas Wnt signaling and autoregulation have been
suggested to be involved at both early and later stages
(Lickert and Kemler, 2002;
Beland et al., 2004;
Prinos et al., 2001). In
agreement with this, we found that the LRE mutants exhibited skeletal defects
affecting the whole cervical region that essentially phenocopied the
malformations observed in Cdx1-null mutants. Consistent with these
observations, in situ hybridization analysis revealed that Cdx1
expression was markedly reduced throughout its normal window of expression.
These data further suggest that Wnt signaling might be required even before
RA, as loss of the LRE impacts on C1 patterning, whereas C2 is the first
vertebra affected in RARE mutants. However, as other RAREs might be involved
in regulating Cdx1 (Houle et al.,
2003; Gaunt et al.,
2003), the full import of retinoid signaling as regards
Cdx1 expression is likely to remain unresolved at present.
Irrespective, these findings underscore a crucial role for Wnt signaling in
regulating Cdx1.
|
|
;
Ikeya and Takada, 2001).
Wnt3a-null mutants also exhibit a reduction in Cdx1
expression and a completely penetrant C2 to C1 transformation
(Ikeya and Takada, 2001).
However, other vertebral defects observed in Cdx1-null mutants are
not manifested in either Wnt3avt-vt or Wnt3a-null
offspring. This is in contrast to the phenotype of LRE mutants, suggesting
that Cdx1 is regulated by other Wnts present in the primitive
streak/tail bud, such as Wnt3 or Wnt8
(Bouillet et al., 1996;
Liu et al., 1999;
Takada et al., 1994).
Conversely, the residual Cdx1 expression in LRE-null offspring, and
the occasional minimal response to exogenous Wnt3a seen in embryo culture
(data not shown), suggests the presence of other functional motifs conveying
the Wnt signal. Indeed, additional potential LREs have been described in the
5' promoter region of the Cdx1 locus
(Lickert et al., 2000). These
other putative elements are, however, clearly unable to effectively compensate
in the absence of the proximal LRE sequences.
Combinatorial signaling and Cdx1 expression
The importance of the Cdx1 RARE and LRE in directing expression in
vivo has been suggested by transgenic studies
(Lickert and Kemler, 2002),
and a definitive role for the RARE has been further demonstrated by targeted
mutagenesis (Houle et al.,
2003). In this regard, loss of the RARE has a limited effect on
Cdx1 expression in vivo and affects morphogenesis of only the second
cervical vertebra. By contrast, mutation of the LRE impacted on Cdx1
expression at all stages and resulted in a nearly complete recapitulation of
the Cdx1-null phenotype. Subsequent loss of the RARE in an LRE mutant
background resulted in only a slight additional effect on expression and
vertebral patterning, as expected from the dominant impact of the LRE
mutation. The combinatorial contribution of Wnt and retinoid signaling to
Cdx1 expression and function was, however, clearly evidenced by
comparison of LRE+/- and LRE+RARE+/- skeletons, where a
more complete C2 to C1 transformation was observed in the latter
background.
A strong synergy between retinoid and Wnt signaling in inducing
transcription from the Cdx1 promoter has been observed in tissue
culture models, and this interaction requires both the RARE and the LRE
(Prinos et al., 2001).
Although RA still induces Cdx1 in vivo in RARE mutants, perhaps owing
to other RAREs (Houle et al.,
2003; Gaunt et al.,
2003), the response to RA was greatly attenuated in the LRE mutant
background in the present study. This suggests that retinoid signaling is
reliant on the LRE to mediate full induction of Cdx1 transcription.
Although the mechanism underlying this cooperativity is speculative, it might
relate to the architectural role of Lef/Tcf transcription factors
(Dragan et al., 2004), or
might be indicative of chromatin modification events affected by Wnt
signaling, which is essential for productive interaction of the retinoid
receptors with the promoter. In any event, this finding clearly underscores a
cooperative role for Wnt and RA in the regulation of Cdx1 in
vivo.
Cdx1 and Hox gene expression
In situ hybridization analysis of LRE-null and LRE+RARE-null embryos
revealed identical posterior shifts in expression of Hox paralog group 3 and 4
genes, as has been observed in Cdx1-null mutants
(Houle et al., 2003;
van Den et al., 2002). By
contrast, only Hox group 4 genes are affected in RARE mutants. In this regard,
Cdx1 expression in RARE-null mutants is properly initiated but
transcript levels are reduced (Houle et
al., 2003). This suggests Hox paralog group 3 genes might require
a lower threshold of Cdx1 activity than group 4 genes, leading to
normal C1 patterning in RARE mutants, as previously discussed
(Houle et al., 2003) (see also
Charité et al., 1998;
Gaunt et al., 2003;
Gaunt et al., 2004;
Marom et al., 1997;
Deschamps and van Nes, 2005).
However, timing of expression is also an essential component of Hox-mediated
skeletal patterning (Juan and Ruddle,
2003; Dollé et al.,
1989). The delay in onset of Cdx1 transcription seen in
the LRE and LRE+RARE mutants might therefore underlie the defects in
morphogenesis of C1, although it is difficult to differentiate such a
mechanism from dosage effects.
Is Cdx1 function linked to somite segmentation?
LRE and LRE+RARE mutants exhibit a high incidence of fusions between the
first three cervical vertebrae, suggesting a link between Cdx1 and
somitogenesis. The lack of similar defects in Cdx1-null mutants might
be related to residual function in the LRE and LRE+RARE mutant backgrounds,
eventually impacting on events related to sclerotome patterning. In this
regard, it has been shown that Hox gene expression is subject to
Notch-mediated signaling in the anterior presomitic mesoderm
(Zakany et al., 2001), and
also that Notch functions downstream of Wnt signaling in this domain.
Moreover, haploinsufficiency of Dll1, as well as gain or loss of Lfng
function, results in homeotic transformations affecting the cervical and
thoracic vertebrae (Cordes et al.,
2004). As Cdx1 is a direct Wnt target, it is conceivable
that its product could contribute to Wnt-dependent regulation of Notch
signaling processes involved in somitogenesis. However, in situ hybridization
analysis did not reveal any overt alterations in Delta1, Notch1, Hes5
or Lfng in LRE+RARE mutant embryos, although we cannot exclude the
possibility that their oscillatory patterns of expression might be subtly
affected.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/134/12/2315/DC1
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ACKNOWLEDGMENTS |
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REFERENCES |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Allan, D., Houle, M., Bouchard, N., Meyer, B. I., Gruss, P. and Lohnes, D. (2001). RAR gamma and Cdx1 interactions in vertebral patterning. Dev. Biol. 240, 46-60.[CrossRef][Medline]
Altucci, L. and Gronemeyer, H. (2001). Nuclear receptors in cell life and death. Trends Endocrinol. Metab. 12,460 -468.[CrossRef][Medline]
Aulehla, A. and Herrmann, B. G. (2004).
Segmentation in vertebrates: clock and gradient finally joined.
Genes Dev. 18,2060
-2067.
Bastien, J. and Rochette-Egly, C. (2004). Nuclear retinoid receptors and the transcription of retinoid-target genes. Gene 328,1 -16.[CrossRef][Medline]
Beck, F., Erler, T., Russell, A. and James, R. (1995). Expression of Cdx-2 in the mouse embryo and placenta: possible role in patterning of the extra-embryonic membranes. Dev. Dyn. 204,219 -227.[Medline]
Bel-Vialar, S., Itasaki, N. and Krumlauf, R. (2002). Initiating Hox gene expression: in the early chick neural tube differential sensitivity to FGF and RA signaling subdivides the HoxB genes in two distinct groups. Development 129,5103 -5115.[Medline]
Beland, M., Pilon, N., Houle, M., Oh, K., Sylvestre, J. R.,
Prinos, P. and Lohnes, D. (2004). Cdx1 autoregulation is
governed by a novel Cdx1-LEF1 transcription complex. Mol. Cell.
Biol. 24,5028
-5038.
Blomhoff, R. and Blomhoff, H. K. (2006). Overview of retinoid metabolism and function. J. Neurobiol. 66,606 -630.[CrossRef][Medline]
Bouillet, P., Oulad-Abdelghani, M., Ward, S. J., Bronner, S., Chambon, P. and Dollé, P. (1996). A new mouse member of the Wnt gene family, mWnt-8, is expressed during early embryogenesis and is ectopically induced by retinoic acid. Mech. Dev. 58,141 -152.[CrossRef][Medline]
Burke, A. C., Nelson, C. E., Morgan, B. A. and Tabin, C. (1995). Hox genes and the evolution of vertebrate axial morphology. Development 121,333 -346.[Abstract]
Charité, J., de Graaff, W., Consten, D., Reijnen, M. J., Korving, J. and Deschamps, J. (1998). Transducing positional information to the Hox genes: critical interaction of cdx gene products with position-sensitive regulatory elements. Development 125,4349 -4358.[Abstract]
Chawengsaksophak, K., James, R., Hammond, V. E., Kontgen, F. and Beck, F. (1997). Homeosis and intestinal tumours in Cdx2 mutant mice. Nature 386,84 -87.[CrossRef][Medline]
Christ, B. and Ordahl, C. P. (1995). Early stages of chick somite development. Anat. Embryol. 191,381 -396.[CrossRef][Medline]
Christ, B., Jacob, H. J. and Jacob, M. (1974). Somitogenesis in the chick embryo. Determination of the segmentation direction. Verh. Anat. Ges. 68,573 -579.[Medline]
Condie, B. G. and Capecchi, M. R. (1993). Mice homozygous for a targeted disruption of Hoxd-3 (Hox-4.1) exhibit anterior transformations of the first and second cervical vertebrae, the atlas and the axis. Development 119,579 -595.[Abstract]
Cordes, R., Schuster-Gossler, K., Serth, K. and Gossler, A.
(2004). Specification of vertebral identity is coupled to Notch
signalling and the segmentation clock. Development
131,1221
-1233.
Deschamps, J. and van Nes, J. (2005).
Developmental regulation of the Hox genes during axial morphogenesis in the
mouse. Development 132,2931
-2942.
Dollé, P., Izpisua-Belmonte, J. C., Falkenstein, H., Renucci, A. and Duboule, D. (1989). Coordinate expression of the murine Hox-5 complex homoeobox-containing genes during limb pattern formation. Nature 342,767 -772.[CrossRef][Medline]
Dragan, A. I., Read, C. M., Makeyeva, E. N., Milgotina, E. I., Churchill, M. E., Crane-Robinson, C. and Privalov, P. L. (2004). DNA binding and bending by HMG boxes: energetic determinants of specificity. J. Mol. Biol. 343,371 -393.[CrossRef][Medline]
Duboule, D. (1998). Vertebrate hox gene regulation: clustering and/or colinearity? Curr. Opin. Genet. Dev. 8,514 -518.[CrossRef][Medline]
Duboule, D. and Dollé, P. (1989). The structural and functional organization of the murine HOX gene family resembles that of Drosophila homeotic genes. EMBO J. 8,1497 -1505.[Medline]
Dubrulle, J. and Pourquié, O. (2004).
Coupling segmentation to axis formation. Development
131,5783
-5793.
Dubrulle, J., McGrew, M. J. and Pourquié, O. (2001). FGF signaling controls somite boundary position and regulates segmentation clock control of spatiotemporal Hox gene activation. Cell 106,219 -232.[CrossRef][Medline]
Dupé, V., Davenne, M., Brocard, J., Dollé, P., Mark, M., Dierich, A., Chambon, P. and Rijli, F. M. (1997). In vivo functional analysis of the Hoxa-1 3' retinoic acid response element (3'RARE). Development 124,399 -410.[Abstract]
Epstein, M., Pillemer, G., Yelin, R., Yisraeli, J. K. and Fainsod, A. (1997). Patterning of the embryo along the anterior-posterior axis: the role of the caudal genes. Development 124,3805 -3814.[Abstract]
Featherstone, M. S., Baron, A., Gaunt, S. J., Mattei, M. G. and
Duboule, D. (1988). Hox-5.1 defines a homeobox-containing
gene locus on mouse chromosome 2. Proc. Natl. Acad. Sci.
USA 85,4760
-4764.
Ferrier, D. E. K. and Holland, P. W. H. (2001). Ancient origin of the Hox gene cluster. Nat. Rev. Genet. 2,33 -38.[Medline]
Folberg, A., Kovacs, E. N., Huang, H., Houle, M., Lohnes, D. and Featherstone, M. S. (1999). Hoxd4 and Rarg interact synergistically in the specification of the cervical vertebrae. Mech. Dev. 89,65 -74.[CrossRef][Medline]
Gamer, L. W. and Wright, C. V. (1993). Murine Cdx-4 bears striking similarities to the Drosophila caudal gene in its homeodomain sequence and early expression pattern. Mech. Dev. 43,71 -81.[CrossRef][Medline]
Gaunt, S. J. (1988). Mouse homeobox gene transcripts occupy different but overlapping domains in embryonic germ layers and organs: a comparison of Hox-3.1 and Hox-1.5. Development 103,135 -144.[Abstract]
Gaunt, S. J. (1994). Conservation in the Hox code during morphological evolution. Int. J. Dev. Biol. 38,549 -552.[Medline]
Gaunt, S. J., Krumlauf, R. and Duboule, D. (1989). Mouse homeo-genes within a subfamily, Hox-1.4, -2.6 and -5.1, display similar anteroposterior domains of expression in the embryo, but show stage- and tissue-dependent differences in their regulation. Development 107,131 -141.[Abstract]
Gaunt, S. J., Drage, D. and Cockley, A. (2003). Vertebrate caudal gene expression gradients investigated by use of chick cdx-A/lacZ and mouse cdx-1/lacZ reporters in transgenic mouse embryos: evidence for an intron enhancer. Mech. Dev. 120,573 -586.[CrossRef][Medline]
Gaunt, S. J., Cockley, A. and Drage, D. (2004). Additional enhancer copies, with intact cdx binding sites, anteriorize Hoxa-7/lacZ expression in mouse embryos: evidence in keeping with an instructional cdx gradient. Int. J. Dev. Biol. 48,613 -622.[CrossRef][Medline]
Greco, T. L., Takada, S., Newhouse, M. M., McMahon, J. A.,
McMahon, A. P. and Camper, S. A. (1996). Analysis of the
vestigial tail mutation demonstrates that Wnt-3a gene dosage regulates mouse
axial development. Genes Dev.
10,313
-324.
Hogan, B., Constantini, F., Lacy, P. and Beddington, R. S. P. (1994). Manipulating the Mouse Embryo. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press.
Houle, M., Prinos, P., Iulianella, A., Bouchard, N. and Lohnes,
D. (2000). Retinoic acid regulation of Cdx1: an indirect
mechanism for retinoids and vertebral specification. Mol. Cell.
Biol. 20,6579
-6586.
Houle, M., Sylvestre, J. R. and Lohnes, D.
(2003). Retinoic acid regulates a subset of Cdx1 function in
vivo. Development 130,6555
-6567.
Ikeya, M. and Takada, S. (2001). Wnt-3a is required for somite specification along the anteroposterior axis of the mouse embryo and for regulation of cdx-1 expression. Mech. Dev. 103,27 -33.[CrossRef][Medline]
Isaacs, H. V., Pownall, M. E. and Slack, J. M. (1998). Regulation of Hox gene expression and posterior development by the Xenopus caudal homologue Xcad3. EMBO J. 17,3413 -3427.[CrossRef][Medline]
Iulianella, A. and Lohnes, D. (2002). Chimeric analysis of retinoic acid receptor function during cardiac looping. Dev. Biol. 247,62 -75.[CrossRef][Medline]
Iulianella, A., Melton, K. R. and Trainor, P. A. (2003). Somitogenesis: breaking new boundaries. Neuron 40,11 -14.[CrossRef][Medline]
Juan, A. H. and Ruddle, F. H. (2003). Enhancer
timing of Hox gene expression: deletion of the endogenous Hoxc8 early
enhancer. Development
130,4823
-4834.
Kessel, M. and Gruss, P. (1991). Homeotic transformations of murine vertebrae and concomitant alteration of Hox codes induced by retinoic acid. Cell 67, 89-104.[CrossRef][Medline]
Kieny, M., Mauger, A. and Sengel, P. (1972). Early regionalization of somitic mesoderm as studied by the development of axial skeleton of the chick embryo. Dev. Biol. 28,142 -161.[CrossRef][Medline]
Kmita, M. and Duboule, D. (2003). Organizing
axes in time and space; 25 years of colinear tinkering.
Science 301,331
-333.
Lemons, D. and McGinnis, W. (2006). Genomic
evolution of Hox gene clusters. Science
313,1918
-1922.
Lickert, H. and Kemler, R. (2002). Functional analysis of cis-regulatory elements controlling initiation and maintenance of early Cdx1 gene expression in the mouse. Dev. Dyn. 225,216 -220.[CrossRef][Medline]
Lickert, H., Domon, C., Huls, G., Wehrle, C., Duluc, I., Clevers, H., Meyer, B. I., Freund, J. N. and Kemler, R. (2000). Wnt/(beta)-catenin signaling regulates the expression of the homeobox gene Cdx1 in embryonic intestine. Development 127,3805 -3813.[Abstract]
Liu, P., Wakamiya, M., Shea, M. J., Albrecht, U., Behringer, R. R. and Bradley, A. (1999). Requirement for Wnt3 in vertebrate axis formation. Nat. Genet. 22,361 -365.[CrossRef][Medline]
Logan, C. Y. and Nusse, R. (2004). The Wnt signaling pathway in development and disease. Annu. Rev. Cell Dev. Biol. 20,781 -810.[CrossRef][Medline]
Lohnes, D. (2003). The Cdx1 homeodomain protein: an integrator of posterior signaling in the mouse. BioEssays 25,971 -980.[CrossRef][Medline]
Manley, N. R. and Capecchi, M. R. (1995). The role of Hoxa-3 in mouse thymus and thyroid development. Development 121,1989 -2003.[Abstract]
Mark, M., Ghyselinck, N. and Chambon, P. (2006). Function of retinoid nuclear receptors: lessons from genetic and pharmacological dissections of the retinoic acid signaling pathway during mouse embryogenesis. Annu. Rev. Pharmacol. Toxicol. 46,451 -480.[CrossRef][Medline]
Marom, K., Shapira, E. and Fainsod, A. (1997). The chicken caudal genes establish an anterior-posterior gradient by partially overlapping temporal and spatial patterns of expression. Mech. Dev. 64,41 -52.[CrossRef][Medline]
Meyer, B. I. and Gruss, P. (1993). Mouse Cdx-1 expression during gastrulation. Development 117,191 -203.[Abstract]
Mic, F. A., Molotkov, A., Benbrook, D. M. and Duester, G.
(2003). Retinoid activation of retinoic acid receptor but not
retinoid X receptor is sufficient to rescue lethal defect in retinoic acid
synthesis. Proc. Natl. Acad. Sci. USA
100,7135
-7140.
Moon, R. T., Bowerman, B., Boutros, M. and Perrimon, N.
(2002). The promise and perils of Wnt signaling through
beta-catenin. Science
296,1644
-1646.
Mustonen, T., Tummers, M., Mikami, T., Itoh, N., Zhang, N., Gridley, T. and Thesleff, I. (2002). Lunatic fringe, FGF, and BMP regulate the Notch pathway during epithelial morphogenesis of teeth. Dev. Biol. 248,281 -293.[CrossRef][Medline]
Nowicki, J. L. and Burke, A. C. (2000). Hox genes and morphological identity: axial versus lateral patterning in the vertebrate mesoderm. Development 127,4265 -4275.[Abstract]
Prinos, P., Joseph, S., Oh, K., Meyer, B. I., Gruss, P. and Lohnes, D. (2001). Multiple pathways governing Cdx1 expression during murine development. Dev. Biol. 239,257 -269.[CrossRef][Medline]
Sham, M. H., Hunt, P., Nonchev, S., Papalopulu, N., Graham, A., Boncinelli, E. and Krumlauf, R. (1992). Analysis of the murine Hox-2.7 gene: conserved alternative transcripts with differential distributions in the nervous system and the potential for shared regulatory regions. EMBO J. 11,1825 -1836.[Medline]
Subramanian, V., Meyer, B. I. and Gruss, P. (1995). Disruption of the murine homeobox gene Cdx1 affects axial skeletal identities by altering the mesodermal expression domains of Hox genes. Cell 83,641 -653.[CrossRef][Medline]
Takada, S., Stark, K. L., Shea, M. J., Vassileva, G., McMahon,
J. A. and McMahon, A. P. (1994). Wnt-3a regulates somite and
tailbud formation in the mouse embryo. Genes Dev.
8, 174-189.
van Den, A. E., Forlani, S., Chawengsaksophak, K., de Graaff,
W., Beck, F., Meyer, B. I. and Deschamps, J. (2002). Cdx1 and
Cdx2 have overlapping functions in anteroposterior patterning and posterior
axis elongation. Development
129,2181
-2193.
van Nes, J., de Graff, W., Lebrin, F., Gerhard, M., Beck, F. and
Deschamps, J. (2006). The Cdx4 mutation affects axial
development and reveals an essential role of Cdx genes in the ontogenesis of
the placental labyrinth in mice. Development
133,419
-428.
Yamaguchi, T. P., Harpal, K., Henkemeyer, M. and Rossant, J.
(1994). fgfr-1 is required for embryonic growth and mesodermal
patterning during mouse gastrulation. Genes Dev.
8,3032
-3044.
Zakany, J., Kmita, M., Alarcon, P., de la Pompa, J. L. and Duboule, D. (2001). Localized and transient transcription of Hox genes suggests a link between patterning and the segmentation clock. Cell 106,207 -217.[CrossRef][Medline]
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