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First published online 25 May 2006
doi: 10.1242/dev.02422
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1 Division of Mammalian Development, National Institute of Genetics, Yata 1111,
Mishima 411-8540, Japan.
2 Division of Developmental Genetics, RIKEN Research Center for Allergy and
Immunology (RCAI) RIKEN Yokohama Institute, 1-7-22 Suehiro, Tsurumi-ku,
Yokohama 230-0045, Japan.
* Author for correspondence (e-mail: ysaga{at}lab.nig.ac.jp)
Accepted 2 May 2006
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SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: Mesp2, Epha4, Somitogenesis, Segmental border, Mox1, mouse
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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;
Bessho and Kageyama, 2003;
Maroto and Pourquié,
2001; Pourquié,
2003; Rida et al.,
2004; Saga and Takeda,
2001). Notch signaling is suppressed in the anterior PSM by a bHLH
protein, Mesp2, and the anterior limits of the Mesp2 expression domain
demarcate the next segmental border
(Morimoto et al., 2005). Mesp2
is a key transcription factor for both segment border formation and for the
generation of rostrocaudal patterning within somites
(Saga et al., 1997;
Takahashi et al., 2000). The
expression of many genes is affected in the Mesp2-null embryo, in which the
genes required for rostral property are suppressed but those required for the
development of caudal properties are enhanced. Among these genes, only lunatic
fringe (Lfng) has so far been shown to be a direct target of Mesp2
(Morimoto et al., 2005).
Since the Mesp2 expression domain is very similar to that of
Epha4, and this gene is also suppressed in the Mesp2-null embryo
(Nomura-Kitabayashi et al.,
2002), it was probable that Mesp2 directly activated
Epha4 in the rostral compartment of the somites. Furthermore, Epha4
is implicated in segmental border formation in zebrafish
(Cooke et al., 2005;
Barrios et al., 2003;
Durbin et al., 1998), although
gene knockout studies indicate that Epha4 is not the sole protein required for
segmental border formation in the mouse, as no somitic phenotype has been
reported (Dottori et al., 1998;
Kullander et al., 2001) (M.
Asano, personal communication). The identification of target genes for a
transcription factor is necessary to understand fully the genetic networks
involved in a particular biological system. However, it is very difficult to
achieve these using straightforward methods such as SELEX or
immunoprecipitation, particularly in embryonic tissues. As an alternative
method, we attempted to first identify the Epha4 enhancer and then
test whether Mesp2 directly binds to this region; if it does not bind, we can
search the binding protein that might be a direct target of Mesp2.
Fortunately, the Epha4 enhancer elements identified showed direct
binding to Mesp2, together with E47 (Tcfe2a-Mouse Genome Informatics) in
vitro. Moreover, the forced expression of Mesp2 resulted in the reverse
phenotype of the Mesp2-null embryo, whereby Epha4 is activated,
Uncx4.1 is suppressed and ectopic epithelialization could be observed
in the transgenic embryos.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). E-box deletion and mutant constructs were subsequently
generated via PCR using the 630 bp enhancer region (HindIII cut) as a
template (Imai et al.,
1991).
Formation of E-box multimer constructs
Synthetic oligonucleotides were designed to generate two repeats of 20 bp
containing an E-box when annealed (Fig.
2C). These E-box-containing sequences were flanked by
BglII and BamHI sites. The complementary oligonucleotides
were annealed and phosphorylated with T4 polynucleotide kinase prior to
ligation. Ligated DNA were digested with BamHI and BglII,
and separated on 2% agarose gels. Multimerized bands were excised and
subcloned into the pBluescript vector.
Generation of transgenic mice
All constructs were digested with restriction enzymes to remove vector
sequences and then gel purified. Transgenic mice were generated by
microinjection of fertilized eggs. Microinjected eggs were transferred into
the oviducts of pseudopregnant foster females. The genotypes of the embryos
were identified by PCR using DNA prepared from the yolk sac.
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Electrophoretic mobility shift assay (EMSA)
For protein preparation, NIH3T3 cells were grown at 80% confluency in 10 cm
dishes and transfected with expression vectors containing either
3xFLAG-tagged Mesp2 or Myc-tagged E47. Nuclear extracts were prepared
using Nuclear Extract Kit (Active Motif). The protein concentrations were
measured by the Bradford assay (Pierce). EMSA was performed using a DIG
gelshift and detection kit (Roche). Binding reactions were carried out by
mixing DIG-labeled and unlabeled (for competition experiments) probes with
nuclear extracts from NIH3T3 cells. In experiments using antibodies, the
nuclear extracts were preincubated with the antibody for 1 hour on ice.
Generation of CAG-CAT-Mesp2 transgenic and Mox1-cre knock-in mice
A targeting vector was designed to introduce the Cre gene near to
the translational initiation site of the Mox1 gene (see Fig. S1 in
the supplementary material) and used to establish the Mox1-cre mouse
line, in which Cre recombinase is expressed instead of Mox1 and the gene
activity is examined by crossing with a reporter line, R26R
(Zambrowicz et al., 1997). To
achieve the ectopic expression of Mesp2, a CAG-floxed-CAT-Mesp2
transgene was constructed (Yamauchi et
al., 1999), in which CAT gene can be excised by Cre
recombinase and thus the Mesp2 gene comes under the control of the
CAG promoter (see Fig. S1 in the supplementary material). Transgenic
mouse lines were established by microinjection of CAG-floxed
CAT-Mesp2 DNA as described above.
Analyses of embryos by LacZ staining, in situ hybridization, skeletal and the histological methods
Embryos were fixed and stained in X-gal solution for the detection of
ß-gal activity, as described previously
(Saga et al., 1999). For
histology analyses, samples stained by X-gal were postfixed with 4%
paraformaldehyde, dehydrated in an ethanol series, embedded in paraffin and
sectioned at 6 µm. Whole-mount in situ hybridization was performed using
InsituPro robot (Intavis). The transcripts were visualized using
anti-digoxigenin (DIG) antibodies conjugated to alkaline phosphatase. Color
reactions were performed using BM Purple (Roche). Methods employed for section
in situ hybridization and for the immunohistological detection of Mesp2 have
been previously described (Morimoto et
al., 2005). Skeletal preparations by Alcian Blue/Alizarin Red
staining have also been described previously
(Saga et al., 1997;
Takahashi et al., 2000).
Probes used for the in situ hybridization detection of Uncx4.1 and
Sox9 were kindly provided by Dr Peter Gruss and Dr Veronique
Lefebvre, respectively. For the detection of actin filaments, frozen sections
were stained with AlexaFluor 488-conjugated phalloidin (Molecular Probes)
according to the manufacturer's protocol.
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). To identify the somite specific enhancer region of the
Epha4 gene, we focused on the more upstream region of the gene. We
subsequently found the enhancer within an 8.8 kb fragment, beginning 8 kb
upstream of the Epha4 transcriptional start site
(Fig. 1A). ß-Gal activity
was observed in the rostral compartment of the segmented somites
(Fig.1B,C) and to confirm that
this reflects endogenous Epha4 expression, we compared the expression
of lacZ RNA with the endogenous Epha4 transcripts. Among
several expression domains of endogenous Epha4, such as limb buds,
branchial arches and rhombomeres, an identical expression pattern was revealed
in both the anterior PSM and the rostral compartment of the S1 somites
(Fig. 1D,E). Further transgenic
analyses were conducted using DNA fragments that had been generated by several
restriction enzymes. Although we detected one embryo which showed
somite-specific expression using Sw-X region, we concentrated our analyses on
the P-Sw region, which showed most consistent results. Further deletion
identified a HindIII fragment of 630 bp that could sustain endogenous
pattern of somite-specific Epha4 expression
(Fig. 1A). We established a
permanent transgenic line using a lacZ reporter with the 630 bp
enhancer. The somite-specific expression was observed during somitogenesis
from 8.5 to 11.5 days post-coitum (dpc) as similar to the endogenous one (see
Fig. S2 in the supplementary material). However, the transgene expression
became weaker in the later stage embryo at 11.5 dpc and the somite-specific
expression was not observed with both probes for endogenous Epha4 and
lacZ transgene after 13.5 dpc (data not shown). When the expression
was examined in the Mesp2-null genetic background
(Mesp2L/L)
(Takahashi et al., 2000), no
ß-gal activity was detected in the Mesp2L/L embryos
(Fig. 1F,G), which confirms
that the enhancer contains cis elements required for the Epha4
activation downstream of the Mesp2.
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|
),
we analyzed reporter activity with or without E47, which is a typical A-type
bHLH factor. As shown in Fig.
3A, Mesp2 and E47 alone exhibited weak transactivating activities
when these expression vectors were separately transfected with the reporter
construct, whereas strong activity was observed when Mesp2 and E47 were
co-transfected. The transactivating activity of Mesp2 alone can be ascribed to
its association with endogenous E47. Next, we constructed reporters with six
repeats of each of the Epha4 enhancer E-boxes (E1 to E4) used in our
transgenic analyses (Fig. 2C).
Interestingly, very strong activity was observed when the E3 and E4 constructs
were used, and this was only observed upon cotransfection with E47
(Fig. 3B). This specificity was
confirmed by the lack of activity resulting from a construct with a
mutant-type E3 enhancer element. E2 showed no activity, which is consistent
with the findings of our transgenic analysis. E1 did not have strong activity,
although we obtained positive activity for this E-box via transgenic
analysis.
|
|
;
Cooke et al., 2005;
Durbin et al., 1998). However,
loss-of-function experiments have failed to show any functional relevance for
Epha4 in the mouse. In order to investigate whether Mesp2 functions as an
activator of Epha4, and possibly to reveal the role of Epha4 during
somitogenesis, we established a system that achieves the conditional
expression of Mesp2 using Cre-loxP. A transgenic mouse line
CAG-floxed-CAT-Mesp2 was established in our laboratory, in which the
CAT gene is inserted between two loxP sites and can
therefore be excised by Cre recombinase. Hence, the Mesp2 gene in
this system will come under the control of the CAG promoter after
this excision. To activate Mesp2, we generated and then used a
Mox1-Cre mouse (see Fig. S1 in the supplementary material).
Mox1 expression initiates just prior to segment border formation, in
a similar manner to endogenous Mesp2, but its expression persists
after segmentation and is relatively higher in the caudal half of the somites
(Mankoo et al., 2003;
Saga et al., 1997)
(Fig. 5A,B). The Cre
expression was detected as early as 8.5 dpc and showed the similar pattern to
the Mox1 (Fig.
5C-E). To confirm the presence of Cre recombinase activity, we crossed the Mox1-Cre mouse with the R26R reporter line and examined ß-gal activity during the period 8.5-11.5 dpc (Fig. 5F-I; data not shown). The expression of the reporter was found to begin in the paraxial mesoderm and the most prominent levels were restricted to the somitic derivatives, at least up to 11.5 dpc. Some reporter expression in the rostral neural tube and in the intermediate mesoderm was also observed. We detected differences in the initial expression domain between the Mox1 (Fig. 5B) or Cre transcripts (Fig. 5E), and ß-gal reporter activity (Fig. 5G), which most likely reflects a time-lag for the activation of the reporter gene following the excision of the CAT gene by Cre recombinase. Histological sections revealed that the reporter activation was initiated in only a few somitic cells just after segmentation, but that the ß-gal expression gradually expanded throughout the entire components of somite derivatives. Hence, this Cre line is a useful system to drive genes in the somitic cell lineage.
Mesp2 activation induces abnormal epithelialization
To activate Mesp2 expression in the somitic lineage, we crossed
the CAG-CAT-Mesp2 and Mox1-cre lines. The double
heterozygous mice died shortly after birth and their skeletal specimens
revealed strong malformations (see below), indicating abnormal somitogenesis.
Under a dissection microscope, the morphology of the somites was not found to
have been disrupted, which was unexpected from the observations of the
skeletal phenotype. Segmental boundaries were observed, although their width
was not perfectly equal to the wild type and the surface appeared to be rough.
At first, we analyzed Mesp2 expression at 10.5 dpc
(Fig. 6A,B). In the wild-type
and single heterozygous embryos, Mesp2 is expressed in the anterior
PSM as a single band, although the width and the strength of this
expression differs from embryo to embryo as shown previously
(Fig. 6A)
(Takahashi et al., 2000). In
double heterozygotes, however, the ectopic expression of Mesp2 could
be observed throughout the entire somitic region, in addition to its normal
expression pattern in the anterior PSM
(Fig. 6B). Moreover, the
Mesp2-positive cells often formed clusters and were not localized in
specific regions of somites (Fig.
6C,D).
A similar ectopic expression pattern was observed for Epha4, although the levels of ectopic expression were much lower than the endogenous gene expression (Fig. 6E-H). The spotty expression pattern in both Mesp2 and Epha4 indicates that the expression is suppressed or the transcripts are destabilized in many cells and only parts of cells maintain the expression. To further investigate the characteristics of the gene expression profiles and morphologies, serial sections were prepared and subjected to staining for Mesp2 protein, Epha4 transcripts and actin filaments (Fig. 6I-N). The segmental borders were found to have generated but fluorescent phalloidin staining revealed cells showing abnormal epithelialized features and broken epithelial sheaths were also evident (Fig. 6N). In the cells nearby, both ectopic Mesp2 (Fig. 6K) and Epha4 expression (Fig. 6L) could be observed. Although we could not conclude that Mesp2 directly induced Epha4 using the serial sections, these observations indicate that the cells may have acquired repulsive properties that enable them to form abnormal cell borders within somites (Fig. 6O).
Mesp2 activation induces skeletal malformation
The CAG-CAT-Mesp2/Mox1-Cre double heterozygous fetus showed strong
skeletal defects, which are restricted in the ribs and vertebra as expected
from the somite-specific Mox1 expression
(Fig. 7A-H)
(Mankoo et al., 2003). The
vertebral bodies and the lamina of neural arches were present in these
fetuses, although they displayed severe defects in both their morphology and
patterning. By contrast, the pedicles of the neural arches were largely lost
(Fig. 7C,G). In addition, the
proximal region of the ribs did not form properly
(Fig. 7D,H). This phenotype
contrasts with Mesp2-null embryos and is somewhat similar to Psen1-null
mutants (Takahashi et al.,
2000), indicating that it is a rostralized phenotype. To gain
insight into the morphogenetic failure underlying the skeletal defects
observed in the double transgenic mice, cartilage formation was examined by
whole-mount staining with Alcian Blue. Strikingly, rib as well as pedicle
cartilages were severely affected even in the 13.5 dpc embryo
(Fig. 8A,B).
|
;
Mansouri et al., 2000). In the
CAG-CAT-Mesp2/Mox1-Cre double heterozygotes, the caudally restricted
expression pattern was not disrupted but the levels of expression were much
lower and the stripes were often interrupted
(Fig. 8D,F). The histological
section revealed the presence of signal-negative regions in the caudal
compartments, which was often accompanied by the morphological abnormalities.
We noticed local fusion between cells in the caudal compartment and the
rostral ones in the posterior somite (Fig.
8H). Such a fusion was never observed in wild-type or single
heterozygous embryos (Fig.
8G).
To explore more genes affected in Mesp2-activated embryos and to understand
the cause of abnormalities, we examined expressions of several somitic markers
at 11.5 dpc. The segmental expression of Pax3 that is the marker of
dermomyotome (Fig. 8I)
(Denetclaw and Ordahl, 2000)
was expanded in the double transgenic embryo
(Fig. 8J), which may indicate
expansion of the dermomyotomal progenitor. By contrast, Sox9-positive
cell lineage appeared to be relatively reduced in the transgenic embryo
especially in the thoracic region (Fig.
8K,L), which may account for the underdevelopment of the rib
cartilage. The expansion of the rostral compartment of somites was indicated
by the expression of Tbx18 (Fig.
8M-P), which is another target candidate of Mesp2 as its
expression is lost in the Mesp2-null embryo
(Bussen et al., 2004) (Y.T.,
unpublished).
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|
DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The core E-box sequence appears to be CAAATG/CATTTG and synthetic enhancers
generated by six repeats of the Epha4 enhancer E1, E3 and E4 motifs,
and flanking sequences, can recapitulate the segmental expression pattern of
this gene in vivo. The differences that we observed in the measured luciferase
activities for the multiple E-boxes may reflect the involvement of the
sequences flanking the core enhancer region in promoting the binding of
bHLH-type transcription factors, which has been observed in other cases
(Powell et al., 2004). In
addition, other factors may modulate the interactions between Mesp2/E47 and
its target sequences. It has been reported that the phosphorylation of E47 is
required for the formation of heterodimers with Myod1 and for the subsequent
binding to the target sequence (Lluis et
al., 2005). The methylation state of target sequences has also
been implicated in the binding by another bHLH heterodimer, Max/Myc, in which
methylation of the CpG dinucleotide within the E-box has been shown to prevent
the access of the bHLH proteins (Perini et
al., 2005). Further studies will be required to determine whether
such modulations are involved in the binding of the Mesp2/E47 heterodimer to
its target sites.
Epha4 is implicated in segmental border formation via its interaction with
the Eph ligand ephrin, which is expressed in apposed cells in zebrafish
(Barrios et al., 2003;
Durbin et al., 1998). However,
there is no direct evidence for this in the mouse, as the loss of Epha4 failed
to reveal any role for this protein during somitogenesis, which may be due to
functional redundancy among the several Eph and ephrin family proteins. In
such a situation, a transgenic strategy of forced gene expression is an
alternative and effective method. In the current study, we have tried the
forced expression of Mesp2 with expectation that Epha4
should be induced under the control of Mesp2. The forced expression of Mesp2
not only activates Epha4 expression but also induces the local
segregation of somitic cells. Recently, we showed that Mesp2 establishes the
segmental boundary by suppressing Notch signaling, which then generates a
boundary between the Notch-active and Notch-negative domains
(Morimoto et al., 2005). We
have also shown that this boundary forms the next somite border. However, the
precise molecular mechanisms involved in the generation of these morphological
boundaries are not yet fully understood, although Cdc42 and Rac1 are known to
play important roles in subsequent epithelial somite formation
(Nakaya et al., 2004).
Although the direct evidence was not presented, our current data indicate that
Mesp2 activates Epha4 in the anteriormost cells in the PSM and that
this may activate reverse signaling though ephrin expression in opposing cells
and generate a gap during normal somitogenesis. A similar mechanism has
previously been proposed for the epithelialization of boundary cells in
zebrafish (Barrios et al.,
2003; Cooke et al.,
2005). Nevertheless, we can not exclude the possibility that
pathways other than Epha4 activation by Mesp2 are required for the induction
of epithelialization.
Mesp2 is also known as a strong suppressor of the establishment of caudal
properties, which is mediated by the suppression of both Notch signaling and
Dll1 and Uncx4.1 expression
(Nomura-Kitabayashi et al.,
2002; Takahashi et al.,
2000). We actually did observe suppression of Uncx4.1 in
our double heterozygotes, but the segmental pattern of Uncx4.1
expression at 10.5 dpc was not found to be severely disrupted. Therefore, our
finding of an extremely defective skeletal phenotype in the
CAG-CAT-Mesp2/Mox1-Cre mice was somewhat surprising. We postulate
that prolonged Mesp2 expression, driven by the CAG promoter, must continuously
attenuate Uncx4.1 and the corresponding downstream gene expression in
the later stages of development, which would lead to the almost complete
suppression of chondrogenesis, as observed in the case of loss of Uncx4.1
(Leitges et al., 2000;
Mansouri et al., 2000). One of
target genes activated by Uncx4.1 and responsible for the phenotype would be
Sox9, the product of which is known to be a key regulator of
chondrogenesis (Akiyama et al.,
2005). However, it remains to be investigated whether this
suppression is directly mediated by Mesp2 or by other transcriptional
suppressors that are activated by Mesp2.
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).
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/133/13/2517/DC1
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ACKNOWLEDGMENTS |
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