RESEARCH ARTICLE 2517

Development 133, 2517-2525 (2006) doi:10.1242/dev.02422

Identification of Epha4 enhancer required for segmental
expression and the regulation by Mesp2
Yoshiro Nakajima1, Mitsuru Morimoto1, Yuki Takahashi1, Haruhiko Koseki2 and Yumiko Saga1,*
Somites provide the basic body plan for metameric axial structures in vertebrates, and establish the segmental features through thesequential gene expression in the presomitic mesoderm (PSM). A crucial protein for segment border formation is the bHLH
transcription factor Mesp2, the expression of which is restricted to the anterior PSM. A gene candidate that is activated by Mesp2 is
Epha4, as its expression pattern resembles Mesp2 and is absent in Mesp2-null embryos. We have analyzed the enhancer region of
Epha4, which is responsible for its expression in the anterior PSM, and identified an E-box containing region. Subsequent transgenic
and transient luciferase analyses successfully determined that the presence of repeated E-box sequences is a minimum essential
requirement for the expression in the anterior PSM. We also show that Mesp2 directly binds to the enhancer sequence of Epha4.
Furthermore, the forced expression of Mesp2 in somitic cells results in the activation of Epha4 and repression of the caudal geneUncx4.1, which may trigger the events leading to the formation of abnormal somites and rostralized vertebra. In addition, ectopic
Mesp2 expression induces abnormally epithelialized structures, which support to the idea that Mesp2 induces the formation ofsegmental borders by activating genes that play roles in cellular epithelialization.
KEY WORDS: Mesp2, Epha4, Somitogenesis, Segmental border, Mox1, mouse

Durbin et al., 1998), although gene knockout studies indicate thatEpha4 is not the sole protein required for segmental borderformation in the mouse, as no somitic phenotype has beenreported (Dottori et al., 1998; Kullander et al., 2001) (M. Asano,personal communication). The identification of target genes for atranscription 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 inembryonic 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, theEpha4 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 thereverse phenotype of the Mesp2-null embryo, whereby Epha4 isactivated, Uncx4.1 is suppressed and ectopic epithelialization
could be observed in the transgenic embryos.
MATERIALS AND METHODS
Construction of lacZ reporter constructs

An 8.8 kb fragment (NheI-XbaI) was isolated from an Epha4-containing
bacterial artificial chromosome clone (415B3) and subcloned into thepBluescript vector (Stratagene). A series of deletion constructs were thengenerated using the appropriate restriction enzymes (Fig. 1A). These
genomic fragments were inserted into a lacZ reporter vector, upstream of thehsp promoter (Kothary et al., 1989). 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

1

Division of Mammalian Development, National Institute of Genetics, Yata 1111,
Mishima 411-8540, Japan. 2Division of Developmental Genetics, RIKEN ResearchCenter for Allergy and Immunology (RCAI) RIKEN Yokohama Institute, 1-7-22
Suehiro, Tsurumi-ku, Yokohama 230-0045, Japan.
*Author for correspondence (e-mail: ysaga@lab.nig.ac.jp)
Accepted 2 May 2006

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 complementaryoligonucleotides 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 andsubcloned into the pBluescript vector.

DEVELOPMENTINTRODUCTION
Somites are basic structures that underlie the segmental bodyarchitecture in vertebrates. The mechanisms involved in thegeneration of serially segmental units are a fascinating model systemthat has been used by many developmental biologists to further our
understanding of the temporal and spatial control of gene
expression. Somite precursors are derived as paraxial mesoderm
from the primitive streak or tailbud region and these cells then comeunder the control of the segmentation clock, in which Notch signal
oscillation generates the periodicity (for reviews, see Aulehla andHerrmann, 2004; Bessho and Kageyama, 2003; Maroto andPourquié, 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 borderformation and for the generation of rostrocaudal patterning withinsomites (Saga et al., 1997; Takahashi et al., 2000). The expressionof many genes is affected in the Mesp2-null embryo, in which thegenes required for rostral property are suppressed but those required
for the development of caudal properties are enhanced. Among thesegenes, only lunatic fringe (Lfng) has so far been shown to be a directtarget 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 thesomites. Furthermore, Epha4 is implicated in segmental borderformation in zebrafish (Cooke et al., 2005; Barrios et al., 2003;

2518 RESEARCH ARTICLE

Development 133 (13)Fig. 1. Identiﬁcation of a somitespeciﬁc Epha4 enhancer. (A) The
lacZ transgene constructs used to
identify the cis-acting somite
enhancer within the Epha4 genomic
region. The numbers of transgenic
mouse embryos that expressed ␤-gal
in the somites, among thetransgene-positive embryos, areindicated on the right. E, EcoRI; H,
HindIII; N, NheI; Sa, SacI; Sw, SwaI; P,
PmaCI; X, XbaI. (B,C) Lateral view of
␤-gal activity driven by the 630 bp(HindIII) Epha4 enhancer fragment in
a 10.5 dpc embryo. A magniﬁed
image in the somitic region of B isshown in C. (D,E) Comparison by insitu hybridization at 10.5 dpc of thetransgene expression (lacZ) in atransgenic embryo (D) withendogenous Epha4 expression in thewild-type embryo (E). In situ signals
in the anterior PSM are indicated byarrows in the lower panels showing
magniﬁed images. (F,G) ␤-Gal
activity driven by the 630 bp
enhancer in 10.5 dpc wild-type (F)and Mesp2L/L embryos (G).

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 theembryos were identified by PCR using DNA prepared from the yolk sac.
Luciferase assay

An Epha4 somite enhancer insert (630 bp HindIII-HindIII fragment) and Ebox multimers were cloned into the pGL3-Promoter vector (Promega).
NIH3T3 cells were grown at 80% confluency in 24-multiwell plates andtransfected with luciferase gene constructs using Lipofectamine Plus
(Invitrogen). Cells were harvested 36 hours after transfection and luciferase
activities were measured using a Dual Luciferase Assay Kit (Promega). The
transfection efficiency was normalized by co-transfection of the Renillaluciferase expression vector pRL-TK (Promega), and the relative luciferase
activity was determined as recommended by the manufacturer.
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
3⫻FLAG-tagged Mesp2 or Myc-tagged E47. Nuclear extracts wereprepared 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 withthe 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 thetranslational initiation site of the Mox1 gene (see Fig. S1 in the
supplementary material) and used to establish the Mox1-cre mouse line, inwhich 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 comesunder the control of the CAG promoter (see Fig. S1 in the supplementarymaterial). 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 ␤-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 ␮m.
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 beenpreviously described (Morimoto et al., 2005). Skeletal preparations by
Alcian Blue/Alizarin Red staining have also been described previously (Sagaet 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.

RESULTS
Enhancer analysis of Epha4
Previous enhancer studies have indicated that a somite specificenhancer is not contained within the 7.5 kb region upstream of theEpha4 transcriptional start site, in which only rhombomere-specificenhancer activity has been identified (Theil et al., 1998). To identify
the somite specific enhancer region of the Epha4 gene, we focusedon the more upstream region of the gene. We subsequently found theenhancer within an 8.8 kb fragment, beginning 8 kb upstream of theEpha4 transcriptional start site (Fig. 1A). ␤-Gal activity wasobserved in the rostral compartment of the segmented somites (Fig.

DEVELOPMENT

Generation of transgenic miceRegulation of Epha4 by Mesp2

RESEARCH ARTICLE 2519

1B,C), and to confirm that this reflects endogenous Epha4
expression, we compared the expression of lacZ RNA with theendogenous Epha4 transcripts. Among several expression domainsof endogenous Epha4, such as limb buds, branchial arches andrhombomeres, an identical expression pattern was revealed in boththe 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-specificexpression 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 wasobserved 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-specificexpression was not observed with both probes for endogenous
Epha4 and lacZ transgene after 13.5 dpc (data not shown). When theexpression was examined in the Mesp2-null genetic background(Mesp2L/L) (Takahashi et al., 2000), no ␤-gal activity was detectedin the Mesp2L/L embryos (Fig. 1F,G), which confirms that theenhancer contains cis elements required for the Epha4 activation
downstream of the Mesp2.
Subsequent sequence analysis revealed that this 630 bp region
contained eight E-boxes. As Mesp2 belongs to the bHLH family oftranscription factors, which are known to bind E-box or N-box
motifs, we initially performed deletions of some of the Epha4 Eboxes. We generated four deletion constructs that lack a region(fragments 1-4) of the E-boxes (Fig. 2A) and examined the enhancer
activities. Our results clearly showed that the enhancer activity wascompletely lost when using both fragment 2- and 3-deletion
constructs, indicating that both regions are necessary for its
expression (Fig. 2A). Both fragments 2 and 3 contain two E-boxes,
designated E1- E4 (Fig. 2B). The core sequences of E1, E3 and E4
are identical (CAAATG or CATTTG) but only E1 and E3 areconserved in the human EPHA4 gene. To determine whether these
E-boxes are crucial, we next introduced mutations into theirconsensus sequences and transgenic analysis was conducted using
the full 630 bp enhancer as a wild-type activity control. The E2
mutation did not affect enhancer activity but a considerable
reduction in the activity was observed for the E1 and E3 mutations.
In addition, mutation of E4 substantially disrupted enhancer activity,
indicating that a mutation in a single E-box abrogates the enhancer

DEVELOPMENT

Fig. 2. Identiﬁcation of the functional E-box motifs responsible for the somite-speciﬁc activation of Epha4. (A) Sequence of the 630 bp
Epha4 somite enhancer (core enhancer) region. The four indicated fragments (fragment 1-4) represent deleted sequences from the core enhancer
region. The results of transgenic analyses using these deleted constructs are shown in parentheses. (B) Sequence alignment of somite enhancer
regions (fragment 2 and fragment 3) of mouse Epha4 with the corresponding regions of human Epha4. The mutations introduced in each E-box
and the results of the subsequent transgenic analyses are shown. (C) Schematic representation of artiﬁcially constructed enhancers, containing six Ebox motif repeats. (D) The artiﬁcial enhancers were cloned upstream of a lacZ reporter vector and the results of the subsequent transgenic analyses(representative images are shown) are indicated in parentheses. For the [E2]6-lacZ artiﬁcial enhancer, no somite expression was observed among the
11 transgene-positive embryos. Blue letters indicate putative E-box motifs.

Fig. 3. Transactivation of the Epha4 enhancer by the Mesp2/E47
heterodimer. Epha4 somite enhancers (A, 630 bp core sequence; B, sixtandem repeats of E-box sequences) were ligated to the pGL3
luciferase vector. Luciferase activity was measured at 36 hours after
transfection into NIH3T3 cells. (A) The presence (+) or absence (–) of
either Mesp2 or E47 are indicated in each column. (B) Luciferase
activity was compared with (black bars) and without (white bars)
Mesp2/E47. Mutations in E3 (5⬘-CATTTG-3⬘), that give rise to E3m (5⬘ACGGGT-3⬘), results in the loss of reporter activity. The results shown
are the mean values from three independent experiments. Standard
deviations are indicated by error bars.

activity and that the presence of tandem repeats of the E-box
consensus sites is important for the activity. To confirm this, we
generated reporter constructs with artificial enhancers, composed ofsix tandem repeats of the E1, E2, E3 or E4 boxes and flanking
sequences (Fig. 2C). Transient transgenic analysis revealed that eachof these regions, with the exception of E2, showed weak but specificexpression in the somitic region (Fig. 2D). This confirmed that these
E-boxes are a minimum requirement for the specificity of Epha4
expression in the somitic region and that the consensus sequence is
CAAATG.
The Epha4 enhancer is activated by Mesp2 incultured cells
The identification of E-boxes as a vital component of the Epha4
enhancer strongly indicates that this gene is directly regulated by
Mesp2, as the bHLH-type transcription factor is known to bind E-

Development 133 (13)boxes. In order to further elucidate whether Mesp2 can activate theEpha4 enhancer, we established a luciferase assay system using
NIH3T3 cultured cells. The reporter gene was constructed byligating the 630 bp Epha4 enhancer region to a luciferase gene. As
B-type bHLH transcription factors are known to function asheterodimers with the so called A-type bHLH factors (Ledent andVervoort, 2001), 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 andE47 were co-transfected. The transactivating activity of Mesp2 alone
can be ascribed to its association with endogenous E47. Next, weconstructed 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 E3enhancer element. E2 showed no activity, which is consistent withthe findings of our transgenic analysis. E1 did not have strong
activity, although we obtained positive activity for this E-box via
transgenic analysis.
The binding abilities of Mesp2 and E47 to the Epha4 E-boxes
were then analyzed using electrophoresis mobility shift assays(EMSA). Nuclear extracts were prepared from NIH3T3 cells,
transfected with either FLAG-tagged Mesp2 or Myc-tagged E47,
and these were used in the experiments either separately or in
combination. The E3 motif was used in the EMSA experiments as
it gave the most consistent results in both the transgenic andluciferase assays. As expected, a strong bandshift was observed
when both Mesp2 and E47 were combined, although a faint band
was detectable when E47 was incubated alone, indicating that it may
form a homodimer that can bind the E3 E-box (Fig. 4A). The
sequence specificity of the protein-DNA interactions was confirmed
by competition assay using both intact and mutated sequences (Fig.
4B). The specificity of the heteroduplex complex was also confirmedby supershift experiments with anti-FLAG and anti-Myc antibodies(Fig. 4C). We also examined the binding specificity by applying
other bHLH proteins. A family protein, Mesp1 also showed strong

Fig. 4. The Mesp2/E47 heterodimer binds to the E3 site of the Epha4 enhancer. (A) The results of EMSA using nuclear extracts from NIH3T3
cells transfected with FLAG-Mesp2 and/or Myc-E47 and incubated with E3 probe. The quantities of nuclear extracts used (␮g) are indicated. (B) A
competition assay indicating the speciﬁcity of the binding of the Mesp2/E47 (2 ␮g each) complex to the E3 probe. The addition of 100-fold excess
of unlabeled E3 probe, but not the E3m mutant probe, abolished the binding. (C) Evidence for the heterodimer formation of FLAG-Mesp2/MycE47. The band containing E3 (arrow) was supershifted by the addition of either anti-FLAG or anti-Myc antibodies. The oligonucleotides used wereas follows: E3, 5⬘-TGGGTCACATTTGTCCAAAA-3⬘; E3m, 5⬘-TGGGTCAACGGGTTCCAAAA-3⬘ (E-box is shown in the bold; altered nucleotides in
the mutant probe are underlined).

DEVELOPMENT

2520 RESEARCH ARTICLE

Regulation of Epha4 by Mesp2

RESEARCH ARTICLE 2521

binding, but other bHLH proteins such as paraxis (Tcf15 – Mouse
Genome Informatics), Myod1 and Twist (Twist1 – Mouse Genome
Informatics) did not show significant binding to the E3 probe (seeFig. S3 in the supplementary material). These data strongly suggestthat Mesp2 forms a heterodimer with E47, binds to the E-boxes
within the Epha4 enhancer and then activates Epha4 transcription in
the rostral region of somites.

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 ofthe 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

Fig. 5. Mox1 and Mox1-cre expression and the lineage analysis.
Whole-mount in situ hybridization analysis of Mox1 expression in 9.5
dpc embryos (A,B) and Mox1-cre in 8.25 (C), 8.5 (D) and 11.5 (E) dpc
embryos. (F-I) Whole-mount expression patterns and sagittal sections of
␤-gal-stained R26R/Mox1-cre double heterozygous embryos at 10.5
dpc. (H,I) The sections were counterstained with Eosin. Arrowheads
indicate somite borders forming between S0 and S-1.

the wild-type and single heterozygous embryos, Mesp2 is expressedin the anterior PSM as a single band, although the width andthe 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 beobserved 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 theendogenous gene expression (Fig. 6E-H). The spotty expression
pattern in both Mesp2 and Epha4 indicates that the expression issuppressed or the transcripts are destabilized in many cells and only
parts of cells maintain the expression. To further investigate thecharacteristics 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 fluorescentphalloidin staining revealed cells showing abnormal epithelializedfeatures 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 notconclude 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).

DEVELOPMENT

The overexpression of Mesp2 leads to theactivation of Epha4
Epha4 has been implicated in border formation via the repulsive
interaction with ephrin molecules, and this occurs during theformation of segmental boundaries in the hindbrain and in thesomites (Barrios et al., 2003; 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 toinvestigate whether Mesp2 functions as an activator of Epha4, andpossibly to reveal the role of Epha4 during somitogenesis, weestablished a system that achieves the conditional expression of
Mesp2 using Cre-loxP. A transgenic mouse line CAG-floxed-CATMesp2 was established in our laboratory, in which the CAT gene isinserted between two loxP sites and can therefore be excised by Cre
recombinase. Hence, the Mesp2 gene in this system will comeunder the control of the CAG promoter after this excision. To
activate Mesp2, we generated and then used a Mox1-Cre mouse (seeFig. S1 in the supplementary material). Mox1 expression initiates
just prior to segment border formation, in a similar manner toendogenous Mesp2, but its expression persists after segmentationand 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 theMox1 (Fig. 5C-E).
To confirm the presence of Cre recombinase activity, we crossed
the Mox1-Cre mouse with the R26R reporter line and examined ␤gal activity during the period 8.5-11.5 dpc (Fig. 5F-I; data notshown). The expression of the reporter was found to begin in theparaxial mesoderm and the most prominent levels were restricted tothe 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 initialexpression domain between the Mox1 (Fig. 5B) or Cre transcripts(Fig. 5E), and ␤-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. Histologicalsections revealed that the reporter activation was initiated in only afew somitic cells just after segmentation, but that the ␤-gal
expression gradually expanded throughout the entire components ofsomite derivatives. Hence, this Cre line is a useful system to drive
genes in the somitic cell lineage.

2522 RESEARCH ARTICLE

Development 133 (13)Mesp2 activation induces skeletal malformation
The CAG-CAT-Mesp2/Mox1-Cre double heterozygous fetus showedstrong 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 neuralarches were present in these fetuses, although they displayed severe
defects in both their morphology and patterning. By contrast, thepedicles 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 issomewhat similar to Psen1-null mutants (Takahashi et al., 2000),
indicating that it is a rostralized phenotype. To gain insight into themorphogenetic failure underlying the skeletal defects observed in
the double transgenic mice, cartilage formation was examined bywhole-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).
Mesp2 is required for the establishment of the rostral properties
within somites via the suppression of caudal genes. Therefore,
we anticipated that the forced expression of Mesp2 may lead tothe suppression of caudal properties, which would be the cause of

the skeletal malformation. Uncx4.1 is a molecular marker for
caudal somites (Fig. 8C,E,G) and this gene is also known to berequired for the pedicle formation of the neural arch (Leitges etal., 2000; Mansouri et al., 2000). In the CAG-CAT-Mesp2/Mox1Cre 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 therostral ones in the posterior somite (Fig. 8H). Such a fusion wasnever observed in wild-type or single heterozygous embryos (Fig.
8G).
To explore more genes affected in Mesp2-activated embryos andto understand the cause of abnormalities, we examined expressionsof several somitic markers at 11.5 dpc. The segmental expression of
Pax3 that is the marker of dermomyotome (Fig. 8I) (Denetclaw andOrdahl, 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

DEVELOPMENT

Fig. 6. Ectopic Mesp2 expression in somites leads to the activation of Epha4. Expression of Mesp2 in 10.5 dpc wild-type (A) and CAG-CATMesp2; Mox1-Cre double heterozygous embryos (B-D). In addition to the normal Mesp2 expression in the anterior PSM, ectopic expression of
Mesp2 was observed throughout the entire somitic region (B-D). The expression pattern of Epha4 at 11.5 dpc in wild-type (E,G) and double
heterozygous embryos (F,H) is also shown. An expression pattern for Epha4 that was similar to Mesp2 was observed in the double heterozygote(F,H). Histological analyses of 10.5 dpc wild-type (M) and double heterozygous (I-L,N) embryos. Ectopic Mesp2 protein (I,K) and Epha4 expression(J,L) were evident in serial sections of double heterozygotes. Magniﬁed images of square parts of I and J are shown in K and L, respectively.
Another consecutive section was stained with phalloidin (N) and a similar region of the wild-type embryo is shown in M. In double heterozygotes,
abnormal epithelial cells were observed within the somite (N). A paraffin section stained with nuclear Fast Red revealed gaps in epithelialized
somites in the double heterozygote at 10.5 dpc (O). Black arrows in I and J indicate the endogenous expression of Mesp2 (I) or Epha4 (J). Red
arrows in K,L,N indicate separated cell clusters. White arrow in N indicates an abnormal epithelialized feature. Black arrows in O indicate local
gaps. Scale bar: 100 ␮m.

Fig. 7. Ectopic expression of Mesp2 leads to skeletal
malformations. Skeletal preparations of 18.5 dpc wild-type (A-D) and
CAG-CAT-Mesp2; Mox1-Cre fetuses (E-H) stained with Alcian Blue andAlizarin Red. Lateral (A,E) and dorsal (B,F) views of whole skeletons areshown. Higher magniﬁcations of the lumber region from wild-type(C,D) and double heterozygous fetuses (G,H) are also shown. The lackof pedicles of the neural arches could be observed in the double
heterozygotes (G,H). The rib structure was also severely affected in the
double heterozygotes (G,H). pd, pedicle; lm, lamina.

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 ofsomites was indicated by the expression of Tbx18 (Fig. 8M-P),
which is another target candidate of Mesp2 as its expression is lostin the Mesp2-null embryo (Bussen et al., 2004) (Y.T., unpublished).
These observations are consistent with the idea that ectopic
Mesp2 expression is of sufficient strength to activate rostral genes
such as Epha4 and Tbx18, and to suppress expression of the caudal
gene Uncx4.1.
DISCUSSION
In our current study, we have identified a cluster of E-boxes in theenhancer region of Epha4, incorporating the Mesp2 binding site, inwhich at least three crucial E-boxes (E1, E3 and E4 in Fig. 2C) arepresent. The loss of these motifs results in a substantial reduction ingene reporter activity, in both luciferase and transgenic reporter
assays, indicating that there is an essential requirement of multiple
E-boxes for Epha4 activation by Mesp2. Interestingly, the coexpression of Mesp2 and E47 resulted in higher luciferase activity(tenfold) (Fig. 3B), whereas only weak activity (twofold) was
obtained with Mesp2 alone (data not shown). Mesp2 alone could

RESEARCH ARTICLE 2523

also not bind to E-box containing DNA sequences (Fig. 4A).
Therefore, Mesp2 alone or Mesp2 homodimers appear to be inactive
on Epha4 somite enhancer.
The core E-box sequence appears to be CAAATG/CATTTG andsynthetic enhancers generated by six repeats of the Epha4 enhancer
E1, E3 and E4 motifs, and flanking sequences, can recapitulate thesegmental expression pattern of this gene in vivo. The differencesthat we observed in the measured luciferase activities for themultiple E-boxes may reflect the involvement of the sequences
flanking the core enhancer region in promoting the binding ofbHLH-type transcription factors, which has been observed in othercases (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 theformation of heterodimers with Myod1 and for the subsequentbinding to the target sequence (Lluis et al., 2005). The methylation
state of target sequences has also been implicated in the binding byanother bHLH heterodimer, Max/Myc, in which methylation of the
CpG dinucleotide within the E-box has been shown to prevent theaccess 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 inapposed 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 duringsomitogenesis, which may be due to functional redundancy
among the several Eph and ephrin family proteins. In such asituation, a transgenic strategy of forced gene expression is analternative 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 bysuppressing 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 molecularmechanisms involved in the generation of these morphologicalboundaries 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 epithelializationof boundary cells in zebrafish (Barrios et al., 2003; Cooke et al.,2005). Nevertheless, we can not exclude the possibility thatpathways other than Epha4 activation by Mesp2 are required forthe induction of epithelialization.
Mesp2 is also known as a strong suppressor of the establishmentof caudal properties, which is mediated by the suppression of both
Notch signaling and Dll1 and Uncx4.1 expression (NomuraKitabayashi et al., 2002; Takahashi et al., 2000). We actually did
observe suppression of Uncx4.1 in our double heterozygotes, but thesegmental pattern of Uncx4.1 expression at 10.5 dpc was not foundto be severely disrupted. Therefore, our finding of an extremely
defective skeletal phenotype in the CAG-CAT-Mesp2/Mox1-Cre

DEVELOPMENT

Regulation of Epha4 by Mesp2

2524 RESEARCH ARTICLE

Development 133 (13)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 tothe 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 forthe phenotype would be Sox9, the product of which is known to bea 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.
We also show in our present study that the Mox1-Cre mouse is a
useful tool for inducing either the disruption or activation of genesthat are components of the somitic cell lineage. However, there is adelay in gene activation and the activation of Mesp2 was ‘spotty’ andthese may be the reason why we did not observe strong segmental
defects. Gene reporter activity was also observed in other lineages,
including parts of the neural tube and the intermediate mesoderm.
Therefore, although a detailed lineage study will be required infuture studies, the activity that we observed in our CAG-CATMesp2/Mox1-Cre transgenics proved to be useful for the
manipulation of gene expression, at least in the somitic cell lineages.
Recently, a similar Cre line (Meox1cre) was reported by anotherlaboratory and the results of their study were consistent with ourcurrent data (Jukkola et al., 2005).
We are grateful to Dr Baljinder S. Mankoo for generously providing the
genomic DNA clones for Mox1 and Drs Alan Rawls, Sachiko Iseki and Atsuko
Sehara for cDNA clones encoding paraxis, twist and Myod1, respectively. We
also thank Masayuki Oginuma and Dr Kenta Sumiyama for advice on thetransgenic mouse analysis. This work was supported by Grants-in-Aid for
Science Research on Priority Areas (B), the Organized Research Combination
System and National BioResource Project of the Ministry of Education, Culture,
Sports, Science and Technology, Japan.
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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Regulation of Epha4 by Mesp2