Identification of Epha4 enhancer required for segmental expression and the regulation by Mesp2

Somites are basic structures that underlie the segmental body architecture in vertebrates. The mechanisms involved in the generation of serially segmental units are a fascinating model system that 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 come under the control of the segmentation clock, in which Notch signal oscillation generates the periodicity (for reviews, see Aulehla and Herrmann, 2004; 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.

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

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.

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.

An Epha4 somite enhancer insert (630 bp HindIII-HindIII fragment) and E-box multimers were cloned into the pGL3-Promoter vector (Promega). NIH3T3 cells were grown at 80%confluency in 24-multiwell plates and transfected 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 Renilla luciferase expression vector pRL-TK (Promega),and the relative luciferase activity was determined as recommended by the manufacturer.

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 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.

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-Mesp2transgene 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.

Embryos were fixed and stained in X-gal solution for the detection ofβ-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.

Previous enhancer studies have indicated that a somite specific enhancer is not contained within the 7.5 kb region upstream of the Epha4transcriptional start site, in which only rhombomere-specific enhancer activity has been identified (Theil et al., 1998). 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). β-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(Mesp2^L/L)(Takahashi et al., 2000), noβ-gal activity was detected in the Mesp2^L/L embryos(Fig. 1F,G), which confirms that the enhancer contains cis elements required for the Epha4activation downstream of the Mesp2.

Subsequent sequence analysis revealed that this 630 bp region contained eight E-boxes. As Mesp2 belongs to the bHLH family of transcription factors,which are known to bind E-box or N-box motifs, we initially performed deletions of some of the Epha4 E-boxes. 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 was completely 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 are conserved in the human EPHA4 gene. To determine whether these E-boxes are crucial, we next introduced mutations into their consensus 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 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 of six tandem repeats of the E1, E2, E3 or E4 boxes and flanking sequences(Fig. 2C). Transient transgenic analysis revealed that each of these regions, with the exception of E2, showed weak but specific expression in the somitic region(Fig. 2D). This confirmed that these E-boxes are a minimum requirement for the specificity of Epha4expression in the somitic region and that the consensus sequence is CAAATG.

The identification of E-boxes as a vital component of the Epha4enhancer strongly indicates that this gene is directly regulated by Mesp2, as the bHLH-type transcription factor is known to bind E-boxes. In order to further elucidate whether Mesp2 can activate the Epha4 enhancer, we established a luciferase assay system using NIH3T3 cultured cells. The reporter gene was constructed by ligating the 630 bp Epha4 enhancer region to a luciferase gene. As B-type bHLH transcription factors are known to function as heterodimers with the so called A-type bHLH factors(Ledent and Vervoort, 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 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.

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 and luciferase 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 confirmed by 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 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 (see Fig. S3 in the supplementary material). These data strongly suggest that 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.

Epha4 has been implicated in border formation via the repulsive interaction with ephrin molecules, and this occurs during the formation of segmental boundaries in the hindbrain and in the somites(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 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 Creexpression 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 β-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 Cretranscripts (Fig. 5E), andβ-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 theβ-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.

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 Epha4indicates 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 Epha4expression (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).

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).

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 to the 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 be required for the pedicle formation of the neural arch(Leitges et al., 2000; 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).

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.

In our current study, we have identified a cluster of E-boxes in the enhancer region of Epha4, incorporating the Mesp2 binding site, in which at least three crucial E-boxes (E1, E3 and E4 in Fig. 2C) are present. The loss of these motifs results in a substantial reduction in gene 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 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 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 Epha4should 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 Uncx4.1expression 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.

We also show in our present study that the Mox1-Cre mouse is a useful tool for inducing either the disruption or activation of genes that are components of the somitic cell lineage. However, there is a delay in gene activation and the activation of Mesp2 was `spotty' and these 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 in future studies, the activity that we observed in our CAG-CAT-Mesp2/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 (Meox1^cre) was reported by another laboratory and the results of their study were consistent with our current 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 the transgenic 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.