Developmental Biology 300 (2006) 687 – 698
www.elsevier.com/locate/ydbio

Cooperative Mesp activity is required for normal somitogenesis along theanterior–posterior axis
Mitsuru Morimoto a , Makoto Kiso a , Nobuo Sasaki a , Yumiko Saga a,b,⁎aDivision of Mammalian Development, National Institute of Genetics, Yata 1111, Mishima 411-8540, Japanb
SOKENDAI, Yata 1111, Mishima 411-8540, Japan
Received for publication 20 March 2006; revised 10 July 2006; accepted 19 August 2006
Available online 24 August 2006

Abstract
Mesp2 is a bHLH-type transcription factor that plays a key role during somitogenesis. Mesp2 is transiently expressed and is quickly degraded
once translated. In our current study, we find that Mesp2 contains a degradation domain, which acts as a target of proteasome-mediated proteolysis
and appears to play this role in vivo. We have also defined the nuclear localization signals (NLS) and constructed a minimum Mesp2 protein(P2-HD) composed of the NLS, bHLH and the degradation domains. The ability of the P2-HD as a transcription factor in vivo was examined.
Some of the defects that had been previously observed in the Mesp2-null mice were rescued in the knock-in mice but only in the posterior halfof the body, indicating differential effects of P2-HD along the anterior–posterior (AP) axis. In addition, quantitative analysis of the expression
along the AP axis revealed that the relative levels of Mesp2 increased, whereas Mesp1 is down-regulated in the later stages of development by
the activities of Mesp2 in the wild-type embryo. Moreover, we have found that somitogenesis in the early stages is more susceptible to changes
in the Mesp gene dosage, indicating that a threshold level of Mesp activity must be required for the progression of normal somitogenesis.
© 2006 Elsevier Inc. All rights reserved.
Keywords: Mesp1; Mesp2; Somitogenesis; Vertebra; Protein degradation; Proteasome; Knock-in mouse; Nuclear localization; Basic helix–loop–helix;
Transcriptional factor

Introduction
Somitogenesis is a transient dynamic morphogenetic processthat generates somites in a rostro-caudal progression by
segmenting the paraxial mesoderm (Dubrulle and Pourquie,
2004). The somites provide basic axial structures that underlie
the segmental architecture of not only the vertebra, ribs andmuscles, which are all somite derivatives, but also of thevascular and nervous systems (Brand-Saberi and Christ, 2000;
Monsoro-Burq and Le Douarin, 2000; Borycki and Emerson,2000). The associated metamerism is regulated by periodic gene
expression, that functions as a so-called segmentation clock in
the presomitic mesoderm (PSM) in mouse (Aulehla andJohnson, 1999; Aulehla and Herrmann, 2004; Aulehla et al.,
2003; Jouve et al., 2000; Bessho et al., 2001; Saga and Takeda,
2001; Giudicelli and Lewis, 2004). The periodicity is generated⁎ Corresponding author. Division of Mammalian Development, National
Institute of Genetics, Yata 1111, Mishima 411-8540, Japan. Fax: +55 981 6828.
E-mail address: ysaga@lab.nig.ac.jp (Y. Saga).
0012-1606/$ - see front matter © 2006 Elsevier Inc. All rights reserved.
doi:10.1016/j.ydbio.2006.08.043

via a negative feed back loop of Notch signaling that activates
the transcription of Hes7, which is a negative regulator of itself,and L-fng, which is a negative regulator of Notch signaling, andthis results in the oscillation of Notch activity (Bessho et al.,
2003; Rida et al., 2004). In addition, to achieve strict temporalcontrol of the clock, the stabilities of both the mRNA and proteins have to be precisely regulated. Indeed, it has been shownthat the Hes7 protein is actively degraded via an ubiquitin–
proteasome pathway, which generates Hes7 protein oscillationin the PSM. Furthermore, somitogenesis is disrupted by the introduction of stabilized Hes7, in place of the wild-type protein.
In knock-in mice, the periodic gene expression of both Hes7 andL-fng is destroyed, which is consistent with the notion that stability of these proteins has to be precisely regulated in addition
to their transcription and translation (Hirata et al., 2004).
We have been investigating the in vivo function of the bHLH
transcription factor, Mesp2, for which the periodic expression ofboth mRNA and protein appears in the anterior PSM (Saga et al.,1997). Mesp2 defines the segmental border by suppressing Notch
signaling via the activation of L-fng and the suppression of Dll1

688

M. Morimoto et al. / Developmental Biology 300 (2006) 687–698(Takahashi et al., 2000; Morimoto et al., 2005). Moreover, Mesp2
also defines the rostral identity of the somites by suppressing theexpression of Dll1 and Uncx4.1, which is required for thedevelopment of their caudal properties (Takahashi et al., 2003).
Thus, Mesp2 plays key roles in the anterior PSM, at the point oftransition of the paraxial mesodermal cells from the PSM tosomites. In addition, we have previously shown that Mesp1 is a
closely related member of the Mesp family of genes, which sharesignificant sequence homology in their bHLH regions (Saga et al.,1997). These genes are co-expressed in the early mesoderm, justafter gastrulation, and in the paraxial mesoderm during somitogenesis. They also show a good degree of functional redundancy,
since a knock-in of Mesp1 into the Mesp2 locus can rescue most of
the defects that are observed in the Mesp2-null mouse (Saga,
1998). However, the functions of Mesp1 and Mesp2 duringsomitogenesis are likely to be slightly different as chimericanalyses have revealed that Mesp1 contributes to the epithelialization of the somites but that Mesp2 is essential for establishing
rostro-caudal patterning (Takahashi et al., 2005).
In our current study, we have attempted to define the functional domains of the Mesp2 protein by focusing on the vertebral development reflecting rostro-caudal properties of somites
in vivo. After determining both the degradation domain and thenuclear localization signal of Mesp2, we constructed a possible
minimum functional Mesp2 protein containing the conservedbHLH domain and assessed the ability of this truncated factor tofunction in vivo by generating a Mesp2 locus knock-in of theconstruct. The knock-in mice showed a hypomorphic phenotype
in which the defects are confined within the anterior half of
the axial skeleton. Comparative analyses of the expression of
the Mesp family genes subsequently revealed that the relative
levels of Mesp1 and Mesp2 change during somitogenesis,
and that a compensatory increase in Mesp1 may contribute torescue events in the posterior half of the axial skeleton.
Material and methods
Plasmid construction
Various constructs of Mesp2 were generated according to standard
molecular techniques. Deletion mutant constructs were created by PCRmediated methods, followed by ligation to native flanking DNA fragments. The
wild-type and deletion mutants were cloned into the p3xFLAG-CMV vector(Sigma). EGFP-tagged Mesp2 variants were generated by inserting the Mesp2
coding sequence between EGFP and the poly-A signal.

Expression experiments using cultured cells
COS7 and NIH3T3 cells were maintained in Dulbecco's modified Eagle's
medium (DMEM) supplemented with 10% fetal bovine serum (FBS). Cells weretransfected with 3xFLAG-Mesp2 wild-type or mutant constructs using FuGene
6 (Roche) according to the manufacturer's instructions. The cells were harvested
24 h after transfection and equal numbers of cells were replated with or without
cycloheximide (30 μg/ml) and with the protease inhibitors, MG132 (50 μM),
Z-Leu-Leu-Z (50 μM), PMSF (1 mM) or Pepstatin A (50 μM).

Immunoblotting of 3xFLAG-Mesp2 proteins in vivo
Western blot analysis was conducted using standard procedures. The PSM
region of five embryos of each genotype was collected from same litters at

E10.5. The samples were lysed in buffer containing 50 mM Tris (pH 7.4), 0.2 M
NaCl, 1% Nonidet P40, 0.1% deoxycholate and a protease inhibitor cocktail(Roche). The tissue extracts were separated by SDS-PAGE on 7.5% gels. The
3xFLAG-Mesp2 signals were detected by anti-FLAG antibody F3165 (Sigma)
and quantified using NIH Image. The signals were normalized to the expression
levels of β-actin which were detected by anti-β-actin antibody A2066 (Sigma).

Gene knock-in strategy
To generate several knock-in vectors for Mesp2 variants, we initiallyconstructed a targeting vector cassette with the gateway system (Invitrogen), in
which attL1 and attL2 were included in the site where the Mesp2-cDNA variants
to be inserted. The Mesp2 cDNAs (P2-full, P2-ΔD, P2-HD) were subcloned into
the entry vectors for the gateway system and the final constructs were generated
by in vitro homologous recombination (Supplementary Fig. S1). Introduction of
the targeting vectors into ES cells (TT2) and subsequent screenings wereconducted as previously described (Yagi et al., 1993; Kitajima et al., 2000). The
chimeric mice were then crossed with ICR female mice to establish each mouseline.

Histology, histochemistry and gene expression analysis
Section in situ hybridization and immunohistochemical detection of proteins
were performed as previously described (Morimoto et al., 2005). The methods
used for gene expression analysis by in situ hybridization of whole-mountsamples and skeletal staining have been described previously (Saga et al., 1997;
Takahashi et al., 2000).

Real-time PCRThree primer pairs, one specific for Mesp1, one for Mesp2 and G3PDH,
were designed: Mesp1-L1 (5′-CCTTCGGAGGGAGTAGATC-3′) and Mesp1R1 (5′-AAAGCTTGTGCCTGCTTCA-3′); Mesp2-L2 (5′-GACTGGACACTGGACACAATCCACT-3′) and Mesp2-R2 (5′-GGCCATAGCCAAGCAGACCTCAAA-3′); G3PDH-Fw (5′-ACCACAGTCCATGCCATCAC-3′)
and G3PDH-Rv (5′-TCCACCACCCTGTTGCTGTA-3′). The housekeeping
G3PDH gene was used as “reference” gene. PCR reactions were carried out in
48-well microtiter plate wells in a 20 μl reaction volume with SYBR premix Ex
Taq (TAKARA) with optimized concentrations of specific primers. An BIORAD MiniOpticon Real Time PCR Detection System (BIO-RAD) was
programmed for an initial step of 10 s at 94°C, followed by 39 thermal cyclesof 15 s at 94°C, 15 s at 60°C, 30 s at 72°C. Every assay was run in triplicate andnegative controls (no template, template produced with no RT enzyme) werealways included. For each sample 1 μg of total RNA was reverse transcribed. A
1:25 dilution of each cDNA was used as template in real-time PCR. Specificityof PCR amplification of each primer pair was confirmed by melting curve
analysis (Ririe et al., 1997). Standard curves were generated by measuring
threshold cycle (CT) values of ten-fold serial dilutions of the calibrator cDNAs
for Mesp1 and Mesp2. The relative transcript level was determined by a method
and formula as described (Pfaffl, 2001). To compare the level of Mesp1
expression in different genotypes, embryos were collected and pooled after
genotyping. Mesp1 expression level of each sample was normalized against
G3PDH. For the measurement of relative expression levels between Mesp1 andMesp2, three independent embryo samples were prepared from different stages
and the value of Mesp2 was normalized against Mesp1.

Results
A mechanism to destabilize Mesp2 protein
A striking feature of the Mesp2 protein is its restricted
expression pattern in the anterior PSM every 2 h duringsomitogenesis. Since the Mesp2 levels quickly dissipate oncethe segmental border is established, we speculated that there
must be specific mechanisms that destabilize the protein. To

M. Morimoto et al. / Developmental Biology 300 (2006) 687–698

elucidate these possible mechanism(s), we examined thestability of Flag-tagged Mesp2 protein, expressed in COS7
cells, with a number of protein inhibitors. Mesp2 protein is
detectable as several bands, due to phosphorylation byendogenous kinases (Fig. 1 and Supplementary Fig. S2), andappears to be stabilized in the presence of MG132 (proteasome
inhibitor), but not by Z-Leu-Leu-H, PMSF or Pepstatin A(a calpain, serine protease or aspartic protease inhibitors,
respectively). To confirm this observation, a chase experiment
was conducted in the presence of cycloheximide, in which thehalf life of Mesp2 protein was determined to be approximately
1.3 h in COS7 cells. The degradation rate, however, wasremarkably inhibited in the presence of MG132, whereby thehalf life was extended to more than 4 h (Fig. 1B). These results
indicate that Mesp2 is mainly degraded by the proteasome
pathway, but also suggests the involvement of anothermechanism because the complete inhibition of Mesp2 degradation was not achieved by MG132 treatment (Fig. 1B).
To identify the Mesp2 domain required for its degradation, aseries of deletion mutants were individually expressed in COS7
cells and assayed for stability (Fig. 1C). A Δ311–330 mutant
was found to be the most stable in our assay system andsubsequent deletion analysis revealed that the region containing
amino acids 326–330 is the most likely to confer sensitivity tothe proteasome. We have thus designated this motif as the“degradation domain”. To compare the effects of MG132 uponfull-length and mutant Mesp2 (Δ311–330) in cultured cells, theprotein levels were visualized by immunoblotting in thepresence of either MG132 or DMSO (Fig. 1D). The Δ311–
330 mutant protein was found to accumulate even in theabsence of MG132, indicating that it is stabilized by its
resistance to the proteasome pathway.
To confirm the destabilizing effect of the degradation domain
of Mesp2 in vivo, we generated knock-in mice expressing
FLAG-tagged -Mesp2 Δ326–330 (F-Mesp2ΔD) (Fig. 2A)
instead of the wild-type protein (Supplementary Fig. S2). As acontrol, we also generated a knock-in mouse expressing FLAGtagged full-length-Mesp2 (F-Mesp2-full). The heterozygous
mice showed no abnormalities and the homozygous mice werealso fertile in the cases of both F-Mesp2ΔD and F-Mesp2-full.
To assess the stability of the introduced proteins, theneighboring sections of the PSM region were subjected to insitu hybridization analysis for Mesp2 transcripts and immunohistochemical detection of Mesp2 protein. We found that the
localization of the knock-in Mesp2 was similar to wild type(Fig. 2B). Unexpectedly, however, the mRNA expression levelsof the introduced Mesp2s (both full-length and mutant) werelower than wild type (Fig. 2B). Since Mesp2 expression levels
change cyclically during somitogenesis, we prepared several
repeat samples and confirmed this lower transcription result.
The reason for this effect is not clear but may be due to theunrelated nucleotide sequences in the 5′-untranslated region of
the introduced by targeting vectors.
The level of protein expression was therefore also reduced in
the knock-in mouse embryos and we noticed a broad expression
domain for F-Mesp2ΔD. To determine the effects of a lack of
the Mesp2 degradation domain in vivo, the protein expression

689

levels were compared by anti-FLAG Western blot analysis
using 5 tails from homozygous F-Mesp2ΔD and F-Mesp2-fullE10.5 embryos (Fig. 2C). The subsequent results indicated that
the F-Mesp2ΔD protein appears to be stabilized by the lack of adegradation domain in vivo, since these protein levels are
relatively higher than F-Mesp2. In addition, comparative
analyses of the skeletal specimens at E17.5 suggest that thedegradation domain might be functional in vivo. The homozygous F-Mesp2-full mouse shows a fused rib phenotype at a high frequency (Fig. 2D). Interestingly, however, theF-Mesp2ΔD fetus shows this rib defect at lower frequency.
We speculate that this situation may reflect the estimatedlower production of F-Mesp2-full under knock-in conditions
and that the defect is rescued by the stabilized F-Mesp2ΔD.
To further confirm this rescue effect, we reduced the genedosage by crossing F-Mesp2-full or F-Mesp2ΔD with
Mesp1/Mesp2 double KO (dKO) mice and compared theskeletal phenotype in the presence of a single knock-in
allele. The homozygous dKO mice die before E9.5 due tothe lack of embryonic mesoderm (Kitajima et al., 2000) but
the heterozygous mouse has no abnormality in the skeletal
morphology (no rib fusion was detected among 6 fetuses
examined). F-Mesp2-full/dKO embryos showed more severe
abnormalities in the ribs and vertebra, with an average numberof fused ribs of 16, whereas the average number of fused ribs
was 6.5 in the F-Mesp2ΔD/dKO mice (Fig. 2D). These results
indicate that the stability of F-Mesp2 ΔD may positively affectthe function of Mesp2 and result in the suppression of thedefect caused by a lower dosage of Mesp alleles.
Generation of a minimum functional Mesp2 protein
Mesp2 localizes to the nucleus in a cell as a transcription
factor. To identify the minimum regions of Mesp2 that are
required for a functioning protein, we screened for a putative
nuclear localization signal (NLS) of Mesp2. Two arginine
clusters (R70,71,73 and R145–150) that could be implicated asa potential NLS were identified outside of the basic helix–loop–
helix (bHLH) domain. We replaced these amino acids withalanine residues and generated EGFP-tagged Mesp2 variants(Supplementary Fig. S3A). The cellular localization of theresulting Mesp2 proteins was then examined using confocal
microscopy (Supplementary Fig. S3B). Wild-type Mesp2EGFP was found to localize in the nucleus as expected, but
the R70,71,73A and R145–150A variants were observed not
only in the nucleus but also in the cytoplasm. Furthermore, theR70,71,73,145–150A variant was in fact mainly localized in thecytoplasm. We confirmed that these GFP fusion proteins wereexpressed as the correct size in the culture cells by the Western
blotting (Supplementary Fig. S3B). These data suggest that
R70,71,73 and R145–150 function as an NLS in the Mesp2
protein.
In addition to the degradation domain and the NLS, thebHLH domain is known to be required for DNA binding
and for homo- or heterodimer formation. Hence, we tried toconstruct a minimal but functional Mesp2 (P2-HD protein)by combining the 3xFLAG tag with the regions 61–160aa

690

M. Morimoto et al. / Developmental Biology 300 (2006) 687–698

Fig. 1. The Mesp2 protein is degraded by the proteasome via its “degradation domain”. (A) Cos7 cells transfected with 3xFLAG-Mesp2 (F-Mesp2) were incubated in
the presence of cycloheximide and protease inhibitors, MG132, Z-L-L-H, PMSF or Pepstatin A for 2 h. The targets of these inhibitors are the proteasome, calpain,
serine proteases and aspartic proteases, respectively. The F-Mesp2 proteins were detected by immunoblotting with anti-FLAG antibodies. (B) F-Mesp2 protein
stability was analyzed in the presence of cycloheximide by treatment with either DMSO or MG132. The degradation process was then monitored by immunoblottingwith anti-FLAG antibodies and was found to be inhibited by MG132 but not DMSO (vehicle control). (C) Functional mapping of the degradation domain of Mesp2.
The degradation process was compared among the Mesp2 mutants shown in the figure by the same method used in panel B to identify the domains required for the
degradation of Mesp2. Stabilized mutants are indicated by the red circles on the right. (D) Either full-length or D311–330 mutant F-Mesp2 was transfected into Cos7
cells and cultivated for 6 h with or without MG132. F-Mesp2 proteins were then detected by Western blotting (left panel) and quantified using NIH Image software(right panel).

M. Morimoto et al. / Developmental Biology 300 (2006) 687–698(bHLH and NLS) and 311–338aa (Degradation domain) of
Mesp2 (Supplementary Fig. S3C). We confirmed that thisprotein localized to the nucleus (Supplementary Fig. S3C).
To investigate the function of the P2-HD in vivo, we thengenerated a P2-HD knock-in mouse using a similar method
to that described in Supplementary Fig. S1. P2-HD heterozygous mice showed completely normal metameric vertebrae
at E17.5 (Fig. 3A upper). In contrast, the P2-HD homozygous mice exhibited skeletal defects in the anterior region of691

the vertebra along the A–P axis, the cervical and thoracic
vertebrae, and the rib (Fig. 3A middle). However, this phenotype was much milder than that of GFP-KI mice (Mesp2-nullmice), in which the pedicles of the neural arches in the entire
vertebra and the ribs are fused to each other as a result of thecaudalization of somites, as shown previously (Fig. 3A lower,
Takahashi et al., 2005). These data suggest that the P2-HD
protein partially rescues the skeletal defects observed in the
Mesp2-null mouse.

Fig. 2. A Mesp2 mutant protein that lacks the degradation domain is stabilized in vivo. (A) Schematic representation of a 3xFLAG-Mesp2Δ326–330 mutant (F-Mesp2
ΔD). (B) Comparison of the Mesp2 expression levels (protein and mRNA) among wild-type (WT) Mesp2, F-Mesp2 and F-Mesp2ΔD. Neighboring sections were
subjected to detection by either anti-Mesp2 antibodies or RNA probes. Among specimens examined, representative samples containing the highest signals are shown.
(C) Western blot analysis of Mesp2 proteins in vivo. Sample lysates were prepared from five PSM tissues of WT, F-Mesp2 or F-Mesp2ΔD embryos at E11.0 and
Mesp2 proteins were then detected using anti-FLAG antibodies. Wild-type embryos served as a negative control (we have never successfully detected endogenous
Mesp2 protein with anti-Mesp2 antibodies). The signal intensities were quantified using NIH Image and normalized to the expression levels of β-actin. (D)Comparative analyses of skeletal defects among different Mesp genotypes. The skeletal specimens were prepared at E17.5 and the defects observed in the rib were
quantified by determining the number of fused sites. No fusions were detectable in the wild-type specimens. Fused ribs were detected in the homozygous F-Mesp2
mice at a higher frequency than in the homozygous F-Mesp2ΔD mice. A much more severe skeletal phenotype was observed in both the F-Mesp2/dKO and FMesp2ΔD/dKO embryos due to the lower Mesp dosage.

692

M. Morimoto et al. / Developmental Biology 300 (2006) 687–698

Fig. 3. The phenotype of a minimum Mesp2 functional mutant, P2-HD. (A–C) Comparative analyses of the skeletal morphologies at E17.5 (A) and the expression of
Uncx4.1 at E9.5 (B) in wild-type, P2-HD-KI or GFP-KI mice. (A) The homozygous P2-HD knock-in mouse exhibits skeletal defects in the anterior part of the axial
skeleton, the cervical and thoracic vertebra and in the rib (middle panels in A). Much stronger defects are observed in the GFP-KI (Mesp2-null) mouse. Red lines
indicate the skeletal defect region. (B) Metameric expression pattern of Uncx4.1 is disrupted in Mesp2-null embryo (right panel). This abnormal expression is rescuedin the homozygous P2-HD knock-in mouse (middle panel). (C) Mesp1 expression is increased in the P2-HD embryo at E10.5. The expression levels of the P2-HD
protein are lower than that of the wild-type (left panels). In contrast, the expression levels of Mesp1 were found to be up-regulated in the P2-HD KI embryo (middle
panels).

M. Morimoto et al. / Developmental Biology 300 (2006) 687–698

The expression of Uncx4.1, a caudal somite marker gene
(Mansouri et al., 1997), was observed in the caudal compartments of the somites of both wild-type and P2-HD embryos,
whereas an extended expression pattern was observed in the
Mesp2-null embryo due to the lack of the rostral properties ofthe somites (Fig. 3B). Therefore, judging by the expression
pattern of Uncx4.1, the minimal Mesp2 protein, P2-HD, cansubstitute for Mesp2 in vivo. However, the expression of P2HD may not be sufficient to facilitate the gene regulation
required during subsequent vertebral development, particularlyin the anterior part of the axial skeleton. To determine themechanism underlying this region-specific defect, we first693

analyzed Mesp2 expression using specific antibodies and foundthat the levels of P2-HD protein are lower than wild type(Fig. 3C). This phenomenon is consistent with our analyses of
F-Mesp2 and Mesp2ΔD KI mice, which showed lower expression of both protein and mRNA for these species. On theother hand, using neighboring sections we have observed an upregulation of Mesp1 in the P2-HD homozygotes (Fig. 3C). In
the GFP-KI homozygous embryo, Mesp1 expression was alsoenhanced (Supplementary Fig. S4A). Since we have found that
Mesp1 expression is enhanced, and that Mesp1 also plays animportant role in somitogenesis when the Mesp2 gene is defective (Takahashi et al. manuscript in preparation), we

Fig. 4. The Mesp1 expression levels increase as somitogenesis progresses in P2-HD KI mice. (A) Whole-mount in situ hybridization was performed with Mesp1
probes using early (8 somites) to late (36 somites) somite-stage embryos. The Mesp1 expression profile in the P2-HD KI embryo is stronger than wild type throughout
somitogenesis. (B) The levels of Mesp1 expression were compared among wild-type (+/+), P2HD heterozygous (+/HD) and P2HD homozygous (HD/HD) embryos
using samples derived from two litters at two different developmental stages (E8.5 and E9.75). Total RNA isolates were prepared from pooled whole embryos and realtime PCR analysis was then conducted for each genotype. The numbers of embryos used in the analyses varied from 2 to 5 as indicated in the panel. The level of Mesp1
mRNA was normalized to G3PDH. (C) The relative ratio of the Mesp2 expression levels to that of Mesp1 were examined at the early (E8.5) and later (E9.75) somite
stages by real-time PCR with three different sets of cDNAs derived form wild-type embryos. These data were normalized to the levels of Mesp1 expression. The
numbers of embryos used were indicated in the panel.

694

M. Morimoto et al. / Developmental Biology 300 (2006) 687–698

hypothesized that the relative contributions of Mesp1 andMesp2 might be regulated during somitogenesis and the regionspecific defect that we observed in these experiments mightreflect such a functional bias.
Differential expression of Mesp1 and Mesp2 duringsomitogenesis
Mesp1 and Mesp2 are transiently expressed prior tosegmental border formation. Therefore, the downstream target
genes must be involved in the subsequent chondrogenesis
pathways that are required for skeletal development (Takahashi
et al., 2000). Somites are sequentially generated from a rostral toa caudal direction and it is known that their regional specificity
is defined when each somite is generated during development.
Cervical vertebra are derived from 5–12 somites generated
during E8.0–8.5, thoracic vertebrae are generated from 12–25
somites at E8.5–9.5, the lumbar are produced from 25–31
somites at E9.5–10.0, the sacral types are derived from 31–36
somites at E10.0–10.5 and caudal vertebrae are formed from
36–65 somites at E10.5–13.5 (Gossler and Tam, 2002).
To elucidate the contribution of Mesp1 to somitogenesis, andthe reason why the P2-HD proteins show region-specific defects
in the axial skeleton, the expression pattern of Mesp1 was
examined by whole-mount in situ hybridization from E8.5 (8–9
somites, cervical level) to E10.5 (35–36 somites, caudal
vertebral level) (Fig. 4A). In P2-HD homozygous embryos,
the expression level of Mesp1 was found to be higher than thatof the wild-type embryos throughout each of the stages
examined. In addition, it appears that the level of Mesp1
expression increases in the P2-HD embryo at the later stages,
whereas it was relatively constant in wild-type embryos. We
therefore reasoned that the normal vertebral formation from theposterior to the lumbar levels is probably due to the upregulation of Mesp1 during somitogenesis. To confirm thisobservation, we used quantitative RT-PCR to compare the levelsof Mesp1 during somitogenesis among wild-type and P2-HD
hetero- or homozygous littermates at E8.5 and E9.75 (Fig. 4B).
Since the embryo size differs from stage to stage, only the ratio
among different genotypes was compared. At E8.5, Mesp1
expression in P2-HD homozygous embryo was about 3.2-foldhigher than wild type. At E9.75, this higher level of Mesp1 wasincreased to about 7.6 fold above wild type. As previously
reported, both Mesp1 and Mesp2 are expressed in the early
nascent mesoderm but the expression is transient and become
confined in the paraxial mesoderm after E8.25 just before
somitogenesis (Saga et al., 1999). These data indicate that thedifferences in the expression levels of Mesp1 between wild-type
and P2-HD embryos increase with the progression ofsomitogenesis.
Since Mesp1 and Mesp2 have some similar functions, andthe expression levels of both Mesp2 and Mesp1 are likely to beimportant, we next analyzed the relative expression levels ofboth genes using real-time PCR analysis with cDNAs from thewild-type embryos. Because both the Mesp1 and Mesp2
expression patterns change cyclically at every somite formation,
we initially tested whether those expression patterns change
synchronously during somitogenesis. Bisected tail samples
were subjected to in situ hybridization using Mesp1 or Mesp2
probes. As shown in Supplementary Fig. S4B, the expression
levels of both signals do appear to change synchronously.
Consequently, we speculated that the relative RNA levels for
Mesp1 and Mesp2 could be analyzed using pooled RNA fromseveral embryo samples. Subsequently, the value of Mesp2 wasnormalized to Mesp1 and we then compared the values duringsomitogenesis. As anticipated by the cyclic expression pattern,
the value of Mesp1 and Mesp2 and the ratio between them
revealed by the real-time PCR were varied even in the samedevelopmental stage. However, we could confirm that the ratio
became increased as somitogenesis progresses. The meandifference we have detected between E8.5 and E9.75 were2.0-fold (Fig. 4C). These results suggest that the relative levelsof Mesp2 increase as somitogenesis proceeds, while the levelsof Mesp1 might be simultaneously reduced, in wild-type
embryos.
Functional contributions of Mesp1 and Mesp2 duringsomitogenesis
Based on the above observations we hypothesized that (1)Mesp2 suppresses Mesp1 expression in the later stage embryo,(2) the reduction of Mesp2 results in the de-repression of Mesp1
and (3) a particular level of Mesp activity (Mesp1 + Mesp2) isrequired for the normal progression of somitogenesis (Figs. 4

Fig. 5. A working hypothesis for the skeletal phenotypes of the Mesp2-mutants, depicted in the right panels. By comparative analysis of Mesp1 and Mesp2 expression
levels in the wild-type and mutant embryos, we speculate that the expression of Mesp2 increases as somitogenesis progresses and that the levels of Mesp1 are reducedin tandem in the wild-type embryo. A reduction of Mesp2 function thus results in the de-repression of Mesp1 expression. Somitogenesis proceeds normally when thefunctional levels of Mesp1 and Mesp2 are maintained above a specific threshold (indicated by the dotted line in the right panels). Green lines indicate abnormal
skeletal regions. (A) In the wild-type embryo, normal skeletal formation is maintained by the collaborative expression and function of Mesp1 and Mesp2. The levels ofthese two proteins are above the required functional threshold throughout somitogenesis. (B) In the P2-HD homozygous embryo, the collaborative activity of Mesp1
and Mesp2 is not sufficient to form the normal skeleton at the early stages of development since the level of P2-HD is low. However, the P2-HD expression increases ina manner similar to wild-type Mesp2, and the resulting Mesp activity reaches the required threshold at E9.5, which causes the restoration of normal somitogenesis
thereafter. The skeletal defects are then restricted to the anterior half of the body, including the cervical and thoracic vertebrae, and the ribs. (C) In the presence of only asingle P2-HD allele, there is some delay in the Mesp levels reaching their functional threshold (after E10.0), which results in the extension of the skeletal defects to thelumber vertebral level. (D) Further reduction of the Mesp1 gene dosage compared with Mesp2 HD/GFP causes a further extension of the defect into the sacralvertebrate. The collaborative activity of the Mesp gene reaches the required threshold at E10.5. (E) In the Mesp2-null mouse, Mesp1 activity cannot reach thefunctional threshold during somitogenesis and the skeletal defects extend to the most posterior vertebra. (F) More severe defects are observed in the Mesp2 GFP/dKO
mouse, which has only a single Mesp1 allele. The fetus thus shows severe truncation of the vertebra that might be due to the atrophy of somite derivatives. These datasuggest that the relative contributions of Mesp1 and Mesp2 differ during somitogenesis.

M. Morimoto et al. / Developmental Biology 300 (2006) 687–698

and 5A), and skeletal defects will thus only be observed in theregion where Mesp function is below this threshold level.
According to this hypothesis, the phenotype of P2-HD can beinterpreted as a lower Mesp activity (below the threshold) onlyin the early stage embryo (Fig. 5B). Since we do not know thedefinite values of Mesp1 and Mesp2 expression levels, we havearbitrarily defined the threshold level for Mesp activity in the695

wild-type embryo as the activity required for establishingnormal rostro-caudal patterning, and we have assumed that only
the relative ratios of Mesp1 and Mesp2 change duringsomitogenesis (Fig. 5A, right panel).
To evaluate our hypothesis, we genetically manipulated
somitogenesis by changing the dosage of Mesp1 and Mesp2
and examining the resulting skeletal morphologies at E17.5. By

696

M. Morimoto et al. / Developmental Biology 300 (2006) 687–698

crossing P2-HD with either Mesp2 GFP or dKO mice we wereable to compare the Mesp1 dosage effects with the impact of asingle P2-HD allele (Figs. 5C and D). At first, we found that the
reduction of the P2-HD allele to a single dosage resulted in theextension of the skeletal defects in the posterior part of the body,which is visualized by the fusion of the pedicles of the neuralarches (Fig. 5C, left panel). The Mesp2 HD/dKO mouse, which
has both a single Mesp1 and P2-HD allele, showed a moreposterior expansion of this defect; both the fusion of thepedicles and malformation of the caudal vertebra were evident(Fig. 5D, left panel). Mesp2 GFP/GFF (Mesp2-null) mice
showed defects in their entire vertebra, and in this case we
predict that the levels of Mesp1 will not reach the thresholdlevel required for normal rostral–caudal patterning (Figs. 3B,
5E). The further reduction of Mesp1 levels in the Mesp2 GFP/dKO mouse resulted in the shortening of the trunk region, as
well as more severe fusion of the vertebra (Fig. 5F). Thesephenotypes can be explained for each genotype by our current
hypothesis (Fig. 5 right panel).
Discussion
Mesp2 protein stability is primarily regulated by aproteasome-mediated pathway
The most notable feature of the Mesp2 expression profile is
the restriction of its expression domain in the anterior PSM,
with a clear boundary formed that will demarcate the next
segmental border (Morimoto et al., 2005). To establish thesegmental boundary, at intervals of 2 h in mice, the protein
would most likely need to be degraded once it achieves its
function. In our current experiments, a molecular mapping
study was successful in defining the “degradation domain” toamino acids 326–330 of the Mesp2 protein, which is requiredfor proteolysis mediated by the proteasome in cultured cells(Fig. 1). However, the proteasome inhibitor MG132 did notcompletely abrogate Mesp2 proteolysis (Fig. 1B), indicating the
involvement of additional proteolytic pathways in this regulation. We next attempted to determine the effects of this Mesp2“degradation domain” in vivo by utilizing a knock-in strategy
for the stabilized construct into the endogenous locus (Fig. 2).
Unfortunately, however, this approach resulted in the reductionin the levels of transcription and we observed relatively severe
defects in the control embryo into which a full-length Mesp2
fragment was introduced by a similar targeting strategy. These
defects could be ascribed to the lower levels of expression of
Mesp2 protein that are shown in Fig. 2B. In such a situation, amore highly stable Mesp2 variant might compensate for the lackof endogenous Mesp2 function. Thus we could not observe
anomalies that were due to a more highly stabilized Mesp
protein.

would be the minimum requirements for a functional protein(Fig. S3C). The resulting knock-in mice displayed fused
pedicles of the neural arches in both the cervical and thoracic
regions (Fig. 3A), indicating that the somites were caudalized
during the early stages of somitogenesis at E8.0–9.5 due to thelack of Mesp2 function. However, we could not detect any
abnormalities in the expression pattern of Uncx4.1, which is aknown downstream gene target of Mesp2 (Fig. 3B). This raised
the interesting issue of why the region-specific defects of P2HD occurred. The bHLH-type transcription factors are known
to work as homo- or heterodimers via the binding to target geneenhancers. The targets may be selected by partnering factors
and the transcriptional activity might then be determined byother interacting proteins recruited to the target sequences. In
this regard, we have recently shown that Mesp2 forms aheterodimer with E47 and activates EphA4 transcription via thebinding of multiple E-boxes in the EphA4 enhancer (Nakajima
et al., 2006). It is possible that P2-HD may not have sufficientability to recruit other transcription factors required for full
activity. However, in the later stage embryo (after E9.5), thedeficiencies of the P2-HD protein might be compensated for byincreased levels of Mesp1, whereby Mesp function must reach asufficient level to fulfill its functional role. Since the targets of
Mesp2 that involved in pedicle formation have not yet been
identified, other than Uncx4.1, it is not yet feasible to analyze
the changes in the activity of these genes, the levels of which
would be expected to be low in the early stages but sufficiently
higher in the later stages of somitogenesis.
The gene dosage of Mesp1 and Mesp2 is critical for skeletal
development
The Mesp1 gene shows a similar expression pattern to
Mesp2 in the anterior PSM. In the Mesp2-null embryo, as wellas in other knock-in mice in which Mesp2 function is expected
to be reduced, the expression levels of Mesp1 increase,indicating that Mesp2 generally suppresses Mesp1 transcription.
We have now found that the level of Mesp2 expression
increases as somitogenesis progresses, and that Mesp1 expression consequently decreases during normal somitogenesis (Fig.
5A). In the P2-HD embryo, Mesp1 expression was found toincrease although it could not be determined whether this was
because of the lower levels of P2-HD expression in our current
knock-in strategy or due to the lesser ability of P2-HD to
suppress Mesp1 transcription. Unfortunately, we have noantibody to detect Mesp1 protein. Nevertheless, we speculatethat P2-HD embryos can proceed with normal somitogenesis in
the later stages of development because of the rescue of Mespfunction by Mesp1 (Fig. 4). Since this compensatory phenomenon is mainly observed in later stage embryos, the dosage
effect is detected from the anterior part and then spreads to theposterior region (Fig. 5).

The P2-HD Mesp2 variant rescues the null defects in laterstage embryos

The regulation of Mesp1 and Mesp2 during somitogenesis

We constructed a truncated Mesp2 protein composed of the
NLS, bHLH and degradation domain, which we predicted

Somitogenesis continues from stages E8.0 to E13.5 and thebasic regulatory mechanisms underlying somite segmentation

M. Morimoto et al. / Developmental Biology 300 (2006) 687–698

are most likely similar but may have some variation during thisprocess. It was recently shown in zebrafish that mutation of
integlinα5 affects only the early stages of somitogenesis,
although it is required for the maintenance of epithelial somites(Julich et al., 2005a; Koshida et al., 2005). The reverse
phenomenon has been reported for mutants of Notch signaling
pathway genes. In both zebrafish (Holley et al., 2002; Julich etal., 2005b) and mice (Conlon et al., 1995; Wong et al., 1997;
Zhang and Gridley, 1998; Bessho et al., 2001), Notch signaling
is involved in clock gene regulation and rostro-caudal
patterning (Rida et al., 2004). In either case, the defects in
Notch mutants become more severe as somitogenesis progresses. These data indicate that there could be some differencesin the regulatory mechanisms along the A–P axis. Thesefindings could also be explained by different contributions ofsimilar factors or by specific developmentally regulated
molecules. In the case of P2-HD homozygous mice, in which
the defects are observed only in the anterior part of the axial
skeleton (Figs. 3B and 5B), we do not think that the functions ofMesp1 and Mesp2 change during somitogenesis. We insteadfavor the idea that the levels of Mesp1 and Mesp2 change, andthat a critical balance is maintained, during somitogenesis in thewild-type mouse.
Acknowledgments
We especially thank Yuki Takahashi for many technicalsupports and maintaining the mice used in this study. 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.
Appendix A. Supplementary data
Supplementary data associated with this article can be found,in the online version, at doi:10.1016/j.ydbio.2006.08.043.

References
Aulehla, A., Herrmann, B.G., 2004. Segmentation in vertebrates: clock andgradient finally joined. Genes Dev. 18, 2060–2067.
Aulehla, A., Johnson, R.L., 1999. Dynamic expression of lunatic fringe suggests
a link between notch signaling and an autonomous cellular oscillator driving
somite segmentation. Dev. Biol. 207, 49–61.
Aulehla, A., Wehrle, C., Brand-Saberi, B., Kemler, R., Gossler, A., Kanzler, B.,
Herrmann, B.G., 2003. Wnt3a plays a major role in the segmentation clock
controlling somitogenesis. Dev. Cell 4, 395–406.
Bessho, Y., Sakata, R., Komatsu, S., Shiota, K., Yamada, S., Kageyama, R.,
2001. Dynamic expression and essential functions of Hes7 in somite
segmentation. Genes Dev. 15, 2642–2647.
Bessho, Y., Hirata, H., Masamizu, Y., Kageyama, R., 2003. Periodic repression
by the bHLH factor Hes7 is an essential mechanism for the somite
segmentation clock. Genes Dev. 17, 1451–1456.
Borycki, A.G., Emerson Jr., C.P., 2000. Multiple tissue interactions and signaltransduction pathways control somite myogenesis. Curr. Top. Dev. Biol. 48,
165–224.
Brand-Saberi, B., Christ, B., 2000. Evolution and development of distinct celllineages derived from somites. Curr. Top. Dev. Biol. 48, 1–42.

697

Conlon, R.A., Reaume, A.G., Rossant, J., 1995. Notch1 is required for thecoordinate segmentation of somites. Development 121, 1533–1545.
Dubrulle, J., Pourquie, O., 2004. Coupling segmentation to axis formation.
Development 131, 5783–5793.
Giudicelli, F., Lewis, J., 2004. The vertebrate segmentation clock. Curr. Opin.
Genet. Dev. 14, 407–414.
Gossler, A., Tam, P.P.L., 2002. Somitogenesis: segmentation of the paraxial
mesoderm and the delineation of tissue compartments. In: Rossant, J., Tam,
P.P.L. (Eds.), Mouse Development. Academic Press, pp. 127–149.
Hirata, H., Bessho, Y., Kokubu, H., Masamizu, Y., Yamada, S., Lewis, J.,
Kageyama, R., 2004. Instability of Hes7 protein is crucial for the somite
segmentation clock. Nat. Genet. 36, 750–754.
Holley, S.A., Julich, D., Rauch, G.J., Geisler, R., Nusslein-Volhard, C.,
2002. her1 and the notch pathway function within the oscillator
mechanism that regulates zebrafish somitogenesis. Development 129,
1175–1183.
Jouve, C., Palmeirim, I., Henrique, D., Beckers, J., Gossler, A., Ish-Horowicz,
D., Pourquie, O., 2000. Notch signalling is required for cyclic expression of
the hairy-like gene HES1 in the presomitic mesoderm. Development 127,
1421–1429.
Julich, D., Geisler, R., Holley, S.A., 2005a. Integrinalpha5 and delta/notch
signaling have complementary spatiotemporal requirements during zebrafish somitogenesis. Dev. Cell 8, 575–586.
Julich, D., Hwee Lim, C., Round, J., Nicolaije, C., Schroeder, J., Davies,
A., Geisler, R., Lewis, J., Jiang, Y.J., Holley, S.A., 2005b. beamter/deltaC and the role of Notch ligands in the zebrafish somite
segmentation, hindbrain neurogenesis and hypochord differentiation.
Dev. Biol. 286, 391–404.
Kitajima, S., Takagi, A., Inoue, T., Saga, Y., 2000. MesP1 and MesP2 areessential for the development of cardiac mesoderm. Development 127,
3215–3226.
Koshida, S., Kishimoto, Y., Ustumi, H., Shimizu, T., Furutani-Seiki, M.,
Kondoh, H., Takada, S., 2005. Integrinalpha5-dependent fibronectin
accumulation for maintenance of somite boundaries in zebrafish embryos.
Dev. Cell 8, 587–598.
Mansouri, A., Yokota, Y., Wehr, R., Copeland, N.G., Jenkins, N.A., Gruss,P., 1997. Paired-related murine homeobox gene expressed in thedeveloping sclerotome, kidney, and nervous system. Dev. Dyn. 210,
53–65.
Monsoro-Burq, A.H., Le Douarin, N., 2000. Duality of molecular signaling
involved in vertebral chondrogenesis. Curr. Top. Dev. Biol. 48, 43–75.
Morimoto, M., Takahashi, Y., Endo, M., Saga, Y., 2005. The Mesp2
transcription factor establishes segmental borders by suppressing Notch
activity. Nature 435, 354–359.
Nakajima, Y., Morimoto, M., Takahashi, Y., Koseki, H., Saga, Y., 2006.
Identification of Epha4 enhancer required for segmental expression and theregulation by Mesp2. Development 133, 2517–2525.
Pfaffl, M.W., 2001. A new mathematical model for relative quantification in
real-time RT-PCR. Nucleic Acids Res. 29, e45.
Rida, P.C., Le Minh, N., Jiang, Y.J., 2004. A Notch feeling of somite
segmentation and beyond. Dev. Biol. 265, 2–22.
Ririe, K.M., Rasmussen, R.P., Wittwer, C.T., 1997. Product differentiation by
analysis of DNA melting curves during the polymerase chain reaction. Anal.
Biochem. 245, 154–160.
Saga, Y., 1998. Genetic rescue of segmentation defect in MesP2-deficient miceby MesP1 gene replacement. Mech. Dev. 75, 53–66.
Saga, Y., Takeda, H., 2001. The making of the somite: molecular events invertebrate segmentation. Nat. Rev., Genet. 2, 835–845.
Saga, Y., Hata, N., Koseki, H., Taketo, M.M., 1997. Mesp2: a novel mouse gene
expressed in the presegmented mesoderm and essential for segmentation
initiation. Genes Dev. 11, 1827–1839.
Saga, Y., Miyagawa-Tomita, S., Takagi, A., Kitajima, S., Miyazaki, J., Inoue, T.,1999. MesP1 is expressed in the heart precursor cells and required for theformation of a single heart tube. Development 126, 3437–3447.
Takahashi, Y., Koizumi, K., Takagi, A., Kitajima, S., Inoue, T., Koseki, H.,
Saga, Y., 2000. Mesp2 initiates somite segmentation through the Notch
signalling pathway. Nat. Genet. 25, 390–396.
Takahashi, Y., Inoue, T., Gossler, A., Saga, Y., 2003. Feedback loops comprising

698

M. Morimoto et al. / Developmental Biology 300 (2006) 687–698

Dll1, Dll3 and Mesp2, and differential involvement of Psen1 are essential for
rostrocaudal patterning of somites. Development 130, 4259–4268.
Takahashi, Y., Kitajima, S., Inoue, T., Kanno, J., Saga, Y., 2005. Differential
contributions of Mesp1 and Mesp2 to the epithelialization and rostro-caudal
patterning of somites. Development 132, 787–796.
Wong, P.C., Zheng, H., Chen, H., Becher, M.W., Sirinathsinghji, D.J.,
Trumbauer, M.E., Chen, H.Y., Price, D.L., Van der Ploeg, L.H., Sisodia,

S.S., 1997. Presenilin 1 is required for Notch1 and DII1 expression in theparaxial mesoderm. Nature 387, 288–292.
Yagi, T., Tokunaga, T., Furuta, Y., Nada, S., Yoshida, M., Tsukada, T., Saga, Y.,
Takeda, N., Ikawa, Y., Aizawa, S., 1993. A novel ES cell line, TT2, withhigh germline-differentiating potency. Anal. Biochem. 214, 70–76.
Zhang, N., Gridley, T., 1998. Defects in somite formation in lunatic fringedeficient mice. Nature 394, 374–377.