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First published online October 10, 2008
doi: 10.1242/10.1242/dev.027144
1 Division of Cellular and Molecular Toxicology, National Institute of Health
Sciences, 1-18-1 Kamiyoga, Setagaya-ku, Tokyo 158-8501, Japan.
2 Division of Mammalian Development, National Institute of Genetics, 1111 Yata,
Mishima, Shizuoka 411-8540, Japan.
3 Research Institute for Cell Engineering, National Institute of Advanced
Industrial Science and Technology, 3-11-46 Nakoji, Amagasaki, Hyogo 661-0974
Japan.
* Authors for correspondence (e-mails: yasuhiko{at}nihs.go.jp; ysaga{at}lab.nig.ac.jp)
Accepted 8 September 2008
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Key words: T-box transcription factor, Enhancer, Targeted disruption, Somitogenesis
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). This
process proceeds through the interaction of a number of signaling cascades,
including Notch, Wnt and Fgf (Delfini et
al., 2005; Dunty et al.,
2008; Galceran et al.,
2004; Hofmann et al.,
2004; Moreno and Kintner,
2004; Takahashi et al.,
2000). Thus, somitogenesis could be a very useful model system in
which to study the interactions among the various signaling cascades that
facilitate periodic pattern formation.
The basic helix-loop-helix transcription factor Mesp2 plays a crucial role
in both somite segment border formation and in the establishment of the
rostrocaudal patterning of each somite
(Saga et al., 1997).
Mesp2 shows dynamic and periodic expression in the anterior PSM. This
expression pattern defines the positioning of the newly forming somite by
suppressing Notch signaling, in part through the activation of lunatic fringe
(Lfng) (Morimoto et al.,
2005). Genetic analyses have revealed that Mesp2
expression is itself controlled by Notch signaling, indicating the existence
of complicated feedback circuitry
(Takahashi et al., 2003;
Takahashi et al., 2000). We
have previously identified the minimal PSM-specific Mesp2 enhancer
(denoted P2PSME) that is sufficient to reproduce the normal Mesp2
expression pattern in transgenic animals
(Haraguchi et al., 2001). We
have also demonstrated that the T-box transcriptional regulator Tbx6 directly
binds to P2PSME and is essential for P2PSME activity
(Yasuhiko et al., 2006). We
also showed that Notch signaling strongly enhanced Mesp2 activation
via Tbx6 and we identified the sequences that are important for this
enhancement using an in vitro reporter assay
(Yasuhiko et al., 2006).
However, the question of whether P2PSME is indispensable for Mesp2
expression during somitogenesis remained to be addressed. Because of
differences in the expression patterns of Mesp2 and Tbx6 - Tbx6 is
expressed throughout the PSM and tailbud
(Chapman et al., 1996;
White and Chapman, 2005)
whereas Mesp2 expression is observed only in the anterior PSM
(Saga et al., 1997) - another
open question was whether Tbx6 actually binds to P2PSME.
The evolutionary aspect of this system is also noteworthy. We previously
identified the mespb PSM-specific enhancer in the teleost fish
medaka, and reported that the mutation of two T-box binding sites therein
diminished its PSM-specific enhancer activity in transgenic embryos
(Terasaki et al., 2006).
However, definitive evidence as to whether the T-box-factor-dependent
regulation is a conserved mechanism among vertebrates remains elusive.
In this study, we established Mesp2 enhancer knockout mice and confirmed that Tbx6 binding sequences are essential for Mesp2 expression. The in vivo association of Tbx6 with P2PSME was confirmed in chromatin immunoprecipitation assays, and reporter assays further showed that the number and spatial organization of Tbx6 binding sites are important for P2PSME activity. Furthermore, using a knock-in mouse that harbors the medaka mespb enhancer in place of the mouse Mesp2 enhancer, we show that the T-box-factor-dependent regulation of the Mesp gene is evolutionally conserved between fish and mice.
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) with
the following primers (mutated nucleotides in lower case): mB1,
5'-CCTTCGAGGGGTCAGAATCgAtAtCTCTGCAAATGGGCCCGCTTT-3'; mB2,
5'-CCTTCGAGaGtaCtGAATCCACACCTCTGCAAATGGGCCCGCTTT-3'; mD,
5'-AACCTGGCAGGGGACCACCTCgCgaCTTTAGTCCAGATAAAAGCT-3'; mG,
5'-CTGGGCTCTGTGGGTTTTGAattCTCTCTGCAACCTGGCA-3'. The mutated Tbx6
binding sites are indicated for each construct, such that P2EmB1D represents a
P2PSME containing both mB1 and mD.
Gene targeting
For targeted disruption of P2PSME, a 356-bp DNA fragment containing mutated
Site B and Site D was generated by PCR using primers mB1 and mD. As a negative
control, the wild-type P2PSME fragment was also generated by PCR. To construct
the targeting vectors, a floxed PGK-neoR selection marker cassette was
inserted between a 6-kb long arm and the 356-bp DNA fragment with or without
mutations (Fig. 1A). The region
corresponding to Mesp2 exon 1, intron 1 and a part of exon 2 served
as the short homology arm. The targeting vector was introduced into mouse ES
cells (strain TT2) by electroporation. Resulting G418-resistant ES clones were
characterized by PCR using primers: Fesneo,
5'-CGCCTTCTATCGCCTTCTTGACGAG-3' and RP213,
5'-CAGGACAGCCACTGAGCTGCAGGCCTGA-3'. Southern blots were performed
to confirm homologous recombination. Positive ES clones were then aggregated
with 8-cell stage ICR mouse embryos in order to produce chimeric mice. The ES
selection marker PGK-neoR was removed by crossing the chimeric mice with
CAG-Cre mice, which express Cre recombinase ubiquitously. The resulting mouse
strains, with insertions of either mutated P2PSME or wild-type P2PSME, were
designated P2EmB1D or P2EmCont, respectively. Although the knockout mice were
established using an ES cell line (TT2) obtained from a C57BL/6 x CBA
cross (Yagi et al., 1993),
mice were maintained in an ICR background unless otherwise stated.
Skeletal preparation
Embryonic day 17.5 (E17.5) mouse embryos were obtained by crossing the
mutants of interest. Embryos were then fixed with 90% ethanol. For genotyping,
PCR was performed using a piece of embryonic liver digested with proteinase K
(Roche). Alcian Blue and Alizarin Red staining were performed as described
(Saga et al., 1997;
Takahashi et al., 2000).
Generation of anti-Tbx6 antibody
His-tagged fragments of Tbx6 protein (N-terminal antigen, amino acids 2-78;
internal antigen, amino acids 311-408)
(White and Chapman, 2005) were
produced using the pET system (Novagen) and Escherichia coli
Rosetta-gamiB (Novagen) as a host strain. The Tbx6 fragments were extracted
from bacterial culture using the MagneHis system (Promega), purified by
thrombin digestion to remove the His-tag, followed by affinity column
purification (Novagen) and dialysis using a semipermeable membrane cassette
(Pierce). Rabbits (two animals for each antigen) were immunized with the
purified Tbx6 fragments and processed for antibody purification following the
standard procedures of Hokudo Bio (Abuta, Hokkaido, Japan).
Protein and mRNA expression analyses
Whole-mount RNA in situ hybridization was performed as described
(Saga et al., 1997).
Whole-mount immunohistochemistry and simultaneous staining of Mesp2
mRNA and Tbx6 protein were as previously described
(Morimoto et al., 2005;
Oginuma et al., 2008).
Chromatin immunoprecipitation (ChIP) assay
Embryonic tails were dissected along the anteroposterior axis into three
parts using a tungsten needle. Somite part (s) corresponds to SIV to SII,
anterior PSM (ap) is from SI to S-1, and posterior PSM (pp) corresponds to the
region posterior to S-2. A total of 120 embryos were dissected, the samples
treated with trypsin and dispersed cells counted (around
1x106 cells for each sample). Cells were fixed in 1%
formaldehyde in PBS for 10 minutes at 37°C. The preparation of cell
lysates and ChIP assay were performed using the Chromatin Immunoprecipitation
Assay Kit (Upstate biology) according to the manufacturer's protocol. PCR
primers used for ChIP assays were: LP286,
5'-AGACATCCAGGTACCTCGAGGTC-3'; LP287,
5'-CGGGATAGACATCCAGGTACCCA-3'; and RP287,
5'-GGCTGGTGTGACTCTGGGAAGCT-3'. LP286 and RP287 were used for
detection of mutated P2PSME, whereas LP287 and RP287 were used for detection
of wild-type P2PSME. As a positive control, the Dll1 mesoderm (msd)
enhancer was amplified using the following primers: LP259,
5'-CCCAACACAGATGATTCTGCCCAGTAACT-3'; and RP255,
5'-GCTTTGTGTTGAGCATGCCATGAGCTGTA-3'. A sequence 22 kb from P2PSME
was amplified by PCR as a negative control, using the following primers:
LP285, 5'-GGTCTGTTTGCAGCTGATTCTGAA-3'; and RP286,
5'-CAGTTCTCACCTTGCTTCCATGT-3'.
Electromobility shift assay (EMSA)
The full-length Tbx6 ORF was obtained from the pACT-Tbx6
construct, which was previously isolated from a yeast one-hybrid screen
(Yasuhiko et al., 2006). After
ligation to a 3xFLAG tag (Sigma), the tagged Tbx6 insert was
cloned into pCS2+ (Rupp et al.,
1994). In vitro transcription/translation was then performed using
the TNT In Vitro Translation Kit (Promega) according to the manufacturer's
protocol. Oligonucleotide probes were labeled with DIG-11-ddUTP using
recombinant TdT (Roche Diagnostics). Five microliters of crude in vitro
translated product was subjected to EMSA. As a negative control, reticulocyte
lysate without Tbx6 template was used. The oligonucleotide probes are
as follows (mutated nucleotides are indicated in lower case): SiteF,
5'-GCTAAATTACGGGTATATGGACCACACCTGTATCAGTCCC-3'; SiteG,
5'-CTGGGCTCTGTGGGTTTTGACACCTCTCTGCAACCTGGCA-3'; SiteGmut,
5'-CTGGGCTCTGTGGGTTTTGAattCTCTCTGCAACCTGGCA-3'; SiteB,
5'-CCTTCGAGGGGTCAGAATCCACACCTCTGCAAATGGGCCCGCTTT-3'; T1,
5'-CAAGTGCTGGTCTTGGCATCACACCTCTTTATTTGTTTCCATAC-3'; T2,
5'-GCAGAATCTGCAGAGGTGTCACTTCACACCTCTGTGGCCTGGCT-3'; and T3,
5'-GCTCTCACAGCTGAGGTGTGAAGCGACACCTCCAGGCTCATAAG-3'.
EMSA was performed as described
(Yasuhiko et al., 2006).
Anti-Tbx6 antibody (3.5 µg) was added to the reaction to assess the
specificity of the protein-DNA interaction. As a competitor, a 100-fold excess
of unlabeled oligonucleotide corresponding to the probe was added to the
reaction.
Transgenic assay
DNA fragments with and without mutations in conserved upstream sites were
generated from an Mesp2 genomic fragment using a standard PCR-based
protocol. Each transgene comprised the lacZ reporter and a 6-kb
genomic fragment upstream of the Mesp2 first ATG, including P2PSME
with and without mutated Tbx6 binding sites. The transgenes were injected into
the male pronucleus of a fertilized egg as described
(Hogan et al., 1994). Embryos
recovered at E9.5-10.5 were analyzed for lacZ expression by X-Gal
staining (Saga et al., 1992)
and were subsequently examined for the presence of the transgene by PCR
(Sasaki and Hogan, 1996).
Luciferase assay
The KpnI-NcoI fragments (356 bp) corresponding to P2PSME,
with and without mutations in the Tbx6 binding sites, were subcloned into the
pGL3-Basic (Promega) vector to generate luciferase reporter constructs. The
expression vectors for the proteins to be assessed were constructed in the
same way as those used in the EMSA assays described above. The luciferase
assay using COS-7 cells was conducted as described previously
(Yasuhiko et al., 2006). Each
assay was performed in triplicate and repeated at least twice.
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). To establish the function of these Tbx6 binding sites in
vivo, we introduced nucleotide substitutions into the mouse genome using a
gene-targeting technique. These mutations disrupted two Tbx6 binding sites,
denoted Site B and Site D, that were shown to be sufficient to activate
Mesp2 expression in vitro
(Yasuhiko et al., 2006)
(Fig. 1A). After the
establishment of a neoP2EmB1D mouse line, the neoR cassette was
removed (
neo) by a cross with the deleter mouse line CAG-Cre.
Interbreeding of the neo mutants gave rise to homozygotes
(P2EmB1D/P2EmB1D) that retained a loxP site after neoR removal
(Fig. 1B). This residual loxP
site appears to have no effect on Mesp2 expression or somitogenesis
because another knock-in mouse, P2EmCont, in which wild-type P2PSME
is knocked-in using the same strategy, had viable homozygous offspring without
any morphological defects (data not shown).
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neo-type
Mesp2-null mouse (Fig.
1C, P2MCM/P2MCM). As expected from the phenotype,
Mesp2 expression in P2EmB1D/P2EmB1D embryos was eliminated
(Fig. 1D). Segmental borders
were generated during an early stage of somitogenesis
(Fig. 1D, right-hand panels).
However, the borders were unlikely to be maintained because the vertebral
bodies were fused along the anteroposterior axis at later developmental stages
(Fig. 1C).
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;
Takahashi et al., 2007).
Mesp1 expression was upregulated in the P2EmB1D/P2EmB1D
embryo, but its expression domain was broader than normal
(Fig. 1D). Epha4,
which is required for proper border formation, was expressed normally.
However, Tbx18, which is implicated in the maintenance of segmental
border and somite patterning (Bussen et
al., 2004), was not expressed
(Fig. 1D). These gene
expression patterns were similar to those reported for the neo-type
Mesp2-null mouse (Morimoto et
al., 2006; Takahashi et al.,
2007). These results confirmed that the Tbx6 binding sites are
bona fide enhancer elements required for Mesp2 expression.
Tbx6 binds the Mesp2 PSM enhancer in vivo
The Tbx6 protein is normally broadly distributed in the PSM and tailbud
(White and Chapman, 2005),
whereas Mesp2 is expressed only in the anterior PSM
(Saga et al., 1997). This
discrepancy between the Tbx6 and Mesp2 expression patterns prompted
us to investigate whether Tbx6 actually binds to the Mesp2 enhancer
in vivo. We raised an anti-Tbx6 antibody using two different antigens: an
N-terminal portion of Tbx6 and an internal portion. The internal antigen
yielded an antibody with good specificity and sensitivity. Embryo whole-mount
immunohistochemistry confirmed the previously reported distinct Tbx6 staining
pattern in the PSM and tailbud (Fig.
2A) (White and Chapman,
2005). Western blot analyses further revealed that this antibody
identifies a single band of approximately 58 kDa in cell lysates prepared from
the posterior region (PSM and tailbud), but not from the anterior region
(formed somite), of E11.5 tails (Fig.
2B). We also performed double staining of Mesp2 mRNA and
Tbx6 protein and confirmed colocalization only in the anterior-most region of
the PSM (Fig. 2C).
For ChIP assays, we dissected E11.5 embryo tails into three regions: the
tailbud and posterior PSM (pp), the anterior PSM and newly formed somites
(ap), and formed somites (s). Protein-DNA complexes were prepared from each
pool and used in ChIP assays, which revealed that Tbx6 binds to the
Mesp2 PSM enhancer in the ap and pp regions, but not in the s region,
which is consistent with the expression pattern of Tbx6
(Fig. 2E). These results
indicate that Tbx6 binds to the P2PSME uniformly in its expression domain,
suggesting that Tbx6 alone cannot activate Mesp2 in the posterior PSM
where it binds. Dll1 is known to be a downstream target of Tbx6 and
putative binding sites have been identified in its mesoderm (msd) enhancer
(White and Chapman, 2005).
ChIP assays using the putative Dll1 msd enhancer
(Fig. 2E, column Dll1) revealed
that Tbx6 also binds to the Dll1 enhancer in both the ap and pp
regions, which is consistent with the expression pattern of Dll1. In
all cases, the negative control (PCR amplification of an unrelated sequence in
the mouse genome) gave no signal in ChIP assays with the anti-Tbx6 antibody
(Fig. 2E,F, column NC). Thus,
these results confirm our previous finding that Tbx6 binding is required for
Mesp2 expression, but is not sufficient for full transcriptional
activation.
Mutated P2PSME contains Tbx6 binding sites that are inactive in vivo
We next applied the ChIP assay system to confirm that the phenotype of our
enhancer-specific knockout mouse was due to the lack of Tbx6 binding in the
Mesp2 enhancer region. We performed ChIP assays using the tails of
P2EmB1D heterozygous embryos and specific primer sets in order to
distinguish the mutated DNA fragment from its wild-type counterpart, expecting
that Tbx6 would not bind to the mutated enhancer. Surprisingly, mutated
P2PSME, which has no PSM-specific transcriptional activity
(Fig. 1), gave rise to a band
that co-precipitated with the anti-Tbx6 antibody. This indicated that Tbx6
still binds to the mutated PSME in vivo
(Fig. 2F). To identify the Tbx6
binding site within the mutated P2PSME, we re-examined this region for a
consensus Tbx6 binding sequence (White and
Chapman, 2005) and found two additional candidate sites, denoted
Site F and Site G, in and upstream of P2PSME
(Fig. 3A). EMSA demonstrated
that Site G was strongly associated with Tbx6 in vitro
(Fig. 3B).
The number and spatial organization of the T-box binding sites are important for initiating Mesp2 transcription via Notch signaling
We reported previously that the simultaneous mutation of two Tbx6 binding
sites, Site B and Site D, eliminates PSM-specific activation of a reporter
gene by P2PSME in transgenic embryos
(Yasuhiko et al., 2006). To
confirm this finding and also investigate the possible involvement of the new
Tbx6 binding site, Site G, in enhancer activity, we generated a series of
reporter constructs with P2PSME harboring serial mutations in the Tbx6 binding
sites. We tested two types of reporter assay: a luciferase assay using
cultured cells, and transgenic analyses. In the luciferase assay, the loss of
any single Tbx6 binding site among Sites B, D and G, caused a 10-fold
reduction in Tbx6-dependent and Tbx6 plus Notch signaling-dependent reporter
activation (Fig. 3C, right).
Conversely, expression of a lacZ reporter in transgenic embryos was
not markedly affected by the loss of any individual Tbx6 binding site
(Fig. 3C, left). These results
suggested that each Tbx6 binding site contributes equally to P2PSME activity,
but that the loss of a single site is not sufficient to disrupt the in vivo
function of P2PSME.
We next examined the effects of systematically removing multiple Tbx6 binding sites. Removal of two Tbx6 binding sites from P2PSME resulted in a further decrease in luciferase reporter activity (Fig. 3C, lane P2EmDG, and Fig. 3D). lacZ expression in transgenic embryos was also diminished, both in intensity and frequency. Out of nine transgene-positive embryos, only one showed weak lacZ expression with the P2EmB1 reporter, which has two intact Tbx6 binding sites (Fig. 3D, left). When three out of four Tbx6 binding sites were eliminated, the synergistic effects of Tbx6 and Notch signaling on P2PSME activation were no longer observed and mutants resembled P2EmB1DG, which has lost Tbx6 binding capability at all four sites (Fig. 3D, right). In transgenic embryos, lacZ expression was not activated by any single Tbx6 binding site (Fig. 3D, left, P2EmB1D). These results strongly suggest that the PSM-specific expression of Mesp2 requires at least two Tbx6 binding sites in P2PSME. Notably, the P2PSME reporters with two intact Tbx6 binding sites (P2EmDG, P2EmB1, P2EmB2D, P2EmB2G) showed variable levels of activity in the luciferase assay. This finding contrasts with the uniform reporter activity found with either one or three mutated Tbx6 binding sites (Fig. 3C,D). P2EmDG, with two Tbx6 binding sites in Site B intact, displayed a more than 2-fold stronger activity than P2EmB2G, which harbors single Tbx6 binding sites within Site B and Site D. P2EmB2G activated the luciferase reporter at levels comparable to those of reporters with a single Tbx6 binding site and showed no synergistic activation when Notch signaling was applied (Fig. 3D). Taken together, these data indicate that the four Tbx6 binding sites have equal importance in regulating P2PSME activity, and at least two neighboring sites are required for the Notch signaling-dependent induction of Mesp2 expression.
The medaka mespb PSM enhancer regulates Mesp2 expression and normal somite formation in the mouse embryo
mespb, the zebrafish homolog of Mesp2, shows a similar
expression pattern to mouse Mesp2 during embryogenesis and we
speculated that it might exert a similar function in the mouse
(Nomura-Kitabayashi et al.,
2002). We have previously identified the PSM-specific enhancer of
medaka mespb, which contains T-box binding sites. Two of these sites,
T1 and T2, are important for PSM-specific mespb expression
(Terasaki et al., 2006)
(Fig. 4A). These data suggest
that the T-box-protein-dependent expression mechanism is evolutionally
conserved between mammals and teleosts (zebrafish, medaka). We demonstrated
that zebrafish Tbx24, a T-box protein that is homologous to mouse Tbx6 and is
responsible for the fused somite (fss) mutant phenotype,
binds to the medaka mespb PSME
(Fig. 4B). A sequence
comparison revealed three putative T-box binding sites in the medaka
mespb PSME (Fig. 4A).
Two of these had the ability to bind two Tbx24 molecules each, whereas in the
mouse P2PSME, only Site B can bind two Tbx6 molecules
(Fig. 4B).
To more directly demonstrate the evolutionary conservation of this regulatory mechanism, we generated a knock-in mouse with a medaka mespb upstream sequence inserted in place of the endogenous Mesp2 PSME. For this purpose, we substituted the 356-bp sequence upstream of the Mesp2 first ATG with 2.8 kb of sequence upstream of the mespb first ATG, generating a medakaP2 mouse (Fig. 4C). Heterozygous mice (medakaP2/+) were viable and appeared normal (data not shown). Homozygous mice (medakaP2/medakaP2) were also viable and showed no physical malformations (Fig. 4D). In skeletal preparations, we observed that medakaP2 homozygous fetuses were indistinguishable from heterozygous or wild-type littermates (Fig. 4E), indicating that the PSMEs of medaka mespb and mouse Mesp2 are functionally equivalent, despite some differences in their structural features.
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). Taken
together, these data suggest that it is highly likely that the restricted
expression pattern of Mesp2 in the anterior PSM is regulated by a
combination of Tbx6 and Notch signaling in vivo. Similarly, Tbx6 activates
Dll1 together with Wnt signaling
(Hofmann et al., 2004) and
activates Ripply in cooperation with Mesp2
(Hitachi et al., 2008).
Although the molecular mechanisms by which Tbx6 regulates its target genes
together with various partners remain elusive, it is possible that the number
and spatial organization of Tbx6 binding sites facilitate the response of
P2PSME to Notch signaling.
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The loss of two or more of the four Tbx6 binding sites greatly diminishes
P2PSME activity in both luciferase and transgenic assays
(Fig. 3D). Interestingly, the
reporters with two intact Tbx6 binding sites showed varied levels of activity
depending upon the position of the intact sites. Two intact Tbx6 binding sites
in Site B resulted in the highest reporter activity
(Fig. 3D). These data indicate
that Site B may be of predominant importance in the function of P2PSME,
implying that its two neighboring Tbx6 binding sites play a central role in
regulating the activation of Mesp2. The binding of Tbx6 to one of the
two binding sites in Site B depends on the presence of another Tbx6 molecule
binding to this site (Yasuhiko et al.,
2006). This property might be related to the unique
palindrome-like sequence of this site. Although several T-box binding sites
have been identified in the upstream region of other Tbx6-downstream genes,
such as Dll1 (Hofmann et al.,
2004) and Msgn1
(Wittler et al., 2007), the
palindrome-like site has thus far been found only in the PSME of
Mesp2 and its medaka ortholog mespb
(Fig. 4A). It is therefore
possible that two neighboring Tbx6 molecules on the palindrome-like site are
specifically recognized by as yet unidentified factor(s)
(Fig. 5, `X') that together
with Tbx6 constitutes an RBPJ-
(Rbpj)-independent Notch signaling
machinery [disruption of potential RBPJ- binding sites does not affect
P2PSME activity in transgenic embryos
(Yasuhiko et al., 2006)].
Further analyses of Mesp2 PSME might shed light on these novel
regulatory mechanisms that operate during development.
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An evolutionally conserved mechanism regulating Mesp expression through multiple T-box binding sites
We previously found that the deletion of two T-box binding sites in the
mespb PSME greatly reduced its PSM-specific enhancer activity in
transgenic medaka embryos (Terasaki et
al., 2006), similar to our findings in transgenic mouse embryos.
The medaka mespb PSME harbors three T-box binding sites (T1-T3),
which is similar to the complement of the mouse Mesp2 PSME
(Fig. 4A). However, the total
length of the PSME is very different between mouse Mesp2 and medaka
mespb (356 bp versus 2.8 kb, respectively)
(Terasaki et al., 2006). The
number of T-box proteins that bind to the medaka and mouse PSMEs is also
different (Fig. 4A,B), and the
distance between each element is greater in the mespb PSME than in
its mouse counterpart.
We have demonstrated, however, that the medaka mespb PSME is
functionally equivalent to the mouse Mesp2 PSME. In our transgenic
assay, a mutation in the double T-box binding site (Site B in mouse and Site
T2 in medaka) had the most profound effect upon PSME activity. Consistent with
these results, deletion of medaka Site T1 (harboring a single T-box binding
sequence) did not affect reporter gene expression. However, deletion of one of
the sites within the double T-box binding sequence (T2) caused a 50% decrease
in reporter expression (Terasaki et al.,
2006), again demonstrating the importance of the binding to the
double T-box site for PSM enhancer function.
In the teleost fish, zebrafish, the T-box transcription factor Tbx24 was
identified as responsible for the fused somite (fss) mutant
phenotype. Tbx24 has a T-box domain that is homologous to that of mouse Tbx6
(Nikaido et al., 2002). The
segmentation of somites and expression of mespb are eliminated in the
fss mutant (Sawada et al.,
2000), implying that mespb is a downstream target of
Tbx24, similar to the relationship between Mesp2 and Tbx6 in mice.
However, fss mutant fish are viable and fertile
(van Eeden et al., 1996),
whereas Tbx6-null mouse embryos fail to form a mesoderm and die early
in development (Chapman and Papaioannou,
1998). This difference might be due to the presence in zebrafish
of a Tbx6 counterpart gene, spadetail, which supports
paraxial mesoderm formation. Despite this difference, our data clearly
demonstrate that the mechanism regulating the PSM-specific expression of
Mesp2 and mespb is evolutionarily well conserved between
fish and mice.
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Bussen, M., Petry, M., Schuster-Gossler, K., Leitges, M.,
Gossler, A. and Kispert, A. (2004). The T-box transcription
factor Tbx18 maintains the separation of anterior and posterior somite
compartments. Genes Dev.
18,1209
-1221.
Chapman, D. L. and Papaioannou, V. E. (1998).
Three neural tubes in mouse embryos with mutations in the T-box gene Tbx6.
Nature 391,695
-697.[CrossRef][Medline]
Chapman, D. L., Agulnik, I., Hancock, S., Silver, L. M. and
Papaioannou, V. E. (1996). Tbx6, a mouse T-Box gene
implicated in paraxial mesoderm formation at gastrulation. Dev.
Biol. 180,534
-542.[CrossRef][Medline]
Delfini, M. C., Dubrulle, J., Malapert, P., Chal, J. and
Pourquie, O. (2005). Control of the segmentation process by
graded MAPK/ERK activation in the chick embryo. Proc. Natl. Acad.
Sci. USA 102,11343
-11348.
Dunty, W. C., Jr, Biris, K. K., Chalamalasetty, R. B., Taketo,
M. M., Lewandoski, M. and Yamaguchi, T. P. (2008).
Wnt3a/β-catenin signaling controls posterior body development by
coordinating mesoderm formation and segmentation.
Development 135,85
-94.
Galceran, J., Sustmann, C., Hsu, S. C., Folberth, S. and
Grosschedl, R. (2004). LEF1-mediated regulation of
Delta-like1 links Wnt and Notch signaling in somitogenesis. Genes
Dev. 18,2718
-2723.
Haraguchi, S., Kitajima, S., Takagi, A., Takeda, H., Inoue, T.
and Saga, Y. (2001). Transcriptional regulation of Mesp1 and
Mesp2 genes: differential usage of enhancers during development.
Mech. Dev. 108,59
-69.[CrossRef][Medline]
Hitachi, K., Kondow, A., Danno, H., Inui, M., Uchiyama, H. and
Asashima, M. (2008). Tbx6, Thylacine1, and E47
synergistically activate bowline expression in Xenopus somitogenesis.
Dev. Biol. 313,816
-828.[CrossRef][Medline]
Hofmann, M., Schuster-Gossler, K., Watabe-Rudolph, M., Aulehla,
A., Herrmann, B. G. and Gossler, A. (2004). WNT signaling, in
synergy with T/TBX6, controls Notch signaling by regulating Dll1 expression in
the presomitic mesoderm of mouse embryos. Genes Dev.
18,2712
-2717.
Hogan, B., Beddington, R., Costantini, F. and Lacy, E.
(1994). Manipulating the Mouse Embryo: a Laboratory
Manual. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory
Press.
Moreno, T. A. and Kintner, C. (2004).
Regulation of segmental patterning by retinoic acid signaling during Xenopus
somitogenesis. Dev. Cell
6, 205-218.[CrossRef][Medline]
Morimoto, M., Takahashi, Y., Endo, M. and Saga, Y.
(2005). The Mesp2 transcription factor establishes segmental
borders by suppressing Notch activity. Nature
435,354
-359.[CrossRef][Medline]
Morimoto, M., Kiso, M., Sasaki, N. and Saga, Y.
(2006). Cooperative Mesp activity is required for normal
somitogenesis along the anterior-posterior axis. Dev.
Biol. 300,687
-698.[CrossRef][Medline]
Nikaido, M., Kawakami, A., Sawada, A., Furutani-Seiki, M.,
Takeda, H. and Araki, K. (2002). Tbx24, encoding a T-box
protein, is mutated in the zebrafish somite-segmentation mutant fused somites.
Nat. Genet. 31,195
-199.[CrossRef][Medline]
Nomura-Kitabayashi, A., Takahashi, Y., Kitajima, S., Inoue, T.,
Takeda, H. and Saga, Y. (2002). Hypomorphic Mesp allele
distinguishes establishment of rostrocaudal polarity and segment border
formation in somitogenesis. Development
129,2473
-2481.[Medline]
Oginuma, M., Niwa, Y., Chapman, D. L. and Saga, Y.
(2008). Mesp2 and Tbx6 cooperatively create periodic patterns
coupled with the clock machinery during mouse somitogenesis.
Development 35,2555
-2562.
Rupp, R. A., Snider, L. and Weintraub, H.
(1994). Xenopus embryos regulate the nuclear localization of
XMyoD. Genes Dev. 8,1311
-1323.
Saga, Y. and Takeda, H. (2001). The making of
the somite: molecular events in vertebrate segmentation. Nat. Rev.
Genet. 2,835
-845.[CrossRef][Medline]
Saga, Y., Yagi, T., Ikawa, Y., Sakakura, T. and Aizawa, S.
(1992). Mice develop normally without tenascin. Genes
Dev. 6,1821
-1831.
Saga, Y., Hata, N., Koseki, H. and Taketo, M. M.
(1997). Mesp2: a novel mouse gene expressed in the presegmented
mesoderm and essential for segmentation initiation. Genes
Dev. 11,1827
-1839.
Sasaki, H. and Hogan, B. L. (1996). Enhancer
analysis of the mouse HNF-3 beta gene: regulatory elements for node/notochord
and floor plate are independent and consist of multiple sub-elements.
Genes Cells 1,59
-72.[Abstract]
Sawada, A., Fritz, A., Jiang, Y. J., Yamamoto, A., Yamasu, K.,
Kuroiwa, A., Saga, Y. and Takeda, H. (2000). Zebrafish Mesp
family genes, mesp-a and mesp-b are segmentally expressed in the presomitic
mesoderm, and Mesp-b confers the anterior identity to the developing somites.
Development 127,1691
-1702.[Abstract]
Takahashi, Y., Koizumi, K., Takagi, A., Kitajima, S., Inoue, T.,
Koseki, H. and Saga, Y. (2000). Mesp2 initiates somite
segmentation through the Notch signalling pathway. Nat.
Genet. 25,390
-396.[CrossRef][Medline]
Takahashi, Y., Inoue, T., Gossler, A. and Saga, Y.
(2003). Feedback loops comprising Dll1, Dll3 and Mesp2, and
differential involvement of Psen1 are essential for rostrocaudal patterning of
somites. Development
130,4259
-4268.
Takahashi, Y., Yasuhiko, Y., Kitajima, S., Kanno, J. and Saga,
Y. (2007). Appropriate suppression of Notch signaling by Mesp
factors is essential for stripe pattern formation leading to segment boundary
formation. Dev. Biol.
304.593
-603.[CrossRef][Medline]
Terasaki, H., Murakami, R., Yasuhiko, Y., Shin, I. T., Kohara,
Y., Saga, Y. and Takeda, H. (2006). Transgenic analysis of
the medaka mesp-b enhancer in somitogenesis. Dev. Growth
Differ. 48,153
-168.[CrossRef][Medline]
van Eeden, F. J., Granato, M., Schach, U., Brand, M.,
Furutani-Seiki, M., Haffter, P., Hammerschmidt, M., Heisenberg, C. P., Jiang,
Y. J., Kane, D. A. et al. (1996). Mutations affecting somite
formation and patterning in the zebrafish, Danio rerio.
Development 123,153
-164.[Abstract]
White, P. H. and Chapman, D. L. (2005). Dll1 is
a downstream target of Tbx6 in the paraxial mesoderm.
Genesis 42,193
-202.[CrossRef][Medline]
Wittler, L., Shin, E. H., Grote, P., Kispert, A., Beckers, A.,
Gossler, A., Werber, M. and Herrmann, B. G. (2007).
Expression of Msgn1 in the presomitic mesoderm is controlled by synergism of
WNT signalling and Tbx6. EMBO Rep.
8, 784-789.[CrossRef][Medline]
Yagi, T., Tokunaga, T., Furuta, Y., Nada, S., Yoshida, M.,
Tsukada, T., Saga, Y., Takeda, N., Ikawa, Y. and Aizawa, S.
(1993). A novel ES cell line, TT2, with high
germline-differentiating potency. Anal. Biochem.
214, 70-76.[CrossRef][Medline]
Yasuhiko, Y., Haraguchi, S., Kitajima, S., Takahashi, Y., Kanno,
J. and Saga, Y. (2006). Tbx6-mediated Notch signaling
controls somite-specific Mesp2 expression. Proc. Natl. Acad. Sci.
USA 103,3651
-3656.
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