Vol 435|19 May 2005|doi:10.1038/nature03591

LETTERSThe Mesp2 transcription factor establishes
segmental borders by suppressing Notch activity
Mitsuru Morimoto1, Yu Takahashi3, Maho Endo1 & Yumiko Saga1,2
The serially segmented (metameric) structures of vertebrates are
based on somites that are periodically formed during embryogenesis. A ‘clock and wavefront’ model has been proposed to
explain the underlying mechanism of somite formation1, in
which the periodicity is generated by oscillation of Notch components (the clock) in the posterior pre-somitic mesoderm(PSM)2–6. This temporal periodicity is then translated into thesegmental units in the ‘wavefront’7,8. The wavefront is thought toexist in the anterior PSM and progress backwards at a constantrate; however, there has been no direct evidence as to whether thelevels of Notch activity really oscillate and how such oscillation istranslated into a segmental pattern in the anterior PSM. Here, wehave visualized endogenous levels of Notch1 activity in mice,
showing that it oscillates in the posterior PSM but is arrested in
the anterior PSM. Somite boundaries formed at the interface
between Notch1-activated and -repressed domains. Genetic andbiochemical studies indicate that this interface is generated bysuppression of Notch activity by mesoderm posterior 2 (Mesp2)
through induction of the lunatic fringe gene (Lfng). We proposethat the oscillation of Notch activity is arrested and translated in
the wavefront by Mesp2.
Mesp2—a basic helix–loop–helix-type transcription factor—andits related proteins have an essential role in both segmentation androstro-caudal patterning of somites in the anterior PSM9–12. To
understand further the involvement of Mesp2 in boundary formation, we generated a Mesp2–venus knock-in mouse (Supplementary Fig. 1) in which Mesp2 localization is detected in live embryos
using confocal microscopy (Fig. 1a–g). As expected, the Mesp2–
venus fusion protein was detected in the nuclei of cells (Fig. 1d).
Furthermore, in embryos at 8.75 days post coitus (d.p.c.), two to
three segmental stripes are detectable, showing an anterior toposterior gradient for each segment (Fig. 1a–d). The signal in themost anterior segment is very weak but a second stripe has a clear
anterior boundary that perfectly matches the segmental border;
indeed, it lines the entire border. The strongest third stripe also hasa clear anterior border in the PSM in which no sign of a morphological boundary can be detected. This band may therefore demarcate
the next segmental border. In later-stage embryos, one clear stripe
was detected, although we occasionally observed an additional signal
in the newly formed somite (Fig. 1e– g).
We have previously shown that the expression domain of Mesp2
transcript changes during somitogenesis13, appearing at S21 or S22
as a single band of one somite in length, and then graduallyshrinking, leaving the rostral half of its expression domain intact.
To elucidate further the dynamic behaviour of Mesp2, we haverecorded its expression pattern using time-lapse microscopy. A
new Mesp2 expression domain is evident as a single band of about
one somite in length, whereas the expression domain of the preexisting band undergoes changes such that the rostral part of theband is maintained longer before finally disappearing (Supplementary Movie 1 and Supplementary Fig. 2). This dynamic change isconsistent with the expression profile of Mesp2 messenger RNA,
previously obtained using explant cultures13. However, because
Mesp2–venus is a fusion protein, it may not faithfully reproduce
the pattern of endogenous protein distribution. This prompted us
to generate antibodies against the Mesp2 protein. The antibodystaining pattern reveals that the distribution of endogenous Mesp2 issimilar to that of Mesp2–venus (Fig. 1h–j). The neighbouring section
was subjected to in situ hybridization analysis. The mRNA localization pattern was found to be consistent with the immunohistochemical results, indicating that message is translated immediately and thatthe protein is rapidly degraded but the transcript disappears earlier,as expected (Fig. 1j). We occasionally observed two bands of Mesp2
protein: an anterior band located in the segmented border and an
posterior one in the next presumptive border. In this case, transcript
was only observed in the posterior region (Fig. 1h). Taken together,
we conclude that the anterior border of the expression domain of the
Mesp2 protein coincides with the next segmental border in the PSM.
The expression of Notch components, such as Hes7 (hairy andenhancer of split 7) and Lfng, oscillates in the PSM3,4,6, whereas
Notch1 is expressed throughout the entire PSM and at higher levels in
the anterior PSM9. We attempted to visualize the activation of Notch
signalling by detecting a processed NICD (Notch intracellulardomain) using a specific antibody. Unlike its RNA expression
pattern, Notch1 activity exhibits a dynamic pattern in the PSM
during somitogenesis. We analysed 52 embryos, which exhibited four
distinct patterns; the positive signals were detected in the tailbud(Fig. 1k), the middle of the PSM (Fig. 1l, m) or more anteriorly
within the PSM (Fig. 1n). Notably, in most of the embryos, Notch
activation is in a similar phase to the transcript of Lfng, which
encodes a glycosyltransferase that can modify Notch activity14,15(Fig. 1k–n). The expression domain of Lfng appears slightly later
than Notch activation, supporting the idea that Lfng transcription is
activated by Notch signalling, as previously indicated16. These results
indicate that the activity of Notch oscillates in the posterior PSM. In aprevious report, Hes7 was shown to function as a suppressor of Hes7
and Lfng transcription17, forming a feedback loop in the coreoscillator. We thus tested the pattern of Notch activation in mice
defective in Notch pathway components (Fig. 2a–e). In Dll1-nullembryos18, no Notch1 activity is found anywhere in the PSM(Fig. 2c), whereas in Lfng-null embryos19, Notch1 activity is detected
throughout the entire PSM but does not seem to oscillate (Fig. 2d).
These observations indicate that the Dll1 ligand is required for
Notch1 activation, and that Lfng functions as a suppressor of
Notch activity, thus generating the oscillatory activation of Notch1.
In the anterior PSM, Notch oscillatory activity is arrested andlocalizes in the caudal portion of the S0 somite, whereas Lfng
transcripts are detectable in the rostral part of S 2 1 and their

1
Division of Mammalian Development, National Institute of Genetics, and 2Sokendai, Yata 1111, Mishima 411-8540, Japan. 3Cellular & Molecular Toxicology Division, National
Institute of Health Sciences, 1-18-1 Kamiyoga, Setagaya-ku, Tokyo 158-8501, Japan.

354© 2005 Nature Publishing GroupLETTERS

Vol 435|19 May 2005|NATURE

expression seldom overlaps with the Notch1 activation domain(Fig. 1k–n). Of note, Notch activation and Lfng expression domains
almost co-occur in the posterior PSM, but are segregated into therostral and caudal parts in the anterior PSM. This suggests that Lfng
expression is activated by an additional factor in the anterior PSM.
Mesp2 may well be this factor, as it is co-expressed with Lfng in thisregion (refs 20, 21; see also Supplementary Fig. 3). We propose that in
the anterior PSM, Mesp2 may downregulate Notch activity throughthe activation of Lfng, which is a Notch modulator. To address thispossibility, we first examined the relationship between Mesp2 andNotch activity in the anterior PSM. Double staining with anti-activeNICD and anti-Mesp2 antibodies enabled us to examine the spatial
relationship between the localization of these two factors in a single
section (Fig. 2f–u). At the initial phase of Mesp2 expression the two

expression domains partially overlap in such a way that cells in theposterior Notch activation domain co-express Mesp2 (Fig. 2f–i). The
overlapping domain gradually reduces as expression levels of Mesp2
increase (Fig. 2j–m). Ultimately, the two expression domains arecompletely separated from each other and form a clear boundary(Fig. 2n–q). Notably, the strongest detectable signals of both Notch
activity and Mesp2 are found in close proximity to the boundary on
both sides. This boundary will form the next segmental border, as
indicated by the expression of Mesp2–venus (Fig. 1a–g). Once a newborder is generated, next Notch activity appears in the anterior PSM
and Mesp2 protein appears just in the middle of the Notch activation
domain, to generate a next segmental border (Fig. 2r–u).
We next examined whether downregulation of Notch activity by
Mesp2 is mediated by Lfng. In Lfng-null embryos19, Mesp2 protein is

Figure 1 | Visualization of the Mesp2 protein and oscillation of Notch1
activity and Lfng transcripts. a–g, Expression of the Mesp2–venus fusion
protein is shown in 8.75 d.p.c. (a–d) and 10.5 d.p.c. (e–g) embryos asconfocal images (a, e; green), bright-field images (b, f) and merged images(c, g). The boxed area in c is shown at higher magnification in d. Stronger
Mesp2–venus signals are localized at the anterior border of the predictednext somite (next borders are indicated by arrowheads). h–j, Mesp2
transcript and protein (using anti-Mesp2 antibody) expression wascompared using three sets of serial sections of 11.5 d.p.c. embryos.
k–n, Notch1 activity periodically oscillating in the PSM may activate Lfng
transcription. Notch1 activity was revealed by immunostaining with
antibodies against active-NICD and the adjacent sections were stained byin situ hybridization with Lfng probe. Solid and dotted lines indicate Sþ1and S0 somite regions, respectively, and coloured bars represent the stained
domain. n ¼ 52 embryos. Each expression pattern is summarized on the topof each panel.

© 2005 Nature Publishing Group355

LETTERS

NATURE|Vol 435|19 May 2005

Figure 2 | Analyses of gene and protein expression critical for clock
oscillation and its arrest. a–e, Serial sections were prepared from 11.5 d.p.c.
wild type (a), Mesp2 L/L (b), Dll1 2/2 (c) or Lfng 2/2 (d, e) embryonic tails.
Each section was stained with anti-Mesp2 or anti-active-NICD antibodies,or with Dll1 or Lfng RNA probe. Coloured bars represent stained domains.
In Mesp2 L/L embryos, Notch1 activity is expanded in the anterior PSM, andexpression of Dll1 is also strongly expanded. In Dll1 2/2 embryos,
expression of Mesp2 is decreased and Notch1 activity is almost completely
lost. In all Lfng 2/2 embryos (n ¼ 15), Notch1 activity is strongly detected356

throughout the PSM but its oscillatory expression is lost. Each expression
pattern is summarized in the right panels. f–u, Mesp2 protein (red) and
Notch1 activity (green) were compared by double immunostaining. A
merged set of four images representing different patterns is shown (f, j, n, r).
The border regions (boxed areas) are magnified and the signals are showneither separately (g, h, k, l, o, p, s, t) or merged (i, m, q, u). Dotted lines
indicate the boundaries, and the expression patterns are summarized in theright panels. Asterisk indicates nonspecific immunofluorescence of blood
cells.

© 2005 Nature Publishing GroupLETTERS

Vol 435|19 May 2005|NATURE

expressed in the appropriate location, although the expression isbroad and does not show a normal anterior–posterior gradient(Fig. 2d). Hence, Lfng is required for normal Mesp2 protein
localization. Importantly, the anterior limit of the Notch1 activation
domain, which encompasses the PSM and does not have oscillatory
Notch1 activity, overlaps with the Mesp2 expression domain, indicating that Mesp2 fails to suppress Notch activity and thus theNotch activation domain does not segregate in Lfng-null embryos(Fig. 2d, e).
To study further the function of Mesp2 in the control of both
Notch activity and Lfng expression, a number of neighbouringsections of Mesp2-null embryos were subjected to NICD and Lfng
staining. We observed that Notch1 activity and Lfng expression
oscillate normally in the posterior PSM in Mesp2-null embryos;
however, their expression is abnormal and expands anteriorly inthe anterior PSM, and they do not make narrow stripes (Fig. 3a).
This was more clearly shown in whole-mount staining of Lfng
expression in 9.5 d.p.c. embryos (Supplementary Fig. 4). The
expanded signals could be sustained by ectopic expression of Dll1,
which extends anteriorly in Mesp2-null embryos (Fig. 2b and ref. 9).

More importantly, the two domains of Notch1 activation andLfng expression are never segregated and stay in unison (compare
Figs 1k–m and 3a). We interpret this to mean that in wild-type
embryos, Mesp2 disrupts the relationship between Notch activity
and Lfng by stably activating Lfng, a Notch repressor, in the anterior
PSM. In order to determine whether Mesp2 acts on the Lfng enhancer
and regulates its gene expression, we generated a reporter constructthat contained a 2.3-kilobase (kb) upstream enhancer sequence(defined by transgenic analyses reported in refs 20 and 21) linked
to a luciferase gene (Fig. 3b). As shown in Fig. 3c, both Mesp2 andNICD act as activators in this reporter system, and a synergistic effect
was also observed. However, these activities are strongly suppressedby Hes7, which is active in the posterior PSM and is downregulated in
the anterior PSM22. Furthermore, we found that Mesp2 specificallybinds to a portion of the Lfng enhancer that contains a putative
N-box (Fig. 3d). These results strongly suggest that Lfng is activatedby NICD and suppressed by Hes7 in the posterior PSM, whereas it isactivated by Mesp2 and hence its expression is restricted andconfined to the Mesp2 expression domain, thereby inhibiting andarresting the oscillation of Notch activity (Fig. 4 and ref. 16). This

Figure 3 | Mesp2 may activate Lfng to arrest the oscillation of Notch
activity in the anterior PSM. a, Serial sections of mouse tail regions wereprepared from Mesp2 L/L embryos (n ¼ 11) and stained with either antiactive-NICD antibody (top) or Lfng probe (bottom). Notch1 activity and
Lfng signal oscillate in the posterior PSM but do not arrest in the anterior
PSM. b, A schematic representation of a luciferase reporter construct withan Lfng 5 0 upstream region (top). DNA fragments A-E, B-N and B-E are used
for gel mobility shift assays. c, Luciferase reporter assay showing that NICD
and Mesp2 are strong activators of the Lfng enhancer, whereas Hes7 is astrong repressor of the Lfng enhancer. d, Mesp2 specifically binds only to the
N-box-containing B-N fragment.

© 2005 Nature Publishing Group357

LETTERS

NATURE|Vol 435|19 May 2005

result is consistent with previous studies indicating that the regulatory region of Lfng is separable into a region A that regulates the cyclic
expression in the posterior PSM, and a region B that regulates
expression in the anterior PSM20,21.
The PSM can be divided into two regions based on the finding thatthe clock oscillates in the posterior PSM whereas it is slowed down
and arrested in the anterior PSM. A switch between these two states isthought to be triggered by FGF signalling23,24, and Mesp2 is crucial inthis process. In the absence of Mesp2, the Dll1-dependent relationship between Notch activity and Lfng is maintained; that is, thenormal wavefront is not generated, even if the clock system functions
normally. We have previously shown that a major role of Mesp2 is togenerate rostro-caudal polarity within the somite through suppression of the Dll1-dependent Notch pathway in the presumptive rostral
domain13. We now propose that Mesp2 has an additional role insegment border formation by restricting Lfng expression in theanterior PSM. In both cases, Mesp2 functions as a strong suppressorof Notch activity.
We have shown that Lfng is a negative regulator of Dll1–Notch
signalling. The results are consistent16 or contrast25,26 with previous
findings. In the Drosophila wing disc, Fringe inhibits Serratedependent but potentiates Delta-dependent Notch signalling,whereas in somite segmentation, Lfng suppresses Dll1-dependent
Notch activation (Fig. 4 and ref. 16). In a mammalian cell
culture system, Dll1–Notch signalling is enhanced by Lfng27. These

Figure 4 | Schematic representation of the regulatory mechanism
underlying the clock system and of the implications of Mesp2 function inestablishing the segmental boundary. In the posterior PSM, theDll1–Notch signal initially activates both Lfng and Hes7. Hes7 is a strongtranscriptional repressor of Lfng and of the Hes7 gene itself, whereas Lfng is anegative modulator of the Notch receptor in this cellular context. The
positive and negative feedback loops thus generates oscillation of Notch1
activity. In the anterior PSM, the Notch1 activity and Lfng waves are nolonger subject to negative regulation by Hes7, as Mesp2 now becomes amajor regulator of Lfng activation and Dll1 suppression. As a result, both
Notch1 activity and Lfng waves are arrested and generate a clear boundary
between the Notch1 activity domain and the Mesp2 expression domain,which produce the next segmental boundary.
358

discrepancies could be due to different cellular contexts and different
biological systems, emphasizing variation of modulating mechanisms for Notch signalling.
We have now provided direct evidence that Notch activity constitutes a core of the segmentation clock and that Mesp2 arrests
the oscillation of Notch activity and initiates a segmentation programme. The next crucial question would be the mechanism bywhich Mesp2 is turned on in the anterior PSM. Our analyses haverevealed a strict spatial relationship between stabilized Notch activation domains and the emergence of Mesp2 expression (Fig. 2f–u;
see also Supplementary Fig. 5). In the absence of Notch signalling,
however, the Mesp2 expression region is approximately normal in theanterior PSM (Fig. 2c), and thus its initial activation depends on
positional information such as the Fgf8 (refs 23, 24) and retinoic acid(RA) gradients28. However, the role of RA remains elusive because no
severe effect on Mesp2 expression is observed in a targeted disruptionof CYP26 (ref. 29), a degrading enzyme of RA (Supplementary Fig. 6).
Detailed enhancer analysis of Mesp2 will provide further insights
into the mechanism of where and how a sharp NICD–Mesp2
boundary forms.
METHODS
Time-lapse recording using confocal microscopy. Heterozygous Mesp2–venus
mouse embryos were collected at 10.5 d.p.c. The tail region was excised andcultured with DMEM/F12 medium (without Phenol red) containing 10% fetal
calf serum on a 35-mm glass-bottomed dish (Matsunami Glass) at 37 8C. For theculturing of embryos, the Heating Insert P and Incubator S systems (Zeiss) wereused to obtain optimal growth conditions. Scanning for the fluorescence protein
venus30 (excitation at 514 nm) and bright imaging of the tail region was
performed with an Axiovert 200M confocal laser scanning microscope (Zeiss).
Three-dimensional (x, y, t) image data sets were recorded at 10-min intervals as
typical time-lapse imaging sessions (n ¼ 30). The colour images were captured
using an Axiocam (Zeiss) and converted to pseudo-colours using Zeiss Axiovision software.
In situ hybridization and immunohistochemistry. The tails were dissected at
10.5–11.5 d.p.c., fixed with 4% PFA and embedded in OCT. For in situ
hybridizations, frozen sections (6–8 mm) were hybridized with digoxigeninlabelled antisense cRNA probes (Roche). Hybridized probes were detected using
AP-conjugated anti-DIG sheep antibody (1:1,000, Roche) and visualized with
NBT/BCIP (1:50, Roche).
For immunohistochemistry analysis, frozen sections (6–8 m m) were
immersed in unmasking solution (Vector Laboratories) and autoclaved at
105 8C for 15 min to enable antigen retrieval. Immunostaining was thenperformed following a standard method with either anti-Mesp2 (1:500) or
anti-NICD (1:500, Cell signaling technology) as primary antibodies, biotinylated
anti-rabbit IgG goat antibody (1:200, Vector Laboratories) as a secondary
antibody and an ABC kit (Vector Laboratories) for signal detection.
For double-immunofluorescent staining, antibody reactions and the detection of Notch1 activity and Mesp2 were separately conducted after antigen
retrieval. Detection of Notch1 activity was performed first with anti-activeNICD (1:200, Cell signaling technology) primary antibody, horseradish peroxidase-conjugated anti-rabbit IgG donkey antibody (1:100, Amersham
Pharmacia Biotech) secondary antibody and fluorescein isothiocyanateconjugated Tyramid (Perkin Elmer) signal detection. Detection of Mesp2 wasthen performed with anti-Mesp2 (1:200) biotinylated anti-rabbit IgG goat
antibody (1:200, Vector Laboratories) and Alexa Flour-594-conjugated Streptavidin (Molecular Probes). The corresponding signals were observed with an
Olympus BX61 fluorescence microscope system using an ORCA-ER digital
camera (HAMAMATSU photo) and analysed with MetaMorph software (Universal Imaging).
Luciferase assay. For luciferase reporter analysis under the control of the Lfng
upstream 2.5-kb (EcoRI–NotI) enhancer (0.2 mg), the reporter construct wastransfected with or without the expression vectors for NICD–venus (50 ng),
3 £ Flag-tagged Hes7 (20 ng) or 3 £ Flag-tagged Mesp2 (50 ng) into NH3T3
cells (0.25 £ 105 cells per well in 24-multiwell plates) using Lipofectamine Plus
(Invitrogen) following the manufacturer’s instructions. The vector containing
the Renilla luciferase gene under the control of the thymidine kinase promoter(10 ng) was co-transfected as an internal standard to normalize for transfection
efficiency. After 36 h, luciferase activities were measured using a Dual Luciferase
Assay Kit (Promega).
Gel-shift assay. A 6 £ His-Mesp2 protein was produced using a baculovirus© 2005 Nature Publishing GroupLETTERS

Vol 435|19 May 2005|NATURE

expression system with Sf-9 cells, and then purified through a TALON metal
affinity resin (Clontech) with 100 mM imidazole as the elution buffer. The
double-stranded DNA oligonucleotide probes were end-labelled with DIG andprotein–DNA complexes were detected using a Roche DIG gel shift kit. Binding
reactions were carried out for 30 min on ice, and protein–DNA complexes wereanalysed on 6% native-polyacrylamide gels. The sense strand sequences of theoligonucleotide probes used were: A-E 5 0 -GGGCACAGCCAGCACCATCTGCTTAGGCATCTGCCTACCAGAGGTGGTGCCTCC-3 0 ; B-N 5 0 -GCCCCCGCT
CGTGCTAGGCGCAGGCAAGGCCCCCGCCCGTGGGAAGGGGGCCG-3 0 ;B-E 5 0 -GGCGCACAGCTGGGTCACATCTGGTAGGAGTGCGGAGGAACCAAATGGAACAG-3 0 .
Received 3 January; accepted 31 March 2005.
1.

2.

3.

4.

5.
6.
7.
8.
9.

10.

11.

12.

13.
14.
15.
16.

Cooke, J. & Zeeman, E. C. A clock and wavefront model for control of thenumber of repeated structures during animal morphogenesis. J. Theor. Biol. 58,
455–-476 (1976).
Palmeirim, I., Henrique, D., Ish-Horowicz, D. & Pourquie, O. Avian hairy gene
expression identifies a molecular clock linked to vertebrate segmentation andsomitogenesis. Cell 91, 639–-648 (1997).
McGrew, M. J., Dale, J. K., Fraboulet, S. & Pourquie, O. The lunatic fringe geneis a target of the molecular clock linked to somite segmentation in avian
embryos. Curr. Biol. 8, 979–-982 (1998).
Forsberg, H., Crozet, F. & Brown, N. A. Waves of mouse Lunatic fringe
expression, in four-hour cycles at two-hour intervals, precede somite boundary
formation. Curr. Biol. 8, 1027–-1030 (1998).
Jiang, Y. J. et al. Notch signalling and the synchronization of the somite
segmentation clock. Nature 408, 475–-479 (2000).
Bessho, Y. et al. Dynamic expression and essential functions of Hes7 in somite
segmentation. Genes Dev. 15, 2642–-2647 (2001).
Saga, Y. & Takeda, H. The making of the somite: molecular events invertebrate segmentation. Nature Rev. Genet. 2, 835–-845 (2001).
Pourquie, O. The segmentation clock: converting embryonic time into spatial
pattern. Science 301, 328–-330 (2003).
Saga, Y., Hata, N., Koseki, H. & Taketo, M. M. Mesp2: a novel mouse gene
expressed in the presegmented mesoderm and essential for segmentation
initiation. Genes Dev. 11, 1827–-1839 (1997).
Buchberger, A., Seidl, K., Klein, C., Eberhardt, H. & Arnold, H. H. cMeso-1, anovel bHLH transcription factor, is involved in somite formation in chicken
embryos. Dev. Biol. 199, 201–-215 (1998).
Sparrow, D. B. et al. Thylacine 1 is expressed segmentally within the paraxial
mesoderm of the Xenopus embryo and interacts with the Notch pathway.
Development 125, 2041–-2051 (1998).
Sawada, A. et al. Zebrafish Mesp family genes, mesp-a and mesp-b aresegmentally expressed in the presomitic mesoderm, and Mesp-b confers theanterior identity to the developing somites. Development 127, 1691–-1702(2000).
Takahashi, Y. et al. Mesp2 initiates somite segmentation through the Notch
signalling pathway. Nature Genet. 25, 390–-396 (2000).
Bruckner, K., Perez, L., Clausen, H. & Cohen, S. Glycosyltransferase activity ofFringe modulates Notch-Delta interactions. Nature 406, 411–-415 (2000).
Moloney, D. J. et al. Fringe is a glycosyltransferase that modifies Notch. Nature
406, 369–-375 (2000).
Dale, J. K. et al. Periodic notch inhibition by lunatic fringe underlies the chicksegmentation clock. Nature 421, 275–-278 (2003).

17. Bessho, Y., Hirata, H., Masamizu, Y. & Kageyama, R. Periodic repression by thebHLH factor Hes7 is an essential mechanism for the somite segmentation
clock. Genes Dev. 17, 1451–-1456 (2003).
18. Hrabe de Angelis, M., McIntyre, J. II & Gossler, A. Maintenance of somite
borders in mice requires the Delta homologue DII1. Nature 386, 717–-721(1997).
19. Evrard, Y. A., Lun, Y., Aulehla, A., Gan, L. & Johnson, R. L. lunatic fringe is an
essential mediator of somite segmentation and patterning. Nature 394,
377–-381 (1998).
20. Cole, S. E., Levorse, J. M., Tilghman, S. M. & Vogt, T. F. Clock regulatory
elements control cyclic expression of Lunatic fringe during somitogenesis. Dev.
Cell 3, 75–-84 (2002).
21. Morales, A. V., Yasuda, Y. & Ish-Horowicz, D. Periodic Lunatic fringe
expression is controlled during segmentation by a cyclic transcriptional
enhancer responsive to notch signaling. Dev. Cell 3, 63–-74 (2002).
22. Bessho, Y. et al. Dynamic expression and essential functions of Hes7 in somite
segmentation. Genes Dev. 15, 2642–-2647 (2001).
23. Sawada, A. et al. Fgf/MAPK signalling is a crucial positional cue in somite
boundary formation. Development 128, 4873–-4880 (2001).
24. Dubrulle, J., McGrew, M. J. & Pourquie, O. FGF signaling controls somite
boundary position and regulates segmentation clock control of spatiotemporalHox gene activation. Cell 106, 219–-232 (2001).
25. Panin, V. M., Papayannopoulos, V., Wilson, R. & Irvine, K. D. Fringe modulates
Notch–-ligand interactions. Nature 387, 908–-912 (1997).
26. Haines, N. & Irvine, K. D. Glycosylation regulates Notch signalling. Nature Rev.
Mol. Cell Biol. 4, 786–-797 (2003).
27. Hicks, C. et al. Fringe differentially modulates Jagged1 and Delta1 signallingthrough Notch1 and Notch2. Nature Cell Biol. 2, 515–-520 (2000).
28. Moreno, T. A. & Kintner, C. Regulation of segmental patterning by retinoic acidsignaling during Xenopus somitogenesis. Dev. Cell 6, 205–-218 (2004).
29. Sakai, Y. et al. The retinoic acid-inactivating enzyme CYP26 is essential forestablishing an uneven distribution of retinoic acid along the anterio-posterior
axis within the mouse embryo. Genes Dev. 15, 213–-225 (2001).
30. Nagai, T. et al. A variant of yellow fluorescent protein with fast and efficientmaturation for cell-biological applications. Nature Biotechnol. 20, 87–-90(2002).

Supplementary Information is linked to the online version of the paper at
www.nature.com/nature.
Acknowledgements We thank S. Kitajima and E. Ikeno for their assistance ingenerating the Mesp2–venus knock-in mouse; M. Ikumi and Y. Takahashi fortechnical assistance and maintaining the mice used in this study; A. Gossler and
R. Johnson for providing the Dll1and lunatic fringe knockout mice; andH. Hamada for CYP26a-null embryos. We also thank H. Takeda for criticalreading of this manuscript. We are grateful to H. Tanaka for preparing purified
Mesp2 protein. 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 andTechnology, Japan.
Author Information Reprints and permissions information is available at
npg.nature.com/reprintsandpermissions. The authors declare no competing
financial interests. Correspondence and requests for materials should be
addressed to Y.S. (ysaga@lab.nig.ac.jp).

© 2005 Nature Publishing Group359