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Somitogenesis, the sequential formation of a periodic pattern along the antero-posterior axis
of vertebrate embryos, is one of the most obvious examples of the segmental patterning
processes that take place during embryogenesis and also one of the major unresolved
events in developmental biology.
Somitogenesis is a series of dynamic morphogenetic events that involve cyclicalsignaling. The periodicity of somitogenesis is controlled by segmentation clock
operating in the presomitic mesoderm (PSM), the precursor of somites. Notch
signaling plays important roles not only in the segmentation clock mechanism
but also as an output signal of the clock to induce Mesp2 transcription that
controls somite formation. n the present revie!, recent advances in the
understanding of the molecular mechanisms underlying the translation of clock
information into the spatial patterning of segmental somites in mice are
discussed. Particular attention is paid to the interplay bet!een t!o the distinct
signaling path!ays of Notch and "#" and the Mesp$ transcription factor acting
as an e%ector molecule during mouse somitogenesis.
During vertebrate embryogenesis, paraxial mesoderm gives rise to somites, which
subsequently develop into the dermis, skeletal muscle, ribs and vertebrae of the adult.
utations that disrupt the patterning of individual somites have dramatic effects on these
tissues, including fusions of the ribs and vertebrae. !he !-box transcription factor, !bx", is
expressed in the paraxial mesoderm but is downregulated as somites develop. #t is essential
for the formation of posterior somites, which are replaced with ectopic neural tubes in !bx"-
null mutant embryos. $e show that partial restoration of !bx" expression in null mutants
rescues somite development, but that rostrocaudal patterning within them is defective,ultimately resulting in rib and vertebral fusions, demonstrating that !bx" activity in the
paraxial mesoderm is required not simply for somite specification but also for their normal
patterning. Somite patterning is dependent upon %otch signaling and we show that !bx"
genetically interacts with the %otch ligand, delta-like & 'Dll&(. Dll& expression, which is
absent in the !bx"-null mutant, is restored at reduced levels in the partially rescued mutants,
suggesting that Dll& is a target of !bx". $e also identify the spontaneous mutation rib-
vertebrae as a hypomorphic mutation in !bx". !he similarity in the phenotypes we describe
here and that of some human birth defects, such as spondylocostal dysostosis, raises the
possibility that mutations in !bx" or components of this pathway may be responsible for
these defects.
Organisms are composed of a number of cells arranged in a well-
coordinated manner. A fertilized egg repeats cell division and differentiates
into animal body in the embryogenesis, in which various phenomena take
place in a predestined order that the inherent “biological clock” in living
body controls. e attempt to clarify the principle of animal morphogenesis
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through investigating the mechanism of the “biological clock” that
controls various life phenomena in the embryonic development.
!esea ch fo somitogenesis in the ve teb ates as a model system fo the
biological clock
"he mouse body is composed of metameric structure along the
anteroposterior a#is. $or e#ample, the spine is made up of accumulation of
multiple vertebrae, each of which is similar in shape. %uch metamerism is
based on the somite, which is a transient structure in the mid-embryogenesis. %omites are symmetrically arranged on the both side of the
neural tube as even-grained epithelial spheres that give rise to vertebrae,
ribs, muscles and skins.
"he primordium of somite, located at the caudal tip of the mouse embryo,
e#tends posteriorly. "he anterior e#tremity of the somite primordium is
pinched off to generate a pair of somite in a two-hour cycle, resulting in the
formation of repeats of a similar size structure. On the basis of this finding, it
has been considered that there is a biological clock, which determines the
two-hour cycle, in the primordium of somite. "he e#pression of several
genes oscillates in the primordium of somite, corresponding the cycle of the
somite segmentation, that serves as a molecular evidence of the biological
clock. e are e#ploring the mechanism for biological block on the basis of
such oscillatory gene e#pression.
"ranscription factor &es' is specifically e#pressed in the primordium of
somite ($ig. )* and in a cyclic manner ($ig. +*. y genetic and biochemical
e#periments, we have shown that &es' is involved as a principal factor in
the mechanism for the biological clock that determines the two-hour cycle
($ig. +, $ig. *. e are conducting studies to understand the biological clock
in a comprehensive manner.
The number of vertebrae is defined strictly for a given species and
depends on the number of somites, which are the earliest metameric
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structures that form in development. Somites are formed by sequential
segmentation. The periodicity of somite segmentation is orchestrated by
the synchronous oscillation of gene expression in the presomitic
mesoderm (PSM, termed the !somite segmentation cloc",# in which$otch signaling plays a crucial role. %ere we show that the cloc" period
is sensitive to $otch activity, which is fine&tuned by its feedbac"
regulator, $otch®ulated an"yrin repeat protein ($rarp, and that
$rarp is essential for forming the proper number and morphology of
axial s"eleton components. $ull&mutant mice for Nrarp have fewer
vertebrae and have defective morphologies. $otch activity is enhanced in
the PSM of the Nrarp') embryo, where the &*&h segmentation period is
extended by + min, thereby forming fewer somites and their resultant vertebrae. educed $otch activity partially rescues
the Nrarp') phenotype in the number of somites, but not in morphology.
Therefore we propose that the period of the somite segmentation cloc" is
sensitive to $otch activity and that $rarp plays essential roles in the
morphology of vertebrae and ribs.
)o to*
INTRODUCTION
The somite is the earliest discernible metameric structure in vertebrates.
-t gives rise to vertebrae, ribs, and s"eletal muscles, thereby providing a
segmental pattern along the anterior)posterior axis. Somites are aligned
along both sides of the neural tube. pair of somites buds off
sequentially from the anterior extremity of the presomitic mesoderm
(PSM in a rhythmic manner (Pourquie, *//0 . The periodicity of this
repetitive process is orchestrated by the synchronous oscillation of gene
expression in the PSM, termed the !somite segmentation cloc"#
(Pourquie, *//1 . Some of the genes that are cyclically activated and
deactivated, including Hes7 and Lunatic fringe ( Lfng, are components
of $otch signaling (2orsberg et al ., 0334 5 Mc6rew et al ., 0334 5 ulehla
and 7ohnson, 0333 5 8essho et al ., *//0b .
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3172277/#ui-ncbiinpagenav-2http://www.ncbi.nlm.nih.gov/pubmed/11687492http://www.ncbi.nlm.nih.gov/pubmed/12869750http://www.ncbi.nlm.nih.gov/pubmed/9740806http://www.ncbi.nlm.nih.gov/pubmed/9740806http://www.ncbi.nlm.nih.gov/pubmed/9740806http://www.ncbi.nlm.nih.gov/pubmed/9742402http://www.ncbi.nlm.nih.gov/pubmed/9742402http://www.ncbi.nlm.nih.gov/pubmed/9742402http://www.ncbi.nlm.nih.gov/pubmed/10049564http://www.ncbi.nlm.nih.gov/pubmed/10049564http://www.ncbi.nlm.nih.gov/pubmed/11641270http://www.ncbi.nlm.nih.gov/pubmed/11641270http://www.ncbi.nlm.nih.gov/pubmed/11641270http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3172277/#ui-ncbiinpagenav-2http://www.ncbi.nlm.nih.gov/pubmed/11687492http://www.ncbi.nlm.nih.gov/pubmed/12869750http://www.ncbi.nlm.nih.gov/pubmed/9740806http://www.ncbi.nlm.nih.gov/pubmed/9742402http://www.ncbi.nlm.nih.gov/pubmed/10049564http://www.ncbi.nlm.nih.gov/pubmed/10049564http://www.ncbi.nlm.nih.gov/pubmed/11641270
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$otch signaling is cell)cell contact)dependent and plays a ma9or role in
development (8olos et al ., *//: . fter $otch binds to its ligand, ;elta,
on an ad9acent cell, it is activated and undergoes limited proteolysis,
which is dependent on vrard et al ., 0334 5 ?hang and6ridley, 0334 5 8essho et al ., *//0b . Therefore $otch signaling
regulates the segmentation cloc", which in turn orchestrates somite
patternings and the resultant vertebrae.
-n addition to $otch components, the amount of $-=; oscillates in
synchrony with Hes7 and Lfngexpression in the PSM. This periodicity of
$otch activity contributes to periodic somite segmentation (Morimoto et
al., *//+ . $-=; activates Lfng transcription, and @fng inhibits $-=;
production to form a negative feedbac" loop (;ale et al ., *//1 .
8ecause Lfng expression is cyclical in the PSM, this negative feedbac"
loop probably contributes to oscillatory $-=; production. The dynamic
$otch activity that is produced by the compound feedbac" loops may
play a crucial role in regulating the segmentation cloc" because it acts
upstream of Hes7 and Lfng.
$otch®ulated an"yrin repeat protein ( Nrarp encodes a small 00A&
amino&acid residue protein with two an"yrin repeat motifs in the
carboxy terminus. This protein is highly conserved among
vertebrates. Nrarp is induced by $otch signaling (Brebs et al ., *//0
5 @amar et al ., *//0 5 TopcCews"a et al ., *//1 5 Pirot et al ., *//A , and
it promotes $-=; loss by forming a ternary complex with $-=; and
8P9 (@amar et al ., *//0 . Thus $rarp acts as a feedbac" regulator in
$otch signaling. -n addition, it is expressed periodically in the PSM
http://www.ncbi.nlm.nih.gov/pubmed/17409286http://www.ncbi.nlm.nih.gov/pubmed/17409286http://www.ncbi.nlm.nih.gov/pubmed/17409286http://www.ncbi.nlm.nih.gov/pubmed/11260262http://www.ncbi.nlm.nih.gov/pubmed/11260262http://www.ncbi.nlm.nih.gov/pubmed/11260262http://www.ncbi.nlm.nih.gov/pubmed/11260262http://www.ncbi.nlm.nih.gov/pubmed/12110169http://www.ncbi.nlm.nih.gov/pubmed/12110169http://www.ncbi.nlm.nih.gov/pubmed/12110169http://www.ncbi.nlm.nih.gov/pubmed/12110168http://www.ncbi.nlm.nih.gov/pubmed/12110168http://www.ncbi.nlm.nih.gov/pubmed/12110168http://www.ncbi.nlm.nih.gov/pubmed/12783854http://www.ncbi.nlm.nih.gov/pubmed/12783854http://www.ncbi.nlm.nih.gov/pubmed/12783854http://www.ncbi.nlm.nih.gov/pubmed/9690473http://www.ncbi.nlm.nih.gov/pubmed/9690473http://www.ncbi.nlm.nih.gov/pubmed/9690473http://www.ncbi.nlm.nih.gov/pubmed/9690472http://www.ncbi.nlm.nih.gov/pubmed/9690472http://www.ncbi.nlm.nih.gov/pubmed/11641270http://www.ncbi.nlm.nih.gov/pubmed/11641270http://www.ncbi.nlm.nih.gov/pubmed/11641270http://www.ncbi.nlm.nih.gov/pubmed/15902259http://www.ncbi.nlm.nih.gov/pubmed/15902259http://www.ncbi.nlm.nih.gov/pubmed/15902259http://www.ncbi.nlm.nih.gov/pubmed/15902259http://www.ncbi.nlm.nih.gov/pubmed/12529645http://www.ncbi.nlm.nih.gov/pubmed/12529645http://www.ncbi.nlm.nih.gov/pubmed/12529645http://www.ncbi.nlm.nih.gov/pubmed/11783997http://www.ncbi.nlm.nih.gov/pubmed/11783997http://www.ncbi.nlm.nih.gov/pubmed/11783997http://www.ncbi.nlm.nih.gov/pubmed/11485984http://www.ncbi.nlm.nih.gov/pubmed/11485984http://www.ncbi.nlm.nih.gov/pubmed/11485984http://www.ncbi.nlm.nih.gov/pubmed/12711545http://www.ncbi.nlm.nih.gov/pubmed/12711545http://www.ncbi.nlm.nih.gov/pubmed/12711545http://www.ncbi.nlm.nih.gov/pubmed/15325262http://www.ncbi.nlm.nih.gov/pubmed/15325262http://www.ncbi.nlm.nih.gov/pubmed/15325262http://www.ncbi.nlm.nih.gov/pubmed/11485984http://www.ncbi.nlm.nih.gov/pubmed/11485984http://www.ncbi.nlm.nih.gov/pubmed/11485984http://www.ncbi.nlm.nih.gov/pubmed/17409286http://www.ncbi.nlm.nih.gov/pubmed/11260262http://www.ncbi.nlm.nih.gov/pubmed/11260262http://www.ncbi.nlm.nih.gov/pubmed/12110169http://www.ncbi.nlm.nih.gov/pubmed/12110168http://www.ncbi.nlm.nih.gov/pubmed/12783854http://www.ncbi.nlm.nih.gov/pubmed/9690473http://www.ncbi.nlm.nih.gov/pubmed/9690472http://www.ncbi.nlm.nih.gov/pubmed/9690472http://www.ncbi.nlm.nih.gov/pubmed/11641270http://www.ncbi.nlm.nih.gov/pubmed/15902259http://www.ncbi.nlm.nih.gov/pubmed/15902259http://www.ncbi.nlm.nih.gov/pubmed/12529645http://www.ncbi.nlm.nih.gov/pubmed/11783997http://www.ncbi.nlm.nih.gov/pubmed/11485984http://www.ncbi.nlm.nih.gov/pubmed/12711545http://www.ncbi.nlm.nih.gov/pubmed/15325262http://www.ncbi.nlm.nih.gov/pubmed/11485984
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(Sewell et al ., *//3 5 Dright et al ., *//3 . The roles of Nrarp in the
segmentation cloc" and somitogenesis, however, remain largely unclear.
To investigate the roles of $rarp in these processes, we generated
an Nrarp "noc"out mouse and inspected its segmentation cloc" and
somitogenesis. %ere we show that $rarp is essential for proper axial
s"eleton formation. $ull&mutant Nrarp mice have small but substantial
defects in their vertebrae and ribs. -n addition, Nrarp mutants lose two
vertebrae as a consequence of the extended somite segmentation cloc"
period. The loss of $rarp increases $otch signaling but does not affect
any other signaling, including Dnt signaling in the PSM. emar"ably, an
inhibitor of $otch signaling partially rescues the cloc" period phenotype.-t failed, however, to improve the defects in the axial s"eleton
components. Thus our studies suggested that $rarp plays indispensable
roles in securing the number and morphology of axial s"eleton
components.
)o to*
RESULTS
Nrarp−/ + mutant mice had defects in axial skeletons and fewer vertebrae
Nrarp is strongly expressed in the PSM in a cyclical manner during
mouse development (Sewell et al ., *//3 5 Dright et al ., *//3 . De
expected it to play a role in vertebrae and rib formation, because the
PSM is the primordial somite, which gives rise to the vertebrae and ribs.
To investigate the role of $rarp, we disrupted the Nrarp gene
(Supplemental 2igure S0. Twenty&two of ++ Nrarp&homoCygous mice
had "in"ed tails (2igure 0 . -nterestingly, the Hes7 &heteroCygous
mutants had similarly "in"ed tails (8essho et al ., *//0b ,
although Nrarp&homoCygous mutants express normal levels of Hes7 in
the PSM (2igures 1; and andA8. A8. ccording to this phenotype, we
anticipated defects to be present in the axial s"eleton. Thus we visualiCed
the bones and cartilage of newborn mice, and inspected the shape and
numbers of their vertebrae and ribs. -n all of the Nrarp') mutant mice, afew vertebrae and ribs had small defects, which included partial fusion of
http://www.ncbi.nlm.nih.gov/pubmed/19268448http://www.ncbi.nlm.nih.gov/pubmed/19268448http://www.ncbi.nlm.nih.gov/pubmed/19268448http://www.ncbi.nlm.nih.gov/pubmed/19882724http://www.ncbi.nlm.nih.gov/pubmed/19882724http://www.ncbi.nlm.nih.gov/pubmed/19882724http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3172277/#ui-ncbiinpagenav-2http://www.ncbi.nlm.nih.gov/pubmed/19268448http://www.ncbi.nlm.nih.gov/pubmed/19268448http://www.ncbi.nlm.nih.gov/pubmed/19268448http://www.ncbi.nlm.nih.gov/pubmed/19882724http://www.ncbi.nlm.nih.gov/pubmed/19882724http://www.ncbi.nlm.nih.gov/pubmed/19882724http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3172277/figure/F1/http://www.ncbi.nlm.nih.gov/pubmed/11641270http://www.ncbi.nlm.nih.gov/pubmed/11641270http://www.ncbi.nlm.nih.gov/pubmed/11641270http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3172277/figure/F3/http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3172277/figure/F4/http://www.ncbi.nlm.nih.gov/pubmed/19268448http://www.ncbi.nlm.nih.gov/pubmed/19882724http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3172277/#ui-ncbiinpagenav-2http://www.ncbi.nlm.nih.gov/pubmed/19268448http://www.ncbi.nlm.nih.gov/pubmed/19882724http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3172277/figure/F1/http://www.ncbi.nlm.nih.gov/pubmed/11641270http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3172277/figure/F3/http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3172277/figure/F4/
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the ribs and abnormally shaped vertebrae (2igure 08. The ma9ority of
the vertebrae and ribs were normal. De next examined the s"eletal
patterns of adult mice by x&ray computed tomography (=T. Similar to
the newborns, Nrarp')
mutant adult mice had a few abnormal vertebraeand ribs (2igure 0=. Therefore $rarp was essential for minute
configuration of the axial s"eleton.
2-6E> 0F
S"eletal defects of $rarp&null mice. ( "in"ed tail that was typical of
adult homoCygous mutants. (8 ;orsal views and high magnifications
(right of s"eletal preparations at postnatal day 0. The arrows and the
asteris" are used to indicate the location ...
2-6E> 1F
@oss of Nrarp specifically increases Hes5 expression but does not affect
cyclical gene expression patterns. ( Guantification of $-=; by
immunoblotting (n H 1. The ratios of $-=; to I&actin are indicated. (=
-mmunoreactivity of $-=; in the ...
2-6E> AF
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=omparison of gene expression levels between wild&type
and Nrarp−/– embryos. ( =omparison of gene expression levels in the
PSM between the Nrarp') and the wild&type embryos (n H 1. -n this
microarray analysis, the average ...
De counted the number of vertebrae. The total number of vertebrae was
+:.A J /.* and +3./ J /./ in the Nrarp') mutant mice (n H 3 and in
their wild&type littermate mice (n H :, respectively (Table 0. Most of
the Nrarp') mice lost a lumber vertebra and a caudal vertebra (Table 0.
These results were consistent with the results for the newborn mice, for
which all of the Nrarp') had five lumbar vertebrae, whereas their wild&
type littermate newborn mice had six (2igure 08 and Supplemental TableS0. De did not observe total fusion of the vertebrae or ribs in
the Nrarp') mice5 even some of them had small defects. Therefore it was
not li"ely that the fusion of two ad9acent vertebrae reduced the number
of vertebrae. Thus $rarp was essential for securing the number and
configuration of axial s"eleton components.
T8@> 0F
K&ray analyses of adult mice vertebrae.
Nrarp+ mutant mice had fewer somites
Somites give rise to vertebrae in a one&to&one ratio5 the caudal half of a
pair of somites and the rostral half of the next pair merge into a vertebra
(6ossler and %rabe de ngelis, 0334 . Thus the number of vertebrae
corresponds to the number of somites. Somites are transient structures
in development and differentiate subsequently into bones, muscle, and
s"in. Therefore it was difficult to count the total number of somites. De
compared the number of somites located between the fore& and hind&
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3172277/table/T1/http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3172277/table/T1/http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3172277/figure/F1/http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3172277/table/T1/http://www.ncbi.nlm.nih.gov/pubmed/9399080http://www.ncbi.nlm.nih.gov/pubmed/9399080http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3172277/table/T1/http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3172277/table/T1/http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3172277/figure/F1/http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3172277/table/T1/http://www.ncbi.nlm.nih.gov/pubmed/9399080
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limb buds (2@8s and %@8s, respectively at embryonic day (> 0/.+.
The Nrarp') embryos had 0: somites between the limb buds (n H 01,
whereas their wild&type littermate embryos had 04 somites (n H 1A
(2igure * . This result was consistent with the s"eletal phenotype, andalso suggested that the positions of the limb buds were not affected in
the absence of $rarp.
2-6E> *F
$rarp&null mice have smaller numbers of somites. ( =omparison of the
numbers of somites between the 2@8 and the %@8. The dashed lines are
used to indicate the rostral borders of the 2@8 and the
%@8. Uncx4.1 expression was detected in the posterior half ...
7udging from the expression of Uncx4.1, which was exclusively restrictedto the posterior compartment of each somite, at > 4.+, > 0/.+, and > 00.+
(2igure * and Supplemental 2igure S*, the morphology of the somites
was almost normal. De did not find any defects in their shape, so we
ruled out the possibility that the fusion of two ad9acent somites caused a
reduction in the number of somites. -n addition, because
the Nrarp') embryos were not distinguishable from the NrarpL' or wild&
type embryos, and because they showed similar expression pattern
of Fgf8 (Supplemental 2igure S1, we ruled out the possibility of a small
delay in the general development of the Nrarp') embryos.
!he segmentation clock period was longer in Nrarp+ mutant mice
De showed that Nrarp') mice had fewer numbers of somites and
vertebrae, respectively. This result led us to two possibilitiesF The somite
formation period was longer andor the total duration of somite
formation was shorter in the Nrarp') embryos. Thus we counted the
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number of somites at various stages to measure the somite formation
period. Somite formation starts between > :.+ and > 4./. De failed to
detect a significant difference in the number between the genotypes
throughout the initial stage of somite formation at the early stage, > 4.+(approximately 0*5 2igure *8. Thus we presumed that the start of
somite formation was not affected. t > 0/.+ and > 00.+, the number of
somites in the Nrarp')embryos was significantly lower than that of their
wild&type littermate embryos (2igure *, = and ;. $umerically spea"ing,
the 3+ confidence interval (=- of mean decrease in the somite number
of Nrarp') from that of wild type was ()*.N+, )0.0/ at > 00.+. 8ecause
the average number of somites formed during the :* h from > 4.+ to >
00.+ was A/.N in the wild&type embryos, on average somites of (1:.:+,13.1/ were produced (3+ =- during the :* h in the Nrarp') embryos,
to correspond to the above =- of the mean decrease. This difference in
number of somites between the Nrarp') embryos and their wild&type
littermate embryos was consistent with the difference in the total
number of vertebrae (+3./ vs. +:.A. Esing this result, we estimated that
the period of somite formation was &0/N min and 000 min in the wild&
type embryos and the Nrarp−/– embryos, respectively (2igure *>.
Therefore the loss of $rarp extends the segmentation cloc" period by &+
min, and this extension resulted in fewer numbers of somites and
vertebrae.
%otch activity was up-regulated in the Nrarp+ S
$rarp reduces $otch activity by enhancing $-=; degradation (@amar et
al., *//0 . Thus we predicted that $otch activity was increased in the
PSM cells of the Nrarp') embryos. De collected the PSM of several
embryos and determined the quantity of $-=; to measure the $otch
activity in the PSM. The amount of $-=; increased 0.3&fold in the PSM
of the Nrarp−/– embryos at > 0/.+ (2igure 1 over that in their wild&type
littermate embryos. %owever, the cyclical pattern of $-=;
immunoreactivity was not affected in the mutant PSM (2igure
1=. Hes5 expression, another target gene of $otch, was up®ulated
(2igure 18. Similar to the $-=;immunoreactivity, Hes5 , Hes7, and Lfng expression was cyclical in
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the Nrarp') PSM in the same way as in wild&type PSM (2igure 1, ; and
>. Therefore the loss of $rarp enhanced $otch activity but did not affect
its cyclical pattern of activity.
8ecause $rarp has been reported to up®ulate Dnt signaling
(-shitani et al ., *//+ 5 Phng et al ., *//3 , we chec"ed the expression
of Axin, which acts downstream of Dnt signaling. De detected the
same level of Axin m$ in the Nrarp') PSM as in the wild&type PSM,
and the cyclical pattern was not affected (2igure 12. De next carried out
a microarray analysis to compare comprehensively the gene expression
in the Nrarp') PSM to that in their wild&type littermate PSM. De
detected an increase in Hes5 expression as well as a dramatic decreasein Nrarp expression (2igure A . The expression of the Dnt target genes
(including Axin, Lef1, and !es"genin was not altered, however, in
the Nrarp')PSM (2igure A8 and Supplemental Table S*. -n addition,
the expression of target genes including #uspand spr"ut$ of fibroblast
growth factor (262 signaling was not altered (2igure A8 and
Supplemental Table S*, although 262 signaling plays an important role
in somitogenesis. Thus we found that a loss of $rarp leads to a
remar"able increase in $otch activity in the PSM.
/educed %otch activity shortened the period of the segmentation clock
De found that the period of the somite segmentation cloc" was longer
in Nrarp') mutant cells and that $otch activity in the PSM cells was
enhanced. De assumed that the period of the somite segmentation cloc"
was sensitive to $otch activity5 higher $otch activity leads to a longer
cloc" period, and lower $otch activity leads to a shorter cloc" period. Totest this assumption, we decreased $otch activity in the embryo PSM by
administering a $otch inhibitor to pregnant female mice. De
administered the :.+ and
then determined the amount of $-=; in the PSM and examined the
number of somites on > 0/.+. dministration of /.0, /.1, and 0./ mg"g
of @OA00,+:+ reduced $otch activity in the PSM by 0/, 1/, and 4/,
respectively (2igure + . 0/ reduction in $otch activity resulted in an
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extra somite formingF 03 somites between the limb buds at > 0/.+ (n H
A:, in comparison with 04 in the control (2igure +, 8 and =. larger
reduction in $otch activity disrupted the somite pattern, however, and
we could not count the number of somites (Supplemental 2igure SA.The mice treated with /.0 mg"g @OA00,+:+ had consistently seven
lumbar vertebrae (n H A00 compared with six in the control, and they
had some malformation in axial s"eletons (2igure +, ; and >, and
Supplemental Table S1. Thus these results suggested that a mild
reduction in $otch activity in the PSM led to a shorter cloc" period. The
possibility that the $otch inhibitor affects other phenomena could not be
excluded, however, because even lower dose administration of the $otch
inhibitor resulted in malformation in axial s"eletons (2igure +>.
2-6E> +F
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configurations than did the untreated Nrarp') neonates or inhibitor&
treated wild&type neonates. Thus, because reduced $otch activity did not
correct the defective axial s"eleton morphology of Nrarp'), $rarp itself
was li"ely essential for proper morphogenesis. 2urthermore, the s"eletalmorphology of the Nrarp') neonates was more severe than that of the
wild&type neonates for both conditions of $otch inhibitor administration
and no treatment. Therefore $rarp may have contributed to maintaining
proper s"eletal morphology.
!he segmentation clock period was sensitive to %otch activity
De found that the segmentation cloc" period is sensitive to $otch
activity from our genetic and pharmacological experiments. The
segmentation cloc" is regulated by synchronous gene oscillation in the
PSM. -n PSM cells, $otch signaling activates Hes7 transcription, and
%es: inhibits transcription of its own gene and provo"es the oscillatory
gene expression over a *&h cycle in the mouse, where the %es: negative
feedbac" loop provides a core mechanism for the cloc" to run on (2igure
N . mathematical model was constructed based on this negative
feedbac" loop, and was used to successfully reproduce the oscillatorygene expression (@ewis, *//1 5 %irata et al ., *//A . Model analysis
revealed that the oscillation amplitude becomes small when $otch
activity is low (@ewis, *//1 . De carried out further analyses of the
model and found that the oscillation period also decreases with low
$otch activity, because a smaller amplitude means that less time is
required for a cycle to pass (see Supplemental $ote 0. Stri"ingly, we
found from a simulation based on the model that the oscillation period is
sensitive to the average level of $otch activity in the modelF %igher
$otch activity extends the period and gradually increases it with
extremely large activity, whereas lower $otch activity shortens it (2igure
N8. This result supports the experimental results that the cloc" period
was sensitive to $otch activity. -n addition, by using the model analysis
we predicted that the %es: negative feedbac" loop provides a molecular
mechanism for cloc" period sensitivity to $otch activity.
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2-6E> NF
Mathematical analyses of the effect of $otch activity on the oscillation
period. ( The molecular mechanism of generation of oscillatory gene
expression in the mouse PSM cell. ctivated $otch receives limited
proteolysis, which is dependent on 4.+. Therefore the loss of $rarp may not have affected the
timing of the initiation of somite formation, because > 4.+ is quite an
early stage of somitogenesis (6ossler and %rabe de ngelis, 0334 . -n
addition, the numbers of somites increased proportionally in bothgenotypes, and the number of somites in the Nrarp') embryo was
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significantly lower than that in the wild&type embryos at > 0/.+ and >
00.+, respectively. Therefore the period of somite formation was longer in
the absence of $rarp. De could not rule out the possibility that the
duration of somite formation in the Nrarp')
was shorter than in the wild&type embryos, so the numbers of somites and vertebrae were lower in
the Nrarp') embryos because it was not possible to detect the end of
somite formation. This possibility did not li"ely happen, however,
because the loss of two vertebrae (+3./ vs. +:.A, wild&type and Nrarp'),
respectively is consistent with the number of somites that formed
between > 4.+ and > 00.+ (A/.N vs. 14.3. Thus we conclude that
extending the period of somite formation was the ma9or cause of the
lower number of vertebrae in the Nrarp') embryos.
De showed that the cloc" period was sensitive to $otch activity. -t was
reported that other signaling, Dnt or retinoic acid, affects the pace of the
segmentation cloc" (Bawa"ami et al ., *//+ 5 ermot et al ., *//+
5 ermot and Pourquie, *//+ 5 6ibb et al ., *//3 . -n addition, the
period of the somite segmentation cloc" is reported to be sensitive to the
surrounding temperature in Cebrafish development (Schroter et al .,
*//4 . To our "owledge our report here, however, is the first for which
the correlation between a fluctuating segmentation period and the
intensity of molecular signaling was analyCed quantitatively. -n addition,
we interpreted the correlation by using mathematical analyses. -n the
mouse, the %es: negative feedbac" loop, which acts downstream of
$otch, is essential for oscillatory gene expression in the PSM (8essho et
al., *//1 . The results of our mathematical simulation led us to
speculate that increasing $otch activity leads to extending the oscillationperiod. This speculation is in agreement with our experimental results.
The relationship between the somite formation period and $otch activity
in Cebrafish was reported recentlyF ttenuation of $otch activity led to a
longer somite formation period (%errgen et al ., */0/ , and this
observation is opposite to our findings. -n Cebrafish, $otch signaling
plays a ma9or role in the synchroniCation of the oscillatory gene
expression between each PSM cell (7iang et al ., */// 5%ori"awa et al .,*//N . The authors of the recent study focused on this $otch&
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dependent synchroniCation between the PSM cells, and, by using
mathematical analyses, they accounted for the delay in the somite
formation period by the attenuation of $otch activity. Their model
assumes, however, that the oscillation period is not affected by thechange in $otch activity in each PSM cell. 8y contrast, we conducted
mathematical analyses of the oscillatory gene expression in each cell,
although we did not consider the synchroniCation between cells because
the role of $otch signaling in the synchroniCation remains to be
elucidated in the mouse. 2urther study will be essential to gaining a
comprehensive understanding of the mechanism of tuning of the somite
formation period in vertebrates.
The small defects in the morphologies of the vertebrae and ribs in
the Nrarp') mice were not repaired by using the $otch inhibitor.
Therefore they were not caused by higher $otch activity, but rather
$rarp was essential for proper axial s"eleton morphogenesis. 2eedbac"
regulation of $otch activity via $rarp may have contributed to axial
s"eleton morphogenesis. $rarp mediates Dnt&signaling)dependent
neural&crest&cell development by stabiliCing @>20, which is a
downstream effector of Dnt signaling (-shitani et al ., *//+ . $rarp also
coordinates $otch and Dnt signaling in Cebrafish and mouse endothelial
cells, thereby controlling angiogenesis (Phng et al ., *//3 . -n contrast,
we detected enhanced $otch activity but failed to detect a change in Dnt
signaling. De could have inferred this from the expression of Dnt
downstream genes in our "noc"out mice. =onsistent with this
observation, the extended period of somite segmentation was restored by
using the $otch inhibitor. Thus $rarp seemed to regulate $otchsignaling but not Dnt signaling in somitogenesis, in which $rarp
maintained the proper period of somite segmentation and the proper
numbers of somites and vertebrae.
De failed to detect abnormalities in the morphology of the somites,
although we found defects in the vertebrae and ribs in
the Nrarp') embryos. -t is possible that there were minute discrepancies
in the siCe of the somites or in their gene&expression pattern. 2urtherinvestigation is necessary to clarify this. De found that administering the
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$otch inhibitor caused vertebra and rib malformation. This result is
consistent with the results of a report in which cyclical $otch activity was
crucial for somite formation (Morimoto et al ., *//+ . The $otch
inhibitor may have perturbed the cyclical $otch activity in PSM so thatthe morphology of the vertebrae and ribs was affected. De could not,
however, rule out the other possibility that the $otch inhibitor may
affect the ossification or some other process of s"eletal morphogenesis
but the somite segmentation cloc", because lower dose administration of
the $otch inhibitor affected the morphology of the axial s"eleton but not
the somite morphology. Stri"ingly, the $otch inhibitor disturbed the
configuration of the axial s"eleton more severely in the absence of $rarp
than in the wild&type condition. Thus we speculate that $rarp maycontribute to the robustness against environmental perturbation of
somite formation or oscillatory gene expression. 2urther study is needed
to elucidate the role of $rarp in somite formation.
)o to*
MATERIALS AND METHODS
)eneration of Nrarp-knockout mice and genotyping
Nrarp%deficient mice were generated by homologous recombination
with a targeting vector in which the Nrarp coding region was replaced
with ->S& Lac& and P6B&ne" (Supplemental 2igure S*a. TT* >S cell
lines ( Oagi et al ., 0331 that were missing Nrarp were identified by
using Southern blot analysis with +Q and 1Q probes (Supplemental 2igure
S*b. =himeric mice were generated as described previously (8essho etal., *//0b . Nrarp%deficient mice were bac"&crossed with =;0 more
than six times. De crossed pairs of 4& to +/&w"&old NrarpL) mice and
collected their embryos at a certain embryonic stage to compare $rarp
mutants with their wild&type littermates. =himera, 20, and 2* mice were
initially genotyped by Southern blot analysis (Supplemental 2igure S*b
and allele&specific P= of somatic cell (s"in ;$. >mbryos were
genotyped by P= of yol"&sac ;$ by using the primers $rarp&2
=T=T=TTT6=T6T66T66666=T6= (which binds to the
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+QET region of $rarp and $rarp&
=6=6==TT=T=66666==T= (which binds to the +QET
region and downstream of $rarp&2, which detect a 1*/&base&pair
fragment indicative of the wild&type allele, and primers $eo&2 6===66=6666=6=T6=TT and $rarp&, which
detect a +*/&base&pair fragment indicative of the mutated allele.
#mmunoblotting
PSMs were dissected from the embryos at > 4.+ or >0/.+ and lysed in a
buffer that contained 0 $P&A/, +/ mM Tris&%=l (p%:.+, 0+/ mM
$a=l, and + mM >;T. The tissue lysates were fractionated by using
S;S)P6> and then blotted onto %ybond&P membranes (mersham,
Piscataway, $7. $ext they were probed with anti&cleaved $otch0
monoclonal antibody (=ell Signaling Technology, ;anvers, M or anti&
I&actin monoclonal antibody (Sigma&ldrich, St. @ouis, MR, and signals
were then detected with a chemiluminescence detection system
(mersham. Guantification was performed using the public domain
$ational -nstitutes of %ealth ($-% image program, -mage7. $otch
signaling activity was evaluated by measuring the amount of $-=; that was normaliCed to the amount of I&actin, which was used as an internal
control.
icroarray analysis and quantitative /!-0/
PSMs were dissected from the > 0/.+ embryos of 4& to 0*&mo&old NrarpL
' female mice that were crossed with NrarpL' male mice. The part of the
neural tube that was attached to the PSM was removed. The PSMs werethen stored immediately in liquid nitrogen for several days. Total $
was extracted using the S Total $ -solation System (Promega,
Madison, D-. De prepared three independent biological replicates.
Microarray analysis was performed as described previously ( Datanabeet
al., *//3 . The microarray data set that we analyCed (6S>04A03 is
available through the $ational =enter for 8iotechnology -nformation
($=8- 6ene >xpression Rmnibus. everse transcription was performed
by using SuperScript -- reverse transcriptase (-nvitrogen, =arlsbad, =.
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Guantitative P= reactions were performed by using BP SO8 2ST
Eniversal qP= (Bapa 8iosystems, Doburn, M. >ach reaction was
carried out in triplicate using gene&specific primers. The expression level
of each gene was first normaliCed to that of 61P;%. The primers for themouse genes that we used in the quantitative P=s were as follows.
'(p#)* 61pdh&S, +Q&===6T==T6==T==&1Q, and 61pdh&S,
+Q&T=======T6TT6=T6T&1Q5 Nrarp* $rarp&S, +Q&
=T=6==TT666666&1Q, and $rarp&S, +Q&
===6==TTTT==&1Q5 Hes5* %es+&S, +Q&
6=6=T66=6=T66&1Q, and %es+&S, +Q&
666=TTT6=T6T6TTT=&1Q5 +ar4* =arA&S, +Q&6T=T666T6==6&1Q, and =arA&S, +Q&
TT6T==TT=66T==T==TT&1Q5 'pr1((* 6pr011&S, +Q&
T=TT=T66==TT66&1Q, and 6pr011&S, +Q&
6666==6T666TT6&1Q5 )"x5*hox+&S, +Q&
=66TT666=T6666T&1Q, and hox+&S, +Q&
6=T6TT=TT==66T=TT==&1Q5 Hes7* %es:&S, +Q&
T666=T666=T6=T6&1Q, and %es:&S, +Q&
=TTT=T==6=TT=666TT&1Q5 Lfng* @fng&S, +Q&
T6TTT66=6=66=&1Q, and @fng&S, +Q&
=666T6T6T=T666T=6&1Q5 -usp* ;uspN&S, +Q&
66T66==T66T66&1Q, and ;uspN&S, +Q&
66====TT==66T6&1Q5 pr$* Spry*&S, +Q&
6666TT=666666&1Q, and Spry*&S, +Q&6T=TT66=&
6T6T6TT===&1Q5 Axin* xin*&S, +Q&=T66=T==66T==&
1Q, and xin*&S, +Q&T=6=T==T==T6TT66&1Q5 N0#1* $"d0&S, +Q&=6T66=T666666=&1Q, and $"d0&S, +Q&
=66T=T66T6T66TTT=T==&1Q.
Skeletal preparation, x-ray 0!, and whole-mount in situ hybridi1ation
The cartilage and bones of newborn mice were stained with alcian blue
and aliCarin red, respectively, after being fixed in 3+ ethanol (8essho et
al., *//0b . The s"eletal structures of N& to 0*&w"&old mice were
examined by using a @aTheta @=T&0// Series x&ray =T (lo"a,
http://www.ncbi.nlm.nih.gov/pubmed/11641270http://www.ncbi.nlm.nih.gov/pubmed/11641270http://www.ncbi.nlm.nih.gov/pubmed/11641270http://www.ncbi.nlm.nih.gov/pubmed/11641270http://www.ncbi.nlm.nih.gov/pubmed/11641270http://www.ncbi.nlm.nih.gov/pubmed/11641270
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Dallingford, =T. Dhole&mount in situ hybridiCations were carried out
(8essho et al ., *//0a with minor modifications.
#mmunostaining
Dhole&mount immunostaining was carried out as described previously
(8essho et al ., *//1 with minor modifications. > 0/.+ embryos were
fixed with A paraformaldehyde at A= for 0 h and then treated with N
%*R* for 0/ min and * Triton K&0// for 0/ min. $ext, we incubated the
embryos with anti&cleaved $otch0 monoclonal antibody (=ell Signaling
Technology overnight at A=, and then with peroxidase&con9ugated
anti)rabbit immunoglobulin 6 antibody for 1 h. De then visualiCed the
peroxidase activity as deposits of 1,1Q&diaminobenCidine
tetrahydrochloride (Sigma.
0ounting somites and vertebrae and statistical analysis
De detected the expression of Uncx4.1 m$ by using in situ
hybridiCation to count the number of somites. The stain localiCes to the
caudal domain in somites5 therefore it was used as a landmar" of the
somite caudal border. De conducted experiments that compared wild
type, NrarpL and Nrarp')using littermates and counted the somites for
each embryo before genotyping. The bud positions were determined as
described previously (=han et al ., *//A*//+ for counting somites
that were located between the 2@8s and %@8s. 2or accuracy, we laterally
and dorsally observed embryos by using high&magnification fields.
2ollowing this, we counted the number of somites of each embryo and
calculated the average number of somites for each genotype forindividual pregnant females. This process allowed us to determine the
changes in the numbers of somites for Nrarp') or NrarpL' mice in
comparison to their wild&type littermates. Then, we determined the
average number of somites for each genotype of the embryos that we
collected from multiple pregnant females and compared the number
with the one that was determined for the wild&type embryos by using the
paired t test. De performed this comparison at > 4.+ (n H N pregnant
mothers, > 0/.+ (n H 04, and > 00.+ (n H 4. De also estimated a 3+ =-
http://www.ncbi.nlm.nih.gov/pubmed/11260262http://www.ncbi.nlm.nih.gov/pubmed/11260262http://www.ncbi.nlm.nih.gov/pubmed/11260262http://www.ncbi.nlm.nih.gov/pubmed/11260262http://www.ncbi.nlm.nih.gov/pubmed/12783854http://www.ncbi.nlm.nih.gov/pubmed/12783854http://www.ncbi.nlm.nih.gov/pubmed/12783854http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3172277/#B6http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3172277/#B6http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3172277/#B6http://www.ncbi.nlm.nih.gov/pubmed/11260262http://www.ncbi.nlm.nih.gov/pubmed/12783854http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3172277/#B6
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of the segment cloc" extension in Nrarp') embryos relative to the wild
type. The =- was estimated by applying maximum li"elihood estimation
to a hierarchical generative model of the somite number (see
Supplemental $ote * for details. lthough mice treated with /.0 mgml@OA00,+:+ had substantial malformation of the axial s"eleton, we did not
observe a split vertebral body. Thus we counted their vertebral bodies.
2or small or malformed vertebral bodies, we chec"ed the vertebral
arches, which should be on either side of each vertebral body.
2nimals and 3-secretase inhibitor administration
De then administered @OA00,+:+ to pregnant female mice per os
following a 0*&h fasting period. The @OA00,+:+ was formulated as a 1/
mgml solution in dimethyl sulfoxide and was diluted in + ethanol in
sunflower seed oil. -t was administered every *A h from > :.+ to > 3.+.
The amount of $-=; in the PSM was quantified *A h after administering
the @OA00,+:+ or the vehicle. Rur experiments were approved by the
nimal =are =ommittee of $ara -nstitute of Science and Technology.
They were conducted in accordance with guidelines that were
established by the Science =ouncil of 7apan.
)o to*
SUPPLEMENTARY MATERIAL
Supplemental Materials:
=lic" here to view.
)o to*
ACKNOWLEDGMENTS
De than" Shigeru Bondo, $aoyu"i -naga"i, Ta"ashi Bondo, Bentaro
%irata, Miguel Maroto, Bim ;ale, lexander ulehla, and Siripong
Thitamadee for discussion5 ;avid -sh&%orowicC, chim 6ossler, Rlivier
Pourquie, Thomas $. Sato, Ou"ie Ta"abata"e, 7an Moren, and -an Smith
for critically reading the manuscript5 and Michi"o Saitou for generatingthe "noc"out mice.
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3172277/#ui-ncbiinpagenav-2http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3172277/bin/supp_22_18_3541__index.htmlhttp://www.ncbi.nlm.nih.gov/pmc/articles/PMC3172277/#ui-ncbiinpagenav-2http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3172277/#ui-ncbiinpagenav-2http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3172277/bin/supp_22_18_3541__index.htmlhttp://www.ncbi.nlm.nih.gov/pmc/articles/PMC3172277/#ui-ncbiinpagenav-2
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This wor" was supported by a Ba"enhi (6rant&in&id for Scientific
esearch for the priority area !Systems 6enomics#5 Ba"enhi 8 and
Da"ate -nitiatives and 8 from the Ministry of >ducation, =ulture,
Sports, Science and Technology (M>KT, 7apan5 and by the EeharaMemorial 2oundation. This wor" was also supported in part by the
6lobal =R> Program in $-ST (2rontier 8iosciencesF strategies for
survival and adaptation in a changing global environment, M>KT,
7apan.
%es: controls the cyclic expression of pr"ut$4 via
transcriptional inhibition
The expression of pr"ut$4 oscillates in *&h cycles in phase with
the $otch regulated cyclic gene, Lfng
The expression of pr"ut$4 oscillates in the mouse PSM
=yclic expression of pr"ut$4 is not observed in the Cebrafish
PSM
Finally, we examined the expression profile of the orthologue of Sprouty4 in the
zebrafish PSM. Some genes in the Notch pathway
including her1, her7 and deltaC show cyclic expression in the zebrafish[!"
[#!, [$!, but other genes which cycle in the mouse PSM do not display cyclic
expression or ha%e not been well examined in zebrafish [&'!.
SproutyA is a strong candidate for the mediator that integrates
spatiotemporal information during somitogenesis
(n the PSM, F)F signaling establishes a posterior*to*anterior gradient, which is
in%ol%ed in the positioning of somite boundaries
M+-(+/S +N0 M-120S op
n itu %ybridiCation and 6raphical nalysis
http://www.plosone.org/article/info:doi/10.1371/journal.pone.0005603#pone.0005603-Holley1http://www.plosone.org/article/info:doi/10.1371/journal.pone.0005603#pone.0005603-Henry1http://www.plosone.org/article/info:doi/10.1371/journal.pone.0005603#pone.0005603-Holley2http://www.plosone.org/article/info:doi/10.1371/journal.pone.0005603#pone.0005603-Prince1http://www.plosone.org/article/info:doi/10.1371/journal.pone.0005603#tophttp://www.plosone.org/article/info:doi/10.1371/journal.pone.0005603#pone.0005603-Holley1http://www.plosone.org/article/info:doi/10.1371/journal.pone.0005603#pone.0005603-Henry1http://www.plosone.org/article/info:doi/10.1371/journal.pone.0005603#pone.0005603-Holley2http://www.plosone.org/article/info:doi/10.1371/journal.pone.0005603#pone.0005603-Prince1http://www.plosone.org/article/info:doi/10.1371/journal.pone.0005603#top
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3hole mount in situ hybridization were carried out as pre%iously
described[$4!, ['$!. he following regions were used as probes5
mouse Sprouty4, nucleotide residues *67*&&885 Lfng, &8*&$#65Uncx4.1, *&'*
, zebrafish Sprouty4, $6*#$4. 3e detected expressionof Sprouty4 or Lfng in -&4.7 mouse PSM by 9:(P;N9. he expression pattern
was digitally scored Sion image software. he relati%e positions in the PSM were
normalized by the lengths between the newest somite boundary , for
which we used Uncx4.1 probe as a mar?er, and posterior end of embryo .
he %alues from each assessment were analyzed graphically.
-xperiments were appro%ed by the +nimal :are :ommittee of Nara (nstitute of
Science and echnology and were conducted in accordance with guidelines
established by the Science :ouncil of @apan.
>xplant =ulture
he posterior part of embryo was bisected at -&4.7. 2ne half was fixed
immediately, and the other half was cultured in &4= F9S*0M-M;F&6 for 4 or
&64 min before fixation. Hes7 *null mutants and its littermates were bisected and
cultured for ' h in &= F9S*0M-M;F&6 with or without bF)F .
+fter fixation, the expression of Sprouty' was detected by in situ hybridization.
Transgenic mice
For transient transgenic experiment, we used a %ector containing the 7.'
?b Hes7 promoter followed by exonic regions of Hes7 with (-S*Aenus and SA'4
polyadenylation signal [!.
@uciferase ssays
For promoter analysis, a luciferase reporter under
the control of theSprouty4 promoter were transfected into
N(1$$ cells, which were plated in 6'*well plates, with 4, 67, 74 nanogram of
expression %ector for 1es8 as pre%iously described[$4!. he
%ector for enilla luciferase gene under the control of the SA'4 promoter was co*transfected as an internal standard to normalize the
http://www.plosone.org/article/info:doi/10.1371/journal.pone.0005603#pone.0005603-Bessho4http://www.plosone.org/article/info:doi/10.1371/journal.pone.0005603#pone.0005603-Matsui1http://www.plosone.org/article/info:doi/10.1371/journal.pone.0005603#pone.0005603-Niwa1http://www.plosone.org/article/info:doi/10.1371/journal.pone.0005603#pone.0005603-Bessho4http://www.plosone.org/article/info:doi/10.1371/journal.pone.0005603#pone.0005603-Bessho4http://www.plosone.org/article/info:doi/10.1371/journal.pone.0005603#pone.0005603-Matsui1http://www.plosone.org/article/info:doi/10.1371/journal.pone.0005603#pone.0005603-Niwa1http://www.plosone.org/article/info:doi/10.1371/journal.pone.0005603#pone.0005603-Bessho4
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transfected efficacy. +fter 6' h, bF)F were added to the
culture media and incubate for 6' h. hen, the cells were har%ested and
luciferase acti%ities were measured. o introduce mutations to the promoter of
Sprouty', site*directed and semi*random mutagenesis was performed aspre%iously described[''!. Primer seDuences . N*box& MutantE:+)++))::+++::+)):++)++)+:+:, -*
box& MutantE:):::+:::+:)::+)::+::::+, N*box6
MutantE+++))))+)+))):::+))+++:+++))::)), -*box6
MutantE::+:):+):++):)):+:):+):)::)::), -*box$ and N*box$
MutantE):):):+:)))))):)+::::+:::+:++.
Destern 8lotting
N(1 $$ cells were transfected with 4, 67, 74 nanogram of Hes7 expression
%ector. +fter '# hour, they were lysed in lysis buffer