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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, $otch&regulated an"yrin repeat protein ($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 .

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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&regulated 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

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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

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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&

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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&regulated

(2igure 18. Similar to the $-=;immunoreactivity, Hes5 , Hes7, and Lfng expression was cyclical in

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3172277/figure/F2/http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3172277/figure/F2/http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3172277/figure/F2/http://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/11485984http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3172277/figure/F3/http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3172277/figure/F3/http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3172277/figure/F3/http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3172277/figure/F3/http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3172277/figure/F2/http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3172277/figure/F2/http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3172277/figure/F2/http://www.ncbi.nlm.nih.gov/pubmed/11485984http://www.ncbi.nlm.nih.gov/pubmed/11485984http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3172277/figure/F3/http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3172277/figure/F3/http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3172277/figure/F3/http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3172277/figure/F3/


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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&regulate 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,

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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, *&'*

&#4, 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

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