3 与体节形成相关基因 The Genetics and Embryology of Zebrafish

SPECIAL ISSUE REVIEWS–A PEER REVIEWED FORUM

The Genetics and Embryology of Zebra?sh Metamerism

Scott A.Holley*

Somites are the most obvious metameric structures in the vertebrate embryo.They are mesodermal segments that form in bilateral pairs?anking the notochord and are created sequentially in an anterior to posterior sequence concomitant with the posterior growth of the trunk and tail.Zebra?sh somitogenesis is regulated by a clock that causes cells in the presomitic mesoderm(PSM)to undergo cyclical activation and repression of several notch pathway genes.Coordinated oscillation among neighboring cells manifests as stripes of gene expression that pass through the cells of the PSM in a posterior to anterior direction.As axial growth continually adds new cells to the posterior tail bud,cells of the PSM become relatively less posterior. This gradual assumption of a more anterior position occurs over developmental time and constitutes part of a maturation process that governs morphological segmentation in conjunction with the clock.Segment morphogenesis involves a mesenchymal to epithelial transition as prospective border cells at the anterior end of the mesenchymal PSM adopt a polarized,columnar morphology and surround a mesenchymal core of cells.The segmental pattern in?uences the development of the somite derivatives such as the myotome, and the myotome reciprocates to affect the formation of segment boundaries.While somites appear to be serially homologous,there may be variation in the segmentation mechanism along the body axis.Moreover, whereas the genetic architecture of the zebra?sh,mouse,and chick segmentation clocks shares many common elements,there is evidence that the gene networks have undergone independent modi?cation during evolution.Developmental Dynamics236:1422–1449,2007.?2007Wiley-Liss,Inc.

Key words:somite;segmentation;somitogenesis;notch;delta;clock;oscillator;wavefront;somite morphogenesis; myotome,sclerotome

Accepted22March2007

INTRODUCTION

The dorsal neural tube,notochord, pharyngeal pouches,and somites are the de?ning features of the vertebrate phylotype.The metameric pattern of the somites is inherited by the ribs and vertebral column and in?uences the development of other somite deri-vates such and the skeletal muscle and dermis.Somite number varies signi?cantly among the vertebrates, as adult frogs have6–9presacral ver-tebrae,certain amphibians form15or

fewer while some snakes have several

hundred.However,somite number

within a species is relatively constant

(Richardson et al.,1998).Zebra?sh

make30–32somites at a rate of

roughly one bilateral pair every30

min at28°C(Hanneman and Wester-

?eld,1989).Broadly speaking,seg-

mentation processes in higher ani-

mals can proceed by either of two

modes:simultaneous or sequential.As

the name suggests,simultaneous seg-

mentation generates all segments at

once.Thus,this process only subdi-

vides fully formed?elds of cells.Si-

multaneous segmentation is exempli-

?ed by the formation of rhombomeres

in the vertebrate hindbrain and seg-

mentation of long germ band insects

such as Drosophila.Sequential seg-

mentation is conceptually quite differ-

ent in that it involves the progressive

subdivision of a?eld of cells that is

The Supplementary Material referred to in this article can be found at https://www.doczj.com/doc/2a14796399.html,/jpages/1058-8388/suppmat Department of Molecular,Cellular and Developmental Biology,Yale University,New Haven,Connecticut

Grant sponsor:NICHD;Grant number:R01HD045738;Grant sponsor:the American Cancer Society;Grant number:RSG-07-05001-DDC.

*Correspondence to:Scott A.Holley,Department of Molecular,Cellular and Developmental Biology,Yale University,New Haven,CT06520.E-mail:scott.holley@https://www.doczj.com/doc/2a14796399.html,

DOI10.1002/dvdy.21162

Published online8May2007in Wiley InterScience(https://www.doczj.com/doc/2a14796399.html,).

DEVELOPMENTAL DYNAMICS236:1422–1449,2007

?2007Wiley-Liss,Inc.

3.084

segmentation occurs concomitant with the posterior growth of the em-bryo in short germ band insects,in-cluding the?ower beetle tribolium, and during vertebrate somitogenesis. Strikingly,the vertebrate embryo has the capacity to regulate somitogenesis to ensure that the appropriate num-ber of somites is generated even when the number of somite progenitors var-ies signi?cantly.For example,am-phibian blastula,in which up to one third of the blastomeres have been re-moved,can still give rise to a com-plete,yet diminutive,embryo.These smaller embryos form the same num-ber of somites,at the same rate,as unmanipulated sibling embryos,but each segment contains fewer cells. Thus,there is not a physical con-straint within the segmentation mechanism stipulating that a seg-ment must contain a speci?c number of cells(Cooke,1975).These observa-tions led to the“clock and wavefront”model of somitogenesis(Cooke and Zeeman,1975;Cooke,1981,1998).In this model,the clock causes oscilla-tions in gene transcription within the cells of the presomitic mesoderm (PSM),and cells are competent to form a somite only at a particular phase of the clock.The wavefront rep-resents the anterior to posterior pro-gression of development of the em-bryo.Thus,the wavefront sweeps along the body axis once and is corre-lated with extension of the trunk and tail.The wavefront is often referred to as the maturation front in that it gov-erns the maturation of the PSM.In the clock and wavefront model,the wavefront drives somite morphogene-sis,but its activity is gated by the clock:a somite border forms only when the wavefront reaches a group of cells in the appropriate phase of the clock.Thus,in this model,the size of each somite and the rate of somite formation are determined by the speed of the wavefront and the fre-quency of the oscillator(Supplemen-tary Movie S1,which can be viewed at https://www.doczj.com/doc/2a14796399.html,/ jpages/1058-8388/suppmat).

Other models have been proposed to explain somitogenesis by invoking Turing equations(Meinhardt,1982, 1986),a variation of the progress zone model of limb development(Kerszberg duction”model(Schnell and Maini,

2000),and models linking somite pe-

riodicity to the cell cycle(Primmett et

al.,1989;Collier et al.,2000;Stern

and Vasiliauskas,2000;McInerney et

al.,2004).More recently,models spe-

ci?c to the zebra?sh clock and incor-

porating increasingly detailed experi-

mental data have been published

(Lewis,2003;Horikawa et al.,2006;

Cinquin,2007).While it is not yet

clear if any of these models precisely

describe what happens in vivo,the in-

teraction between theory and experi-

mentation in the?eld of segmentation

has been unusually intense.

Here,a review of our understanding

of zebra?sh somitogenesis is pre-

sented,starting with speci?cation of

the somite progenitors in the blastula,

creation of the segmental pattern by

means of the somite clock,stabiliza-

tion of the segmental pattern by the

wavefront,consummation of segment

polarity,and execution of somite mor-

phogenesis.The relationships be-

tween segmentation and the differen-

tiation of the myotome and sclerotome

are reviewed.Subsequently,differ-

ences in somitogenesis along the ante-

rior–posterior(A-P)axis are discussed

and then mechanisms that ensure bi-

lateral symmetry in vertebrate seg-

mentation are reviewed.Finally,ze-

bra?sh somitogenesis is compared

with segmentation of other chordate

model organisms.

SPECIFICATION AND

MIGRATION OF THE

SOMITE ANLAGEN

Progenitors of the somites arise from

the ventral and lateral margin of the

zebra?sh blastula.While fate map-

ping studies show that the precursors

to the trunk and tail somites are in-

termingled,genetic studies demon-

strate that the anlagen of the anterior

trunk(somites1–9),posterior trunk

(somites10–15),and tail(somites

(16–30)are speci?ed before gastrula-

tion(Fig.1A;Kimmel et al.,1990;

Warga and Nusslein-Volhard,1999;

Szeto and Kimelman,2006).These an-

lagen are set-aside early in develop-

ment,and the cells remember when

they are allowed to enter the segmen-

tation program several hours later.

Direct reception of nodal is required

while bmp signaling speci?es the tail

somites(Szeto and Kimelman,2006).

nodal has an indirect role in specify-

ing posterior trunk fates by promoting

fgf expression in the margin(Mathieu

et al.,2004;Szeto and Kimelman,

2006).In turn,fgf appears to promote

posterior trunk by repressing bmp ex-

pression(Furthauer et al.,2004;Szeto

and Kimelman,2006).Later in devel-

opment,fgf signaling is required to

maintain the fates of most of the

somite anlagen as fgf8;fgf24double

morphants lack all but the anterior

2–3somites(Draper et al.,2003).wnt

signaling is also needed to maintain

posterior trunk and tail somite fates

as embryos lacking both wnt3a and

wnt8form only10–12somites

(Thorpe et al.,2005).These loss-of-

function studies are complemented by

experiments that have shown that ec-

topic tails can be induced by injection

of bmp4,nodal,and wnt8mRNA into

blastomeres at the animal pole,which

would normally give rise to ectoderm

(Agathon et al.,2003).Patterning by

nodal,fgf,and bmp signaling is medi-

ated largely through the t-box genes:

spadetail,no tail,and tbx6.spadetail

speci?es the anterior and posterior

trunk somites while no tail speci?es

the tail somites(Ho and Kane,1990;

Halpern et al.,1993;Amacher and

Kimmel,1998;Grif?n et al.,1998;

Grif?n and Kimelman,2002;Goering

et al.,2003).tbx6may function

semiredundantly with both spadetail

and no tail(Grif?n et al.,1998;Goer-

ing et al.,2003).Later still,fused

somites/tbx24interacts with the clock

and is required for somite maturation

while tbx15and tbx18are expressed

in the somites after segmentation

(Holley et al.,2000;Begemann et al.,

2002;Nikaido et al.,2002).Thus,

nodal,fgf,wnt,and t-box genes act in

succession,with some reiteration,to

link early patterning and mainte-

nance of the mesoderm to the subse-

quent segmentation program(re-

viewed in(Holley,2006a).

Fate mapping experiments indicate

that in the late blastula,the precur-

sors of trunk somites are located in

the dorsal–lateral margin,while the

tail somites arise from the ventral

margin(Fig.1A;Kimmel et al.,1990;

Warga and Nusslein-Volhard,1999).

During gastrulation,the more dorsal

and lateral cells converge toward the dorsal midline,while the ventral cells make up a“no convergence zone”that becomes the posterior tail bud as gas-trulation is completed(Myers et al., 2002).Fate mapping studies suggest that the anterior12somites are de-rived from the somite anlagen that underwent some dorsal convergence and were already in the segmental plate at the end of gastrulation(Mu¨l-ler et al.,1996;Kanki and Ho,1997; Ju¨lich et al.,2005a).Some of the pos-terior trunk somite anlagen and all of the tail somite precursors pass through the posterior tail bud or pro-genitor zone(Fig.1A–C).During the segmentation period,some somite precursor cells form a dorsal–medial domain above the notochord,continue to migrate posteriorly,and will dive ventrally into the progenitor zone at the posterior tip of the tail(Kanki and Ho,1997).Cells moving into the pro-genitor zone remain there for variable amounts of time.The progenitors of the posterior trunk somites13–15and the tail somites appear to be intermin-gled in the progenitor zone.Indeed, there is extensive cell mixing in both the progenitor zone and the initiation zone.It is not known how these dis-tinct progenitor populations maintain their positional identities as the two anlagens within the tail bud cannot be distinguished by patterns of gene ex-

pression(Kanki and Ho,1997;Mara

et al.,2007).

TAIL BUD AND PSM

The tail bud can be divided into four

regions:progenitor zone,initiation

zone,posterior PSM,and anterior

PSM(Fig.1C).The progenitor zone

is immediately posterior to the

chordal–neural hinge,while the ini-

tiation zone is lateral to the progen-

itor zone and posterior to the tip of

the notochord.The progenitor zone

contains the precursors for the more

posterior somites,and cells in the

progenitor zone appear to transcribe

the oscillating genes her1,her7,and

deltaC in a steady,nonoscillating

manner.Cells move laterally from

the progenitor zone into the initia-

tion zone where the her1,her7,and

deltaC oscillations seem to begin.In

10somite stage embryos,the poste-

rior PSM extends25cells anterior to

the posterior tip of the notochord.

This anterior boundary corresponds

to the point(?two cells)where

deltaC expression levels increase in

wild-type embryos and the anterior-

most extent of her1expression in

fss/tbx24?/?embryos.At the10

somite stage,the posterior PSM con-

tains the anlagen for approximately

?ve somites that each are roughly

?ve cells in length along the A-P axis

(Fig.1E).The anterior PSM contains

all cells lying anterior to the poste-

rior PSM and contains enough cells

for approximately two to three

somites(Mara et al.,2007).By con-

vention,the most recently formed

somite is the SI,while the second-

newest somite is the SII.In the PSM,

the S0,S-I,and S-II will sequentially

give rise to the next three segments

(Fig.1D;Pourquie and Tam,2001).

THE NOTCH PATHWAY

AND ZEBRAFISH

SEGMENTATION

The notch signaling pathway is one

of the most broadly used cell:cell

communication mechanisms in

metazoan development.Notch is a

large transmembrane receptor with

multiple extracellular EGF repeats

and a cytoplasmic domain with six

ankrin repeats and a RAM domain.

Notch binds to its transmembrane

ligands Delta/Jagged/Serrate on the

surface of adjacent cells.Ligand

binding causes Notch to be cleaved

in its transmembrane domain by

?-Secretase,releasing the cytoplas-

mic domain in the receiving cell.The

cytoplasmic domain translocates to

the nucleus where it interacts with

Fig.1.An overview of zebra?sh somitogenesis.A:The progenitors for the tail somites(yellow)and trunk somites(teal)are speci?ed in the late blastula4hours postfertilization(hpf),dorsal is right and ventral left(adapted with permission from the Company of Biologists(Warga and Nusslein-Volhard,1999).During gastrulation(8hpf),the anlagen for the anterior trunk somites(green)and some of the posterior trunk somite progenitors(orange)converge toward the dorsal midline,while some of the posterior trunk somite progenitors and the tail progenitors remain ventral.The arrows indicate the direction of epiboly toward the vegetal pole.Somites form sequentially with the anterior somites(green)forming before the posterior trunk somites(orange)and the tail somites(yellow).In the17hpf embryo,the unsegmented yellow region in the tail bud is the presomitic mesoderm.The anlagen of the posterior somites form a progenitor zone (red)in the tail bud posterior to the chordal–neural hinge.Somitogenesis is complete at25hpf.All views are lateral.B,C:Dorsal views of the tail bud.The progenitors of the posterior somites will exit the progenitor zone laterally(small arrows)to enter the initiation zone as the tail extends posteriorly(large arrow). Cells attenuate their movement and are relatively sessile in the PSM.The PSM is bilaterally symmetric and?anks the notochord and neural tube(not shown). D:A time series representing a single somite cycle showing the posterior displacement of the progenitor zone(red)as the tail extends.Oscillating gene expression(blue)sweeps from posterior to anterior.These oscillations cease in the anterior PSM,and expression of the gene disappears as morphological somite formation occurs.Note that the most-recently formed somite is called the SI while the region in the anterior PSM that will give rise to the next somite is the S0.As the S0becomes the SI,the old SI becomes the SII(Pourquie′and Tam,2001).E:Morphological somite formation occurs as mesenchymal PSM cells(light purple)undergo a mesenchymal to epithelial transition along the somite boundary(yellow).The somite consists of epithelial border cells(purple) and an internal core of mesenchyme(light blue).The adaxial cells(aquamarine)are a morphologically distinct row of medial cells that elongate along the anterior–posterior axis of each segment after somite formation and later give rise to slow muscle?bers.

Fig.2.Notch pathway genes and somitogenesis.Whether expression of the gene oscillates,yes(y)or no(n),is indicated.The expression patterns are depicted.The three most recently formed somites are shown with the anterior(a)and posterior(p)halves of each somite labeled.The tail bud is divided into the anterior presomitic mesoderm(PSM;a psm),posterior PSM(p psm),initiation zone(iz),and progenitor zone(pz).The bd indicates that the gene is required for normal oscillating gene expression or for segment polarity but that the phenotype is background dependent.The regions of the body axis that are affected in the mutant or morphant are indicated.Segments in green,yellow,or orange form normally in the mutant or morphant, whereas uncolored segments are missing or abnormal.The segments in the three shades of gray indicate an absence of the relevant loss-of-function data.References:a(Bierkamp and Campos-Ortega,1993),b(van Eeden et al.,1996),c(van Eeden et al.,1998),d(Holley et al.,2002),e(Westin and Lardelli,1997),f(Kortschak et al.,2001),g(Dornseifer et al.,1997),h(Holley et al.,2000),i(Smithers et al.,2000),j(Jiang et al.,2000),k(Ju¨lich et al., 2005b),l(Prince et al.,2001),m(Qiu et al.,2004),n(Topczewska et al.,2003),o(Ishitani et al.,2005),p(Jiang et al.,1996),q(Itoh et al.,2003),r(Sieger et al.,2003),and s(Echeverri and Oates,2007).

1424HOLLEY

the DNA binding protein Su(H)/ RBPJ?,converting it from a tran-scriptional repressor to an activator. notch signaling frequently activates the transcription of members of the

hairy/enhancer-of-split family of

transcriptional repressors.The abil-

ity of Delta to activate Notch re-

quires the ubiquitinylation of the cy-

toplasmic domain of Delta by an E3

ubiquitin ligase of either the Mind-

bomb or Neuralized family.This

ubiquitinylation causes the sending

cells to internalize Delta and the ex-

tracellular domain of Notch from the

adjacent cell.There is evidence that

Delta can also cell-autonomously in-

hibit the ability of a cell to receive a

Delta signal from an adjacent cell

(Bray,2006).The mouse Delta-

like-3,which is required for murine

segmentation,appears to speci?cally

function in this inhibitory manner

(Ladi et al.,2005).The fringe genes,

such as lunatic fringe,are glycosyl-

transferases that modify the EGF

domain of Notch while the receptor

passes through the Golgi.This mod-

i?cation biases Notch’s af?nity for

its different ligands.There are addi-

tional genes within the notch path-

way,but those mentioned above are

the ones that will be discussed here

in the context of zebra?sh segmenta-

tion.For a more detailed review of

notch signaling,see Bray(2006).

The?rst suggestion that the notch

pathway may play a role in zebra?sh

segmentation came from expression

analysis.

notch1a is expressed in the

hypoblast of the germ ring during

gastrulation and is later expressed

in the progenitor zone,initiation

zone,PSM,and posterior of each

somite(Fig.2)(Bierkamp and Cam-

pos-Ortega,1993).notch2,formerly

notch6,is expressed in the PSM,but

not in the posterior tail bud(progen-

itor zone and initiation zone).In the

most recently formed somites,

notch2is transcribed in the anterior

half of each segment and along the

dorsal and ventral surface of the

somite(Fig.2;Westin and Lardelli,

1997;Kortschak et al.,2001).

notch3,formerly notch5,is ex-

pressed in stripes in the anterior

PSM and in the posterior of each

somite(Fig.2;Westin and Lardelli,

1997;Kortschak et al.,2001).

notch1b is expressed in the posterior

of each somite but not the PSM(Fig.

2;Westin and Lardelli,1997).Two

notch ligands,deltaD and deltaC,

are important for zebra?sh somito-

genesis.deltaD is expressed in the

germ ring during gastrulation and

later in the progenitor zone,initia-

tion zone,and throughout the PSM Fig.1.

Fig.2.

GENETICS AND EMBRYOLOGY OF ZEBRAFISH METAMERISM1425

(Dornseifer et al.,1997).In the an-terior PSM,deltaD is up-regulated in a broad domain that re?nes into strong stripes of expression(Dornse-ifer et al.,1997;Holley et al.,2000, 2002).After segmentation,deltaD expression is enriched in the ante-rior half of each somite(Fig.2;Dorn-seifer et al.,1997).deltaC is ex-pressed in the germ ring during gastrulation,in the progenitor zone at the posterior tail bud and in a striped pattern in the PSM(Smith-ers et al.,2000).In the nascent somite,deltaC is transcribed in the anterior half of the segment,but during somite morphogenesis, deltaC expression switches polarity to be expressed in the posterior of each somite(Fig.2;Smithers et al., 2000;Ju¨lich et al.,2005b).lunatic fringe,is transcribed in a segmental pattern in the anterior PSM and in the anterior half of the nascent somites(Fig.2;Prince et al.,2001; Qiu et al.,2004).Zebra?sh have two homologues of nrarp,an intracellu-lar protein containing two ankrin re-peats,which acts as a negative reg-ulator of notch signaling(Lamar et al.,2001;Topczewska et al.,2003). The zebra?sh nrarp a mRNA is seen in the germ ring during gastrulation and throughout the progenitor zone, initiation zone,and posterior PSM.In the anterior PSM,nrarp a is ex-pressed in segmental stripes in the anterior half of the nascent and form-ing somite.nrarp b mRNA is observed in one segmental stripe in the anterior PSM but not elsewhere in the somitic mesoderm(Fig.2;Topczewska et al., 2003).mib and the two zebra?sh Su(H)homologues,Su(H)1and2,are each ubiquitously expressed during gastrulation and segmentation(Fig.2; Itoh et al.,2003;Sieger et al.,2003; Echeverri and Oates,2007).

The?rst functional evidence for the role of notch signaling in ze-bra?sh segmentation came from gain-of-function studies.Ectopic ex-pression of an activated form of notch consisting solely of the intra-cellular domain,called NIC or NICD (2007),perturbs the normal pattern of her1expression,segment polarity, and morphological somite formation (Takke and Campos-Ortega,1999). Similarly,overexpression of a domi-nant-negative form of the Xenopus suppressor of hairless homologue

that cannot bind DNA,X-Su(H)DBM,

perturbs morphological somite forma-

tion(Wettstein et al.,1997;Ju¨lich et

al.,2005a).It has been reported that

ectopic expression of either deltaD or

deltaC can perturb zebra?sh somito-

genesis(Takke and Campos-Ortega,

1999).Others have been unable to ob-

serve such an effect by overexpressing

either wild-type deltaD or deltaC,but

have observed segmentation defects

when overexpressing a chimeric delta

containing the extracellular and

transmembrane domain of deltaC and

the cytoplasmic domain of deltaD

(Mara et al.,2007).

TU¨BINGEN SEGMENTATION

MUTANTS

In the large-scale genetic screens per-

formed in Tu¨bingen and Boston,?ve

genes were found that are required for

normal segmentation of the meso-

derm:fused somites(fss),after eight

(aei),deadly seven(des),beamter

(bea),and white tail/mindbomb(mib;

Jiang et al.,1996;Schier et al.,1996;

van Eeden et al.,1996).These mu-

tants displayed defects in morpho-

logical segmentation and segment

polarity.The segmentation defects

subsequently led to mispatterning of

primary motoneurons and malformed

vertebral bodies(van Eeden et al.,

1996).mib embryos were shown to

have lost the segmental expression of

Myf-5and notch1a(Jiang et al.,1996).

The gene affected in each of these mu-

tants has been identi?ed and four of

the?ve are notch pathway genes.after

eight and beamter encode the Notch

ligands deltaD and deltaC,respec-

tively(Holley et al.,2000;Ju¨lich et al.,

2005b).deadly seven is the receptor

notch1a(Holley et al.,2002).mind-

bomb encodes an E3ubiquitin ligase

required for notch signaling(Itoh et

al.,2003).fused somites,the only gene

of the?ve that is not a notch pathway

gene,encodes the t-box family tran-

scription factor tbx24(Nikaido et al.,

2002).

SEGMENTATION CLOCK

The segmentation clock creates oscil-

lations in gene expression that mani-

fest as stripes that traverse the PSM

in a posterior to anterior direction

(Fig.1D).The initial genes shown to

oscillate were vertebrate orthologues

of the Drosophila pair-rule gene hairy,

a target of notch signaling.hairy or-

thologues in zebra?sh and mouse are

called her(hairy and enhancer of split-

related)or hes(hairy enhancer of split)

genes,respectively.The existence of a

segmentation clock was?rst demon-

strated in the chick by dissecting and

culturing the bilateral halves of the

PSM.Half was cultured for a longer

period of time before?xation and in

situ hybridization.These experiments

demonstrated that the position of the

stripes of c-hairy in the PSM changed

over time and that the cyclical charac-

teristic of these changes in gene ex-

pression correlated with the length of

the somite cycle(Palmeirim et al.,

1997).Due to the small size and mor-

phogenesis of the zebra?sh tail bud,

the bilateral culturing technique has

not yet been successfully applied to

zebra?sh.Instead,her1expression

was shown to oscillate by measuring

the distances between the myoD and

her1stripes in double in situ hybrid-

ization experiments.The distances be-

tween the myoD stripes,representing

the segmental pattern of the formed

somites,does not vary signi?cantly,

while the distances between the her1

stripes showed a large variation.

When embryos were staged at the

early or late12somites stage before

?xation,and the distances between

the her1stripes measured,it was

found that the stripes moved anteri-

orly and that distance between the

her1stripes decreased over develop-

mental time(Fig.3).These changes in

gene expression could not be ex-

plained by cell migration or cell death

(Holley et al.,2000).A similar,but

nonquantitative,set of experiments

suggested that her1expression oscil-

lated by analyzing the position of the

her1stripes relative to the segmental

stripes of mesp-a in the anterior PSM

(Sawada et al.,2000).deltaC expres-

sion was shown to oscillate using a

different methodology than the“stag-

ing-measurement”method applied to

her1.Jiang and colleagues used a

“heat gradient”to disjoin the rates of

development of the bilateral halves of

the PSM.Because the rate of ze-

bra?sh development is dependent

upon temperature,this caused the

warmer side of the tail bud to develop

1426HOLLEY

faster and the bilateral stripes of deltaC expression to be uncoupled.By incubating embryos in the heat gradi-ent for different lengths of time,the phases of bilateral deltaC expres-sion shifted in a reproducible pat-tern,indicating that deltaC expres-sion oscillated(Jiang et al.,2000).A subsequent study using the“staging-measurement”method examined deltaC and deltaD expression and showed that expression of the former oscil-lates while the latter does not(Fig.3; Holley et al.,2002).Subsequent stud-ies showed that her7,her11,her12, and her15oscillate by performing dou-ble in situ hybridization with her1and observing cyclical changes in expres-sion in correlation with changes in her1expression(Fig.3;Oates and Ho, 2002;Gajewski et al.,2003,2006; Sieger et al.,2004;Shankaran et al., 2007).

deltaC,her1,her7,her11,her12and her15all generally oscillate in phase, and they are all regulated by notch signaling.her1and her7are located head to head in the zebra?sh genome separated by11kb,and the two genes exhibit similar oscillating expression (Henry et al.,2002;Gajewski et al., 2003).However,there are notable dif-ferences in the expression of her11, her12,and her15.her11appears only to oscillate in the anterior half of the tail bud,while her12and her15oscil-lations are con?ned to the posterior half of the tail bud(Fig.3;Gajewski et al.,2006;Sieger et al.,2006;Shanka-ran et al.,2007).Control of oscillating gene expression differs between the anterior PSM and the rest of the tail bud.This observation was?rst re-vealed through study of the fss/tbx24 mutant,which only affects the oscil-lating expression in the anterior PSM, while the notch pathway mutants ex-hibit aberrant expression throughout the tail bud(van Eeden et al.,1998; Holley et al.,2000).Furthermore, transgenic analysis of the her1cis reg-ulatory elements demonstrates that distinct elements are responsible for driving expression in the anterior and posterior PSM(Gajewski et al.,2003). Fluorescent in situ hybridization using Tyramide Signal Ampli?cation (TSA)allows the identi?cation of dif-ferent phases of oscillation by means of the subcellular localization of https://www.doczj.com/doc/2a14796399.html,ing this method,one can see robust transcription of the chro-

mosomal loci at the anterior of each

stripe of her1,her7,and deltaC

expression(Ju¨lich et al.,2005b;

Horikawa et al.,2006;Mara et al.,

2007).More posteriorly,the nuclei?ll

with transcript and then the whole

cell?lls with the oscillating mRNA.In

the posterior of each stripe,cells with

no nuclear and decreasing levels of

cytoplasmic staining can be observed

as cells have ceased active transcrip-

tion and the mRNAs are degraded

(Ju¨lich et al.,2005b;Mara et al.,

2007).The oscillations of her1,her7,or

deltaC were characterized in detail by

examining the expression of each gene

in a large number of cells.In the pro-

genitor zone,her1,her7,and deltaC

begin to be expressed,but this expres-

sion does not appear to oscillate.Ini-

tiation of oscillation seems to be cou-

pled to exit from the progenitor zone

laterally into the initiation zone.How-

ever,oscillations in the initiation zone

do not appear to be as well synchro-

nized among neighboring cells as they

are in the posterior PSM.The appear-

ance of synchronized oscillations cor-

relates with an attenuation in cell

movement,suggesting that cell move-

ment is a source of noise for the seg-

mentation clock(Mara et al.,2007).

Mitosis also produces noise with

which the system must contend to es-

tablish and maintain synchrony

among the clocks of neighboring cells.

Visualization of her1mRNA with TSA

concomitantly with a green?uores-

cent protein(GFP)-tagged histone2B

showed that condensed or segregating

chromatin does not exhibit active

transcription of the clock genes,while

nondividing neighboring cells are

transcriptionally active.During every

30-min somite cycle,10–15%of cells

in the PSM undergo mitosis,which

minimally takes15min.Therefore,a

signi?cant portion of cells in the PSM

will have their clocks shifted out of

phase with their neighboring cells due

to mitosis.The segmentation clock

must buffer against this noise to

create the segmental prepattern

(Horikawa et al.,2006).

Notch Function in the

Segmentation Clock

The zebra?sh notch pathway mu-

tants,bea/deltaC,aei/deltaD,des/

notch1a,and mib,each form the ante-

rior three to nine somites before

segmentation breaks down(Jiang et

al.,1996;van Eeden et al.,1996).Ac-

cordingly,oscillating gene expression

is normal during anterior somitogen-

esis but gradually deteriorates before

the onset of the morphological defects

(van Eeden et al.,1998;Jiang et al.,

2000).By the eight somite stage,the

expression of her1,her7,or deltaC is

no longer in a striped pattern but is in

a disorganized“salt and pepper”pat-

tern.Double knockdown of Su(H)1

and2,disrupted the striped expres-

sion of her1,her7,and deltaC,al-

though the overall level of expression

was higher than observed in des/

notch1a mutants.The elevated ex-

pression suggests that,in addition to

their positive function in notch signal-

ing,the Su(H)homologues likely func-

tion as a repressor in the absence of

activated notch signaling(Sieger et

al.,2003;Echeverri and Oates,2007).

Double knockdown of both nrarp a

and nrarp b leads to elevated levels of

her1mRNA but has no apparent af-

fect on the oscillation of her1expres-

sion or on morphological segmenta-

tion(Ishitani et al.,2005).Morpholino

inhibition of notch3/5has no sig-

ni?cant effect on somitogenesis by

itself and can only cause a slight en-

hancement of the segmentation defect

seen in des/notch1a mutants(Susan

Truong and Scott Holley,unpublished

observations).

In aei/deltaD embryos,the salt and

pepper pattern is con?ned to the an-

terior PSM,while in bea/deltaC em-

bryos the salt and pepper expression

is found throughout the PSM(van Ee-

den et al.,1998;Holley et al.,2000,

2002;Jiang et al.,2000;Oates and Ho,

2002).The two delta mutants also dif-

fer in the time of onset of the segmen-

tation defect in that bea/deltaC em-

bryos form only three to?ve normal

anterior somites while aei/deltaD em-

bryos generate seven to nine proper

somites(van Eeden et al.,1997).

These differences suggest that these

two deltas have distinct functions in

the segmentation clock.Elucidation of

these functional differences provides

a resolution to the debate about

whether notch signaling exclusively

synchronizes the oscillations between

neighboring cells(Jiang et al.,2000)

or if notch also is fundamentally re-GENETICS AND EMBRYOLOGY OF ZEBRAFISH METAMERISM1427

sponsible for generating the oscilla-tions(Holley et al.,2000,2002).In short,the answer appears to be that some notch pathway genes drive the clock while others primarily act to synchronize the clocks of adjacent cells(Mara et al.,2007).

Both deltaD and deltaC are homo-logues of mammalian delta-like-1(un-published observations;Ladi et al., 2005).Nonetheless,they have differ-ent mutant phenotypes and a series of pharmacogenetic experiments using the deltaD and deltaC mutants indi-cate that the two deltas represent dis-tinct signals within the segmentation clock.deltaC cannot substitute for deltaD,and chimeric analysis sug-gests that functional differences be-tween the ligands are conferred by the coding sequence of both the extracel-lular and intracellular domain(Mara et al.,2007).

Examination of the expression of her1,her7,and deltaC expression us-ing high resolution?uorescent in situ hybridization strongly suggests that these genes do not oscillate in the pos-terior PSM of aei/deltaD embryos but that asynchronous oscillation persists in bea/deltaC embryos.Moreover,aei/

deltaD embryos show a decrease in

expression of her1,her7,and deltaC in

the progenitor zone.These data sug-

gest that the two deltas perform dis-

tinct functions in that deltaD drives

the oscillations in the initiation zone

and posterior PSM.deltaC appears to

primarily function in synchronizing

the oscillations among neighboring

cells in the PSM(Mara et al.,2007).

Given that the expression of deltaC

oscillates,it is well suited to couple

the oscillations of neighboring cells

(Jiang et al.,2000).In contrast,deltaD

expression does not oscillate,meaning

that it is likely a continuous signal

that helps to drive the oscillations

while deltaC levels?uctuate on top of

the basal level of deltaD.Experiments

combining genetic mosaics with high

resolution in situ hybridization sug-

gest that a cell needs both deltaD and

deltaC to provide a suf?ciently strong

signal to affect the clocks of neighbor-

ing cells.In these experiments,clones

of cells lacking her1and her7,and

thus de?cient in cyclic gene expres-

sion,shifted the phase of oscillation in

their neighboring wild-type cells.This

experiment demonstrated the cou-

pling of the clocks of adjacent cells,

and additional experiments showed

that the donor clones required both

deltaD and deltaC to in?uence the os-

cillations of their neighbors.Further-

more,the phase shift created by the

donor clones was successfully recapit-

ulated in a computational model of the

clock(Horikawa et al.,2006).

Additional evidence of the coupling

of the oscillations among neighboring

cells comes from mosaic experiments

in which wild-type cells from the pos-

terior PSM of one embryo are trans-

planted into the PSM of a host em-

bryo.Because the two embryos may

not be in the same phase of the somite

cycle at the time of transplantation,

the donor cells often show different

patterns of her1expression than the

host embryo when the mosaics are ex-

amined immediately after transplan-

tation.In contrast,mosaics examined

three somite cycles after transplanta-

tion showed synchronous oscillation of

donor and host cells(Horikawa et al.,

2006).

In models of the segmentation clock,

the instability of both the oscillating

Fig.3.hairy/enhancer of split-related genes in somitogenesis.Whether expression of the gene oscillates,yes(y)or no(n),is indicated.The expression patterns are depicted.The three most recently formed somites are shown with the anterior(a)and posterior(p)halves of each somite labeled.The tail bud is divided into the anterior presomitic mesoderm(PSM;a psm),posterior PSM(p psm),initiation zone(iz),and progenitor zone(pz).The bd indicates that the gene is required for normal oscillating gene expression or for segment polarity but that the phenotype is background dependent.The regions of the body axis that are affected in the mutant or morphant are indicated.Segments in green,yellow,or orange form normally in the mutant or morphant,whereas uncolored segments are missing or abnormal.The segments in the three shades of gray indicate an absence of the relevant loss of function data.Note that there are a variety of independently reported morphological defects caused by loss of her1function.Loss of her1in combination with loss of other genes can perturb all somites.References:a(Holley et al.,2000),b(Sawada et al.,2000),c(Holley et al.,2002),d(Henry et al.,2002),e(Oates and Ho,2002),f (Gajewski et al.,2003),g(Sieger et al.,2004),h(Sieger et al.,2006),i(Takke et al.,1999),j(Gajewski et al.,2006),k(Pasini et al.,2001),l(Pasini et al.,2004), m(Oates et al.,2005a),n(Shankaran et al.,2007),o(Kawamura et al.,2005b),and p(Winkler et al.,2003).

Fig.4.Gene networks governing zebra?sh segmentation.A:The oscillator gene network in the posterior tail bud is depicted.Through Notch1a,DeltaD drives expression of the oscillating her genes and deltaC.RPTP?acts upstream of,or in parallel to,the notch pathway.The Her genes act in a negative feedback loop to block transcription of the oscillating genes.After the Her proteins are degraded,transcription of the oscillating genes is reinitiated.Her proteins can act as homodimers or heterodimers,and possible dimerization partners are shown in brackets.The oscillating her genes are in blue.fgf signaling affects the clock by promoting the expression of her13.2in the posterior tail bud(red).Other,nonoscillating her genes are in gray.deltaC acts in a feedback loop to coordinate oscillations among neighboring cells.B:In the anterior tail bud,fgf and her13.2no longer in?uence the clock circuitry,but the oscillations do become dependent upon fss/tbx24(yellow).Note that the color-coding corresponds to that of Figure5.C:The clock,in conjunction with fss/tbx24, establishes segment polarity.mespb appears to promote anterior half segment fates and repress posterior half segment fates.Anterior and posterior half segments may interact to stabilize the segmental pattern.Segment polarity in?uences somite morphogenesis by means of transcriptional control of ephrin/eph genes and posttranscriptional regulation of Integrins and Cadherins.Genes differentially expressed in the anterior-half somite are notch2/6, deltaD,lfng,nrarp-a,nrarp-b,her6,her11,ripply1,papc,snail2,tbx15,ephA4,ephrinB2b,and fgf8.Genes differentially expressed in the posterior-half somite are notch1a,notch1b,notch3/5,deltaC,her4,hey1,ephrinA1,ephrinB2a,myoD,uncx4,and snail1a.

Fig.5.The wavefront and somitogenesis.The tail buds are depicted with the oscillating genes on the left half(blue)and wavefront genes on the right half(red and yellow).Note that the color-coding corresponds to that of Figure4.A:The wavefront consists of a gradient of fgf signaling and her13.2 expression in the posterior tail bud(red).In the anterior presomitic mesoderm(PSM),the wavefront is characterized by a gradual decrease in fgf and her13.2and the emerging activity of fss/tbx24(yellow).The fgf?her13.2and fss/tbx24domains progress posteriorly with the extension of the body axis.B:In the absence of fss/tbx24,oscillations occur normally in the posterior tail bud but disappear in the anterior PSM.Subsequently,there is no attempt to make segment borders.C:In the absence of normal clock function in the aei/deltaD mutant,oscillations of her1,her7,and deltaC expression do not occur in the posterior PSM.fss/tbx24promotes the anterior expression of the oscillating genes in a disorganized salt and pepper pattern.The salt and pepper pattern may represent asynchronous oscillations or an attempt to establish segment polarity in the absence of an oscillator-generated prepattern.This salt and pepper pattern arises in an anterior to posterior direction along the receding wavefront.A wave of irregular boundary formation follows the salt and pepper pattern.

1428HOLLEY

mRNAs and proteins is an important feature,an idea supported by com-putational analysis (Lewis,2003;Cinquin,2007).Interestingly though,embryos homozygous for the tortuga mutation display a posttranscrip-tional stabilization of the oscillating mRNAs her1,her7,and deltaC as re-vealed with in situ hybridization with an exon probe,but still have normal transcriptional oscillation of her1and deltaC as indicated by in situ hybrid-ization with an intron probe.In tor-tuga embryos,peaks and valleys of ex-pression are still observed,and these differential levels may be suf?cient to maintain the oscillating pattern even with persistent low levels of mRNA in the interstripe regions.Consistent with the normal transcriptional oscil-lations,tortuga mutants have no seg-mentation defect.her1,deltaC and her7oscillating mRNAs are differen-tially affected in the tortuga mutant in that the defects in her1expression are ?rst seen at the three somite stage,while the elevated levels of deltaC and her7mRNAs are ?rst observed at the 6and 16somite stage,respectively.With each mRNA,the persistent lev-els of mRNA gradually increase with each successive somite cycle (Dill

and

Fig.

3.

Fig.

4.

Fig.5.

GENETICS AND EMBRYOLOGY OF ZEBRAFISH METAMERISM

1429

?ect diminishing quantities of mater-nally supplied tortuga,whose molecu-lar identity is currently unknown,or may be indicative of changes in the clock function over time.

Her Function in the Segmentation Clock

her1and her7have been the most ex-tensively studied of the oscillating her genes.The functional analysis of her1 has been complicated by differing phe-notypes being reported for the her1 morphants.Injection of a translation-blocking morpholino against her1sta-bilizes the her1mRNA in addition to blocking production of protein.The initial report of the her1morphant phenotype did not recognize the mRNA stabilization(Holley et al., 2002),while subsequent studies did (Henry et al.,2002;Oates and Ho, 2002).A later study demonstrated the stabilization de?nitively by examin-ing her1morphants with both a probe against the full-length cDNA,which recognizes all transcripts,and with an intron probe,which recognizes only nascent unspliced transcripts.This analysis suggests that oscillations in her1transcription still occur in the posterior PSM of her1morphants,but that her1expression disappears in the anterior PSM(the same effect was seen on her7expression;Gajewski et al.,2003).Oates and Ho reported very weak effects on both her1,her7,and deltaC expression in the her1mor-phants(Oates and Ho,2002).In con-trast,two other reports showed aboli-tion of deltaC oscillation in the her1 morphants(Holley et al.,2002;Gajew-ski et al.,2003).Despite these differ-ences,it is clear that her1morphants have a weaker phenotype than her7 morphants,which lose the striped pat-tern of her1,her7,and deltaC(Fig.3; Oates and Ho,2002;Gajewski et al., 2003).Moreover,concomitantly elimi-nating both her1and her7,either by means of morpholinos(Oates and Ho, 2002)or chromosome deletion(Henry et al.,2002),leads to a stronger phe-notype.This result suggests that her1 and her7have partially overlapping functions within the zebra?sh somite clock.A similar result was attained with the functional analysis of her11. Knockdown of her11alone had very combination with inhibition of either

her1or her7led to a strong perturba-

tion of the oscillating expression of

her1and her7(Sieger et al.,2004).

Knockdown of her12perturbs the os-

cillating expression of her1,her7,and

deltaC,indicating a nonredundant

function for her12within the segmen-

tation clock.In contrast,inhibition of

her15alone or in combination with

other genes did not lead to a segmen-

tation defect(Shankaran et al.,2007).

Because the Her proteins function

as dimers,distinct species of homo-

and heterodimer could exist in differ-

ent regions of the tail bud and these

dimers could vary in their binding

speci?city,repressive activity,and

stability(Fig.4).Based on the pat-

terns of mRNA expression,Her1,

Her7,Her12,and Her15are present

throughout the tail bud.Her4.1ex-

pression should overlap with these

proteins in the progenitor zone(Fig.3;

Gajewski et al.,2006),while Her13.2

should be coexpressed with them

throughout the posterior tail bud(Fig.

3;Kawamura et al.,2005b).Her11

should overlap with the other oscillat-

ing Her proteins in the anterior and

posterior PSM.Experimentally,Her1

has been shown to interact with

Her13.2in a GST pull-down experi-

ment,and a luciferase reporter assay

demonstrated that cotransfection of

both her1and her13.2had a synergis-

tic effect in repressing reporter gene

expression driven by the her1en-

hancer.Thus,the Her1:Her13.2het-

erodimer may have a stronger repres-

sor activity than the Her1or Her13.2

homodimer.In vivo,morpholino

knockdown of her13.2leads to a

strong perturbation of the oscillating

expression of her1,her7,and deltaC,a

phenotype similar to that of the her7

morphant(Fig.3;Kawamura et al.,

2005b).It has also been noted that

knockdown of her7leads to a general

elevation in transcription of her1,sug-

gesting that her7is a transcriptional

repressor(Oates et al.,2005a).While

loss-of-function phenotypes suggest

that her7and her13.2act as repres-

sors in vivo,the her1morphant phe-

notype is more complicated.Given

that her1inhibits its own expression

in the luciferase assay,it is odd that

knockdown of her1leads to a loss of

her1and her7expression in the ante-

elimination of a repressor would lead

to elevated expression of its tar-

get genes(Gajewski et al.,2003;

Kawamura et al.,2005b).A resolution

to this apparent paradox is suggested

by computational modeling of the ze-

bra?sh clock.In testing13parame-

ters that were independently varied,

the her1morphant phenotype can be

accounted for if Her1and Her7form a

heterodimer and if this heterodimer

has a weaker repressive activity than

either homodimer or heterodimer

with Her13.2.In this model,the Her1/

Her7heterodimer acts as a“protective

species”that would bind to cis regula-

tory elements and prevent binding of

the more robust repressors(Cinquin,

2007).

The roles of her4and her6in somi-

togenesis are less clear.The expres-

sion of neither gene oscillates as her4

is expressed in the posterior of the tail

bud in the progenitor zone and in a

transient stripe in the nascent somite

(Fig.3;Takke et al.,1999;Gajewski et

al.,2006).her6is expressed in the an-

terior-most PSM and in the posterior

half of each somite(Fig.3;Pasini et

al.,2001).Ectopic expression of either

her4or her6has been shown to per-

turb somitogenesis(Takke and Cam-

pos-Ortega,1999;Pasini et al.,2004).

However,loss-of-function data for

these genes are less clear.It has been

reported that knockdown of either

her6alone or her6in combination with

her4leads to a perturbation of the os-

cillating expression of her1and deltaC

(Pasini et al.,2004).How loss of her6

could have this effect when the gene is

not expressed in the posterior PSM is

unclear.

hairy is a Drosophila pair-rule gene

meaning that it is expressed in every-

other segment and is required for the

formation of alternating segment

boundaries in the Drosophila embryo.

her1was initially reported to be

expressed in a pair-rule pattern

raising the possibility that Drosophila

and vertebrates shared segmentation

mechanisms(Mu¨ller et al.,1996).

However,a more recent study compar-

ing her1expression to the segmental

pattern of myoD showed that her1is

expressed in the anlagen of consecu-

tive somites(Holley et al.,2000).In

the initial study,Mu¨ller and col-

leagues labeled cells in the PSM that

that were in consecutive her1stripes and found that these cells were subse-quently incorporated into alternating somites.In retrospect,these results are likely due to the fact that,in the PSM,distances larger than a single somite can separate consecutive oscil-lating her1stripes.In a genetic screen for mutants with aberrant her1ex-pression,a mutant with a deletion of her1and the adjacent her7gene was isolated.The authors reported that al-ternating somite borders were af-fected in the her1;her7deletion mu-tant,resurrecting the idea that her1 (and perhaps her7)have pair-rule-like functions(Henry et al.,2002).In con-trast,others examining the knock-down phenotypes of her1and/or her7 have not observed a pair-rule-like phenotype(Holley et al.,2002;Oates and Ho,2002;Gajewski et al.,2003). In theory,the discrepancies in ob-served phenotypes could be due to dif-ferences between the phenotype of the deletion allele and morphants.While many consider this issue resolved in favor of non–pair-rule function,it re-mains a point of contention.In fact, her15has recently been reported to be expressed in the anlagen of alternat-ing segments(Fig.3;Shankaran et al.,2007).

Wnt Signaling and the Clock In the mouse,the wnt pathway plays a role in segmentation,and the wnt pathway genes axin2and nkd1oscil-late in the PSM.While axin2expres-sion does not appear to be under the control of notch,oscillating ndk1ex-pression is dependent upon hes7 (Aulehla et al.,2003;Ishikawa et al., 2004).Conversely,wnt3a is required for the oscillating expression of luna-tic fringe in the mouse(Aulehla et al., 2003).In zebra?sh,there is no direct evidence that wnt plays a role in gov-erning segmentation.The strongest indication that wnt signaling may have a function in zebra?sh segmen-tation comes from the analysis of re-ceptor protein tyrosine phosphatase?, RPTP?,whose human and mouse ho-mologues have been shown to bind to and dephosphorylate?-catenin. RPTP?is broadly expressed during the segmentation period.Knockdown of RPTP?abolishes the striped ex-pression of her1,her7,and deltaC and subsequently leads to a loss of seg-

mental expression of mesp-a,mesp-b,

and papc(Fig.6;Aerne and Ish-

Horowicz,2004).RPTP?is also re-

quired for normal convergence exten-

sion,a process controlled by

noncanonical wnt signaling,perhaps

suggesting that RPTP?in?uences

both branches of wnt signaling(Topc-

zewski et al.,2001;Jessen et al.,2002;

Aerne and Ish-Horowicz,2004).

THE WAVEFRONT

The wavefront represents the anterior

to posterior progression of develop-

ment during the segmentation period.

Mechanistically,it can be thought of

as the link between axis elongation

and morphological somite formation.

In zebra?sh,there are three genes

that participate as part of the wave-

front:fgf8,her13.2,and fss/tbx24

(Figs.4,5;Holley et al.,2000;Sawada

et al.,2001;Kawamura et al.,2005b).

fgf8and her13.2act in the posterior

tail bud(red in Figs.4,5),while fss/

tbx24functions in the anterior PSM

(yellow in Figs.4,5).A uni?ed model

for the action of these genes suggests

that fgf signaling couples growth of

the tail with the segmentation pro-

gram.fgf in the posterior keeps the

cells in an immature state and when

the cells escape in?uence of fgf in the

anterior PSM,the oscillations cease

and cells acquire a segmental identity.

her13.2links fgf signaling to the clock

in the posterior PSM,while the clock

becomes dependent upon fss/tbx24in

the anterior PSM(Fig.4A,B).fss/

tbx24is required to stabilize the oscil-

lations,establish segment polarity,

and initiate somite morphogenesis

(Fig.5B).

As in the mouse and the chick,fgf8

is expressed in a gradient in the pos-

terior tail bud with the highest levels

in the posterior(Dubrulle et al.,2001;

Sawada et al.,2001;Dubrulle and

Pourquie′,2004).The low end of the

gradient is in the rostral end of the

posterior PSM.This pattern of fgf8

results in a corresponding gradient of

fgf signaling as indicated by the dis-

tribution of phosphorylated ERK in

the tail bud.In the model,this gradi-

ent keeps cells oscillating and pre-

vents them from?xing their segmen-

tal identity in the posterior tail bud.

Evidence for this model comes from

chemical inhibition of fgf signaling us-

ing SU5402(Dubrulle et al.,2001;

Sawada et al.,2001).Transient addi-

tion of SU5402for8min at the two

somite stages causes the seventh and

eighth somite to be larger than usual,

because the gradient of fgf signaling

would have suddenly shrunk toward

the posterior.The reason that this

shift in the gradient does not affect

the third through sixth somites is

likely because(1)there is a time delay

before the SU5402can get into the

embryo to ef?ciently block fgf signal-

ing and(2)the anlagen for the third

through sixth somite would have al-

ready escaped the fgf gradient by the

time the drug took effect.This poste-

rior displacement of the fgf gradient

causes the oscillations of her1to cease

earlier/more posteriorly.The expres-

sion of anterior-speci?c genes such as

mesp-a is also moved to the posterior.

A complementary experiment in

which an?broblast growth factor-8

(FGF8)-soaked bead was implanted

next the PSM produced smaller

somites because it extended the FGF8

gradient anteriorly(Sawada et al.,

2001).In all,the drug and bead exper-

iments provide strong support to the

idea that a gradient of fgf signaling

controls the maturation of the PSM

(Dubrulle et al.,2001;Sawada et al.,

2001).However,there is no genetic

evidence for this model,as the ace/fgf8

mutant does not have a strong somite

defect and neither does a conditional

knockout of fgf8in the mouse(Reifers

et al.,1998;Perantoni et al.,2005).

There are several possible explana-

tions for the mutant phenotypes.First

is genetic redundancy,and there is

clear evidence for this in zebra?sh,as

an fgf8;fgf24double morphant does

not even form a tail(Draper et al.,

2003).Because the double morphant

lacks posterior mesoderm,the identi-

?cation of a speci?c effect on the seg-

mentation program is not possible.

However,it is easy to imagine that

fgf24could substitute for loss of fgf8

both in promoting tail formation and

in regulating somite maturation.A

second explanation for the phenotype

of the single mutants is that this pat-

terning system is regulative:a muta-

tion that constantly reduces the fgf

gradient can be compensated for,

while a sudden perturbation with

SU5402can produce a somite defect GENETICS AND EMBRYOLOGY OF ZEBRAFISH METAMERISM1431

planation for the lack of a segmenta-tion phenotype in the fgf8mutants is that other factors such as wnt signal-ing may be the operative molecule in vivo or that wnt may function in par-allel with fgf signaling to regulate pro-gression of the wavefront(Aulehla et al.,2003).

In the posterior tail bud,her13.2 connects fgf signaling to the clock (Figs.4A,5,red).her13.2is expressed in virtually the same domain in which phosphorylated ERK is observed:in the progenitor zone,initiation zone, and caudal half of the posterior PSM (Fig.3).her13.2expression is reduced in SU5402-treated embryos and ex-panded by implantation of an FGF8-soaked bead.Unlike her1,her13.2 expression is not effected by perturba-tion of notch signaling(Kawamura et al.,2005b).However,like the notch pathway mutants,morpholino knock-down of her13.2perturbs somite for-mation after the seventh to ninth somite and strongly disrupts the oscil-lating expression of her1,her7and deltaC(Fig.3;Holley et al.,2000; Jiang et al.,2000;Oates and Ho,2002; Kawamura et al.,2005b).Thus,while her13.2is regulated by fgf signaling, her13.2functions like the notch path-way components of the clock.Indeed, knockdown of her13.2has no effect on the expression of no tail or spadetail, both targets of fgf signaling (Kawamura et al.,2005b).

In the anterior PSM as cells escape the in?uence of fgf8and down-regu-late her13.2,the expression of the os-cillating genes becomes dependent upon fss/tbx24(Figs.4B,5,yellow; van Eeden et al.,1998;Holley et al., 2000).fss/tbx24is broadly expressed in the posterior and anterior PSM and in the anterior half of the two most recently formed somites(Fig.6;Ni-kaido et al.,2002).In fss/tbx24mu-tants,the oscillations occur normally in the posterior PSM but expression of these genes disappears in the anterior PSM(Fig.5B;van Eeden et al.,1998; Holley et al.,2000).This loss of ex-pression appears to be due to a failure to reinitiate expression of the oscillat-ing genes after?nishing the normal “off”phase of the cycle.These embryos do not establish segment polarity and do not make any somites(van Eeden ular borders in the posterior trunk

and tail,cells in the paraxial meso-

derm of fss/tbx24embryos do not even

make irregular borders and in fact fail

to undergo a mesenchymal to epithe-

lial transition(Fig.5B;Durbin et al.,

2000;Holley et al.,2000;Barrios et

al.,2003).

As a cell enters the segmentation

program in the initiation zone and be-

comes relatively less posterior as the

tail continues to extend,the architec-

ture of the oscillator circuit changes.

Initially,oscillating Her1will het-

erodimerize with Her13.2,which is

under the control of fgf8.This het-

erodimer acts in a negative feedback

loop to repress the expression of clock

genes(Fig.4A;Kawamura et al.,

2005b).As cells enter the anterior

PSM,other species of homo-and het-

erodimer,which may differ in their

repressive activity,are predicted to

replace Her13.2-containing het-

erodimers.At the same time,the os-

cillations become dependent upon fss/

tbx24(Fig.4B;van Eeden et al.,1998;

Holley et al.,2000).The maturation

front that controls somite formation

can be shifted posteriorly by tran-

siently inhibiting fgf activity while in

the absence of fss/tbx24somite matu-

ration does not occur at all(Holley et

al.,2000;Sawada et al.,2001).In the

absence of oscillations in the posterior

of aei/deltaD embryos,the wavefront

creates a salt and pepper pattern of

expression of the oscillating genes in

the anterior PSM followed by irregu-

lar border morphogenesis(Fig.4C;

Holley et al.,2000,2002).If fgf signal-

ing is inhibited in an aei/deltaD em-

bryo,this wavefront of salt and pepper

expression is again shifted to the pos-

terior(Sawada et al.,2001).In aei/

deltaD;fss/tbx24embryos,this salt

and pepper expression is lost(Holley

et al.,2002).Examination of her1,

her7,and deltaC expression in the an-

terior PSM of aei/deltaD embryos sug-

gests that her1and her7may oscillate

asynchronously but that deltaC ex-

pression does not(Mara et al.,2007).

(Decoupling of deltaC expression from

her1and her7is also seen when the

retinoic acid catabolizing enzyme,

cyp26a1,is inhibited(Echeverri and

Oates,2007)).A salt and pepper pat-

tern is not inherently indicative of os-

pepper pattern in aei/deltaD embryos

(Durbin et al.,2000;Sawada et al.,

2000).

SEGMENT POLARITY

Within the anterior PSM,segment po-

larity is established by means of con-

summation of the pattern created by

the clock(Fig.4C,D).Not surpris-

ingly,mutations that affect clock func-

tion also perturb segment polarity,

with markers of anterior and posteri-

or-half somites generally expressed in

a disorganized manner throughout

the somitic mesoderm(van Eeden et

al.,1996;Durbin et al.,2000).Three

genes,fss/tbx24,ripply1,and foxc1a,

play early roles in establishing seg-

ment polarity,with fss/tbx24and rip-

ply1also having effects on oscillating

gene expression in the anterior PSM

(Fig.4C;van Eeden et al.,1998;Hol-

ley et al.,2000;Topczewska et al.,

2001a;Kawamura et al.,2005a).

Downstream of fss/tbx24,the pathway

to segment polarity branches with fss/

tbx24,ripply1,and foxc1a upstream of

mesp-b,which appears to promote an-

terior half-somite identity.mesp-a ex-

pression overlaps that of mesp-b and

is downstream of fss/tbx24but not rip-

ply1or foxc1a(Fig.4C;Durbin et al.,

2000;Sawada et al.,2000;Topcze-

wska et al.,2001a;Kawamura et al.,

2005a;Oates et al.,2005b).

fss/tbx24,which is broadly ex-

pressed in the PSM,is required for the

expression of ripply1,foxc1a,her4,

mesp-b,and mesp-a(Fig.6;unpub-

lished observations;Durbin et al.,

2000;Sawada et al.,2000;Topcze-

wska et al.,2001a;Kawamura et al.,

2005a;Oates et al.,2005b).Further-

more,some markers of anterior half-

somites such as ephA4,ephrinB2b,

lfng,fgf8,and papc and a few markers

of the posterior half-somite such as

ephrinA1and notch3/5are not ex-

pressed in fss/tbx24mutants.Other

genes that are normally segmentally

expressed in the posterior half-somite,

including myoD,ephrinB2a,deltaC,

notch1a,and notch1b,and anterior-

half somites such as notch2and

deltaD are expressed throughout the

somitic mesoderm(unpublished ob-

servations;van Eeden et al.,1996;

Durbin et al.,2000;Holley et al.,2000;

al.,2001a;Barrios et al.,2003; Kawamura et al.,2005a;Oates et al., 2005b).

Genetic mosaic experiments indi-cate that the dependence of her1ex-pression on fss/tbx24is cell autono-mous as is the expression of mesp-b, papc,and fgf8(Holley et al.,2000; Oates et al.,2005b).However,wild-type cells transplanted into an fss/ tbx24?/?embryo can induce sur-rounding host cells to express notch3/5.mesp-b,papc,and fgf8are expressed in the anterior half-somite, while notch3/5is a posterior marker. Thus,fss/tbx24may function primar-ily cell-autonomously to promote ex-pression of anterior half-somite fates which then induce or promote the adoption of posterior fates in neigh-boring cells(Oates et al.,2005b). Therefore,there is cell–cell signaling in the S-II,S-I,and S0that helps con-vert the oscillator-generated pattern into segment polarity.This fss/tbx24-dependent,anterior to posterior wave of cell–cell communication may con-tribute to the salt and pepper pattern of gene expression in the anterior PSM of the notch pathway mutants such as aei/deltaD?/?,as this salt and pepper pattern is missing in fss/ tbx24;aei/deltaD double mutants(Hol-ley et al.,2000).

Ripply1is a nuclear protein with a WRPW domain,which allows it to physically interact with Groucho,a corepressor that also binds to the WRPW domain of Hairy-related pro-teins(Paroush et al.,1994;Kawamura et al.,2005a).ripply1is expressed in a segmental pattern in the anterior PSM and in the anterior half the most recently formed somites.A related gene,ripply2shows only the striped PSM expression(Fig.6).ripply1 mRNA is largely absent in fss/tbx24 mutants and is not segmented in aei/ delta?/?,mib?/?,or mesp-b morpho-lino-injected embryos.Ectopic expres-sion of ripply1repressed mesp-b expression,but not mesp-a.Inhibition of ripply1does not eliminate the striped pattern of mesp-b,but does cause mesp-b expression to persist throughout the somitic mesoderm. These results suggest that Ripply1 acts as a transcriptional repressor and that ripply1and mesp-b regulate each other’s expression.Genetic mosaics by ripply1is cell autonomous.By con-

trast,overexpression or inhibition of

ripply1has little effect on mesp-a,

again underscoring the difference in

regulation of the two mesp homo-

logues(Fig.4C).ripply1does not af-

fect the oscillations but does cause ex-

pression of her1to persist within the

somitic mesoderm.deltaC,deltaD,

fgf8,and myoD are also expressed

throughout the somitic tissue.ripply1

appears to function downstream of

fss/tbx24in promoting the maturation

of PSM to somitic mesoderm(Fig.4C;

Kawamura et al.,2005a).

foxc1,a forkhead/winged helix class

transcription factor,is broadly tran-

scribed throughout the PSM with par-

ticularly strong,somewhat segmental

expression in the S0and S-I(Fig.6;

Topczewska et al.,2001b).Foxc1a pro-

tein is evenly distributed in the nuclei

of the PSM and nascent somites.The

transcription of foxc1a in the anterior

PSM is dependent upon fss/tbx24.

Like fss/tbx24and ripply1,inhibition

of foxc1a perturbs formation of all

somites.Knockdown of foxc1a leads to

a loss mesp-

b expression but has no

affect on mesp-a.In the anterior PSM

and nascent somites of the mor-

phants,stripes of ephrinB2a were

fused but papc expression was nor-

mal.notch3/5and notch2/6expression

was strongly reduced.Oscillating ex-

pression of her1and deltaC was unaf-

fected,but the expression of deltaC in

the somite was missing(Topczewska

et al.,2001a).foxc1a,therefore,specif-

ically affects the transition in deltaC

expression from the anterior half of

the S0to the posterior half of each

somite(Ju¨lich et al.,2005b).In sum-

mary,foxc1a is a broadly expressed

gene that acts downstream of fss/

tbx24to help establish segment polar-

ity(Fig.4C;Topczewska et al.,2001a).

mesp-a and mesp-b are basic helix–

loop–helix(bHLH)genes,homologous

to the thylacine genes of Xenopus and

mesp genes in the mouse,the latter of

which have been shown to play a ma-

jor role in establishing segment polar-

ity during mouse somitogenesis(Saga

et al.,1997;Sparrow et al.,1998;

Durbin et al.,2000;Sawada et al.,

2000;Takahashi et al.,2000,2003).

mesp-a and b are expressed in a seg-

mental pattern in the S0,S-I,and

S-II.mesp-b stripes are in the future

expression initially encompasses all of

the S-II and re?nes to only the ante-

rior half of the S-I(Fig.6;Durbin et

al.,2000;Sawada et al.,2000).mesp-a is

expressed in a faint,disorganized man-

ner in aei/deltaD?/?,bea/deltaC?/?,

des/notch1a?/?,and mib?/?embryos

and exhibits a stronger salt and pepper

pattern in her7morpholino-injected em-

bryos(Durbin et al.,2000;Sawada et

al.,2000;Oates et al.,2005b).mesp-b

mRNA is in a salt and pepper pattern in

bea/deltaC?/?and mib?/?(Sawada et

al.,2000).Expression of both genes is

absent in fss/tbx24?/?(Durbin et al.,

2000;Sawada et al.,2000).Ectopic ex-

pression of mesp-a causes a defect in

gastrulation precluding an analysis of

its effect on segmentation.Ectopic ex-

pression of mesp-b blocks somite forma-

tion,inhibits the expression of myoD

and notch3/5,which are normally ex-

pressed in the posterior of each somite,

and expands the expression of fgfr1,

notch2/6,and papc,which are nor-

mally expressed in the anterior half of

each somite(Sawada et al.,2000).

Overexpression of mesp-b did not af-

fect either her1or mesp-a expression.

mesp-b appears to act downstream of

fss/tbx24and foxc1a to promote ante-

rior-half somite and repress posterior-

half somite fates(Fig.4C).

Two zebra?sh gadd45?(Growth Ar-

rest and DNA Damage)homologues

are expressed in a single stripe in the

anterior PSM(Fig.6).Mammalian

gadd45?has been implicated in cell

cycle control.Simultaneous knock-

down of both homologues blocks for-

mation of all somites and perturbs the

segmental expression of mesp-a and

myoD.her1expression is also affected

in the morphants,but still appears to

oscillate.Interestingly,the posterior

domain of fgf8expression is expanded

anteriorly but the expression of fss/

tbx24and the posteriorly expressed

genes such as wnt3a and tbx6are un-

affected.Overexpression of gadd45?

represses mesp-a and myoD transcrip-

tion(Kawahara et al.,2005).The ex-

act role of gadd45?in somitogenesis is

currently unclear.It could be involved

in either establishing segment polar-

ity or regulating the maturation pro-

gram.

There are indications that speci?c

delta/notch signals may help establish

anterior and posterior half-somite

identities by means of cell:cell commu-nication.For example,deltaD and deltaC are expressed in the anterior and posterior half-somite,respectively (Dornseifer et al.,1997;Smithers et al.,2000).notch3/5expression in the posterior half-somite is lost in em-bryos lacking deltaC but not in aei /deltaD ?/?embryos (Oates et al.,2005a).Similarly,the hairy/enhancer of spilt orthologue,hey1is differen-tially affected in these mutants.hey1is expressed in the posterior half of each somite and in the anterior PSM.In wild-type embryos,the level of ex-pression in the most recently formed one to two somites is always

weaker

Fig.

6.

Fig.7.

Fig.6.Nonnotch pathway genes involved in pat-terning the presomitic mesoderm (PSM)and/or establishing segment polarity.Whether expres-sion of the gene oscillates,yes (y)or no (n),is indicated.The ?y indicates that fss/tbx24is only necessary for normal expression of the oscillating genes in the anterior PSM.The ?y indicates that loss of fgf8may have a mild effect on somite pattern.The expression patterns are depicted.The three most recently formed somites are shown with the anterior (a)and posterior (p)halves of each somite labeled.The tail bud is divided into the anterior PSM (a psm),posterior PSM (p psm),initiation zone (iz),and progenitor zone (pz).The regions of the body axis that are affected in the mutant or morphant are indicated.Segments in green,yellow,or orange form nor-mally in the mutant or morphant,whereas uncol-ored segments are missing or abnormal.The seg-ments in the three shades of gray indicate an absence of the relevant loss of function data.References:a (Aerne and Ish-Horowicz,2004),b (van Eeden et al.,1996),c (van Eeden et al.,1998),d (Durbin et al.,2000),e (Holley et al.,2000),f (Nikaido et al.,2002),g (Reifers et al.,1998),h (Kawamura et al.,2005a),i (Topczewska et al.,2001b),j (Topczewska et al.,2001a),k (Durbin et al.,2000),l (Sawada et al.,2000),and m (Kawa-hara et al.,2005).

Fig.7.Segmentally expressed genes involved in somite morphogenesis and/or commonly used as markers for somite pattern.Whether expression of the gene oscillates,yes (y)or no (n),is indi-cated.The expression patterns are depicted.The three most recently formed somites are shown with the anterior (a)and posterior (p)halves of each somite labeled.The tail bud is divided into the anterior presomitic mesoderm (PSM;a psm),posterior PSM (p psm),initiation zone (iz),and progenitor zone (pz).References:a (Begemann et al.,2002),b (Durbin et al.,1998),c (Barrios et al.,2003),d (Yamamoto et al.,1998),e (Sawada et al.,2000),f (Weinberg et al.,1996),g (Kawakami et al.,2005b),h (Hammerschmidt and Nu ¨sslein-Vol-hard,1993),i (Thisse et al.,1993),j (Nisha Tam-hankar and Scott Holley,unpublished observa-tions),k (Kudoh et al.,2001),and l (Clements and Kimelman,2005).

1434

HOLLEY

expression domain,albeit disorga-nized,is seen in aei/deltaD?/?em-bryos but not in bea/deltaC or des/ notch1a mutants(Winkler et al., 2003;Sieger et al.,2004).In contrast, her11,which is expressed in an oscil-lating pattern in the PSM and in the anterior of the nascent somites,dis-plays a similar expression pattern in the these three notch pathway mu-tants.her11expression is sometimes missing in the region of the nascent somites in aei/deltaD?/?,bea/ deltaC?/?,and des/notch1a?/?em-bryos(Sieger et al.,2004).This vari-able expression domain is too far anterior to likely be due to oscillations and probably results from an attempt to generate some segment polarity in these mutant embryos.Note that ex-pression of hey1and notch3/5are dif-ferentially affected in the mutants while her11is not.This difference is perhaps due to hey1and notch3/5be-ing normally expressed in the poste-rior half-somite,while hey1is ex-pressed in the anterior half of each somite.Together,these segment po-larity phenotypes suggest that deltaD and deltaC represent distinct signals within the somites that help establish segment polarity by means of cell:cell communication.

SOMITE MORPHOGENESIS As somite polarity is established,mor-phological segmentation commences. Somite morphogenesis involves a mes-enchymal to epithelial transition (MET)of the boundary cells.The typ-ical trunk somite averages around?ve cells in length and consists of epithe-lial anterior and posterior boundary cells separated by internal mesenchy-mal cells(Fig.1E).The internal mes-enchymal cells are not needed for seg-mentation to occur as knypek;trilobite double mutants form somites consist-ing of only a row of anterior boundary cells and a row of posterior boundary cells.In wild-type embryos,there is little cell movement in the anterior PSM and only local cell jostling among nascent boundary cells(Henry et al., 2000).The initiation of border mor-phogenesis is marked by the cluster-ing Integrin?5,along the basal side of the prospective boundary cells(Fig. 8A1)and alignment of the nuclei(not thereafter,a?bronectin matrix begins

to form between adjacent segments

(not shown;Crawford et al.,2003;

Ju¨lich et al.,2005a).Cells then begin

to adopt a columnar morphology with

basally aligned nuclei,a process that

continues as the somite matures to be

the SIII(Fig.8A2;Henry et al.,2000).

Filamentous actin becomes enriched

at the boundary(Fig.8A2;Barrios et

al.,2003).Paxillin and activated,and

phosphorylated Focal Adhesion Ki-

nase(Fak)also accumulates along the

basal side of the boundary cells(Fig.

8A3;Henry et al.,2001;Crawford et

al.,2003).The centrosomes localize

apically(Fig.8A2),and?-catenin is

enriched along the apical cortex of the

boundary cells(Fig.8A3;Barrios et

al.,2003).Several hours after the

somite initially forms,a Laminin ma-

trix is assembled along the somite

boundary(Crawford et al.,2003).

Somite morphogenesis appears to

depend upon the coordinated action of

eph/ephrin,integrin,cadherin,and

notch signaling.Eph proteins are re-

ceptor tyrosine kinases that bind to

their ligands,Ephrins on the surface

of adjacent cells.Ephrins exist in two

forms,a six glycosylphosphatidyli-

nositol(GPI)-linked Ephrin-A class,in

which the GPI tethers the protein to

the plasma membrane,or the trans-

membrane Ephrin-B class ligands.

The Ephrin-B class can activate recep-

tors and also transduce a signal cell-

autonomously by means of its cyto-

plasmic domain.There are two classes

of Eph receptors,EphA and EphB,

that bind to either the Ephrin-A or

Ephrin-B ligands,respectively,al-

though there are examples of promis-

cuous interactions between the two

classes(Pasquale,2005).In the ze-

bra?sh,ephA4and ephrinB2b are ex-

pressed in the PSM,and in the ante-

rior of the S-I,S0,and nascent

somites.ephrinA1mRNA is seen in

the posterior tail bud,throughout the

S-I,and in the posterior of the S0and

each somite,while ephrinB2a is ex-

pressed in the posterior of S-I,S0and

each segment(Fig.7;Durbin et al.,

1998;Barrios et al.,2003).EphA4can

bind to EphrinB2a and EphrinA1,

while an EphB can only interact with

EphrinB2a.Ectopic expression of

ephrinB2a,but not ephA4or ephB,

causes a morphological segmentation

expression of myoD,fgf8,deltaD,

paraxis,and her1.Secreted forms of

the Ephrins or variants of the recep-

tors lacking the cytoplasmic/kinase

domain have a dominant-negative ac-

tivity when ectopically expressed and

produced somite defects.Together,

these experiments suggest that eph/

ephrin signaling is involved in regu-

lating somite morphogenesis and

somite polarity(Durbin et al.,1998).

The paraxial mesoderm cells in fss/

tbx24embryos do not attempt somite

morphogenesis and do not undergo

the mesenchymal to epithelial transi-

tion(Durbin et al.,2000;Holley et al.,

2000;Barrios et al.,2003).Expression

of zebra?sh paraxis,the homologue of

a mouse gene required for somite epi-

thelialization,is maintained in fss/

tbx24?/?embryos(Burgess et al.,

1996;Shanmugalingam and Wilson,

1998;Topczewska et al.,2001a).fss/

tbx24mutant embryos do lack expres-

sion of ephA4,and genetic mosaic ex-

periments show that clones of ephA4-

expressing cells in fss/tbx24?/?embryos

form borders at the interface of the

donor and host cells(Durbin et al.,

2000;Barrios et al.,2003).ephA4

causes the expressing cells to adopt

a columnar morphology and to api-

cally localize?-catenin.Additionally,

ephA4induces the nuclei of adjacent

host cells to align basally and centro-

somes to reside apically(Barrios et al.,

2003).Other aspects of normal border

formation are absent from the ephA4-

expressing clones,suggesting that ad-

ditional factors downstream of fss/

tbx24are necessary for a complete

MET.

Cadherins are located around the

cell cortex of cells in the PSM and in

the mature somites(Crawford et al.,

2003).Paraxial protocadherin,papc,

mRNA is seen throughout the tail

bud and S-II but re?nes to the ante-

rior half of S-I,S0,SI,and SII(Fig.

7;Yamamoto et al.,1998;Sawada et

al.,2000).Ectopic expression of a

full-length papc does not affect seg-

mentation,but injection of a se-

creted form encoding three extracel-

lular cadherin domains perturbs

somite morphogenesis and myoD ex-

pression(Yamamoto et al.,1998).

These experiments suggest that,

while regulation of papc expression

is not needed for normal somitogen-

esis,homophilic interactions among cadherins are necessary.

Integrins are heterodimeric trans-membrane proteins,consisting of an ?and a?subunit,that link the extracellular matrix to the actin cy-toskeleton.Integrins can signal bidi-rectionally to modify the extracellu-lar matrix or to alter the polarity of the cystoskeleton and affect gene ex-pression(Hynes,2002).Intracellular effectors of Integrins include the adapter protein Paxillin and Fak, both of which are localized to the basal side of the somite boundary cells(Fig.8A;Hynes,2002;Craw-ford et al.,2003).Integrin?5?1is the primary receptor for?bronectin, and Integrin?6?4is the Laminin receptor(Hynes,2002).

mRNAs for the integrin-associated genes are generally not observed in a segmental pattern,suggesting that regulation of these genes is largely posttranscriptional.The exceptions to this are fak1a and?bronectin1b (fn1b),formerly?bronectin3.fak1a is transcribed in the anterior PSM and in a segmental pattern in the posterior of each somite,and this pattern is perturbed in fss/tbx24?/?embryos and in the notch pathway mutants(Henry et al.,2001).fak1b, in contrast,is ubiquitously ex-pressed during somitogenesis(Craw-ford et al.,2003).Indeed while Fak protein is seen in all cells in the pre-somitic and somitic mesoderm,Fak and phosphorylated/activated Fak is enriched along the somite boundary (Henry et al.,2001;Crawford et al., 2003).fn1b is expressed in the PSM and in the somites,while fn1a is ex-pressed in the posterior tail bud and ubiquitously during gastrulation.in-tegrin?5is transcribed ubiquitously at the shield stage,but is restricted to the posterior tail bud and adaxial cells during somitogenesis(Ju¨lich et al.,2005a;Koshida et al.,2005).in-tegrin?1mRNA is seen in the poste-rior tail bud and throughout the somites(Ju¨lich et al.,2005a). Mutation of either integrin?5or fn1a leads to a failure to maintain somite boundaries in the anterior seven to nine somites(Ju¨lich et al., 2005a;Koshida et al.,2005).Knock-down of fn1b leads to a mild exten-sion defect and a failure to maintain all somite boundaries,while elimi-nation of both fn1a and fn1b leads to

a severe truncation of the body axis,

suggesting that the two genes are

partially redundant(Ju¨lich et al.,

2005a).Loss of integrin?5does not

affect expression of the oscillating

genes or mesp-b but does perturb the

segmental expression of myoD.In in-

tegrin?5?/?embryos,the boundary

cells do not show a polarized distri-

bution of the centrosomes or phos-

phorylated Fak and the Fn matrix is

disjointed.These observations indi-

cate that integrin?5/fn function is

required for the completion and/or

maintenance of MET(Ju¨lich et al.,

2005a;Koshida et al.,2005).Inte-

grin signaling may function with

Eph/Ephrin signaling during somite

morphogenesis as morpholino knock-

down of ephrinB2a only leads to a

short delay in boundary formation,

while inhibition of both ephrinB2a

and integrin?5leads to a broad fail-

ure to maintain the borders of all

somites(Koshida et al.,2005).

Integrin?5-GFP clusters along the

basal side of the somite boundary

cell in wild-type embryos and in the

irregular boundaries that form in

the posterior of the notch pathway

mutants(Fig.8A).These irregular

boundaries require integrin?5,as

they do not form in the double mu-

tants between integrin?5and either

aei/deltaD,bea/deltaC,or des/

notch1a.In fact,in the double mu-

tants,cells of the paraxial mesoderm

fail to undergo any MET.Because

MET occurs in the posterior of the

integrin?5mutant and along the ir-

regular boundaries in the posterior

of the notch pathway mutants,the

absence of MET in the double mu-

tants is a synergistic genetic interac-

tion between the mutations.This

synergy suggests that notch and in-

tegrin?5signaling may work in con-

cert to cause MET during somite

morphogenesis.The exact relation-

ship between delta/notch,ephrin/

eph,and integrin?5signaling in

somite border morphogenesis are not

understood.The parsimonious ex-

planation is that notch de?nes dis-

crete domains of ephrin/eph expres-

sion,which then leads to local

activation of integrin?5in the pro-

spective somite boundary cells

(Ju¨lich et al.,2005a).

SEGMENTATION AND

DIFFERENTIATION OF THE

SOMITE DERIVATIVES

Myotome

After segmentation,the zebra?sh

somite is subdivided into myotome,

sclerotome,and dermomyotome,which

give rise to the musculature,verte-

brae,and myogenic progenitors that

facilitate subsequent growth of the

skeletal muscle(Morin-Kensicki and

Eisen,1997;Stickney et al.,2000;

Morin-Kensicki et al.,2002;Devoto et

al.,2006;Hammond et al.,2006;Holl-

way et al.,2007;Stellabotte et al.,

2007).Here,aspects of the somite de-

rivatives that speci?cally relate to

segmentation are reviewed in detail.

The zebra?sh myotome differenti-

ates into the muscle pioneers,slow

muscle and fast muscle,with the bulk

of the myotome becoming fast muscle.

The muscle pioneers are early-devel-

oping,Engrailed-expressing slow

muscle?bers that are located at the

level of the horizontal myoseptum

that divides the myotome into dorsal

and ventral halves(Waterman,1969;

van Raamsdonk et al.,1978,1982;

Felsenfeld et al.,1991;Hatta et al.,

1991).The muscle pioneers,slow mus-

cle precursors,and horizontal myosep-

tum are missing in embryos in which

transduction of hedgehog signaling

to the somites is perturbed,and

the somites in these embryos are

u-shaped,rather than the chevron

shape seen in wild-type embryos(Cur-

rie and Ingham,1996;Devoto et al.,

1996;van Eeden et al.,1996;Blagden

et al.,1997;Schauerte et al.,1998;

Karlstrom et al.,1999;Lewis et al.,

1999;Barresi et al.,2000;Chen et al.,

2001;Coutelle et al.,2001;Du and

Dienhart,2001;Roy et al.,2001;Bax-

endale et al.,2004;Hirsinger et al.,

2004;Sekimizu et al.,2004;Wilbanks

et al.,2004;Wolff et al.,2004;

Kawakami et al.,2005a;Woods and

Talbot,2005;Feng et al.,2006;van

der Meer et al.,2006).

The slow muscle?bers,including

the muscle pioneers,exhibit an inter-

esting developmental progression in

that they are derived from the adaxial

cells in the medial somites but mi-

grate through the somite to the lateral

surface(Figs.1E,8).After segmenta-

tion,the15–20columnar adaxial cells

1436HOLLEY

in a somite are arranged roughly 4?4along the anterior–posterior and dor-sal–ventral axes.In the SI and SII,the adaxial cells can be seen rearrang-ing such that they stack along the dor-sal–ventral axis and each cell extends along the entire anterior–posterior length of the somite,a process that takes approximately 2hr.Over the next 3–4hr,the slow muscle ?bers migrate laterally through the somite to the lateral surface (Fig.8B;Devoto et al.,1996).As the slow muscle ?bers migrate,they induce a medial to lat-

eral wave of fast muscle ?ber differen-tiation (Blagden et al.,1997;Henry and Amacher,2004).The migration of the slow muscle cells to the lateral surface requires the function of two cadherins ,n-cadherin and m-cad-herin ,which display complementary changes in expression during the

slow

Fig.

8.

Fig.9.

Fig.8.Somite and myotome morphogenesis.A:A rendering of a dorsal view of one bilateral half of the paraxial mesoderm.Medial is down,and lateral is up.Note that the boundary cells become gradually more columnar as the somite matures.Inset 1,depicts clustering of Inte-grin ?5(green)along the nascent somite bound-ary at the basal surface of the boundary cells.Inset 2,as the boundary matures,?lamentous actin (yellow)concentrates along the boundary and the nuclei (red)localize basally.The centro-somes (green)adopt an apical position.Inset 3,Integrin-associated proteins such as Focal Ad-hesion Kinase and Paxillin (red)are localized to each somite boundary,while adherins junctions proteins such as ?-catenin (green)are enriched at the apical side of the boundary cells.B:A schematic time series of myotome morphogen-esis adapted with permission from the Com-pany of Biologists (Stellabotte et al.,2007).In each diagram,anterior is up and medial is to the right.Slow muscle ?bers (teal)originate medi-ally and are the ?rst to elongate and migrate to the lateral edge of the myotome.This lateral migration induces the differentiation of fast ?-bers (purple).Myogenic precursor cells (orange)responsible for later growth of the myotome,derive from the anterior somite.These cells ro-tate to the lateral surface,divide,intercalate into the myotome,and give rise to additional fast muscle ?bers in the lateral myotome.

Fig.9.The relationship between zebra?sh somites and vertebrae.A:The anterior limit of expression of hox genes in the zebra?sh trunk is indicated.References:a (Prince et al.,1998),b (Sordino et al.,1996),and c (van der Hoeven et al.,1996).Zebra?sh have 30–32somites,which align to the posterior three quarters of one ver-tebral body and the anterior one quarter of the next posterior vertebra.The total number of vertebrae range from 29–33,with the variability arising within the transition from rib-bearing to hemal-arch bearing vertebrae (Morin-Kensicki et al.,2002).The different vertebral subtypes are indicated.The two cervical and the ?rst two rib-bearing vertebrae contribute to the Webe-rian apparatus (Morin-Kensicki et al.,2002;Bird and Mabee,2003;Fleming et al.,2004).Ambi-guity regarding the alignment and total number of somites and vertebrae is indicated by the hatched posterior two somites.Adapted with permission from the Company of Biologists from (Morin-Kensicki et al.,2002).B:Ichthyo-logical convention classi?es the ?rst four verte-brae as “Weberian,”the rib-bearing vertebrae as “precaudal,”the rib-and hemal arch-bearing as “transitional,”and the hemal arch-bearing as “caudal.”Adapted with permission from (Bird and Mabee,2003).

GENETICS AND EMBRYOLOGY OF ZEBRAFISH METAMERISM 1437

muscle migration period.n-cadherin is initially expressed only in the me-dial somite,but the expression ex-pands laterally to encompass the en-tire somite.n-cadherin,in contrast,is initially expressed throughout the somite but disappears in a medial to lateral wave and is ultimately only expressed in the postmigratory slow ?bers on the lateral surface of the myotome.In embryos lacking either of the two cadherins,the lateral migra-tion is perturbed(Cortes et al.,2003). The period of migration of the slow ?bers and elongation of the fast?bers has been termed the transitory phase in myotome border formation.The ini-tial phase is the formation of the epi-thelial somite with a Fibronectin ma-trix at the border.The third phase of myotome border morphogenesis is marked by the completion of myo?ber development and the production of a Laminin matrix.The10th somite reaches this third phase around24 hours postfertilization(hpf;Henry et al.,2005).

Each of the Tu¨bingen segmentation mutants are viable to some extent and actually display segmented myo-tomes,although the borders are some-what irregular(van Eeden et al., 1996).These boundaries are hedge-hog-dependent:they are missing when hedgehog signaling is inhibited in the segmentation mutants(van Ee-den et al.,1998;Henry et al.,2005). Inhibition of hedgehog signaling does not affect the initial irregular bound-aries that form in the posterior of the notch pathway mutants(Henry et al., 2005).The earliest evidence of seg-mentation recovery in fss/tbx24mu-tant embryos can be seen in the seg-mental alignment of the nuclei of the slow muscle cells before their lateral migration(van Eeden et al.,1996). Given that the border rescue is seen after the slow muscle migration occurs at24hpf,it is likely that the migrat-ing slow muscle?bers are responsible for the boundary recovery in these mutants.The length of slow muscle ?bers appears to help de?ne the length of the fast?bers and collec-tively they may create a myotome boundary.These boundaries,in turn, help to de?ne or stabilize the length of the myo?bers(Henry et al.,2005). The rescue of the myotome bound-aries in fss/tbx24and the notch path-way mutants also requires integrin?5.

integrin?5mutants have u-shaped

somites,but in contrast to the hedge-

hog pathway mutants,the horizontal

myoseptum is formed and the speci?-

cation and lateral migration of the

slow muscle?bers is normal.inte-

grin?5expression is elevated in the

adaxial cells,and this Integrin?5,in

conjunction with the Integrin?5ex-

pressed in the fast?bers,is likely re-

quired for the formation of the extra-

cellular matrix that constitutes the

myotome boundary(Ju¨lich et al.,

2005a).These Integrins would nor-

mally help to anchor the myo?bers to

the myotome boundary,which acts as

a tendon to transfer the force gener-

ated by the contracting?bers.If the

migrating slow muscle?bers can

cause the fast?bers to coordinately

differentiate and elongate,collectively

these?bers may be able to generate a

myotome border de novo,by means of

their Integrins,in the segmentation

mutants.

The?rst fast?bers to differentiate

in each somite derive from the cells of

the posterior somite expressing myoD.

Anterior–posterior patterning of the

somite also affects later myogenesis in

that myogenic progenitors that con-

tribute to the later growth of the myo-

tome are derived from the anterior of

each somite.These fast-?ber myo-

genic progenitors ultimately express

pax3/pax7and are located at the lat-

eral surface of the mature myotome.

These anterior cells rotate laterally af-

ter somite formation and before the

migration of the slow muscle?bers

(Fig.8B).During later growth of the

myotome,these lateral progenitors

appear to intercalate into the myo-

tome,suggesting that the later growth

of the myotome proceeds by lateral ad-

dition of fast?bers(Fig.8B;Hollway

et al.,2007;Stellabotte et al.,2007).

Sclerotome and Vertebral

Column

While most of the zebra?sh somite

forms myotome,a small portion is

fated to be sclerotome.In contrast,the

amniote somite is mostly sclerotome.

The zebra?sh sclerotome is located in

the ventral–medial portion of the

somite and contributes to the axial

skeleton.Fate mapping has shown

that the posterior ventral–medial

somite gives rise to both sclerotome

and myotome,while the anterior ven-

tral–medial somite only becomes scle-

rotome(Morin-Kensicki and Eisen,

1997).After somite formation,the

sclerotome cells undergo an epithelial

to mesenchymal transition and mi-

grate dorsally to surround the noto-

chord.These cells express the bHLH

gene twist,and the pattern of migra-

tion can be seen in transverse sections

of progressively more mature somites.

As the adaxial cells rearrange and be-

gin to migrate laterally as slow muscle

?bers,the twist-expressing cells begin

their dorsal migration.As the slow

muscle?bers reach the lateral myo-

tome,the twist-expressing cells reach

the dorsal side of the notochord(Stick-

ney et al.,2000).The migration path

of the anterior sclerotome is less vari-

ant than that of the posterior scle-

rotome and colocalizes with ventrally

migrating neural crest cells and spe-

ci?c motor axons within each somite

(Morin-Kensicki and Eisen,1997).

The segmental relationship be-

tween the somites and the vertebrae is

thought to be offset by one half seg-

ment,meaning that one somite con-

tributes to the posterior portion of one

vertebra and the anterior portion of

another vertebra(Fig.9A;Remak,

1855).While the idea of resegmenta-

tion has its detractors,it is generally

accepted that it does occur in am-

niotes(Verbout,1976;Stern and

Vasiliauskas,2000).Fate mapping

experiments suggest that,while re-

segmentation may occur in the ze-

bra?sh,it appears to be leaky in that

cells from the anterior or posterior

half of a somite may contribute to two

consecutive vertebrae rather than

strictly giving rise to a speci?c portion

of a single vertebra(Morin-Kensicki et

al.,2002).

Fate mapping studies indicate that

sclerotome may contribute to the ver-

tebral arches and the centra(verte-

bral bodies;Morin-Kensicki et al.,

2002).However,the centra may de-

rive largely from the notochord and

not the sclerotome.Notochords ex-

cised before any evidence of vertebra

formation and cultured in isolation

can form a segmented bone matrix.

Moreover,histological analysis sug-

gests that the centra do not contain

osteoblasts and may not form by

means of a cartilage intermediate.

1438HOLLEY

The centra appear to form from bone matrix secreted by the notochord (Fleming et al.,2004).In fss/tbx24?/?embryos,somite borders do not form and the neural and hemal arches are malformed,but the vertebral bodies are not fused(van Eeden et al.,1996; Fleming et al.,2004).Thus,disruption of somitic metamerism does not elim-inate segmentation of the vertebral bodies.In contrast,laser ablation of notochord cells does lead to fusion of the centra(Fleming et al.,2004).In counterpoint to the fss/tbx24mutant, mouse mesp2knockouts exhibit exten-sive vertebral fusion(Saga et al., 1997).The difference in severity could be due to the fact that the mouse somite is mostly sclerotome and loss of somitic segmentation in this larger population of cells may not be rescued by any pattern present within the no-tochord.

Zebra?sh usually form32verte-brae,but the number may vary from 29–33(Fig.9;Morin-Kensicki et al., 2002).There are generally2cervical vertebrae,10rib bearing vertebrae,2 rib and hemal arch-bearing vertebrae, 14hemal arch-bearing vertebrae,and 4tail?n vertebrae(Morin-Kensicki et al.,2002;Bird and Mabee,2003).The variability in total vertebrae occurs in the number of posterior rib-bearing and anterior hemal arch-bearing ver-tebrae(Fig.9A;Morin-Kensicki et al., 2002).The?rst four vertebrae,encom-passing the two cervical vertebrae and the?rst two rib-bearing vertebrae, contribute to the Weberian apparatus, which is thought to transmit vibra-tions from the swim bladder to the otic vesicle(Fig.9;Morin-Kensicki et al., 2002;Bird and Mabee,2003;Fleming et al.,2004).In the ichthyological lit-erature,the term“precaudal verte-brae”is used to describe the rib-bear-ing vertebrae posterior to the “Weberian vertebrae,”the rib-and he-mal arch-bearing vertebrae are called “transitional,”and“caudal vertebrae”are the hemal arch-bearing vertebrae (Fig.9B;Bird and Mabee,2003).The developing centra can be seen at day9 by Alizeran red staining for bone,and most structures of the adult axial skeleton are completed by day21(Du et al.,2001;Morin-Kensicki et al., 2002;Bird and Mabee,2003). Alignment of the myotome with the vertebrae indicates that each myo-tome spans the posterior three quar-

ters of one vertebral body and one

quarter of the next posterior vertebra.

The?rst two to three somites do not

align next to vertebrae,suggesting

that they may contribute to the poste-

rior skull by analogy to the occipital

somites of amniotes.Myotome5,

which is derived from somite5,aligns

with the second and third vertebra

(Fig.9A;Morin-Kensicki et al.,2002).

Thus,as in the mouse and chick,the

anterior limit of hox6paralogue group

expression coincides with the transi-

tion from cervical vertebrae to rib-

bearing/thoracic vertebrae(Fig.9A).

By contrast,the anterior limit of hox9

paralogue group expression is somite

7or8,within the precaudal/rib-bear-

ing/thoracic region,while in mouse

and chick the hox9boundary coincides

with the thoracic/lumbar transition

(Fig.9A;Burke et al.,1995;Prince et

al.,1998;Morin-Kensicki et al.,2002).

Somite16should give rise to the last

rib-bearing vertebra,and somite17

should contribute to the?rst caudal

vertebrae.This transition corresponds

to the anterior limit of hoxd12a ex-

pression and is also the?rst of the

nodal-independent somites speci?ed

during the late blastula(Fig.9A;van

der Hoeven et al.,1996;Morin-Ken-

sicki et al.,2002;Szeto and Kimel-

man,2006).

ANTERIOR–POSTERIOR

DIFFERENCES IN

SOMITOGENESIS

Distinct anlagen for the anterior

trunk,posterior trunk,and tail

somites are speci?ed in the late blas-

tula before gastrulation(Szeto and

Kimelman,2006).These subdivisions

in the somite primordia are re?ected

in the loss of portions of the paraxial

mesoderm when various t-box genes,

nodals,wnts,or fgfs are perturbed(re-

viewed in Holley,2006a).The transi-

tions from the anterior trunk(somites

1–9),posterior trunk(somites10–15),

and tail(somites16–30)are also re-

?ected in differences in the develop-

ment and segmentation of these re-

gions of the body axis,with the most

prominent differences occurring at the

progression from anterior to posterior

trunk.

Three genes are known to be exclu-

sively expressed in the anterior

somites,tbx15,a nanos-related gene,

and soxlla(de Martino et al.,2000;

Begemann et al.,2002;Ju¨lich et al.,

2005a).Notably,there is no known

function for any of these genes in

somitogenesis,and none of the genes

that show anterior-or posterior-spe-

ci?c somite defects are differentially

expressed along the body axis.

In several respects,the develop-

ment of the anterior trunk somites oc-

curs more synchronously than the

more posterior segments.The seg-

mental expression of myoD,snail1a,

and engrailed arises simultaneously

in the?rst roughly six somites at the

six to seven somite stage,while ex-

pression of these genes in the remain-

ing somites arises sequentially with

the formation of each segment(Hatta

et al.,1991;Ekker et al.,1992;Ham-

merschmidt and Nu¨sslein-Volhard,

1993;Thisse et al.,1993;Weinberg et

al.,1996;Ju¨lich et al.,2005a).These

differences in gene expression mirror

the morphogenesis of the adaxial

cells.The adaxial cells begin to rear-

range simultaneously in the anterior

?6somites at the7–10somite stage,

but rearrange sequentially in the pos-

terior somites after segment morpho-

genesis is initiated(Felsenfeld et al.,

1991;van Eeden et al.,1996).As dis-

cussed above,the adaxial cells are ca-

pable of generating a segmental pat-

tern in the absence of fss or notch

pathway signaling(van Eeden et al.,

1998;Henry et al.,2005).Therefore,

there is a greater temporal separation

between this secondary,adaxial cell-

mediated segmentation and initial

border morphogenesis during anterior

somitogenesis,relative to posterior

somitogenesis,where adaxial cell re-

arrangement appears more as a con-

tinuation of somite border formation

and epithelialization.

fss/tbx24,foxc1a,and ripply1are re-

quired for formation of somites along

the entire body axis,but the formation

of the anterior?ve to seven somites is

generally resistant to perturbation

(van Eeden et al.,1996;Topczewska et

al.,2001a;Kawamura et al.,2005a).

Mutations in the notch pathway genes

bea/deltaC,aei/deltaD,des/notch1a,

and mib do not affect the anterior

somites(van Eeden et al.,1996;Hol-

ley et al.,2000,2002;Itoh et al.,2003;

Ju¨lich et al.,2005b).The formation of

the anterior somites in these mutants GENETICS AND EMBRYOLOGY OF ZEBRAFISH METAMERISM1439

is not due to functional redundancy between these genes or to maternal contribution(van Eeden et al.,1996; Ju¨lich et al.,2005b).The anterior somites are also resistant to dominant perturbation of notch signaling by means of injection of low levels of a dominant negative Su(H)or a consti-tutively active form of notch(Ju¨lich et al.,2005a).Moreover,treatment of embryos with a chemical inhibitor of notch signaling,DAPT,does not affect the anterior somites(Geling et al., 2002;Mara et al.,2007).Inhibition of non-notch pathway genes also tends to affect all but the anterior trunk somites.Morpholino knockdown of RPTP?does not affect the formation of the?rst seven somites.The anterior trunk somites are also normal when RPTP?function is inhibited in aei/ deltaD embryos(Aerne and Ish-Horowicz,2004).Additionally,while fss/tbx24?/?embryos typically lack all somites,there are alleles of fss/ tbx24that form one or two somites (unpublished observations).Collec-tively,these phenotypes suggest that the genetic control of anterior somito-genesis differs in some way from the control of posterior somitogenesis.fss/ tbx24?/?;aei/deltaD?/?;des/notch1a?/?embryos display defects centered around somites7–9,suggesting that the transition from anterior trunk to posterior trunk somitogenesis is par-ticularly sensitive to the dosage of these genes(Ju¨lich et al.,2005a). Only two zebra?sh mutants are known that speci?cally affect the anterior somites.integrin?5?/?and ?bronectin1a?/?embryos fail in the morphogenesis of the anterior trunk somites(Ju¨lich et al.,2005a;Koshida et al.,2005).Double mutants between in-tegrin?5and the notch pathway mu-tants display defects along the entire body axis(Ju¨lich et al.,2005a).One pos-sible explanation for the difference in anterior somite formation is that,at the time that the?rst few somites are form-ing,the paraxial mesoderm is still un-dergoing dorsal convergence,and the tissue is relatively shallow along the dorsal–ventral axis and broad along the medial–lateral axis.Thus,aspects of somite formation may be delayed or reg-ulated differently to allow for the con-tinuation of dorsal convergence. Although not affected by inhibiting notch signaling,anterior trunk somito-genesis can be perturbed by blocking

the function her genes,either alone or

in concert with other genes.Morpholino

knockdown of her1has been reported to

cause,at most,a very mild perturbation

of the anterior trunk somites,and there

is debate about the effect,if any,inhibi-

tion of her1has on the posterior trunk

and tail somites(Holley et al.,2002;

Oates and Ho,2002;Sieger et al.,2006).

Inhibition of her7affects somites poste-

rior to somites7–10,but knockdown of

both her7and deltaC affects all somites

(Oates and Ho,2002;Oates et al.,

2005a).Elimination of her1and her7

also affects all somites(Henry et al.,

2002;Oates and Ho,2002).In other ge-

netic combinations,her1and her7do

not behave equivalently.Knockdown of

her13.2affects the posterior somites,

but knockdown of both her1and her13.2

affects all somites and abolishes oscil-

lating expression of her1,her7,and

deltaC by the tail bud stage.In contrast,

knockdown of her7or both Su(H)para-

logues in conjunction with her13.2or

knockdown of her13.2in bea/deltaC

mutants does not affect formation of the

anterior somites(Kawamura et al.,

2005b;Sieger et al.,2006).Knockdown

of her1with both Su(H)genes affects

oscillating gene expression from the be-

ginning of somitogenesis(Sieger et al.,

2006).Why does elimination of notch

target genes in combination with other

genes perturb anterior somitogenesis

while double mutants among notch

pathway genes themselves,or chemical

inhibition of notch signaling,do not af-

fect the?rst few somites?Perhaps these

results suggest that there are other sig-

nals that activate the expression of the

her genes during the?rst rounds of seg-

mentation.

There are con?icting reports re-

garding the phenotype of the embryos

lacking both Su(H)1and2.The initial

study focused on Su(H)1,but the mor-

pholino used in this study also effec-

tively inhibits Su(H)2due to high

sequence homology around the trans-

lational start site.The authors found

that injection of this morpholino abol-

ished oscillating expression of her1,

her7,and deltaC and that,like the

notch pathway mutants,the segmen-

tation defect started around the7th—

9th somite(Sieger et al.,2003,2006).

A second study used two morpholinos,

one for each Su(H)paralogue.They

observed a similar effect on gene ex-

pression but found that the anterior

somites were perturbed(Echeverri

and Oates,2007).This difference may

be due to different ef?cacies of the

morpholinos or to the genetic back-

ground in which the experiments were

performed.However,it would be curi-

ous if knockdown of the two Su(H)

paralogues affects the anterior

somites,while otherwise inhibiting

notch signaling does not.The answer

to this puzzle may be that the pertur-

bation of the anterior somites is due to

elimination of the repressive function

of Su(H),which operates in the ab-

sence of notch signaling.That basal

levels of her1and her7expression are

higher in the Su(H)morphant than in

the des/notch1a embryos,indicates

that the Su(H)paralogues do function

as default repressors in the PSM

(Sieger et al.,2003).

That the anterior somites form in

the notch pathway mutants has been

explained as a gradual desynchroniza-

tion of the clock over the?rst few

somite cycles(Jiang et al.,2000).This

model correlates well with the gradual

breakdown in expression of her1,her7,

and deltaC,but as discussed above,is

inconsistent with many other aspects

of the notch mutant phenotypes.Im-

plicit in this model is the idea that all

of the somite precursors begin syn-

chronized oscillations at the same

time,early in development.However,

a detailed examination of her1,her7,

and deltaC expression in the posterior

somite precursors strongly suggests

that the expression of these genes do

not oscillate in the progenitor zone.

The precursors to the posterior?18

somites appear to initiate oscillations

at different times as the cells leave the

medial progenitor zone into the lateral

initiation zone(Mara et al.,2007).

As somitogenesis proceeds,the tail

bud shrinks in size such that two to

three her1stripes are typically seen in

a7–8somite stage embryo,while

most15somite stage embryos have

only one to two stripes(Mu¨ller et al.,

1996;Holley et al.,2000).At the7–8

somite stage,the anterior and poste-

rior PSM combined have the anlagen

for roughly10future somites.By the

12somite stage,the PSM has the pre-

cursors for?8somites and by the15

somite stage the PSM has shrunk to

less than7future somites.The de-

cease in the number of cells in the

1440HOLLEY

PSM may make the system more sen-sitive to noise generated by mitosis, cell movement,and stochastic gene expression.Thus,any perturbation of the genetic circuitry of the clock may have a proclivity to cause segmenta-tion defects during posterior trunk and tail somitogenesis.

Differences between the anterior approximately seven somites and the more posterior segments are also observed in amphioxus,Xenopus, mouse,and human.Genetic perturba-tion of notch signaling in mice and humans(Conlon et al.,1995;Oka et al.,1995;Hrabe′Angelis et al.,1997; Wong et al.,1997;Evrard et al.,1998; Kusumi et al.,1998;Zhang and Grid-ley,1998;Bulman et al.,2000;Bessho et al.,2001;Dunwoodie et al.,2002)or wnt3a(Takada et al.,1994;Aulehla et al.,2003)and mesp2(Saga et al., 1997)in mice leads to a somite defect in the posterior but not the anterior somites.Mice mutant for either of the transcription factors mesogenin or tbx6form only the anterior paraxial mesoderm,revealing genetic differ-ences in the speci?cation of anlagen of the anterior somites(Chapman and Papaioannou,1998;Yoon and Wold, 2000).Anterior-speci?c defects are ob-served in mice mutant for PDGFR?, which is believed to mediate signaling between the myotome and sclerotome. Mice mutant for this receptor show extensive fusion of the cervical verte-brae but more subtle defects in tho-racic and lumbar vertebrae(Soriano, 1997;Tallquist et al.,2000).This phe-notype is similar to congenital human defects known as Klippel–Feil syn-drome,in which the cervical vertebrae are fused but the rib cage is only mod-erately affected,if at all(Clarke et al., 1998;Pourquie′and Kusumi,2001). Anterior somitogenesis in the mouse and the cephalochordate amphioxus occurs more rapidly than posterior somitogenesis(Tam,1981;Schubert et al.,2001).Conversely,the rotation of the anterior seven to nine somites during Xenopus segmentation is slower than the rotation in the poste-rior somites(Afonin et al.,2006). LEFT–RIGHT SYMMETRY

OF SEGMENTATION

The left–right patterning of the verte-brate trunk establishes the asymmet-ric position/morphology of the heart,

lung,liver,and other viscera(Raya

and Belmonte,2006).Signals that es-

tablish or maintain left–right asym-

metry also act on the paraxial meso-

derm,and if these signals are not

buffered in some way,somitogenesis

will occur asymmetrically,with one

side of the embryo forming somites

more rapidly than the other.This

asymmetry only effects somites

?8–15and is transient,as the bilat-

eral alignment of the somites recovers

later during the segmentation period

(Kawakami et al.,2005b;Vermot et

al.,2005).Neither the mechanisms of

this recovery nor the exact cause of

the initial asymmetry are fully under-

stood,although it is clear that retinoic

acid has a roll in balancing the bilat-

eral rate of somite formation(Diez del

Corral et al.,2003;Moreno and Kint-

ner,2004;Kawakami et al.,2005b;

Vermot et al.,2005;Vermot and Pour-

quie,2005;Echeverri and Oates,

2007;Sirbu and Duester,2006).

raldh2is the enzyme that catalyzes

the last step in the biosynthesis of

retinoic acid.During the segmenta-

tion period,raldh2is expressed in the

anterior PSM and somites in the ze-

bra?sh,Xenopus,mouse,and chick

(Begemann et al.,2001;Diez del Cor-

ral et al.,2003;Moreno and Kintner,

2004;Sirbu and Duester,2006).It has

been proposed that retinoic acid pro-

motes the transcription of bHLH

genes in anterior PSM of the Xenopus

embryo(Moreno and Kintner,2004).

In the chick,somite explants treated

with a retinoic acid agonist cease to

express fgf8and reciprocally,FGF8

represses the expression raldh2in ex-

plants.These results led to the hy-

pothesis that reciprocal gradients of

retinoic acid and fgf exist in the PSM

(Diez del Corral et al.,2003).In

raldh2-de?cient mid-somitogenesis

stage embryos,fgf8expression in the

PSM is expanded anteriorly,particu-

larly on the right side.The timing of

the asymmetric fgf8expression corre-

lates with the appearance of asym-

metric somite formation(Kawakami

et al.,2005b;Vermot et al.,2005;Ver-

mot and Pourquie,2005;Sirbu and

Duester,2006).During the early somi-

togenesis stages in raldh2?/?mouse

embryos,fgf8expression is normal in

the PSM but expanded anteriorly in

the overlying neural ectoderm(Sirbu

and Duester,2006).Moreover,seg-

mentation in raldh2?/?mouse em-

bryos can be rescued if retinoic acid is

provided through the mother’s diet up

until the beginning of somitogenesis

(day E8.25).Thus,retinoic acid is not

required throughout the segmenta-

tion period for normal somitogenesis

to occur,arguing that retinoic acid is

not needed to continually regulate the

fgf gradient or to induce gene expres-

sion in the anterior PSM.Moreover,

using a retinoic acid responsive trans-

gene,the raldh2?/?embryos rescued

by dietary supplement,only showed

evidence of retinoic acid signaling in

the neural plate and cranial meso-

derm not in the somites or PSM.

These results suggest that retinoic

acid produced in the paraxial meso-

derm primarily acts to regulate fgf8

expression in the neural ectoderm and

that deregulation of the neural ex-

pression of fgf8may later affect the

mesodermal expression of fgf8in some

way that is not understood(Sirbu and

Duester,2006).This explanation con-

trasts with the model that opposing

gradients of retinoic acid and fgf8reg-

ulate somite maturation throughout

somitogenesis.Obviously,more exper-

iments are required to better under-

stand the relationship between reti-

noic acid,left–right patterning and

somitogenesis.

Perturbation of early establishment

of left–right asymmetry by inhibiting

H?/K?-ATPase activity,left–right dy-

nein,notch signaling,or the transcrip-

tion factor terra,leads to asymmetric

somite formation(Kawakami et al.,

2005b;Saude et al.,2005;Vermot and

Pourquie,2005;Echeverri and Oates,

2007).Similarly,inhibition of raldh2,

by means of mutation,antisense,or

pharmacology,results in asymmetric

somitogenesis in the zebra?sh,mouse,

and chick(Kawakami et al.,2005b;

Vermot et al.,2005;Vermot and Pour-

quie,2005;Sirbu and Duester,2006).

In each case,asymmetric somite for-

mation is preceded by a loss of bilat-

erally symmetric activity of the somite

clock(Kawakami et al.,2005b;Ver-

mot et al.,2005;Vermot and Pour-

quie,2005;Echeverri and Oates,

2006).Antisense inhibition of ze-

bra?sh cyp26a1,a retinoic acid catab-

olizing enzyme expressed in the poste-

rior tail,also leads to asymmetric

oscillation of the somite clock(Echev-GENETICS AND EMBRYOLOGY OF ZEBRAFISH METAMERISM1441