COMMENT

TIG MARCH 1998 VOL. 14 NO. 3

85Copyright © 1998 Elsevier Science Ltd. All rights reserved. 0168-9525/98/$19.00PII: S0168-9525(98)01396-1

A gene that resuscitates a theory – somitogenesis and a molecular oscillator

JONATHAN COOKEj-cooke@nimr.mrc.ac.uk

NATIONAL INSTITUTE FOR MEDICAL RESEARCH, THE RIDGEWAY, MILL HILL, LONDON, UK NW7 1AA.

Many embryo types exhibit globalregulation during early development;the capacity to achieve constant pro-portions, and other features of theirspecies’ body pattern, despite widevariation among individuals in thetotal amount of tissue available whilethe pattern is forming. But explainingthis has understandably fallen lowon the agenda of molecular develop-mental biology. The contemporaryview tends to be that with satisfyingexplanations of more local develop-mental mechanisms being added tothe stockpile almost weekly, the engi-neering or design principles that linkthese mechanisms into a globallyregulative system will surely ‘fall out’,or become apparent, as the list of‘local’ mechanisms nears completion.However, a recently published paper1appears directly to link earlier ideasof global organization in embryoswith modern gene expression data. Itconcerns the extraordinary transcrip-tion dynamics of a vertebrate (chick)homologue of the Drosophila pair-rulegene hairy, chairy1, whose mRNAlevels cycle dramatically and regularlyin a widespread and spatially organ-ized way, in time with the formationof successive somites. This behaviouris highly reminiscent of one of thecomponents of an abstract modelproposed more than 20 years ago2,3

to explain global regulation of verte-brate somite number. And quite apartfrom this connection, the paper isexciting in revealing how muchremains to be understood about themolecular organizations that are avail-able within and between cells, andare deployed in control of a demand-ing pattern like that of the vertebratesomite series.

SomitogenesisSomite segments are reiterated,

obviously developmentally equivalentunits. They form in a head-to-tail se-quence by the successive develop-ment, at regular time intervals, of regu-larly spaced fissures that subdivide thecolumns of paraxial mesoderm. Thus

over the great majority of the series,somite number increases smoothlywith development, with successiveintersegmental fissures smoothlyspaced along the head-to-tail dimen-sion. Regulation is global in the sensethat, even when individual embryosof a species differ by as much as 35% in the extent of available tissue(cell number) in this long dimension,the species-characteristic number ofsomites (on the order of 50) is arrivedat with a variability of 5% or less4. Inother words, the cellular distance be-tween successive fissures, thus cellnumber per somite, is closely adjustedin relation to overall size of each indi-vidual at the onset of somitogenesis.However, some conditions, such asextreme temperatures5,6 or partialsubstitution of ambient H2O with D2O,do slightly but systematically changethe final somite number found, inrelation to major landmarks in theother (non-segmental) aspects of bodypattern. The impression is that thenormal remarkable number controlmight result from, as it were, the beat-ing of a short regularly repeated timeinterval against another, much longer

time period. These two componentprocesses, normally quite tightly inte-grated, are experimentally separableby their slightly different response co-efficients to temperature and so on.

Although the cellular details of seg-mentation vary between vertebratetypes, somite formation is similarlyorganized in all. In birds, the subjectof the recent paper, somitogenic tis-sue forms bilateral ribbons, the seg-mental plates. These extend from theflanks of the regressing primitivestreak, where new tissue is fed intothem posteriorly, up to the position ofthe most recently segmented somite.The segmental plates incorporate, atany given time in development, thetissue of the next 10–12 somites thatwill segment. This tissue is known to be autonomously programmed,segmenting in its pre-arranged spatiotemporal order even when the entire plates or parts of them aresurgically isolated and explanted, orpolarity-reversed in situ and so on.Figure 1 attempts to show somite for-mation in an embryo of this generaltype, in relation to the model mecha-nism that follows.

S1

SP

S1

S13

Regressingstreak

0 h 18 h1.5 h 3 h 4.5 h 6 h 7.5 h 9 h 10.5 h 12 h 13.5 h 15 h 16.5 h A

P

FIGURE 1. Somite formation in a generalized vertebrate embryo. Cells are recruited intothe segmental plate as they leave the sides of the regressing streak. The dot markssuccessive positions occupied by one particular cell or cell group at time intervals (90 min)equal to that taken to form successive somites, until the incorporation of this cell or cellgroup into a forming somite (final diagram, S13). For much of development, there is aconstant number of somites-worth of tissue in the segmental plate (approximately 12 inchick), because recruitment at the posterior keeps pace with segmentation at theanterior, as the wavefront of somite formation passes down the total body pattern.Abbreviations: A, anterior; P, posterior; S1, first somite; S13, thirteenth somite; SP, segmentalplate. (Redrawn, with permission, from Ref. 1.)

COMMENT

A theoretical modelThe clock and wavefront model2,3,

in the absence at that time of anydirect molecular clues to mechanism,simply took account of the above fea-tures of somitogenesis. It proposedthat all the relevant (somite-forming)cells share an ‘oscillator’, a regularlyrepeated cycle of states of durationequal to the time between segmen-tation of successive somites, and areentrained or phase-linked with respectto this oscillator. That is, cell inter-communication at some point hasensured that cells relatively closelypositioned within the embryo areoscillating near-synchronously. Thiswas the ‘clock’. The model’s ‘wave-front’ was simply an expression of theobservation that embryos (includingexperimentally extreme ‘small’ and‘large’ ones) that begin anterior seg-mentation synchronously, also fin-ish synchronously. ‘Wavefront’ waschosen in important contradistinctionto ‘wave’, because experimentationhad revealed this passage of visiblecell change down the body to be anautonomous expression of smoothlygraded rates of development, between‘head’ and ‘tail’ cells. In other wordsall somitogenic cells are undergoinga prolonged, ‘hidden’ developmentalprogression towards the sudden onsetof rapid change in properties thatconstitutes somite formation, but theyreach this onset after times that in-crease smoothly with relative positionin the head–tail axis. This graded pro-gramme is set up along the somito-genic tissue by some much earlierregulatory process, and does not resultfrom propagating signals that passjust ahead of the observed changes.Thus, a nerve impulse propagates asa wave along an axon, whereas a col-lection of alarm clocks, originally setto ring at one-minute intervals in thefuture and then appropriately orderedalong a table top, produce in duecourse a wavefront of ringing.

How are these two aspects of cellorganization combined to completethe segmentation and number-controlmechanism? It was postulated thatonly when in a certain phase sectorof their ‘clock’ oscillator, can cellsexpress a competence for the rapidchanges of somite formation – ineffect, for sudden change to the epi-thelial phenotype – that they havejust acquired because the ‘wavefront’has reached and passed their positionin the body. Change will then occur

synchronously within successive,regular-sized blocks of tissue in thehead-to-tail axis, each time the clockenters its permissive phase. The con-stant of somite number is in effect thequotient of dividing the species-specific clock period into the equallyspecies-specific and regulated wave-front passage time.

chairy1 reveals a molecularoscillator

chairy1, encoding a basic-helix–loop–helix-containing putative tran-scription factor, is distinctively morerelated to the Drosophila hairy anddeadpan genes than to other verte-brate and insect genes within thegeneral hairy family, and its role inthe insect segmentation process pro-vides an obvious incentive to study it.Nevertheless, these observations fromchick do little specifically to supportthe current wave of speculation thatvertebrate and arthropod segmen-tations are at depth homologous.There is clear central involvement ofthe Delta/Notch system in vertebratesomitogenesis7 but not in insect seg-ment patterning. Furthermore, whilevarious insects’ hairy homologues8,and the more diverged vertebraterelative Her1 (zebrafish)9, show spa-tially periodic expression either syn-chronously or sequentially duringprolonged segmentation, none showsanything corresponding to whatPalmeirim et al.1 have observed. Onlyreading their beautiful paper can ad-equately convey this, but the essenceis that individual segmental platecells, during their progress from thestreak to incorporation into a somite,pass through some 10 rhythmic cyclesof chairy1 mRNA synthesis with degra-dation to undetectable levels in be-tween. Crucially, the cells are phase-organized with respect to this processsuch that at each tissue location, thecycles are experienced as alternating‘waves’ (really, wavefronts, see abovesection) of new mRNA expressionand then degradation, sweeping fromposterior to anterior.

Although each cycle within eachcell lasts for the time that elapses be-tween formation of successive inter-segmental fissures, progressive phase-shifting with respect to the cyclebetween the ‘youngest’ cells (firstcycle, near the streak) and those aboutto segment (say, cycle 11), amountsto nearly a cycle’s worth of time.Thus the global ‘time-lapse’ illusion,

from the in situ data recordingchairy1 RNA signal, is of wavefrontspassing up the plate once per roundof segmentation, with each new wavebeginning posteriorly as the previousone arrives at the position of newlysegmenting tissue. This makes it quitehard to keep clearly in mind what islogically established by the data, andre-illustrated from the original paperin Fig. 2; that each cell experiences,during its residence in the plate, not just one cycle but approximatelythe number of cycles that there are‘somitesworth’ of tissue in the plate.The appearance that the RNA bandsshorten as they progress anteriorlycould be due to increase in relativephase difference between successivecells, or to relatively shorter synthesisbursts within an unaltered total cycletime, at more anterior locations (thisappears experimentally unresolved).

Anteriorly, within cells of each seg-ment at its formation, the alternatingstates of chairy1 expression ‘freeze’,such that the posterior part of theblock maintains the gene expressionwhile the anterior part permanentlyswitches it off. chairy1 could thus beinvolved, finally, in maintenance of the sharp transition in cell propertynecessary to stabilize the somites’integrity. Several other known genesshow one or two stripes of expression,differentiating presumptive anteriorand posterior domains at and near thepoint of segmentation (e.g. Refs 10,11), but only chairy1 activity as yet isknown to exhibit such widespread,prolonged and organized temporalcycling on a morphogenetic time-scale. The paper also shows that thisphased oscillatory arrangement isautonomous to segmental plate. Justas for segmentation itself, it is not the immediate result of activelypropagated posteroanterior waves ofintercellular signal, because isolatedposterior and anterior half platesessentially stick to their scheduledwavefronts of chairy1 expression overthe term of a cycle or two in culture.

ImplicationsA first point is the extreme dyna-

mism of intracellular control, over lev-els of a specific mRNA, revealed bythe detailed results. Interestingly fromthe cell biological standpoint, theauthors show this cycle is not de-pendent upon new protein synthesisfor continuation in the short term. Theappearance once each 90 minutes of

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COMMENT

shifting, but sharp, wave-fronts between ‘off’, intense‘on’ and then ‘off again’ sig-nal levels, across particularlythe more mature anteriorparts of the plate, must re-quire both very rapid tran-scription and apparentlyequally rapid downregu-lation and specific mRNAdegradation. But to allowsuch control, is the RNA half-life very short throughoutthe cycle? Palmeirim et al.take the independence fromongoing protein synthesisto suggest that chairy1mRNA oscillations are just a passively driven output,from an oscillator whosecentral mechanism is other-wise still hidden from us.This raises the possibilitythat the cells, possibly evenall cells in the embryo, havebeen ‘oscillating’ and achiev-ing or maintaining large-scale phase-organizationsince well before onset ofchairy1 expression.

A possible confusionarises because the phase-shift of the ‘clock’ revealedby chairy1 causes posterior-to-anterior wavefront ap-pearances, whereas thewavefront of the original‘clock and wavefront’ modelis required to be from anterior to pos-terior and slow, advancing just oncedown the body during development.This wavefront is a redescription of thesurface observation that tissue devel-ops on a rapid schedule anteriorly,smoothly and continuously gradingto a slow schedule posteriorly, in a waythat is clearly set up before gastru-lation. Its mysterious property, namelythat both in smaller and in largerindividual embryos its passage downthe complete body pattern occupiesthe same amount of real time, is at theheart of global regulation. At the mostobvious level, the Cell paper does notaddress this wavefront (but insteadoffers direct evidence that the phaseorganization of the clock is driven fromposteriorly; see below). At anotherlevel, however, the molecular obser-vations suggest novel more integratedversions of clock and wavefrontideas, that make the required globalmeasurement of developmental timewithin embryos dependent upon

the same ‘clock’ oscillator both forrepeated (segmentation) and slow-smooth graded (wavefront) com-ponents. Thus the reported increasingintensity, and perhaps temporal sharp-ness, of episodes of chairy1 mRNAsynthesis in successive cycles mightbe a clue that this oscillator is, like atraditional clock pendulum, also anescapement. That is, successive cyclesare not the same for each cell andcycle number may measure elapsedtime. Such a view opens the way forthe idea that the embryo’s time, fromrapidly developing (anterior) toslower-developing (posterior) bodyregions, is itself measured out in‘ticks’ of the same clock that makesthe formation of somite fissures alocally periodic event. The originalconcept2,3 was of a less integrated sortthan this, having ‘clock’ and ‘wave-front’ merely passing like ships in thenight to leave the somite pattern, buthaving little else to do with eachother during development.

The coherently phase-shifted chairy1 oscillationsare a long-cherished dreamcome true for certain theo-reticians about tissue inembryos12,13. Our thinkingabout their mechanism mustbe guided by SherlockHolmes’ remark that ‘Oncethe impossible has beeneliminated, whatever re-mains, however improbable-seeming, must be the truth’.Only large-scale phase co-herence in tissue, as is ob-served here, would allow acellular oscillator to be usedin generating a segment pat-tern in the way proposed.Indeed heat shocks to Xen-opus gastrulae, proposed toact by phase-randomizingthe then hypothetical (pre-chairy1) clock, were oncecharmingly reported as lead-ing to ‘somite Balkaniz-ation’14. But total phase-coherence, that is, synchronyacross distance, is in prin-ciple as impossible to main-tain in an embryo as in auniverse; the required com-munication of state takesappreciable time (in ab-sence as yet of neural inte-gration)! Thus, the most fas-cinating aspect of embryoorganization suggested by

chairy1 expression is a widespread,organized cell intercommunication.Palmeirim et al. rightly emphasizehow their short-term division and cul-ture experiments show that all seg-mental plate cells are autonomouslycycling, rather than being driven tomake each cycle only by a signalpropagated from posterior regions.But how does their cycling get, andremain, spatially organized in thefirst place?

As proposed above, an oscillatordriving chairy1 expression could havebeen running from earlier in devel-opment. Tissue at the streak could inreality be continuing to drive phasecoherence along segmental plate via an oscillator-linked propagatedsignal (as in a field of slime mouldamoebae13). The reported experi-ments, of short-term observation inculture, would not have the resol-ution to detect the slight falling behindschedule in anterior halves followingtransection and isolation, that would

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(a)

(b)

A

P

0 1 2 3 4 5 6Time

Anterior halfof somite

Posterior halfof somite

7 8 9 10 11 12

90 min (1 somite formation)

S1S2 S2

S3

S1S1S2

S1

Stage 1 Stage 1Stage 2 Stage 3

S2S3

chairy1mRNA

FIGURE 2. The expression of the chairy1 gene during somito-genesis. (a) The continuous cycle of chairy1 expression, relatedto the formation of one somite. Constrictions denote nascentsomite material (e.g. Stage 2, S2). Waves marking onset and offsetof gene expression seem to pass anteriorly along the segmentalplate, narrowing [and probably intensifying, see (b)] as they go.This is unrelated to movement of individual cells up or down theplate, which is minimal. (b) As each cell resides in the plate while11 or 12 somites form, it experiences the same number of pulsesof chairy1 mRNA synthesis alternating with degradation. Wavesof expression appear to pass forward because the pulses areslightly phase-shifted between posterior and successively moreanterior cells, and culminate in longer-term expression in just theposterior half of each newly formed somite. The sharpness andintensity of RNA bands in the real data, especially in the anteriorplate, reveal the exactness of the intracellular controls (and probablyintercellular communication, see text) involved in maintainingthis organization. Abbreviations: A, anterior; P, posterior; S1–S3,first to third somites. (Redrawn, with permission, from Ref. 1.)

MEETING REPORTS

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It’s a dog’s lifeSECOND MOLECULAR GENETICS AND CANINE GENETIC HEALTH CONFERENCE, ST LOUIS, MO, USA, 31 OCTOBER–1 NOVEMBER 1997.

One-hundred and forty scientists,researchers and veterinarians fromaround the globe gathered in St Louisfor a couple of days and nights toponder and review the state of workon the canine genome. Just threeyears earlier, Jasper Rine (Univ. ofCalifornia, Berkeley, USA) stood upat the First Molecular Genetics andCanine Genetic Health Conference(NJ, USA) and appealed for moreopenness and collaboration. Rine andothers drew parallels between theplans being laid then in the dogworld and the dead-ends already en-countered by entrepreneurs in thehuman arena. Pleas to avoid the samemistakes seemed to fall on deaf ears.

What a gratifying contrast a fewshort years have made! The AmericanKennel Club (AKC) and the AKCCanine Health Foundation (Aurora,OH, USA) put on an excellent two-dayworkshop. Robert Wayne (Univ. ofCalifornia, Los Angeles, USA) energeti-cally keynoted the conference byreviewing his work on mitochondrialDNA sequence differences betweenwolves and dogs, supporting the

hypothesis that gray wolves were theancestors of dogs some 100 000 yearsago. He also described newer workusing microsatellites (short tandem re-peats or STRs) that extend the mtDNA

data and provide a potential tool tovalidate breed congruity. And thenthe attendees got down to business.

Six sessions spanned the two daysand covered topics from ‘Genetic Vari-ation in Purebred Dogs’, ‘Towards anIntegrated Linkage and Physical Mapof the Canine Genome’, ‘Molecular

Tools and Comparative MappingMethods’, and ‘Mapping Canine Dis-ease Genes and Canine HealthDisorders’. As the sessions started, itbecame obvious that some of thebest work arose from collaborationsthat were international in scope.

All the tools and techniques wereborrowed from work on other speciesand from the Human Genome Project.But limited resources mean that theborrowed tools must be carefullyselected and expertly applied. MatthewBreen (Animal Health Trust, UK) andCornelia Langford (Sanger Centre, UK)described the development of cyto-genetic resources that will facilitategreatly the physical placement ontothe canine genome of hundreds ofSTRs as well as ‘type I’ markers (codingsequences). Chromosome paints havealso been developed and are alreadyhelping to standardize the caninekaryotype (38 pairs of acrocentricautosomes + X,Y). Francis Galibert(CNRS Recombinaisons Genetiques,Rennes Cedex, France) reported onthe construction and characterizationof canine radiation hybrid cell lines

Copyright © 1998 Elsevier Science Ltd. All rights reserved. 0168-9525/98/$19.00PII: S0168-9525(98)01423-1

be predicted from this idea. Obser-vations relevant to such an idea haveindeed been made15, in that gapjunctional communication exists alongsegmental plate but is interrupted forcells within each nascent somite asfissure formation cuts them off frommore posterior tissue.

The alternative is that the signal-ling between cells necessary toachieve initial phase coherence mightfinish as they leave the streak. Thenin seeing expression ‘waves’, whoseschedule is maintained even withinanterior and posterior isolated halfsegmental plates, we would simplybe observing how an inherently veryaccurate clock, within each independ-ent cell, slows a little with each cycle(accounting for the later onset andoffset times of chairy1 anteriorly andthus the ‘waves’). But if this is indeedthe explanation, the sharpness and

coherence of the RNA signal wavesreveal a mindblowing accuracy attain-able by the intracellular ‘watches’. Infact, for me, the sharpening appear-ance of the wavefronts anteriorly (thuswith successive cycles) rules out thispossibility and demands intercellularcommunication. With the onset ofmolecular observations like this theremay again be justification for think-ing about large-scale, and even glo-bal, dynamic organization underlyingaspects of embryo patterning.

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