Fish genomics and biology

Here we review the impact of the genome sequence for those fish species for which it is already available, and we examine how the combination of genomics ... Skiptomainpagecontent HOME ABOUT ARCHIVE SUBMIT SUBSCRIBE ADVERTISE AUTHORINFO CONTACT HELP SearchforKeyword: GO AdvancedSearch Fishgenomicsandbiology HuguesRoestCrollius1,3and JeanWeissenbach2 1DyogenLab,CentreNationaldelaRechercheScientifiqueUMR8541,EcoleNormaleSupérieure,75005Paris,France 2GenoscopeandCentreNationaldelaRechercheScientifiqueUMR8030,91057EvryCedex,France  NextSection Abstract Thelastcommonancestorbetweenfishandmammalsdatesbacktotheveryoriginofthevertebratelineageandtoday,half ofmodernvertebratesarefish.Itisthusnotsurprisingthatseveralfishspecieshaveplayedimportantrolesinrecent yearstoadvanceourunderstandingofvertebrategenomeevolution,toinformusonthestructureofhumangenes,and,somewhat moreunexpectedly,toprovideleadstounderstandingthefunctionofgenesinvolvedinhumandiseases.Genomesequencecomparisons betweensuchdistantlyrelatedorganismsarehighlyinformativeduetotheaccumulationofneutralmutationsinnonfunctional regions.Yethumansandfishessharemanydevelopmentalpathways,organsystems,andphysiologicalmechanisms,makingconclusions relevanttohumanbiology.Therespectiveadvantagesofzebrafish,medaka,Tetraodon,orTakifuguhavebeenwellexploitedsofarwithbioinformaticsanalysesandmolecularbiologytechniques.Howeverthefullpotential offishgenomicsisabouttobeunleashedwiththeintegrationofmoretraditionaldisciplinessuchasbiochemistryandphysiology, withthestudyofadditionalspeciessuchascarp,trout,ortilapiaandabroadeningofitsapplicationstoenvironmental genomicsoraquaculture. PreviousSectionNextSection “Therethenetsbroughtupbeautifulspecimensoffish:Somewithazurefinsandtailslikegold,thefleshofwhichisunrivalled; somenearlydestituteofscales,butofexquisiteflavour;others,withbonyjaws,andyellow-tingedgills,asgoodasbonitos; allfishthatwouldbeofusetous.”WhilethegastronomicqualitiesoffishdidnotescapeJulesVerneinhis187020,000LeaguesUndertheSea,fisharenolessputtogooduseintwenty-firstcenturybiology.Inthisnewcontext,onecouldeasilyreplacefincolor andfleshqualitybygenomesizeandembryotransparencyinasimilarenumerationoftheadvantagesoftheseanimalsforbiology ingeneralandmoleculargeneticsinparticular.IfCaptainNemowasinapositiontooffersuchvarietyonhismenu,itis partlybecausefishcomprisemorethan25,000species,byfarthemostsuccessfulvertebrategroup.Indeedfewaquaticecosystems haveeludedcolonizationbyatleastsomefishspecies,fromTibetanstreamstotheabyssoftheoceansviasub-zeroAntarctic seas(Nelson1994).Ofthesespecies,manyhavelongbeenusedasmodelsindifferentdisciplinesofbiology(Fig.1)becauseofthisverydiversity:Theatrophyorexaggerationofimportantanatomicalorphysiologicalfunctionsoccurwith sufficientfrequencytohaveattractedbiologiststofishmodels(EpsteinandEpstein2005).Thisincludesmoleculargeneticsandgenomeresearch,forwhichfishalsopossessinterestingandoutstandingfeatures, ifnotall-timerecords,amongvertebrates. About30yearsago,apopulartropicalaquariumfishnamedDaniorerio(zebrafish)wasalreadyseenasendowedwithmanyadvantagesforgeneticanalysis:ashortgenerationtime(about3mo), largeeggclutchesallyearround,easymaintenance,andexternaldevelopmentofatransparentembryo(Streisingeretal.1981).Combinedwithlarge-scalemutagenesisscreensinitiatedintheearly1990s(Haffteretal.1996;Stainieretal.1996),zebrafishfilledagapingholeinvertebratedevelopmentalbiology:theabilitytostudygenesviatheirmutantphenotypes onalargescaleasinDrosophilamelanogasterorCaenorhabditiselegans.Sincethen,capitaldiscoveriesforourunderstandingofvertebratedevelopmentandhumandiseasehavealreadyemergedfrom zebrafishstudies.HoweverifoneconsidersgenomeanalysisaquestionofDNAsequenceacquisitionand“mining,”thenfishes reallybecameamajorplayerin1993whenSydneyBrennersuggestedanewspeciesasagenomemodel,themarinepufferfish Takifugurubripes(fugu)(Brenneretal.1993).AsidefromitsgastronomicdelicacystatusinJapanandChina,fugupossessoneofthesmallestvertebrategenomes.Thisfeature,alreadyrecordedforitsfreshwaterrelativeTetraodonnigroviridisin1968(Hinegardner1968),isamajoradvantagetorapidlygainaccesstoalargecatalogofgenesinavertebrateatacostcomparativelysmaller thanforthemuchlargergenomeofamammalianspecies.However,bothpufferfisharespeciesforwhichweknowlittleinterms ofphysiology,reproduction,orlifecycle.Sincebiologyisstillasciencelargelydrivenbythequalityanddepthofthe experimentaldataandourabilitytoextractmeaningfromit,theoutcomeofbothpufferfishgenomeprogramshasuntilrecently beenconfinedtodiscoveriesonthestructureandevolutionofgenesandgenomes,withfewconnectionstodevelopment,cell biology,orphysiology. Howeverthisisabouttochange,withtheemergenceofnetworksofscientistsandcross-disciplinaryplatformswhereprecise biologicalquestionsareexaminedwithanarrayoftoolsandresourcesthatincludegenomesequencesandgenomictechniques. Herewereviewtheimpactofthegenomesequenceforthosefishspeciesforwhichitisalreadyavailable,andweexamine howthecombinationofgenomicswithmoretraditionaldisciplinesmightpavethewayforamuchwiderimpactonbiology. PreviousSectionNextSection Anglinginthegenomicaquarium Thesequenceofthehumangenomewasstilladistantgoalin1993,buttheprojectofsequencingtheentiregenomeofamulticellular eukaryote,thatofthenematodewormC.elegans,waswellonitstrack(Sulstonetal.1992).Asithappens,manyimportanteukaryoticmodelorganismsthatwerealreadybeingstudiedwithmolecularbiologyapproaches hadgenomesizeswithinthereachofsequencingtechnologiesofthetime:Saccharomycescerevisiae(14Mb),D.melanogaster(180Mb),C.elegans(100Mb),andArabidopsisthaliana(125Mb).Butnoneofthesewerevertebrates,asituationwhichmotivatedapilotprojecttoevaluatetheusefulnessoffuguasamodelvertebrategenome(Brenneretal.1993).Thisinfluentialanalysisshowedthatthegenomewasindeedabout400Mb,oreighttimessmallerthanthehumanormouse genomes.Justasimportant,exon–intronboundariesseemedconserved,suggestingthatgenestructureswerelikelytobevery similar,albeitinamuchmorecompactsequence.Rapidly,otherstudiesstrengthenedthenotionthatpufferfishDNAcould helpidentifyandbetterunderstandthestructureandsometimesthefunctionofmammaliansequences. Viewlargerversion: Inthiswindow Inanewwindow DownloadasPowerPointSlide Figure1. Consensusphylogeneticrelationshipsbetweenfish(simplifiedfromNelson1994;Inoueetal.2003)andtetraopods,includingfishconsideredasbiologicalmodels(underlined),andthoseforwhichasequencehasbeenpublished (boxed,continousline)orisunderway(boxed,dashedline).Thethickbranchindicatesthemostlikelypositionofthewhole-genome duplicationattherootoftheteleosts,basedonHoeggetal.(2004). Inonesuchearlyattempt,afugusequencenexttotheHoxb4ageneandconservedwithmouse,showedenhanceractivityintransgenicmice(Aparicioetal.1995).ThepowerofcomparativegenomicsinvertebrateswasalsosoonillustratedwhenthecompleteHuntingtondisease(HD)gene fromfuguwassequencedandcomparedtoitshumanortholog(Baxendaleetal.1995).TheanalysisshowedthatthefuguHDgenepossessesafour-glutaminerepeat,whereasmousehassevenandhealthyhumansaminimalofeight.Becauseatract offourglutaminesisunlikelytoformafunctionalsiteinitself(suchasapolarzipper-,Perutzetal.1994),thefuguproteinsupportedtheviewthatitisonlyafterexpandingtoover37residuesinHDpatientsthatitsomehowgainedanew pathogenicfunction.Thisinitialsequencingprojectinfuguwasfollowedbymanymorethatgenerallyshowedlimitedlong-rangeconservationofgeneorder,rarelymorethanfourgenes perconservedsyntenyblock,thusdampeningdowntheinitialhopethatthiscompactgenomewasavaluabletooltoaccelerate themappingofhumangenes,thenapriorityintheHumanGenomeProject(Gilleyetal.1997).Howevertherewerestillmajorreasonstoestablishcompletesequencesoffishgenomes:(1)theseearlystudieshadshown theusefulnessofcomparativesequencingwhenconstructinggenemodelsonthehumangenome,(2)theinitiationoflargescale mutagenesisprojectsonzebrafishwascallingforaglobalgenomeeffortalreadyinpreparationwiththeconstructionofgenetic maps(Shimodaetal.1999;Kellyetal.2000)andradiationhybridmaps(Geisleretal.1999;Hukriedeetal.2001). PreviousSectionNextSection Trawlingforwholegenomes Reevaluatingthenumberofhumangenes Fuguisarelativelylargemarinefishthatcontainselevateddosesoftetrodotoxincausinglivespecimenorfrozensamplesto bethesubjectofrestrictiveimportationlawsinmostcountriesoutsideofAsiaandthusposingpracticalproblemsforgenomic analyses.Adifferentpufferfish,Tetraodonnigroviridis(Greenspottedpuffer,sometimesconfusedintheaquariumfishmarketwithTetraodonfluviatilis,whichisadifferentspecies)wasproposed(Crnogorac-Jurcevicetal.1997)thatalleviatesthisrestriction:Tetraodonalsopossessesasmallcompactgenome(Hinegardner1968)butitisapopularaquariumfishthatcanliveinfreshwater(Ebert2001).Incontrasttofugu,fewspecificTetraodonlociweresequencedandstudiedincomparisontotheirhomologsinotherspecies.Fromthebeginninginstead,TetraodongenomicDNAwasexploitedinlarge-scalecomparisonsbetweendifferentvertebrategenomes.Theinitialrationalebehindthis secondpufferfishprojectwastoassistintheannotationofhumangenes,aslowandfastidioustaskwhenperformedbyhumans, oftenunreliablewhenperformedbyautomaticapproaches,andyetoneoftheprimarygoalsoftheHumanGenomeProject.On thebasisofaninitialsamplingofrandomsequencesfromtheTetraodongenome(about30%),atoolnamedExofishthatwasbasedonBLASTsequencealignmentswasdevelopedtoidentifyconserved regionsinhumangenomicDNAthatcorrespondtocodingexons,rapidlyandwithhighspecificity(RoestCrolliusetal.2000).Asurprisingoutcomeofthisfirstexampleofaglobalsequencecomparisonbetweenlargesamplesoftwovertebrategenomes isthatthenumberofconservedsequencesidentifiedbyExofishinthehumangenomewasnotcompatiblewiththe60,000to 150,000genesthatitwasthoughttopossessatthetime.IndeedcomparisonswiththeTetraodonsequencesampleindicatedthattheentirehumangenomewouldcontainabout88,000evolutionaryconservedregions(termed ecores)correspondingtohumanexons,whileknownhumangenespossessedonaveragebetween2.6and3.2ecores.Asimpleratio betweenthesefiguresyieldsatotalofabout30,000humangenes.Thisnewestimate,confirmedlaterbytheinitialanalysis ofthehumangenomesequence(Landeretal.2001),challengedthenotionthatthecomplexityofgeneticinformationcontainedinagenomeisafunctionofthenumberofprotein-coding genes. Pufferfishgenomefeaturesindraftsequences Thefugugenome,thefirstvertebrategenometobesequencedafterhuman,wasobtainedusingthewhole-genomeshotgunmethod(Aparicioetal.2002).Thissequencedraftenabledanumberofinterestingobservations,suchasdifferencesinspecificproteinfamiliesbetween humanandfugu.TheTetraodongenomesequencewassubsequentlyproduced(Jaillonetal.2004),alsowiththewhole-genomeshotgunmethodalbeitwithahigherredundancyinsequencereads(8.3vs.5.6).Bothpufferfish possessabout70differentfamiliesoftransposableelementsagainstonly20forhumanormouse,butinpufferfishtheycomprise twotothreeordersofmagnitudefewercopies.InterestinglyinTetraodon,SINEandLINEfamiliesaredistributedinoppositeregionsofthegenomecomparedtohumanormouse:SINEsaremoreabundant inG+C-richsequencesinmammals,andinA+T-richregionsinTetraodon,andviceversaforLINEelements.Moresurprisingly,theseinitialstudiesofTetraodonandfugushowedanumberofdifferencesintheirgenomes.ForinstanceaG+C-richregionpresentinbothTetraodonandmammalgenomesisabsentinfugu.AlsosomegenefamiliessuchastypeIcytokinesandtheirreceptors,presentinallvertebratesstudiedsofar,werenotably difficulttofindinfugu,whileover30membersofthefamilycouldbeidentifiedinTetraodon.Thesediscrepanciesaremostlikelyattributabletobiasesinclonelibrariesordifferencesinmethodologies,andhopefully shouldberesolvedasthegenomesreachcompletion.Whencomparingfishandmammalgenecatalogs,surprisinglyfewmajordifferences couldbedocumentedwhenusingtheGeneOntology(Harrisetal.2004)classificationsystem.Morestrikingdifferencescouldbeseenusingproteindomaincomparisons:Proteinsinvolvedinsodium transportaremoreabundantinfish,whichalsocontainanallantoinpathwayforpurinedegradationthatisabsentinhumans. Neutralnucleotidicsequenceevolutionperyearwasfoundtobetwiceasfastinpufferfishasbetweenhumanandmouse,and proteinevolutionalsoappearstoproceedatafasterrateinfish,althoughthereasonsforthisarestillunclear.Itshould benotedthattheseresultsdependonthedatingofthedivergencebetweenTetraodonandfugu(18–30Mya)(Crnogorac-Jurcevicetal.1997). Insightsintovertebrategenomeevolution Perhapsoneofthemajordifferencesinthetwopufferfishdraftsequencesresidesinthefactthatthefugugenomesequencewasassembledpurelybythewhole-genomeshotgunmethodwithnophysicalmapping,whereasmost(64%)ofthe Tetraodongenomicsequenceisanchoredoneachofthechromosomes,providingalong-rangeviewofgeneorganizationinthegenome. Thisaddedinformationprimarilyresolvedalong-standingissueontheoccurrenceofawhole-genomeduplicationinthefish lineage. Remarkably,theideathatanincreaseinchromosomenumbersmaybeasourceofphenotypicnoveltyisnearlyacenturyold. In1911,Kuwadaalreadyobservedthatsomevarietiesofmaizeweretetraploidandsuggestedthatthismaybethesourceof “innumerableraces”(TaylorandRaes2004).Duringthefollowing60years,theoccurrencesandconsequencesofgeneandgenomeduplicationscontinuedtobediscussed (TaylorandRaes2004),withforinstancetheproposalbyStephensin1951thatincreasingthenumberofgeneticlociwastheonlypathto“evolutionary progress”andhissuggestionthatgenomeduplicationcouldbeonewayofachievingthis(Stephens1951).Thesetheoriesreachedahighpointin1970withthepublicationofSusumoOhno'sbook(Ohno1970)thatstatedseverallandmarknotions:(1)withoutduplicatedgenes,theemergenceofmetazoans,vertebrates,andmammals fromunicellularorganismswouldhavebeenimpossible,(2)thisprocessrequiredthecreationofnewlociwithpreviously nonexistentfunctions,and(3)hepostulatedthatatleastonewhole-genomeduplicationfacilitatedtheevolutionofvertebrates. Whiletheseideasweremetwithmildenthusiasmatthetime(Lewin1971;Spofford1972),itistodayawidelyacceptednotionthattocreatefunctionalnovelty,geneduplicationsareatleastasimportantas pointmutationsinindividualloci.Moreoverwhentheduplicationaffectstheentiregenomeatonce,thispotentialfornovelty istheoreticallyamplifiedbyallowingtheduplicationandretentionofpartialorcompletemetabolicpathways. Viewlargerversion: Inthiswindow Inanewwindow DownloadasPowerPointSlide Figure2. (A)Aschematicmodelofwhole-genomeduplicationwithfourchromosomes,followedbymassivegeneloss,chromosomefusionsand fissions,inter-andintrachromosomalrearrangements.Eachcoloredrectangleisachromosome,andlinesaredrawnbetween duplicatecopiesofgenespresentonsisterchromosomesorchromosomesegments.Thetoppanelstartswiththesisterchromosomesfacingeachotherandillustratesthechangesinducedbychromosomerearrangements, whilethelowerpanelshowsthesituationinacircularrepresentation,whichassumesonedoesnotknowtherelationshipsbetweenchromosomes apriori.Afterseveralmillionyearsofevolution,thedistributionofthefewduplicategenesthatremaindonotbeara traceoftheancientduplicationevent.(B)Thesamerepresentationasinthelowerpanelin(A),butwithrealdatafromtheTetraodongenome:Despitemorethan300millionyearssincetheduplication,thedistributionofabout2%ofTetraodongenesthatremainstrictlyintwocopies(joinedbybluelines)inthegenomeshowsastrikingpatternwherechromosomes areassociatedinpairs(e.g.,chromosome9and11,or10and14),orsometimesintriplets(e.g.,chromosomes5,13,and 19).Theformersuggeststhatnointerchromosomalrearrangementshaveoccurredonthesechromosomepairssincetheduplication, whilethelatterisreminiscentofachromosomefusionorfission. Howeverthefateofmostduplicatecopiesofgenes,overtensorhundredsofmillionyears,istobeeliminatedfromthegenome inaglobalprocesscalleddiploidization(Wolfe2001;Jaillonetal.2004;Kellisetal.2004)(Fig.2).Thefactthatmostduplicatecopiesofgeneshavelongdisappearedfromancientlyduplicatedgenomesisonereasonwhy providingproofoftheduplicationisoftendifficult.Inthecaseoffish,astrongindicationinsupportoftheduplication camefromtherevelationthatzebrafishpossesssevenHOXclustersonsevendifferentchromosomes,insteadofthefourclusters foundinmammals(Amoresetal.1998).Thisobservationimmediatelysuggestedthatthesefourchromosomesatleast—butmostlikelytheentiregenome—duplicated onceinanancientteleost,followedbythelossofoneHOXclusterinthezebrafishlineage.Thisconclusionwassustained byexamplesinotherfishspecies(MeyerandSchartl1999)butstrongersupportcamefromcomparativeanalysesinzebrafishusingmanymoregeneloci(Postlethwaitetal.2000)aswellasresultsfromExpressedSequenceTags(ESTs)positionedonthezebrafishgeneticlinkagemap(Woodsetal.2000),whereitwasclearthatthisgenomecontainedlargeduplicatedsegmentsthatdidnotexistinhumanormouse.However, thepossibilitythatthefrequentoccurrencesofduplicatedgenesinfishoriginatedfromahighlevelofsegmentalorlocal duplicationscouldnotbeentirelyruledout(Robinson-Rechavietal.2001).Thesestudieswerefollowedbyseveralattemptsatdatingtheemergenceofduplicategenecopiesinfugu(Christoffelsetal.2004;Vandepoeleetal.2004)butwitharelativelylargeuncertainty. Theultimatedemonstrationrestedonthelong-rangecontinuityoftheTetraodonsequenceassemblycoveringpartsofallchromosomes(Jaillonetal.2004).Althoughtheparalogs(pairsofgenesthatappearbyduplicationofanancestralgene)thatwereidentifiedusingvery conservativecriteriarepresentlessthan2%ofthecurrentsetofgenes,theirdistributioninthegenomeclearlyassociates chromosomesinpairsorintriplets,asituationexpectedifasingletetraploidizationeventtookplaceatsomepointin thepast,followedbyafewchromosomefissionsorfusions(Fig.2).AsecondandevenmoretellingsignatureoftheduplicationwasfoundinthealternatingpatternofTetraodonsyntenicgroupsalonghumanchromosomes.Indeed,thereturntoadiploidstateafterthewhole-genomeduplicationmostly affectsgenes,notnecessarilychromosomes.Whilethetwoduplicatedsisterchromosomesremainanintegralpartofthegenome, theyeachgraduallylooseabout50%oftheirgenes,insmallclustersthatalternatebetweenthetwo(Fig.3A).Toobservetheresultofthisprocess,oneneedstocomparethepositionofgenesonachromosomeofaspeciesthatdid notduplicateduringthesameperiodoftime,withorthologousgenesofthespeciesthatdidduplicate.Thecharacteristic patternthatresultsseemsquiteuniversal,fromsingle-celleukaryotes(Kellisetal.2004)tovertebrates(Jaillonetal.2004),andisillustratedinFigure3B.Thedeletionprocesstakesplacebecausethesupernumerarygenecopiesarenotunderselectionandtheythusrapidlyacquire deleteriousmutations.Thechoiceofwhichcopywillbedeletedisdrivenbythefirstmutationtooccurinoneofthetwo copies,aprocessthatmustberandomsinceultimatelyeachsisterchromosomeinheritsabouthalfoftheinitialgenecomplement. Howeversomeduplicatecopiesarenotdeleted,inthiscaseimplyingeitherthatbothcopiesareimmediatelyplacedunder selection,thatadeleteriousmutationpartiallyobliteratesthefunctionofonecopymakingtheothergeneessential,or thatthefirstmutationisnotdeleteriousbutfavorable.Becauseitisagenome-wideprocessofgeneselectionaffecting allfunctionalclassesatthesametime,itisofinteresttoinvestigate,inthecaseoffish,whichclasseshaveemerged asadvantageouscomparedtogenomesthatdidnotduplicate.Ideally,thequestionwouldbebestaddressedusingoneofthe livingfishspeciesthatdivergedjustbeforethewhole-genomeduplication,suchassturgeons(acipenseriformes)orgar(semionotiformes) (Hoeggetal.2004)(Fig.1).Untilthegenomesequenceofsuchaspeciesbecomesavailable,acomparisonwiththegenecatalogsofmuchmoredistant speciessuchasmammalsusingGeneOntologyannotations(Harrisetal.2004)andphylogeneticclassificationsrevealssignificantdifferences:Development,celldifferentiationandcellcommunication classesareenrichedingeneduplicates(F.G.Brunet,H.RoestCrollius,M.Paris,J.M.Aury,P.Gibert,O.Jaillon,V.Laudet, andM.Robinson-Rechavi,inprep.).Thisresultisinterestinginthelightoftheteleostradiationthattookplaceapproximately atthetimeofthegenomeduplication.Alsostrikinglyandaspreviouslyshowninyeastandnematode(DavisandPetrov2004),fishgenesthatevolveslowlypriortotheduplicationseemtobepreferentiallyretainedintwocopiesaftertheduplication: butwhatoftherateofevolutionbetweenthetworetainedcopies?Whereasthesubfunctionalizationmodel(Forceetal.1999)predictsthatthetwocopieswillsharetheancestralfunctionandthusevolveunderthesameconstraints,theneofunctionalization model(Ohno1970)proposesthattheemergenceofanewfunctioninonecopyoccursunderpositiveselection,i.e.,onecopywillevolvefaster thantheother.Interestingly,intheabove-mentionedstudy,Tetraodongeneduplicatesrecurrentlyshowamarkedlyacceleratedevolutionofoneofthetwocopies,inagreementwiththelatter modelandsupportingtheviewthatgenome(andhencegene)duplicationisadrivingforcebehindtheemergenceoffunctional novelty.Theancestralteleostgenomeduplicationprovideditsownerwithapowerfultoolkittoadaptanddiversify:twice asmanygenesasanyotheremergingvertebrate.Itistemptingtoproposethataconsequenceofthisgenomedoublingisto befoundintherichdiversityofextantfishspecies,unparalleledtodayamongvertebrates(Amoresetal.1998;MeyerandSchartl1999).Ultimately,theavailabilityofmorefishgenomesequenceswillhelpdistinguishbetweenteleost-wideandlineage-specific strategiesfortheretentionofbeneficialduplicatefunctionalclasses.Butfornow,theteleostgenomeduplicationprovides adirectentrypointintoanotherexcitingtheme:thereconstructionoftheancestralgenomepriortotheduplication,which wouldcloselyresemblethatoftheancestralbonyvertebrategenome.Studiesinzebrafish(Postlethwaitetal.2000),medaka(Naruseetal.2004),andTetraodon(Jaillonetal.2004)havealreadydelineatedtheprobable12proto-chromosomeswithincreasingprecision. Zebrafishandmedaka:Biologicalmodelscomeback Thezebrafishasamodelsystemhasaccompaniedthedevelopmentofmolecularbiologyfromthe1960stothegenomicrevolution ofthe1990s(GrunwaldandEisen2002).Followingthepublicationoftheresultsfromtwolarge-scalegeneticscreensin1996inaspecialissueofDevelopment(1996;123:1–461),zebrafishwaspropelledattheforefrontofdevelopmentalbiologyresearch.Whilesuchscreensarecurrently beingexpandedtotackleawiderrangeofmutationsinembryonicandadultstages(seebelow),manyresearchersarealsostudying zebrafishmodelsofhumandiseases(DooleyandZon2000)includingblooddiseases(deJongandZon2005),heartdisorders(MacRaeandFishman2002),andcancer(Amatrudaetal.2002).Asmentionedabove,genomeresearchonzebrafishstartedinparalleltothesedevelopments.Earlyeffortstomapgenes andgeneticmarkersservedtoidentifythelociofspecificmutantgenes,andtocomparetheorganizationofgeneswithother vertebrates(Barbazuketal.2000;Woodsetal.2000).Microarraytechnologyhasalsobeenapplied,incombinationwithsinglenucleotidepolymorphism(SNP)identification,to acceleratethelinkingofmutantphenotypestotheirgenotypes(Stickneyetal.2002).Thezebrafishgenomeisabout1700Mb,andthatoftheTübingenstrainisnowbeingsequencedattheSangerInstituteusing amixedstrategywherebothlargeinsertclonesandtheentiregenomearesubmittedtothewhole-genomeshotgunmethod.Today thezebrafishcommunityworldwidehasexpandedtremendouslywithover3500scientistsregisteredintheZFINcommunitydatabase (zfin.org).Theavailabilityofthegenomesequencewillprovideaunifyingreferencetointegratethewealthoffunctionaldataaccumulated sofar. Viewlargerversion: Inthiswindow Inanewwindow DownloadasPowerPointSlide Figure3. (A)Duplicationleadstodouble-conservedsynteny.Afterspeciation,achromosomeorchromosomesegmentfromanancestralspecies isduplicatedinonelineagebutnotintheother.Intheformer(e.g.,Tetraodon),supernumerarycopiesofgenesareprogressivelydeletedfromeachoftheduplicatedsegmentsinapproximatelyequalproportion (diploidization).Ultimatelythetwoduplicatedchromosomesonlycontain50%oftheinitialgenecomplementandarethusvery differentfromeachother.Thedifficultyoffindingtheoriginalpairofsisterchromosomescanbealleviatedbyacomparison withagenomethatoriginatesfromthesamepre-duplicationancestor,butthatdidnotduplicate(e.g.,human).Thenonduplicated chromosomesegmentshouldcontaingeneswithorthologsalternatingbetweenthetwoduplicatedchromosomes(adaptedfromKellisetal.2004).(B)Exampleofdouble-conservedsynteny.An88-MbregioncoveringthemajorityofthelongarmofhumanchromosomeXcontains 65geneswithorthologsintheTetraodongenome,alternatingbetweenchromosome1andchromosome7.Genesarerepresented bysmallarrowsthatindicatetheorientationoftranscription. TheMedaka(Oryzalatipes)hasalsolongbeenusedasamodelingenetics,datingbacktothebeginningoflastcentury(Wittbrodtetal.2002).Whileinmanyrespectsitmatcheszebrafishinitsadvantagesasalaboratorymodel,Medakapossessesseveralcharacteristics ofitsownasausefulbiologicalmodelsuchastheexistenceofseveralfertileinbredstrainsandembryonicstemcellsthat canbestablyculturedlongenoughtoenablegeneticmanipulation(Hongetal.1996),althoughtheiruseiscurrentlylimitedbythefactthattheydonotcontributetothegermline.Thecomplementaritybetween medakaandzebrafishisobviouswhenconsideringthenumerousmutationsofthesamelocusthataffecteachfishdifferently orevenuniquely,thusenablingthedecipheringofspecies-specificpatternsforthesemutations(Furutani-SeikiandWittbrodt2004).Interestingly,Medakaistheonlyfishsofarforwhichasinglegeneticlocus,DMY,hasbeenfoundtoberesponsiblefor sexdetermination,asinmammals(Matsudaetal.2002;Nandaetal.2002).WhileDMYisarecentinventionintheMedakalineage(Lutfallaetal.2003;Volffetal.2003)andthuswasnotfoundinothermodelfishspecies(Kondoetal.2003),itprovidesthefirstopportunitytostudythemolecularbasisofsexdeterminationinfish.Manyresourcesforgenome researchhavebeendevelopedforMedaka,includinggeneticmaps(Naruseetal.2004),ESTsequences(Kimuraetal.2004),andphysicalmaps(Khorasanietal.2004).The800-MbgenomeofMedakahasnowbeensequencedandassembled,andtheanalysisthatisunderwayshouldrevealexciting insightsintovertebrategenomeevolution. PreviousSectionNextSection Thefutureoffishgenomics Thefutureoffishgenomicsisbright,andthispredictionissustainedbelowbythreeexampleswherespecificcharacteristics offishspeciesweresuccessfullyexploitedtogainpenetratinginsightsinabroadrangeofsubjects. Fish,asahighlydiversifiedgroupofthevertebratefamily,experienceanastonishingrangeofenvironmentalconditions towhichtheirphysiologies,bodyshapes,andlifestyleshaveadapted.However,acommondenominatorofallfishspeciesis theiraquatichabitat,meaningthatwaterisindirectcontactwithseveraltissuesandinternalcompartmentsoftheanimal, potentiallyinducingahighsensitivitytowater-borneparameterssuchastemperature,oxygenlevels,salinity,andsometimes toxicchemicals.Thisintimaterelationshipbetweenanorganismandawiderangeofdifferentenvironmentshasrecentlyprompted theviewthatfishcouldbeusedasmodelsfor“environmentalgenomics”(CossinsandCrawford2005),inotherwordsthestudyoftheinterfacebetweenanorganismanditsenvironmentusinggenomicapproaches.Thisconcept hadrecentlybeenillustratedbyanelegantstudyinvolvingtheexposureofcarptoincreasinglevelsofcold,from30°Cto 10°Candmeasuringthechangeinthelevelofexpressionofseveralthousandgenesusingmicroarraytechniques(Graceyetal.2004).Carp,asmostfish,ispoikilothermic(cold-blooded),whichimpliesthatitsbodytemperaturefollowsthatofthewater itisimmersedin.Intheseventissuesthatweremonitored,thedecreaseintemperaturewascorrelatedwithagradedincrease inexpressionofacoresetof252genespredominantlyfromtranscriptionalregulation,RNAsplicingandtranslationsystems, whileveryfewgenescommontoalltissuesshowedadecreaseinexpression.Conversely,tissuespecificresponsesshowedthat brainmodifiesitsglycolyticactivitywhileaswitchtolipidmetabolismisobservedinliver.Interestinglythisstudywas performedinanonmodelspecies,exemplifyingtheuseofaspecificspeciesoffishwithaspecificfeatureofinterest,in thiscasethecarpwhichtoleratesawiderangeoftemperature,toinvestigateawholebodyphysiologicaladaptationtoa changeinenvironment.Whiletheresultsareusefultobetterunderstandthemolecularbasisoftheresponsetocoldincarp, fishsharesomanyaspectsoftheirdevelopmentalpathways,physiologicalmechanismsandorgansystemswithmammalsthatthese resultsarealsorelevanttohumanphysiology. Thesecondillustrationofthestrengthoffuturegenomicresearchusingfishspeciesisitsrecognitionbyinternational fundingagencies.OnesuchrecentexampleisthemajorimpetustozebrafishgenomicsprovidedbytheEuropeanUnionFramework ProgrammeforResearch.ByfundingtheZF-MODELconsortium(www.zf-models.org),anIntegratedProjectdevotedtothestudyofzebrafishmodelsforhumandevelopmentanddisease,theprojectlinksabout 30groupsfromsevenEuropeancountries(Bradbury2004).Theresearchplanistotakeadvantageofthezebrafishmodelincombinationwithanarrayoflarge-scaletechniquesto identifyzebrafishgenesthatreplicatetosomedegreehumanpathologies,orareinvolvedindevelopmentalpathwayssimilar toours.Thescaleofthisendeavorcanbemeasuredbythethousandsofmutantsalreadygeneratedbytheconsortiummembers usingeitherforwardgenetics(forinstancewithchemicalmutagenesisscreens),orreversegenetics(forinstanceusingthe TILLINGtechnique;McCallumetal.2000)andthescopeofthescreensusedtoanalyzethem.Designedtoidentifymutantsthatresemblehumandisorders,thesescreens arealsocarriedoutonadultfishandencompassforinstancebonemalformation,skindevelopment,eyemovementoraddiction behavior.CombinedwithenhancerdetectiontechniquesusingGreenFluorescentProtein(GFP)transgeniclinesandexpression profilingwithmicroarrayanalysis,theconsortiumwillintegrateabroadrangeofexpertisetotacklethefunctionofthe mutatedgenes,whichhopefullywillleadtoabetterunderstandingofcorrespondinghumandisorders. Inadditiontocountingseveralmemberspecieselevatedtothestatusofgenomicmodelssuchaszebrafishorpufferfish,fish alsorepresentamajorsourceoffoodforhumans.TheEuropeanUnionthusalsorecognizedtheneedforimprovingaquaculture researchbyfundingtheAQUAFIRSTconsortiumtoidentifygenesassociatedwithstressanddiseaseresistanceinseabream, seabass,andrainbowtroutinordertoprovideaphysiologicalandgeneticbasisformarker-assistedselectivebreeding. Genomictechniquesandsequencecomparisonswithgenomicmodelsarecornerstonesofthisproject,whichillustrateshowfish genomicsmaygrowincomingyearsoutsideoffundamentalresearchlabstowardmoreappliedobjectivesnonethelessessential tohumanwelfare. Afinalexampleoftheuseoffishgenomesthatmayexpandinthenearfuturerestsonthephylogeneticpositionoffishspecies incomparisontomammals(Fig.1).Todayagreatdealofattentionisfocusedonthepartofeukaryotegenomesthatdonotcodeforproteinsbutarenevertheless functional.Elementsthatmaycarryoutspecificfunctionsintheseregionsincludenon-codingRNAgenesandregulatorycontrol regions.Oneofthemostpowerfultechniquestoidentifysuchelementsistoaligngenomicsequencesofdistantlyrelated organismsandlookforregionsthathaveremainedsimilarduringevolution,thussuggestingthatafunctionalconstraintis actingtopreservethesequencefrommutations.Theadvantageoffishinthiscontextisthelongevolutionarydistance,approximately 450millionyears,sincetheirlastancestorwithmammals.Neutralmutationshavesincesaturatedthegenometoapointwhere anyconservedregionbetweenforinstancehumanandpufferfishisindicativeofafunctionalconstraint.Thiscomparative approachwasfirstappliedonagenomescaletoidentifycodingexons(RoestCrolliusetal.2000),andmorerecentlytoidentifyultra-conservedregions(UCRs)ofunknownfunction(Sandelinetal.2004;Woolfeetal.2004).HoweveranadditionalassumptionthatcanbededucedfromthediscoveryofUCRsconservedbetweensuchdistantorganisms istheirfundamentalimportanceacrossvertebrates.Inlinewiththis,UCRsfoundinthiswaylieinclustersaroundgenes involvedintheregulationofdevelopment.IndeedwhentheorthologousregionswereassayedusingGFPreporterconstructs inzebrafishembryos,mostshowedsignificantenhanceractivityinoneormoretissues(Woolfeetal.2004).Sofish–mammalsequencealignmentsnotonlyprovidethemeanstoidentifyfunctionalelements,theyalsoactasascreen toselectthoseelementsessentialtovertebrates.Withtheproductionofnewfishgenomesequencesaswellasnewmammalian sequences,suchcomparativestudiesarelikelytoplayanimportantroleinguidingtheidentificationoffunctionalnoncoding elements,andindecipheringthesubtlesequencevariationsthatmightleadtophenotypicchanges(Ahituvetal.2004;Boffellietal.2004). Manyfishspeciesareroutinelybeingstudiedatthemolecularlevelandevenatthegenomiclevelandhavenotbeencited here.ForinstancethousandsofESTsequencesareavailableforcarp,catfish,salmon,trout,killifish,stickleback,ortilapia andlargeinsertBAClibrariesarealsoavailableforseveralofthesespecies,furtherillustratingthewidespreadinterests inusingfishforgenomicresearch.Fourfishgenomesequencesareorwillbeavailablesoon:fugu,Tetraodon,medaka,andzebrafish.Thesticklebackgenome(Gasterosteusaculeatus)isalsowelladvanced(Table1),buttoourknowledgenootheriscurrentlyongoingbeyondthese,althoughobviouslyseveralinterestgroupsareactively workingtowardspromotingcertainspecies.Researchonsalmonandtroutforinstancewouldgreatlybenefitfromtheavailability ofthegenomesequence.Indeedmoreisknownaboutthephysiologyandbiologyofrainbowtroutthananyotherfishspecies, althoughgenomicsequencingcouldbecomplicatedbyanadditionalgenomeduplicationinthesalmonidlineagesome25–100Mya (AllendorfandThorgaard1984).Thesarcopterygianfishcoelacanthisthenearestlivingrelativeoftetrapods(Gorretal.1991)(Fig.1)andthusescapedthewhole-genomeduplicationthataffectedtheteleosts.Thismaybeaseriousadvantagesinceitmight provideaccesstoagenomethatresemblestheearlytetrapodgenome,unaffectedbytheconsequenceofmassivegeneduplications suchasgeneconversion.Altogetherthecoelacanthwouldindeedbeanexcellentcandidateforgenomesequencingasitwould provideareferencegenomefortetrapodswhileallowingtheidentificationofgenomicfeaturesthatdifferentiatethemfrom teleosts(Noonanetal.2004).Forsimilarreasonsbutacrossawiderevolutionaryscale,theelephantfish(Callorhinchusmilii,acartilaginousfish;Fig.1)hasrecentlybeenproposedasagoodmodeltostudythegenomestructureandgenecontentofabasaljawedvertebrate,and provideacommonreferencefortetrapodsandray-finedfishes(Venkateshetal.2005). Viewthistable: Inthiswindow Inanewwindow Table1. Summaryofcurrentgenomicresourcesonfishspecies Uptonowfishgenomicshasbeenabletodrawonthesimilaritiesandthedifferencesbetweenmammalianandfishgenomesto gainprofoundinsightsintotheevolutionofvertebrategenomesingeneral,andintothefunctionofindividualgenesoften associatedwithhumandisordersinparticular.CaptainNemo'sfishingexploitswiththeNautilusmaybehardtomatchbutthenetcastbygenomescientistshasalsoreeledinsomeunexpectedsurprises,andtheendofthis storyiscertainlyalongwayoff. PreviousSectionNextSection Footnotes Articleandpublicationareathttp://www.genome.org/cgi/doi/10.1101/gr.3735805. ↵3Correspondingauthor.E-mailhrc{at}ens.fr;fax33-1-44-32-39-41. ColdSpringHarborLaboratoryPress PreviousSection  References ↵ Ahituv,N.,Rubin,E.M.,andNobrega,M.A.2004.Exploitinghuman—fishgenomecomparisonsfordecipheringgeneregulation.Hum.Mol.Genet.13SpecNo2:R261-R266. 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