Sea!Urchin!Body!Plan!Development!and!Evolution:!An!Integrative! Transcriptomic!Approach! by! Jennifer!Wygoda!Israel!
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1 SeaUrchinBodyPlanDevelopmentandEvolution:AnIntegrative TranscriptomicApproach by JenniferWygodaIsrael UniversityPrograminGeneticsandGenomics DukeUniversity Date: Approved: DavidMcClay,CoHSupervisor GregWray,CoHSupervisor AmyBejsovec BlancheCapel BobGoldstein Dissertationsubmittedinpartialfulfillmentof therequirementsforthedegreeofdoctorofphilosophy intheuniversityprogramingeneticsandgenomics inthegraduateschoolofdukeuniversity 2015
2 ABSTRACT SeaUrchinBodyPlanDevelopmentandEvolution:AnIntegrative TranscriptomicApproach by JenniferWygodaIsrael UniversityPrograminGeneticsandGenomics DukeUniversity Date: Approved: DavidMcClay,CoHSupervisor GregWray,CoHSupervisor AmyBejsovec BlancheCapel BobGoldstein Anabstractofadissertationsubmittedinpartialfulfillmentof therequirementsforthedegreeofdoctorofphilosophy intheuniversityprogramingeneticsandgenomics inthegraduateschoolofdukeuniversity 2015
3 Copyrightby JenniferWygodaIsrael 2015
4 Abstract( Mydissertationworkintegratescomparativetranscriptomicsand functionalanalysestoinvestigategeneexpressionchangesunderlyingtwo significantaspectsofseaurchinevolutionanddevelopment:thedramatic developmentalchangesassociatedwithanecologicallysignificantshiftinlife historystrategyandthedevelopmentoftheunusualradialbodyplanofadult seaurchins. InChapter2,Iinvestigateevolutionarychangesingeneexpression underlyingtheswitchfromfeeding(planktotrophic)tononfeeding (lecithotrophic)developmentinseaurchins.inordertoidentifythesechanges,i usedilluminarnahseqtomeasureexpressiondynamicsacross7developmental stagesinthreeseaurchinspecies:thelecithotrophheliocidaris+erythrogramma,the closelyrelatedplanktotrophheliocidaris+tuberculata,andanoutgroup planktotrophlytechinus+variegatus.myanalysesdrawonawellhcharacterized developmentalgeneregulatorynetwork(grn)inseaurchinstounderstand howtheancestralplanktotrophicdevelopmentalprogramwasalteredduringthe evolutionoflecithotrophicdevelopment.myresultssuggestthatchangesingene expressionprofilesoccurredmorefrequentlyacrossthetranscriptomeduringthe iv
5 evolutionoflecithotrophythanduringthepersistenceofplanktotrophy.these changeswereevenmorepronouncedwithinthegrnthanacrossthe transcriptomeasawhole,andoccurredineachnetworkterritory(skeletogenic, endomesodermandectoderm).ifoundevidenceforbothconservationand divergenceofregulatoryinteractionsinthenetwork,aswellassignificant changesintheexpressionofgeneswithknownrolesinlarvalskeletogenesis, whichisdramaticallyalteredinlecithotrophs.ifurtherexplorednetwork dynamicsbetweenspeciesusingcoexpressionanalyses,whichallowedmeto identifynovelplayerslikelyinvolvedinseaurchinneurogenesisandendoderm patterning. InChapter3,Iinvestigatedevelopmentalchangesingeneexpression underlyingradialbodyplandevelopmentandmetamorphosisinh.+ erythrogramma.usingilluminarnahseq,imeasuredgeneexpressionprofiles acrosslarval,metamorphic,andposthmetamorphiclifecyclephases.myresults presentahighhresolutionviewofgeneexpressiondynamicsduringthecomplex transitionfromprehtoposthmetamorphicdevelopmentandsuggestthatdistinct setsofregulatoryandeffectorproteinsareusedduringdifferentlifehistory phases. v
6 Collectively,myinvestigationsprovideanimportantfoundationfor future,empiricalstudiestoinvestigatethefunctionalroleofgeneexpression changeintheevolutionofdevelopmentaldifferencesbetweenspeciesandalso forthegenerationoftheunusualradialbodyplanofseaurchins. vi
7 Dedication( Tomyfamilyfortheirunconditionalloveandunwaveringsupport. vii
8 Contents( Abstract...iv ListofTables...xii ListofFigures...xiii Acknowledgements...xv 1.Introduction Overview Seaurchinsasamodelsystemforlifehistoryevolution Diversityofseaurchinlifehistorystrategies Theswitchfromplanktotrophictolecithotrophicdevelopment TheevolutionoflecithotrophyinthegenusHeliocidaris Overview Radicalreorganizationofearlydevelopment Extensiveevolutionarychangesinthelarvalsupportsystem Dramaticaccelerationofjuvenilebodyplandevelopment Theroleofgeneexpressionchangeintheevolutionofdevelopmental differencesinheliocidaris Summaryofchapters...22 Chapter2:ReHwiringofaDevelopmentalGeneRegulatoryNetworkDuringthe EvolutionofaNovelLifeHistoryDifferenceinSeaUrchins Introduction...24 viii
9 2.2Results Globalgeneexpressionpatternsduringdevelopmentrecapitulate phylogeny Changesingeneexpressionprofilesweremorenumerousduringthe evolutionoflecithotrophythanduringthepersistenceofplanktotrophy Changesingeneexpressionduringtheevolutionoflecithotrophyare qualitativelydistinctfromthoseduringthepersistenceofplanktotrophy Changesinexpressiondifferbetweenthegeneregulatorynetworkand thetranscriptomeasawhole Distinctpatternsofevolutionarychangeingeneexpressionamong embryonicterritories Coexpressionanalysisidentifiesnovelcandidategenesforgutand neuraldevelopment Discussion Towardsananalyticalframeworkforcomparativedevelopmental transcriptomics Theevolutionoflecithotrophyalterednaturalselectionthroughoutthe transcriptome TheexpressionofGRNgenesevolvesquitedifferentlyfromthe transcriptomeasawhole ChangesintheexpressionofGRNgeneslikelyunderlietheevolution ofspecifictraitsinthelecithotroph Coexpressionanalysesextendthesearchforcandidategenesfromthe GRNtotherestofthetranscriptome Materialsandmethods...77 ix
10 2.4.1Samplecollection RNApreparationandsequencing Transcriptomeassemblyandannotation Readmappinganddatanormalization Clusteranalysisandcategoricalenrichment Cloningofcandidategenes Fixationandwholemountinsituhybridization Acknowledgements...86 Chapter3:TranscriptomicAnalysisoftheHighlyDerivedRadialBodyPlanofa SeaUrchin Introduction Results Ahighlydynamicdevelopmentaltranscriptome ExtensivechangesinfunctionalcategoriesaccompanythepreHtopostH metamorphictransition Broadpatternsofgeneexpressionchangesduringdevelopment Neurogenesistranscriptionfactorshavedistinctphasesofexpression Conclusionsandfuturedirections Materialsandmethods Samplecollection RNApreparationandsequencing x
11 3.4.3Transcriptomeassemblyandannotation Readmapping,datanormalization,anddifferentialexpression Categoricalenrichmentandclusteranalyses Acknowledgements Chapter4:SummaryandFutureDirections Summary Futuredirections Usinginterspecifichybridstouncoverthegeneticbasisforgene expressiondivergence FunctionalcharacterizationofcandidategenesinH.+erythrogramma..132 References Biography xi
12 List(of(Tables( Table1:Planktotrophyvs.lecithotrophy:advantagesanddisadvantages...8 Table2:Planktotrophyvs.lecithotrophy:developmentaldifferences...11 Table3:Impactofinitialclusternumberonexpressionclassification...42 Table4:EnrichedPANTHERcategories:genesupregulatedintheadvanced rudimentstagevs.earlyjuvenilestage Table5:EnrichedPANTHERcategories:genesupregulatedinearlyjuvenile stagevs.advancedrudimentstage Table6:EnrichedPANTHERcategories:genesupregulatedinearlyjuvenile stagevs.latejuvenilestage Table7:EnrichedPANTHERcategories:genesupregulatedinlatejuvenilestage vs.earlyjuvenilestage xii
13 List(of(Figures( Figure1:Planktotrophyvs.lecithotrophy:acomparisonofcellfatespecification atthe32hcellstage...14 Figure2:Planktotrophyvs.lecithotrophy:acomparisonofskeletaldevelopment...17 Figure3:Planktotrophyvs.lecithotrophy:acomparisonofcoelomicpouch development...19 Figure4:Geneexpressionduringseaurchindevelopmentreflectsknown phylogeneticrelationshipsbetweenspecies...31 Figure5:LifeHhistorystrategyisarelativelyminorcomponentofgeneexpression divergenceacrossthetranscriptome...34 Figure6:Geneexpressiondivergenceishighestintheeggandtendstodecrease acrossdevelopment...35 Figure7:FuzzycHmeansclusteringgroupssimilarexpressionprofilesacross developmentintosevendistinctclusters...40 Figure8:Comparativeclusteringstrategyrevealscasesofexpressionprofile conservationandchangewithinaphylogeneticframework...41 Figure9:Subtleshiftsaremorecommonthandramaticchangesingene expressionbetweenspecies...43 Figure10:LifeHhistorystrategyisamajorcomponentofgeneexpression divergenceacrossthegeneregulatorynetwork...48 Figure11:Networkgenesexhibitbothconservedanddivergentexpression profilesbetweenspecies...56 Figure12:Genescoexpressedduringplanktotrophicdevelopmentsignificantly overlapwiththosecoexpressedduringlecithotrophicdevelopment...62 xiii
14 Figure13:CrossHspeciestranscriptomecomparisonrevealsnovelcandidatesfor planktotrophicdevelopment...63 Figure14:DevelopmentaltranscriptomeinHeliocidaris+erythrogramma...95 Figure15:Metamorphicandpostmetamorphiclifecyclestagesarecharacterized bydynamicgeneexpressionpatterns...96 Figure16:Advancedrudiment,earlyjuvenile,andlatejuvenilesexhibitstageH specificenrichmentofpantherbiologicalprocesscategories Figure17:AdditionalclustersidentifiedwithfuzzycHmeansclustering Figure18:FuzzycHmeansclusteringidentifiesgeneralpatternsofgene expressionacrossdevelopment Figure19:Expressionofneuraltranscriptionfactorsduringjuvenile development xiv
15 Acknowledgements( TherearemanypeopleIwouldliketothankfortheirsupportand guidance.first,myparents,lindaandmark,whoneverceasetoamazemewith theirunconditionalloveandencouragement.wordscannotexpresshowgrateful Iamforyoursupport. IwanttothankmygraduateadvisorsGreg,WrayandDave,McClayfor theirmentorshipandenthusiasmthroughoutmytimeatduke.theybothgave meimmensefreedomtopursuemyownresearchinterestsandhelpedmeto developtheconfidencenecessarytobecomeanindependentscientist.iwould alsoliketothankmycommitteemembers,amy,bejsovec,blanche,capel,and Bob,Goldsteinfortheirguidanceandscientificdirection. Mydissertationworkwouldnothavebeenpossiblewithoutcurrentand formerlabmembers.firstiwanttothankcourtney,babbittforspending countlesshourstotrainme.herpatienceandencouragementmademy transitionfromthebenchtothecomputernotonlypossible,butenjoyable.ialso wanttothankdavid,garfieldanddan,runcieforintroducingmetotheworld ofseaurchinsandforcontinuingtoprovidemewithcriticalfeedbackonmy projects,dede,lyonsforherscientificdiscussionsandinsightfuladvice xv
16 throughoutgraduateschool,andmegan,martikforherfriendshipandher passionforscience thankyouall Finally,Iwanttothankmyhusband,Kevin,forhisloveandfriendship overthepast11years.iamsogratefulforhisencouragementandsteadfast support.ialsowanttothankabbieforthehappinessshebringsuseveryday. xvi
17 1.(Introduction( 1
18 1.1#Overview# Multicellularorganismsexhibitanextraordinarydiversityoflifehistories (i.e.,organismalpatternsofdevelopment,growth,reproduction,andsurvival). Howanorganismallocateslimitedresourcestooptimizesurvivaland reproductivesuccessisakeydriveroflifehistoryevolutionandinvolves importanttradehoffsinopposingdemographictraits(e.g.,fecundityandparental investment)(flatt&heyland,2011).whilesignificantprogresshasbeenmadein understandingtheecologicalfactorsthatshapethesetraitsandthetradehoffs thatconstraintheirevolution,relativelylittleisknownaboutthegeneticor molecularbasisfortheevolutionoflifehistorydifferencesbetweenspecies. Here,Iinvestigatethegeneticanddevelopmentalbasisforanecologically significantlifehistorytransition:theswitchfromfeeding(planktotrophic)to nonfeeding(lecithotrophic)developmentinseaurchins. 2
19 1.2#Sea#urchins#as#a#model#system#for#life#history#evolution# Seaurchinsareanidealsystemtoinvestigatetheevolutionofalternative lifehistorystrategies.first,anabundantfossilrecordanddetailedphylogeny provideavaluablecontextforunderstandingwhenandhowoftenalternative developmentalstrategiesevolved(a.b.smithetal.,2006;wray,1996).second, seaurchinsareawellhstudied,experimentallytractablemodelsystemfor development(mcclay,2011).seaurchineggscanbeinjectedwithawiderange ofmolecularreagents,includingmorpholinos,whichblockthetranslationof specificmrnas,orexpressionconstructs,whichinduceexogenousmrna expression(ettensohn,wessel,&wray,2004).followinginjection,larvaecanbe easilyassayedforchangesinmorphologyandtheexpressionofdownstream targetgenes.thesemanipulationsallowfortestablehypothesesaboutthe specificgeneticchangesthatmayhaveaccompaniedtheevolutionofan alternativedevelopmentalstrategy.finally,thedevelopmentalgeneregulatory network(grn)inseaurchins,mostlybuiltfromstudiesinstrongylocentrotus+ purpuratus,lytechinus+variegatus,andparacentrotus+lividus,isthemostthorough GRNofanyanimaltodateandcoverskeyaspectsofseaurchindevelopment: fromtheunfertilizedeggthroughtheformationofaplanktonicfeedinglarva 3
20 (Barsi,Li,&Davidson,2015;BenHTaboudeHLeon,Su,Lin,Li,&Davidson,2013; Croceetal.,2011;E.Li,Materna,&Davidson,2013;McIntyre,Seay,Croce,& McClay,2013;Oliveri,Tu,&Davidson,2008;Peter&Davidson,2010,2011; Rafiq,Cheers,&Ettensohn,2012;Rafiq,Shashikant,McManus,&Ettensohn, 2014;Range,Angerer,&Angerer,2013;Rho&McClay,2011;Rottingeretal., 2008;Sethi,Angerer,&Angerer,2009;Sethi,Wikramanayake,Angerer,Range,& Angerer,2012;Suetal.,2009;Wei,Yaguchi,Yaguchi,Angerer,&Angerer,2009). Importantly,thisnetworkprovidesavaluableframeworkforinvestigatinghow geneexpressiondivergenceduringdevelopmentcontributestophenotypic evolutionbetweenspecieswithalternativedevelopmentalstrategies. 4
21 1.3#Diversity#of#sea#urchin#life#history#strategies# Seaurchinsemployawiderangeofreproductiveanddevelopmental strategies,whichcanbebroadlycharacterizedaccordingtothreecomponents:(1) locationofembryonicdevelopment(e.g.,planktonicorbroodingdevelopment), (2)thepresenceorabsenceofacomplexlarvalstage(e.g.,indirectordirect development),and(3)theassociatedlarvalnutritionalmode(e.g.,feeding, nonfeeding,orfacultativefeeding)(mcedward&janies,1997).mostseaurchin specieshaveembryosthatdevelopexternallyinthewatercolumn(planktonic development),althoughsomespecieswillbroodembryosinspecializedpouches oramongspinesoftheadultfemale(broodingdevelopment).indirect developershaveacomplexlarvalstagethatprecedesmetamorphosis,whereas directdevelopersdonotpassthroughadistinctlarvalstage.instead,theyhave anabbreviatedembryonicdevelopmentandrapidlypatterntheadultbodyplan. Seaurchinspeciesalsovaryintheirnutritionalrequirementsduring development.feeding,or planktotrophic,speciesmustobtainexogenousfood tofuelgrowthanddevelopment,whilenonfeedingor lecithotrophic species obtainalloftheirnutritionalcontentfrommaternallyprovisionedeggs.notably, switchesbetweenthesestrategiesoccurrednumeroustimeswithinseaurchins 5
22 andinvolvedclassiclifehistorytradehoffsinfecundity,parentalinvestment, dispersal,ageandsizeatmaturity,andlifespan(mcedward&janies,1997). 6
23 1.4#The#switch#from#planktotrophic#to#lecithotrophic# development# TheancestrallifeHhistorystrategyinseaurchinsisplanktonic,indirect developmentthroughafeeding(planktotrophic)larvalstage,oftenreferredtoas a pluteus larva(mcedward&janies,1997;strathmann,1985).however, nonfeeding(lecithotrophic)developmenthasindependentlyevolvedmultiple timeswithinthisclade(jeffery,emlet,&littlewood,2003;wray,1996). Comparedtoplanktotrophs,lecithotrophsproducefewer,largereggsthatare richinmaternalproteinsandlipidstores,allowingthesespeciestoomitthe larvalfeedingstage(byrneetal.,1999;villinski,villinski,byrne,&raff,2002). Thesenonfeedinglarvaehaveoftenreducedorlostseveralkeymorphological featuresassociatedwithfeeding(e.g.,thelarvalgut)andundergoaccelerated juveniledevelopment(parks,parr,chin,leaf,&raff,1988;williams& Anderson,1975).Althoughtheselectiveforcesdrivingtheevolutionof lecithotrophicdevelopmenthavereceivedwidespreadattention(hart&marko, 2010;Pechenik,1999;Strathmann,1990;Wray&Raff,1991a)(summarizedin Table1),muchlessisknownaboutthemolecularunderpinningsofthis substantialshiftinlifehistorystrategy. 7
24 8 Table,1:,Planktotrophy,vs.,lecithotrophy:,advantages,and,disadvantages, Advantages, Disadvantages, Planktotrophy,, Reducedintraspecific resourcecompetition betweensiblingsand generations Reducedbenthicmortality Increaseddispersalcapacity decreaseslikelihoodof inbreeding Increasedabilitytowithstand localextinction Lowertotalenergycostto produce Increaseddispersalcapacity mayresultinmovement awayfromfavorablehabitat Increasedplanktonic mortality Environmentalstress experiencedbylarvaemay reduceposthmetamorphic performance Lecithotrophy,, Reduceddispersalcapacity allowsmaintenanceof favorablehabitat Reducedplanktonic mortality Environmentalstress experiencedbylarvaeis unlikelytoreduceposth metamorphicperformance Increasedintraspecific resourcecompetition betweensiblingsand generations Increasedbenthicmortality Reduceddispersalcapacity increaseslikelihoodof inbreeding Reducedabilitytowithstand localextinction
25 1.5#The#evolution#of#lecithotrophy#in#the#genus#Heliocidaris# 1.5.1(Overview( Theswitchfromplanktotrophictolecithotrophicdevelopmentinthesea urchingenusheliocidarisisoneofthemostcomprehensivelystudiedlifehistory transitionsinanyanimal(bisgrove&raff,1989;byrneetal.,1999;ferkowicz& Raff,2001;Haag&Raff,1998;Henry,Wray,&Raff,1990;Kauffman&Raff,2003; Klueg,Harkey,&Raff,1997;Love,Lee,Andrews,&Raff,2008;Love&Raff, 2006;Parksetal.,1988;M.S.Smith,Collins,&Raff,2009;M.S.Smith,Turner,& Raff,2008;Villinskietal.,2002;Wilson,Andrews,&Raff,2005;Wilson, Andrews,RudolfTurner,&Raff,2005;Wray&Raff,1989,1990,1991b;Zhou, Wilson,Andrews,Kauffman,&Raff,2003;Zigler,Raff,Popodi,Raff,&Lessios, 2003).Decadesofresearchhavecontributedtoourunderstandingofthe differencesincelllineagespecification,cellhcellinteractionsandgeneexpression patternsbetweentheplanktotrophheliocidaris+tuberculataandthelecithotroph Heliocidaris+erythrogramma.Notably,developmentalprocessespreviously conservedfor10sh100sofmillionsofyearschangeddramaticallyinh.+ erythrogrammaoverarelativelyshortevolutionarytimescale(divergence=5myr) (Byrneetal.,1999;Henryetal.,1990;Raff,1992;M.S.Smithetal.,2009;Villinski 9
26 etal.,2002;williams&anderson,1975;wray&raff,1989,1990,1991b).these changesaredescribedbelowandsummarizedintable2. 10
27 Table2:Planktotrophyvs.lecithotrophy:developmentaldifferences 11 H.t.(Planktotroph) H.e.(Lecithotroph)' Egg 95µm,lowlevelsofwaxesterlipids 430µm,highlevelsofwaxesterlipids Increaseddepositionofmaternalfactors CellFate Unequal4 th cleavagetoformmicromeres Equal4 th cleavage,micromeresnotformed Radialsymmetryofvegetalfates DorsalHventralsymmetryofvegetalfates Animaloriginofciliaryband Vegetaloriginofciliaryband Skeletogeniclineagesegregatedat5 th Skeletogeniclineagesegregatedafter6 th cleavage cleavage(delayed) Skeleton <60skeletogeniccellsundergoingression >1700skeletogeniccellsundergoingression Characteristicskeletogenicringpattern Randomskeletogenicspicules Gut Gastrulationinvolvessymmetrical,initial Gastrulationinvolvesanasymmetrical, cellinvolutionfollowedbycell continuouscellinvolution rearrangements Archenterondoesnotdifferentiateintoa Formationoftripartitegutfrom tripartitegut,larvalmouthabsent archenteron,larvalmouthpresent Coelomic SmallleftandrightCPsdeveloptowards DevelopmentofanenlargedleftCPis Pouches theendofgastrulation accelerated (CPs) Juvenilebodyplanbeginstoformweeks Juvenilebodyplanbeginstoformwithin28 afterfertilization hoursoffertilization Ectoderm Oralandaboralectoderm Smallpatchoforalectodermlater differentiatesintovestibularectoderm Noovertoraloraboralectoderm Differentiationofvestibularectodermis greatlyaccelerated
28 1.5.2%Radical%reorganization%of%early%development% Theacquisitionofalarge,yolkyeggwasamajorcomponentof lecithotrophicevolutioninthegenusheliocidaris,andthequantitativeand qualitativedifferencesbetweeneggsoftheplanktotrophh.,tuberculataandthe lecithotrophh.,erythrogrammaareprofound(raff,1987,1992).theh., erythrogrammaeggissubstantiallylargerthantheh.,tuberculataegg(430µmvs. 95µm),resultinginpartfromthedepositionofnonIvitellogenicmaterial (maternallipidsandprotein)duringanovelsecondphaseofoogenesis(byrneet al.,1999;raff,1987;williams&anderson,1975).theh.,erythrogrammaeggalso containsadistinctclassofwaxesterlipidsnotpresentintheeggsof planktotrophs(villinskietal.,2002). Aradicallyreorganizedcellfatemapalsoaccompaniedtheswitchfrom planktotrophictolecithotrophicdevelopmentinheliocidaris(figure1)(raff, 1992;Wray&Raff,1989,1990).Mostnotably,whiletheplanktotrophicembryo undergoesanunequal4 th cleavagetosegregatethedistinctivemicromere lineage,theh.,erythrogrammaembryoundergoesanequal4 th cleavageanddoes notproducemicromeres(raff,1992;wray&raff,1989).thisdifferenceis remarkable,giventhatthemicromeresactastheorganizingcenterofthe 12
29 planktotrophicembryo(davidson,cameron,&ransick,1998;oliverietal., 2008).TheabsenceofmicromeresinH.,erythrogrammaalsoresultsinthedelayof skeletogeniclineagespecificationinthisspecies.othernotablechangesincell fateinh.,erythrogrammaincludeadorsaliventralsymmetryofvegetalcellfates, asopposedtotheradialsymmetryobservedinplanktotrophs,andthevegetal originoftheciliaryband,whichhasananimalorigininplanktotrophs (Cameron,Houghevans,Britten,&Davidson,1987;Wray&Raff,1989,1990). 13
30 Figure'1:'Planktotrophy'vs.'lecithotrophy:'a'comparison'of'cell'fate'specification'at' the'32:cell'stage.' Comparedtoplanktotrophs,whichhaveunequal4 th and5 th cleavages, lecithotrophsundergoequalcleavagesanddonotformmicromeres. LecithotrophsalsohaveadorsalIventralsymmetryofvegetalfates(dark grey),asopposedtoaradialsymmetryinplanktotrophs.inaddition,the ciliarybandisofanimaloriginintheplanktotroph,butvegetalorigininthe lecithotroph.thisfigureisadaptedfromraff(1992). 14
31 1.5.3%Extensive%evolutionary%changes%in%the%larval%support%system% Planktotrophiclarvaedeveloptwokeyfeaturesnecessarytosupport planktonicfeeding:anintricatelarvalskeletonandatripartitegut.although developmentofthesetissuesishighlyconservedamongplanktotrophicspecies, eachhaschangedextensivelyinh.,erythrogramma,(loveetal.,2008;parksetal., 1988;Williams&Anderson,1975). Inplanktotrophs,thelargemicromeresaretheskeletogeniclineage foundercellsandaresegregatedat5 th cleavage(davidsonetal.,1998).following specification,skeletogenicprecursorsdetachfromtheepitheliumandingress intotheblastocoel(figure2a).thecellsthenmigratetowardectodermalcues, assembleintoaringpattern,andsecretebiomineralizationproteins,whichare thebuildingblocksofthelarvalskeleton.althoughskeletogenicspecificationis delayedinh.,erythrogramma,thisspeciesspecifiesmanymoreskeletogeniccells comparedtoplanktotrophs(parksetal.,1988).however,thesecellsdonot assembleintoaringpatternandinsteadformafew,randomlyplacedremnant spicules(figure2b).thelarvalguthasalsoundergonewidespreadchanges duringtheevolutionoflecithotrophicdevelopment(loveetal.,2008;wray& Raff,1991b).Inplanktotrophs,gastrulationbeginswitharadiallysymmetric 15
32 involutionofcellstoformashort,widetube(i.e.,thearchenteron),whichislater transformedintoalong,slendertubebycellrearrangements(burke,myers, Sexton,&Jackson,1991;Ettensohn,1985).Afterextendingacrosstheblastocoel totheanimalpole,thearchenteronfuseswiththeoverlyingoralectodermto formthelarvalmouth(hardin,1996).theplanktotrophicgutthendifferentiates intothreecompartments:theforegut,midgut,andhindgut. WhileH.,erythrogrammaalsoexhibitsaninitialphaseofsymmetric invaginationduringgastrulation,thearchenteronisnotextendedbycell rearrangements(wray&raff,1991b).instead,thearchenteronelongatesby prolonged,asymmetricinvolutionofcellsovertheventrallipoftheblastopore. Furthermore,thearchenterondoesnotextendtotheanimalpole itstops elongationonceitreachesmidwayacrosstheblastocoel.followinggastrulation, thearchenterondoesnotdifferentiateintoatripartitegutandthelarvalmouthis notformed(williams&anderson,1975). 16
33 Figure'2:'Planktotrophy'vs.'lecithotrophy:'a'comparison'of'skeletal' development' (A)Inplanktotrophs,asmallnumberofskeletogenicprecursors(red) willingressintotheblastocoelandmigratetowardectodermalcues. Thesecellswillassembleintoaringpatternandsecretea biomineralizedlarvalskeleton.(b)inlecithotrophs,alargenumberof skeletogenicprecursors(red)willingressintotheblastocoelbutwill onlyformrandomlyaplaced,remnantspicules 17
34 1.5.4%Dramatic%acceleration%of%juvenile%body%plan%development%% Neartheendofgastrulationinplanktotrophs,smallcoelomicpouches pinchoffoftheleftandrightsidesofthearchenteron(figure3a)(pehrson& Cohen,1986;M.M.Smith,CruzSmith,Cameron,&Urry,2008).Weekslater, thesepoucheswillsubdivideintothreecompartments(axocoel,hydrocoel,and somatocoel)andasmallregionoforalectodermwilldifferentiateintovestibular ectoderm(m.m.smithetal.,2008).soonafter,thevestibularectodermwill ingressandmakecontactwiththeenlargedlefthydrocoel,initiating developmentofthejuvenilebodyplan(m.m.smithetal.,2008). Oneofmostconspicuousaspectsoflecithotrophyisthedramatic accelerationofthisprocess.insteadofoccurringweekstomonthsafter fertilization,juvenilebodyplandevelopmentbeginswithin28hoursof fertilizationinh.#erythrogramma#(williams&anderson,1975).inthisspecies,a disproportionatelylargeleftcoelomicpouchformsdirectlyfromthearchenteron followedbyasmallerrightcoelomicpouch(figure3b)(m.s.smithetal.,2009). Theleftcoelomicpouchthenquicklydifferentiatesintotheaxocoel,hydrocoel, andsomatocoel.asinplanktotrophs,thehydrocoelinteractswiththeoverlying vestibularectodermtopatternthejuvenilebodyplan(m.s.smithetal.,2009). 18
35 Figure'3:'Planktotrophy'vs.'lecithotrophy:'a'comparison'of' coelomic'pouch'development' (A)Inplanktotrophs,twosmallcoelomicpouchesformonleft (blue)andright'(yellow)sidesofthearchenteron.weekslater, thesepoucheswillsubdivideintothreecompartmentsandasmall regionoforalectodermwilldifferentiateintovestibularectoderm (green).soonafter,thevestibularectodermwillingressandmake contactwiththeenlargedlefthydrocoel(blue),initiating developmentofthejuvenilebodyplan.(b)inlecithotrophs,a disproportionatelylargeleftcoelomicpouch(blue)formsdirectly fromthearchenteronfollowedbyasmallerrightcoelomicpouch (yellow).theleftcoelomicpouchwillquicklydifferentiateintothe axocoel,hydrocoel,andsomatocoel.asinplanktotrophs,the hydrocoelwillinteractwiththeoverlyingvestibularectoderm (green)topatternthejuvenilebodyplan.thisfigureisadapted fromsmithet#al.(2009). 19
36 1.6Theroleofgeneexpressionchangeintheevolutionof developmentaldifferencesinheliocidaris TheprofounddevelopmentaldifferencesbetweenH.#tuberculataandH.# erythrogrammamustresultfromunderlyingchangesingeneexpressionduring development.theshiftsintimingofkeydevelopmentalprocessesinh.# erythrogrammasuggestthatatleastsomeoftheseunderlyingevolutionary changesinexpressionareheterochronicinnature(parksetal.,1988;m.s.smith etal.,2009).yet,otherdevelopmentalchangesinh.#erythrogramma#indicate rewiring ofregulatoryinteractionsorevenlossofgeneexpression(henryet al.,1990;raff,1992;wray&raff,1990,1991b).and,whileahandfulofcase studiessupporteachofthesescenarios(kauffman&raff,2003;kluegetal., 1997;M.S.Smithetal.,2008;Wilson,Andrews,&Raff,2005),ourunderstanding oftheroleofgeneexpressionchangeintheevolutionofdevelopmental differencesbetweenh.#tuberculataandh.#erythrogramma#issignificantlylimited. Importantly,theextentofevolutionarychangeinexpressionacrossthe transcriptomeisunknown.nordoweunderstandhowthesegeneexpression changesaresituated,facilitated,orconstrainedwithinthecontextofthewell^ definedseaurchingeneregulatorynetwork(grn).theselimitationsstemin 20
37 largepartfromthefactthatuntilrecentlyitwasnotfeasibletostudymorethana handfulofgenesineventhebeststudieddevelopmentalsystems,asituation amelioratedbyrecentadvancesinwhole^genomeexpressionprofiling. 21
38 1.7Summaryofchapters Mydissertationresearchiscomprisedoftwomaindatachapters.In Chapter2,Iintegratecomparativetranscriptomicsandfunctionalanalysesto investigatetheroleofgeneexpressionchangeintheevolutionofdevelopmental differencesbetweenthreespeciesofseaurchin:theplanktotrophheliocidaris# tuberculata,thecloselyrelatedlecithotrophheliocidaris#erythrogrammaandan outgroupplanktotrophlytechinus#variegatus.specifically,ifocusonhowchanges tothewell^definedgeneregulatorynetworkofplanktotrophsledtothe evolutionofdramaticdifferencesinheliocidaris#erythrogramma,including developmentalmechanismspreviouslyconservedfor10s^100smillionsofyears. InChapter3,Iuseatranscriptomicapproachtofurtherexploregeneexpression changesrelatedtojuvenilebodyplandevelopmentandmetamorphosisinh.# erythrogramma.inchapter4ipresentasummaryofmydissertationworkand futuredirections. 22
39 Chapter%2:%Re>wiring%of%a%Developmental%Gene% Regulatory%Network%During%the%Evolution%of%a%Novel%Life% History%Difference%in%Sea%Urchins% Israel'JW,'Martik'MM,'Byrne'M,'Raff'R,'McClay'DR,'and'GA'Wray' My'contribution'to'this'work:' 1. Contributedtotheconceptionanddesignofexperiments 2. Performedallcomputationalanalyses 3. Wrotemanuscript 23
40 2.1Introduction Changesinregulatorygeneinteractionsduringdevelopmentareknown toplayamajorroleintheevolutionofphenotypicdifferencesbetweenspecies. Yet,howtheseregulatorychangesaresituated,facilitated,andconstrained withinthecontextofbroadergeneregulatorynetworks(grns)remainsalargely unresolvedquestionforbothevolutionaryanddevelopmentalbiologists. ThedevelopmentalGRNofeuechinoidseaurchins,mainlybuiltfrom studiesinstrongylocentrotus#purpuratus,lytechinus#variegatus,andparacentrotus# lividus,isthemostcomprehensiveandwell^studiedgrnofanyanimaltodate (Annunziata&Arnone,2014;Barsietal.,2015;Ben^Taboude^Leonetal.,2013; Croceetal.,2011;Davidsonetal.,2002;E.Lietal.,2013;Materna,Swartz,& Smith,2013;McIntyreetal.,2013;Oliverietal.,2008;Peter&Davidson,2010, 2011;Rafiqetal.,2012;Rafiqetal.,2014;Rangeetal.,2013;Rho&McClay,2011; Sethietal.,2009;Sharma&Ettensohn,2011;Suetal.,2009;Weietal.,2009)and providesavaluableframeworkforinvestigatinghowgeneexpression divergenceduringdevelopmentcontributestophenotypicevolutionbetween species(davidson&erwin,2006;erkenbrack&davidson,2015;hinman, Nguyen,Cameron,&Davidson,2003;McCauley,Weideman,&Hinman,2010; 24
41 McCauley,Wright,Exner,Kitazawa,&Hinman,2012).Thisnetworkcontains over200experimentallyverifiedregulatoryinteractionsandcoversmajoraspects ofseaurchindevelopment:fromtheunfertilizedeggthroughtheformationofa planktonicfeedinglarva.decadesofcomparativegeneticandbiochemicalwork haveshownthatmuchofthisnetworkexhibitsasurprisinglyhighlevelof conservationamongseaurchinspecies,includingthoseseparatedbymorethan 50millionyears(Myr)(Gildor&Ben^Taboude^Leon,2015;McClay,2011). Furthermore,keyfeaturesoftheseaurchinGRNarepresentevenindistantly relatedechinoderms(hinmanetal.,2003;mccauleyetal.,2010).thisraises questionsabouttheevolvabilityofthenetworkasawhole,particularlyamong euechinoidseaurchins:isthenetworkhighlyconstrainedandtherefore evolutionaryinflexible?oristhenetworksimplyoptimizedtoproducea planktonicfeedinglarvaandthusmaintainedbystabilizingselection? Toexplorethesequestionswechosetoexaminethisnetworkinthe contextofamajorlifehistorytransitioninseaurchinsthatinvolvedextensive anddramaticchangesindevelopmentalmechanisms:theswitchfromfeeding (planktotrophic)tonon^feeding(lecithotrophic)larvaldevelopment(strathmann, 1985).Althoughplanktotrophyistheancestralmodeofdevelopmentinsea 25
42 urchins(mcedward&janies,1997;strathmann,1985),lecithotrophyhasevolved atleast14timesindependentlyinthisclade(jefferyetal.,2003;wray,1996). Lecithotrophsproducefewer,largereggsthatarerichinmaternalproteinsand lipidstores,allowingthesespeciestoomitthelarvalfeedingstage(byrneetal., 1999;Villinskietal.,2002).Thesenon^feedinglarvaehaveoftenreducedorlost severalkeymorphologicalfeaturesandundergoacceleratedjuvenile development(m.s.smith,zigler,&raff,2007;strathmann,1985;williams& Anderson,1975;Wray&Raff,1991a). WefocusourinvestigationonthegenusHeliocidaris,whichencompasses oneofthemostcomprehensivelystudiedlifehistorytransformationsfroma developmentalperspectiveinanyanimal(byrneetal.,1999;haag&raff,1998; Henryetal.,1990;Henry,Wray,&Raff,1991;Kauffman&Raff,2003;Loveetal., 2008;Parksetal.,1988;M.S.Smithetal.,2009;M.S.Smithetal.,2008;Wray& Raff,1989).AwealthofresearchcomparingtheplanktotrophHeliocidaris# tuberculataandthelecithotrophheliocidaris#erythrogrammahascontributedtoour understandingofthedramaticdifferencesincelllineagespecification,cell^cell interactions,andgeneexpressionpatternsthataccompaniedtherapidtransition fromplanktotrophictolecithotrophicdevelopmentinthisgenus(divergence 26
43 time=5myr)(haag&raff,1998;kauffman&raff,2003;kluegetal.,1997;m.s. Smithetal.,2008;Wilson,Andrews,&Raff,2005;Wray&Raff,1989,1990;Zigler etal.,2003).whilesomedevelopmentalprocessesaredelayedinh.# erythrogrammarelativetotheplanktotrophs(e.g.,skeletogeniclineage specification),othersareaccelerated(e.g.,patterningofthejuvenilebodyplan) (Parksetal.,1988;M.S.Smithetal.,2009).Notably,developmentalprocesses previouslyconservedfor10s^100sofmillionsofyearschangeddramaticallyand rapidlyinh.#erythrogramma#andsomeofthesechangesstronglysuggest extensive rewiring ofthewell^defineddevelopmentalgrninseaurchins (Henryetal.,1990;Raff,1992;Wray&Raff,1990,1991b). Animportantgoalforthisstudyistoidentifyevolutionarychangesin geneexpressionunderlyingtheecologicallysignificantshiftindevelopmental strategyfromplanktotrophytolecithotrophyinheliocidaris.thedramatically alteredtimingofkeydevelopmentalprocessesinh.#erythrogrammarelativeto planktotrophssuggestthatatleastsomeoftheunderlyingevolutionarychanges ingeneexpressionareheterochronicshiftsinthetimingofexpressionduring development.still,otherdevelopmentalchangesinh.e.implyalteredregulatory interactionsorevenlossofgeneexpressionduringtheevolutionof 27
44 lecithotrophicdevelopmentandresultsfrompreviousstudiessupporteachof thesescenarios(kauffman&raff,2003;kluegetal.,1997;m.s.smithetal.,2008; Wilson,Andrews,&Raff,2005).Todetecttheseevolutionarychangesingene expressionacrossthetranscriptome,wedevelopedacomparativeclustering strategythatidentifiesstatisticallysupporteddifferencesintheshapeof expressionprofilesduringdevelopment,asopposedtoprioritizingdifferencesat individualtimepoints.importantly,ourmethoddifferentiatessimplecasesof delayedoracceleratedexpressionfrommorecomplexcasesofgeneexpression changeinh.#erythrogramma.akeyaspectofthismethodisouruseofanexplicit phylogeneticframeworkwithanoutgroupplanktotrophicspecies,whichallows ustopolarizedifferencesingeneexpressiontospecificbranchesofthe phylogeny. Inthispaper,weusethecomparativeclusteringstrategynotedaboveto comparegenome^wideexpressiondynamicsacrossdevelopment,froman unfertilizedeggtoanearlylarva,betweenh.#tuberculataandh.#erythrogramma, usingtheplanktotrophl.#variegatusasanoutgroup.wealsoexaminethese evolutionarychangesingeneexpressionwithinthecontextofthewell^defined seaurchingrnandfurtherleverageourcomparisonstoidentifynovelplayers 28
45 likelyinvolvedinseaurchindevelopment.ourresultsrevealthatchangesin geneexpressionprofilesweremorenumerousduringtheevolutionof lecithotrophythanduringthepersistenceofplanktotrophy,andthiscontrastis evenstrongerwhenonlygrngenesareconsidered.wefoundevidenceforboth conservationanddivergenceofgrnlinkagesinh.#erythrogramma,aswellas significantchangesintheexpressionofgeneswithknownrolesinpatterningthe larvalskeleton,whichisgreatlymodifiedinlecithotrophs.collectively,our resultsindicatethatthetransitionfromplanktotrophictolecithotrophic developmentinvolvednumerouschangestokeydevelopmentalprocesses. 29
46 2.2Results Inordertoidentifyevolutionarychangesingeneexpressionunderlying lecithotrophyinheliocidaris,weusedilluminarna^seqtomeasureexpression dynamicsacrossdevelopmentinthreeseaurchinspecies:thelecithotrophh.# erythrogramma,thecloselyrelatedplanktotrophh.#tuberculata#(divergencetime= 5Myr)(Zigleretal.,2003),andanoutgroupplanktotrophL.#variegatus# (divergencetime=35^45myr)(figure4a)(a.b.smith,1989).wesampled7 stagesofdevelopmentintriplicateforeachspecies,fromunfertilizedeggsto earlylarvae(figure4b).thesedataformthebasisfortheanalysesdescribed below. 30
47 Figure'4:'Gene'expression'during'sea'urchin'development'reflects'known' phylogenetic'relationships'between'species.' (A)Ourstudyincludesthreeseaurchinspecies:thesisterspeciesH.#tuberculata (planktotroph)andh.#erythrogramma#(lecithotroph),whichdivergedapproximately5 millionyearsago(myr),andoutgroupspeciesl.#vareigatus(planktotroph),which divergedapproximately35^45myrago.(b)ourdevelopmentaltime^courseincludes sevenstagesacrosseachspecies,fromunfertilizedeggstoearlylarvae.(c)principal^ component(pc)analysisofgeneexpression.pc1explains36%oftheoverallvariation andclearlyseparatesearly(eggthrough32^cellstage)fromlaterdevelopmentalstages (blastulathroughearlylarva),whereaspc2separatedl.#variegatusfromthetwo Heliocidarisspecies,correspondingtotheirphylogeneticrelationships. ' 31
48 2.2.1%Global%gene%expression%patterns%during%development% recapitulate%phylogeny% Giventhestrikingdifferencesbetweenplanktotrophicandlecithotrophic development,wewereinterestedintheextenttowhichvariationinglobalgene expressionpatternswasexplainedbylife^historystrategy,asopposedto phylogeneticrelationshipsbetweenspecies.toaddressthisquestion,we performedaprincipalcomponentanalysis(pca)toidentifythemajorsourcesof varianceinourtranscriptomedataset.thefirstpcexplained36%ofthe variationanddistinguishedearlydevelopmentalstages(eggthrough32^cell stage)fromlaterones(blastulathroughearlylarva)inallthreespecies(figure 4C).Thisseparationcorrespondsroughlytomaternalversus#zygoticgene expressionprofiles.pc2,whichexplained18%ofthevariance,separatedl.# variegatusfromthetwoheliocidarisspecies,correspondingtotheirphylogenetic relationships.frompc2itisfurtherevidentthatoverallexpressionpatterns slightlydivergebetweenthetwoheliocidarisspeciesduringlaterdevelopment, whiletheystronglyconvergeduringlaterdevelopmentbetweenthetwogenera. PC3,whichexplained13%ofthevariation,furtherdistinguishedH.# erythrogrammafromh.#tuberculata,separatingbylife^historystrategy(figure5). Theseresultsidentifydevelopmentalstageandphylogenyasthemainsourcesof 32
49 differencesinglobalgeneexpressionprofilesamongthesamplesweexamined. Incontrast,life^historystrategywasarelativelyminorcontributortodivergence inexpressioninthetranscriptomeasawhole. Inordertogainfurtherinsightintotheextentofgeneexpressionchange betweenspecies,wecomputedthetranscriptomedivergence(1^,spearman s correlationcoefficient)betweeneachspeciespairatindividualstageswithinour time^course(figure6).consistentwithourpcanalysis,ourresultsreflectthe knownphylogeneticrelationshipsbetweenspecies.thedivergenceofboth Heliocidarisspecies,relativetotheoutgroupL.#variegatus,ishighestintheegg andtendstodecreaseacrossdevelopment.earlystages(eggthrough32^cell)are moredivergentthanlaterones(blastulathroughearlylarva),againlikely reflectingmaternalversus#zygoticgeneexpressionprofiles.ourresultsalsoshow thath.#erythrogrammaismoredivergentwithrespecttotheoutgroupthanh.# tuberculataacrossdevelopment,exceptatthegastrulastage. 33
50 Figure'5:'LifeNhistory'strategy'is'a'relatively'minor'component'of'gene'expression' divergence'across'the'transcriptome.' Principalcomponent(PC)analysisofgeneexpression.PC2explains18%ofthe variationandseparatedl.#variegatusfromthetwoheliocidarisspecies,corresponding totheirphylogeneticrelationships.pc3explains13%ofthevariationandseparated speciesbylife^historystrategy. ' 34
51 Figure'6:'Gene'expression'divergence'is'highest'in'the'egg'and'tends'to'decrease' across'development.' Comparisonsofmeanexpressiondivergence(1^,Spearman scorrelationcoefficient) betweenspeciesforeachdevelopmentalstage(e,egg;4c,4cell^stage;16c,16cell^ stage;32c,32cell^stage;b,blastula;g,gastrula;el,earlylarva).errorbarsrepresent 95%confidenceintervalsbasedonbootstrappinganalysisof10,713orthologsrandomly sampledwithreplacement1,000times. ' 35
52 2.2.2%Changes%in%gene%expression%profiles%were%more%numerous% during%the%evolution%of%lecithotrophy%than%during%the%persistence%of% planktotrophy% Animportantgoalforthisstudyistoidentifychangesingeneexpression thatmayhavecontributedtotheevolutionoflecithotrophy.because developmentisadynamic,time^dependentprocess,wesoughtamethodto identifystatisticallysupportedevolutionarydifferencesintheshapesofgene expressionprofilesduringdevelopment.weimplementedacomparative clusteringstrategytodetectsubstantialdifferencesinexpressionprofilesacross developmentregardlessofabsoluteexpressionlevelandwithinanexplicit phylogeneticframework(seemethodsfordetails).first,weusedfuzzyc^means clusteringtogroupsimilarexpressionprofilesinouroutgroupspeciesl.# variegatusinto7distinctclustersrepresentingstage^specificpeaksinexpression profiles(figure7a).next,weseparatelyassignedh.#tuberculataandh.# erythrogrammagenestotheseclustersbasedonthemembershipscoreof individualh.t.andh.e.geneexpressionprofilestothel.#variegatuscluster centroids.withineachspecies,wealsodesignatedaseparateclusterforgenes withverylowexpressionacrossthetime^course,whichwefurtherrefertoasthe VLE cluster.thisstrategyallowedustoeasilyidentifywhichgenesexhibited thesameexpressionprofileamongallthreespecies(conservation),differed 36
53 amongallthreespecies(divergence),orchangedspecificallyalongaparticular branchofthephylogeny,whichwerefertoas clusterjumps (Figure8). Bythesecriteria,moregenesshowconservedexpressionprofilesbetween H.#tuberculataandH.#erythrogrammathanbetweenL.#variegatusandeitherofthe Heliocidarisspecies(Figure7B).Thisresultshowsthatoveralldivergencein embryonicgeneexpressionfollowsphylogeneticrelationshipsratherthan developmentalstrategy,reinforcingourprincipalcomponentsanalysis(figure 1C).UnlikeaPCA,however,thisapproachallowedustopolarizeevolutionary differencesinexpression.weobservedmoreclusterjumpsalongtheh.e.branch thantheh.t.branch,andtheratioofexpressionchangebetweentheheliocidaris branches(~1.3x)remainssimilarregardlessoftheinitialnumberofclusters formed(table3).thisresultsuggeststhat,althoughmostexpressiondifferences appeartoaccumulateneutrally,asubsetarerelatedtotheevolutionofaderived developmentalmodeinh.#erythrogramma. OntheH.#erythrogrammabranch,jumpsoccurredbetweenevery combinationofclusters,whichledustoaskwhethertherewereanysignificant differenceswithrespecttothenatureofgeneexpressionchangesduringthe evolutionoflecithotrophy.wefoundthatsignificantlymoregenesjumpedfrom 37
54 cluster3tocluster4inh.e.comparedtoh.t.(fisher sexacttest,adjustedp=1.23 x10^06 ).Asignificantlyhighernumberofgenesalsojumpedfromcluster6and cluster7intothevlegroupinh.e.(fisher sexacttest,adjustedp=8.98x10^07 andadjustedp=1.72x10^10,respectively). TotestwhethergeneexpressionchangesinH.e.aresimilarinmagnitude tothoseinh.t.,weperformedapcanalysisofexpressionprofilesthatexhibited branch^specificchangeineitherheliocidarisspecies(figure9a).wethen calculatedscaleddistancemeasures,or jumpscores,betweenh.e.andh.t. orthologsfromthefirsttwopcloadings.thismethodenabledusto quantitativelydifferentiatesubtlefromdramaticshiftsinexpression.for example,inh.e.,thebiomineralizationgenemsp130exhibitsaslightdelayin expressioncomparedtotheplanktotrophs(i.e.,msp130jumpsfromcluster6into cluster7inh.e.)andthereforehasarelativelysmalljumpscore.thisdelayin Msp130expressionwasobservedpreviously,andisconsistentwithanaltered skeletogenicprograminthelecithotroph(kluegetal.,1997).comparedto Msp130,thegeneHox9/10exhibitsamoredramaticjumpinexpressionalongthe H.e.branch,movingfromtheVLEgroupintocluster7.Thischangeinexpression isconsistentwiththeroleofhox9/10inpatterningthejuvenilebodyplan,which 38
55 isgreatlyacceleratedinh.e.(arenas^mena,cameron,&davidson,2000; Williams&Anderson,1975).Wenextplottedthedensityofjumpscoresinboth Heliocidarisspecies,andfromtheleft^skeweddistributionsitisevidentthat jumpsbetweensimilarclustersaremorefrequentthanjumpsbetweenhighly distinctclusters(figure9b).thisresultindicatesthatwhilesubtledivergencein geneexpressionprofilesisfrequentbetweenspecies,dramaticchangesareless common. 39
56 Figure'7:'Fuzzy'cNmeans'clustering'groups'similar'expression'profiles'across' development'into'seven'distinct'clusters.' (A)'ClustercentroidsidentifiedintheoutgroupL.v.areshown,alongwiththenumber ofgenesineachspeciesthatwereassignedtoagivencluster.green=l.v.,blue=h.t., andpurple=h.e.#(b)usingacomparativeclusteringstrategy(seefigures3and Methods),wecharacterizedexpressionprofiledivergence(white,29.8%),conservation (darkgrey,19.1%)andgenus^levelchange(lightgrey,19.8%)acrossthetranscriptome. Wealsodetectedbranch^specificchangeinH.t.(blue,13.5%)andH.e.(purple,17.8%). ' 40
57 Figure'8:'Comparative'clustering'strategy'reveals'cases'of'expression'profile' conservation'and'change'within'a'phylogenetic'framework.' Thismethodidentifiesgenesthatexhibitthesameexpressionprofileamongallthree species(conservation),differamongallthreespecies(divergence),differbetweenl.v.# andtheheliocidarisspecies(genus^levelchange),orchangespecificallyalonga particularbranchofthephylogeny(clusterjumpinh.t.#orh.e.). ' 41
58 Table'3:'Impact'of'initial'cluster'number'on'expression'classification' Number'of'Clusters' 5' 7' 9' Conserved'Genes' 2658(24.8%) 2050(19.1%) 1614(15.1%) Diverged'Genes' 2296(21.4%) 3194(29.8%) 4057(37.9%) Genus'Difference' 2221(20.7%) 2121(19.8%) 1995(18.6%) H.t.'Branch'Change' 1549(14.5%) 1447(13.5%) 1288(12.0%) H.e.'Branch'Change' 1989(18.6%) 1901(17.7%) 1759(16.4% Ratio'H.e./H.t.'
59 Figure'9:'Subtle'shifts'are'more'common'than'dramatic'changes'in'gene'expression' between'species.' (A)PCanalysisofstandardizedexpressionprofilesforgenesthatexhibitedacluster jumpineitherh.t.orh.e.allgenesaredepictedingrey,whileclustercentroidsarein green.anexampleofasubtle(msp130)anddramatic(hox9/10)clusterjumpinh.e.are indicated.jumpscoresforindividualgeneswerecalculatedbasedonscaleddistance measuresbetweenh.e.andh.t.orthologsfromthefirsttwopcloadings.(b)kernel densitycurvesofjumpscoresforgenesthatexhibitedaclusterjumpineitherh.t. (blue)orh.e.(purple). ' 43
60 2.2.3%Changes%in%gene%expression%during%the%evolution%of% lecithotrophy%are%qualitatively%distinct%from%those%during%the% persistence%of%planktotrophy% Toexplorewhethertheevolutionaryconservationanddifferenceswe observedindevelopmentalexpressionprofileswerereflectedinhigher^level functionalclassesofgenes,weperformedacategoricalenrichmentanalysis usingahypergeometrictestandthegobiologicalprocessontologydatabase (seemethods).inthedivergencegeneset(profileassignedtoadifferentcluster ineachspecies),wefoundsignificantenrichmentofonemetaboliccategory whencontrollingforafalsediscoveryrate(fdr)of10%:bileacidbiosynthetic process.incontrast,theconservedgeneset(profileinthesameclusterinall threespecies)wasenrichedby237categories(10%fdr)encompassingawide rangeofbiologicalprocesses,includingmanyrelatedtodevelopment(e.g.,cell fatedetermination).wedidnotobserveanysignificantlyenrichedcategoriesfor genesthatchangedspecificallyalongtheh.e.orh.t.branch,norforgenesthat differedbetweenl.v.andtheheliocidaris#species. Weperformedaparallelanalysisinwhichwereducedthegeneset backgroundfromall3^wayorthologstoonlythosethatexhibitedabranch^ specificchangeinexpression(i.e.,divergedandconservedgeneswereexcluded). Similartoourearlieranalysis,wedidnotobserveanysignificantlyenriched 44
61 categoriesforgenesthatchangedspecificallyalongtheh.t.branchorforgenes thatdifferedbetweenl.v.andtheheliocidarisspecies.however,forgenesthat changedspecificallyalongtheh.e.branch,wefoundsignificantenrichmentof4 categorieswhencontrollingforanfdrof10%,allofwhichwererelatedto developmentalprocesses(i.e.,cartilagecondensation,neuronmigration, midbraindevelopment,andnegativeregulationofneurondifferentiation). Severalofthegenesinthesedevelopmentalcategorieshavecritical regulatoryrolesduringembryonicorlarvaldevelopmentinseaurchins(e.g.dlx, gsc,myod,foxc,pax2/5/8).thatsaid,somearenotknowntobeinvolvedinthe specificdevelopmentalprocessesindicatedbythegocategorynameper#se#insea urchins,astheseassociationsarebasedonstudiesindistantlyrelatedmodel organisms.whatisclearfrom ourresultsisthatmajorchangesintheexpression profilesofdevelopmentalregulatorygenesoccurredduringtheevolutionof lecithotrophyinheliocidaris. 45
62 2.2.4%Changes%in%expression%differ%between%the%gene%regulatory% network%and%the%transcriptome%as%a%whole% Tofurtherexploregeneexpressionchangesassociatedwiththeevolution oflecithotrophicdevelopment,weexaminedclusterjumpswithinthecontextof thewell^establishedgeneregulatorynetwork(grn)ofeuechinoids.the networkisdividedintothreemainterritorieswithintheembryothatexecute distinctspecificationanddifferentiationprogramsfortheskeletogenic, endomesodermal,andectodermalcelllineages(davidsonetal.,2002).boththe organizationandfunctionofthegrnishighlyconserved,evenamongdistantly relatedechinodermspecies(gildor&ben^taboude^leon,2015;hinmanetal., 2003;McCauleyetal.,2010).Yetthedramaticallyalteredskeleton,nonfunctional gut,andreorganizedectodermofh.#erythrogrammalarvaesuggestthatchanges mayhaveoccurredinthegrnduringtherelativelyrecentevolutionof lecithotrophicdevelopment(haag&raff,1998;loveetal.,2008;parksetal., 1988;Raff,1992).Thisledustoconsiderthefollowingquestions:towhatextent haveexpressionchangesevolvedinthegrnofh.#erythrogramma?arethese changescharacterizedbyheterochronicshiftsinexpressionand/oristhe expressionofparticulargeneslost?arethesechangeslocalizedtoparticular territoriesorspecificsub^circuitsofthoseterritories(e.g.,specification, 46
63 patterning,differentiation)?andimportantly,dotheseexpressionchangeslikely havedownstreamphenotypicconsequencesfordevelopment? Incontrasttoclusterjumpsacrossthetranscriptome(Figure7B),we observedmoreconservationandlessdivergenceofgeneexpressionprofiles withinthegrn(figure10a).notably,morenetworkgeneshadconserved expressionprofilesbetweentheplanktotrophsthanbetweenthetwoheliocidaris species,suggestingthatlifehistorystrategy,andnotphylogeneticposition,isthe principaldeterminantofinter^speciesdifferencesingeneexpressionwithinthe GRN.Consistentwiththisresult,weobservedamuchhigherratioofexpression changebetweenh.e.andh.t.withinthegrncomparedtothetranscriptomeasa whole(branchlengthratio~5.8x,figure10a).ourresultsalsorevealthat changesalongtheh.e.branchoccurredinallthreeprincipalterritoriesofthe GRN:skeletogenic,endomesodermandectoderm(Figure10B).Takentogether, theseresultsstronglypointtowardevolutionaryre^wiringofgrninteractions duringtherapidevolutionoflecithotrophicdevelopmentinheliocidaris. 47
64 Figure'10:'LifeNhistory'strategy'is'a'major'component'of'gene'expression'divergence' across'the'gene'regulatory'network.' (A)Comparedtothetranscriptome,thegeneregulatorynetwork(GRN)exhibits increasedexpressionprofileconservationamongspecies(darkgrey,41.5%)and reduceddivergence(white,11.3%).genus^levelandh.t.^specificclusterjumpsarealso reduced(lightgrey,8.5%andblue,5.7%,respectively),whileclusterjumpsspecifically alongtheh.e.branchincreasedatthegrnlevelcomparedtothetranscriptome (purple,33%).(b)h.e.specificbranchjumps(purple)occurineachgrnterritory, thoughtheyareconcentratedintheskeletogeniclineage(binomialtest,p<0.01).the associatedjumpscoreforeachh.e.specificbranchjumpisrepresentedbyboxcolor. JumpsintotheVLEgrouparerepresentedbyopenboxes. ' 48
65 2.2.5%Distinct%patterns%of%evolutionary%change%in%gene%expression% among%embryonic%territories% ChangesontheH.e.branchoccurredatalllevelsoftheGRN,fromgenes involvedinearlyfatespecificationprocessestothoseinvolvedinterminal differentiationoflarvalcells.astrikingexampleofearlychangeisthedelayed expressionofwnt8,oneofthefirstzygoticallyactivatedgenesinthegrn (Figure11A).ThisdelaywasalsoobservedbyKauffmanandRaffusing Northernblots(Kauffman&Raff,2003).Inplanktotrophs,Wnt8isinitially expressedinthemicromeres,whichconstitutethemajororganizingcenterofthe embryo(wikramanayakeetal.,2004).wnt8^mediatedsignalinginitiatesaseries ofinductiveinteractionsalongtheanimal^vegetalaxisthatspecifythemajorcell fateswithintheendomesoderm(em).inhibitionofwnt8signalinginspecies withplanktotrophiclarvaeleadstoextensivedevelopmentaldefects (Wikramanayakeetal.,2004).Unlikeplanktotrophs,H.e.hasanequalfourth cleavage,doesnotproducemicromeres,andendomesodermalfatespecification isdelayedandthesequenceisaltered(wray&raff,1989,1990).thedelayed expressionofwnt8islikelyrelatedtothesechangesinfatespecification. Nonetheless,Wnt8#expressionremainsnecessaryforEMspecificationinH.# erythrogramma(kauffman&raff,2003).furthermore,ourresultsshowthat 49
66 componentsofemspecificationdownstreamofwnt8signalinginplanktotrophs, includingbra#andfoxa#exhibitconservedexpressionprofilesbetweenl.v.and theheliocidarisspecies(figure11a).theseresultssuggestthattheem specificationsub^circuithasbeenconservedinfunction,butcompressedin timing,duringtheevolutionoflecithotrophicdevelopmentinheliocidaris. Followingspecification,theEMterritoryseparatesintodistinct endodermalandnon^skeletogenicmesodermal(nsm)lineages,withendoderm contributingtothelarvalgutandthensmlineagecontributingtoblastocoelar cells,pigmentcells,musclecells,andcoelomicpouchcellsofthelarva(cameron, Fraser,Britten,&Davidson,1991;Davidsonetal.,1998;Ruffins&Ettensohn, 1993,1996).ThelarvaofH.e.hasasmall,nonfunctionalgutthatlacksthemouth, tripartitegastrointestinaltract,andassociatedmusclesthatarepresentin planktotrophiclarvae(raff,1992;williams&anderson,1975).inh.e.,we observedchangesintheshapeofexpressionprofilesfortwoendodermalgrn genes,brn1/2/4andabopec(figure11b).inaddition,althoughtheendodermal geneendo16hadaconservedexpressionprofilebetweenallthreespecies,it exhibitedasubstantiallyreducedlevelofexpressioninh.e.comparedtothe 50
67 planktotrophs(figure11b).theseevolutionarychangesingeneexpressionare likelyrelatedtothedevelopmentofthereduced,nonfuctionallarvalgutinh.e. Currently,themostdetailedsub^circuitoftheNSMlineagedetails developmentofthecoelomicpouches,whichformoneithersideofthelarvalgut (Pearse&Cameron,1991).Priortometamorphosisinplanktotrophs,theleft coelomicpouchenlargesandsignalstotheoverlyingectodermtoinitiate patterningofthejuvenilebodyplan(luo&su,2012;pearse&cameron,1991). Thisinteractionusuallyoccursweeksafterfertilizationinplanktotrophicspecies. InH.e.,thedevelopmentoftheleftcoelomicpouchisgreatlyaccelerated,and adultbodyplanformationisinitiatedjustaftergastrulation,lessthan2days afterfertilization(m.s.smithetal.,2009;williams&anderson,1975).our resultsprovideevidenceofbothconservationanddivergencewithinthe coelomicpouchsub^circuitofthegrn.inplanktotrophs,thetranscriptionfactor FoxY,whichislocatedattopoftheregulatoryhierarchyunderlyingcoelomic pouchcellspecification(maternaetal.,2013),isconservedinexpressionbetween allthreespeciesbutexhibitsanelevatedlevelofexpressioninh.e.(figure11c). OtherconservedcoelomicpouchgenesincludeFoxFandSoxE.However, expressionoffoxc,adownstreamtargetoffoxy,isacceleratedandexpressionof 51
68 ScratchX,anotherFoxYtarget,islostinH.e.(Figure11C)(Maternaetal.,2013). Theseobservationssuggestthatbothcompressionandrewiringofthecoelomic pouchsub^circuithavecontributedtothegreatlyaccelerateddevelopmentofthe adultbodyplaninh.e. WhileexpressionchangeswithintheH.e.GRNoccurredinallthree territoriesoftheembryo,clusterjumpswereconcentratedwithinthe skeletogeniclineage(binomialtest,p<0.01).bothplanktotrophsand lecithotrophsspecifyskeletogeniccells,butinh.e.,thesecellsdonotmigrateand assembleintothestereotypicalringpatternofplanktotrophiclarvaeandproduce amuchsmallerandsimplerskeleton(parksetal.,1988).therefore,itwasnot surprisingthatvegf/vegfrsignaling,whichprovidesguidancecuesand differentiationsignalstomigratoryskeletogeniccellsinplanktotrophs (Duloquin,Lhomond,&Gache,2007),appearstobedramaticallyalteredinH.# erythrogramma:expressionofthevegfligand,whichsignalstothemigrating skeletogeniccellsfromtheoverlyingectoderm,isgreatlyreducedandexpression ofitsreceptorvegfrisgreatlymodified(figure11d).theexpressionof severaldownstreameffectorgenesinvolvedinthebiomineralizationand constructionofthelarvalskeletonwasalsolostduringtheevolutionof 52
69 lecithotrophicdevelopmentinheliocidaris,includingsmq49andcqlectin(figure 11E)(Illies,Peeler,Dechtiaruk,&Ettensohn,2002;Killian&Wilt,1996; Livingstonetal.,2006).However,theexpressionofotherskeletogenicgeneswas maintainedatareducedlevelinh.e.(e.g.,smq50andp58qa).thesegenesmay playaconservedroleinlarvalskeletogenesisormaybenecessaryforformation ofthejuvenileskeleton,whichisgreatlyacceleratedinh.e(williams& Anderson,1975). Lastly,weexaminedgeneexpressionchangeswithintheectodermal territory.inplanktotrophs,distinctoralandaboralterritoriesofgeneexpression areestablishedwithintheectoderm,separatedbytheciliaryband(davidsonet al.,1998).inh.e.,theectodermissubstantiallyreorganized,includinglossofan oral^aboraldistinctionandgainofnovelexpressiondomains(haag&raff,1998; Love&Raff,2006).AlthoughdifferencesbetweentheplanktotrophsandH.e. werenotasdramaticintheectodermasinotherterritories,wenonetheless observedseveralexpressionchangesconsistentwiththeevolutionofamodified ectoderminh.#erythrogramma.asnotedpreviously,theectodermofh.e.no longerexpressestheligandvegf,animportantfactorinvolvedinpatterningthe larvalskeleton,atanappreciablelevel.similarly,thetranscriptionfactor 53
70 Pax2/5/8,whichisalsoinvolvedinskeletalpatterning,isdelayedandreducedin H.e(Figure11F)(McIntyreetal.,2013;Rottingeretal.,2008).Inaddition,we observeddelayedexpressionofthetranscriptionfactorsdlxandhox7(figure 11F).BothDlx#andHox7areinvolvedindifferentiationofaboralectodermin planktotrophiclarvae(ben^taboude^leonetal.,2013),andpreviousstudies suggestimportantrolesforthesegenesinformationofthejuvenilebodyplan (Arenas^Menaetal.,2000;Lowe,Issel^Tarver,&Wray,2002). 54
71 55
72 Figure'11:'Network'genes'exhibit'both'conserved'and'divergent'expression' profiles'between'species.' Expressionprofilesforexamplegenecasesinvolvedin(A)'endomesoderm specification(b)endodermdevelopment(c)coelomicpouchdevelopment(d) skeletogenicpatterning(e)skeletogenicbiomineralizationand(f)ectoderm development.biologicalreplicatesarerepresentedascirclesandaverage expressionprofilesacrossreplicatesarerepresentedaslines.expressionvalues belowthehorizontallinearelessthan5cpmandaredesignatedasvery^low expressed(vle). ' 56
73 2.2.6%Coexpression%analysis%identifies%novel%candidate%genes%for%gut% and%neural%development% Becausethegeneticbasisfortheevolutionoflecithotrophyisunlikelyto resideonlyingenesthatarepartofthegrn,wenextexploredwhetherwe couldleverageourknowledgeofdevelopmentaldifferencesbetweenh.e.and theplanktotrophs,alongwithourcomparativetranscriptomicdataset,toidentify novelcandidategenesforfutureempiricalstudies.ourapproachis straightforward,andbelowwedescribethemethodindetail. First,wereexaminedtheresultsfromourcomparativeclusteringanalysis ofgeneexpressionprofilesacrossthetranscriptome(figure7a).however, insteadoftreatinggenesasisolatedunits,wenowconsiderthemasmembersof coexpressiongroups.wefirstcreatedanoverlapmatrixofl.v.andh.t.cluster membershipinordertovisualizethegenesincommonbetweeneach planktotrophclusterpair(figure12a).wealsotestedforsignificantoverlapof eachclusterpairbetweenspecies.thisallowedustoexplorethemovementof groupsofcoexpressedgenes,asopposedtoindividualclusterjumps,between species.clusterpairswithsignificantoverlapbetweentheplanktotrophs (Fisher sexacttest,adjustedp<0.05)primarilyfallalongthematrixdiagonal, indicatingthatgenescoexpressedearlyinl.v.developmentsignificantlyoverlap 57
74 withthosecoexpressedearlyinh.t.development,andthesameistrueforgenes coexpressedduringlaterdevelopmentalstages.genescoexpressedbelowthe matrixdiagonalrepresentgenesthathaveacceleratedexpressionprofilesinh.t. comparedtol.v.,whereasgenescoexpressedabovethematrixdiagonal representgenesthathavedelayedexpressioninh.t.comparedtol.v.wefurther refertothe8coexpressedgroupsspecificallyalongthematrixdiagonalas consensusclusters (e.g.,consensuscluster1contains186genesthatare coexpressedinbothh.t.cluster1andl.v.#cluster1). Next,weexaminedtheoverlapoftheseplanktotrophconsensusclusters withh.e.clusters(figure12b).similartoourpreviousanalysis,clusterpairs withsignificantoverlapbetweentheh.e.andtheplanktotrophs(fisher sexact test,adjustedp<0.05)primarilyfallalongthematrixdiagonal.however, significantoverlapofclusterpairsoff^diagonalarealsopresent,includingthe overlapofconsensuscluster7andthevleclusterofh.e,whichcontains98 genes. Theplanktotroph/H.e.overlapmatrix(Figure12B)providesavaluable frameworktoquerythemovementofcoexpressiongroupsbetweenspeciesin ordertoidentifypromisingcandidatesforfutureinvestigation.fromour 58
75 comparativeanalysisofthegrn,itwasclearthatmanyexpressionchanges withinh.e.occurinafairlypredictablemanner.forexample,h.#erythrogramma doesnotdevelopafunctionallarvalgutorskeletonandtheexpressionofgenes thatunderliegutandskeletaldifferentiationinplanktotrophsisoftenreducedor lostinh.#erythrogramma.therefore,wehypothesizedthatnovelcandidatesfor genesinvolvedingutandskeletaldifferentiationinplanktotrophswouldalsobe reducedorlostduringtheevolutionoflecithotrophy.toexplorethispossibility, weexaminedtheoverlapoftheplanktotrophconsensuscluster7withthevle clusterinh.#erythrogramma,whichcontains98genes(figure12b).ofthesegenes, severalarecurrentlyinthegrnorhaveknownrolesinthedevelopmentof differentiatedskeleton(e.g.,otp)orgut(e.g.,hb9),tissuesthatarehighly conservedinplanktotrophsbutnotinh.#erythrogramma#(parksetal.,1988; Williams&Anderson,1975).Wechosetofurtherinvestigatethetranscription factornkx6.1,whoseexpressionhasbeenobservedinthedevelopinggutofother organisms(nelson,janiesch,&sander,2005;sanderetal.,2000).ourresults showthatinl.#variegatus,nkx6.1expressionisrestrictedtothehindgutofthe larva(figure13a).thisresultsuggestsarolefornkx6.1inplanktotrophicgut differentiationanddemonstratestheutilityofourcomparativeapproachasa 59
76 tooltoidentifynovelcandidategenesforexpressionchangesunderlyingspecific derivedphenotypesinh.#erythrogramma. Tofurtherinvestigatetheusefulnessofourcomparativeapproachasa genediscoverytool,weexaminedthegroupof628genescoexpressedbetween allthreespeciesincluster6.wehypothesizedthatthesegenesarelikely involvedindevelopmentalprocessessharedbybothplanktotrophsandthe lecithotroph.notsurprisingly,manygrngenesareassignedtothisgroup, includingalargeportionoftheectodermalsub^circuit.wechosetofocuson Mab21l2,whichisinvolvedinnervoussystemdevelopmentinotherorganisms (Chow,Hall,&Emmons,1995;Wong&Chow,2002).IntheL.v.#larva,Mab21l2 expressionisrestrictedtotwolateralpatchesintheanteriorneurogenicectoderm (Figure13B).ThisexpressionpatternissuggestiveofaroleforMab21l2inthe developmentofserotonergicneurons,whichdevelopasbilaterallysymmetric, apicalganglioninallthreespecies(bisgrove&raff,1989). 60
77 61
78 Figure'12:'Genes'coexpressed'during'planktotrophic'development' significantly'overlap'with'those'coexpressed'during'lecithotrophic' development.' A)OverlapmatrixofH.t.andL.v.clusters.ThexaxisrepresentsH.t.#clusters andtheyaxisrepresentsl.v.clusters.eachcellcontainsthenumberof intersectinggenesandthecellcolorrepresentsthesignificanceofthe intersection.cellsalongthematrixdiagonalare consensusclusters betweentheplanktotrophicspecies(e.g.,consensuscluster1contains186 genesthatarecoexpressedinbothh.t.cluster1andl.v.#cluster1).consensus clustercellsareoutlinedinblack.genescoexpressedbelowthematrix diagonalrepresentgenesthathaveacceleratedexpressionprofilesinh.t. comparedtol.v.,whereasgenescoexpressedabovethematrixdiagonal representgenesthathavedelayedexpressioninh.t.comparedtol.v.(b)' OverlapmatrixofplanktotrophicconsensusclustersandH.e.clusters.Thex axisrepresentsplanktotrophicconsensusclusters(detailedina),exceptfor thegreycluster,whichrepresentsgenesthatdidnotexhibitconsensus expressionprofilesbetweenplanktotrophs(i.e.,genesthatwereoff^diagonal ina).theyaxisrepresentsh.e.clusters.eachcellcontainsthenumberof intersectinggenesandthecellcolorrepresentsthesignificanceofthe intersection.cellsalongthematrixdiagonalarecoexpressedbetweenall threespeciesandareoutlinedinblack.genescoexpressedbelowthematrix diagonalrepresentgenesthathaveacceleratedexpressionprofilesinthe planktotrophscomparedtoh.e.,whereasgenescoexpressedabovethematrix diagonalrepresentgenesthathavedelayedexpressionintheplanktotrophs comparedtoh.e.# ' 62
79 Figure'13:'CrossNspecies'transcriptome'comparison'reveals'novel'candidates'for' planktotrophic'development.' (A)'ExpressionofNkx6.1isrestrictedtotheendodermintheearlylarvaandtothe hindgutinthelarvainl.#variegatus.(b)expressionof'mab21l2isrestrictedtotwo lateralpatchesintheanteriorneurogenicectoderminl.#variegatuslarvae.' ' 63
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