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Predicting Ancestral Segmentation Phenotypes from Drosophila to Anopheles Using In Silico PDF

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RESEARCHARTICLE Predicting Ancestral Segmentation Phenotypes from Drosophila to Anopheles In Silico Using Evolution JeremyB.Rothschild1,PanagiotisTsimiklis1,EricD.Siggia2,PaulFrançois1* 1PhysicsDepartment,McGillUniversity,ErnestRutherfordPhysicsBuilding,Montreal,Quebec,Canada, 2CenterforStudiesinPhysicsandBiology,TheRockefellerUniversity,NewYork,NewYork,UnitedStates ofAmerica a11111 *[email protected] Abstract Molecularevolutionisanestablishedtechniqueforinferringgenehomologybutregulatory DNAturnsoversorapidlythatinferenceofancestralnetworksisoftenimpossible.Insilico OPENACCESS evolutionisusedtocomputethemostparsimoniouspathinregulatoryspaceforanterior- Citation:RothschildJB,TsimiklisP,SiggiaED, posteriorpatterninglinkingtwoDipterianspecies.Theexpressionpatternofgapgeneshas FrançoisP(2016)PredictingAncestralSegmentation PhenotypesfromDrosophilatoAnophelesUsingIn evolvedbetweenDrosophila(fly)andAnopheles(mosquito),yetoneoftheirtargets,eve, SilicoEvolution.PLoSGenet12(5):e1006052. hasremainedinvariant.Ourmodelpredictsthatstripe5inflydisappearsandanewposte- doi:10.1371/journal.pgen.1006052 riorstripeiscreatedinmosquito,thusevestripemodules3+7and4+6inflyarehomologous Editor:ClaudeDesplan,NewYorkUniversity, to3+6and4+5inmosquito.WecanplaceClogmiaonthisevolutionarypathwayandit UNITEDSTATES sharesthemosquitohomologies.Toaccountfortheevolutionoftheotherpair-rulegenesin Received:February24,2016 theposteriorwehavetoassumethattheancestralDipterianutilizedadynamicmethodto Accepted:April23,2016 phasethosegenesinrelationtoeve. Published:May26,2016 Copyright:©2016Rothschildetal.Thisisanopen accessarticledistributedunderthetermsofthe AuthorSummary CreativeCommonsAttributionLicense,whichpermits unrestricteduse,distribution,andreproductioninany Thelastcommonancestorofthefruitfly(Drosophila)andmosquito(Anopheles)lived medium,providedtheoriginalauthorandsourceare morethan200Millionyearsago.Canweuseavailabledataoninsectsalivetodaytoinfer credited. whattheirancestorlookedlike?Inthismanuscript,wefocusonearlyembryonicdevelop- DataAvailabilityStatement:Allrelevantdataare ment,whenstripesofgeneticexpressionappearanddefinethelocationofinsectsegments withinthepaperanditsSupportingInformationfiles. (“segmentation”).Weuseanevolutionaryalgorithmtoreconstructandpredictdynamics Funding:ThisworkwassupportedbytheSimons ofgenescontrollingstripesinthelastcommonancestorofflyandmosquito.Wepredicta Investigatormathematicalmodellingofbiological newanddifferentcombinatoriallogicofstripeformationinmosquitocomparedtofly, systems,https://www.simonsfoundation.orgtoPF,the whichisfullyconsistentwithdevelopmentofintermediatespeciessuchasmoth-fly(Clog- NationalScienceFoundationgrantPHY-1502151 mia).Oursimulationsfurthersuggestthatthedynamicsofgeneexpressioninthislast http://www.nsf.gov/toEDS,theFondsdeRecherche duQuébecNatureetTechnologies,Programe commonancestorweresimilartootherinsects,suchaswasps(Nasonia).Ourmethod NouveauxChercheurshttp://www.frqnt.gouv.qc.ca/ illustrateshowcomputationalmethodsinspiredbymachinelearningandnon-linearphys- accueiltoPF,andtheNaturalScienceand icscanbeusedtoinfergenedynamicsinspeciesthatdisappearedmillionsofyearsago. EngineeringResearchCouncilofCanada, UndergraduateSummerResearchAwardhttp://www. nserc-crsng.gc.ca/index_eng.asptoPT.Thefunders PLOSGenetics|DOI:10.1371/journal.pgen.1006052 May26,2016 1/19 PredictingAncestralSegmentationPhenotypesfromDrosophilatoAnophelesUsingInSilicoEvolution hadnoroleinstudydesign,datacollectionand Introduction analysis,decisiontopublish,orpreparationofthe Molecularphylogeniesbasedonproteincodinggeneshavegreatlyenhancedevolutionarythe- manuscript. ory,andinfavorablecasesevenallowareconstructionofthelastcommonancestralgeneor CompetingInterests:Theauthorshavedeclared evenfullevolutionarypathways[1].Howeverregulatorysequenceevolvesmorerapidlythan thatnocompetinginterestsexist. codingsequenceandthefunctionalbindingsitescanmovearoundwithoutimpactingthe functionofa*1kbfunctionalregulatorymodule[2,3].Thusoneisofteninthesituation wheregenehomologiesareobvious,yetthereisnovisiblesequencehomologyintheregulatory regions.Atthephenotypiclevel,geneexpressiondomainscanbeeasilymappedbyin-situ hybridizationyetamolecularunderstandingislimitedoutsideofmodelorganisms.Thereis considerableneedforacomputationaltoolthatcantakesparsephenotypicinformation,e.g., broadlydefinedspace-timegeneexpression,andconstructthesimplestphylogeneticrelation- shipsconsistentwithdata,therebyhighlightinginterestingeventsformolecularfollowup. Drosophilasegmentationisaparadigmaticexampleofdynamicdevelopmentalnetwork. Positionalinformationpropagatesfrommaternalgradientssuchasbicoid(bcd)andcaudal (cad)togapgenessuchashunchback,giant,knirpsandKruppel(respectivelyhb,gt,kni,Kr), andthentothestripedexpressionofprimarypair-rulegenessuchaseven-skipped(eve),hairy (h),runt(run),andpartiallyfushi-tarazu(ftz)[4,5].Thepair-rulegenesinturncontrolthe segmentpolaritygenesthatarebroadlyconservedacrossthearthropods[6].Mutagenesisand bioinformaticsstudieshaverevealedthemainDNAmotifscontrollingtheexpressionofgap andpair-rulegenes[7]whilesystematicquantitativeimaginghasledtophenomenological modelsforsegmentationdynamics[8,9]. Recentevo-devostudieshavestartedtomapthesegmentationhierarchyinotherdipterans (Anopheles[10],Clogmia[11],Megaselia[12]).Almostallinformationcomesfromlocalizing therelevantmRNAbyin-situhybridization,andknockingdown(KD)varioustranscriptswith RNAinterference.Informationineachofthesethreespeciesisstillverysparse:whileweknow thepositionofthegapgenesandthesinglepair-rulegeneeve,thereisonlyfewinformationon thephasingoftheotherpair-rulegenesrelativetoeve.Whethertheyarepositionedbythegap genesorotherso-calledprimarypair-rulegenesisnotknowninthoseDipterans.Thereareno definedgeneregulatorymodulesinthesespecies,soallinformationaboutgapgeneregulation isinferredfromtheirpositionandshiftsinputativetargetsunderKD. Inspiteofthissparseinformation,someinterestingquestionscanbeposed.Theanterior gapgenepatternappearsinvariantinallspeciesasdotheevestripes,thoughthereareonlysix inClogmiabeforegastrulationvs7inDrosophilaandupto8inAnopheles.Thereismorevari- abilityintheposterior.Therelativepositionsoftheposteriordomainsofhbandgtareinverted inAnopheleswithrespecttoDrosophila,whileinClogmia,neitherofthesegapgenesare expressedposteriorlybeforegastrulation.Itisreasonabletoassumethattheprimarypair-rule stripesarepositionedbygapgenerepression,sotheevolutionaryinterchangeoftheposterior hbandgiantdomainsposesproblemsforindividualevestriperegulatorymodules.For instance,eve5inDrosophilaisrepressedposteriorlybygtsoiftheposteriorgtdomainis removed,eve5extendsbroadlyposteriorlyinDrosophila[13].Sohowcangtdomainbemuch moreposteriorinAnopheles,andvirtuallynonexistentinClogmia?Similarlythetwonested moduleseve3+7and4+6arebothdefinedbyknirepressionfromtheinteriorandhbrepression fromtheexterior[14],whichseemslessplausibleinAnophelesbasedontherelativepositions oftheevestripesandgapgenes. Howiscomputationalmodelingbestharnessedtothetaskofinferringtheevolutionary pathbetweenflyandmosquitowithsuchsparseinformationaboutoneendpointandinterme- diates?Oneverygenerallessonfromthemachinelearningfieldistoavoidoverfitting[15] [16].Moreparametersmakelesspredictive,“hairball”models[17]thatcanalwaysbe PLOSGenetics|DOI:10.1371/journal.pgen.1006052 May26,2016 2/19 PredictingAncestralSegmentationPhenotypesfromDrosophilatoAnophelesUsingInSilicoEvolution complexifiedratherthanfalsified.Thetemptationinthepresentinstanceistoimportintothe evolutionarysimulationallthemoleculardetailswehaveaccumulatedaboutDrosophila.A realisticmodelfortheAPpatterninginDrosophilawithmultiplefactors,shortrangerepres- sionandcooperativity,wasformulatedin[18],andappliedtotheevolutionofnewenhancers in[3].Whenguidedbystrongselectionforthecorrectdomainofexpression[3],newmodules canevolveonthetimescaleof107years[19].Thekeypointmadeintheseandrelatedpapers isthatdenovoevolutionofenhancersisfastbecausetheirgenotypetophenotypemapcanbe optimizedbypointmutationsandhillclimbing.Thesepapersalsoobservethatunderthe quickandsloppylogicofevolution,theexcessofbindingsitesortheprevalenceofgenericacti- vatorsandpositionspecificinhibitorscanallbeunderstoodasthemostquicklyrealizedsolu- tionstothefitnessoptimizationproblem. Wedonotseethecreationofnewmodulesinresponsetostrongselectionasnecessaryfor thetransitionfromflyandmosquitobacktotheirlastcommonancestor(LCA).Ratherviathe logicofevolutionarybricolage[20],organicevolutionandthuscomputation,shouldseekthe mostquicklyevolvedrepurposingofexistingcomponentsthatconnectsthetwodefinedend- pointssubjecttotheconstraintofviabilityforallintermediates.Wewillshowthatgapand pair-ruleregulationinflycanbecontinuouslyadjustedtoaccommodatetheobservedchanges intheposteriorgapgeneexpressionpatterns.Giventherangeoftimeswehavetocover,the highrateofchurninregulatorysequenceamongtheDrosophlids[19](withlittleeffectonphe- notype),andthechangesinregulatoryfactorssuchastheabsenceofbicoidinAnopheles,itis thusmostpracticalandinformativetosimulatethephenotypeandignorethemolecularlevel. Phenotypicmodelshavebeeninformativeinotherareas[21,22]andinthepresentcontext fitquantitativegeneticdataastohowexpressiondomainsshiftwhenupstreamfactorsare altered.Similarapproachesarefoundin[23],and[8].Wethenuseanevolutionarycomputa- tionthatinitializesthenetworkmodelwithDrosophilaparameters,andmutatesandselects witha‘fitness’thatdirectsthemodeltowardsAnopheles.Puttingasidethespecificmolecular informationwehaveforDrosophilamakesourapproachapplicabletoawiderrangeof problems. Invariablywefind,evestripe5disappearsandeither(orboth)theeve4+6or3+7modules addathirdposteriorstripetocompensate.Thustheposteriorevestripesarenothomologous inDrosophilaandAnopheles.Whenweconsiderregulationoftheotherprimarypairrule genesinfly,weconcludethatthemostplausiblecommonlong-germancestorofflyandmos- quitoemployedadynamicpatterningsystembasedonaforwardshiftintheevepatternas observedinClogmiaandDrosophila[24]toimposephaserelationshipsontheremainingpair rulegenes.Thusthereshouldbenohomologyintheposteriorgapgeneregulationofrun,h,or ftzbetweenflyandmosquito. Weemphasizethatnocomputation,nomatterhowcomplex,willeverproveoneevolution- aryscenariooveranother.Computationisatbestaheuristictooltouncoverinteresting hypothesisthatonecouldnotguess,andbuttressthosehypothesisbytheirfidelitywithaquan- titativephenotypicmodelforregulation.Thecomputationislikeascreenforallsolutionsto anevolutionaryproblemgivendefinedrules.Totheextenttheingredientsofthephenotypic modelareplausibleandtransparent,andthepredictionsintuitive,theymaystimulate experiments. MaterialsandMethods Evolutionaryalgorithm ThemainlessonoftwodecadesofquantitativeanalysisofDrosophilasegmentationisthat positionalinformationofpair-rulestripesisessentiallydefinedbygap-generepression(seee.g. PLOSGenetics|DOI:10.1371/journal.pgen.1006052 May26,2016 3/19 PredictingAncestralSegmentationPhenotypesfromDrosophilatoAnophelesUsingInSilicoEvolution reviewin[5]).Gapgenesthemselvesarepositionedbyamixtureofcross-repression[9,25] andactivationprovidedbymaternalgradients.Wewillbuildourgeneticorphenotypicmodel forDrosophilabydefininganinteractionkernelforeachgapgeneandpair-ruleregulatory module.Thekerneltakesthenumericalvaluesoftheinputsandoutputstheexpression.The generalfunctionalformisgivenintheS1Text,andthespecificinputsshowninthenext subsection. TheevolutionaryalgorithmthatwillproducetheAnophelesnetworkisallowedtochange onlythenumericalparameterswithinthekernelfunctions.Thustheparametersthatdefine thematernaltogapregulation,interactionsamongthegapgenes,andtheirregulationofthe pairrulegenesallchange.Thealgorithmdoesnotcreatenewkernelsnoraddnewinputsto existingkernels,butitisimportanttoincludefromthestartallpotentialregulatoryinputsthat mightplayaroleduringevolution,eveniftheireffectisminorinDrosophila.Theoutputofa kernelisallowedtobecome0signifyingitselimination. Thisconservativechoicefortheallowed‘mutations’,wasmotivatedabove,andjustified here.Firstlyweshowthatthedesiredconversionfromflytomosquitocanberealizedwithout addingnewkernels,andmerelymodifyingexistingones.Bindingsitesturnoverrapidlyin modulessoparameterevolutioninexistingkernelsshouldbefast,whilecreatingkernelsinthe absenceofdirectionalselectioninanticipationofafutureneedismorespeculative,andargu- ablyslower.Secondlytheanterior(roughlyevestripe4andforward)gapgenepatternin Anophelesandtheintermediatespeciesislargelyinvariant,whileseveralofthepair-rulegene modulescontrolbothananteriorandaposteriorstripe.Sincewewillimposethattheanterior regulationisinvariant,itwasmostlogicaltokeeptheinputstothesetwostripekernelsinvari- antalso.Oncetheallowedmutationsaredefined,thealgorithmproceedsbyroundsofmuta- tion-selection.ApopulationofnetworksisinitializedtotheDrosophilaparameters,each networkismutatedandretainedifitismorefitthanitsparent.Themostfithalfofthepopula- tionisduplicatedandformsthenextgeneration.Detailsonthecodecanbefoundin[26,27], andourcodeisavailableuponrequest. Thefunction(negativefitness)thatwewanttominimizeforeachnetworkisasumofterms measuring(1)deviationoftheposteriorhbandgtprofilesfromtheAnophelespattern(2)devi- ationoftheanterioreveprofilefromDrosophila.Inadditiontheremustbeatleast7evestripes. From[10]weknowhbmovesforwardinmosquito,whileposteriorgtisweakandprobably playsnoroleinpatterningsoweassumeit’sabsent.Noteweconstraintheexpressionprofiles, soevolutionhastofindawaytoaltertheposteriorhbkerneltomoveitsexpressionforward andmatchAnopheles.Whenweincludeasecondpairrulegene,ftz,todefinethe14stripeseg- mentpolaritypattern,weinsistitsstripesalternatewiththoseofeve.Nothingaboutintermedi- atespeciessuchasClogmiaisassumed.OncetheevolutionarypathtoAnophelesis understood,andwithittheregulation,thehomologyofthe6Clogmiastripesbecomesobvious withoutanyfurthercomputationasweexplainbelow. Wedonotimposethattheevestripesbeofequalwidth,thoughinanumberofinstances wecheckedthatlocalparameteroptimizationcanreadilysatisfythisconstraint,seee.g.S4 Video.NormalDrosophilasegmentationisknowntobeextremelyprecise,[28,29].However considerablechangeineveexpressionintheblastulaisnotincompatiblewithadultviability. Anearlyexamplewasinducedbyvariablebcddosage[30].Laterexamplesincludelossofpara- segments7and11[31],andevenabdominalsegmentA5[32,33],withfurtherdetailsleftfor thediscussion.Somevariationinphenotypeisessentialforevolution.Sinceonecanonlyclaim heuristicvalueforourevolutionarycomputations,tryingtobetterdefinethefitnesscostsof quantitativelyimperfectpatternsaddsmoreuncertaintythanitresolvesandencumbersasim- plestory. PLOSGenetics|DOI:10.1371/journal.pgen.1006052 May26,2016 4/19 PredictingAncestralSegmentationPhenotypesfromDrosophilatoAnophelesUsingInSilicoEvolution Fig1.SimplifiedmodelofDrosophilanetwork.A-B:simulatedmaternalandgapgeneprofiles.C:simulatedpair-rulegeneprofiles.A’-C’Summary ofinteractionsusedtogeneratetheseprofiles.EquationsandreferencesfortheinteractionsaregivenintheS1Text.Agenericspatiallyuniform activatorisassumedwhereneeded. doi:10.1371/journal.pgen.1006052.g001 IdealizedDrosophilanetwork ThestartingpointofoursimulationsisanidealizedDrosophilashowninFig1.S1Textdetails ourassumptions,wesummarizetheirmainfeaturesbelow. Therearematernalinputgradients,bcdanteriorandcadposterior,repressedbybcd.bcdis frozenthroughoutthesimulation.Whilethereisnobcdinmosquito,weassumesomeother genesuchasotdtakesitsplace[34].Inadditionwehavefixedprofilesoftailless(tll)andhucke- bein(hkb)intheposterior.Thosegradientssupplypositionalinformationtothegapgenes,hb, gt,kni,Kr,whicharetheonlyonesweneedtofollowduringtheevolution.Atthephenotypic levelweconsider,gapdomainslookverysimilarinDrosophilaandAnopheles,themaindiffer- encebeingtheposteriorexchangesbetweenhbandgt. Ourdescriptionofthekernelsdefininggapterritoriesincorporatesregulatoryinteractions inferredfromgenetics,thatarepresumablyconservedinevolutiongiventheobservedsimilari- tiesofthegappatterns(seedetailsinS1Text).Repressioncomesfrommorethantheimmedi- atelyadjacentgapgenes,sincewhenthesearemutated,expressiontypicallydoesnotextendto theanteriororposteriorpoleoftheembryo.Weomitotherpotentialinteractionsbecausethey donotimpacttheconservedqualitativegappatternandwouldfurtherrequiredetailedmolecu- lardatatobefitinaspecies-specificmanner[9,35].Thereaderwillobservethatallgapgene expressionpatternsalongthecomputedpathwaysfromflytomosquitoremainfixedinsize, suggestingwearenotomittinganyessentialinteractionsasgtandhbinterchange. Foreveweincludeonlystripes2to7.(Wedonotsimulateevestripe1becausewefocuson theposteriorregulation,anditsregulationisdecoupledfromtheotherstripes.)andthushave fourevemodulestoconsidereve2[36],eve3+7,eve4+6[14,37]andeve5[13].Thereisgood geneticevidence,reinforcedbybioinformaticstudies[5,7],thattheirpositionislargelydefined bygapgenerepression.Weincludemorethantheminimalinteractionsrequiredtofitthewild typeeveandgapgenepatternsintheposteriorsincehbandgtdomainsinterchangeaswe evolvetomosquito,andmutagenesisexperimentsinflysuggeststriperegulationbymorethan theclosestgapgenes.Forinstance,inahbmutantbackground,neithereve6norevestripe7 expandmuchintheposterior[13]andinagtmutant,eve5stripeonlyextendsposteriortoeve 7stripe[13].Thustheremustbeadditionalrepressionfromtheposteriorthatweassume comesfromtll.Weallowauniformactivatorforstripes3–7and4–6(suppliedbyDSTAT[7, 37]orZelda[38,39]),butinourframeworknopositionalinformationisgivenbyactivators. PLOSGenetics|DOI:10.1371/journal.pgen.1006052 May26,2016 5/19 PredictingAncestralSegmentationPhenotypesfromDrosophilatoAnophelesUsingInSilicoEvolution ThereareasimilarsetofftzmodulesdefinedbygapgenerepressioninFig1.ftz4represents aspecialcaseinthatthereisnostripespecificelementanditappearsthatftzstripe4isonly expressedaspartofthe7stripe‘zebra’element[5].Thusftzhaspartiallythecharacterofasec- ondarypairrulegenethattakesinputfromotherprimarygenes,afactthatwillbeimportant inthefollowing. Results GapgeneAnophelespatternhasbeendescribedbyGoltsevetal.[10].Asexplainedbefore,the maindifferencebetweenDrosophilaandAnophelesgappatternsisintherelativepositioningof hbandgt.SpecificallyhbmovesforwardandthegtdomainbecomessoposteriorinAnopheles thatitisunlikelytosetstripeboundaries. Thisinterchangeoflocalizationinthecourseofevolutionposesaproblemforeve5whose posteriorboundaryisregulatedbygtinDrosophilawhichisimplausibleinAnopheles,andalso inClogmiawhereposteriorgtisabsent(andthereforeintheLCAofthesethreeinsects).Soit isveryplausiblethateveregulationhaschangedbetweentheseinsects.Similarly,therelation betweenevestripesandgapgenesintheposteriorisratherdifferent:forinstance,Anopheles eve6/7aresymmetricaloneithersideofposteriorhb(Fig.6in[10]),whiletheyarebothante- riortoDrosophilahb.Finally,Anophelesevenhasaweak(andlate)extra8thevestripecom- paredtoDrosophila. InthefollowingweusecomputationtoevolveevolutionarypathwaysbetweenDrosophila andAnophelesandinferaLCA.(Aswithsimplemodelsofmolecularevolution,ourmutation ratesarethesameforwardorbackwardintime.)Allsolutionsdescribedherewerefoundsev- eraltimesandforvaryingparametersoftheviabilityfunctionsortheinitialnetworkitself.S1 andS2Videossummarizetheevolutionarypathways.Predictedevolutionarypathwaysaredis- playedonasimplifiedinsectevolutionarytreeinFig2A. Variationinevestripesandsubsequentlossofeve5 SimulationsbeginfromtheDrosophilanetworkinFig1andtargettheAnophelesgappattern asanendpoint.Thenumberofevestripes(including1)mustbeatleast7,andthereisno restrictionontheirrelativesizeorposition. AtypicalexampleofsuchsimulationisprovidedonFig3,withintermediatestepspictured onthephylogenyinFig2A.Astheposteriorhbdomainmovesforwarditsplitsevestripe7. Therepressionfromhbthatdefinedtheposteriorboundariesofeve6–7graduallyshiftstotll. Stripe5transientlyfragmentsintotwoadditionaldomains,Fig3B,neitherofwhichemergesas andistinctstripe.Butonceeve7splitsintwo,thestripe5elementcandisappearwhilerespect- ingourconstraintofatleast7stripes.Afteritdisappearsposteriorgt,issuperfluous. AvariationonthispathwayispresentedonFig4.Thistimetheevolutionoftheposterior hbdomainanterior,splitseve6tocreateaneweve8.Onceanewevestripeappearsinthepos- terior(Fig4B),posteriorgtfirstdisappearssothateve5expandsposteriorly,fusingwitheve6 (thuseffectivelydisappearing,(Fig4C).Thustheeve5stripemoduleisnolongerneededand disappears,leadingtoafinalconfigurationsimilartoFig3D. Theevolutionaryscenariowithcreationofanewevestripeintheposteriorandsubsequent removalofeve5andgtposteriorishighlyreproducibleinoursimulationsforavarietyofcon- ditionsthatimplementthesameevolutionarypressures.Thusstripes4+6and3+7inDrosoph- ilabecomestripes4+5and3+6inAnopheles.FurthermoreoneoftheseDrosophilamodules controls3stripesinAnopheles.OnFig3,Drosophilaeve3+7givesrisetostripes3,6,7,whileon fig4,Drosophilaeve4+6givesrisetostripes4,5,7.Inbothcases,oneevestripeissplitbyhbto givetwostripesintheposteriorthataresymmetricallypositionedaroundthehbdomain.The PLOSGenetics|DOI:10.1371/journal.pgen.1006052 May26,2016 6/19 PredictingAncestralSegmentationPhenotypesfromDrosophilatoAnophelesUsingInSilicoEvolution Fig2.Summaryofourpredictions.APredictedevolutionarypathwaysfromdifferentsimulationsdetailedinFig3(labelF1),Fig4(labelF2),Fig5 (labelFtz),Fig7(labelLC).Thetimesshownfortheintermediatesareonlyschematic.Gapandevepatternsinthreeinsectspeciesandtheinferredlast commonancestor(LCA)areindicated.BSummaryofhomologybetweenEvemodulesindifferentspeciespredictedbyourevolutionarysimulations. doi:10.1371/journal.pgen.1006052.g002 8stripesinAnopheleswouldbemosteasilyexplainedifbothmodulesgrowanotherstripewith Drosophilaeve4+6(resp.3+7)becoming4,5,8(resp.3,6,7)inmosquito. Oursimulationsalsopredictthatintermediatedipteransmusthaveretainedthislogic whereeve4+5(resp.3+6)arehomologoustotheeve4+6(resp.3+7)moduleinDrosophila. Bothmodulesarerepressedbykniandhb,soinparticularstripes4–5and3–6shouldbelaid symmetricallywithrespecttokniinthoseintermediatedipterans.Thisisapredictionofour computation,thatexploitstheknowngapgeneregulation,butwasinnowayimposed.Strik- ingly,evestripes4+5and3+6inbothClogmiaandAnophelesareindeedlaidrathersymmetri- callywithrespecttoknicontrarytothesituationinDrosophila.FurthermoreClogmiahasonly 6evestripespriortogastrulationandconsistentwithourmodel,lacktheposteriorhbdomain thatgeneratedthetwoadditionalposteriorstripesinAnopheles. Keepingsegmentationlogicbyincludingftz Wecouldnotfindexamplesofviablemutantfliesmissing2consecutivesegments(corre- spondingtoonefullpair-ruleperiod).Thissuggeststhat,evenifinsomemutants(e.g.thehop- scotchmutant[32]),whenoneevestripedisappears,theembryoneedstokeepsomepolarity informationrequiredforthedefinitionofparasegments. Sinceeve5overlapsanddefinesA4pandA5a,properparasegmentdefinitionmeansthat thepolarityofA4aandA5pmustbemaintained(andsubsumecellsthatwereinA4pand A5a).Anaturalhypothesisisthentoassumeanotherpairrulegene,outofphasewitheve, mustpersistwhenevestripe5disappearstoprovideinputtothesegmentpolaritysystem.We chosetoaddftztoourmodel.Werecognizethattheproximateinputtothesegmentpolarity genesisnotdirectlyfromeveandftzbutwehavetoinsistthatthemodelrespecttheminimal PLOSGenetics|DOI:10.1371/journal.pgen.1006052 May26,2016 7/19 PredictingAncestralSegmentationPhenotypesfromDrosophilatoAnophelesUsingInSilicoEvolution Fig3.Simulatedevolutionarypathway(labelF1onFig2A)fromDrosophilatoAnopheles,withsalientchangesdiscussedinthemaintext.For eachtranscriptionalevemoduleonlythegapgenesthatregulateitareshownwiththesamecolorschemeasFigs1and2A.evestripe1isnotshown andthemaximumexpressionofeachmoduleisnormalizedto1exceptwhenitdipsbeneathathresholdequivalenttoitsloss. doi:10.1371/journal.pgen.1006052.g003 informationlogicallyrequiredforthesegmentpolaritypattern.Thuswhenanevestripedisap- pears,theneighboringftzstripesmergeandonlyoneparasegmentdisappears.(Wewillcon- siderbelowhowtheconstraintsonevolutionimposedbytheotherprimarypair-rulegenesin Drosophila[5]canbesatisfied,ifweinsistthattherelativephaseamongthepairrulesgenesis maintained.) Inafirstroundofsimulationswherewemodelftzasaprimarypair-rulegene(andpostulate apureftzstripe4moduledelimitedbyhbonitsanteriorsideandgtposteriorly),theevolution- arypathwayobservedintheprevioussectiondies.Thereareseveralreasonsforthis:firstgt controlsbotheve5andtheputativeftz4,soitisverydifficulttohaveitdisappeargiventhis dualrolewhilekeepingtheftz/evealternationthatweimpose.Second,ifanewevestripe appearsintheposteriorasbefore,ithasnoreasontobecoupledproperlytoacorresponding alternatingftzstripe.Essentially,ifftzisprimary,simulationsfailtoevolveneweveposterior stripeswithoutbreakingthealternationofftz/eve. Itisthusinterestinginthiscontextthatftzstipe4appearsonlytogetherwiththe7stripe zebraelement[5].Thusifweallowrepressionofftz4-zebrabyeveandnotgt,itbecomesslaved PLOSGenetics|DOI:10.1371/journal.pgen.1006052 May26,2016 8/19 PredictingAncestralSegmentationPhenotypesfromDrosophilatoAnophelesUsingInSilicoEvolution Fig4.Simulatedevolutionarypathway(labelF2onFig2A)fromDrosophilatoAnopheles,followingtheconventionsofFig3.InCtheposterior eve5stripeiscountedasmergedwithstripe6. doi:10.1371/journal.pgen.1006052.g004 toeveintheposteriorandfunctionsasasecondarypairrulegene.Inthesimulation,Fig5,ftz stripes5,6,7thatarepositionedbygapgenes,graduallydisappearinfavorofthezebraelement. Themodulesthatcontrolledpairsofstripes1+5,2+7and3+6nowcontrolonlytheanterior memberandcanevolvetointerdigitatewiththeevestripes.Withtheposteriorftzstripescon- trolledbyeverepression,thepatterncanevolvetotheAnophelesconfigurationasbeforewhile preservingeveandftzalternationthroughout,Fig5.Wheneve5disappearsftz4andftz5 merge(sinceeverepressioniskeepingthemdistinct)Fig5Band5C,thuspreservingtheeve,ftz alternation. Istheancestralpair-rulepatterningdynamicallygenerated? Thefactthatourevolutionarysimulationsfailwhenftzispurelyprimaryandsucceedwhenftz ismoresecondarysuggeststhatitwillbethesameforotherprimarypair-rulegenessuchash andrunt.Weneverthelessneedtoaskhowtherelativephaseoftheprimarypairrulecouldbe conservedintheevolutionaryscenariospresentedhere.Wepropose,bymeansofaquantita- tivemodel,thatpair-ruleregulationintheposterioroftheLCAismoredynamicthan PLOSGenetics|DOI:10.1371/journal.pgen.1006052 May26,2016 9/19 PredictingAncestralSegmentationPhenotypesfromDrosophilatoAnophelesUsingInSilicoEvolution Fig5.Simulatedevolutionarypathway(labelftzonFig2A)fromDrosophilatoAnopheles,includingftz.ConventionsofFig3foreveandftz stripesareused. doi:10.1371/journal.pgen.1006052.g005 conventionallyassumedinDrosophila.Thiswillimplythatthereisnohomologybetweenthe posteriorregulationofthepair-rulegenesbygapgenes,otherthanforeveitself. SpecificallybothinDrosophila[40]andClogmia[24]evestripesmovefromposteriorto anteriorpriortogastrulation.Ourideaisthatsuitablecombinationsofstrongandweakrepres- sionamongrun,h,ftz,andevecanreadthisphaseinformationandstabilizethepair-rulepat- ternweobserveinDrosophila,withoutdirectgapgeneinput.Themodelisrelatedtothepair- rulegeneoscillatorthatpatternstheposteriorofshortgerminsects,aspreviouslysuggestedin [41].(Howeverthemodelisnotcapableofintrinsicoscillationssinceeveisdrivenbygapgenes andnotbyotherpairrulegenes,thoughitiseasytoenvisagehowintrinsicpairrulefeedback onevecouldbegraduallyreplacedbyextrinsicgapregulationduringtheshorttolonggerm bandtransition.)Viewedwithinasinglecell,theforwarddisplacementofeveappearsasone completetemporalcycle,thusageneregulatorynetworkderivedfromadelayednegativefeed backoscillatoramongthepairrulegenescanusethesameinteractionstoproducestable phasesinspace.IncertainrespectsourconjecturedLCAresemblesNasonia[42]wheretheseg- mentsposteriortoA5arepatterneddynamicallyaswereconsiderinmoredetailinthe Discussion. PLOSGenetics|DOI:10.1371/journal.pgen.1006052 May26,2016 10/19

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To account for the evolution of the other pair-rule genes in the position of the gap genes and the single pair-rule gene eve, there is only few information on Krotov D, Dubuis JO, Gregor T, Bialek W. Morphogenesis at criticality.
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