Predicting Ancestral Segmentation Phenotypes from Drosophila to Anopheles Using In Silico

19 Pages·2016·5.14 MB·English

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
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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.
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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.
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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.
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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
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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
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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
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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.
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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