Retinal Expression of the Drosophila eyes absent Gene Is Controlled by Several Cooperatively

31 Pages·2015·3.73 MB·English

RESEARCHARTICLE
Retinal Expression of the Drosophila eyes
absent Gene Is Controlled by Several
Cooperatively Acting Cis-regulatory Elements
BonnieM.Weasner1,BrandonP.Weasner1,SarahD.Neuman2,ArashBashirullah2,Justin
P.Kumar1*
1 DepartmentofBiology,IndianaUniversity,Bloomington,Indiana,UnitedStatesofAmerica,2 Divisionof
PharmaceuticalSciences,UniversityofWisconsin,Madison,Wisconsin,UnitedStatesofAmerica
*[email protected]
a11111
Abstract
Theeyesabsent(eya)geneofthefruitfly,Drosophilamelanogaster,isamemberofanevo-
lutionarilyconservedgeneregulatorynetworkthatcontrolseyeformationinallseeingani-
mals.Thelossofeyaleadstothecompleteeliminationofthecompoundeyewhileforced
OPENACCESS expressionofeyainnon-retinaltissuesissufficienttoinduceectopiceyeformation.Within
Citation:WeasnerBM,WeasnerBP,NeumanSD, thedevelopingretinaeyaisexpressedinadynamicpatternandisinvolvedintissuespecifi-
BashirullahA,KumarJP(2016)RetinalExpression
cation/determination,cellproliferation,apoptosis,andcellfatechoice.Inthisreportwe
oftheDrosophilaeyesabsentGeneIsControlled
explorethemechanismsbywhicheyaexpressionisspatiallyandtemporallygovernedin
bySeveralCooperativelyActingCis-regulatory
Elements.PLoSGenet12(12):e1006462. thedevelopingeye.Wedemonstratethatmultiplecis-regulatoryelementsfunctioncoopera-
doi:10.1371/journal.pgen.1006462 tivelytocontroleyatranscriptionandthatspacingbetweenapairofenhancerelementsis
Editor:ClaudeDesplan,NewYorkUniversity, importantformaintainingcorrectgeneexpression.Lastly,weshowthatthelossofeya
UNITEDSTATES expressioninsineoculis(so)mutantsistheresultofmassivecelldeathandaprogressive
Received:June2,2016 homeotictransformationofretinalprogenitorcellsintoheadepidermis.
Accepted:November4,2016
Published:December8,2016
Copyright:©2016Weasneretal.Thisisanopen AuthorSummary
accessarticledistributedunderthetermsofthe
CreativeCommonsAttributionLicense,which Activationofagenerequiresinteractionsbetweenenhancerandpromoterelements.It
permitsunrestricteduse,distribution,and hasbeenknownforsometimethattranscriptionofageneexpressedinacomplexpattern
reproductioninanymedium,providedtheoriginal orinmultipletissuesisregulatedbyanarrayofenhancers.Recentstudieshavealsodem-
authorandsourcearecredited.
onstratedthatmultipleenhancerscanregulateasingleexpressionpatternwithinasingle
DataAvailabilityStatement:Allrelevantdataare tissue.Inthisstudyweaskedhowtheexpressionpatternofeyesabsent(eya)isregulated
withinthepaperanditssupportinginformation attheleveloftheenhancerinthedevelopingretina.Wefoundthatseveraladjacentlyposi-
files.
tionedenhancerelementsfunctioncooperativelytocontroltemporalandspatialexpres-
Funding:Theworkissupportedbygrantsfromthe sionofeyaandthatthespacingbetweentwoofthesecis-regulatoryelementsisimportant
NationalInstituteofGeneralMedicalSciences(R01 totheirfunction.Thisstudyshowstheimportanceofenhancercooperationandarchitec-
GM095944)toAB,theNationalScience
tureinregulatingcomplexanddynamicallychangingexpressionpatterns.
FoundationGRFP(DGE-125659)toSDN,andthe
NationalEyeInstitute(R01EY014863)toJPK.The
fundershadnoroleinthestudydesign,data
collectionandanalysis,decisiontopublish,or
preparationofthismanuscript.
PLOSGenetics|DOI:10.1371/journal.pgen.1006462 December8,2016 1/31
RegulationofeyaExpressionintheDrosophilaRetina
CompetingInterests:Theauthorshavedeclared Introduction
thatnocompetinginterestsexist.
Constructionofaproperlyfunctioningorganortissueisdependentupontheactivityofhun-
dredsofgenesthatcanbeconceptuallyorganizedintoageneregulatorynetwork(GRN)[1–
4].Thesegenescontrolthespecification/determination,patterning,differentiation,andphysi-
ologyofallcelltypeswithinthedevelopingandadultorgan.Thedevelopmentoftheretinain
thefruitfly,Drosophilamelanogaster,iscontrolledinpartbyanevolutionarilyconservedgene
regulatorynetworkcalledtheretinaldetermination(RD)network[5].Thecoremembersare
twoPAX6genes,twinofeyeless(toy)andeyeless(ey),theSIXgenesineoculis(so),theEYA
familymembereyesabsent(eya),andtheSKI/SNOproto-oncogenedachshund(dac)[6–11].
Inadditiontothesecoremembers,theflyversionofthisnetworkcontainsanadditionalnine
genesofwhichsomearefunctionallyconservedwithinthevertebrateeye[5].Mutationsinthe
flyRDgenesleadtodrasticreductionsofthecompoundeyeswhileforcedexpressioninnon-
oculartissuessuchasthewings,antennas,andlegsleadstotheformationofstructurallycom-
pleteectopiceyes.Theseobservationssuggestthatthesefactorsoccupythehighestpositions
withinthelargereye/lensgeneregulatorynetwork.Inadditiontotheeye,thecoremembers
areusedreiterativelyduringdevelopmenttoalsodeterminethefateofmanynon-oculartis-
suessuchasthemusculature,skeletalsystem,nose,ear,pancreas,andkidney[12–14].Studies
oftheRDnetworkcanthereforeprovideinvaluableinsightsintothespecificationandpattern-
ingofawiderangeoftissuesandorgansbeyondtheeye.
TheRDnetworkhasbeenbeststudiedinDrosophilawithaquartercenturyofinvestigation
havingidentifiedawealthofgenetic,biochemical,andmolecularinteractionsamongstthe
differentmembers.Numerousreviewarticlesovertheyearshavesummarizedthesefindings
instaticcircuitmaps[5,12,15–17].Whiletheseinteractiondiagramshavebeenhelpfulin
understandingtherelationshipamongstnetworkmembers,theycanbemisleadingsincethe
networkgenesareexpressedindynamicpatternsthatchangebothspatiallyandtemporally
[18].Inaddition,individualgenesinitiateexpressionatdifferenttimesindevelopment
[6,8,9,11,17,19],areco-expressedwithothernetworkgenesinsomecellsbutnotinothers
[18],andappeartointeractdifferentlydependingupontheexactspatial,temporal,anddevel-
opmentalcontext[20,21].Asaresultthestaticmapsofregulatoryinteractionsdonotneces-
sarilyreflecttherealityofwhatishappeningthroughouttheeyeineitherspaceortime.Inthis
report,wehavefocusedonunderstandinghow,atthelevelofcis-regulatoryelements,theeya
geneisregulatedtemporallyandspatiallyinthedevelopingretina.Wethenusethisinforma-
tiontoevaluateonetenantoftheRDcircuitmap–namelywetestthepotentialregulationof
eyabytheSotranscriptionfactor.
TheEyaproteinfunctionsasatranscriptionalco-activatorandproteintyrosinephospha-
tase[22–24],althoughthelatteractivityappearsdispensableforeyedevelopmentinDrosophila
[25].WithinthenucleusEyainteractswithmembersoftheSIX/Sofamilyofhomeodomain
containingDNAbindingproteins[22].Together,SIX-EYAcomplexesfunctionasbipartite
transcriptionfactorstoactivatetargetsnecessaryforthespecification,differentiation,and
growthoftheretina[22,23,26].Recentreportsindicatethatthesecomplexesalsofunctionas
transcriptionalrepressorsalthoughtheexactmechanismunderlyingthisactivityhasyettobe
determined[19,20,27,28].Bothgenesareexpressedinnearlyidenticalspatialpatternswithin
thedevelopingeye[10,11].Expressionofbothgenesislostinbotheyaandsomutants[29].
ThesepropertieshaveledtotheproposalthattheSo-Eyacomplexregulatestheexpressionof
bothgenes.
Inthewildtypeeyeeyaexpressionistemporallyandspatiallydynamic[11].Thisexpres-
sioniscompletelyeliminatedfromtheretinaofeya2mutants,whichareviablebutcompletely
lacktheadultcompoundeyes[11].Thesefliesharbora322bpdeletion,whichlies576bp
PLOSGenetics|DOI:10.1371/journal.pgen.1006462 December8,2016 2/31
RegulationofeyaExpressionintheDrosophilaRetina
upstreamofthetranscriptionalstartsite[11].Whenmultimerizedthis322bpfragmentdrives
expressionofatranscriptionalreporterinapatternthatapproximatesthewildtypegene
[30,31].Italsocontainssufficientactivitytopartiallyrestoreeyedevelopmenttoeya2mutants
whendrivingexpressionofarescuingtransgene[30,31].Basedonthisevidencethisenhancer,
formanyyears,wasthoughttobethesolecis-regulatoryelementcontrollingeyaexpression
withinthedevelopingeye.SequenceanalysisidentifiedthepresenceofacanonicalSobinding
sitewithinthisenhancertherebyraisingthepossibilitythateyaexpressionintheeyeiscon-
trolledbySo[31,32].Morerecently,severalstudiesoftheeyalocushaveidentifiedtwoaddi-
tionalretinalenhancers,thepresenceofadditionalSobindingsites,andmultiplegenomic
positionswhereSoappearstobindineye-antennaldiscs[33–35].Togetherthesedatahave
beenusedtosupportthepremisethattheinitiationandmaintenanceofeyaexpressionis
underthecontrolofSo.
Inthispaperwereporttheidentificationofseveralcis-regulatoryelementswithintheeya
locusthatcontributetoitsexpressioninthedevelopingeye.Threeoftheseenhancerslieadja-
centtoeachotherandwedemonstratethattheyfunctioncooperativelytoregulatethetempo-
ralandspatialexpressionpatternofeyaduringeyedevelopment.Wealsoshowthatthe
spacingbetweentwooftheseenhancersisimportantfortheactivityofeachcis-regulatoryele-
ment.Andfinally,weshowthateachoftheretinalenhancers(thoseidentifiedinthisand
otherstudies)remainactiveinsoloss-of-functionmutants.Thisisatoddswiththemodelin
whicheyaisregulatedbySo.Weshowthatthelossofeyaexpressioninsomutantsisactually
theresultofcelldeathandaprogressivefatetransformationoftheretinaintoheadepidermis.
OurfindingsdonotsupportaroleforSointheinitiationofeyaexpression.Howeverwedo
notruleoutthepossibilitythatSofunctionstomaintaineyatranscriptionintheretina.
Results
So-VP16partiallyrestoreseyaexpressionandrescueseya2mutants
Inthirdlarvalinstarretinaseyaisexpressedinasmallstripeofcellsaheadoftheadvancing
morphogeneticfurrow,indifferentiatingphotoreceptor,cone,andpigmentcells,andinthe
developingocelli(Fig1Aand1B)[11].Intheeya2mutanteyaexpressioniscompletelylost
fromtheeyefield(Fig1Cand1D).WefirstsetouttodetermineiftheSoconsensussitesand
regionsofSoChIPpeaksthatarefoundoutsideoftheoriginal322bpenhancerarefunctional.
Todothisweattemptedtorescuetheeya2mutantbyforciblyexpressingaSo-VP16chimeric
constructinthedevelopingeyewithaney-GAL4driver.Thisproteiniscapableoffullyrestor-
ingeyedevelopmenttoso1mutants[27]andactivatesaluciferasereporteratlevelsthatare
20-foldhigherthanSoaloneand5-foldhigherthantheSo-Eyacomplex(Fig1G).Basedon
thesedatawereasonedthatSo-VP16servesasastrongtranscriptionalactivatorandtherefore
isasuitablesubstitutefortheSo-Eyacomplex(So-VP16=So-Eya).ExpressionofSo-VP16
partiallyrestoresbotheyaexpressionandeyedevelopmentto62%ofthe57animalsthatwe
examined(Fig1Eand1F;S1A–S1CFig).Consistentwithbeingaveryweakactivator,expres-
sionofwildtypeSoaloneisinsufficienttorestoreeithereyaexpressionoreyedevelopmentto
eya2mutants(Fig1G;S1DandS1EFig)[27].Theseresultsledustoinitiallyconcludethat
additionalSo-responsiveenhancerelement(s)arepresentwithintheeyalocus.
Newlyidentifiedregulatoryelementsaredynamicallyregulatedduring
larvaleyedevelopment
InordertoidentifyregulatoryelementsthatareresponsivetotheSo-Eyacomplexweusedthe
osm-6geneandaCTSFinsulatorsitetodefinethe5‘and3‘boundariesrespectivelyoftheeya
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RegulationofeyaExpressionintheDrosophilaRetina
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RegulationofeyaExpressionintheDrosophilaRetina
Fig1.So-VP16reactivateseyaexpressionintheretinaofeya2mutants.(A,C,E)SEMimagesofadult
femaleDrosophilacompoundeyesandheads.(A)wildtype.(C)eya2.(E)eya2mutantsinwhichexpression
ofUAS-So-VP16partiallyrestoreseyedevelopment.(B,D,F)Lightmicroscopeimagesofthirdinstareye-
antennaldiscs—eyaexpressionisdetectedbyantibodystainingagainstEyaprotein.(B)Wildtypeexpression
ofeyainthecompoundeyeandocelli.(D)Lossofeyaexpressionintheeyeportionofdiscineya2mutants.
Expressionwiththeocelliismaintainedinthemutant.(F)Partialrestorationofeyaexpressionintheeye
portionofdiscuponexpressionofUAS-So-VP16.Anterioristotherightinadultheadandimaginaldisc
images.Atleast30adultfliesanddevelopingimaginaldiscswereexaminedforeachgenotypewith57adult
eya2;ey-GAL4,UAS-So-VP16fliesbeingscoredforrescueofeyestructure(G)Luciferaseassayquantifying
activationstrengthofSo-VP16.Y-axisisrelativeluciferaseunits(RLU).Threebiologicalreplicateswere
conductedforeachexperiment.ErrorbarsinpanelGrepresentstandarddeviation.Scalebar,100μm
doi:10.1371/journal.pgen.1006462.g001
locusandthenclonedfragmentsofDNAbetweenthesetwogenomicmarkersaheadofamini-
malhsp70promoterandalacZreporter(Fig2A).Theseconstructswereinsertedintothe
samegenomiccoordinates(attP-3BVK00033—cytologicalposition65B2)usingthePhiC31
integrasesystemtomaintainsimilarexpressionlevelsacrossreporters.Wanderingthirdinstar
eye-antennalimaginaldiscswerethenexaminedforlacZreporterexpression.Weidentified
sixgenomicfragmentsthatarecapableofdrivingexpressionofthereporterinportionsofthe
endogenouseyapattern(Fig2B–2G).Threeofthesefragments(PSE,1,andE)havebeenpre-
viouslyidentifiedasenhancerscontrollingeyaexpressionintheretina[30,31,35].ThePSE,
whichstandsforphotoreceptorspecificenhancer,drivesexpressionsolelyincellsbehindthe
morphogeneticfurrow(Fig2Aand2B)[35]whilefragment1(alsocalledIAMforimmedi-
atelyanteriortothemorphogeneticfurrow)drivesexpressionaheadoftheadvancingmor-
phogeneticfurrowandindifferentiatingcells(Fig2Aand2C)[35].FragmentE(forextant)is
theenhancerthatisdeletedineya2mutants(Fig2Aand2D)[30,31].Oursequenceanalysis
indicatesthatthefragmentis319bpinlength(andnot322bpasoriginallyreported).Frag-
ments2,3and4arethreenewretinalenhancersthatcontroleyaexpressioninthedeveloping
eye(Fig2Aand2E–2G).
Wenextdeterminedthetemporalandspatialexpressionpatternsofeachindividualfrag-
mentandcomparedthesepatternstoendogenouseyaexpression.Eyaproteinispresentinthe
wildtypeeyediscasearlyas48hrsAEL(early2ndinstar,Fig3A)andcontinuestobeexpressed
broadlyat72hrsAEL(early3rdinstar,Fig3B).Bythelatethirdlarvalinstarstageeyaexpres-
sionisrestrictedtoanarrowbandofcellsaheadofthemorphogeneticfurrowandtoalldiffer-
entiatingphotoreceptorandconecells(Fig3C).Nosingleindividualfragmentfully
recapitulatestheendogenouseyaexpressionpattern.Forexample,reporterexpressiondriven
byfragment1istemporallyandspatiallydelayedcomparedtowildtypeeyaexpressionmean-
ingthatalthoughitisactivatedinafeweyaexpressingcellsearlyindevelopment,itisnotuntil
latethirdinstarthatexpressionstartstocoincidewiththespatialpatternofendogenouseya
(Fig3D–3F,Table1).Incontrast,whilereporterexpressiondrivenbyfragmentEcoincides
withearlyendogenouseya,itslateexpressionisweakinintensityandappearsmottled(Fig
3G–3I,Table1).Lastly,thebulkoffragment2drivenexpressionwithinyoungerdiscsisineya
negativecellswhileinlaterdiscsreporterexpressiondoescoincidewiththeendogenouseya
gene(Fig3J–3L,Table1).
Sinceeachofthesethreefragments(1,E,2)doesmimicaspecifictemporaland/orspatial
aspectofeyaexpressionwehypothesizedthattheseenhancers,whichlieadjacenttoeach
other,mightfunctioncooperativelytocontrolalltemporalandspatialaspectsofeyaexpres-
sion.Totestthismodelwegeneratedasingle1181bpfragmentconsistingoffragments1,E,
and2andaspredictedthiscompositeenhancerfullyrecapitulatesthetemporalandspatial
expressionpatternofeyawithinthedevelopingeye(Fig3M–3O,Table1).Toruleoutposition
dependenteffectsweinsertedthisconstructintoasecondgenomiclandingsite(attP-9A
VK00019—cytologicalposition68D2)andobservethattheexpressionpatternofthisinsertion
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RegulationofeyaExpressionintheDrosophilaRetina
Fig2.Multipleenhancerscontrolexpressionofeyainthedevelopingeye.(A)Illustrationofthegenomicmapoftheeyalocus—representationisnotto
scale.SequencesandgenomiclocationofeachfragmentareprovidedinsupplementarymaterialsandmethodsS1-3.Sizeofeachfragment(bp)isindicated
withineachbar.Bluebars=individualretinalenhancers.PSE=photoreceptorspecificenhancerpreviouslyidentifiedandnamedbyGraemeMardon’sgroup
in[20].E=319bpextantenhancerpreviouslyidentifiedin[11].Enhancer1(IAM)=immediatelyaheadofmorphogeneticfurrowenhancerwaspreviously
identifiedandnamedbyGraemeMardon’sgroupin[20].Werefertothisenhanceras1asitshowsadifferentexpressionpatternthanpreviouslyreported.
2–4representnewlyidentifiedenhancerelements.Orangebar=compositeenhancer,purplebar=enhancer1+E,greybarsindicateregionsthatdonotdrive
expressionintheretinaincludingthefragmentusedasthe319bpspacer,asterisks=Sobindingsites,redbars=regionsofSoChIPpeaks.eya1andeya2
deletionsareindicatedbyredlinesimmediatelyaheadofexon1(B-G)Lightmicroscopeimagesofthirdinstareye-antennaldiscs.AllimagesrepresentlacZ
reporterexpressioninawildtypegeneticbackground.LacZreporteractivationisindicatedbyantibodystainingagainstβ-galactosidase.Whitearrowheads
markthepositionofthemorphogeneticfurrow.(B)ThePSEenhancerdrivesexpressionofthereporteronlyincellsthatlieposteriortothemorphogenetic
furrow.(C)Enhancer1(alsocalledIAM)drivesexpressionincellsaheadandbehindthemorphogeneticfurrow.(D)The319bpextantenhancerdrivesweak
reporterexpressionincellsaheadandposteriortothemorphogeneticfurrow.(E)Enhancer2drivesexpressionofthereporterincellsanteriorandposteriorto
furrow.(F)Enhancer3drivesexpressionincellsaheadandbehindthemorphogeneticfurrow.(G)Enhancer4drivesexpressiononlyincellsposteriortothe
morphogeneticfurrow.NosingleenhancerelementfullyrecapitulatesendogenousEyaexpression.Anterioristotherightinimaginaldiscimages.Atleast30
imaginaldiscswereexaminedforeachgenotype.Scalebar,100μm
doi:10.1371/journal.pgen.1006462.g002
isidenticaltotheoriginalinsertionandrecapitulatesendogenouseyaexpression(S2Fig).It
appearsthatthetemporalexpressionofthecompositeenhanceristhesumoradditionofthe
individualelements.Andinterestingly,recreatingthegenomicorganizationofthesethreecis-
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RegulationofeyaExpressionintheDrosophilaRetina
Fig3.Thecompositeenhancercontrolsalleyaexpressionwithinthedevelopingeye.(A-U)Light
microscopeimagesofdevelopingeye-antennaldiscs.Imagesofimaginaldiscsat48hrsand72hrsAELwere
takenat20Xwhileimagesofwanderingthirdinstarlarvaeweretakenat10X.AEL=afteregglaying.
Red=Eyaprotein,green=β-galactosidase,yellow=positionsofco-localizationbetweenEyaandβ-
galactosidase.Arrowheadmarksthepositionofthemorphogeneticfurrow.Allenhancer-lacZreportersare
placedinawildtypegeneticbackground.(A-C)LocalizationofEyaproteinindevelopingwildtyperetinasat
differentdevelopmentaltimepoints.(D-F)Enhancer1dependentexpressionisactivatedinafewEya
expressingcellsearlyindevelopmentandco-localizeswithEyaposteriortothemorphogeneticfurrowlatein
development.(G-I)Extantenhancerdependentexpressionco-localizeswithEyaandisrobustearlyin
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RegulationofeyaExpressionintheDrosophilaRetina
developmentbutbecomesweakerandsparseasdevelopmentproceeds.(J-L)Enhancer2-dependent
expressionislargelypresentinnon-eyaexpressingcellsearlyindevelopment.Co-localizationwithEyacan
beseenincellsanteriorandposteriortothefurrowlaterindevelopmentbutasignificantportionofreporter
expressionstillpresentinnon-eyaexpressingcells.(M-O)Compositeenhancer-dependentexpression
showsco-localizationwithEyaproteinthroughoutallstagesoflarvaleyedevelopment.Thisistheonly
constructtofullyrecapitulatetemporalandspatialeyaexpression.(P-R)Enhancer3-dependentexpressionis
largelypresentinnon-eyaexpressingcellsthroughoutdevelopment.Someco-localizationwithEyaproteinis
seenatlaterstagesincellsanteriorandposteriortothefurrow.(S-U)Enhancer4-dependentexpressionco-
localizeswithafewEyaexpressingcellsposteriortothefurrowlateindevelopment.Anterioristotherightin
imaginaldiscimages.Atleast30imaginaldiscswereexaminedforeachgenotypeanddevelopmentaltime
point.Scalebar,50μm
doi:10.1371/journal.pgen.1006462.g003
regulatoryelementseliminatestheectopicexpressionfromtheeye-antennaldisc(Fig3J–3O,
Table1).Sincethecompositeenhancerrecapitulatestheentireeyaexpressionpatternitispos-
siblethatfragments3,4,andPSEarefunctionallyredundant.Consistentwiththismodel,the
expressionpatternscontrolledbythesefragmentsarefullycoveredbythecompositeenhancer
(Fig3P–3U,Table1).
Thecompositeeyaenhancercanfullyrescueeyedevelopmentineya1
andeya2mutants
Wethensetouttotestifthecompositeenhancerissufficienttofullyrescuetheno-eyepheno-
typesofeya2andeya1mutants.Theoriginalcharacterizationoftheeya1mutantindicated
twochromosomalaberrationsareassociatedwiththismutation.First,achromosomalre-
arrangementcompletelyinvertstheorientationoftheeyalocuswithintheleftarmofthesec-
ondchromosome.Thisisnotthoughttointerferewithnormaleyaexpression.Second,an
approximately1.5kbdeletionwasdetectedatthe5‘endofthegene.The319bpdeletionineya2
lieswithinthelarger~1.5kbdeletionineya1.Thustheno-eyephenotypeofeya1andeya2is
thoughttoresultfromthedisruptionofthesameregulatorysites[11,31].Toprecisely
Table1. EnhancerExpressionandRescue. Foreachrescueexperimenttwo-threefemaleeyeswerephotographedwithascanningelectronmicrograph.
Wemanuallycountedthenumberofommatidiaforeacheyeandcalculatedbothaveragesandstandarddeviations(listedwithintable).Therawommatidia
countsfortherescueexperimentsareasfollows:eya1;enhancer1—eyaRBcDNA(141,235,190),eya2;enhancer1—eyaRBcDNA(322,245),eya1;
enhancerE—eyaRBcDNA(39,38,35),eya2;enhancerE—eyaRBcDNA(358,372,362),eya1;enhancer1+E+2—eyaRBcDNA(792,730,762),eya2;
enhancer1+E+2—eyaRBcDNA(757,825,829),eya1;enhancer1+E—eyaRBcDNA(376,324,298),eya2;enhancer1+E—eyaRBcDNA(635,584,636),
eya1;enhancer1+spacer+2—eyaRBcDNA(433,433,432),eya2;enhancer1+spacer+2—eyaRBcDNA(443,448,556).
Enhancer LacZReporterExpression cDNARescueAverage#ofommatidia
48hrsAEL 72hrsAEL Late3rdInstar eya1 eya2
Enhancer1 - † ‡ 189±47 284±54
EnhancerE ‡ ‡ † 37±2 364±7
Enhancer2 † † † - -
Enhancer1+E+2 ‡ ‡ ‡ 761±31 804±40
Enhancer1+E ‡ ‡ ‡ 333±40 618±30
Enhancer1+2 - - † - -
Spaceralone N/A N/A - N/A -
Enhancer1+spacer+2 † † ‡† 432±1 482±64
Enhancer1+5bp+2 † † ‡† - -
Enhancer3 † † † - -
Enhancer4 - - † - -
-Noexpressionorrescue
‡Recapitulateseyaexpression
†Expressesinnon-eya+cellsoronlyinafewEya+cells
doi:10.1371/journal.pgen.1006462.t001
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RegulationofeyaExpressionintheDrosophilaRetina
Fig4.Thecompositeenhancerfullyrestoreseyedevelopmenttoeya1andeya2mutants.(A)qRT-PCR
quantificationofeyaRAandRBtranscriptlevelsineye-antennaldiscs.Y-axismeasurestherelativeexpression
levelsofeachtranscript.Rawdatafromsinglerunsfromthreebiologicalreplicateswereusedtogeneratethe
graph.Errorbarsrepresentstandarderror.(B-O)SEMimagesofadultDrosophilacompoundeyesandheadsfrom
enhancercDNAfusionrescueexperiments.EachenhancerisdrivingexpressionoftheeyaRBisoformwithinthe
developingeyeofeya1andeya2mutants.(B,I)Enhancer1—eyaRBcDNAfusionpartiallyrescues100%of
animalsexamined.(C,J)EnhancerE—eyaRBcDNAfusionpartiallyrescues100%ofanimalsexamined.Rescue
efficiencyissignificantlyreducedineya1background.(D,K)Enhancer2—eyaRBcDNAfusiondoesnotrescue
eithereya1oreya2mutants.(E,L)Compositeenhancer—eyacDNAfusionfullyrescues100%ofanimalsexamined
towildtypeeyesize.(F,M)Enhancer1+E—eyacDNAfusionpartiallyrescues100%ofanimalsexamined.(G,N)
Enhancer3—eyacDNAfusiondoesnotrescueeya1oreya2mutants.(H,O)Enhancer4—eyacDNAfusiondoes
notrescueeya1oreya2mutants.Anterioristotherightinalladultheadimages.Atleast100adultflieswere
examinedqualitativelyforeachgenotype.Quantificationofrescue(assayedbynumberofommatidia)ofasubsetof
adultsisprovidedinTable1.Scalebar,100μm.
doi:10.1371/journal.pgen.1006462.g004
determinethebreakpointsoftheeya1deletioninrelationtothecompositeenhancerweiso-
latedandre-sequencedtheregionaroundthetranscriptionalstartsiteanddeterminedthatthe
deletionisactually1826bpinlengthwiththedeletionextending581bpupstreamoftheeya2
deletionand344bpdownstreamofthetranscriptionalstartsite.Thisdeletioncompletely
deletesthecompositeenhancer,thetranscriptionalstartsite,andalargeportionoftheeyaRB
transcript5‘UTR(Fig2A).UsingqRT-PCRweconfirmedthattheRBtranscriptiscompletely
eliminatedineya1mutantsanddrasticallyreducedineya2mutants(Fig4A).TheRAtran-
scriptisalsogreatlyreduced,butnoteliminated,inbothmutantallelessuggestingthatthe
compositeenhancerregulatesbotheyapromoters(Figs2Aand4A).
Totestwhetherfragments1,E,and2aresufficienttorescuethetwoeyamutants,each
enhancerelement,aswellasthefullcompositeenhancer,wasclonedupstreamofaminimal
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RegulationofeyaExpressionintheDrosophilaRetina
hsp70promoterandtheeyaRBcDNA.UsingthePhiC31integrasesystemtheseconstructs
wereinsertedintothesamegenomiclocationthatweusedfortheoriginallacZreporter
expressionanalysis(attP-3BVK00033—cytologicallocation65B2).Forallrescueexperiments
atleast100adultflieswereinitiallyassayedqualitativelyfortherestorationofeyedevelopment.
FortherescuequantificationinTable1thenumberofommatidiainadultrighteyesfrom2–3
individualfemaleflieswerecountedandcomparedtowildtype.Thenumberofommatidia
perrescueispresentedasanaverageofthe2–3individuals.Awildtypeeyefromafemaleflyis
definedashavingbetween750and800ommatidia[36].
Bothfragments1andEarecapableofpartiallyrestoringeyedevelopmentin100%ofeya2
andeya1mutants.Fragment1restoreseyesizetoapproximately38%ofwildtypeineya2and
25%ineya1(Fig4Band4I,Table1).EnhancerErestoreseyesizetoapproximately49%of
wildtypeineya2butlessthan1%ineya1(Fig4Cand4J,Table1).Expressionfromfragment2,
onitsown,failstorescueeithermutant(Fig4Dand4K,Table1).Consistentwithourexpres-
sionanalysis,thefullcompositeenhancerfullyrestoreseyedevelopmentto100%ofbotheya
mutants(Fig4Eand4L,Table1).Andfinally,neitherfragment3nor4arecapableofrescuing
theno-eyephenotypeofeithermutant(Fig4G,4H,4Nand4O,Table1).Themajorityoffrag-
ment3drivenexpressionisoutsideoftheendogenouseyaexpressionpatternandwould
thereforenotbepredictedtorestoreeyedevelopmenttoeyamutants(Fig3P–3R,Table1).
Theinabilityoffragment4torescueeyedevelopmentstemsfromthefactthatitisnormally
expressedonlyindifferentiatingcellsposteriortothemorphogeneticfurrow(Fig3S–3U,
Table1).Neitherthefurrownordifferentiatedphotoreceptorcellsarepresentineithereya1or
eya2mutants[11].
Thelackofanydiscernablerescuebyfragment2anditsinappropriateexpressionpattern
initiallyindicatedthatitmaynotfunctionasanenhancer.Instead,itsproximitytothetran-
scriptionalstartsiteofeyaRB,suggestedthatitmightserveasabasalcorepromoter.Totest
thisideaweplacedfragment2andthecompositeenhancerintoaplasmidthatcontainsalacZ
reporterbutlacksaminimalpromoter.Undertheseconditionsfragment2isstillcapableof
drivinglacZexpressioninthedevelopingeyebutonlyindevelopingphotoreceptors(S3Aand
S3BFig).Incontrast,lacZreporterexpressiondrivenbythecompositeenhancerisidenticalto
theconstructthatcontainedtheminimalhsp70promoterfragment(S3CandS3DFig).These
datasupporttheproposalthatfragment2functions,inpart,asabasalpromoter.Assuchwe
thentestedthemodelthatallpertinentregulatoryinformationmayresideonlyinfragments1
andE.WefirstexaminedlacZreporterexpressionwithafragmentthatcontainedsegments1
andEonlyandasexpectedthisconstructfullyrecapitulatesendogenouseyaexpression(S3E
andS3FFig).Wenextattemptedtorescuebotheya1andeya2mutantswiththisshorterfrag-
ment.Whileweobservedrescuein100%ofanimalsitonlyrestoreseyesizeto82%ofwild
typeineya2and44%ineya1mutants(Fig4E,4F,4Land4M,Table1).Thisisunlikethecom-
positeenhancer,whichcompletelyrestoreseyesizetothebotheyamutants.Thissuggeststhat,
inadditiontofunctioningasabasalcorepromoter,fragment2doesindeedcontainregulatory
informationthatisnecessaryforrobusteyaexpression.
Wewereintriguedbythedifferencesinrescueefficiencyofourconstructsineya1andeya2
mutants.SincetheendogenoustranscriptionalstartsitefortheRBtranscriptisintactinthe
eya2mutantbutisdeletedintheeya1mutantwehypothesizedthatthehigherdegreeofrescue
intheeya2mutantisduetoareactivationoftheendogenouseyagene.UsingqRT-PCRwe
measuredeyaRBtranscriptlevelswithineyamutantsthathavebeenrescuedbyexpression
fromenhancerE.Thisenhancerwaschosensinceitshowedthemostdramaticdifferencein
rescueefficiency.Aspredicted,weobservethatexpressionoftheeyaRBcDNAinitiatesaposi-
tivefeedbackloopontheendogenouslocusandreactivateseyaexpressionineya2butnoteya1
(Fig4A,4Cand4J).Intheeya2mutant,fragments1(IAM),2,3,4,andPSEarepresentand
PLOSGenetics|DOI:10.1371/journal.pgen.1006462 December8,2016 10/31