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PLOSONE:TheRoleofARF6inBiliaryAtresia

The Role of ARF6 in Biliary Atresia

MylarappaNingappa

, JuhoonSo

, JosephGlessner, ChethanAshokkumar, SarangarajanRanganathan, JunMin,

BrandonW.Higgs, QingSun, KimberlyHaberman, LoriSchmitt, SilviaVilarinho, PramodK.Mistry, GerardVockley,

AnilDhawan, GeorgeK.Gittes, HakonHakonarson, RonaldJaffe, ShankarSubramaniam, DonghunShin, RakeshSindhi
Published:September17,2015

http://dx.doi.org/10.1371/journal.pone.0138381

Abstract
Background&Aims

Alteredextrahepaticbileducts,gut,andcardiovascularanomaliesconstitutethevariablephenotypeofbiliaryatresia(BA).
Methods

Toidentifypotentialsusceptibilityloci,Caucasianchildren,normal(controls)andwithBA(cases)attwoUScenterswerecompared
at>550000SNPloci.Systemsbiologyanalysiswascarriedoutonthedata.Inordertovalidateakeygeneidentifiedinthe
analysis,biliarymorphogenesiswasevaluatedin25daypostfertilizationzebrafishembryosaftermorpholinoantisense
oligonucleotideknockdownofthecandidategeneADPribosylationfactor6(ARF6,Moarf6).
Results

Among39and24casesatcenters1and2,respectively,and1907controls,whichclusteredtogetheronprincipalcomponent
analysis,theSNPsrs3126184andrs10140366ina3flankingenhancerregionforARF6demonstratedhigherminorallele
frequencies(MAF)ineachcohort,and63combinedcases,comparedwithcontrols(0.286vs.0.131,P=5.94x107,OR2.660.286
vs.0.13,P=5.57x107,OR2.66).Significancewasenhancedin77totalcases,whichincluded14additionalBAgenotypedat
rs3126184only(p=1.58x102,OR=2.66).Pathwayanalysisofthe1000toprankedSNPsinCHPcasesrevealedenrichmentof
genesforEGFregulators(p<1x107),ERK/MAPKandCREBcanonicalpathways(p<1x1034),andfunctionalnetworksforcellular
developmentandproliferation(p<1x1045),furthersupportingtheroleofEGFRARF6signalinginBA.Inzebrafishembryos,Mo

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arf6injectionresultedinasparseintrahepaticbiliarynetwork,severalbiliaryepithelialcelldefects,andpoorbileexcretiontothe
gallbladdercomparedwithuninjectedembryos.BiliarydefectswerereproducedwiththeEGFRblockerAG1478aloneorwithMo
arf6atlowerdosesofeachagentandrescuedwitharf6mRNA.
Conclusions

TheBAassociatedSNPsidentifyachromosome14q21.3susceptibilitylocusencompassingtheARF6gene.arf6knockdownin
zebrafishimplicatesearlybiliarydysgenesisasabasisforBA,andalsosuggestsaroleforEGFRsignalinginBApathogenesis.
Citation:NingappaM,SoJ,GlessnerJ,AshokkumarC,RanganathanS,MinJ,etal.(2015)TheRoleofARF6inBiliary
Atresia.PLoSONE10(9):e0138381.doi:10.1371/journal.pone.0138381
Editor:GianfrancoAlpini,TexasA&MHealthScienceCenter,UNITEDSTATES
Received:December15,2014Accepted:January22,2015Published:September17,2015
Copyright:2015Ningappaetal.ThisisanopenaccessarticledistributedunderthetermsoftheCreativeCommons
AttributionLicense,whichpermitsunrestricteduse,distribution,andreproductioninanymedium,providedtheoriginalauthor
andsourcearecredited
DataAvailability:Allrelevantdataarewithinthepaper,itsSupportingInformationfiles,andavailableviadbGaP
(http://www.ncbi.nlm.nih.gov/projects/gap/cgibin/study.cgi?study_id=phs000960.v1.p1).
Funding:ThisstudywasfundedbytheHillmanFoundationofPittsburgh,TheAmericanLiverFoundation,TeamTransplant,
theHerridgeandGiventerBrafffamilies.Thefundershadnoroleinstudydesign,datacollectionandanalysis,decisionto
publish,orpreparationofthemanuscript.
Competinginterests:Theauthorshavedeclaredthatnocompetinginterestsexist.

Introduction
Biliaryatresia(BA),whichischaracterizedbyjaundiceandlossofextrahepaticbileducts(EHBD)atbirth,affects1:18000
Caucasianchildrenandisfatalwithoutsurgicalintervention[1].TheBAdiseasephenotypeisbroadandunderstandingthe
molecularanddevelopmentalbasisofBAcanimprovediagnosisandselectionoftreatment.Thistaskposessignificantchallenges,
becauseBApresentsshortlyafterbirthsuggestinganoriginearlyduringfetaldevelopmentintheembryologictypeofBA,or
perinatallyintheisolated,potentiallyinfectious/inflammatorytypeofBA.Eithertypeprecludestheelucidationofconclusive
evidence.Thisevidencewouldideallydocumentdiseaseprogressioninsequentialfetaltissue.Phenotypicheterogeneityconsisting
ofseveralassociatedextrahepaticanomaliesfurtherdefieseitheraunifiedmechanistichypothesisoraperfectexperimentalmodel,

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butsuggeststhatmultiplesusceptibilitylocimaybeinvolved.Extrahepaticmanifestationscanincludegutorcardiovascular
anomaliesincludinglateralitydefectssuchasasplenia/polyspeniaandheterotaxytovaryingdegrees[2,3].Diseasecomplexityis
compoundedfurtherbyhistologicalfeatureswhichcanoverlapwiththoseofothercholestaticconditionssuchasneonatal
cholestasis,ductalplatemalformation,andCarolisdisease[2,4,5].Acommonpathophysiologicfindingisfailuretoexcretebile
fromtheliveronnuclearimaging,mandatingsurgicaldrainagewithportoenterostomy.Thisprocedurefailsinnearlyhalfofall
patientsleadingtobilestasisandcirrhosis.Asaresult,BAaccountsforoverathirdofallpediatriclivertransplants,worldwide[6].
Poorlydevelopedintrahepaticbileductscanalsoexplainfailureofportoenterostomy.Suchalesioncannotbeidentifiedwith
certaintyamidsthistologicalsequelaeofbiliaryobstructioninmostcases.Thesefeaturesincludereactiveintrahepaticbileduct
proliferation,cholestasis,fibrosisandcirrhosis,whichcandistortliverarchitecturebeyondrecognition[7].
TheroleofenvironmentalfactorsisunderscoredbytheassociationofBAwithrotavirus,reovirus,andcytomegalovirusinfections,
andthefactthatthesevirusesinducecomparablelesionsexperimentally.However,thesemodelsdonotprovideinsightintothe
developmentalbasisofBAsuggestedbythepresenceofminorextrahepaticanomaliesevenintheisolatedvarietyofBA
[8,9,10,11].
Towardgeneratingnovelunbiasedhypotheseswithevidencefromdiseasedhumansubjects,twosetsofgenomewideassociation
studies(GWAS)haveprovidednewdirections.IntwoNorthAmericanchildrenwithBA,LeyvaVegaetal.[12]observedacommon
1.76heterozygousdeletioninchromosome2q37.3.Thedeletionwasinheritedfromthefatherwhodidnothaveanyliverdiseasein
oneofthetwoaffectedchildren,confirmingthatotherlociand/orenvironmentalfactorswerealsonecessaryinBApathogenesis.
ThesefindingswereconfirmedbyCuietal.in6of61childrenwithBAfromthesameinvestigatorgroup,inwhomthedeletionwas
confinedtotheGPC1genewithinthesamelocus[13].Anoveldevelopmentalmodelwasusedtogeneratesupportiveevidence,
whichconsistedofimpairedbiliarynetworkdevelopmentaftergpc1knockdownwithmorpholinoantisenseoligonuleotide(MO)in
zebrafishembryos.In339ChinesechildrenwithBA,Chengetal.[14]confirmedtheassociationbetweenBAandanintergenic
chromosome10q24SNPlocus,rs17095355,whichhadbeenreportedpreviouslybyGarciaBarceloetal.[15]fromthesame
group.ThisSNPlocusindicatedtheinvolvementoftheneighboringADD3geneinBA,becauseofdifferentialimmunostainingof
theADD3proteinwhendiseasedandnormallivertissuewascompared.Recently,Tsaietal.[16]alsofoundanassociation
betweenBAandoneof333SNPsinthechromosome10q24locusin171NorthAmericanBApatients.However,thebest
associatedSNP,rs7099604,waslocatedinthefirstintronofADD3inthislocus.Interestingly,knockdownofadd3inzebrafishalso
impairedbiliarynetworkformationprovidingsupportiveevidenceforyetanothercandidategeneidentifiedwithGWAS.
Together,theseobservationsreinforcetheideathatmultiplesusceptibilitylocialongwithenvironmentalfactorsmaybeinvolvedin
BA,andsuggestthatreplicatingaGWASfindingmaybechallenging.AddingtothisdifficultyisthefactthattheSNPsbest
associatedwithBAwerelocatedindifferentregionsinthechromosome10q24susceptibilitylocusinChineseandNorthAmerican
patients.Finally,unrecognizedminorextrahepaticanomalies,whichareassociatedevenwiththeisolatednonsyndromicvariety
ofBA,canintroducetraitheterogeneityinanytestcohort.Unrecognizeddiseasetraitheterogeneityisacommoncauseoffailureto
replicateanassociation[17].TheselimitationsindeterminingBApathogenesiswithGWASstudiesarewellservedbythe
availabilityofawellcharacterizedzebrafishmodelofbileductdevelopment.Thismodelcanprovidesupportiveevidencefor
additionalnovelsusceptibilitygenesalsoidentifiedbyGWAS.

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ToidentifyadditionalsusceptibilitylociweperformedGWASinCaucasianBAcasesandcontrolswhopresentedtotheChildrens
HospitalofPittsburgh(CHP)andtheCenterforAppliedGenomics(CAG)attheChildrensHospitalofPhiladelphia.Thisanalysis
identifiedARF6asaleadingcandidategeneinvolvedinbileductdevelopment,basedonSNPassociations.ToidentifyotherARF6
relatedpathwaysmembers,thetoprankedSNPsidentifiedbyGWASwerefurtherevaluatedwithasystemsbiologyanalysis.
SimilartopreviousGWASstudies,weusedMOmediatedtranslationblockadetoobtainsupportiveevidencefortheroleofarf6in
theearlybileductdevelopmentinzebrafish.

MaterialsandMethods
ChildrenwithBAwereenrolledatCHPafterobtainingwrittenconsentfromparentsfortheUniversityofPittsburghIRBapproved
protocolnumber0405628.AtCAG,allsubjectswereenrolledafterobtainingwrittenconsentfromparentsperChildrensHospitalof
PhiladelphiaIRBapprovalnumber06004886.GenotypingofDNAobtainedfrombloodwasperformedwiththeInfinium
HumanHap550KBeadChip(Illumina,SanDiego,CA).SNPgenotypefrequencieswerecomparedbetweencasesandcontrolswith
achisquareteststatisticappliedinPlink[18]forSNPswithatleast90%callrateand1%minorallelefrequency(MAF).Fourteen
additionalpatientsweregenotypedwithTaqManSNPGenotypingAssay(LifeTechnologiesIDC_11918520_10)forthe
candidateSNPrs3126184.Taqmangenotypingassaywasnotavailableforrs10140366.Allzebrafishexperimentswereperformed
withapprovalfromtheInstitutionalAnimalCareandUseCommittee(IACUC)oftheUniversityofPittsburgh,approvalnumber
13102738.
ARF6immunostainingofliverexplantsfromchildrenwithBA

Formalinfixed,paraffinembedded(FFPE)sectionswerestainedontheVentanaBenchmarkUltraIHCstainingplatformusing
ARF6polyclonalantibody(Abcam,Cambridge,MA).AntigenretrievalwasperformedusingVentanasproprietaryultraCC1cell
conditioningreagentandvisualizedwiththeuvDABdetectionsystem.Sectionswerecounterstainedwithhematoxylin.
SNPandPathwayAnalysis

AmongtoprankedSNPloci,weexaminedenrichmentforothergenesrelatedtoARF6anditsassociatedEGFRpathwaygenes
[19,20,21].NetworkandenrichmentanalysesusingCytoscape,apopularnetworkvisualizationandanalysistool,andIngenuity
PathwayAnalysis(IPA)wereperformed[22,23].First,alargehumannetworkwascreatedinCytoscapebyintegratingprotein
proteininteractiondatafromBIOGRIDandSTRINGdatabases,transcriptionfactorinteractionfromTRANSFAC,andpathway
informationfromKEGGpathways[24,25,26,27].FromtheSTRINGdatabasethatusesascoringschemebetween0and1based
onpredictedandexperimentallyvalidatedproteinproteininteractions,a0.9cutoffwasusedtoextracthighlyprobableprotein
proteininteraction.Then,asmallernetworkwascreatedusingonlythefirstneighborsofgenesthatcouldbemappedfromthetop
1000significantSNPsfromtheGWASCHPcohort.ForSNPtogenemapping,+/20kbwindowwasappliedonthemappingfile
providedbythemanufacturerfortheInfiniumHumanHap550KBeadChip(Illumina,SanDiego,CA).Atotalof2506genesinthe
newlocalnetworkwassubmittedtoIngenuityPathwayAnalysis(IPA)forupstreamregulatoranalysisandenrichmentofcanonical
pathwaysandbiologicalfunctionalcategories.

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Zebrafishstrains

Embryosandadultfishwereraisedandmaintainedunderstandardlaboratoryconditions.Weusedthefollowingtransgeniclines:
Tg(EPV.Tp1Mmu.Hbb:EGFP)um14[28],Tg(EPV.Tp1Mmu.Hbb:hist2h2lmCherry)s93[29],Tg(fabp10a:dsRedela3l:EGFP)gz[30],
[referredtohereasTg(Tp1:GFP),Tg(Tp1:H2BmCherry),andTg(fabp10a:dsRed),respectively],Tg(ins:dsRed)m1018[31],and
Tg(sox17:GFP)s870[32].
Morpholino,mRNA,andDNAinjections

Embryoswereinjectedattheonecellstagewith0.5or2ngofarf6ATGMO(5GATCTTGGAAAGCATCTTCCCCATG3)[33],3
ngofarf6aUTRMO(5GTGCAAGACTTAGTCGCTTTTCCGA3)or3ngofarf6bUTRMO(5GTTCATAAATGGTCAATTCCCTCCA
3).120pgofarf6amRNAor25pgofplasmidscontainingCMV:GFPconstructswasinjectedintothecellattheonecellstage.
Chemicaltreatment

A3.17mMstockoftheEGFRinhibitorAG1478(CaymanChemical)waspreparedin100%dimethylsulfoxide(DMSO)anddiluted
to1or4Mwitheggwater.Asacontrol,0.1%DMSOsolutionineggwaterwasused.
Wholemountinsituhybridizationandimmunostaining

Wholemountinsituhybridizationwasperformedaspreviouslydescribed[34].Wholemountimmunostainingwasperformedas
previouslydescribed[35],usingthefollowingantibodies:antiGFP(1:1000AvesLabs,Inc.,Tigard,OR),antiProx1(1:1000
Millipore,Billerica,MA),mousemonoclonal2F11(1:100Abcam,Cambridge,MA),antiAbcb11(1:200KamiyaBiomedical,Seattle,
WA),antidsRed(1:200Clontech,MountainView,CA),antiArf6(1:400Abcam,Cambridge,MA),anticatenin(1:200Sigma
Aldrich,St.Louis,MO)andAlexaFluor488,568,and647conjugatedsecondaryantibodies(1:500LifeTechnologies,Grand
Island,NY).Hoechst33342(2.5ng/mlSigmaAldrich,St.Louis,MO)wasusedforDNAstaining.
Timelapseimaging

Timelapseimagingwasperformedaspreviouslydescribed[36].Briefly,Tg(Tp1:GFP)Tg(Tp1:H2BmCherry)larvaeat74hpfwere
partiallymountedon0.5%lowmeltingagaroseina35mmpetridishcontainingeggwatersupplementedwith0.2mM1phenyl2
thiourea(SigmaAldrich)and2g/mltricaine(SigmaAldrich).ConfocalZstackimageswerecapturedevery15minutesfor10
hoursusinga20xwaterdippinglens.
PED6andproliferationassay

PED6assaywasperformedbytreatinglarvaewith0.3g/mlPED6(LifeTechnologies)for3hoursasdescribedpreviously[37].
Proliferationassaywasperformedbytreatinglarvaewitheggwatercontaining10mMEdU(LifeTechnologies)and1%DMSO.
AfteronehourEdUtreatment,thelarvaewereharvestedandprocessedforEdUlabelingusingtheClickitEdUImagingkit(Life
Technology).

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GenerationofGFPexpressionconstructsformorpholinovalidation

TheGFPcodingregionwasamplifiedbyPCRwithaforwardprimerthatcontainsamorpholinotargetsequenceinfrontofGFP
startcodonandareverseprimerthatcontainsitsstopcodon.ThePCRproductswereclonedintothepGEMTvector(Promega,
Madison,WI)andtheirsequenceswereanalyzed.Cloneswithoutanymutationswerechosenandusedforsubcloningintothe
pCS2+expressionvectorthatcontainstheCMVpromoterandtheSV40polyAsite.PlasmidscontainingthefinalCMV:GFP
constructswereusedforinjection.
qRTPCRmethods

TotalRNAwasextractedfrompoolof100liversofuninjectedcontrolandarf6ATGMOinjectedlarvaeat4.5dpfbyusingRNeasy
MiniKit(Qiagen,Valencia,CA).Wholetranscriptomeamplificationsystem(WTA2)(SigmaAldrich,St.Louis,MO)wasusedto
synthesizegquantitiesofamplifiedcDNAstartingwith~50ngoftotalRNA.QuantitativePCRwasmeasuredinSYBR
Green/ROXqPCRmastermix(Fermentas,GlenBurnie,MD)withprimers(IDT,Coralville,IA)showninS1Table.qPCRwas
performedusing7300RealTimePCR(AppliedBiosystems,FosterCity,CA).Therelativeexpressionlevelofgeneswasshownin
foldchangeasnormalizedtothehousekeepinggene,eef1a1l1byusingtheCtmethod.
Imageacquisition,processing,andstatisticalanalysis

ZeissLSM700confocalandLeicaM205FAepifluorescencemicroscopeswereusedtoobtainzebrafishimagedata.Confocal
stackswereanalyzedusingtheZen2009software.ThelengthofBECfilopodiaandinterconnectingbilepreductuleswas
measuredusingtheImageJsoftwareandwasshownasmeansSEM(standarderrorofthemean).ThenumberofBECswas
alsocountedusingtheImageJsoftwareaspreviouslydescribed[38].UnpairedtwotailedStudentsttestwasusedforstatistical
analysisp<0.05wasconsideredstatisticallysignificant.

Results
GWASidentifiesARF6asaBAsusceptibilitylocus

AllDNAsampleshadexcellentdataqualitywithcallrates>98%.Of50CHPand30CAGBAcasesand2818controls,39CHPand
24CAGcases,and1907controlsclusteredtogetheronprincipalcomponentanalysisof>550000SNPs,anddemonstratedavery
lowgenomicinflationfactorof1.00627(Fig1A)[39].IncomparisonofCHPcaseswithcontrols,the1,000mostsignificantSNPs
wererankedfurtherbytheirproximitytoothersignificantSNPsinthetop1000in10kbwindows,toboostconfidenceinthe
association(S2Table).rs3126184andrs10140366,whichranked7thand8thoverallinsignificanceemergedasthetopranked
pairdemonstratingsignificantlyhigherMAFsinCHPcases,inCAGcases,and63combinedcasesfrombothcenters,compared
withcontrols(Table1).FourteenadditionalBAcasesgenotypedatrs3126184demonstratedMAF0.286,andenhancedthe
significanceoftheassociationfurther,whenaddedto63casesabove(p=4.19x108,OR=2.66).rs3126184andrs10140366map
to14q22.1atpositions4944228549445256inlinkagedisequilibriumwitheachotherandthe8.763kbupstreamgene,ARF6(Fig

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1B).ARF6regulatesembryonicliverdevelopmentinmice[40].ARF6expressionisnegativelycorrelatedwiththeminorallelesof
rs1040366(r=0.1346,p=0.023)andrs3126184(r=0.1128,p=0.061)inthecombinedHapMappopulationsfromSNPexpv1.2
[41].Together,thesefindingssuggestthatARF6proteinexpressionmaybeimpairedinBAlivertissue.

Fig1.EigenstratPrincipalComponentsAnalysis.

(A)Boxedregionshowswellstratifiedcasesandcontrolscarriedforwardinanalysis.(B)TheBAsusceptibilitylocusdefined
byrs3126184andrs10140366liesinanenhancerregioninthe3flankingregionoftheARF6gene(UCSCgenomebrowser
evaluationshowenrichedhistonemarksH3K4me1overlayedwithH3K27Acisanindicativeofenhancerregion).(C)iivshow
ARF6immunostaininginliverexplantsfromnormalchildrenwithintactbileducts(BD)(i),childrenwithBAwithbileduct
paucityinportaltracts(PT)(ii)orcirrhosis(iii),andachildwithhepatocellularcarcinoma(iv).T=tumorcells,L=lobule.
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Table1.Associationtestingresultsforrs3126184andrs10140366for77BAcasesand1907controls.

http://dx.doi.org/10.1371/journal.pone.0138381.t001
GWASpathwayanalysesfurthersupporttheimportanceofARF6andEGFRsignaling

Amongthe14SNPsthatfallintothecodingregionofgenes,twomappedtothegenes,CREB3L4andCRTC2.Thesegenesare
relatedtocAMPresponseelementbindingprotein(CREB)pathway,whichcanbeactivatedbyEGFRsignalingthroughthe
ERK/MAPKpathway[42,43,44,45].WealsoperformednetworkandenrichmentanalysesusingCytoscapeandIngenuityIPASNP
togenemappingin20kb+/windowsrevealed299uniquegenesassociatedwith419ofthe1000torankedSNPs(S2Table).
Ingenuitysupstreamregulatoranalysisonthe2506genes(S3Table)fromthefirstneighbornetworkcreatedinCytoscape
th
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revealedthatoutof632potentialupstreamregulators,the35thrankedregulator,sortedbyenrichmentpvalue,wasEGF(p=1.26
x107).OthersignificantEGFrelatedupstreamregulatorswereTNF(p=3.14x1025)andERK1/2(p=3x1014).Thesignificant
canonicalpathwayswereERK/MAPK(p=5.74x1037)andcAMPmediatedsignaling(p=1.5x1035)pathwayswhileenriched
functionalcategorieswerecellularproliferation(p=1.85x10103)andcellulardevelopment(p=1.63x1046).
PoorARF6staininginhumanBAliverexplantswithintrahepaticbileductpaucity

Explantsfrom29of39CHPcaseswereimmunostainedforARF6withpositiveandnegativecontrols.Hepatocytesandbileducts
showedfinegranularimmunostaininginnormallivertissue(Fig1Ci).PoorARF6stainingwasseenintwoexplantswithbileduct
paucitywithminimalfibrosis(Fig1Cii).Inoneoftheseexplants,paucitywasconfinedtotheleftlobe.Therightlobeofthisandthe
remaining27explantswithdiffuseductalhyperplasia,cholestasis,andfibrosis,demonstratedARF6stainingwhichwassimilarto
normallivertissue(Fig1Ciii).Intensestainingwasobservedinhepatocellularcarcinoma(positivecontrol,Fig1Civ).Therefore,
ARF6proteinexpressionwasimpairedinthepresenceofbileductpaucity.
ThezebrafishorthologuesofhumanARF6,arf6a,andarf6bareexpressedinthedevelopingliverandendodermderivedorgans

Twoarf6genes,arf6aandarf6b,withidenticalaminoacidsequences,arelocatedonchromosomes20and17,respectively,in
zebrafishtheiraminoacidsequencesare99percentidenticaltohumanARF6.Usinganinsituprobeforthearf6acodingregion
whosenucleotidesequenceis81%identicaltothatofarf6b(S1Fig,blackbox),wefoundthatarf6a/bexpressionappeared
ubiquitousat30hourspostfertilization(hpf),whentheliverformingregionconsistsofhepatoblasts,butisrestrictedtotheliverat
42hpf,whenhepatoblastsdifferentiatetobiliaryepithelialcells(BECs)[46](Fig2A,arrows).Asembryosfurtherdevelop,arf6a/b
expressionwasclearlyobservedintheliverandotherendodermderivedorgansincludingthepancreasandtheintestinalbulb(Fig
2A).Weusedarf6aandarf6binsituprobestargetingnonidentical3UTRregionsofthesegenestodeterminetheirrelativeliver
expression(S1Fig,greenandredboxes,respectively).Bothgenesweresimilarlyexpressedintheliverandotherendoderm
derivedorgansat72hpf(Fig2B).At96hpf,arf6bexpressionpersistedwhereasarf6aexpressiondeclinedintheliver(Fig2B).

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Fig2.arf6knockdownresultsindevelopmentalbiliarydefectsinzebrafish.

(A,B)Wholemountinsituhybridizationshowingarf6aandarf6bexpressionindevelopingembryos/larvae.Arrowspointto
theliverarrowheadsthepancreasbracketsmarktheintestinalbulb.(C)TheTg(Tp1:GFP)andTg(fabp10a:dsRed)lines
revealtheintrahepaticbiliarystructureandliversize,respectively.Epifluorescenceimagesshowingtheexpressionofthese
transgenesrevealedadefectintheintrahepaticbiliarystructureinarf6ATGMOinjectedlarvae.Basedontheseverityofthe
biliarydefect,larvaeweredividedintothreegroups:normal,intermediate,andsevere.Graphshowsthepercentageoflarvae
ineachgroup.Arrowspointtotheliverdottedlinesoutlinetheliver(AC).(D)EpifluorescenceimagesshowingPED6
accumulationinthegallbladder(arrows).BasedonPED6levelsinthegallbladder,larvaeweredividedintothreegroups:
absent,small/faint,andnormal.Graphshowsthepercentageoflarvaeineachgroup.n,thenumberoflarvaeexamined
scalebars,100m.
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arf6knockdownresultsinbiliarydefects

Toinvestigatetheroleofbothgenesinliverdevelopment,wedesignedthreetranslationblockingantisensemorpholino
oligonucleotides(MO).arf6aUTRandarf6bUTRMOstargetednonidentical5UTRsequencesofcorrespondinggenesarf6ATG
MOtargetedthearf6aATGstartcodonregionwhichdemonstratedhighsequencehomologywiththestartcodonforarf6b(S1Fig,
underlines).Theirefficacywasevaluatedbyexamininggreenfluorescentprotein(GFP)expressionfromtheCMV:GFPconstructs
containingeachMOtargetsequenceinfrontoftheGFPstartcodon(S2Fig).Thearf6ATGMOblockedGFPexpressionfromthe
CMV:GFPconstructcontainingthecorrespondingarf6aorarf6bsequences(S2BFig)becauseofhighsequencehomologyinthe
startcodonregionsofthesetwogenes,andwasthereforeusedtoknockdownbothgenes.Thearf6ATGMOwasfurthervalidated
bywholemountimmunostainingwithantiArf6antibody.Arf6expressionwasgreatlyreducedintheMOinjectedembryosand
um14

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increasedinarf6amRNAinjectedembryos(S3Fig).TheTg(Tp1:GFP)um14line,whichexpressesGFPinBEC[46,38],andthe
Tg(fabp10a:dsRed)gz15line,whichexpressesdsRedinhepatocytes[47],wereusedtoevaluateintrahepaticbiliarystructuresand
liversize,respectively.Liversizewassmallerandimportantlyintrahepaticbiliaryductswererelativelysparseinarf6ATGMO
injectedlarvae,comparedwithcontrols(Fig2C).Thesephenotypeswererescuedwitharf6amRNAinasignificantnumberof
larvae(S4AFig).Wenextexaminedwhetherhepatocytesecretionintobileductswasdefectiveinarf6ATGMOinjectedlarvae
usingthefluorescentlylabeledfattyacidreporterPED6,whichaccumulatesinthegallbladderafterbiliarysecretion[37].PED6
accumulationinthegallbladderwasgreatlyreducedintheMOinjectedlarvaecomparedtocontrols(Fig2D).Thisdefectwas
rescuedwitharf6amRNAinasignificantnumberoflarvae(S4BFig).Singleinjectionofarf6aorarf6bUTRMOproducedbiliary
defectsinasmallernumberoflarvaethancoinjectionsofthesetwoUTRMOs,orinjectionwitharf6ATGMO(S5Fig),indicating
thatbotharf6aandarf6bregulatebiliarydevelopment.
Detailedcharacterizationofbiliarydevelopmentaldefectswasperformedatmultiplestagesfrom40hpfto5dayspostfertilization
(dpf)usingwholemountimmunostainingandconfocalmicroscopy.Comparedwithcontrols,arf6ATGMOinjectedembryos/larvae
demonstrated1)smallerliversize,asassessedbytheexpressionofthehepatoblastmarkerProx1[48]orthehepatocytemarker
fabp10a:dsRed(Fig3A),2)reducednumberofBECs,asassessedbyTp1:GFPexpression,withreduceddispersionandenhanced
clustering(Fig3A),3)reducedfilopodialprotrusionofBECsat72and96,butnot62,hpfinfixedlarvae(Fig3B)andfrom74to84
hpfinlivelarvae(S6Fig),4)reducedlengthofinterconnectingbilepreductulesat5dpf,asrepresentedbythedistancebetween
twonucleiofBECs(Fig3C).Inparticular,althoughliversizeintheMOinjectedembryosat62hpfwassimilartothatincontrolsat
48hpf,theirBECdistributionwasverydifferentfromeachother,suggestingthatthebiliarydefectsobservedintheMOinjected
embryoswerenotsimplyduetodevelopmentaldelay.Wefurtherconfirmedtheclusteringphenotype,usingtheTg(Tp1:H2B
mCherry)s939linethatexpressesmCherryinBECnuclei[38].ThequantificationofthepercentageofsingleBECsversuscellsin
clusteroftwo,three,orfourandmorecellsrevealedasignificantdecreaseinthepercentageofBECspresentassinglecellsand
doubletsintheMOinjectedlarvaecomparedtocontrols,concomitantwithasignificantincreaseinthepercentageofcellsin
clustersoffourandmorecells(Fig3D).ThisclusteringphenotypewasnotduetoincreasedBECproliferation.Inaproliferation
assay,therateofBECproliferationwasnotsignificantlydifferentbetweencontrolsandtheMOinjectedlarvaeat75hpf(S7CFig),
althoughthetotalnumberofBECsineachliverwassmallerintheMOinjectedlarvaethanincontrols(S7BFig).Sincebilefirst
secretesintobilecanaliculithatconnectbilepreductulesinzebrafishandbileductulesinmammals,wealsoexaminedthe
expressionpatternsofAbcb11,abilesaltexportpumppresentinthebilecanaliculiofhepatocytes[49].Thecanaliculiweremuch
shorterintheMOinjectedlarvaethanincontrols(Fig3E).

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Fig3.arf6regulatesintrahepaticbiliarymorphogenesis.

(AC)Confocalimagesshowingthedevelopmentoftheintrahepaticbiliarynetwork.Tg(Tp1:GFP)embryos/larvaewere
processedforimmunostainingwithantiGFP(green).For40and48hpf,antiProx1staining(red)wasalsoused.Brackets
delineatethelengthofBECfilopodiaat62,72,and96hpf,andthelengthofinterconnectingbilepreductulesat5dpf(A)
graphsshowtheirquantitation(B,C).(D)ConfocalimagesshowingthelocationofBECnucleiintheentireliver.The
Tg(Tp1:H2BmCherry)linerevealsBECnuclei.DashedlinesoutlineclusterswithfourormoreBECs.Graphshowsthe
percentageofBECspresentassinglecells,doublets,triplets,orinclustersoffourormorecells.(E)Confocalimagesofthe
liverimmunostainedforGFP(green)andAbcb11(red).TheTg(Tp1:GFP)lineandantiAbcb11revealtheintrahepaticbiliary
structureandbilecanaliculi,respectively.Alldottedlinesoutlinetheliver.Asterisks,statisticalsignificance(*p<0.0001)error
bars,SEMscalebars,25m.
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arf6knockdownresultsindefectsinotherendodermderivedorgans

Sincearf6aandarf6barealsoexpressedinotherendodermderivedorgans(Fig2B),weinvestigatedwhetherotherendoderm
derivedorgansaredefectiveinarf6ATGMOinjectedembryosusingtheTg(sox17:GFP)s870linethatexpressesGFPinall
endodermalcells[32].ThepancreasandtheintestinalbulbweresmallerintheMOinjectedembryosthanincontrols(S8AFig,

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arrowheadsandbrackets,respectively).Inaddition,thebuddingoftheswimbladder,thezebrafishequivalentofthemammalian
lung,wasnotevidentintheMOinjectedembryosat48hpf,whereasitwasevidentincontrols(S8AFig,openarrowhead).In
contrasttothesedefects,thegallbladder,EHBD,andtheextrapancreaticductappearednormalintheMOinjectedlarvaeat75hpf
(S8BFig),asassessedbythe2F11antibody,whichtargetsthehepatopancreaticductalsystem[35].
BlockingEGFRsignalingresultsinbiliarydefects

BecauseEGFRsignalingactivatesARF6andinducesbreastcancerinvasion,wetestedwhetherimpairedEGFRsignalingcould
reproducetheintrahepaticbiliarydefectsobservedinarf6ATGMOinjectedlarvae[50].Theexpressionofegfra,azebrafish
orthologueofhumanEGFR,wasclearlyobservedintheliverat72hpf(Fig4A).ComparedwithDMSOtreatedcontrols,larvae
treatedwith4MAG1478,apotentEGFRinhibitor[51],from48hpfonwardsdemonstrated1)reducedBECfilopodiallength(Fig
4B),bilecanaliculilength(Fig4C),andPED6accumulationinthegallbladder(Fig4E),2)enhancedclusteringofBECs(Fig4D)
withoutdifferencesinproliferationat75hpf(S7CFig).Therefore,EGFRArf6signalingpathwaymayregulateintrahepaticbiliary
morphogenesis.

Fig4.EGFRsignalingregulatesbiliarymorphogenesis.

(A)Wholemountinsituhybridizationimageshowingegfraexpressionintheliverat72hpf.(B)Confocalimagesoftheliver
showingtheintrahepaticbiliarystructure,asrevealedbyTp1:GFPexpression.Tg(Tp1:GFP)embryosweretreatedwith
DMSOor4MAG1478from48to96hpf,andprocessedforwholemountimmunostainingwithantiGFPantibody.The
lengthofBECfilopodiawasquantifiedasshowninagraph.BracketsdelineatethelengthofBECfilopodia.(C)Confocal
imagesofthelivershowingtheexpressionofTp1:GFP(green)andAbcb11(red)forbiliarystructureandbilecanaliculi,
respectively.(D)ConfocalimagesofthelivershowingthelocationofBECnucleiintheentireliver,asassessedbyTp1:H2B

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mCherryexpression.DashedlinesoutlineclusterswithfourormoreBECs.GraphshowingthepercentageofBECspresent
assinglecells,doublets,triplets,orinclustersoffourormorecells.(E)EpifluorescenceimagesshowingPED6accumulation
inthegallbladderinDMSOorAG1478treatedlarvaeat5dpf.Graphshowingthepercentageoflarvaeexhibitingdifferent
levelsofPED6accumulationinthegallbladder.Arrowspointtothegallbladder.Alldottedlinesoutlinetheliver.nindicates
thenumberoflarvaeexaminedasterisksindicatestatisticalsignificance(*p<0.0001).Errorbars,SEMscalebars,25m.
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WenextinvestigatedwhetherEGFRsignalingandArf6actedinthesamepathwaytoregulateintrahepaticbiliarymorphogenesis.
Atfourfoldlowerdoses(0.5ngand1M,respectively),arf6ATGMOorAG1478aloneproducedminimalornodefectsin
intrahepaticbiliarystructureat75hpforinPED6accumulationinthegallbladderat5dpf.Whenthesetworeagentswere
combined,biliarystructurewasgreatlyaffected(Fig5Aand5B)andPED6accumulationgreatlyreduced(Fig5C),similarto
embryosinjectedwith2ngofarf6ATGMO(Fig2Cand2D).ThesedatasuggestthatEGFRandArf6likelyregulateintrahepatic
biliarymorphogenesisviathesamepathway.

Fig5.EGFRsignalingandArf6actinthesamepathwayintheregulationofintrahepaticbiliarymorphogenesis.

Insteadof4MAG1478and2ngofarf6ATGMO,1MAG1478and0.5ngofarf6ATGMOwereused.(A)Epifluorescence
imagesshowingtheexpressionofTp1:GFPandfabp10a:dsRedrevealedaseveredefectintheintrahepaticbiliarystructure
onlywhentheMOinjectionwascombinedwiththeAG1478treatment.Basedontheseverityofthebiliarydefect,larvaewere
dividedintothreegroups:normal,intermediate,andsevere.Arrowspointtotheliveranddottedlinesoutlinetheliver.Scale
bars,100m.(B)GraphshowingthepercentageoflarvaeineachgroupshowninA.(C)Graphshowingthepercentageof
larvaeexhibitingdifferentlevelsofPED6accumulationinthegallbladderat5dpf.nindicatesthenumberoflarvaeexamined.
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EGFRGEP100ARF6pathwaygenesareupregulatedinarf6MOinjectedlarvae

Wenextexamined,byquantitativePCR,theexpressionlevelsofEGFRGEP100ARF6pathwaygenes.ARF6isnecessaryfor
membranepseudopodformationviaASAP1[52]andRAC1andalsoactivatesERK/MAPKsignalingviaRAC1[53,54],Wealso
examinedtheexpressionofhedgehogsignalinggeneswhichwereupregulatedinzebrafishembryosafterknockdownofgpc1ina
previousstudy[13].
Dissectedlivertissuefrom4.5dpfzebrafishlarvae,injectedwitharf6ATGMOinthreebatchesof~100larvaeeach,showedthat
egfra,gep100,arf6,asap1andrac1wereupregulatedinallreplicatesinarf6morphantscomparedwithuninjectedembryos(Fig6).
Amongthehedgehogsignalinggenes,onlyptch1,butnotgli1orgli2ademonstratedconsistentupregulationinallthreereplicates
(S4Table).Thetranscriptionfactors,gli1,andgli2amediatetheeffectsofthehedgehogreceptorptch1ondownstreamgenesin
thecanonicalhehdgehogsignalingpathway.Theseobservationssuggestedthatarf6knockdownaffectsEGFR,butnotcanonical
hedgehogsignalingintheliver.

Fig6.QuantitativeRTPCRanalysisofEGFRpathwaygenesinlivertissuefromzebrafish.

Therelativeexpressionlevelofgeneswasshowninfoldchangeinarf6morphantsoveruninjectedcontrols.Errorbars
shown,SEM(averageofthreeindependentexperiments).
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Discussion
Ourstudyidentifiesthechromosome14q21.3locus,definedbythehighlyassociatedSNPsrs3126184andrs10140366,asa
susceptibilitylocusforBA.This3flankingregionincludestheupstreamARF6gene,becauseminorallelesatbothSNPlociare
associatedwithdecreasedARF6expressioninthecombinedHapMappopulations(SNPexpv1.2),andrs10140366liesinan
enhancerregionenrichedforthehistonemarksH3K4me1andH3K27Ac(Fig1B).Further,Arf6/knockoutmiceshowimpaired
liverdevelopment[40].AlthoughdistinctfromGPC1andADD3,twogenesassociatedwithBAinrecentGWAS,theARF6genehas
similarfunctionalanddevelopmentalconsequences[13,16].Knockdownofbothgpc1andarf6inzebrafishimpairsbiliarynetwork
formation.ThisisexpectedbecauseGPC1andARF6mediatefibroblastandepidermalgrowthfactorsignaling,respectively,which

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arenecessaryforthegrowthanddevelopmentoforgansbyepithelialbranchingmorphogenesis[55,56].ADD3mayalsohavea
developmentalrolebecauseitisexpressedtoagreaterextentinfetalliverthaninadultliver[57].ARF6andADD3bothregulate
actincytoskeletalremodeling,andaffectcellmobilityandcellcellcontact,whichfacilitateorganogenesis[52,58].Amongthetop
ranked1000SNPsinourstudy,rs17127145closesttotheADD3generanked676thnonewasinproximitytotheglypican1gene.
Therefore,theassociationbetweenbileductpaucityandpoorARF6immunostaininginexplantswithBAledustofurtherask
whetherarf6knockdowninzebrafishcouldaffectearlybiliarymorphogenesisandexplaintheclinicalmanifestationsofBA.
PoorintrahepaticbiliarynetworkformationandpoorbileexcretionmanifestedasabsenceofPED6accumulationinthegallbladder
aredominanteffectsofarf6knockdown.Clinicalcorrelatesincludefailuretoexcreteradionuclideintotheextrahepaticbileducton
hepatobiliaryscanning,failureofsurgicaldrainage,andbileductpaucityinsomeBApatients[59,60].Inarf6ATGMOinjected
zebrafishembryos,poorbileexcretionalsoresultsfromreducedbiliarycanalicularlengthandenhancedclusteringofBEC,
presumablyduetoreducedBECmobilityduringmorphogenesis.AlteredepithelialcellpolarityisaknowneffectofARF6deficiency
inMDKcaninekidneycellline[61].Clinicalcorrelatesincludecanaliculardilatationwithcysticbilelakescharacteristicoftheductal
platemalformationinuptoafourthofBApatients[62].BecauseMOisdilutedoutbycelldivision,arf6MOeffectsareshortlived.
arf6mutantlinesareneededtoinvestigatelongtermconsequences.Otherpathwaysmayalsofunctionatsubsequent
developmentalstagesandexplainwhyEHBDlossandatreticgallbladderscharacteristicofhumanBAwereabsentinarf6MO
injectedzebrafish.
ARF6isacriticalmemberoftheEGFRpathway,whichfacilitatesorgandevelopment,growthandregenerationbyregulating
epithelialbranchingmorphogenesis[56].ARF6isactivatedbythebindingofEGFRtoitsactivator,GEP100andhasseveral
downstreameffects.ActivationofthedownstreameffectorASAP1isnecessaryforanormalactincytoskeletonandcellmembrane
function[52].AnupregulatedEGFRGEP100ARF6pathwayfacilitatesinvadopodiaformationininvasivebreastcancer.ARF6is
alsorequiredforRAC1mediatedepithelialcellpolarity[61]andmembranepseudopodformation[53].RAC1activationalso
regulatesERKsignaling[54].SequentialactivationofERK/MAPKandCREBsignalingpathwaysisrequiredfornormalcellular
developmentandproliferation,andisanotherdownstreameffectofEGFRGEP100ARF6signaling[43,44,45,52].Systemsbiology
analysisofGWASresultssupportstheroleofthismolecularpathwayinBAbyshowingenrichmentofEGFregulatorygenes,the
canonicalERK/MAPKandCREBsignalingpathways,andthefunctionalcategoriesofcellulardevelopmentandproliferation(Fig
7).

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Fig7.AproposedmechanismforpoorbileductdevelopmentinBA.

LigationofactivatedEGFRtoGEP100initiatessequentialactivationofARF6,ERK/MAPKandCREBsignalingproteins
resultingincellulardevelopmentandproliferation.NegativeregulationofARF6originatingfromtheregulatoryregionsdefined
byrs3126184andrs10140366inhumanBA,andarf6knockdownorEGFRinhibitionwithAG1478inzebrafishembryoslead
topoorbileductdevelopment.
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Therefore,wehypothesizedfurtherandfoundthatEGFRsignalingblockadewiththeinhibitorAG1478reproducedallbiliarydefects
associatedwitharf6ATGMOinducedknockdowninzebrafish.Further,reduceddosesofAG1478andarf6ATGMOalso
reproducedthesedefectswhenusedincombination,butnotwhenusedalone.TheseobservationssuggestthatEGFRARF6
signalingregulatesearlybranchingmorphogenesisofthebiliarytree,andthatfunctionallyimpairedpathwaymemberscouldalso
contributetopoorbiledrainagelikelyfromabnormalintrahepaticbiliarynetworkinchildrenwithBA.Supportiveevidenceconsists
ofpoorARF6immunostaininginthoseliverexplants,whichmanifestedbileductpaucitywithoutfibrosis,butnotintheremainder,in
whichadvancedcirrhosiswithmarkedcholestasisprecludedanevaluationofintrahepaticbileductspriortothedevelopmentof
chronicobstructivechanges(Fig1C).
Thecomplexmoleculareffectsofarf6knockdowninzebrafishalsoprovidepotentialexplanationsforthefibrosisthatisseeninBA.
arf6knockdowninzebrafishwasassociatedwithintrahepaticupregulationofegfra,gep100,arf6,andgenesfordownstream
effectorsofArf6suchasasap1andrac1(Fig6).Amongthecanonicalhedgehogsignalingpathwaygenes,whichhavebeen
implicatedpreviouslyinbiliarydysgenesisonlyptch1,whichinducesseveralhedgehogtargetgenesviaeffectsontheglifamilyof
receptors,andnotthegli1orgli2genes,wasconsistentlyupregulatedinarf6morphants(S4Table)(13).ActivationofErkbysonic
hedgehogcanoccurindependentofcanonicalhedgehogsignalingviaPTCH[63]furtherconfirmingthatARF6dysregulationmay

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haveseveraldownstreameffectsoncellmembranefunction,cellproliferationandcelldevelopmentviaeffectsondownstream
effectors,ASAP1,RAC1andPTCH1.Inexperimentalmodels,upregulatedEGFRpathwaymembersleadtofibrosisandcirrhosis,
acommonfindingatlivertransplantationafterfailedportoenterostomyforBA[64].
Theseobservationsfromanestablishedzebrafishmodelofbileductdevelopmentprovideadditionalsupportiveevidenceforthe
biologicalrelevanceofARF6inbiliaryatresiainthisfirstreport.Obviousfuturedirectionsalsoincludereplicationofthese
experimentalfindings.Thistaskrequiresthattheeffectsofaninducedperturbationbeobservedoverseveraldaysininvitro
mammaliancelllines,becausecompletedisruptionoftheARF6geneinArf6/miceleadstoalmostcompletelethality[40].
BecausepreviousstudiesconductedinpatientswithBAindicateahighlikelihoodofmultiplesusceptibilitylociandtheenvironment
indiseasepathogenesis,futuremodels,whichcannotdemonstratesuchadditiveeffectsarelikelytomaintainasupportiverole
placingthelargerburdenofproofbackonthehumanevidencefromdiseasedsubjects.
Itisgenerallyassumedthattheintrahepaticbiliarytreeisalsoabnormalinextrahepaticatresia[65,59,60].However,intrahepatic
ductscannotbeexaminedbeforetheonsetofjaundiceandclinicalevidenceofextrahepaticobstructioninhumanBA.Anecdotally,
serialbiopsiesfromfourchildrenwithconfirmedextrahepaticbiliaryatresiahavedemonstratedthatintrahepaticbileductpaucity
withoutfibrosisprecededobstructiveductalproliferationandcirrhosisatportoenterostomyorlivertransplantation[66].Features
indicativeofintrahepaticductinvolvementarepredominantlyclinicaldiseaseprogressiondespiteportoenterostomy,anattenuated
intrahepaticpictureoncholangiographyandhistopathologiclymphocytesaroundandwithinintrahepaticBECs[67,68].Inthe
RhesusrotavirusmousemodelofBA,intrahepaticportalareasmaycontainlymphocytesthoughtherelevanceofthemodelto
humanBAisunclear[11,69].AlsonotevidentiswhetherintrahepaticchangesinhumanBAareprimary,orsecondaryto
extrahepaticobstruction.Landing,in1974,proposedaunitarianhypothesisofinfantileobstructivecholangiopathiesinwhichthe
entirebiliarytreewasvulnerablebutextrahepaticorintrahepaticdiseasedependedonthedevelopmentaltimingandnatureofthe
insult[70].Theconceptlapsedforlackofproofuntilnow.Thezebrafishmodelfromtwoindependentstudies,includingours,
suggestsagainthattheentirebiliarytreeisgeneticallysusceptibleandthatinthisinstance,theoutcomeofARF6dysregulation
involvestheintrahepatictree.
Insummary,GWASstudiesandanalysisofSNPdataforfunctionsandpathwayspostulatestheroleofARF6,involvedintheEGFR
pathway,indefectivebileductformation.Wevalidatethishypothesisinazebrafishmodel,whichunambiguouslyshowsthatarf6
knockdownimpactedtheEGFRpathwayintheliverandleadtodefectivebileductformation.

SupportingInformation
S1Fig.Sequencealignmentbetweenarf6aandarf6bcDNA.

Sequenceschosenforthedesignofarf6ATG,arf6aUTR,andarf6bUTRMOsareunderlinedblack,green,andred,respectively.
Thetargetregionsofarf6,arf6a,andarf6binsituprobesareboxedblack,green,andred,respectively.Thestart(ATG)andstop
(TAA)codonsofthesetwogenesareredcolored.
doi:10.1371/journal.pone.0138381.s001
(TIF)

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S2Fig.Morpholinovalidation.

(AD)CMV:GFPconstructscontainingthetargetsequenceofeachMOinfrontoftheGFPstartcodonwereinjectedaloneor
togetherwiththecorrespondingMO.GFPexpressionwasbarelydetectedinthecoinjectedembryos.Graphsshowthepercentage
ofembryosexhibitingstrong,weak,ornoGFPexpression.arf6ATGMOalsoblockedGFPexpressionfromtheCMV:GFP
constructscontainingthearf6bregioncorrespondingtotheMOtargetregioninarf6a(B),indicatingthatarf6ATGMOblocksboth
arf6aandarf6btranslation.nindicatesthenumberofembryosexamined.Scalebars:200m.
doi:10.1371/journal.pone.0138381.s002
(TIF)
S3Fig.Arf6expressionisgreatlyreducedinarf6ATGMOinjectedembryos.

Wildtypeembryoswereinjectedattheonecellstagewith2ngofarf6ATGMOor120pgofarf6amRNA,harvestedat7hpf,and
processedforwholemountimmunostainingwithantiArf6(green)andanticatenin(red)antibodies.DNAwasalsostainedwith
Hoechst33342(gray).cateninexpressionrevealsthecellmembrane.ConfocalimagesshowedthatArf6expressionwasgreatly
increasedinarf6amRNAinjectedembryos,whereasitwasgreatlyreducedinarf6ATGMOinjectedembryos,validatingthe
efficacyoftheMO.Dorsalviewsscalebar,25m.
doi:10.1371/journal.pone.0138381.s003
(TIF)
S4Fig.arf6amRNAinjectionpartiallyrescuesbiliarydefectsinarf6ATGMOinjectedlarvae.

(A)TheTg(Tp1:GFP),Tg(fabp10a:dsRed),andTg(ins:dsRed)lineswereusedtorevealtheintrahepaticbiliarystructure,theliver,
andthedorsalpancreas,respectively.Epifluorescenceimagesshowingtheexpressionofthesetransgenesrevealedthatadefect
intheintrahepaticbiliarystructureinarf6ATGMOinjectedlarvaewaspartiallyrescuedbyarf6amRNAinjection.Basedonthe
severityofthebiliarydefect,larvaeweredividedintothreegroups:normal,intermediate,andsevere.Graphshowingthe
percentageoflarvaeineachgroup.Arrowspointtotheliverarrowheadspointtothedorsalpancreas.Dottedlinesoutlinetheliver.
Lateralviews,anteriortotheleft.(B)EpifluorescenceimagesshowingPED6accumulationinthegallbladderrevealedthatthe
PED6accumulationdefectinarf6ATGMOinjectedlarvaewasalsopartiallyrescuedbyarf6amRNAinjection.BasedonPED6
levelsinthegallbladder,larvaeweredividedintothreegroups:absent,small/faint,andnormal.Graphshowingthepercentageof
larvaeineachgroup.Arrowspointtothegallbladder.Lateralviews,anteriortotheright.nindicatesthenumberoflarvaeexamined.
Scalebars,100m.
doi:10.1371/journal.pone.0138381.s004
(TIF)
S5Fig.Simultaneousknockdownofarf6aandarf6bresultsinmoreseverebiliarydefectsthantheirsingleknockdown.

(A)TheTg(Tp1:GFP),Tg(fabp10a:dsRed),andTg(ins:dsRed)lineswereusedtorevealtheintrahepaticbiliarystructure,theliver,
andthedorsalpancreas,respectively.Epifluorescenceimagesshowingtheexpressionofthesetransgenesrevealedthatasevere
biliarydefectwasobservedmoreofteninlarvaecoinjectedwith3ngofarf6aUTRand3ngofarf6bUTRMOsthaninsingly

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injectedlarvae.Basedontheseverityofthebiliarydefect,larvaeweredividedintothreegroups:normal,intermediate,andsevere.
Graphshowingthepercentageoflarvaeineachgroup.Arrowspointtotheliverarrowheadspointtothedorsalpancreas.Dotted
linesoutlinetheliver.Lateralviews,anteriortotheleft.(B)EpifluorescenceimagesshowingPED6accumulationinthegallbladder.
BasedonPED6levelsinthegallbladder,larvaeweredividedintothreegroups:absent,small/faint,andnormal.Graphshowing
thepercentageoflarvaeineachgroup.Arrowspointtothegallbladder.Lateralviews,anteriortotheright.nindicatesthenumber
oflarvaeexamined.Scalebars,100m.
doi:10.1371/journal.pone.0138381.s005
(TIF)
S6Fig.ThemovementofBECfilopodiaisgreatlyreducedinarf6ATGMOinjectedlarvae.

(A,B).TimelapseconfocalimagesshowingBECbehaviorsinarf6ATGMOinjectedandcontrollarvae.Thebehaviorswere
assessedbyTp1:GFP(green)andTp1:H2BmCherry(red)expressioninBECcytoplasmandnuclei,respectively.Confocalimages
every60minutesfrom74to84hpfwerepresented.ArrowspointtoBECfilopodia.Scalebars,25m.
doi:10.1371/journal.pone.0138381.s006
(TIF)
S7Fig.Proliferationisnotaffectedinarf6ATGMOinjectedorAG1478treatedlarvaeat75hpf.

(A)ConfocalimagesshowingEdU+proliferatingcells(gray)andH2BmCherry+BECs(red)intheliverofcontrol,arf6ATGMO
injected,orAG1478treatedTg(Tp1:H2BmCherry)larvae.ForEdUlabeling,thelarvaeweretreatedwithEdUforonehourpriorto
harvest.Dottedlinesoutlinetheliver.Scalebar,50m.(B)GraphshowingthetotalnumberofBECsineachliver.Asterisks
indicatestatisticalsignificance:*p<0.0001,**p<0.005.(C)GraphshowingthepercentageofEdU+BECsamongBECs.Errorbars,
SEM.nindicatesthenumberoflarvaeexamined.
doi:10.1371/journal.pone.0138381.s007
(TIF)
S8Fig.Theeffectofarf6knockdownonthedevelopmentofendodermderivedorgans.

(A)Confocalimagesshowingtheendodermandendodermderivedorgansat48hpf.TheTg(sox17:GFP)linewasusedtoreveal
theendodermandendodermderivedorgans.Arrows,arrowheads,andopenarrowheadpointtotheliver,thepancreasandthe
swimbladder,respectivelybracketsmarktheintestinalbulb.(B)Confocalimagesshowingthehepatopancreaticductalsystemat
75hpf.Tg(Tp1:GFP)Tg(fabp10a:dsRed)larvaewereprocessedforwholemountimmunostainingwith2F11(gray),GFP(green),
anddsRed(red)antibodies.Incontrasttotheintrahepaticbiliarydefect,thehepatopancreaticductalsystem,revealedby2F11
antibody,appearedtobenormalinarf6ATGMOinjectedlarvae.Arrows,arrowheads,openarrowheads,andopenarrowspointto
thegallbladder(gb),theextrahepaticduct(ehd),thecommonbileduct(cbd),andtheextrapancreaticduct(epd),respectively.The
hepatopancreaticductalsystemisschematicallyillustrated.Ventralviews,anteriorup.Scalebars,50m.
doi:10.1371/journal.pone.0138381.s008
(TIF)

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S1Table.qPCRprimersequencesforgeneexpressionstudiesinlivertissuefromzebrafish.

doi:10.1371/journal.pone.0138381.s009
(DOCX)
S2Table.FourhundrednineteenSNPsmappedtounique299geneswithin20kb+/windowsamongthetop1000significantSNPsinCHPcohortwith
BAinthegenomewideassociationstudy.

doi:10.1371/journal.pone.0138381.s010
(XLS)
S3Table.Amongthe1000toprankedsignificantSNPs,419mappedto299uniquegenesasdescribedinthemethods.

Amongthesegenes,231showedinteractionwithothergenesfromthelargehumannetworkconsistingmainlyofproteinprotein
interaction.Thefirstneighbornetworkwascreatedbyassimilatingallofthefirstinteractinggeneswiththese231genesfromthe
largehumannetwork.Thetotalof2506genesinthenewsmallernetworkislistedinthetable.Moreinformationcanbefoundinthe
tablethatincludestheaveragePvaluescalculatedfrommultiplesSNPsandtheinteractinggenes.
doi:10.1371/journal.pone.0138381.s011
(XLSX)
S4Table.QuantitativeRTPCRanalysisofhedgehogtargetgenesinlivertissuefromzebrafish.

doi:10.1371/journal.pone.0138381.s012
(DOCX)

Acknowledgments
Obtainedfunding:DS(TheAmericanLiverFoundation)RSHillmanFoundationofPittsburgh,TeamTransplant,andtheHerridge
andGiventerBrafffamilies.Studysupervision:RS.WethankMs.DaleZeccaformanuscriptpreparation.
Presentedatthe64thAnnualMeetingoftheAmericanAssociationfortheStudyofLiverDiseases:TheLiverMeeting2013on
November3,2013.

AuthorContributions
Conceivedanddesignedtheexperiments:JGHHDSRS.Performedtheexperiments:MNJSCA.Analyzedthedata:MNJSJG
CASRBWHQSGVKHLSSVPKMGKGRJHHJMDSRS.Contributedreagents/materials/analysistools:MNJSCA.Wrotethe
paper:MNJSJGCASRBWHQSKHLSSVPKMGKGRJHHDSADSSRS.Rereviewofbiopsies:RJ.

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