The cavefish genome reveals candidate genes for eye loss


previous item | next item

Citation
The cavefish genome reveals candidate genes for eye loss

Material Information

Title:
The cavefish genome reveals candidate genes for eye loss
Series Title:
Nature Communications
Creator:
McGaugh, Suzanne E.
Gross, Joshua B.
Aken, Bronwen
Blin, Maryline
Borowsky, Richard
Chalopin, Domitille
Hinaux, Hélène
Jeffery, William R.
Keene, Alex
Ma, Li
Minx, Patrick
Murphy, Daniel
O'Quin, Kelly E.
Rétaux, Sylvie
Rohner, Nicolas
Searle, Steve M. J.
Stahl, Bethany A.
Tabin, Cliff
Volff, Jean-Nicolas
Yoshizawa, Masato
Warren, Wesley C.
Publisher:
Springer Nature
Publication Date:
Language:
English

Subjects

Subjects / Keywords:
Evolution ( local )
Genomics ( local )
Retina ( local )
Genre:
serial ( sobekcm )

Notes

Abstract:
Natural populations subjected to strong environmental selection pressures offer a window into the genetic underpinnings of evolutionary change. Cavefish populations, Astyanax mexicanus (Teleostei: Characiphysi), exhibit repeated, independent evolution for a variety of traits including eye degeneration, pigment loss, increased size and number of taste buds and mechanosensory organs, and shifts in many behavioural traits. Surface and cave forms are interfertile making this system amenable to genetic interrogation; however, lack of a reference genome has hampered efforts to identify genes responsible for changes in cave forms of A. mexicanus. Here we present the first de novo genome assembly for Astyanax mexicanus cavefish, contrast repeat elements to other teleost genomes, identify candidate genes underlying quantitative trait loci (QTL), and assay these candidate genes for potential functional and expression differences. We expect the cavefish genome to advance understanding of the evolutionary process, as well as, analogous human disease including retinal dysfunction.
Original Version:
Nature Communications, Vol. 5, no. 5307 (2014-10-20).

Record Information

Source Institution:
University of South Florida Library
Holding Location:
University of South Florida
Rights Management:
Unknown
Resource Identifier:
K26-05222 ( USFLDC: LOCAL DOI )
k26.5222 ( USFLDC: LOCAL Handle )

USFLDC Membership

Aggregations:
University of South Florida
Karst Information Portal

Postcard Information

Format:
serial

Downloads

This item is only available as the following downloads:


Full Text

PAGE 1

ARTICLEReceived31Jul2014 | Accepted17Sep2014 | Published20Oct2014Thecave“shgenomerevealscandidategenes foreyelossSuzanneE.McGaugh1, w,JoshuaB.Gross2,BronwenAken3,4,MarylineBlin5,RichardBorowsky6, DomitilleChalopin7,He ´ le ` neHinaux5,WilliamR.Jeffery8,AlexKeene9,LiMa8,PatrickMinx1,DanielMurphy3,4, KellyE.OQuin10,SylvieRe ´ taux5,NicolasRohner11,SteveM.J.Searle3,BethanyA.Stahl2,CliffTabin11, Jean-NicolasVolff7,MasatoYoshizawa9&WesleyC.Warren1Naturalpopulationssubjectedtostrongenvironmentalselectionpressuresofferawindow intothegeneticunderpinningsofevolutionarychange.Cave“shpopulations, Astyanax mexicanus (Teleostei:Characiphysi),exhibitrepeated,independentevolutionforavarietyof traitsincludingeyedegeneration,pigmentloss,increasedsizeandnumberoftastebudsand mechanosensoryorgans,andshiftsinmanybehaviouraltraits.Surfaceandcaveformsare interfertilemakingthissystemamenabletogeneticinterrogation;however,lackofareference genomehashamperedeffortstoidentifygenesresponsibleforchangesincaveformsof A.mexicanus. Herewepresentthe“rst denovo genomeassemblyfor Astyanaxmexicanus cave“sh,contrastrepeatelementstootherteleostgenomes,identifycandidategenes underlyingquantitativetraitloci(QTL),andassaythesecandidategenesforpotential functionalandexpressiondifferences.Weexpectthecave“shgenometoadvanceunderstandingoftheevolutionaryprocess,aswellas,analogoushumandiseaseincludingretinal dysfunction. DOI:10.1038/ncomms6307 OPEN 1TheGenomeInstitute,WashingtonUniversity,CampusBox8501,StLouis,Missouri63108,USA.2DepartmentofBiologicalSciences,Universityof Cincinnati,711BRieveschlHall,312CollegeDrive,Cincinnati,Ohio45221,USA.3WellcomeTrustSangerInstitute,WellcomeTrustGenomeCampus,Hinxton, CambridgeCB101SA,UK.4EuropeanMolecularBiologyLaboratory,EuropeanBioinformaticsInstitute,WellcomeTrustGenomeCampus,Hinxton, CambridgeCB101SD,UK.5DECAgroup,NeurobiologyandDevelopmentLaboratory,CNRS-InstitutdeNeurobiologieAlfredFessard,91198Gif-sur-Yvette, France.6DepartmentofBiology,NewYorkUniversity,NewYork,NewYork10003-6688,USA.7InstitutdeGe ´ nomiqueFonctionnelledeLyon,EcoleNormale Supe ´ rieuredeLyon,CNRS,UMR5242,UCBL,46alle ´ edItalie,LyonF-69364,France.8DepartmentofBiology,UniversityofMaryland,CollegePark, Maryland20742,USA.9DepartmentofBiology,UniversityofNevada,Reno,Nevada89557,USA.10DepartmentofBiology,CentreCollege,600West WalnutSt,Danville,Kentucky40422,USA.11HarvardMedicalSchoolDepartmentofGenetics,77AvenueLouisPasteur;NRB360,Boston,Massachusetts 02115,USA. w Presentaddress:Ecology,Evolution,andBehavior,UniversityofMinnesota,100EcologyBuilding,1987UpperBufordCir,FalconHeights, Minnesota55108,USA.CorrespondenceandrequestsformaterialsshouldbeaddressedtoS.E.M.(email:smcgaugh@umn.edu). NATURECOMMUNICATIONS |5:5307|DOI:10.1038/ncomms6307|www.nature.com/naturecommunications1& 2014 MacmillanPublishersLimited.Allrightsreserved.

PAGE 2

Someofthemostfundamentalquestionsinevolutionarybiologyinvolvehoworganismscanadapttonewenviron-ments.Naturalpopulationsunderstrongselectionmaybeespeciallyusefulindecipheringthegeneticvariantsunderpinningtheseevolutionaryresponses.Yet,fewsystemspossessdramaticphenotypicchangesthatcanbede“nitivelyattributedtoselectionpressuresofanewenvironment.Evenfewerspeciescanbeusedtounderstandhowevolutionproceedswhenrepeatedinseparatepopulations.Caveanimalsofferoneofthemostexcitingsystemsinwhichtostudythesequestions1.Speci“cally,surfaceformsoftheMexicantetra,Astyanaxmexicanus,colonizedmultiplecavesinnortheasternMexicoandevolvedextremecave-associatedtraitsatleastfourindependenttimesoverthepast2…3Myr(refs2,3).Cave“shpopulationsexhibitrepeatedmorphologicalevolutionforavarietyoftraitsincludingeyedegeneration2,4,pigmentloss5,6,increasedsizeandnumberofspecializedmechanosensoryorganscalledneuromasts7andincreasednumbersoftastebuds4.Cave“shhavealsoevolvedbehaviouraldifferencesrelativetotheirsurface-dwellingcounterpartsincludingincreasedattractiontovibrations7,increasedolfactorycapabilities8,alteredfeedingangles9andlossofschoolingandaggression10,11.Further,cave“shlosebodyweightlessquicklythansurfacemorphs8andshowdramaticsleepreductionscomparedtosurface“sh12.Thepolarityofthesetraitchangesisknown(derivedincave“sh).Therefore,thesenaturalreplicatesofferauniqueopportunitytostudythegeneticbasesofparallelandconvergentevolutionarychanges1.Further,A.mexicanusisamenabletomoleculargeneticmanipulationinthelab13,14,andpriorQTL(quantitativetraitlocus)analysesofsurfaceandcave“shcrossesidenti“edgenomicregionsregulatingnumerousbehaviouralandmorphologicaltraits4,7…10,15.Inthiswork,wepresentthe“rstdenovogenomeassemblyforA.mexicanuscave“shtoallowformorepreciseidenti“cationofcandidategenesunderlyingQTLthanwaspreviouslypossiblewithsynteniccomparisonstozebra“sh15,16.Wedemonstratetheeffectivenessofthisapproachbyidentifyingcandidategenesforeyedevelopmentandothercave-derivedtraits.WefurtheranalyzeRNAseqdatatosurveythesecandidategenesforpotentialcodingandexpressionleveldifferencesbetweenthesurfaceandcavepopulations.Formanytraits,weexpectthatthecave“shgenomewillprovideatoolfordiscoveryoftheroleofindividualgenesandpathways.ResultsSequencingandannotation.Thesequencedcave“shindividualwasthe“rst-generationoffspringoftwowild-caughtparents,whichoriginatedfromPacho´ncave,Tamaulipas,Mexico.Whilethereareatleast29cavesthatcontainAstyanaxcave“sh,thePacho´ncave“sharethemoststudiedandexhibitthemostextremetroglomorphicphenotypesrelativetotheothercaves2.Thisgenomedraftwasassembledtoasizeof964Mb,whichissimilarinsizetoacongenericinBrazil17.Thedraftgenomecontains10,735scaffolds(N50contig¼14.7kb;N50scaffold¼1.775Mb),andthelongestscaffoldsizewas9.823Mb(SupplementaryData1).UsingtheEnsemblannotationpipeline18andRNAseqtranscriptevidence(eightuniquetissues;SupplementaryData2),wepredictedatotalof23,042protein-codinggenes,similartoothersequencedteleost“shes.Zebra“shistheclosestsequencedrelativetocave“sh(divergedapproximately250Myr)19,andweannotated16,480one-to-oneorthologswithzebra“sh.Toestimategenerepresentationinthedraftgenome,weusedassembledcave“shtranscriptsandevolutionarilyconservedgenemodels.AlignmentoftheAstyanaxbestopenreadingframes(SupplementaryData2)tothegenomescaffoldsfoundthatacrosstissue-speci“ctranscriptomes,amedianof81%oftranscriptsalignoveratleast75%oftheirlengthwithatleast90%identity.Further,CEGMAanalyses20indicatedthat95%ofthe248ultra-conservedcoreeukaryoticgenesarepresentinthegenomeassembly,and69%ofthe248ultra-conservedcoreeukaryoticgeneswereconsideredcompletegenes.Collectively,thissuggeststhattheassemblyhascapturedmuchoftheprotein-codingsequenceinthecave“shgenome.Transposableelementannotation.One-thirdofthecave“shgenomeiscomposedoftransposableelements(TEs)(SupplementaryData3and4).Thisrepetitivecontentiscom-parabletomostpublished“shgenomes(SupplementaryData3),withtheexceptionsofzebra“sh(52.2%TEs)21andFugu(2.8%)(ref.22).Inthecave“sh,DNAtransposonsaremoreabundantanddiversi“edthanretrotransposons,asthereareatleast12differentsuperfamiliesofDNAtransposonsrepresenting12.7%ofthegenome.Incontrast,retrotransposonscompriseonly2.3%ofthegenome(SupplementaryData4).Itappearsthatarecentwaveoftranspositionoccurredinthecave“shgenome(Fig.1)andwascomposedmostlyofTc-MarinerandhATsuperfamilies,whichcurrentlycompriseapproximately9.5%ofthecave“shgenome.Similarly,zebra“shexperiencedarecentlargeexpansionofrepeatfamilies,includingTc-MarinerandhATsuperfamilies,whereasanothercommonmodel,stickleback,hasnot(RepeatMaskerGenomicDatasets).WeestimatedthepotentialageofthedifferentofcopiesforeachTE-relatedsuperfamiliesbycalculatingKimuradistancesassum-ingthatmostofthemutationsinTEcopiesareneutral.Theratesoftransversions(q)andtransitions(p)weretransformedinKimuradistancesusing[K¼½ln(12pq) 14ln(12q)].Thecave“shgenomediffersincomparisonwithzebra“shinthatitappearstolackveryyoungelements(asindicatedbytheKimuradistancefromtheconsensus,Fig.1,RepeatMaskerGenomicDatasets).Giventhecaveatsofpossibleassemblyartefacts,lackofveryyoungelementsindicatesthatitisunlikelythatmanycopiesofTc-MarinerandhATsuperfamiliesarestill 1.00.80.60.40.20.010Kimura distance (from 0 to 50)SINE/tRNA–LysSINE/otherLINE/Rex–BabarLINE/L2RC/HelitronDNA/TcMarinerDNA/hATDNA/KolobokDNA/HarbingerDNA/CMCDNA/othersPercentage of TEs in the Cavefish genome234567891011121314151617181920212223242526272829303132333435363738394041424344454647484950 Figure1|TEsuperfamilieshistoryinthecave“shgenome.Onlysuperfamiliesthatshowcontenthigherthan0.1%inSupplementaryData4wereused.Kimuradistancesarerangedfromvalue0representingrecentTEcopiesto50fortheoldTEinsertions. ARTICLENATURECOMMUNICATIONS|DOI:10.1038/ncomms63072NATURECOMMUNICATIONS|5:5307|DOI:10.1038/ncomms6307|www.nature.com/naturecommunications&2014MacmillanPublishersLimited.Allrightsreserved.

PAGE 3

activeinthecave“shgenome(con“rmedbyanalysesoftranscriptomes,SupplementaryData5…9).Identi“cationofcandidategenesunderQTL.Perhapsthemostdistincttraitincave“shisthereduced,nearlyabsenteye(Fig.2),whichisindependentlyderivedinmultiple,independentcaveinvasions2,3,23.Incave“sh,earlyeyedevelopmentislargelysimilartotheeyedevelopmentinsurface“shinthatlensvesiclesandopticcupsform,albeit,theyaresmallerincave“shevenatveryearlystages(14h.p.f.,hourspostfertilization24).Thelensapoptosisbeginsafter25h.p.f.(refs24,25),andtheretinaundergoessigni“cantapoptosisatabout35h.p.f.(ref.24).Thisapoptosiscontinuesfordaystoweeks,andleadstoanarrestofeyedevelopment25,26.WeexaminethegenomeforgenesunderQTLforeyesizefromPacho´ncave“shsurface“shcrossesfromvariousstudies4,6…8,15,16.Acrossstudies,wecountatotalof15non-overlappingQTLforeye-relatedphenotypesdiscoveredinthePacho´npopulation4,7…10,15,16(SupplementaryFig.1).ScaffoldsoftendidnotspantheentirecriticalregioncomprisingaQTL;thus,eachQTLcriticalregionmaybedistributedacrossseveralscaffolds.AllgenesonascaffoldcontainingamarkerlinkedtotheQTLwereincluded.Intotal,2,408genesoutofthe23,042genesannotatedinthisdraftofthegenomewereassociatedwiththesegenomicregions.Itislikelythatasigni“cantportionofthesegenesthatarephysicallylinkedtothecausalvariantarenotresponsibleforthephenotype.Tonarrowthelistofcandidategenes,weexaminedthegeneexpressioninsurfaceandcavepopulationswithadevelopmentaltimecoursetakenat10h.p.f.,24h.p.f.,1.5dayspostfertilization(d.p.f.),and3d.p.f.RNAfromeachtimeperiodwasextractedfrom50whole,pooledindividualsandIlluminareadsweregeneratedforcave“shandsurface“shpoolsseparately.Animportantcaveattointerpretingthegeneexpressiondataisthatevenearlyindevelopment,cave“sheyesaresmallerthansurface“sheyes,andlowernumbersoftranscriptsmayre”ectsmallereyesandnotnecessarilydownregulation.Thetranscriptsequenceswerealsousedforobtainingcodingvariantdifferencesbetweensurface“shandcave“sh.Duetotheenormityofde“ninggenetoQTLassociationsformanytroglomorphictraits,weprimarilyfocusedontheeyephenotype.Hereweusedexpressiondataandintegratedpathwayanalysis27topredictlikelyphenotypesandthegenespotentiallyunderlyingthosephenotypes.Utilizingpriorknowledgeofpredictedoutcomebetweentranscriptionalregulatorsandtheirtargetgenes27,weimplicate30genesundertheQTLtoresultincongenitaleyeanomalies.Thedirectionofgeneexpressionchangebetweensurface“shandcave“shsupportsanincreasedlikelihoodofeyeanomaliesincave“shrelativetosurface“shat10h.p.f.,24h.p.f.,and1.5d.p.f.(forexample,12/27,12/19,11/30geneshaveexpressiondirectionconsistentwithincreasedcongenitalanomalyoftheeye,respectively;biased-correctedzscoreZ2.266,Po0.0001inallcases,seeref.27fordetailsofzscorecalculation).Atthelastsampledtimepoint(3d.p.f.),theexpressiondataareconsistentwithincreasedcave“sheyeanomalies,butinterestingly,thezscoresbecomesmallerwiththeprogressionofdevelopment(SupplementaryData10…16)andarenotsigni“cantat3d.p.f.WeperformedanenrichmenttestwithdatacombinedacrosstimepointsandfoundthattheQTLwereenrichedforgenesinvolvedincongenitalanomalyoftheeye,(30/1,560relativeto159/12,040inthetotalexpressiondataset;w2-testwithYatescorrectionPvalueo0.034,w2¼4.48,oddsratio¼1.57,95%con“denceintervalofoddsratio¼1.05…2.35).Additionalgenesinvolvedineyedevelopment,functionanddiseasewereenrichedintheQTLset,thoughnotsigni“cantlyso(129/1,560relativeto921/12,040intotaldataset;w2-testwithYatescorrectionPvalue¼0.35,w2¼0.88,oddsratio¼1.10,95%con“denceintervalofoddsratio¼0.91…1.34).Therefore,wecontendthattheeye-relatedQTLarequalitativelyenrichedforeye-relatedgenesrelativetotherestofthegenome,buttheeye-relatedQTLarequantitativelymorelikelytocontaingenesassociatedwithcongenitaleyedefects.Speci“ccandidategenesundereye-relatedQTL.SeveralgenesfoundundertheQTLareclassiccandidatesforeyedevelopment,andwehighlightseveral,whichmaybeparticularlypromising.WenarroweddownthelistofcandidategenesundertheQTLbyfocusingonthosewithexpressiondifferencesbetweencave“shandsurface“sh.StatisticalcomparisonsofgeneexpressionlevelswereperformedusingthemeasureoflogfoldchangeperformedinCuffdiff2.1.0(ref.28)(seeCuffdiff2.1.0documentationforadditionaldetailsoftest).Unlessotherwisenoted,allPvaluesgivenbelowfordifferentialexpressionbetweencaveandsurface“shweregeneratedbythistest.Linkagegroup(LG)namesareinconsistentacrossstudies;thus,theLGsgivenbelowcorrespondtothenamingschemeintheoriginalstudyinwhichtheQTLwasfoundandthosestudiesarecitedaftertheLGname.Oneofthesecandidategenesidenti“edbythismethodiscryaa,anantiapoptoticchaperoneproteinwhoseabsenceofgeneexpressionwashypothesizedtoplayaroleincave“sheyedegeneration26.CryaafallsunderaQTLforeyesizeonLG27(scaffoldcontainingmarkerAm229b)fromProtasetal.4Next,pitx3isessentialforlensdevelopmentinzebra“sh29,30andknockdownexperimentsresultinzebra“shwithdegeneratelensandretinasandmisshapenlowerjaws29.Cave“shexhibitsigni“cantlylowerexpressionofpitx3at24h.p.f.and3d.p.f.(Po0.002atbothtimepoints,qualitativelyloweratalltimes),butthereareonlytwosynonymousdifferencesbetweensurfaceandcave“shpitx3.Pitx3islocatedundertheQTLforlenslengthonLG14(ref.4)andforeyesizeonLG4(ref.7).Similarly,rx3islocatedunderaQTLforeyesizeonLG4(refs4,8)andunderliesalossofeyesinzebra“sh(chokh)andmedaka(eyeless)mutants31,32.Rx3exhibitssigni“cantlylessexpressionincave“shthaninsurface“shat10h.p.f.and3d.p.f.(Po0.0003atbothtimepoints,qualitativelyloweratalltimes)andnocodingvariants.Likewise,undertheQTLforeyesizeonLG4(refs4,8)arethegenesolfm2aandolfml2a.Zebra“shknockdownsofolfm2resultinabnormalitiesintheolfactorypits,eyesandoptictectumaswellasreducedandless-de“nedPax6expressionintheeye33,andolfm2aexhibitedsigni“cantlylowerexpressionincave“shat3d.p.f.(Po0.001).Wedidnotdetectcodingdifferencesinolfm2a,anddatawereunavailableforolfml2a.Lastly,BCoRisfoundonLG19(refs4,8).BCoRislinkedwithocularcolobomas Figure2|Photographsofsurfaceandcave“sh.(a,b)Surface“sh(line152)(c,d)Pacho´ncave“sh(line45).Scalebarfora,cis1cm.Scalebarforb,dis0.25cm.PhotosbyB.A.S. NATURECOMMUNICATIONS|DOI:10.1038/ncomms6307ARTICLENATURECOMMUNICATIONS|5:5307|DOI:10.1038/ncomms6307|www.nature.com/naturecommunications3&2014MacmillanPublishersLimited.Allrightsreserved.

PAGE 4

inhumanandzebra“sh34anditsbindingpartner,BCL6,hasbeen showntocontrolopticcupmorphogenesisthroughregulationof p53 inzebra“sh35.Cave“shexhibitsigni“cantlylowerexpression of BCoR at10h.p.f.( P o 0.013),andfournonsynonymouscoding differencesexistbetweensurfaceandcave“sh,thoughallappear tobeinevolutionarylabilesites.Importantly,thesegenes representonlyasubsetoftheinterestingcandidatesunderQTL. CandidategenesinQTLwithpotentiallypleiotropiceffects . ForseveralQTL,multipletroglomorphicphenotypesco-localize witheyesize,andthisco-localizationhasbeensuggestedasan evidencethatselectionforsomecave-adaptedtraitsresultedin pleiotropicdegenerationofeyes7.OneoftheseQTLisinvolvedin vibrationattractionbehaviour,eyesizeandsuper“cialneuromast numberattheorbitonLG2(ref.7).ThissameQTLforeyesize hasbeenidenti“edinmultiplestudies(LG7(ref.4),LG8(refs4,8) andLG1(ref.16))andaQTLforthethicknessoftheinner nuclearlayeroftheretinaonLG2(ref.15).TheseLGsfrom variousstudiesallcorrespondtothesamegenomicregion,and herewecountthisregionasasingleQTL.Wemainlyconcentrate ongenesthatareexpressedinbothcaveandsurface“shand appeartohavenotbeenpseudogenizedincave“sh,astheseare genesmostlikelytohavepleiotropiceffectsandtobegood candidatesfordrivingmultiplephenotypesthatco-localizetothe sameQTL. Oneofthemoreinterestingcandidategenesinthisregionis shisa2 ,whichinhibitsWntand“broblastgrowthfactorsignalling byretainingtheirrespectivereceptors,Frizzledand“broblast growthfactorreceptor,intheendoplasmicreticulum36.Cave“sh expressionof shisa2 isqualitativelyhigherthansurface“shatall timepoints(signi“cantlysoat10h.p.f.,24h.p.f.and1.5d.p.f., P o 0.005),but shisa2 containsonlyasinglesynonymouschange betweencaveandsurface“sh.Aduplicatecopyof shisa2 isalso underaneyeQTLfoundonLG6(refs4,8),andthisparalog exhibitsnocodingdifferencesandelevated,butmostlynonsigni“cant,expressionincave“sh(24h.p.f., P o 0.0003,not signi“cantat10h.p.f.and1.5d.p.f.)andlowerexpressionin cave“shat3d.p.f.( P o 0.0002). Because shisa2 interactswithmajordriversofdevelopment,we furtherassessedquantitativeandspatialdifferencesofexpression forthetwo shisa2 genes(LG2andLG6)byquantitativePCR (qPCR)and insitu hybridizationon Astyanax embryos.Forboth genes,qPCRexperimentsdidnotdetectsigni“cantdifferencesat 36h.p.f.betweenthetwomorphs,suggestinganexpression differenceoflessthantwofold(Fig.3a;Mann…Whitney U -test, P 4 0.05).Atthisstage, shisa2 -LG2wasexpressedthroughoutthe epidermisaswellasintheolfactoryepitheliumandthelensin surface“sh,butlensexpressionwasnotablymissingincave“sh (Fig.3b). Shisa2 -LG6hadamorecomplexexpressionpattern, reminiscentofwhatwasdescribedin Xenopus37andzebra“sh38, andincludedexpressioninthebranchialarches,cranialganglia, epidermis,olfactoryepithelium(like shisa2 -LG2),retinaandlens (Fig.3c).Noobviousdifferencewasobservedbetweensurface“sh andcave“shembryosconcerning shisa2 -LG6expressionpattern. Insum,anatomicalanalysisdetectedalackofs hisa2 (LG2) expressioninthecave“shlens,whichsuggestschangesinthe regulatoryregionofthisgenemaycontributetothelossofeyesin cave“sh. In Xenopus embryos,s hisa2 morpholinoknockdownormRNA injectionelicittheexpressionchangesfor otx2, akeyhomeobox geneforheadandeyedevelopment36.We,therefore,also compared otx2 expressionpatternsandlevelsin Astyanax cave“shandsurface“shembryos,thoughwecannotlocalize thisgeneontoaspeci“cLG.While otx2 patternissimilarinthe twomorphsduringheadandbraindevelopment(Fig.4b),lens expressionismuchweakerincave“shat48h.p.f.(Fig.4c),aswell aswhenassessedbywhole-organismsemi-quantitativereverse transcriptase-PCR(Fig.4a,SupplementaryFig.2).Wehave, therefore,identi“edapotentialdevelopmentalregulatorycascade thatmayleadtothecave“sheyelossandthatwouldinvolve shisa2 and otx2 inthedevelopinglens. Inadditionto shisa2 ,weidenti“edcandidategenesunderthis potentiallypleiotropicQTL.Severalgenesmeetingourcriteria underthisparticularQTLinclude prox1 and AIFM1 .Two additionalgenesfoundintheQTLanalysisofOQuin etal.15, crxa and Tbx2a ,arealsopresentunderthisQTLinouranalysis. Prox1 regulatesmanyprocessesindevelopmentincludinglens “breelongationanddifferentiationandtheexitofretinal progenitorcellsfromthecellcyclereviewedinref.39.The knockdownof prox1 resultsinthedisruptionofthelens-speci“c g -crystallinexpressionandsubsequentlensapoptosis40.Cave“sh expressionof prox1 exhibitsasimilarspatialpatterntosurface “shinthedevelopinglens,andforthisreason prox1 was previouslyconsideredunlikelytoplayaroleinthecave-speci“c eyedegeneration41. Prox1 isexpressedinsensoryhaircellsofthe neuromastandtastereceptorcellsoftastebuds,bothofwhichare morenumerousincave“shrelativetosurface,but prox1 expressioninthesestructuresdoesnotoccuruntil96h.p.f. (ref.41).Wedetectednosequencedifferencesbetweencaveand surface“shfor prox1 .However,inourwhole-organismRNAseq data,signi“cantlylowerexpressionincave“shwasobservedat 24h.p.f.,1.5d.p.f.and3d.p.f.( P o 0.022inallcases),while marginallynon-signi“canthigherexpressionincave“shwas observedattheearliestsampledtimepoint(10h.p.f., P ¼ 0.083). Signi“cantlylowerexpressionof prox1 duringthese developmentaltimepointsisconsistentwithincreasedlens apoptosisincave“sh.Therefore,are-examinationofthe contributionof prox1 ,inlightofitslocationunderthisQTL forsuborbitalneuromastcellnumber,VABandeyesize7andits quantitativeexpressiondifferences,maybewarranted. AIFM1 isimplicatedinsigni“cantandprogressiveoptic atrophyinmutantHarlequinmice,andthismutantphenotype canberescuedbyinjectionofanexpressionvectorcontaining AIFM1 (ref.42).Cave“shexhibitsigni“cantlylowerexpression of AIFM1 at24h.p.f.( P o 0.003)thansurface“sh,andthis geneexhibitsanintronicspliceregionvariantand“ve nonsynonymousvariants,twoofwhichappearderivedin cave“sh.Thesevariantswereallpredictedtobetoleratedbya computationalmethodthatattemptstodetermineifanamino acidsubstitutionisdetrimentaltoproteinfunction(SIFT43). Interestingly,theparalogofthisgene, AIFM2 ,isalsolocated undertheQTLforeyesizefoundonLG14(ref.4)andLG4 (ref.7). AIFM2 hassigni“cantlyreducedexpressionincave“sh relativetosurface“shatmosttimepoints(10h.p.f.,24h.p.f., 3d.p.f.; P o 0.022inallcases)withqualitativelylowerexpression at1.5d.p.f.( P ¼ 0.095).Further,thetwospliceregionvariantsare “xedbetweenthesurfaceandcave“sh,oneofwhichalsoresults inanonsynonymouschangethatisputativelyderivedincave“sh, thoughthischangeispredictedbySIFTtobetolerated. Crxa inducesretinalstemcellstodifferentiateintofunctional photoreceptors44.Whenknockeddowninzebra“sh, crxa promptsthedownregulationofgenesinthephototransduction cascade45,andisimplicatedineyereductionexperiencedby anothertroglomorphic“sh, Sinocyclocheilusanophtalmus46.This geneexhibitssigni“cantlyreducedexpressionincave“shat1.5 and3d.p.f.( P o 0.001;attheothertimepoints,expressioncould notbetested). Crxa containednosequencedifferencesbetween caveandsurface“sh. Tbx2a exhibitslocalizedexpressioninzebra“shmainlyinthe oticplacode,opticvesicle,oticvesicleandretina(alsoinventral mesodermandpectoral“nbud)38.T bx2 resultsinsmalleroptic ARTICLENATURECOMMUNICATIONS|DOI:10.1038/ncomms63074NATURECOMMUNICATIONS |5:5307|DOI:10.1038/ncomms6307|www.nature.com/naturecommunications& 2014 MacmillanPublishersLimited.Allrightsreserved.

PAGE 5

cupswhenmutatedinmice47,andtwocopiesexistinzebra“sh.Tbx2aisinvolvedincraniofacialandpharyngealarchdevelopment48,anditsparalogTbx2bisrequiredforproperretinalneuronalformationinzebra“sh49.Tbx2aexhibitslowerexpressionincave“shatalltimepoints(Po0.07at1.5d.p.f.,Po0.001atallothertimepoints)andthreenonsynonymousdifferencesbetweencaveandsurface.Onlyonenonsynonymousdifferenceisputativelyderivedincave“sh(D401E),andsuchanaminoacidreplacementispredictedbySIFTtobetolerated.Underthesecondco-localizingQTLforthetraitsvibrationattractionbehaviour,super“cialneuromastnumberatorbitandeyesizelocatedonLG17inYoshizawaetal.7,welackedscaffoldcoverageforseveralmarkersinthecenteroftheQTL(208e,205dand221a;SupplementaryData17).Thereareseveralinterestinggenesinthisregion(SupplementaryData11),butfewareascompellingasgenesfoundontheco-localizingQTLonLG2ofYoshizawaetal.7Weexpectfuturedraftsofthegenometouncoveradditionalcandidategenesinthisregion.Candidategenesforadditionalcavephenotypes.Lastly,wesoughttobrie”yinvestigateotherdistinctivetraitsforcave“sh,includingreducedpigmentation5,6.First,wefoundthatoneofthemostfamouspigmentationgenes,mc1r,knowntobemutatedinPacho´ncave“sh5(thepopulationfromwhichtheQTLweremapped),islocatedunderthecriticalregionoftheQTLfor 2abchijlmkdfge10Shisa2LG2(QTL)Shisa2LG6(paralog)SF, 36 h.p.f.EpidEpidEpidEpidEpidOlf epitOlf epitOlf epitOlf epitEpidOlf epitLensLensLensLensLensRetinaRetinaLensRetinaLensRetinaCF, 36 h.p.f.qPCR on Shisa2 genes at 36 h.p.f.Relative mRNA levelsShisa2 (LG2)Shisa2 (LG6)SFCF Figure3|Expressionpatternsofshisa2.(a)QuantitativePCRforshisa2geneson36h.p.f.wholelarvaeofsurface“sh(blue)andcave“sh(red).Nosigni“cantdifferencewasfoundbetweencave“shandsurface“shexpression(Mann…WhitneyU-test,P40.05).Theerrorbaristhes.e.ofthemean,andthesamplesizeisthreeineachcase(eachtriplicateisapoolof10…1536h.p.f.larvae).Photographsofinsituhybridizationfortheindicatedshisa2mRNAat36h.p.f.onsurface“sh(b…d,h…j)andcave“sh(e…g,k…m)embryos,focusingonheadandeyeexpression.Thebottompictures(c,d,f,g,i,j,l,m)arecenteredontheeyeregion,withfocuseitheronthelens/retinaorontheoverlyingskin.Inallpanels,anteriorisleftanddorsalisup.Inb,e,h,k,thephotographsweretakenfromlightlylabelledembryos(theepidermisisbarelylabelled)andinc,d,f,g,i,j,l,m,thephotographsweretakenfrommorestronglylabelledembryos(epidermisexpressionisvisible).Epid,epidermis;olfepit,olfactoryepithelium(nose).Thescalebarsare25mmforpanelsb,e,h,kand10mmfortheotherpanels. NATURECOMMUNICATIONS|DOI:10.1038/ncomms6307ARTICLENATURECOMMUNICATIONS|5:5307|DOI:10.1038/ncomms6307|www.nature.com/naturecommunications5&2014MacmillanPublishersLimited.Allrightsreserved.

PAGE 6

numberofmelanocytesinfourregionsofthebody(LG9(refs4,8)).Second,cave“shhaveanincreasednumberoftastebudsandincreasednumberofmaxillaryteeth4,8.AQTLfornumberoftastebudscontainstheserotoninreceptorhtr2a(LG5(refs4,8)),andtastecelldevelopmentandsignaltransductioninvolvesserotoninsignalling50,51.Third,aQTLforthenumberofmaxillaryteethincave“sh(LG13(refs4,8))containsdact2,whichsigni“cantlyinhibitsdlx2duringtoothformationinmouse52.Whenknockedoutinmice,dlx2producesadecreaseinthenumberofmolars53,supportingthenotionthatthisgenemayhaveaconservedroleintheregulationoftoothformation.Analysesofputativegenelosses.Weinvestigatedgenesthatwereputativelylostinthecave“shlineagesincethedivergenceofcave“shandzebra“sh,byexamininggenesthatwerepresentinzebra“shandeightadditionalactinopterygianteleostsavailableinEnsembl(SupplementaryData18).Thesegeneswerenotenrichedfor305geneontologyaccessionsrelatedtoeyedevelopmentorfunction,andsimilarresultswereobtainedforZFINanatomicalexpressiondataandZFIN-predictedphenotype.Transcriptomedatafromtheeighttissuesusedforgeneannotationandthedevelopmentalsurface“shandcave“shtimeserieswereassembledusingTrinity54foratotalof10separatetranscriptomes.OpenreadingframeswerepredictedfromtheseassembliesusingTransdecoderintheTrinitypackage.WeconstructedaBLASTdatabasefromthecodingregionsofzebra“shfromEnsemblGenes74andqueriedthisdatabaseusingeachofthetranscriptsinthelongest_orfs.cds“leswithBLASTn.Weusedastronge-valuecutoff(cutoffo1E-100),andresultswererobustforallvaluesweexaminedfrom1E-20to1E-100.Inthisway,weidenti“edwhethertheputativelymissinggeneinthecave“shgenome(butpresentinthezebra“shgenome)waspotentiallypresentinthesurfaceorcave-derivedRNAseqdata.Forseveralgenesthatwerepotentialcandidatesforloss,wecouldnot“ndarepresentativetranscriptforcave“shbutcould“ndatranscriptcopyamongthesurface“shtranscriptomedata(SupplementaryData19).Weattemptedtocon“rmthelackofatranscriptincave“shusingreversetranscription.However,forallcasesthatwetriedtocon“rmaputativelymissingcave“shtranscript,acave“shtranscriptwasdetected.Althoughnotaddingevidenceforcave“sh-speci“closs,forseverallargegenefamilies,oneorseveralmemberswerenotannotatedinthegenomesequenceandwerenotdetectedinsurfaceorcave“shtranscriptomedata.Whiletheseresultsareverypreliminary,potentialcandidatesforgenelossincludemembersofgenefamiliesinvolvedinvisionsuchasretinoldehydrogenases,crystallins,sineoculishomeoboxes,opsins/rhodopsins(includingmelanopsinwhosetruncatingmutationisimplicatedinthelossofalight-entrainableclockinSomaliancave“sh55),development,regulationofsleepandcircadianclocks(including“broblastgrowthfactors,gamma-aminobutyricacidAreceptors,anddopaminereceptors).Likewise,cave“shexhibitexcessivelocomotoractivitycomparedwithsurface“sh56,andseveralgenesthatinducehyperlocomotionwhenknockedoutorblockedinmiceorzebra“shdonotappeartobepresentinthecurrentcave“shgenomeannotationortranscriptomedata(SupplementaryData19).Interestingly,thenakedmolerat,aspeciesthatalsolivesindarknessandhasreducedeyes,hasalsoexperiencedlossesinsimilargenefamilies57.Assemblyandannotationerrorsoflargegenefamiliesarecommonindraftgenomes;thus,amoreextensiveandde“nitiveexplorationofthesecomplexgenefamiliesawaitsfuturestudies.Weprovidealistfromtheinitial,preliminaryanalysis(SupplementaryData19)tofacilitatefuturestudies.DiscussionInthiswork,wepresentadraftgenomeoftheMexicancave“sh,Astyanaxmexicanusandidentifycandidategenesforsomeofthespeciesmosticonicphenotypes.Pasteffortshavefocusedonmappingtraitstogenomicregions4,7…10,15.Byleveragingthesepaststudies,wedemonstratetheutilityofthegenomeforcandidategenediscovery,andhighlightseveralpotentialregulatorsofeyedevelopmentthatwerepreviouslynotimplicatedincave“sheyedegeneration.WealsoanalysedRNAseqdatatoidentifycodingvariantsbetweencave“shandsurface“shandnarrowthelistofcandidategenesthatpotentiallyimpactdegenerationoftheeye.Identi“cationofcandidategenesfrompastQTLworkisespeciallyexcitinginA.mexicanusbecausecave“shareamenabletoahostofmoleculargenetictechniquesthatcanbeusedtovalidatealleliceffects(forexample,injectionofmessengerRNAintodevelopingembryo13,meganuclease-andtransposase-basedtransgenesis14)andadditionalexperimentaltechniquescanbeaccomplishedusingthecloserelativeandlaboratorymodelzebra“sh5(forexample,geneeditingtechnologiessuchasTALENs).Thus,cave“shrepresentapowerfulsystemforexaminingthegeneticbasesofevolutionarychange,andweexpectprogressincandidate aSemi quantitative PCR on otx2 gene at 40 h.p.f.otx2 mRNA18S rRNASFCFSF10 h.p.f.12.5 h.p.f.40 h.p.f.48 h.p.f.40 h.p.f.48 h.p.f.10 h.p.f.12.5 h.p.f.CFSFLensLensCFbc Figure4|Expressionpatternsofotx2.(a)Semi-quantitativereversetranscriptase-PCRfortheoxt2geneson40h.p.f.wholeembryos.Cave“sh(CF)otx2transcriptsareslightlylessabundantthanthoseofsurface“sh(SF)comparedwithan18SrRNAstandard.(b)Photographsofinsituhybridizationsforotx2mRNAat10,12.5,40and48h.p.f.onsurface“sh(SF)andCFembryos,focusingonheadandeyeexpression.Inallpanels,anteriorisontheleft.Inlowerpanels,dorsalisup.Scalebarsare100mmforpanelslabelled40and48h.p.f.in(b).Scalebarsare250mmforpanelslabelled10and12.5h.p.f.inb.(c)Sectionsofinsitu-hybridizedSFandCFlarvaeat48h.p.f.showstrongotx2downregulationinthecave“shlens.Scalebarsare100mmforpanelsinc. ARTICLENATURECOMMUNICATIONS|DOI:10.1038/ncomms63076NATURECOMMUNICATIONS|5:5307|DOI:10.1038/ncomms6307|www.nature.com/naturecommunications&2014MacmillanPublishersLimited.Allrightsreserved.

PAGE 7

geneidenti“cationwillbemoreef“cientwiththeadditionofthe genome. Whileacandidategeneapproachhasstrongpotentialinthe cave“shsystem,ourexpressionanalyseshighlightthediscoveries enabledbyapathwayapproach.Inoneexample,ouranalysis predictedthereduced-eyephenotypeincave“shrelativeto surface“shbyutilizingthedirectionofexpressionwithineye developmentpathways.Notably,thisresultwasoneofthemost signi“cantphenotypespredictedasadownstreamphenotypic effectfromourdevelopmentalgeneexpressiontimecourseof wholeembryos(SupplementaryData13…16).Nonetheless,many databases,includingtheonesusedinthisstudy(Ingenuity PathwayAnalysis(IPA)),containonlyorthologuesfromhuman, mouseandrat,andapproximately89%ofthegenes(2013/2408) inourQTLdatasetcontainedmatchesintheIPAdatabase.This underscorestheneedforfutureiterationsofthesedatabasesto includenonmammalianmodelspecies(forexample,zebra“sh, Drosophila )toincreasehomologymatchesand,therefore,enable pathwayanalysisforalargeswathoforganisms. Weanticipatethatthisgenomicresourcewillbecoupledwith oneofthelargeststrengthsofthesystem,therepeatedevolution ofsimilarcave-associatedtraitsinindependentlyderivedcave populations2.Crossesbetween“shfromdifferentcaves complementandrestorecertaincave-derivedphenotypes(for example,rudimentaryeyes)23;thus,atleastsomeofthegenetic changesaccountingforcave-associatedtraitsareuniquetoeach cavelineage9.Inaddition,surfacepopulationsprovideapoolof standinggeneticvariationforthecaves58,andthecave“shsystem offersaninterestingsystemforstudyingadaptationinthefaceof gene”ow2.Toinvestigatethesequestions,ongoingworkthatis beyondthescopeofthispaperincludesapopulationgenomic effortfromseveralcaveandsurfacelocalities.Toenhancethese efforts,thecave“shgenomewillneedtobeanchoredtoa physical,chromosome-scalemap.Worktoproduceahigherqualitydraftusinglong-readtechnologyandagenotyping-bysequencinglinkagemapiscurrentlyunderway.Weexpectthat thisupcoming,reviseddraftwillfurtheraidinvestigationsofthe impactofselection,drift,migrationandgeneticarchitecturein creatingthesesreplicatedphenotypes. Inconclusion,the Astyanax genomepresentedherewillallow fordissectionofthegeneticbasesofconstructiveanddegenerativetraitsthatmakethecave“shdistinctive,willfacilitate futurestudiesinvestigatingthepathsofrepeatedevolutionand mayadvanceunderstandingofhumanmaladies(forexample, sleepdisorders,congenitaleyedefects)forwhichthecave“shcan serveasapowerfulnaturalmodelsystem. MethodsSourcematerial.SourceDNAwasobtainedfromtheJefferyLab.DNAwas collectedfromheart,liver,spleenandgillofasingle7…year-oldadultfemale cave“sh(Pacho ´ n)usingtheGenomic-TipTissueMidikits(Qiagen,Valencia,CA). RNAfromeighttissueswasextractedwithRNALipidMidikitsandRNeasykits (Qiagen).AnimalusecompliedwithethicalstandardsandwasapprovedbyThe UniversityofMarylandInstitutionalAnimalCareandUseCommitteeprotocol numberR-12…53toW.R.Jeffery. Genomesequencingandassembly.Usingagenomesizeestimateof1.19Gb, totalrawsequencecoverageofIlluminareadswas B 95 (shortinsertpaired-end reads,3,8and40kbmate-pairedlibraries,SupplementaryData1).Thecombined sequencereadswereassembledusingALLPATHSsoftware59andtheassembled coveragewas70 .Thisdraftassemblywasreferredtoas Astyanaxmexicanus 1.0.2.Thisassemblyhasbeengap“lledwithaversionofImage60,modi“edfor largegenomesandcleanedofcontaminatingcontigsbyperforminga MegaBLAST61ofthecontigsagainstadapter,bacterialandvertebratedatabases. IdentifyinglocationsofQTLingenomeassembly.Manymarkersoverlapped betweenpreviouslypublishedQTLanalyses,renderingitpossibletocompare coarseQTLlocationsacrossreplicatedmaps4,7,8,15,16,eventhoughLGnameswere notconsistentbetweenstudies.Markers”ankingtheQTLwerelocalizedto scaffoldsviabestBLASThit.Brie”y,acombinationof689RAD-tagsequences, microsatellitemarkersandcDNAswithlinkagemappositions15werealignedto thescaffoldsusingBLASTn. Astyanaxmexicanus hasahaploidchromosome numberof25(ref.62),andtherewereatotalon24LGsrepresentedbythese markers(SupplementaryFig.1). Allmarkerswererequiredtohaveane-valueof1E-20exceptforasubsetof microsatelliteandcDNAmarkerswhereonlytheforwardprimer,reverseprimer andsometimestherepeatmotifsequencewereavailable.Forthesemicrosatellites, wordsizewasreducedto7,andhitswererequiredtohaveane-valueoflessthan 1E-1andidentityofgreaterthan99%.TwocDNAstakenfrom Daniorerio were alsoallowedweakeridentity(85%orgreater)andane-valuecutoffof1E-1. OnlythetopBLASThitforeachmarkerwasrecorded.Scaffoldsthatmappedto differentLGswereexcluded( n ¼ 9).Inthecasewherethreeormoremarkers mappedthescaffoldtooneLGandonlyasinglemarkermappedthescaffoldtoa differentLG( n ¼ 3),onlytheincongruentmarkerwasexcluded.Intotal,340 scaffoldswerelocalizedtoLGsrepresenting574Mbofsequence(Supplementary Data17).Scaffoldswithonlyasinglemarker(219scaffolds)wereorderedalongthe chromosomeinlinewiththegeneticmapasdescribedinref.15andthe orientationwasassignedrandomly.Orientationwasassignedforscaffoldswithtwo ormoremarkers(121scaffolds, B 339Mb)physicallyandgeneticallymapped (SupplementaryData17;SupplementaryFig.1).Suchamapisverysimilartowhat isgivenintheSupplementaryMaterialsofref.9. Repetitivelandscape.Repeats,includingTEs,wereidenti“edandannotated usingRepeatModelersoftwarewithdefaultparameters.Theannotationfollowsthe universalclassi“cation63.Theautomaticlibrarywasscreenedto“lteranddiscard sequencessharinghighsimilaritieswithUniprotprotein.Paralleltotheautomatic annotation,potentiallyabsentfamiliesthatwerenotfoundusingRepeatModeler weremanuallysearchedbyBLASTusingknownTEproteins.Theunplaced scaffoldsweremaskedusingRepeatMasker3.3.0(http://www.repeatmasker.org/) withthecave“sh-speci“crepeatedlibraryusing…liband…alignfunctions.Results wereparsedtodeterminecopynumberandcoverageofTEsuperfamiliesusingthe RepeatMaskerout“les. ToinvestigatethelevelofTEtranscription,weanalysedthreedifferent assembledtranscriptomes:muscle,brainandwhole-eyesurface“sh. TranscriptomesweremaskedusingRepeatMasker3.3.0usingthespeci“ccave“sh repeatlibrary,aswasdoneforthegenomicanalyses.Theproportionofthevarious classes(forexample,DNA,LINE,SINE,LTRandunknownelements)were comparedwiththeirrespectiveproportionsinthegenome(camembertgraphs). Weassayedforover-andunder-representationofTEsuperfamiliesbycomparing therespectiveproportionofeachfamilyandsuperfamilyinthegenomeandinthe transcriptomes.Thefollowingequationwasused:(percentageoftheTEfamilyin thegenome[ortranscriptome]*100)/Totalrepeatcontentinthegenome[or transcriptome]). Genepredictionandannotation.Iterativestepsthatrelyonsimilarityevidence frompriorteleostgenemodelsand abinitio genepredictionalgorithmswere followedtobuildgenemodelsaccordingtoestablishedmethodsatEnsembl18. Protein-codingmodelswereextendedintoUTRregionsandcompletedexon modelswerevalidatedwithRNAseqdata(RNAseqanalysessectionbelow)from diversetissuetypes.Additionalmethodsfollowedhereforgeneratinggenebuilds byEnsemblarelocatedat:http://useast.ensembl.org/info/genome/genebuild/ 2013_10_cave“sh_genebuild.pdf.Althoughnotusedinthesestudies,asecondgene setproducedwiththeNCBIgeneannotationpipelineisavailableat:ftp:// ftp.ncbi.nlm.nih.gov/genomes/Astyanax_mexicanus/. RNAseqanalyses.RNAseqdatawasobtainedfromtwodifferentsequencing efforts.The“rstconsistedoftissue-speci“c100-bppaired-endIlluminareads whichwereusedforgenomeannotationbyEnsembl(SupplementaryData2). Theseincludedsamplesfrombrain,heart,kidney,liver,muscle,nasalcavityand skinfromadultPacho ´ ncave“shandeyesfromadultsurface“shfromTexas.For alltissues,multiple(onetosix)individualswerepooled,exceptforeyeswhereone surfaceindividualwasused. ThesecondRNAseqsequencingeffortconsistedofadevelopmentaltimecourse ofembryostakenfrom10h.p.f.,24h.p.f.,1.5d.p.f.and3d.p.f.Fiftyindividuals werepooledforeachtimepoint.ThreeseparateTruSeq2Illuminalibrarieswere madeforeachtimepointfromthesamepoolofRNA,providingtechnical replicates. Time-courseRNAseqdatawerecleanedbytrimmingthe“rstbasewith Fastx_trimmer(http://hannonlab.cshl.edu/fastx_toolkit/),trimmingwith Trimmomaticv0.30(ref.64)usingtheadapterlibraryforTruSeq2,allowinga qualityscoreof30acrossa4-bpslidingwindowandremovingallreads o 30 nucleotidesinlengthafterprocessing.Readswerealignedtothereferencegenome usingTopHat2(ref.65)withdefaultparametersexceptthatthemaximumintron lengthwassetto10,000.Cuf”inks2.1.0(ref.28)wasusedtocalculatedifferencesin expressionbetweencaveandsurfaceRNAseqdata.Cuf”inkswasusedwiththe parameters:--frag-bias-correct--multi-read-correct--upper-quartile-norm-compatible-hits-norm(withthegtf“leforthegenome).Cuffdiffwasusedwiththe NATURECOMMUNICATIONS|DOI:10.1038/ncomms6307ARTICLENATURECOMMUNICATIONS |5:5307|DOI:10.1038/ncomms6307|www.nature.com/naturecommunications7& 2014 MacmillanPublishersLimited.Allrightsreserved.

PAGE 8

parameters:--frag-bias-correct--multi-read-correct--FDR0.1--dispersionmethodper-condition. Time-courseRNAseqworkwasperformedunderaprotocolapprovedbythe InstitutionalAnimalCareandUseCommittee(IACUC)oftheUniversityof Cincinnati;ProtocolNumber10-01-21-01toJ.B.G.andcompliedwithethical regulationfortreatmentofanimals.Samplesforgenomeannotationweretakenin theJefferylabunderanimalcareprotocolsreferencedabove. Transcriptvariantanalyses.Atmost,15Pacho ´ ncave“shcontributedtothe offspringusedintheRNAseqexperiment(thesameninebreedingindividualsfrom Pacho ´ nLine163wereusedtogenerateembryosfor10h.p.f.,24h.p.f.,3d.p.f.time points;sixbreedingindividualsfromPacho ´ nLine138wereusedfor1.5d.p.f.)and atotalofthreeindividualscontributedtothesurfaceembryosfromwhichRNAseq datawerecollected.These“sharelaboratorystocksandlikelysomewhatinbred; thus,wehadlittlepowertoassignallelefrequenciesbetweencaveandsurface pooledsamples.Forallanalyses,weconcentrateondifferencesthatwerecompletely“xedinourdatasetbetweenthesurfaceRNAseqgenotypesandthecave referencegenome þ caveRNAseqgenotypes. Forvariantcalling,weconcatenatedallexpressiondataforsurfaceand separatelyconcatenatedallexpressiondataforcaveandmappedthesesurfaceand cavereadstothereferencegenomeusingTopHat2.Thesealignmentswerepassed toSamtoolsv0.1.19(ref.66)tocreatempileup“lesforcaveandsurfacewhichwere usedbyVarScanv2.3.6(ref.67)tocallsomaticmutationswithsurfacepileups designatedasnormalandcaveastumour.Variantswerethenusedasinputfor EnsemblsstandaloneVariantEffectPredictorv73(ref.68)topredicttheclass (forexample,synonymous,nonsynonymous)ofeachvariant.Todetermineifthe substitutionsidenti“edbyVariantEffectPredictorv73werelikelyderivedin cave“sh,peptidesequencefromorthologuesofzebra“sh,coelcanth,spottedgar, sticklebackandplaty“shwereobtainedfromBiomartandalignedwithClustalW69. Inmostcases,itwasstraightforwardtoclassifywhetherthesurfaceorcaveamino acidwaslikelyderived,andinallothercasesthesiteappearedevolutionarilylabile. Thisanalysisshouldbeinterpretedwiththecaveatthatitdoesnotaccountforthe possibilitythatthevariantclassi“edasderivedincave“shisactuallypresentinthe standinggeneticvariationofthesurface“shwhichwasnotsampledinourdata. SIFTSequencewasusedtopredictthefunctionalimpactofnonsynonymous substitutions43. Identi“cationofcandidategenes.Allpathwayanalyseswereperformedwith theIPAsuiteoftoolsavailableathttp://www.ingenuity.com/products/ipa.The entireanalysis-readypoolcontainedonly65%ofgenesinourQTLdataset (1,560/2,408)(SupplementaryData12)assomeofthegenesunderourQTLandin theIPAdatabasedidnothavesuf“cientexpressiondataforanalysis.Forall enrichmenttests,weusedonlytheanalysis-readygeneset“lteredbyIPA,which doesnotincludemultiplegeneswiththesameEntrezgenenameorgeneslacking expressiondatainourdataset. InadditiontotheIPAanalyses,whichdidnotannotateallofthegenesunder theQTL,weconductedindependentliteraturesearchesongenesandprioritized thosethatwere(1)differentiallyexpressedinatleastoneofthedevelopmentaltime points;(2)containedatleastone“xednonsenseormissensedifferencebetween caveandsurface“sh;or(3)exhibitedexpressioninaneye-relatedstructureduring developmentofthezebra“sh(ZFINanatomicaldatabase)orhadageneontology annotationordescriptionrelatedtoeye,retina,lensoropticfunction. QuantitativePCRfor shisa2.Forthe shisa2insitu hybridizationandqPCR experiments,laboratorystocksof A.mexicanus surface“shoriginatedfromSan SolomonSpring,BalmorheaStatePark,Texas.Cave“shfromPacho ´ ncavewere obtainedin2004…2006fromtheJefferylaboratoryattheUniversityofMaryland, CollegePark,MD,andweresincethenbredintheGIFanimalfacility. TotalRNAwasextractedfrom36h.p.f.cave“shorsurface“shembryoswith TRIzolreagent(Invitrogen)followedbypuri“cationandDNasetreatmentwiththe MachereyNagelNucleoSpinRNAIIkit.RNAamountsweredeterminedbythe Nanodrop2000cspectrophotometer(ThermoScienti“c).TotalRNA(1 m g)was reversetranscribedina20m l“nalreactionvolumeusingtheHighCapacitycDNA ReverseTranscriptionKit(LifeTechnologies,GrandIsland,NY,USA)withRNase inhibitorandrandomprimersfollowingthemanufacturersinstructions. QuantitativePCRwasperformedonaQuantStudio12KFlexReal-TimePCR SystemwithaSYBRgreendetectionprotocol.cDNA(3ng)wasmixedwithFast SYBRGreenMasterMixand500nMofeachprimerina“nalvolumeof10 m l.The reactionmixturewassubmittedto40cyclesofPCR(95 C,20s;(95 C,1s;60 C, 20s) 40)followedbyafusioncycletoanalyzethemeltingcurveofthePCR products.Negativecontrolswithoutthereversetranscriptasewereintroducedto verifytheabsenceofgenomicDNAcontaminants.Primersweredesignedbyusing thePrimer-BLASTtoolfromNCBIandthePrimerExpress3.0software(Life Technologies).Primerswerede“nedeitherinoneexonandoneexon…exon junctionorintwoexonsspannedbyalargeintron.Speci“cityandtheabsenceof multi-locusmatchingattheprimersitewereveri“edbyBLASTanalysis.The ampli“cationef“cienciesofprimersweregeneratedusingtheslopesofstandard curvesobtainedbyafour-folddilutionseries.Ampli“cationspeci“cityforeach real-timePCRreactionwascon“rmedbyanalysisofthedissociationcurves. Determined Ctvalueswerethenexploitedforfurtheranalysis,withthe Gapdh and Actb1 genesasreferences.Eachsamplemeasurementwasmadeatleastin duplicate.PrimersequencesforLG6shisa2 were0974-AM-LG6-F150-CGCAGTG CCCATCTACGTG-30and0975-AM-LG6-R150-TGTTTGGGTCGCAGAC AGC-30.ForLG2shisa2 ,theprimersequenceswere0982-AM-LG2-F350-GGGCA CCACAGTTTTTCCAA-30and0983-AM-LG2-R350-CTGTCCGTGTGCCTG ACTGA-30.For Gapdh and Actb1 ,primerswere0970-AMgapdh-F150-GTTGGC ATCAACGGATTTGG-30and0971-AMgapdh-R150-CCAGGTCAATGAAGG GGTCA-30and0972-AMactb1-F250-GCCATCATGCGTCTTGACCT-30and 0973-AMactb1-R250-ATCTCACGCTCAGCGGTTGT-30,respectively. For shisa2 work,animalsweretreatedaccordingtotheFrenchandEuropean regulationsforhandlingofanimalsinresearch.Authorizationforuseofanimals forthisworkwasprovidedbyParisCentre-SudEthicCommittee(authorization number2012-0052)toS.R.(number91…116). QuantitativePCRfor otx2.TotalRNAwasisolatedfrom40h.p.f.surface“shand Pacho ´ ncave“shlarvaeusingTRIzol(LifeTechnologies).cDNAwassynthesized usingtheSuperScriptTMIIIFirst-StrandSynthesisSuperMixKitandoligo(dT)20primers(LifeTechnologies).Forsemi-quantitativereversetranscriptase-PCR,part ofthe otx2 codingregionwasampli“edfromcDNAwithprimers50-ATGATGT CGTATCTCAAGCAACC-30(forward)and50-TAATCCAAGCAGTCGGCGTT GAAG-30(reverse)usingPCRMaster(RocheAppliedScience,Indianapolis,IN, USA),whichyieldedan otx2 PCRproductof857bp.ThePCRcyclingconditions were:onecycleofinitialdenaturationat94 Cfor5min,followedby35cyclesof denaturation(94 Cfor30s),annealing(58 Cfor30s)andelongation(72 Cfor 45s)anda“nalelongationstepat72 Cfor7min.Ampli“cationofthecontrol18S rRNAwascarriedoutusing1 m lofthesynthesizedcDNAwithprimersina50m l reactionvolumeusingPCRMaster(Roche).The18SrRNAprimerswere50-GAG TATGGTTGCAAAGCTGAAA-30(forward)and50-CCGGACATCTAAGGG CATCA-30(reverse),whichyieldedaPCRproductof343bp.ThePCRcycling conditionswere:onecycleofinitialdenaturationat94 Cfor5min,followedwith 25cyclesofdenaturation(94 Cfor30s),annealing(at62 Cfor30s)andelongation(at72 Cfor30s),followedbya“nalelongationstepat72 Cfor7min. Whole-mount insitu hybridizationfor shisa2.cDNAswereampli“edbyPCR frompCMV-Sport6plasmidspickedfromourcDNAlibraryusingSP6andT7 primersanddigoxygenin-riboprobesweresynthesizedfromPCRtemplates.A protocolforautomatedwhole-mount insitu hybridization(Intavis)wasperformed. Brie”y,embryoswereprogressivelyrehydrated,permeabilizedbyproteinaseK (Sigma)treatmentbeforebeingincubatedovernightat68 Cinhybridization buffercontainingtheappropriate shisa2 probe.Afterstringentwashes,the hybridizedprobesweredetectedbyimmunohistochemistryusinganalkaline phosphatase-conjugatedantibodyagainstdigoxygenin(Roche)andaNBT/BCIP chromogenicsubstrate(Roche). Whole-mount insitu hybridizationfor otx2.Forprobepreparation,the otx2 codingregionfragmentwasampli“edfromsurface“shcDNAwithPCRMaster (Roche)accordingtotheHotstartPCRprotocolusingthe otx2 primersdescribed above.ThePCRcyclingconditionswere:onecycleofinitialdenaturation(94 C) for2min,followedby32cyclesofdenaturation(94 Cfor30s),annealing(58 C for30s)andelongation(72 Cfor45s)anda“nalelongationstep(72 Cfor 7min).The“rstPCRproductwasusedasthetemplateforasecondcycleof PCRampli“cationusingsameconditions.Theresulting857bpPCRproductwas clonedintotheTOPOvectorintheTPOTACloningKitDualPromotor(Life Technologies)andcon“rmedbysequencing. Insitu hybridizationwasperformedaccordingtoref.70withsome modi“cations.TheplasmidDNAwaslinearizedwithrestrictionenzymes Bam H1 and Xho I(LifeTechnologies)at37 Cfor1handpuri“edwiththeQIAquickPCR Puri“cationKit(Qiagen).Senseandantisensedigoxigenin(DIG)-labelledRNAs weretranscribedwithSP6RNAandT7RNAPolymerases(Roche).The invitro transcriptionreactionswereconductedaccordingtotheDIGRNALabelingMix (Roche)protocol.Thereactionswereterminatedwith0.2MEDTA(pH8.0),and RNAwasprecipitatedwith4MLiClandwashedinprechilled70%ethanol.The RNAprobewasdenaturedfor3minat95 C,quicklycooledonicefor5minand thenaddedtotheHYB þ (seebelow)toobtainaconcentratedstock(10 m gml 1). Theembryoswere“xedwith4%paraformaldehydeinPBSovernightat4 C, dehydratedinanincreasingmethanolseriesandstoredat 20 C.Rehydrated embryosweretreatedwithproteinaseK(10 m gml 1inPBST(PBSplus0.1% Tween20))for5…10minatroomtemperature,washedtwicewithPBST,post“xed for20minwith4%paraformaldehydeinPBSTandwashed5timeswithPBST (5mineach).TheembryoswerepretreatedwithHYB (50%formamide,5 SSC,0.1%Tween20)for5minat60 Cwithoutshaking.TheHYB wasreplaced withHYB þ (HYB ,1mgml 1yeastRNA,50 m gml 1heparin)andthe embryoswereprehybridizedat60 Cfor4hwithgentleshaking.The prehybridizationmixwasremovedandreplacedwith1ng m l 1of otx2 senseor antisenseprobeinHYB þ .Hybridizationwascarriedoutat60 Covernight withgentleshaking.Theembryoswerethenwashedtwiceat60 Cwith50% formamide/2 SSCT(salinesodiumcitrateplus0.1%Tween20)for30mineach, oncewith2 SSCTfor15minat60 C,twicewith0.2 SSCT(20mineach)at ARTICLENATURECOMMUNICATIONS|DOI:10.1038/ncomms63078NATURECOMMUNICATIONS |5:5307|DOI:10.1038/ncomms6307|www.nature.com/naturecommunications& 2014 MacmillanPublishersLimited.Allrightsreserved.

PAGE 9

60 CandtwicewithMABT(150mMmaleicacid,100mMNaCl,pH7.5,0.1% Tween20)for5mineachatroomtemperature.Theembryoswereincubatedwith blockingsolution(MABT,2%blockingreagent)overnightat4 Cwithrockingand thenwithAnti-DIG-APFabfragments(1:5,000;Roche)inblockingsolution overnightat4 Cwithgentlerocking.TheembryoswerewashedoncewithMABT containing10%sheepserumatroomtemperaturefor25minandeightmoretimes (45…60mineach)withMABTatroomtemperaturewithgentlyshaking.Then,the embryoswerewashedwithPBSTandincubatedinBMPurpleAPSubstrate (Roche)atroomtemperatureinthedark.Afterthesignaldeveloped,thereaction wasterminatedbyrinsingtheembryosseveraltimesinPBST.Embryoswere processedthroughanincreasingglycerolseriesinPBS(30…50…80%)andimagedby microscopy. Insitu -hybridizedembryosweredehydratedthroughanethanolseries(from 30,50,70,85,95%,andthree100%steps)for20mineachatroomtemperature. Thedehydratedembryoswereincubatedinethanol:Histo-Clear(1:1)withrotation for20min,intwochangesofHisto-Clearfor30mineach),inparaf“n:Histo-Clear (1:1)at62 Cfor1hand“nally100%paraf“nat62 Cfor2h.Theblocks containingembeddedembryoswerecutinto15m msections,andthesectionswere dewaxedandviewedbymicroscopy.References1.Elmer,K.R.&Meyer,A.Adaptationintheageofecologicalgenomics:insights fromparallelismandconvergence. TrendsEcol.Evol. 26, 298…306(2011). 2.Bradic,M.,Beerli,P.,Leo ´ n,F.G.-d.,Esquivel-Bobadilla,S.&Borowsky,R.Gene ”owandpopulationstructureintheMexicanblindcave“shcomplex( Astyanax mexicanus ). BMCEvol.Biol. 12, 9…9(2012). 3.Coghill,L.M.,DarrinHulsey,C.,Chaves-Campos,J.,Garcš ´ adeLeon,F.J.& Johnson,S.G.Nextgenerationphylogeographyofcaveandsurface Astyanax mexicanus . Mol.Phylogenet.Evol. 79C, 368…374(2014). 4.Protas,M.,Conrad,M.,Gross,J.B.,Tabin,C.&Borowsky,R.Regressive evolutionintheMexicancavetetra, Astyanaxmexicanus . Curr.Biol. 17, 452…454(2007). 5.Gross,J.B.,Borowsky,R.&Tabin,C.J.Anovelrolefor Mc1r intheparallel evolutionofdepigmentationinindependentpopulationsofthecave“sh Astyanaxmexicanus . PLoSGenet. 5, e1000326…e1000326(2009). 6.Protas,M.E. etal. Geneticanalysisofcave“shrevealsmolecularconvergencein theevolutionofalbinism. Nat.Genet. 38, 107…111(2006). 7.Yoshizawa,M.,Yamamoto,Y.,OQuin,K.E.&Jeffery,W.R.Evolutionofan adaptivebehavioranditssensoryreceptorspromoteseyeregressioninblind cave“sh. BMCBiol. 10, 108(2012). 8.Protas,M. etal. Multi-traitevolutioninacave“sh, Astyanaxmexicanus . Evol. Dev. 10, 196…209(2008). 9.Kowalko,J.E. etal. Convergenceinfeedingpostureoccursthroughdifferent geneticlociinindependentlyevolvedcavepopulationsof Astyanaxmexicanus . Proc.NatlAcad.Sci.USA 110, 16933…16938(2013). 10.Kowalko,J.E. etal. Lossofschoolingbehaviorincave“shthroughsightdependentandsight-independentmechanisms. Curr.Biol. 23, 1874…1883 (2013). 11.Elipot,Y.,Hinaux,H.,Callebert,J.&Re ´ taux,S.Evolutionaryshiftfrom“ghting toforaginginblindcave“shthroughchangesintheserotoninnetwork. Curr. Biol. 23, 1…10(2013). 12.Duboue ´ ,E.R.,Keene,A.C.&Borowsky,R.L.Evolutionaryconvergenceon sleeplossincave“shpopulations. Curr.Biol. 21, 671…676(2011). 13.Yamamoto,Y.,Stock,D.W.&Jeffery,W.R.Hedgehogsignallingcontrolseye degenerationinblindcave“sh. Nature 431, 844…847(2004). 14.Elipot,Y.,Legendre,L.,Pe ` re,S.,Sohm,F.&Re´ taux,S. Astyanax transgenesis andhusbandry:howcave“shentersthelaboratory. Zebra“sh 11, 291…299 (2014). 15.OQuin,K.E.,Yoshizawa,M.,Doshi,P.&Jeffery,W.R.Quantitativegenetic analysisofretinaldegenerationintheblindcave“sh Astyanaxmexicanus . PLoS ONE 8, e57281(2013). 16.Gross,J.B. etal. Syntenyandcandidategenepredictionusingananchored linkagemapof Astyanaxmexicanus . Proc.NatlAcad.Sci.USA 105, 20106…20111(2008). 17.Carvalho,M.L.,Oliveira,C.,Navarrete,M.C.,Froehlich,O.&Foresti,F. NuclearDNAcontentdeterminationinCharaciformes“sh(Teleostei, Ostariophysi)fromtheNeotropicalregion. Genet.Mol.Biol. 25, 49…55(2002). 18.Flicek,P. etal. Ensembl2012. NucleicAcidsRes. 40, D84…D90(2012). 19.Nakatani,M.,Miya,M.,Mabuchi,K.,Saitoh,K.&Nishida,M.Evolutionary historyofOtophysi(Teleostei),amajorcladeofthemodernfreshwater“shes: PangaeanoriginandMesozoicradiation. BMCEvol.Biol. 11, 177(2011). 20.Parra,G.,Bradnam,K.&Korf,I.CEGMA:apipelinetoaccuratelyannotate coregenesineukaryoticgenomes. Bioinformatics 23, 1061…1067(2007). 21.Howe,K. etal. Thezebra“shreferencegenomesequenceanditsrelationshipto thehumangenome. Nature 496, 498…503(2013). 22.Aparicio,S. etal. Whole-genomeshotgunassemblyandanalysisofthegenome of Fugurubripes . Science 297, 1301…1310(2002). 23.Borowsky,R.Restoringsightinblindcave“sh. Curr.Biol. 19, R23…R24(2008). 24.Alunni,A. etal. Developmentalmechanismsforretinaldegenerationinthe blindcave“sh Astyanaxmexicanus . J.Comp.Neurol. 505, 221…233(2007). 25.Jeffery,W.R.&Martasian,D.P.Evolutionofeyeregressioninthecave“sh Astyanax :apoptosisandthe Pax-6 gene. Am.Zool. 38, 685…696(1998). 26.Strickler,A.G.,Byerly,M.S.&Jeffery,W.R.Lensgeneexpressionanalysis revealsdownregulationoftheanti-apoptoticchaperone a A-crystallinduring cave“sheyedegeneration. Dev.GenesEvol. 217, 771…782(2007). 27.Kra ¨ mer,A.,Green,J.,Pollard,J.&Tugendreich,S.Causalanalysisapproaches inIngenuityPathwayAnalysis. Bioinformatics 30, 523…530(2014). 28.Trapnell,C.etal. Differentialanalysisofgeneregulationattranscriptresolution withRNA-seq. Nat.Biotechnol. 31, 46…53(2013). 29.Shi,X. etal. Zebra“sh pitx3 isnecessaryfornormallensandretinal development. Mech.Dev. 122, 513…527(2005). 30.Zilinski,C.A.,Shah,R.,Lane,M.E.&Jamrich,M.Modulationofzebra“sh pitx3 expressionintheprimordiaofthepituitary,lens,olfactoryepitheliumand cranialgangliaby hedgehog and nodal signaling. Genesis 41, 33…40(2005). 31.Loosli,F. etal. Lossofeyesinzebra“shcausedbymutationof chokh/rx3 . EMBORep. 4, 894…899(2003). 32.Loosli,F. etal. Medakaeyelessisthekeyfactorlinkingretinaldetermination andeyegrowth. Development 128, 4035…4044(2001). 33.Lee,J.-A.,Anholt,R.R.&Cole,G.J.Olfactomedin-2mediatesdevelopmentof theanteriorcentralnervoussystemandheadstructuresinzebra“sh. Mech.Dev. 125, 167…181(2008). 34.Ng,D. etal. OculofaciocardiodentalandLenzmicrophthalmiasyndromes resultfromdistinctclassesofmutationsin BCOR . Nat.Genet. 36, 411…416 (2004). 35.Lee,J.,Lee,B.-K.&Gross,J.M.Bcl6afunctionisrequiredduringopticcup formationtopreventp53-dependentapoptosisandcolobomata. Hum.Mol. Genet. 22, 3568…3582(2013). 36.Yamamoto,A.,Nagano,T.,Takehara,S.,Hibi,M.&Aizawa,S.Shisapromotes headformationthroughtheinhibitionofreceptorproteinmaturationforthe caudalizingfactors,WntandFGF. Cell 120, 223…235(2005). 37.Silva,A.,Filipe,M.,Vitorino,M.,Steinbeisser,H.&Belo,J.Developmental expressionof Shisa-2 in Xenopuslaevis . Int.J.Dev.Biol. 50, 575…579(2006). 38.Thisse,B.&Thisse,C. FastReleaseClones:AHighThroughputExpression Analysis (ZFINDirectDataSubmission.,2004). 39.Ogino,H.,Ochi,H.,Reza,H.M.&Yasuda,K.Transcriptionfactorsinvolvedin lensdevelopmentfromthepreplacodalectoderm. Dev.Biol. 363, 333…347 (2012). 40.Wigle,J.T.,Chowdhury,K.,Gruss,P.&Oliver,G. Prox1 functioniscrucialfor mouselens-“breelongation. Nat.Genet. 21, 318…322(1999). 41.Jeffery,W.R.,Strickler,A.G.,Guiney,S.,Heyser,D.G.&Tomarev,S.I. Prox1 ineyedegenerationandsensoryorgancompensationduringdevelopmentand evolutionofthecave“sh Astyanax . Dev.GenesEvol.210, 223…230(2000). 42.Bouaita,A. etal. Downregulationofapoptosis-inducingfactorinHarlequin miceinducesprogressiveandsevereopticatrophywhichisdurablyprevented byAAV2AIF1 genetherapy. Brain 135, 35…52(2012). 43.Kumar,P.,Henikoff,S.&Ng,P.C.PredictingtheeffectsofcodingnonsynonymousvariantsonproteinfunctionusingtheSIFTalgorithm. Nat. Protoc. 4, 1073…1081(2009). 44.Jomary,C.&Jones,S.E.Inductionoffunctionalphotoreceptorphenotypeby exogenous Crx expressioninmouseretinalstemcells. Invest.Ophthalmol.Vis. Sci. 49, 429…437(2008). 45.Shen,Y.-c.&Raymond,P.A.Zebra“sh cone-rod ( crx )homeoboxgene promotesretinogenesis. Dev.Biol. 269, 237…251(2004). 46.Meng,F. etal. Evolutionoftheeyetranscriptomeunderconstantdarknessin Sinocyclocheilus cave“sh. Mol.Biol.Evol. 30, 1527…1543(2013). 47.Behesti,H.,Papaioannou,V.&Sowden,J.Lossof Tbx2 delaysopticvesicle invaginationleadingtosmallopticcups. Dev.Biol. 333, 360…372(2009). 48.Thu,H.N.T.,Tien,S.F.H.,Loh,S.L.,Yan,J.S.B.&Korzh,V. tbx2a Is requiredforspeci“cationofendodermalpouchesduringdevelopmentofthe pharyngealarches. PLoSONE 8, e77171(2013). 49.Gross,J.M.&Dowling,J.E. Tbx2b isessentialforneuronaldifferentiation alongthedorsal/ventralaxisofthezebra“shretina. Proc.NatlAcad.Sci.USA 102, 4371…4376(2005). 50.Roper,S.D.Tastebudsasperipheralchemosensoryprocessors. Semin.Cell Dev.Biol. 24, 71…79(2013). 51.Ortš ´ z-Alvarado,R. etal. Expressionoftryptophanhydroxylaseindeveloping mousetastepapillae. FEBSLett. 580, 5371…5376(2006). 52.Li,X.,Florez,S.,Wang,J.,Cao,H.&Amendt,B.Dact2repressesPITX2 transcriptionalactivationandcellproliferationthroughWnt/beta-catenin signalingduringodontogenesis. PLoSONE 8, e54868(2013). 53.Qiu,M. etal. RoleoftheDlxhomeoboxgenesinproximodistalpatterningof thebranchialarches:mutationsofDlx-1,Dlx-2,andDlx-1and-2alter morphogenesisofproximalskeletalandsofttissuestructuresderivedfromthe “rstandsecondarches. Dev.Biol. 185, 165…184(1997). 54.Grabherr,M.G. etal. Full-lengthtranscriptomeassemblyfromRNA-Seqdata withoutareferencegenome. Nat.Biotechnol. 29, 644…652(2011). NATURECOMMUNICATIONS|DOI:10.1038/ncomms6307ARTICLENATURECOMMUNICATIONS |5:5307|DOI:10.1038/ncomms6307|www.nature.com/naturecommunications9& 2014 MacmillanPublishersLimited.Allrightsreserved.

PAGE 10

55.Cavallari,N. etal. Ablindcircadianclockincave“shrevealsthatopsins mediateperipheralclockphotoreception. PLoSBiol. 9, e1001142(2011). 56.Sharma,S.,Coombs,S.,Patton,P.&BurtdePerera,T.Thefunctionof wall-followingbehaviorsintheMexicanblindcave“shandasightedrelative, theMexicantetra( Astyanax ). J.Comp.Physiol.A 195, 225…240(2009). 57.Kim,E.B. etal. Genomesequencingrevealsinsightsintophysiologyand longevityofthenakedmolerat. Nature 479, 223…227(2011). 58.Gross,J.&Wilkens,H.Albinisminphylogeneticallyandgeographically distinctpopulationsofAstyanaxcave“sharisesthroughthesameloss-offunctionOca2allele. Heredity 111, 122…130(2013). 59.Gnerre,S. etal. High-qualitydraftassembliesofmammaliangenomesfrom massivelyparallelsequencedata. Proc.NatlAcad.Sci.USA 108, 1513…1518 (2011). 60.Tsai,I.J.,Otto,T.D.&Berriman,M.Method:Improvingdraftassembliesby iterativemappingandassemblyofshortreadstoeliminategaps. GenomeBiol. 11, R41(2010). 61.Zhang,Z.,Schwartz,S.,Wagner,L.&Miller,W.Agreedyalgorithmfor aligningDNAsequences. J.Comput.Biol. 7, 203…214(2000). 62.Kavalco,K.F.&DeAlmeida-Toledo,L.F.Molecularcytogeneticsofblind mexicantetraandcommentsonthekaryotypiccharacteristicsofgenus Astyanax (Teleostei,Characidae). Zebra“sh 4, 103…111(2007). 63.Wicker,T. etal. Auni“edclassi“cationsystemforeukaryotictransposable elements. Nat.Rev.Genet. 8, 973…982(2007). 64.Lohse,M. etal. RobiNA:auser-friendly,integratedsoftwaresolutionfor RNA-Seq-basedtranscriptomics. NucleicAcidsRes. 40 (WebServerissue): W622…W627(2012). 65.Kim,D. etal. TopHat2:accuratealignmentoftranscriptomesinthepresenceof insertions,deletionsandgenefusions. GenomeBiol. 14, R36(2013). 66.Li,H. etal. TheSequencealignment/map(SAM)formatandSAMtools. Bioinformatics 25, 2078…2079(2009). 67.Koboldt,D. etal. VarScan2:Somaticmutationandcopynumberalteration discoveryincancerbyexomesequencing. GenomeRes. 22, 568…576(2012). 68.McLaren,W. etal. Derivingtheconsequencesofgenomicvariantswiththe EnsemblAPIandSNPEffectPredictor. BMCBioinformatics 26, 2069…2070 (2010). 69.Thompson,J.,Higgins,D.&Gibson,T.CLUSTALW:improvingthesensitivity ofprogressivemultiplesequencealignmentthroughsequenceweighting, position-speci“cgappenaltiesandweightmatrixchoice. NucleicAcidsRes. 22, 4673…4680(1994). 70.Strickler,A.G.,Yamamoto,Y.&Jeffery,W.R.EarlyandlatechangesinPax6 expressionaccompanyeyedegenerationduringcave“shdevelopment. Dev. GenesEvol. 211, 138…144(2001).AcknowledgementsThisworkwassupportedbyNIHgrantR24RR032658-01toW.C.W.andTheGenome InstituteatWashingtonUniversitySchoolofMedicine.Collectionswereconductedwith MexicanPermitNumber040396-213-03grantedtoW.R.J.Thisworkwasalsosupported bytheWellcomeTrust(grantnumbersWT095908andWT098051)andtheEuropean MolecularBiologyLaboratory. Shisa2 qPCRworkbene“tedfromthefacilitiesand expertiseoftheQPCRplatformofIMAGIF(CentredeRecherchedeGif-www. imagif.cnrs.fr).ThisworkwassupportedinpartbytheNationalInstitutesofHealth (NIDCR)grantDE022403toJ.B.G.WethankJ.Tabinfortechnicalassistance.Weare gratefulforresourcesfromtheUniversityofMinnesotaSupercomputingInstitute.AuthorcontributionsS.E.M.andW.C.W.aretheprincipalinvestigatorswhoconceivedtheproject,analysed thedataandwrotethemanuscript.W.C.W.andP.M.sequencedandassembledthe genome.J.B.G.andB.A.S.providedRNAseqdata,identi“edcandidategenesandaidedin writingthemanuscript.C.T.andN.R.validatedgenelosscandidates,identi“edcandidate genesandaidedinwritingthemanuscript.D.C.andJ.-N.V.carriedouttransposable elementanalyses.W.R.J.,K.E.O.andM.Y.providedtissueforDNAandRNAsequencing andaidedinwritingthemanuscript.W.R.J.andL.M.carriedoutthe otx2 validation work.A.K.investigatedcandidategenesandA.K.andR.B.aidedinwritingthe manuscript.S.R.,H.H.andM.B.carriedoutthe shisa2 validationworkandaidedin writingthemanuscript.S.M.J.S.ledtheEnsemblgeneannotationandB.A.andD.M. performedthegenomeannotation.AdditionalinformationAccessioncodes: AllgenomicdataareassociatedwithbioprojectPRJNA89115andhave beendepositedinGenBank/EMBL/DDBJNucleotidedatabaseundertheaccessioncode APWO00000000.RNAseqdatahavebeendepositedintheGenBank/EMBL/DDBJ sequencereadarchiveundertheaccessioncodesPRJNA177689(tissue-speci“ctranscriptomes)andPRJNA258661(developmentaltimecourse).Geneannotationscanbe foundathttp://www.ensembl.org/Astyanax_mexicanus/Info/Indexandhavebeen depositedintheGenBank/EMBL/DDBJAssemblydatabaseundertheaccessioncode PRJNA237016. SupplementaryInformation accompaniesthispaperathttp://www.nature.com/ naturecommunications Competing“nancialinterests: Theauthorsdeclarenocompeting“nancialinterests. Reprintsandpermission informationisavailableonlineathttp://npg.nature.com/ reprintsandpermissions/ Howtocitethisarticle :McGaughS.E., etal. Thecave“shgenomerevealscandidate genesforeyeloss. Nat.Commun. 5:5307doi:10.1038/ncomms6307(2014). ThisworkislicensedunderaCreativeCommonsAttributionNonCommercial-ShareAlike4.0InternationalLicense.Theimagesor otherthirdpartymaterialinthisarticleareincludedinthearticlesCreativeCommons license,unlessindicatedotherwiseinthecreditline;ifthematerialisnotincludedunder theCreativeCommonslicense,userswillneedtoobtainpermissionfromthelicense holdertoreproducethematerial.Toviewacopyofthislicense,visithttp:// creativecommons.org/licenses/by-nc-sa/4.0/ ARTICLENATURECOMMUNICATIONS|DOI:10.1038/ncomms630710NATURECOMMUNICATIONS |5:5307|DOI:10.1038/ncomms6307|www.nature.com/naturecommunications& 2014 MacmillanPublishersLimited.Allrightsreserved.


printinsert_linkshareget_appmore_horiz

Download Options

close


  • info Info

    There are only PDFs associated with this resource.

  • link PDF(s)



Cite this item close

APA

Cras ut cursus ante, a fringilla nunc. Mauris lorem nunc, cursus sit amet enim ac, vehicula vestibulum mi. Mauris viverra nisl vel enim faucibus porta. Praesent sit amet ornare diam, non finibus nulla.

MLA

Cras efficitur magna et sapien varius, luctus ullamcorper dolor convallis. Orci varius natoque penatibus et magnis dis parturient montes, nascetur ridiculus mus. Fusce sit amet justo ut erat laoreet congue sed a ante.

CHICAGO

Phasellus ornare in augue eu imperdiet. Donec malesuada sapien ante, at vehicula orci tempor molestie. Proin vitae urna elit. Pellentesque vitae nisi et diam euismod malesuada aliquet non erat.

WIKIPEDIA

Nunc fringilla dolor ut dictum placerat. Proin ac neque rutrum, consectetur ligula id, laoreet ligula. Nulla lorem massa, consectetur vitae consequat in, lobortis at dolor. Nunc sed leo odio.