1

Geneticscreenforpost-embryonicdevelopmentinthe

zebrafish(Daniorerio):dominantmutationsaffecting

adultform

KatrinHenke*,1,JacobM.Daane*2,M.BrentHawkins*!,ChristopherM.Dooley‡,ElisabethM.

Busch-Nentwich‡§,DerekL.Stemple‡,MatthewP.Harris*,1

*DepartmentofOrthopedicSurgeryResearch,BostonChildren’sHospital;Departmentof

Genetics,HarvardMedicalSchool,Boston,MA02115!DepartmentofOrganismicandEvolutionaryBiology,HarvardUniversity,CambridgeMA,

02138‡WellcomeTrustSangerInstitute,Hinxton,UKCB101SA§DepartmentofMedicine,UniversityofCambridge,Cambridge,UK

1towhomcorrespondenceshouldbesent,khenke@genetics.med.harvard.edu,

harris@genetics.med.harvard.edu

2Currentaddress:DepartmentofMarineandEnvironmentalSciences,Northeastern

UniversityMarineScienceCentre,Nahant,MA01908

Genetics: Early Online, published on August 23, 2017 as 10.1534/genetics.117.300187

Copyright 2017.

2

Abstract

Large-scaleforwardgeneticscreenshavebeeninstrumentalforidentifyinggenesthat

regulatedevelopment,homeostasis,regeneration,aswellasthemechanismsofdisease.

Thezebrafish,Daniorerio,isanestablishedgeneticanddevelopmentalmodelusedin

geneticscreenstouncovergenesnecessaryforearlydevelopment.However,the

regulationofpost-embryonicdevelopmenthasreceivedlessattentionasthesescreensare

morelaborintensiveandrequireextensiveresources.Thelackofsystematicinterrogation

oflatedevelopmentleaveslargeaspectsofthegeneticregulationofadultformand

physiologyunresolved.Tounderstandthegeneticcontrolofpost-embryonicdevelopment,

weperformedadominantscreenforphenotypesaffectingtheadultzebrafish.Inour

screenweidentified72adultviablemutantsshowingchangesintheshapeoftheskeleton

aswellasdefectsinpigmentation.Forefficientmappingofthesemutantsandmutation

identification,wedevisedanewmappingstrategybasedonidentificationofmutant-

specifichaplotypes.Usingthismethodincombinationwithacandidategeneapproach,we

wereabletoidentifylinkedmutationsfor22outof25mutantsanalyzed.Broadly,our

mutationalanalysissuggeststhattherearekeygenesandpathwaysassociatedwithlate

development.Manyofthesepathwaysaresharedwithhumansandareaffectedinvarious

diseaseconditions,suggestingconstraintinthegeneticpathwaysthatcanleadtochangein

adultform.Takentogethertheseresultsshowthatdominantscreensareafeasibleand

productivemeanstoidentifymutationsthatcanfurtherourunderstandingofgene

functionduringpost-embryonicdevelopmentandindisease.

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Introduction

Theuseofsystematicforwardgeneticscreenshasbeeninstrumentalinuncoveringgenes

andpathwaysinvolvedinamultitudeofdevelopmentalprocesses(e.g.Brenner1974;

Nüsslein-VolhardandWieschaus1980;Mayeretal.1991;Haffter,Granato,etal.1996;

Drieveretal.1996).Thisphenotypedrivenapproachallowsfortheunbiasedanalysisof

genefunctionthroughgenerationofrandommutationsthroughoutthegenomeusing

chemicalsorirradiationasmutagens.

Manygenesthatarefoundtobeessentialforearlydevelopmentandfunctional

alterationsareoftennotcompatiblewithviability.Suchlethalityhindersthestudyofgene

functionduringpostembryonicstages.Theestablishmentoftissuespecificorinducible

knock-outlinescircumventsthisproblemandenablesanalysisintissuesofinterestorat

specifictime-pointsduringdevelopment.However,thesemethodsdonotlendthemselves

tobroadunbiasedscreeningindevelopment,asoftenonlyafewlocicanbefeasiblytested

atanyonetime.Differenttypesofmutationssuchaspartialloss-of-functionordominant

mutationscanhelpinelucidatingfunctionsinlatedevelopmenteveningeneswithkey

rolesinembryogenesis.Dominantmutations,inparticular,canberevealingofthefull

rangeofmolecularanddevelopmentalgenefunctions,asincreasedandnovelactionsofa

genecanresultinunexpectedphenotypes.Thesedominantmutationscanalsoexhibit

dosagedependenteffects,showinggradedphenotypicdifferencesbetweenheterozygous

andhomozygousindividuals.Thus,uniquemutationsapartfromcompleteloss-of-function

allelescanbeinformativeaboutmolecularregulationofgenefunctioninpost-embryonic

development.

Thezebrafishisawell-establishedgeneticmodel.Screenshavefocusedonthe

identificationofgenesimportantforearlydevelopmentalprocesses,withmutantsshowing

recessiveinheritanceofthephenotype(e.g.Haffter,Granato,etal.1996;Drieveretal.

1996).Screensforrecessivemutationsrequirebreedingtheinducedmutationsto

homozygosityandthereforemultiplegenerationsneedtoberaisedbeforeaphenotypeis

visibleintheF3generation.Thus,recessivescreensrequiretheabilitytoraiseandscreena

largenumberoffishinordertoscreenthefunctionofgenesaffectingaspecific

developmentalprocessinsufficientdepth.

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Themajorityoftheidentifiedrecessivemutantsdisplayearlyphenotypesandare

embryonicorlarvallethal.Onlyabout3%ofmutantsidentifiedinthesescreensforearly

larvalphenotypesledtoviableadultswithobservablephenotypes(Haffter,Granato,etal.

1996).Thus,muchofthegeneticregulationoflatedevelopmentremainsundescribed.

Fewscreenshavelookedforgenesaffectinglatedevelopmentandmosthavebeen

restrictedindepthandphenotypicbreadth(Haffter,Odenthal,etal.1996;BauerandGoetz

2001;Fisher,Jagadeeswaran,andHalpern2003;Andreevaetal.2011;Saitoetal.2011).

However,largerscreenslookingspecificallyforgenesnecessaryfornormalpatterningand

growthoftheadultfish,demonstratedthatalargenumberofmutantscouldbeidentified,

supportingthatlatedevelopmentalprocessescanbeinvestigatedbyclassicgenetic

approaches(ZFmodels;www.zf-health.org/zf-models).Incomparison,thelargestofthese

screensfromtheZFmodelsconsortiumscoredabout1000genomesforadulttraits,about

1/6ofthetotalpredictednumberofgenomesanalyzedforlarvalphenotypesinthisscreen

aswellasthenumberofcombinedgenomesscreenedintheearlyzygoticscreens(Haffter,

Granato,etal.1996;Drieveretal.1996).Intriguingly,manyofthemutationsidentified

fromtheseadultscreensaffectgeneswhoseorthologuesareassociatedwithdiseasein

humans(fgfr1a,col1a1a,bmp1a,edar)(Rohneretal.2009;Fisher,Jagadeeswaran,and

Halpern2003;Asharanietal.2012,1;Harrisetal.2008).

Whileisolatingmutantlinesthroughgeneticscreenshasbeenverysuccessful,

identificationofthecausativemutationsunderlyingmutantphenotypeswaspreviously

difficult,limitingthebroadanalysisoflargeclassesofmutantsandpreventingdetailingof

theircognizantgeneticpathways.Theadventofnext-generationsequencingtechniquesin

combinationwithtargetedcaptureandmultiplexingofsampleshasallowedforcost-

effectiveandefficientidentificationofmutations(Bowenetal.2012;Leshchineretal.

2012;Vozetal.2012;Obholzeretal.2012;Henke,Bowen,andHarris2013;Kettleborough

etal.2013;Ryanetal.2013).Thishasopenedthepotentialforanalysisofgenetic

networksregulatingspecificdevelopmentalprocesses,aslinkageandpotentialcausative

mutationscanbequicklydefinedinwholesetsofmutants.

Wesoughttotakeadvantageofthesenewsequencingtechnologiestoexplorethe

utilityofzebrafishgeneticscreenstoinvestigatethegenescontrollingpost-embryonic

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development.Towardsthisend,weinitiatedascreenformutationsaffectingtheformof

theadultzebrafish.Tofacilitatedepthofscreening,wefocusedonmutantswitha

dominanteffectonmorphology.Importantly,unlikemostallelesidentifiedorcreatedusing

genomeeditingtechniques,ourfocusondominantmutationswascenteredontheabilityto

provideinsightintothemolecularactionofageneotherthansimpleloss-of-function

allelesthroughidentificationofpotentialgain-of-functionandneomorphicalleles.Herewe

showthefeasibilityoflarge-scaledominantscreensinzebrafishforpost-embryonic

development,includingmethodstosystematicallyidentifylinkageandcausativemutations

underlyingdominantmutantphenotypes.Resultsfromthisscreendefineimportant

diseasemodelsinthezebrafish.Furthermore,byclusteringanalysisofsimilarphenotypes

weshowenrichmentformutationsaffectingextracellularmatrixformationasaregulator

oflatedevelopment.Importantly,thisscreensetsthestagefortheuseofzebrafishasan

experimentaltooltoinvestigategeneticregulationthroughmodifieranalysisandefficient

identificationofgenenetworksregulatinglatedevelopment.

Methods

Husbandryandmanagementofidentifiedmutantlines

Zebrafish(Daniorerio)wereraisedandmaintainedunderstandardconditions(Nüsslein-

VolhardandDahm2002)incompliancewithinternalregulatoryreviewatBoston

Children’sHospital.MutantlineswerenamedfollowingtherulessetoutbyZFIN,where

‘mh’isthedesignationofthefoundinglab(Harrislab)and‘d’indicatesdominant

inheritanceoftheallele.

Mutagenesisandscreendesign

Toidentifydominantmutationsthataffecttheadultformofthezebrafish,mutationswere

inducedbytreatmentof30wildtypeTübingenmaleswithN-ethyl-N-nitrosourea(ENU)

treatmentfollowinganoptimizedprotocol(Rohneretal.2011).Thesurviving14

mutagenizedmaleswerematedtwiceaweekwithwildtypefemalesandover14.000

progenywereraised.At10-12weekspost-fertilization(wpf)F1fishwereanesthetizedin

0.02%MS-222andscreenedunderadissectingscopeformorphologicalchanges.Potential

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mutantswereisolatedandcrossedtowildtypefish.Similarly,F2progenywerescreenedat

10-12wpf.Crossesre-expressingthephenotypeinroughly50%ofprogenywere

maintainedandfurtheranalyzed.Variedwildtypelines,suchasAB,Tübingenandalbino

mutants,wereusedforoutcrossing.

Next-generationsequencinglibrarypreparationandexomecapture

Exomesequencingwasperformedaspreviouslydescribed(Kettleboroughetal.

2013)withminormodifications.Briefly,1-2μgofDNAfromindividualmutantsandpooled

siblingswasfragmentedtoenabletheconstructionof150bpto200bpinsertlibraries

accordingtostandardIlluminaprotocols.Followinggenomiclibraryconstruction,500ngof

DNAwashybridizedfor24hourstotheAgilentSureSelectZv9.2biotinylatedwholeexome

RNAbaits.Thehybridizationwasthenenrichedwithstreptavidin-coatedbeadsandthe

restoftheRNAbaitsweredigested.LibrarieswereamplifiedandbarcodedthroughPCR

(10cycles)andlibrarieswerepaired-endsequenced(50bp)usingHiSeq2000v4

chemistries.AsthisversionofthezebrafishexomecaptureisbasedontheZv9genome

assembly,genesandexonsannotatedsubsequenttotheZv9releasewerenotbecaptured

bythismethod.

Mappingbyassociationandcandidategeneidentification

Sequencingreadswerealignedtothezebrafishgenome(GRCz10)usingNovoalign

software(http://www.novocraft.com/main/index.php)withdefaultsettingsandincluding

3’-adaptortrimming.PCRduplicateswereremovedusingtheMarkDuplicatescommandin

Picard(http://picard.sourceforge.net/).VariantswerecalledusingSAMtoolsandBCFtools

(Lietal.2009;Li2011).ToidentifySNPsusefulformapping,weusedtheGATK

VariantFiltrationWalker(McKennaetal.2010)toexcludethefollowingvariants:(1)SNPs

lyinginlow-complexityregionsorinterspersedrepeats,classifiedbyRepeatMasker;(2)

SNPslyinginaclusterof>3SNPsper10bp;(3)SNPswithaqualityscore<30;(4)SNPs

witharoot-mean-squaremappingqualityofcoveringreads<40;(5)SNPswithatotalread

depth<20or>100;(6)SNPswithatotalreaddepth<40.Furthermore,SNPswithanallele

frequencyof1wereexcluded.Forexclusionofcommonvariants,SNPspresentinatleast2

readsinsiblingsormutantswereidentifiedfromtheindividualvariantfilesusingacustom

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pythonscript.AfterremovingSNPspresentinthesiblings,themutantchromosomewas

identifiedbycountingtheremainingSNPsperchromosome.

Toidentifycandidatemutations,themutantvariantfilewasfilteredusingGATK

VariantFiltrationWalkertoexcludethefollowingvariants:(1)SNPslyinginaclusterof>3

SNPsper10bp;(2)SNPswithaqualityscore<30;(4)SNPswitharoot-mean-square

mappingqualityofcoveringreads<40;and(5)SNPswithatotalreaddepth<3or>60.In

addition,allSNPsthatwerecalledashavingmorethan1non-referenceallelewere

removed.SNPspresentinatleast2readsinwildtypestrains,siblingsorothermutants

fromthescreenwereexcludedusingacustompythonscript.TheremainingSNPswere

analyzedandannotatedusingtheEnsemblVariantEffectPredictor(McLarenetal.2016).

Thisstringentfilteringprotocol,whileneededtoexcludefalsepositivecalls,canleadtothe

exclusionofrealvariants.Forexample,themostlikelycandidatemutationincol2a1ainthe

dmh28mutantwascomputationallyexcludedasacandidatemutationduetoalowPhred-

scaledqualityscore.WhileweidentifiedacloselylinkedSNPinaneighboringgeneused

fortheinitiallinkageanalysis,reanalysisofvariantsincandidategenesintheregionbyeye

ledtotheidentificationofthemutationincol2a1a.

MappingandcandidategenedatawasvisualizedusingPhenoGram(Wolfeetal.

2013).

Confirmationoflinkage

Toconfirmlinkageofthephenotypetoaspecificcandidatemutation,genomicDNA

fromatleast10heterozygousmutantsand4wildtypesiblingswasSangersequencedto

confirmpresenceorabsenceofthemutationrespectively.Asameasureforlinkage,we

calculatedthelogarithmoftheodds(LOD)scoreforeachtestedvariant.

LODscore=log[probabilitythelocusislinked/probabilitythelocusisunlinked]

probabilitylocusisunlinked=0.5^numberofmeiosisanalyzed

probabilitylocusislinked=1-recombinationfrequency

recombinationfrequency=numberofrecombinants/numberofmeiosisanalyzed

Generally,aLODscoreof3oraboveisconsideredasproofoflinkageoftwoloci.Inour

case,ifnorecombinationeventwasdetectedin10heterozygousmutantsanalyzed(10

meiosis),theLODscorewouldequal3.

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Skeletalstainingandmicrocomputedtomography

Fishwereeuthanizedin22%MS-222andfixedin3.7%formaldehydefor24hours

atroomtemperature.Forstainingofmineralizedtissuesfishweretransferredintoa

0.005%alizarinred/0.5%KOHsolutionovernight.Excessstainwasremovedwithseveral

washesin0.5%KOH.Specimensweretransferredthroughaglycerolseriesinto80%

glycerolforstorage.

Forscanning,fishwerewashedinPBSorwaterfollowingfixationandthenplacedin

sampletubesembeddedin1%agarosetoreducemovementduringscanning.Imageswere

obtainedusingaSkyscan1173(Bruker),240-degreescanwith0.2rotationalstep.X-ray

sourcevoltagesetto70kVandcurrentsetto80μA.Exposuretimewas1500ms.

Resolutionofscanwas7.14micronsperpixel.ImageswereprocessedusingParaview

(http://www.paraview.org/overview/)orAmirasoftwarepackage,version6.0(FEI).

Dataavailability

Spermfromthe25mutantlinesdescribedinthispaperispreservedtosecurethe

lines.Mutantlinesareavailableonrequestfromtheauthor.

FileS1containstotalandmutantspecificSNPcountsforeachchromosomeforthe

23mutantsmappedwiththemethoddescribedinthismanuscript.FileS2contains

numbersofuniquenonsynonymousmutationsperchromosomeforthe23mutants.Alist

ofprimersusedforconfirmationoflinkageisprovidedinTableS1.

RawsequencedatawassubmittedtotheEuropeanNucleotideArchive(ENA)under

studyPRJEB13615(http://www.ebi.ac.uk/ena/data/view/PRJEB13615).Accession

numbersforindividualsequencingfilescanbefoundinFileS3.

Customscriptsfordataanalysisareavailablefordownloadfromthelabwebsite

www.fishyskeleton.comoruponrequest.

9

Results

ScreenDesign

Tofacilitateabroadsamplingofgeneticvariationthroughoutthezebrafishgenome,we

usedENUmutagenesisandanF1dominantscreendesign(Fig.1A).Wescreened10424

fish,representingasimilarnumberofhaploidgenomes,forphenotypesaffectingadult

form.Weidentified269potentialmutantsaffectingawidediversityoftraits.These

mutantsallshowedspecific,qualitativelydistinctadultphenotypes.Wefocusedrecovery

andisolationtowardsmutantsthatexhibiteddifferencesintheformationandpatterningof

theadultskeleton,includingfinsandscales,aswellaschangesinpigmentation(Table1).

Screeningforchangesinreproductiveability,anotherkeyadulttrait,wasnotpossible,as

reductionoffertilitywouldprecluderecoveryofthemutantlineinanF1screendesign.

Otherauxiliaryphenotypesincludedefectsindevelopmentoftheeyes,representedbya

largenumberoffishexhibitingcloudylenses.Thesewerenotcollectednorincludedinthe

screenresults.

Toconfirmtruemonogenicinheritanceofaphenotype,isolatedF1mutantfounders

wereoutcrossedtowildtypefishandtheF2generationwasscreenedfortheparental

phenotype.Outofthe269potentialmutantsisolatedintheF1generation,135produced

offspring.Inthese135crosses,72showedtheexpectedphenotypein50%oftheoffspring,

suggestingamonogenictrait.Thesemutantsweregroupedinto5majorclassesbasedon

theirmostprominentphenotype.Agroupof23mutantsshowedchangesinthe

developmentoffins;21mutantsarecharacterizedbychangesinstature;thecraniofacial

skeletonwasaffectedin12mutants;4mutantsshowedabroaderchangeinthedermal

skeletonand12mutantsdisplayedchangesinpigmentation(Table1).Aselectgroupof

25mutantsfromtheseclassesweremaintainedformapping,mutationidentificationand

furtherphenotypicanalysis.

Systematicmappingofdominantmutationsfromthescreen

Acandidategeneapproachwasusedtosuccessfullyidentifymutationsintwoofthe

25mutantsisolated,dm1anddmh3(seemutantphenotypessectionbelow).Forlinkage

10

analysisandmutationidentificationoftheremaining23mutants,DNAwasisolatedfroma

singlemutantcarrierand3-6wildtypesiblingsforeachmutant(TableS2).Mostprogeny

stemmedfromheterozygousmutantoutcrossestowildtypestrains,for6mutantsprogeny

fromheterozygousmutantincrosseswereanalyzed.Sequencinglibrarieswereprepared

foreachsinglemutantandpooledwildtypesiblings.Thesepoolswereenrichedforcoding

regionsusingexomecapture.PairedendIlluminasequencingwasperformedon8libraries

perlane(fourmutant/siblingpairs),resultingin15-29millionreadspersampleand

averageexomecoverageof23x(TableS2).

Twooverlappingbutdistinctmethodswereusedtoroughmapandrefinepotential

causativemutationsunderlyingthedominantphenotypes.First,tolocalizethegenetic

locationofthemutationinthegenome,thechromosomecarryingmutant-specificSNPs

wasidentified(Figure1B).Second,throughidentificationofuniquenon-synonymous

mutationsinthemutant,candidatemutationspotentiallyunderlyingthemutantphenotype

weredefinedforsubsequentconfirmationoflinkage.

Identificationofmutant-specifichaplotypes:Giventhehighlevelofheterogeneity

ofthezebrafishgenomeevenwithinstrains(Bowenetal.2012;Stickneyetal.2002;

Guryevetal.2006;Bradleyetal.2007;Coeetal.2009),causativemutationsshouldbe

associatedwithdiscernablehaplotypeblocksofvariantsuniquetothemutant.Wedefined

mutant-specifichaplotypessolelybythepresenceofSNPsuniquetothemutant.These

SNPscanbeidentifiedbyexcludingallSNPsseeninthesiblingpoolfromSNPspresentin

themutant(Figure1B).Ifallhaplotypespresentinthecrossanalyzedaresampledand

sequencedtosufficientdepthinthesiblingpool(FigureS1),onlythechromosome

carryingthephenotype-causingmutationshouldhaveSNPsleftafterthisanalysis.

Tothisend,sequencingreadswerealignedtothecurrentassemblyofthezebrafish

genome(GRCz10)andhighqualitySNPswereidentifiedthatshowedatleast20xcoverage

inboththemutantandcorrespondingsiblingpool(seemethodsfordetails).Althoughwe

achievedaverageexomecoverageof23x,onlyabout50%oftheexomeiscoveredbymore

than20readsandthusisusedintheanalysis(TableS2).Thisislikelyduetounequal

captureefficiencyofdifferentexonsandpreferentialsequencingofcertainregions.SNPs

thatwerehomogeneousinboththemutantandsiblingpoolwereexcludedfromthe

analysisasuninformative.Thisresultedintheidentificationofbetween450and7000

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SNPsperchromosome(Figure2A-B,FileS1).Inthemutantsanalyzedhere,removalof

SNPsfoundinthecorrespondingsiblingpoolresultedinreductionoftotalSNPsto

between0and1469SNPsperchromosome.ThehighestnumberofmutantspecificSNPs

onachromosomewithinthegenomevariessignificantlybetweendifferentmutantlines

analyzedandrangesfrom106to1469(FileS1).Thisvariationhighlightsthedifferent

levelsofbackgroundheterogeneityinthecrossesanalyzed.Althoughonewouldexpectno

mutantspecificSNPsforunlinkedchromosomes,mostchromosomesshowedarelatively

lowlevelofmutant-specificSNPs.Forexample,analysisofthesequencedatafordmh35

andthecorrespondingsiblingpoolresultedintheidentificationof1-135mutantspecific

SNPsperchromosomeforunlinkedchromosomes(Figure2A-B).Chromosome20showed

with449SNPsthehighestnumberofmutantspecificSNPsandwassubsequentlyshownto

belinkedtothemutantphenotype,asdescribedbelow.

Totesttheboundsofsampling,for2mutants,dmh15anddmh4,wesequencedtwo

poolsof3siblingstoadepthof20xeach.Inbothcases,theremainingnumbersofmutant

specificSNPsperchromosomeweresignificantlylowerwhencomparedtoothermutants

whereonlyonepoolof3siblingsorapoolof6siblingswassequencedto20xdepth(File

S1,TableS3).Thisismostlikelyduetounequalsequencingofhaplotypespresentinthe

poolaswellasinsufficientsequencingdepthtocoverallhaplotypes.Differencesin

samplingofparentalhaplotypeswereobviouswhenrunningtheanalysiswithonlyoneof

thesiblingpools.Forexample,forthedmh15mutant,chromosome5hasthehighest

numberofmutantspecificSNPswith534whenonlyusingsiblingpool1(TableS3).

However,thechromosomeharboringthehighestnumberofSNPsswitchestochromosome

7with866mutantspecificSNPsifonlythesecondsiblingpoolisusedintheanalysis

(TableS3).BothchromosomesshowalmostnomutantspecificSNPswhenbothsibling

poolsareusedincombination,indicatingthatthehaplotypepresentinthemutantfor

thesetwochromosomeswasonlysampledinoneofthesiblingpools.

InadditiontoasecondsiblingDNApool,wealsosequencedasecondmutant

individualfordmh15.Byrepeatingtheexperimentwithadifferentmutant,wenoticedthat

notallchromosomesshowedthesametrendasseenwiththefirstcomparison(TableS3).

Morespecifically,chromosome4showedanextremelyhighnumberofmutantspecific

SNPsinthesecondmutant,whereasthefirstmutantsequencedhadalmostnomutant-

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specificSNPsonchromosome4.Thisindicatesthatthechromosomalhaplotypeofthe

secondmutantwasinsufficientlysampledinthetwosiblingpools.Itshouldbenotedthat

inthisexamplethechromosomecarryingthephenotype-causingmutation(chromosome

19),didnothaveahighnumberofmutantspecificSNPs.Thissuggeststhatthephenotype-

causingmutationdoesnotliewithinadiscernablehaplotypeinthisspecificexample.

IdentifyingcandidatemutationsthroughfilteringrelatedandcommonSNPs:Since

wholeexomesaresequencedbythismethod,thesequencingdatacanatthesametimebe

usedformappingaswellastheidentificationofpotentialcandidatemutationsaffecting

genefunction.First,allvariantswithatleast3xcoverageareidentifiedinthemutant

(Table2).Thiscoveragewasconsistentlyseenoveronaverage88%oftheexomeinall

sequencinglibraries(TableS2).Duetothehighrateofvariationwithinthezebrafish

genome,thisanalysisresultsinahighnumberofpotentialphenotype-causingmutations.

Torefinethislistofpotentialcandidatemutationsfurther,allSNPsthatarepresentinthe

correspondingwildtypesiblingsareexcluded.Thisreducesthenumberofpotential

candidatemutationssignificantly(Table2).Furthermore,takingadvantageofexisting

sequencingdatafromwildtypestrains(Bowenetal.2012),allwildtypevariantsobserved

inthisdatasetareexcludedaspotentialcandidatemutationsaswell.Inaddition,asalarge

numberofmutantsfromthesamedominantscreenweresequencedandshareacommon

setofbackgroundallelesfromthefounders,weusedthisdatatofurtherrefinepotential

candidatesbyexcludingallvariantsdetectedinwildtypesiblingpoolsormutantswith

phenotypesdifferentfromthemutantunderanalysis.ThiswayoffilteringsharedSNPs

reducesthenumberofnonsynonymousmutationsobservedgenome-widetoasmall

numberthatcanthenbeprioritizedbasedonpotentiallinkage,predictedgenefunction

andthenatureofthemutation(Table2,FileS2).Forthedmh35mutant,ouranalysis

identified41nonsynonymousmutationsgenomewide(Figure2C).Twelveofthese

mutationsarelocatedonchromosome20,thechromosomewiththehighestnumberof

mutantspecificSNPs(Figure2A-B).

ToconfirmlinkageofthephenotypetoaspecificSNP,regionscontainingthevariant

wereamplifiedfromgenomicDNAfromanumberofindividualheterozygousmutantsand

wildtypesiblingsandthenSangersequencedtoassesspresenceofthevariant.Withthis

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approach,wewereabletoidentifycloselylinkedmutationsfor>80%ofthemutants

sequenced(20/23;Table3).In14outofthe20mutants(70%),linkagewasconfirmedto

thechromosomewiththehighestorsecondhighestnumberofmutantspecificSNPsinthe

genome(TableS4).Forseveralmutantsthenumberofmutant-specificSNPsonthelinked

chromosomewasverylow(0-56)(FileS1,TableS3),suggestingthattherewasnota

discernablehaplotypeassociatedwiththemutation.Butevenincaseswhereourmapping

approachdidnotcorrectlypredictthechromosomecontainingthemutation,themost

likelycandidatemutationcouldbepredictedfor6ofthemutantsbyanalyzingthegenome-

widecandidatemutationlistafterfiltering(Table3).Asvariantsfoundinsiblingsare

excludedaspotentialcandidates,thistechniquereliesheavilyontheabilityto

phenotypicallydistinguishmutantsfromwildtypesiblings.Formutantswithincomplete

penetranceitcanbedifficulttoidentifycandidatemutationsifamutantwasimproperly

scoredasawildtypesiblingasbothphenotype-linkedSNPsandthecausativemutationwill

beremovedduringtheanalysis.Therefore,formutantswithweakphenotypesor

incompletepenetranceitmaybehelpfultoreanalyzepotentialvariantswithoutexclusion

ofsiblingSNPs,comparingagainstcommonSNPsfromwildtypesandunrelatedmutants.

Forthreeoutof23mutantsanalyzedbymapping,alinkedchromosomewasnot

identifiedattimeofpublication.Furtheranalysiswillbeneededtoassessifalternative

SNPsidentifiedinouranalysisshowlinkageoriftherewassamplingbiasstemmingfrom

theexomedesign.Regardless,over85%ofmutantsanalyzedweremapped.

Identifiedmutantphenotypesandunderlyinggeneticchanges

Formutantcharacterization,wefirstclusteredmutantsintobroadphenotypicclasses.

Followingthisclustering,wethencomparethesuiteofcandidategenesidentifiedineach

classtoassessaffectedgeneticpathwaysleadingtochangeinform.Here,wedetailhere

fourclassesofmutantscontaining25mutantstotal.Foreachmutantwehaveperformed

linkageanalysisandidentificationofpotentiallycausativemutations.Theremaining

mutantsfromthescreenarenolongeravailableandwerethereforeexcludedfromthe

manuscript.

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Mutantswithvertebraldefects

Agroupof11dominantmutants,identifiedbytheirstature,werekeptforfurtheranalysis.

Thesevertebralmutantsweregroupedintofiveclassesdescribedbelow.

ClassImutants:Fourofthesemutants(dmh13,dmh14,dmh15,dmh29)werecharacterized

byshortstature,withnoapparentlarvalphenotypeat5dayspostfertilization(dpf).

Analysisbymicrocomputedtomography(μCT)revealedstrongdeformationsofthe

vertebrae,especiallyofthehemalandneuralarches(Figure3B-D).Inaddition,wefound

excessiveboneformation,withtheformationofosteophytes,especiallyinthecentrumof

thevertebrae.Interestingly,mappingofthecausativechangeineachofthesemutants

indicatedthatall4mutantscarrymissensemutationsincomponentsofCollagentype1,

changingaconservedglycineinthetriple-helicaldomaintoadifferentaminoacid(Table

3).TheCollagen1proteinconsistsofthreeproteinchains,twoCollagen1a1(Col1a1)and

oneCollagen1a2(Col1a2)chainthatformatriplehelix.Properhelixformationrequiresa

glycineresidueineverythirdpositionofthehelicaldomainoftheprotein.Missense

mutationsintheseglycineresiduesareknowntoactinadominantfashion(Gajko-Galicka

2002).Stemmingfromthewholegenomeduplicationintheteleostcommonancestor,

zebrafishhavetwoparalogsofthecollagen1a1gene,col1a1aandcol1a1b.Boththedmh13

anddmh14mutantscarrymutationsinthecol1a1agene(G1093R;G1144E),whichare

closelylinked.Whilethedmh29mutantsexhibitmutationsinaglycineresidueofthe

col1a1bgene(G1123D),leadingtoaverysimilarphenotype.Theobservedphenotypeof

thesemutationsaresimilartobutmoreseverethanpreviouslyidentifiedcol1a1amutants

(Fisher,Jagadeeswaran,andHalpern2003;Asharanietal.2012).Thedmh15mutants

exhibitthestrongestphenotypeinthegroup,asheterozygousmutantsshowthegreatest

reductioninbodylength(Figure3D).Inthesemutants,wedetectedamutant-specific

nonsynonymousmutationincol1a2(G882D)linkedtothephenotype.Thephenotypic

similarity,takentogetherwiththepredictedfunctionalconsequenceoftheidentified

changes,makesthesemutationsincollagen1themostlikelycandidatesforcausingthe

phenotype.Interestingly,inthisphenotypicclasswewereabletoidentifydominant

mutationsinallcomponentsofCollagenI,expandingourknowledgeaboutphenotypic

consequencesofmutationsincollagen1overthepreviouslypublishedcol1a1aalleles.

15

ClassIImutants:Unlikethefirstclassofvertebralmutants,whichprimarily

exhibitedphenotypesarisinglateindevelopment,asecondgroupof4mutants(dmh21,

dmh27,dmh28,dmh30)showedalteredlengthalreadyaslarvaeat5dpf(Figure4B).A

fractionofthemutantprogenyshoweddeformationsofthenotochordat5dpfinaddition

toareductioninlength.Analysisoftheadultspineofthemutantsinthisclassrevealed

mostlynormalvertebralmorphology,withtheexceptionofafewfusedcentra(Figure3E).

Wefoundthatall4mutantsarelinkedtochromosome8.Fortwoofthemutants,dmh27

anddmh28,weidentifiedmissensemutationsinageneencodingacomponentoftypeII

Collagen.Similartocol1a1,zebrafishhavetwocopiesofthecol2a1gene,col2a1aand

col2a1b.Bothdmh27anddmh28werefoundtoharbormutationsincol2a1a.Whilethe

mutationindmh28changesaconservedglycineresiduetoasparticacid(G1141D),similar

tocol1amutantsweidentified,thedmh27alleleleadstoatruncationoftheC-terminalpart

oftheprotein(G1174X)(Table3)thatmayactinadominantnegativemanner.dmh21and

dmh30arelikelyclonalmutationsastheyshowthesamehaplotypepatternsurroundinga

linkedvariantinitpr3onchromosome8.Astheprecisemutationfortheseallelesisnot

known,wemaintaintheirseparatealleledesignation.Itpr3isincloseproximitytocol2a1a.

Thedmh21anddmh30mutantshoweverdonotshowanyobviousnonsynonymousor

splicesitemutationsinthepredictedcol2a1agene.Giventhesimilarityinphenotypeto

dmh27anddmh28,itislikelythatthesetooareallelesofcol2a1aandthatthemutations

areregulatoryorinnotwellannotatedisoformsofthegene.Furtherbiochemicalanalysis

ofCol2a1ainthesealleleswillneedtobecarriedouttotestthepotentialcausativeeffectof

theseunidentifiedmutations.

ClassIIImutants:Thedmh16mutantissuperficiallysimilartothemutantslinkedto

col2a1a,displayingshorteradultbodylengthearlyat5dpfandintheadult(Figure3F;

4C).μCTanalysishoweverrevealedthatthevertebralbodiesinthismutantarehighly

deformed,withcompositevertebraephenotype.Severalvertebraewerefoundtohave2or

morehemi-segments.Fusedvertebraewerealsocommonlynoted(Figure3F).Forthis

mutant,weshowlinkagetoamutationincalymmin(cmn)onchromosome12(Table3).

ThisgenewasdiscoveredinacDNAlibraryscreenthroughitsexpressioninthezebrafish

notochordandisthoughttobeanextracellularmatrixprotein(Cerdàetal.2002).The

16

identifiedmutation,M10R,resideswithinthesignalpeptideandcouldpotentiallyinterfere

withproperproteinlocalization.

ClassIVandVmutants:Twofurthermutantsshowdeformationinthespinebeyond

axialshortening.Thedmh31mutant(classIV)seemstobeproportionallynormalinlength

untilrightbeforethecaudalpeduncle,wherethevertebraearehighlydeformedandbend

(Fig.3G).Thisbendinthebodycanalreadybeseenat5dpfasakinkinthenotochord(Fig.

4D).Thedmh31phenotypeshowsstronglinkagetoamissensemutationinamyosin

heavychaingene,myosin,heavypolypeptide2,fastmusclespecific(myhz2).Themyhz2

geneencodesafastmuscle-specificmyosinheavychainandisexpressedinmusclesofthe

posteriorpartofthetrunkat3dpf(Pengetal.2002;Nordetal.2014),coincidingwiththe

positionofthedeformationsofthetrunkseenindmh31mutants(Fig4D).Theidentified

mutationsubstitutesahighlyconservedmethionineinthemyosintail1domainforan

isoleucine(M543I;Table3)andispredictedtobedeleterious.

TheclassVmutant,dmh4,ischaracterizedbyastrongcurvatureinthespine.

Whereastheembryosappeartobeperfectlynormalat5dpf,multiplebendsindorsal,

ventralandlateralorientationcanbedetectedintheadult(Figure3H).Apartfromthe

strongcurvature,thevertebralbodiesseemtobepatternedcorrectlywhenanalyzedby

μCTimaging.Wedididentifyacloselylinkedpredicteddeleteriousmutationinan

uncharacterizedprotein,CU929145.1,withsequencesimilaritytoapredictedmyloid

differentiationmarker-likeprotein2(XP_005174257.1;Table3).Asforgenesinwhichjust

asingleallelewasidentified,furtherexperimentswillbeneededtoshowcausalityofthe

identifiedchange.

Mutationsbroadlyaffectingthedermalskeleton

Fourmutantswereisolatedwithbroaddefectsontheformationand/orpatterningofthe

dermalskeleton.Oneofthehallmarkcharactersthatdifferentiatesthesemutantsfrom

otherclassessuchasfins,whicharealsoconstrainedcomponentsofthedermalskeleton,is

theireffectonscalesand/ordermalbonesoftheskull,suchasthebonecoveringthegills,

theopercle,subopercleandbranchiostegalrays.

Thefirstmutantinthisclass,dmh19,showsaverymildscalephenotype

characterizedbyirregularlysizedandpatternedscales(Fig.5B).Inaddition,themouthis

17

positionedslightlymoresuperiorlyinmutantswhencomparedtowildtypesiblings.Our

mappingapproachindicatedlinkagetochromosome8andidentifiedasingleunique

variantonthischromosomeinageneencodingProteinkinaseCzeta(prkcz).Sequencingof

individualmutantcarriersshowedstronglinkagetothischange,howeverthemutationis

predictedtobetolerated.Furtheranalysisoftheregionwillbeneededtoprovecausality

ofthischangeortoidentifyalternativepotentialcandidatemutations.

Thedmh3mutantischaracterizedbyshorteranddeformedfinraysandhasfewer

butlargerscales(Fig.5C).Dmh3homozygousmutantsshowastronglyenhanced

phenotypewithacompletelossofthedermalcomponentofthefinsandcompletelossof

scales(Fig.5D).Duetothesimilaritiesinphenotypeofthedmh3mutanttopreviously

publishedmutantswithdefectsinectodysplasinsignaling(Harrisetal.2008),wechoosea

candidategeneapproachtoidentifypotentialphenotype-causingmutations.Weisolated

cDNAfromheterozygousdmh3mutantsandsequencedthecodingregionofectodysplasin

pathwaygenes.Thisledtotheisolationofalinkedmissensemutationintheectodysplasin

Areceptor(edar)gene,changingaconservedasparticacidinthedeath-domaintoa

tyrosine(D409Y).

Heterozygousdm18mutantsshowcharacteristicdefectsinthefronto-nasalaspect

oftheskullleadingtotheformationofanotchrightbehindthemaxilla(Fig.5E).Thescales

areirregularlysizedandshowchangesinpatternandorientation.Someofthefinrays

appeartobetruncated,whileotherraysinthesamefinappearnormalinsize.These

phenotypesarestronglyenhancedinthehomozygousmutants(Fig.5F).Herethescales

aregenerallysmallerthaninwildtypeandgrowinnoobviouspatternororientation.The

skullshowsextremefronto-nasalshorteninggeneratingaverysmallandroundshape.Ina

largepercentageofmutants,thegillcoverisverysmallandfailstocoverthefullextentof

thegills.Inaddition,thefinraysseemtoconvergedistally.

Thelastmutantinthisgroup,dmh22,showsmildirregularitiesinscalesizeand

patterning,mostlylocatedtowardthefrontofthetrunk(Fig.5G).Thefinsdisplayvarying

degreesofdefects,rangingfromareductioninthenumberofraystodeformedraysand

raysegments.Hereagain,thehomozygousmutantphenotypeismoreextreme.Thescales

inhomozygousdmh22mutantsgrowinallorientationsanddon’thaveanypatternor

18

consistentsize(Fig.5H).Thefinphenotypesrangefromcompletelossoffinstoastrong

decreaseinfinrayandraysegmentnumber.

Forboththedmh18anddmh22mutantsavariantlinkedtothephenotypecouldnot

beidentifiedattimeofpublication.

Mutantswithspecificdefectsinfinformation

Fivemutantswerefurtheranalyzedthatshowspecificalterationsinthefinswhen

comparedtotheentireskeleton.Mutantsinthisclasscouldgenerallybegroupedinto2

subclasses:1)mutantswithnormalpatternedbutshorterfinsand2)mutantswith

deformedfins.Nolong-finnedmutantswerefound.

Fromthefirstsubclass,wefurtheranalyzedandsequencedonemutantwith

shorterfins,dmh35.Indmh35boththepairedandmedianfinsarenormallypatternedbut

shorterduetoshorteningofthefinraysegments(Figure6B).Sequencinganalysisof

dmh35revealedamissensemutationinconnexin43(cx43)onchromosome20(Table3).

Connexin43isacomponentofgapjunctionsandmutationsincx43havebeenshown

previouslytocausetheshortfinphenotype(Iovineetal.2005).

Fromthesecondsubclassclassoffinmutantsweanalyzedfourmutantsdmh20,

dmh32,dmh33anddmh34.Here,thepelvicfinsaremostseverelyaffectedinthatthey

showpartialtruncationsoffinraysandgenerallyunequallengthofsegmentswithinafin

(Figure6C,E).Finsingeneralshowbendingoffinraysinvaryingextent.Allfourmutants

inthisclassshowstronglinkagetomissensemutationsinthenotchliganddelta-like4

(dll4)(Table3).Thechangeindmh20mutatesaconservedhistidineatposition190inthe

deltaserrateligand(DSL)domainindll4toanasparagine,whilethemutationsindmh32,

dmh33anddmh34leadtoearlytruncationsoftheprotein,partiallydeletingtheepidermal

growth-factor(EGF)domainsaswellasthepredictedtransmembranedomain.Thesedata

supportpreviousfindingsthathaploinsufficiencyindll4canleadtofindeformationsin

adultzebrafish(Leslieetal.2007).

Pigmentmutants

Theadultpigmentpatternisspecifiedearlyduringpostembryonicdevelopment.The

majorityofpigmentationmutantsweidentifiedexhibitedalteredstripepatterns(Figure

19

7),someofwhichcloselyresembledphenotypesidentifiedinpreviousscreens(Haffter,

Odenthal,etal.1996).Twomutants,dmh1anddmh9,displayedfewermelanophore

stripesandwiderinterstriperegions(Fig.7B),resemblingthedominantphenotype

previouslydescribedfortheobelix/jaguarmutantsalteringkcnj13/kir7.1(Iwashitaetal.

2006).Toidentifypotentialcandidatemutationsindmh1,apoolofninehomozygous

mutantswassequencedandallhomozygousmutationsinthegenomewereidentified.

Afterexcludingknownbackgroundvariantsasdescribedbefore,thelistofgeneswas

scannedforpotentialcandidatemutationsrevealingamutationinkcnj13/kir7.1.Similarly,

mappingofdmh9bytheapproachdescribedbeforealsorevealedamissensemutationin

kcnj13/kir7.1thatiscloselylinkedtothephenotype(Table3).Thedmh7anddmh8

mutantswereidentifiedbytheformationofpigmentspotsinsteadofstripes(Fig.7D),

resemblingthephenotypepreviouslydescribedfortheleopardmutant(Watanabeetal.

2006).Herewefoundmissensemutationsinconnexin41.8(cx41.8)(Table3),agap

junctionproteinshowntocausetheleopardphenotypewhenmutated.Thus,these

phenotypesarelikelycausedbythesemutationsandrepresentadditionaldominant

obelix/jaguarorleopardallelesrespectively.

Theotherpigmentationmutant,dmh11,remainsun-mappedaslinkagehasnotyet

beenidentified.Thephenotypeischaracterizedbymildirregularitiesinthemelanophore

stripe,leadingtoawavyappearanceofthestripes(Fig.7E).Similartotheotherpigment

mutants,thismutantshowsadosedependenteffectashomozygotesexhibitastrikingly

enhancedphenotype(Figure7C,F).

Discussion

Geneticapproachestounderstandthedevelopmentalregulationofadultformare

limitedbytheessentialfunctionofmanygenesduringearlydevelopment.Toexpand

forwardgeneticapproachesforinvestigatinglatedevelopment,weshowthatdominant

screensinzebrafishareefficientinidentifyingawidespectrumofmutantsthatretain

viabilityinadults.Tomakefulluseofthesemutantcollections,itisessentialtobeableto

identifytheunderlyingmutationsinafastandefficientmanner.

20

Establishinghigh-throughputmappingstrategiestosupportsystematicanalysisof

dominantmutations

Thecurrentstrategyforidentifyingmutationsinzebrafishbymassivelyparallel

sequencingreliesonbulkedsegregantanalysis,inwhichalocuscontainingthemutationis

identifiedduetoretainedhomogeneityafterrecombination(Bowenetal.2012;Leshchiner

etal.2012;Obholzeretal.2012;Vozetal.2012).Asmanydominantmutantsdonothavea

discernablehomozygousphenotypeorarehomozygouslethal,typicallythewildtypelocus

ismappedtodefinealinkedintervalbyhomogeneity(reversemapping)(Smithetal.

2016).Thiscanbeproblematicwhenworkingwithhighlypolymorphic,non-inbred

genomes,likethatofthezebrafish,asthewildtypelocuswilllikelybeheterogeneousand

thusnon-informativeforhomogeneitymapping.Toeliminatetheneedtoperformmultiple

andspecificcrossestoreducethepotentialpitfallsofpolymorphicwildtypeloci,we

devisedanewstrategytofacilitateidentificationofdominantmutations.Ourprotocoluses

identificationofphenotype-associatedhaplotypesratherthenhomogeneity,todefinea

linkedchromosomeincombinationwithgenomewideanalysisofpotentialcandidate

mutations.Thisstrategyisfastandpermitssystematicassessmentoflargenumbersof

mutantswithinphenotypicclasses.

Althoughweuseexomesequencing,thisstrategycaneasilybeappliedtowhole

genomesequencingdata.Thehigherlevelofpolymorphismsinnon-codingregionsmay

bolstertheabilitytoidentifymutantspecifichaplotypesandcouldthereforeincreasethe

accuracyoftheapproach.However,gettingcomparablesequencingdepthcriticalforthis

analysiswillsubstantiallyincreasethecostpermutantanalyzed.Performingadditional

wholegenomesequencingonjustthemutantsamplemaybebeneficialincaseswhere

linkageisdetectedbutviablecandidatemutation(s)remainelusive.Somephenotype-

causingmutationsmayresidewithinanon-codingregionorinageneorexonnotincluded

intheexomecapturedesign,thereforenotrecovered.Sincetheapproachdescribedhere

doesnotdefineasmall,linkedregiononachromosome,thehighlevelofpolymorphismsin

non-codingregionswillleadtotheidentificationofalargenumberofpotentially

phenotype-causingvariantswithhardtopredictfunctionalconsequences.Therefore,

identificationofaphenotype-causingnon-codingmutationwiththisapproachwouldnot

bestraightforwardandwouldrequiresubstantialadditionalanalysis.Othertechniques

21

suchasRNASeqmayprovevaluableinthesecircumstances,astheywillidentifyputative

affectedlociforfurtheranalysis.

Usingournewstrategyonasubsetof23mutantsfromourscreen,wefindabiasfor

mutationswithinspecificclassesofgenesandbiologicalmechanismssuggestingconstraint

inthegenesthatcanbealteredwhileretainingviability.

Molecularpathwaysofpost-embryonicdevelopment

ConstraintonGeneFunction

Screensformutationsaffectingpost-embryonicdevelopmentwillbebiasedtowards

geneticchangesthatareconsistentwithviability.Thus,genesthatarenon-essentialare

morelikelytobeidentified.Similarly,theprobabilitytoidentifymutationsthatuncover

haploinsufficiency,orcausedominantnegativeorgain-of-functionchanges,ishigher.Even

giventhemodestsamplingofgenomesdoneinthisstudy,wefoundincertainphenotypes

thatmutationsinparticulargeneswereoverrepresented.Forexample,weidentified4

distinctmutationsofdll4,representingthehighestnumberofalleleswithinonegeneinour

screen.Inthiscase,haploinsufficiencyofallelesincreasedthefrequencyofthisgenebeing

identified,asgenerationofaloss-of-functionalleleismorecommonthangenerationof

gain-of-functionordominantnegativealleles.Butinmostcasesonlyoneortwoalleles

wereidentifiedfordifferentgenessuggestingthatthescreenhasnotreachedsaturation.

Whenweextendthesefindingstopreviousscreens,itisclearthatseveralgene

classes,andspecificgenes,arefoundrepeatedly.Thisisclearforpigmentationmutants

whereadditionaldominantkcnj13/kir7.1/obelixandconnexin41.8/leopardalleleswere

identified.Similarlyinfinanddermalskeletondevelopment,additionalcx43.1/sofand

edar/flsalleleswereidentified.Allthesegeneswereidentifiedasdominantmutationsboth

intheinitialTübingenandZFModelscreens.Thesefrequentlyidentifiedlociprobably

representkeynodesinwhichvariationinformcanarise.

PhenotypicVariabilityandGeneticBackgroundEffects

Notably,nodominantlong-finnedmutantswereobservedinthisscreen,although

thesehavebeenpreviouslyidentifiedasby-productsofrecessivescreens(Haffter,

Odenthal,etal.1996;vanEedenetal.1996).Thissuggeststhateventhoughwescreeneda

22

significantnumberofgenomes,thatwehavenotfullysampledpotentialuniquemutations

ingenesthatcanaffectlatedevelopment.Thelackoflong-finnedmutantsinourscreen

couldalsobeattributedtogeneticbackgroundeffects.Formanyofthephenotypeswe

observedsignificantvariabilityinphenotypicstrengthwhenmutantswerecrossedto

differentwildtypestrains.Thissuggeststhatsomegeneticbackgroundsareabletobuffer

effectsofgeneticmutations.

Systematicscreeningandidentifyinggeneticnetworks

Withuseoftargetedcaptureandnext-generationsequencingtechnologiesitisnow

feasibletoidentifycausativemutationsinlargesetsofmutants.Ifsubstantialnumbersof

genomesarescreened,itisnowpossibleinthezebrafishtosystematicallyapproach

identificationofgeneticnetworkssimilartoscreensroutinelyperformedinDrosophila,C.

elegansoryeast.Weidentified21staturemutantsinourscreen.Ofthe10mutantswe

analyzed,sixhadmutationsinfibrilformingcollagens.Fibrilformingcollagenshavelarge

helicaldomainswithconservedglycineresiduesateverythirdposition.Mutationsofanyof

theseresidueswillleadtostructuralchangesoftheproteinthatactinadominantfashion

andthereforerepresentalargenumberoftargetsforthecreationofphenotype-causing

mutations.Whenthesemutantsarecombinedwithseveralmutantspreviouslyidentified

throughtheirrecessivephenotypesuchasbmp1a(Asharanietal.2012),hsp47/serpinh1b

(BhadraandIovine2015)andplod2(Gistelincketal.2016),acomponentofknowngenes

ofthecollagennetwork,includinggenesregulatingcollagenproteinfunctionand

processing,areidentified.Geneticanalysisofsimilarphenotypicclassesofmutantsshould

allowfurtherrefinementofthisnetwork.

ConstraintonIdentifiedAllelesandIdentificationofDiseaseMechanisms

Mutationrecoveryinadultscreensinthezebrafishcouldbereflectiveofabroader

evolutionaryrestrictionofwhichgenescanbealteredandstillareconsistentwithviability.

Thus,apredictionofperformingascreenonpost-embryonicdevelopmentisthatthe

resultswillbebiasedtowardschangesthatalsowouldbefoundunderlyingdiseasestates

inhumans.

23

Werecoveredmutantsthathavesimilaroranalogouseffectsinhumanorthologs

thatareknowntobeassociatedwithdisease.Sixteenoutofthe22mutantswithpotential

phenotype-causingmutationsidentifiedfromthe25mutantsanalyzedharbormutationsin

knowndiseasegenes(Table4).Thissupportstheutilityofforwardgeneticscreensin

zebrafishtopredictthegeneticcauseofhumandiseasesincaseswheretheunderlying

genemutationshavenotbeendeduced.Further,thisincreasesthepotentialforgenetic

screeninginzebrafishtouncovermodifiersofgenefunctionthatcanserveasabasisfor

discoveryinmedicine.Thesystematicmappingofdominantmutationsasdescribedhere

willgreatlyfacilitatethisapproach.

Acknowledgements

WewouldliketothankAltheaJames,JasonBestandChristianLawrenceattheBoston

Children’sHospitalaquaticsprogramforhelpwithzebrafishhusbandry.Theauthors

wouldalsoliketothankKristinRadcliffeforhelpwithscreeningfishandmaintaining

mutantlines.ThisprojectwasfundedinpartthroughNIDCRU01DE024434granttoMPH

andgeneroussupportfromtheChildren’sOrthopedicSurgeryFoundation.

24

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Figure1.ScreenandmappingstrategyA.WildtypemalesweremutagenizedwithENU

toinducerandommutationsinthegenome.Mutagenizedmaleswereoutcrossedto

wildtypefemalesandthesubsequentgeneration(F1)wasscreenedfordominant

mutationsaffectingadultform.Isolatedmutantfounderswereoutcrossedandthe

phenotypeandpenetrancewasassessedintheirprogeny(F2).B.Formappingand

candidatemutationidentification,onemutantandapoolof3-6wildtypesiblingsfromthe

samecrossweresequenced.Sequencedatawasanalyzedinthreesteps:1.AllSNPsinthe

mutantandsiblingpoolwereidentified(blueandredlines;greybarsrepresentindividual

chromosomes);2.Toidentifythechromosomecarryingthemutation(M),mutantspecific

SNPs(bluebars)wereidentifiedbyremovingallSNPspresentinthewildtypesiblings

fromthesetofpreviouslyidentifiedSNPs;3.Candidatemutationsarethenidentifiedby

excludingallbackgroundSNPsfromwildtypestrainsorothermutants/siblingssequenced.

TheremainingSNPsareclassifiedassynonymousornonsynonymousandrankedbytheir

predictedconsequenceongenefunction(deleterious/tolerated).Bluedotsindicate

nonsynonymouschangesonthelinkedchromosome.

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Figure2.Mappingandcandidategeneidentificationindmh35.Toidentifymutant

specifichaplotypes,firstallSNPsinthegenomeareidentified.AfterexclusionofallSNPs

thatarepresentinthesiblingpool,onlymutantspecificSNPsareleft.Thehighestnumber

ofmutantspecificSNPsonachromosomeshouldindicatethechromosomecarryingthe

mutation.A.TheredbarsinthediagramontheleftrepresentSNPsidentifiedinthe

mutantandsiblingpooloneachofthe25chromosomes.Bluebarsinthediagramonthe

rightrepresentmutantspecificSNPsoneachofthe25chromosomes.B.Summaryofthe

numberoftotalandmutantspecificSNPsperchromosome.C.Inasecondstepall

nonsynonymouschangesareidentifiedinthegenome.Bluecirclesindicatemissense

mutations;greencirclesmarknonsensemutations;blackasterisksmarkmutations

predictedtobedeleterious;theredasteriskindicatesthepositionoftheG138Dmissense

mutationinconnexin43,showntobelinkedtothedmh35mutantphenotype.

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Figure3.Mutantswithvertebraldefects.Representativephotographs(left)andμCT

imagesofthevertebralcolumn(right)ofthe5differentsubgroupsofmutantswith

vertebraldefects.A)wildtypezebrafish,regularlypatternedandshapedvertebraeand

vertebralspines.B-D)Heterozygousdmh13,dmh29anddmh15mutantshaveashorter

trunkandshowstronglydeformedvertebralbodiesandspines,withexcessivebone

formation.E)Heterozygousdmh27fisharealsoshorterthanwtfish,butshowmostly

normalpatternedvertebralbodieswiththeexceptionofacoupleoffusedvertebrae.

Vertebralspinesshowslightchangesinangle.F)Inadditiontoashorterbody,

heterozygousdmh16mutantshavestrongdeformationoftheirvertebrae.Inadditionto

fusionofvertebralbodies,somevertebraearesplitupintomultiplehemisegments.G)The

bodyofdmh31mutantsseemstobeproportionallynormaluntiltheregioninfrontofthe

caudalpeduncle.Here,vertebraearestronglydeformedleadingtoabendinthebodyaxis.

H)Strongcurvatureofthespineindorsal,ventralandlateraldirectionleadstoanoverall

shorterbodylengthofthedmh4mutant.Thevertebralbodiesseemtobemostlynormally

patterned.

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Figure4.Larvalphenotypesofmutantswithvertebraldefects.Representative

photographsofwildtype(A)andmutant(B-D)phenotypesat5dpf.Mutantsshowashorter

bodylengthwhencomparedtowildtypelarvae.Afractionofheterozygousdmh28and

dmh31mutants,inadditionshowdeformationsofthenotochord(redarrows).InsetinD

showsadorsalviewofadmh31/+mutantlarvaillustratingthekinkinthetrunkcoinciding

withdeformationsinthenotochord.

Figure5.Mutantswithdefectsofthedermalskeleton.Representativephotographsof

wtandmutants(left),withcloseupsoftheflankstainedwithalizarinredtovisualize

scales(right).Forbettervisualization,tworowsofscalesareoutlinedinred.A)Inwildtype

fish,thescalesaresimilarinsizeandareveryregularlypatterned.B-H)Allmutantsinthis

33

classshowdifferencesinsize,patternandsomeeveninorientationofscales.D,F,H)These

differencesareevenmoreobviousinhomozygousmutants.Inaddition,allmutantsshow

changesinthepatterningandlengthoftheirfinraystovaryingdegree.

Figure6.Mutantswithfindeformations.Representativephotographsofmutantswith

wt(A)short(B)anddeformed(C)fins.B)Boththemedianandpairedfinsofdmh35

heterozygousmutantsareshorter.Alizarinredstainedpelvicfinsofwt(D)anddmh33/+

mutant(E)fish.Dmh33heterozygousmutantsshowshorterfinrayswithdeformed

segmentsofunequallength.

Figure7.Pigmentmutants.Representativephotographsofpigmentmutantsontheleft

andaclose-upoftheirflankontheright,highlightingthedifferencesinpigmentation.

Homozygousphenotypesareshownfordmh1(C)anddmh11(F).B)dmh1mutantsare

characterizedbyareducednumberofmelanophorestripesonthefinsandwiderstripeson

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thetrunk.C)Homozygousdmh1mutantshaveonlyasinglexanthoporestripeontheir

trunks.D)Stripesindmh7mutantsaredisruptedandshowaspotlikepattern.E)

Heterozygousdmh11mutantsshowmilddisruptionsofthemelanophorestripes,leadingto

awaveappearance.F)Incontrast,homozygousdmh11mutantsshowastrongreductionin

melanophorenumber,whilemostlymaintainingastripelikepattern

Table 1: Screen Results F1 screen

#fish screened #potential mutants

10424 269

F2 confirmation screen

#successful matings #confirmed mutants

135 72

phenotype #mutants

fin 23

dermal skeleton 4

craniofacial 12

stature 21

pigment 12

35

Table 2: Candidate gene identification

SNPs identified in dmh35

genome wide Chromosome 20

coding non-synonymous

coding non-synonymous

# total 159,882 52,562 6,262 1,934

# not present in siblings 7,804 3,138 676 262

# not present in siblings or WT strains 1,345 619 97 41

# not present in siblings, WT strains or other mutants and their siblings

62 41 16 12

36

Table 3: Linked variants Mutant allele

#nonsyn genome

# linked chr

location; bp change gene AA change

LOD score

Stature

dmh14 29 3 3:23104458 G/A X col1a1a G1144E 5.41

dmh13 26 2 3:23104092 G/A col1a1a G1093R 3.61

dmh29 303 17 12:3070126 C/T col1a1b G1123D 7.22

dmh15 17/12 1/2 19:41411721 G/A col1a2 G882D 12

dmh27 27 3 8:21181959 G/T col2a1a G1174X 3.91

dmh28 22 6 8:21181691 G/A X col2a1ac G1141D 4.8

dmh21d 66 8 8:21303580 G/A X itpr3 G2135D 8.4

dmh30d 10 5 8:21303580 G/A itpr3 G2135D 1.5

dmh16 17 4 12:13080359 T/G X cmn M10R 5.11

dmh31 9 5 5:31610564 G/A X myhz2 M543I 5.97

dmh4 10 3 24:39229412 T/A X CU929145.1 I156L 3.61

Fin

dmh35 41 12 20:40820378 C/T X cx43 G138D 3.01

dmh20 38 12 20:28348113 C/A X dll4 H190N 8.4

dmh32 28 3 20:28350489 T/A X dll4 C263X 7.22

dmh33 16 5 20:28352804 C/A X dll4 Y449X 6.02

dmh34 32 17 20:28350403 A/T X dll4 R235X 1.5

Dermal skeleton

dmh3a - - 9:56069237 G/T edar D409Y 14.8

dmh19 23 1 8:17607022 A/G X prkcz S113P 5.7

dmh18 49 N/A

dmh22 66 N/A

Pigment

dmh8 23 3 1:46725420 G/T X cx41.8 N63K 4.82

dmh7 35 5 1:46725024 T/A X cx41.8 R195S 4.52

dmh9 25 6 15:40379722 G/A kcnj13 T128M 3.01

dmh1b 102 6 15:40373740 A/T kcnj13 Y325X N/D

dmh11 12 N/A a identified by candidate gene approach; b identified by whole exome sequencing of a homozygous mutant pool; c mutation in col2a1a was filtered out as a candidate due to a low Phred-scaled quality score of 5.46 of the alternative allele ; d mutants are most likely clonal; N/A for these mutants no linkage was found at time of publication; N/D not determined; X linkage to predicted chromosomes was confirmed;

37

Table 4: Human disease genes Gene Human disease

col1a1 Caffey disease Ehlers-Danlos syndrome, classic and type VIIA Osteogenesis imperfecta, type I, type II, type III, type IV

col1a2 Ehlers-Danlos syndrome, cardiac valvular form and type VIIB Osteogenesis imperfecta, type II, type III, type IV

col2a1 Achondrogenesis, type II or hypochondrogenesis Avascular necrosis of the femoral head Czech dysplasia Epiphyseal dysplasia, multiple, with myopia and deafness Kniest dysplasia Legg-Calve-Perthes disease Osteoarthritis with mild chondrodysplasia Otospondylomegaepiphyseal dysplasia Platyspondylic skeletal dysplasia, Torrance type SED congenita SMED Strudwick type Spondyloepiphyseal dysplasia, Stanescu type Spondyloperipheral dysplasia Stickler sydrome, type I, nonsyndromic ocular and type IVitreoretinopathy with phalangeal epiphyseal dysplasia

cx43 Atrioventricular septal defect 3 Craniometaphyseal dysplasia, autosomal recessive Erythrokeratodermia variabilis et progressiva Hypoplastic left heart syndrome 1 Oculodentodigital dysplasiaOculodentodigital dysplasia, autosomal recessive Palmoplantar keratoderma with congenital alopecia Syndactyly, type III

dll4 Adams-Oliver syndrome 6

edar Ectodermal dysplasia 10A, hypohidrotic/hair/nail type, autosomal dominant Ectodermal dysplasia 10B, hypohidrotic/hair/tooth type, autosomal recessive

kcnj13/kir7.1 Leber congenital amaurosis 16 Snowflake vitreoretinal degeneration

cx41.8/GJA5 Atrial fibrillation, familial, 11 Atrial standstill, digenic (GJA5/SCN5A)