Zebrafish bone and general physiology are differently affected by hormones or changes in gravity.

42 Pages·2015·5.19 MB·English

RESEARCHARTICLE
Zebrafish Bone and General Physiology Are
Differently Affected by Hormones or
Changes in Gravity
JessicaAceto1,RasoulNourizadeh-Lillabadi2,RaphaelMarée3,NadiaDardenne4,
NathalieJeanray1,LouisWehenkel3,PeterAleström2,JackJ.W.A.vanLoon5,6☯,
MarcMuller1☯*
1 LaboratoryforOrganogenesisandRegeneration,GIGA-Research,UniversityofLiège,B-4000,Liège,
Sart-Tilman,Belgium,2 BasAM,NorwegianUniversityofLifeSciences,Vetbio,0033Dep,Oslo,Norway,
3 GIGA&DepartmentofElectricalEngineeringandComputerScience,UniversityofLiège,Liège,Belgium,
4 Unitédesoutienméth.enBiostatistiqueetEpidémiologie,UniversityofLiège,B23,SartTilman,Liège,
Belgium,5 DESC(DutchExperimentSupportCenter),DepartmentofOralandMaxillofacialSurgery/Oral
Pathology,VUUniversityMedicalCenter&AcademicCentreforDentistryAmsterdam(ACTA),Amsterdam,
TheNetherlands,6 ESA-ESTEC,TEC-MMG,NL-2200AG,Noordwijk,TheNetherlands
☯Theseauthorscontributedequallytothiswork.
* [email protected]
OPENACCESS
Citation:AcetoJ,Nourizadeh-LillabadiR,MaréeR,
DardenneN,JeanrayN,WehenkelL,etal.(2015) Abstract
ZebrafishBoneandGeneralPhysiologyAre
DifferentlyAffectedbyHormonesorChangesin Teleostfishsuchaszebrafish(Daniorerio)areincreasinglyusedforphysiological,genetic
Gravity.PLoSONE10(6):e0126928.doi:10.1371/
anddevelopmentalstudies.Ourunderstandingofthephysiologicalconsequencesofal-
journal.pone.0126928
teredgravityinanentireorganismisstillincomplete.Weusedalteredgravityanddrugtreat-
AcademicEditor:PaulEckhardWitten,Ghent
mentexperimentstoevaluatetheireffectsspecificallyonboneformationandmore
University,BELGIUM
generallyonwholegenomegeneexpression.Bycombiningmorphometrictoolswithanob-
Received:December18,2014
jectivescoringsystemforthestateofdevelopmentforeachelementintheheadskeleton
Accepted:April9,2015 andspecificgeneexpressionanalysis,weconfirmedandcharacterizedindetailthede-
Published:June10,2015 creaseorincreaseofboneformationcausedbya5daytreatment(from5dpfto10dpf)of,
respectivelyparathyroidhormone(PTH)orvitaminD3(VitD3).Microarraytranscriptome
Copyright:©2015Acetoetal.Thisisanopen
accessarticledistributedunderthetermsofthe analysisafter24hourstreatmentrevealsageneraleffectonphysiologyuponVitD3treat-
CreativeCommonsAttributionLicense,whichpermits ment,whilePTHcausesmorespecificallydevelopmentaleffects.Hypergravity(3gfrom
unrestricteduse,distribution,andreproductioninany
5dpfto9dpf)exposureresultsinasignificantlylargerheadandasignificantincreasein
medium,providedtheoriginalauthorandsourceare
credited. boneformationforasubsetofthecranialbones.Geneexpressionanalysisafter24hrsat
3grevealeddifferentialexpressionofgenesinvolvedinthedevelopmentandfunctionofthe
DataAvailabilityStatement:Rawdataand
completelistsofanalyzeddataarepubliclyavailable skeletal,muscular,nervous,endocrineandcardiovascularsystems.Finally,weproposea
atArrayexpress(https://www.ebi.ac.uk/arrayexpress/) noveltypeofexperimentalapproach,the"ReducedGravityParadigm",bykeepingthede-
undertheaccessions:E-MTAB-3285,E-MTAB-3286,
velopinglarvaeat3ghypergravityforthefirst5daysbeforereturningthemto1gforonead-
E-MTAB-3289,andE-MTAB-3290.
ditionalday.5daysexposureto3gduringtheseearlystagesalsocausedincreasedbone
Funding:Thisworkwassupportedbythe"Fondsde
formation,whilegeneexpressionanalysisrevealedacentralnetworkofregulatorygenes
laRechercheFondamentaleCollective";2.4555.99/
(hes5,sox10,lgals3bp,egr1,edn1,fos,fosb,klf2,gadd45baandsocs3a)whoseexpres-
2.4542.00/2.4561.10,theSSTC;PAI:P5/35,the
UniversityofLiège;GAMEproject,theEuropean sionwasconsistentlyaffectedbythetransitionfromhyper-tonormalgravity.
SpaceAgencyprojectsAO-99-LSS-003andAO-99-
LSS-006,theBelgianSpaceAgencyProdexprojects
FISH-GSIMandFISH-SIM.JvLreceivedgrantMG-
PLOSONE|DOI:10.1371/journal.pone.0126928 June10,2015 1/42
ZebrafishBoneandGeneralPhysiologyinHyper-Gravity
057fromtheNetherlandsOrganisationforScientific Introduction
(NWO)ResearchEarthandLifeSciencesviathe
Formanyyears,thezebrafishhasbeenrecognizedasanexcellentmodelsystemforvertebrate
NetherlandsSpaceOfficeNSO.
developmentalbiology.Morerecently,itisincreasinglyusedtostudyvertebratephysiology,
CompetingInterests:Co-authorMarcMullerisa pathology,pharmacologyandtoxicology[1–5].Itsmainadvantagesareeasymaintenance,
PLOSONEEditorialBoardmember.Thisdoesnot
highfertility,rapidandexternaldevelopment,easyobservationofalldevelopmentalstages,
altertheauthors'adherencetoPLOSONEEditorial
policiesandcriteria. smallsize,transparencyoftheembryosandclosecontactwithsurroundingmedium(water)al-
lowingeasyadministrationofdrugs.Inaddition,itsgenomeissequencedandextensivelyan-
notatedtogetherwithwellestablishedforwardandreversefunctionalgenomicsandaccessto
alreadygeneratedandcharacterizedmutantsandtransgeniclinesoffish(zfin.org).
Skeletaldevelopmentinzebrafishwasfirstmorewidelyaddressedinlargescalemutagenesis
screeninginitiatives,resultinginidentificationofanumberofgenesrequiredforearlyforma-
tionoftheheadskeleton[6,7].Cranialcartilageisthefirstskeletalstructuretobedetectedas
earlyas3dayspost-fertilization(dpf),whilefirstcalcifiedintramembranousbonestructures
starttoformataboutthesametime.Perichondralboneelementsslowlybuildupontheexist-
ingcartilagematrixduringthefollowingdays.Inmammals,oneofthemajorgenesinvolvedin
osteoblastdifferentiationisRunx2.Inzebrafish,itsorthologrunx2bissimilarlyrequiredforos-
teoblastdifferentiation[8]andtheonsetofosteoblastspecificgenes[9],suchasmembersof
thedlxfamily[10]andosterix(osx)[11,12],againwithmammalianorthologs.Otherexpressed
genescodeforboneextracellularmatrix(ECM)proteinsosteocalcin(Osc2)[13],collagen10a1a
(Col10a1a)[14],Bglap,Spp1andcollagen1a1a(Col1a1a)[9,15,16].Thelatterismutatedin
thechihuahua(chi)mutant,amodelforthehumanconditionofosteogenesisimperfecta.Final-
ly,correctcalcificationoftheboneECMdependsontranscellularepithelialcalciumuptake
throughthecalciumchannelTrpv5/6[17]andtheprecisecontrolofphosphate/pyrophosphate
homeostasisbytheEntpd5diphosphohydrolase,expressedinosteoblasts[18]togetherwith
thewidelyexpressedphosphodiesteraseEnpp1[19].Takentogether,theseobservationsindi-
cateanextensivesimilarityofthemolecularpathwaysgoverningbonephysiologybetweentele-
ostsandmammals,validatingthezebrafishasavertebratemodelinthisfield[16,20–22].
Duringspaceflight,humanpassengersexperienceprofoundalterationsoftheirskeletaland
muscularsystem,aswellasbloodcirculatoryandimmunesystems[23–25].Microgravityis
themaindifferentialfactoroftheenvironmentinspaceandisprobablyresponsibleforthe
rapidboneloss(osteoporosis)observedinspace.Variousfishspecies,suchascarp[26],gold-
fish[27–31],orcichlids[32–39]havebeenutilizedinthepastforevaluatingtheeffectsofal-
teredgravity.Morerecently,smallerfishessuchasswordtail[37,40],medaka[41–46]and
zebrafish[47–51]haveattractedmoreattention.Mostanalysesusingfisheshaveconcentrated
ontheimpactofalteredgravityongraviperception[33,52],thevestibularsystem[37,53,54]
anditsinvolvementinmotionsickness[38,55–57].Severalstudiesalsorevealedthatgeneral
embryogenesisofvariousorganismsisnotaffectedbygravityconditions(reviewin[46,49,50,
58]).
Here,weinvestigatetheeffectofincreasedgravityonthegeneralphysiologyofzebrafishlar-
vaebyusingaLargeDiameterCentrifuge(LDC)[59]tostudywholegenomegeneexpression.
Weinvestigateinmoredetailtheeffectsonheadskeletondevelopmentandwevalidateourap-
proachbystudyingtheeffectsofdrugtreatments(VitD3andPTH)knowntoaffectbonefor-
mation.Finally,weproposeanovelapproachtostudytheeffectsofmicrogravitybygrowing
zebrafishinhypergravityfor5days(from0–5dpf)beforereturningthemtonormalgravity,
theReducedGravityParadigm,RGP[60].Thehypothesisforthisparadigmdictatesthatsimi-
lareffectsasobservedfromthetransitiongoingfrom1gintomicro-gareobservedgoingfrom
ahypergravityleveltowardsa1gacceleration,aspecialkindofsimulatedmicrogravityor
PLOSONE|DOI:10.1371/journal.pone.0126928 June10,2015 2/42
ZebrafishBoneandGeneralPhysiologyinHyper-Gravity
‘relativemicrogravity’.However,itmaybeexpectedthatthemagnitudeoftheeffectsapplying
RGPisreducedascomparedtothe1g-μgtransition.
MaterialsandMethods
Animalprocedures
Zebrafish(Daniorerio)weremaintainedunderstandardconditions[61]intheGIGAzebrafish
facility(licenceLA2610359).Briefly,zebrafish(Daniorerio)oftheABstrainwererearedina
recirculatingsystemfromTechniplast,Italyatamaximaldensityof7fish/l.Thewatercharac-
teristicswereasfollows:pH=7.4,conductivity=500μScm-1,temperature=28°C.Thelight
cyclewascontrolled(14hlight,10hdark).Fishwerefedtwicedailywithdrypowder(ZMfish
food)adaptedtotheirageandoncedailywithfreshArtemiasalinanauplii(ZMfishfood).Lar-
vaeagedlessthan14dayswerealsofedtwicedailywithaliveparameciaculture.Wildtypeem-
bryoswereusedandstagedaccordingto[62].
Thedaybeforebreeding,wild-typeadultmaleandfemalezebrafishweresetupinseveral
breedingtanks,separatedbyaclearplasticwall.Afterthelightwasturnedonthenextmorn-
ing,wallsareremoved,eggsaregeneratedbynaturalmatingandcollectedfrom30minutesto
2hoursafterspawning.Aftersorting,cleaneggsaremovedtoPetridishesandincubatedat
28°CinE3medium(5mMNaCl,0.17mMKCl,0.33mMCaCl ,0.33mMMgSO ,0.00001%
2 4
MethyleneBlue).AllprotocolsforexperimentswereevaluatedbytheInstitutionalAnimal
CareandUseCommitteeoftheUniversityofLiègeandapprovedunderthefilenumbers568,
1074,and1264(licenceLA1610002).
Chemicals
Parathyroidhormone(PTH;Merck-Calbiochem,Overijse,Belgium)stocksolution(1μg/ml)
waspreparedinDMSOandstoredinaliquotsat-20°C.VitaminD3(cholecalciferol,VitD3;
Sigma-Aldrich,Diegem,Belgium)stocksolution(200μl/ml)inDMSOwasstoredinaliquotsat
-20°Cformaximumonemonth.
Chemicaltreatments
ThechemicalprotocolwasinspiredbyFlemingandcollaboratorsexperiments[63].Larvaeat
5dpfweretransferredintoa6wellplate(Millipore)containingE3mediumsupplementedwith
therequiredchemicalorvehicle(DMSO)asnegativecontrol.Themediumwaschangedevery
dayatthesametime.FinalconcentrationsinE3wereat10ng/mlforPTHand200ng/mlfor
VitD3.Eachwellcontained20fishin4ml.Theyweretreatedfor1day(n=50–60larvae)to
performmicroarraysandfor5days,from5to9or10dpf,toobservethelonger-termeffectsof
treatmentsbydifferentstaining(n=20–30larvae).Plateswereplacedintothedarkandincu-
batedat28°C.Thelarvaewereeuthanizedbytricaineoverdose(0.048%w/v)anddirectlysub-
mittedtoanRNAextractionat6dpf(formicroarrays)ora4%para-formaldehyde(PFA;
Sigma-Aldrich,Diegem,Belgium)fixationat6,9or10dpf(forstaining).
HypergravityexperimentsintheLargeDiameterCentrifuge
ALargeDiameterCentrifuge(LDC)wasusedforhypergravityexperiments.Itiscomposedof
acentralaxislinkedto2perpendiculararms,eacharmterminatingin2opposinggondolas
whereitispossibletoinstallanincubatorcontainingthesamples.Thearmsprovidean8mdi-
ameterforrotationandcanprovidecentrifugalforcesofmaximum20g.Thezebrafishlarvae
wereincubatedin20mlE3inaPetridishplacedinanincubatorwithinagondolafor3g
experiments,andplacedeitherinanincubatoronthecentrifugeaxis(axe)oroutsideofthe
PLOSONE|DOI:10.1371/journal.pone.0126928 June10,2015 3/42
ZebrafishBoneandGeneralPhysiologyinHyper-Gravity
centrifugefor1gcontrols.Inthissetting,themediumrepresentslessthen5mmofwatercol-
umnandthusthe3gaccelerationcausesanincreaseinhydrostaticpressureofmaximum
0.0015bar,ascomparedtothe1baratmosphericpressure[64].
Stainingmethods
Acid-freeprotocolswereadapted[65]toperformAlcianblue(8GXSigma-Aldrich,Diegem,
Belgium)stainingofcartilagestructuresandAlizarinredS(Sigma-Aldrich,Diegem,Belgium)
stainingofcalcifiedstructures.At6,9or10dpf,thelarvaewerefixedin4%PFAfor2hatroom
temperatureandrinsedseveraltimeswithPBST.
Cartilagewasstainedovernightin10mMMgCl ,80%EtOHand0.04%Alcianblue.The
2
larvaewerewashedindifferentconcentrationsofethanol(80%,50%,25%)toremoveexcess
staining.PigmentationwasbleachedinaH O solution(H O 3%,KOH0.5%)andfinallythe
2 2 2 2
larvaewererinsed3timesinasolutionof25%glycerol/0.1%KOHand50%glycerol,0.1%
KOHandfinallystoredinthissolutionat4°C.
Duringacid-freebonestructurestainingwithAlizarinred,bleachingwasperformedimme-
diatelyafterfixation,beforethestaining.Afterthebleaching,longrinses(atleast20mineach)
ina25%glycerol,0.1%KOHsolutionarenecessarytopreventthefadingofthestaining.The
larvaearestainedina0.05%Alizarinredsolutioninwaterfor30mininthedarkonlowagita-
tion,rinsedina50%glycerol,0.1%KOHsolutiontoremoveexcessstainingandkeptat4°Cin
thesamesolution.
Imagesofstainedlarvae(n=20–30larvae)wereobtainedonabinocular(Olympus,cellB
software).
Imageanalysis
ImageanalysiswasperformedonthepicturesoflarvaestainedwithAlcianblueforcartilageor
Alizarinredforbone.Individualcartilageandboneelementswereidentifiedaccordingto[10,
15,66–68].Formorphometricanalysis,imageswereuploadedintotheCYTOMINEenviron-
ment[69]andmanuallyannotatedbypositioning21landmarksforlarvaestainedforcartilage
(Fig1A)aspreviouslydefinedintheCYTOMINEontology.29landmarkswereplacedforlar-
vaestainedforboneinhormonaltreatments(Fig1C),ofwhich15wereselectedforthehyper-
gravityexperiments.Theprogramthendefinesthepositionsofallselectedlandmarksand
computesallthedistances(inpixels)andangles(inradian)ofallthepossibilitiesbetweentwo
pointsofinterest.ThesedatawereexportedintoanExcelfileandaselectionofinteresting
measureswasconductedbyperformingprincipalcomponentanalysisondataobtainedfrom
differentlytreatedlarvaetoidentifyinvariableorredundantmeasures.Themeasuresselected
were:forcartilage(Alcianblue):AnteriortoEthmoidplate,AnteriortoPosterior,Articulation
downtoArticulationup,Ceratohyalext.downtoCeratohyalext.up,Ceratohyalext.downto
Ceratohyalint.down,Ceratohyalext.uptoCeratohyalint.up,EthmoidplatetoPosterior,
HyosymplecticdowntoHyosymplecticup;andforbone(Alizarinred):Anguloarticulardown
toAnguloarticularup,AnteriortoNotochord,AnteriortoParasphenoida,Branchiostegalray
1downtoBranchiostegalray1up,EntopterygoiddowntoEntopterygoidup,Maxilladownto
Maxillaup,OpercledowntoOpercleup,ParasphenoidatoParasphenoidb,Parasphenoidbto
Parasphenoidc,areaoftheparasphenoidtriangle:parasphenoida,b,andc,andfinallythean-
glesbetweenparasphenoidaandb,aandc,bandc.
StatisticswereperformedusingGraphPadPrism5.At-testwasusedforcontrolversustreat-
mentexperiments,whileaonewayANOVAwasusedformultiplecomparisons.
Morphometricanalysisdidnotinformabouttheextentofossificationwithineachlarva.
Thus,asystematicstructureanalysiswasgenerated.Eachbonestructurewasclassifiedbased
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ZebrafishBoneandGeneralPhysiologyinHyper-Gravity
Fig1.(A-D)Cartilageandboneelementsoftheheadskeletonin10dpfzebrafish.(A)Alcianbluestainingofheadcartilagerepresentingthelandmarks
usedformorphometry.(B)Schematicrepresentationofthedifferentheadcartilageelements.anteriorlimit(an),articulation(ar),ceratobranchialpairs1to4
(cb1-4),ceratohyal(ch),ethmoidplate(et),hyosymplectic(h),Meckel'scartilage(mk),palatoquadrate(pq),posteriorlimit(po).(C)Alizarinredstainingof
cranialbonesrepresentingthelandmarksusedformorphometry.(D)Schematicrepresentationofthedifferentcranialboneelementswith29landmarksused
forchemicalstreatmentsand15landmarksforthe3gandtherelative-hypergravity.The15landmarksareanguloarticular(aa),anterior(an),branchiostegal
ray1(br1),entopterygoid(en),maxilla(m),notochord(n),opercle(o),parasphenoid(p).Notethattheparasphenoidisatriangularbonedefinedbyitsanterior
summit(a)andtwoposteriorsummits(b,c).The29landmarksincludethe15namedbeforewithbranchiostegalray2(br2),cleithrum(c),ceratobranchial5
(cb),ceratohyal(ch),dentary(d),hyomandibular(hm).(E-J)10dpfzebrafishlarvaeafter5dayschemicaltreatments.(E-G)Alcianbluestainingof
cartilage.(H-J)Alizarinredstainingofbone.(E,H)ControlsinDMSO.(F,G)nosignificanteffectof,respectivelyVitD3andPTHoncartilagedevelopment,nor
onchondrocyteshapeorsize(inlaysshowingclose-up).I:increaseofbonedevelopmentafterVitD3treatment.(J)decreaseofbonedevelopmentafterPTH
treatment.Ventralviews,anteriortotheleft,(E-J)scalebar=250μm.
doi:10.1371/journal.pone.0126928.g001
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ZebrafishBoneandGeneralPhysiologyinHyper-Gravity
ontheprogressofdevelopmentintooneofthefourfollowingcategories:absent,earlyossifica-
tion,advancedossificationandoverossification.Whenvalueswereconsideredasquantitative,
comparisonbetweentwogroups(controlversuschemicaltreatmentorhypergravityin1g>3g)
wasassessedbyaStudentt-test,whilecomparisonbetweendifferenttreatments("relativemi-
crogravity"experiment)wasassessedbyananalysisofvariance(ANOVA).Acontingency
tableconsideredordinalvaluesdistributedamongthe4classes(fromabsenttooverossifica-
tion)oronly3classeswhenoneclasswasnotpresentinthesample.Associationbetweenclas-
sesandtreatmentwasassessedbyX²testandbyanordinallogisticregressionandtheodds
ratio(OR).The"relativemicrogravity"experimentwasanalyzedinadditionbygroupingthe
3g,3g>1gand3g>axeversusthe1gsample.
StatisticalanalyseswereperformedusingtheStatisticaSoftware(version10).Resultswere
consideredstatisticallysignificantatthe5%criticallevel(p<0.05).
RNAextractionandreversetranscription
Larvaeat6dpf,after24htreatment,wereusedfortheRNAextraction.TotalRNAwasex-
tractedof60larvaeperexperimentusingTrizol,followedbytheRNeasyMinikit(Qiagen,Hil-
den,Germany)accordingtothemanufacturer’sinstructionsandconservedat-80degrees
using.TheyweretreatedwithRnase-freeDnaseSet(Qiagen,Hilden,Germany).Afterextrac-
tion,thequalityandconcentrationoftotalRNAwasevaluatedbyelectrophoresisoncapillary
gelandtheratioofabsorbanceat260/280nmbyspectrophotometer(Bioanalyzer2100,Agilent
Technologies,Diegem,Belgium).SynthesisofcDNAwasperformedfrom1μgoftotalRNA,
whichwasreversetranscribed(TranscriptoriScriptcDNASynthesisKit,Bio-Rad,Nazareth,
Belgium)accordingtothemanufacturer’sinstructions.
RealTime-PCR
Gene-specificoligonucleotideprimersweredesignedusingPrimer3softwaretospanexon-
exonjunctionstoavoiddetectionofgenomicDNAcontamination(seeS1Tableforprimerse-
quences)andsynthesizedbyEurogentec(Seraing,Belgium)orIntegratedDNATechnology
(Leuven,Belgium).cDNAwasusedastemplateforquantitativeReal-TimePCRwiththeSensi-
MixSYBRKit(Bioline,London,UK),containingSybrgreen.Reactionswereperformedonan
AppliedBiosystems7900HTsequencesDetectionSystem(AppliedBiosystems,FosterCity,
CA)usingtheonboardsoftware(SDS2.4).Purityoftheampliconswascheckedbymelting
curvesattheendofeachreaction.Ctvalueswereexportedfromtheonboardsoftwareasatext
fileandimportedintoacustomizedMicrosoftexcelspreadsheet.1μloftheRTreaction(1/20
ofthetotalcDNA)wasaddedto1XSYBRgreenmastermix(Bioline,London,UK),150nmol
ofeachprimerin15μltotalvolume.Sampleswererunintriplicateinopticallyclear384-well
plates(ABgene),sealedwithopticaladhesivefilm(AppliedBiosystems)."Notemplate"con-
trolswererunforallreactions,andallRNApreparationsweresubjectedtoshamreversetran-
scriptiontocheckfortheabsenceofgenomicDNAamplification.Therelativetranscriptlevel
ofeachgenewasobtainedbythe2-ΔΔCtmethod[70]andnormalizedrelativetothegapdh
(glyceraldehyde-3-phosphatedeshydrogenase)housekeepinggenechosenfromapanelof3
genes(gapdh,ef1-a,ß-actin)asthemoststablyexpressedthroughoutourexperiments(not
shown).Datafrombiologicalreplicateswereaveragedandshownasmeannormalizedgene
expression±SD.
Cyclingparameters:50°Cx2min,95°Cx10min,then40cyclesofthefollowing95°Cx
15s,62°Cx20s.Ameltingtemperature-determiningdissociationstepwasperformedat
95°Cx15s,60°Cx15s,and95°Cx15sattheendoftheamplificationphase.
PLOSONE|DOI:10.1371/journal.pone.0126928 June10,2015 6/42
ZebrafishBoneandGeneralPhysiologyinHyper-Gravity
Microarrayexpressionexperiments
Formicroarrayexpressionanalysis,fourreplicatesfromeachtreatment(controlanddrugor
gravitytreatment)wereanalyzedin2+2dye-swaphybridizations.OneμgtotalRNAwasline-
arlyamplifiedoneroundandlabeled,usingAminoAllylMessageAmpIIaRNAamplification
kit(Ambion-LifeTechnologies,Gent,Belgium)aspreviouslydescribed[71].Fiveμgofthere-
sultingantisenseRNA(aRNA)fromtheexposedandcontrolgroupswaslabeledeitherwith
Cy3-dUTPorCy5-dUTP(GEHealthcareBio-SciencesAB,Uppsala,Sweden).Thelabeledtar-
getswereexaminedforamplificationyieldandincorporationefficiencybymeasuringthe
aRNAconcentrationat260nm,Cy3incorporationat550nm,andCy5at650nmusingNano-
drop(Thermoscientific,Wilmington,DE,USA).AgoodaRNAprobehadalabelingefficiency
of30–50fluorochromesevery1000bases.Oneto5μgofeachlabeledaRNAtargetwasmixed,
9μl25×fragmentationbuffer(AgilentTechnologies,Diegem,Belgium)added,andthefinal
volumeadjustedto225μlwithRNase-freeH2Ofollowedbyincubationfor30minat60°C.
Thehybridizationsolutionwaspreparedbyadding220.5μlof2×hybridizationbuffer(Agilent
Technologies,Diegem,Belgium)and4.5μlsonicatedherringspermDNA(10μg/μl;Promega,
Madison,WI,USA)tothelabeledtargetaRNA.Microarrayslides(4x44KzebrafishV2orV3,
AgilentTechnologies,Diegem,Belgium)wereprehybridizedat42°C,60minusing0.1%bovine
serumalbumin(BSA)FractionV,5×SSC,and0.1%sodiumdodecylsulfate(SDS).Hybridiza-
tionwasperformedat60°Cin16husinggasketslides,hybridizationchamber,andoven(Agi-
lentTechnologies,Diegem,Belgium)accordingtoAgilent60-meroligomicroarrayprocessing
protocol.Microarrayslideswerethenwashed3×5minin0.5×SSC,0.01%SDS(firstwashat
42°Candnexttwoatroomtemperature).Finally,slideswerewashed3timesinroomtemp
with0.06×SSCanddriedimmediatelywithcentrifugationat800×gfor1min.
MicroarrayslideswerescannedusingaGenePix4000B(Axoninstrument,FosterCity,CA).
Scanningwasperformedataleveljustbeforesaturationofseveralspots.Rawdatagenerated
fromGenepixwereimportedintotheBioconductorpackageLIMMAandcorrectedforback-
ground[72].Forwithin-arrayandbetween-arraynormalization,printtipLoessandscalewere
used,respectively[72].AnempiricalBayesmoderatedt-test[72,73]wasappliedtodetectdif-
ferentlyexpressedgenesacrosstreatedandcontrolsamples.Thepvalueswerecorrectedfor
multipletestingusingtheBenjamini–Hochberg(BH)[74]methodandp-values<0.1werese-
lectedasdifferentlyexpressedgenes.Thegeneratedgenelistwasfurtherfilteredforgeneswith
lowintensityandwithsmallchangesinexpression.IntheaveragednormalizedMA-Plot,the
majorityofgeneswereclusteredinbetweenMvaluesof±0.4(foldchange±1.3)andselected
tobethresholdcriteriafordifferentlyexpressedgenelist.TheVitD3datawereobtainedona
SureScanDxinstrument(AgilentTechnologies,Diegem,Belgium)andanalyzedusingthe
GeneSpringsoftware(AgilentTechnologies,Diegem,Belgium)byapplyingthesamesettings.
RawdataandcompletelistsofanalyzeddataarepubliclyavailableatArrayexpress(https://
www.ebi.ac.uk/arrayexpress/)undertheaccessions:E-MTAB-3285,E-MTAB-3286,E-MTAB-
3289,andE-MTAB-3290.
IngenuityPathwayAnalysis
Forpathwayandbiologicalfunctionanalysisofsignificantlydifferentlyexpressedgenes,
Ingenuitypathwayanalyses(IPA,QIAGENRedwoodCity;http://www.ingenuity.com)were
used.Thelistswithdifferentlyexpressedgenesgeneratedbythemicroarrayanalysiswere
translatedintomammalian(human,mouse,andrat)orthologsusingtheUnigene&Gene
OntologyAnnotationToolanduploadedtoIPA.TheIPAsoftwareisanonlineexploratory
toolwithacurateddatabaseforover20,000mammaliangenesand1.9millionpublishedlitera-
turereferences.IPA’sdatabasetogetherwithEntrezGene,GeneOntology,etc.,integrates
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ZebrafishBoneandGeneralPhysiologyinHyper-Gravity
transcriptomicsdatawithminingtechniquestopredictandbuildgenenetworks,pathways,
andbiologicalfunctionclusters.Theoutputresultsaregivenscoresandp-valuesthatarecom-
putedbasedonthenumberofuploadedgenesintheclusterornetworkandthesizeofthenet-
workorclusterintheIngenuityknowledgedatabase.Fisher’sexacttestisusedtodetermine
theprobabilitythateachassociatedbiologicalfunctionisduetochancealone.ScoresforIPA
networksarethenegativelogarithmofthep-value,indicatingthelikelihoodofthefocusgenes
(genesuploadedtoIPA)inanetworkbeingfoundtogetherduetorandomchance.Scoresof2
orhigherhaveatleasta99%likelihoodofnotbeinggeneratedbychancealone.
Results
Effectsofdrugtreatmentsonheadskeletalformation
Tocharacterizeindetailtheprocessofcartilageandboneformationinzebrafish,wefirst
wantedtoexaminetheeffectsofchemicaltreatmentsknowntoaffectskeletaldevelopment.
TreatmentofzebrafishlarvaewithvitaminD(VitD3)waspreviouslyshowntoresultinen-
hancedboneformation,whilecontinuoustreatmentwithparathyroidhormone(PTH)ledto
decreasedboneformation[63].Wedecidedtoconfirmandextendthesefindingsbycompar-
ingtheeffectsonskeletalformationtothoseongeneexpression.
VitD3andPTHtreatmentswereperformedcontinuouslyfrom5dpfto10dpf.Controland
treatedlarvaewerestainedbyAlcianblueforcartilageextracellularmatrix(ECM)andwith
Alizarinredtodetectthecalcifiedbonematrix.Atthisstage,theheadcartilageiswellformed
andacompletesetofcartilageelementsisobserved(Fig1Aand1B).Incontrast,althoughossi-
ficationbeginsat3dpfandthefirstbonestructuresarevisibleat5dpf,theboneskeletoncon-
tinuesitsformationuntil30dpf[68].Nevertheless,at10dpf,anumberofboneelementsare
observedintheheadregion,thefirstvertebralcentraeareformed,whileothersonlybeginto
becalcified(forexamplethebranchiostegalray2)(Fig1Cand1D).
Inthreeindependentexperiments,27–29ventralviewimagesofAlcianblue-orAlizarin
red-stainedlarvaewereobtained.After5daysofVitD3orPTHtreatment,cartilagestaysun-
changedascomparedtothecontrolbygeneralobservation.Thestructuresarewellformed,
completewiththeglycosaminoglycanspresentinthecartilagematrixjudgingfromthesimilar
stainingintensity(Fig1E–1G).Inaclose-upview(Fig1E–1G,inlays),nodifferencecouldbe
observedincellshapeorsizebetweenthedifferenttreatments.Consideringbonecalcification,
ageneralobservationrevealedaclearincreaseofbonedevelopmentuponVitD3treatment
(Fig1I).Somestructuresappearinadvance,suchastheretroarticular(Fig1Iarrowhead)bone
andthepreopercular(notshown)bone,whilesomeotherstructuresarethickersuchasthe
dentaryortheceratohyal,orlongersuchasthebranchiostegalray2.Nevertheless,thegeneral
morphologywasunchanged.Incontrast,continuousPTHtreatmentledtoageneraldecrease
ofboneformationandtoacompleteabsenceofsomestructures,suchastheanguloarticulars
andbranchiostegalray2(Fig1J).
Basedontheseimages,weappliedtwocomplementaryapproachestoobtainamoreobjec-
tivequalitativeandquantitativedescriptionoftheskeleton.Thefirstoneisamorphometric
approachthatevaluatesthegeneralaspectoftheheadskeletonbymeasuringthedistancesbe-
tweenandtherelativepositionofalldetectedboneelements.Theimageswereintroducedinto
theCYTOMINEsoftware(seeMaterialsandMethods,[69])andeachimagewasannotatedby
positioningspecificlandmarksrepresentingthedifferentskeletalelements.Forlarvaestained
forcartilage,21landmarksweredefined(Fig1A),while29pointsofinterestwerepositioned
withintheAlizarinred-stainedboneskeleton(Fig1C).Inthesepictures,weconsiderthehead
separatedhorizontallyin2parts.Somestructuresareuniqueandlocatedonthesymmetry
axis,whileothersarepairedandlocalizedsymmetrically,suchasthedentary,maxilla,
PLOSONE|DOI:10.1371/journal.pone.0126928 June10,2015 8/42
ZebrafishBoneandGeneralPhysiologyinHyper-Gravity
entopterygoid,andhyosymplectic.Tofacilitaterecognition,thesewerelabelled“up”and
“down”.Thesoftwarethencomputesthedistancesbetweenselectedlandmarksandtheangles
formedbylinesdrawnbetweenselectedpoints.
MorphometricanalysisinVitD3-treatedlarvaecartilagerevealedanincreaseofthedistance
betweenarticulation(ar)"up"and"down",leadingtoabroaderjawascomparedtountreated
animals,whilealltheotherdistancesremainedunchanged(S1AandS1CFig).Morphometric
cartilageanalysisoflarvaetreatedwithPTHfor5daysrevealedanincreaseinlengthofthecer-
atohyalcartilages(ch,S1BFig).AnalysisoftheboneskeletonafterVitD3treatmentrevealeda
significantincreaseofthedistancebetweenmaxillae(m,Fig2A),consistentwithabroaderjaw
asalreadyobservedbycartilagemorphometry.Thelengthoftheheadskeletonisalsoincreased
uponVitD3treatmentwithalongerdistancebetweentheanteriorpartofthehead(an)and
thenotochord(n)ortheparasphenoid(p).Othermeasuresarenotsignificantlymodified(Fig
2Aand2C).PTHtreatmentledtoasignificantdecreaseofthesizeoftheparasphenoid(p,Fig
2C).Somestructuresaremissing,suchastheanguloarticular(aa),branchiostegalray2(br2),
ceratohyal(ch)and/ormaxilla(m)andasignificantbroadeningoftheposteriorheadskeleton
isrevealedbytheincreaseddistancebetweenleftandright("up"and"down")branchiostegal
rays1(br1),entopterygoids(en),andopercula(o)(Fig2B).
Thesecondapproachconsistsintheevaluationoftheintensityandprogressionofbonefor-
mationofthedifferentbonestructures,andtheirlevelofossification.Ineachimage,every
bonestructureisassignedascore,rangingfromabsent(red),earlyossification(yellow),ad-
vanced(green)orover-ossified(purple)incomparisontoatypicalimageofacontrollarvaof
thesameage.ThedistributionofthescoresobtainedforthedifferentelementsinVitD3-or
PTH-treatedlarvaeandthecorrespondingcontrolsisshowninFig3andtheresultsofthesta-
tisticalanalysisaregiveninS2andS3Tables.
After5daysVitD3treatment,allthestructuresarepresentandsomeareover-ossifiedlike
thehyomandibular,theentopterygoid,thedentaryandtheceratohyalbones.Early(delayed)
ossificationisdecreasedforallthestructuresshown,ascomparedtocontrols,whileadvanced
ossificationincreasedinthemaxilla,branchiostegalray1,branchiostegalray2andanguloarti-
cular(Fig3A).Statisticalanalysis(S2Table)revealsthatonlytheanguloarticularandthemax-
illaupdonotchangesignificantlyinthiscondition.Alltheotherstructures(br1,br2,mdown,
ch,d,en,hm)aresignificantlyincreased,withthehyomandibulars,entopterygoidsandcera-
tohyalsdisplayingthemostdrasticeffect.Theseresultsconfirmaverysignificantpositiveeffect
ofVitD3treatmentonboneformation.
PTHtreatmentresultedinnearlyoppositeeffectstoVitD3.Onlytheentopterygoidandthe
branchiostegalray1arepresentineachfish(Fig3B)withthebranchiostegalray1unaffected
andtheentopterygoiddisplaying60%ofearlyossificationinPTH-treatedlarvaecomparedto
3,45%incontrols.Alltheotherstructureswereabsentinatleast20%ofthetotal27fishana-
lyzed.Thestrongesteffectwasseenintheanguloarticularbonewith94%ofabsencecompared
to21%absence,19%earlyossificationand60%ofadvancedossificationinthecontrols.Specif-
icstatisticalanalysisconfirmedthatPTHtreatmentsignificantly(p<0,001)reducednearlyall
thestructuresexceptbranchiostegalray1(S3Table).
Toobtainaglobalscoredescribingtheheadskeletoninthedifferentconditions,theindivid-
ualstructurescoresineachimagewereaddedupandameanglobalscorewasobtainedshow-
ingthatVitD3treatmentsignificantlyincreasesbonedevelopment(fromascoreof26±3inthe
controlsto33±4intheVitD3treatment),whilePTHtreatmentsignificantlydecreasesossifica-
tiontoapproximatelyhalfofuntreatedcontrol(fromascoreof27±4to13±5,5).
Insummary,thesecompleteimageanalysesrevealsthatVitD3treatmentconservesthegen-
eralskeletalmorphology,butleadstoalongerheadandalargerjaw.Bonecalcificationisstron-
gerformostelements,andsomeelementscalcifyearlier.Incontrast,PTHtreatmentconserves
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ZebrafishBoneandGeneralPhysiologyinHyper-Gravity
Fig2.Morphometricanalysisresultsofbonematrixstainingafter5dayschemicaltreatments.Thedistancesaremeasuredinpixels.Mean±SDand
t-testanalysiswerecalculatedforeachmeasureonatleast20individuals.*p<0.05,**p<0.01and***p<0.001.(A)DistancesafterVitD3treatment.(B)
DistancesafterPTHtreatment.(C)Areaoftheparasphenoidboneresultsafter5daysPTHorVitD3treatment.AbbreviationsasinFig1.A)Analysisofthe
PLOSONE|DOI:10.1371/journal.pone.0126928 June10,2015 10/42