Zebrafish skeleton development

19 Pages·2017·18.14 MB·English

RESEARCHARTICLE
Zebrafish skeleton development: High
resolution micro-CT and FIB-SEM block
surface serial imaging for phenotype
identification
JeremieSilvent1*,AnatAkiva1,VladBrumfeld2,NatalieReznikov3,4,KatyaRechav2,
KarinaYaniv5,LiaAddadi1,SteveWeiner1
1 DepartmentofStructuralBiology,WeizmannInstituteofScience,Rehovot,Israel,2 Departmentof
ChemicalResearchSupport,WeizmannInstituteofScience,Rehovot,Israel,3 DepartmentofMaterials,
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InstituteofBiomedicalEngineering,ImperialCollegeLondon,London,UnitedKingdom,4 Departmentof
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Bioengineering,InstituteofBiomedicalEngineering,ImperialCollegeLondon,London,UnitedKingdom,
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5 DepartmentofBiologicalRegulation,WeizmannInstituteofScience,Rehovot,Israel
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a1111111111 *[email protected]
Abstract
OPENACCESS Althoughboneisoneofthemoststudiedlivingmaterials,manyquestionsaboutthemanner
Citation:SilventJ,AkivaA,BrumfeldV,Reznikov inwhichbonesformremainunresolved,includingfinedetailsoftheskeletalstructureduring
N,RechavK,YanivK,etal.(2017)Zebrafish development.Inthisstudy,wemonitoredskeletondevelopmentofzebrafishlarvae,using
skeletondevelopment:Highresolutionmicro-CT
calceinfluorescence,high-resolutionmicro-CT3DimagesandFIB-SEMintheblocksur-
andFIB-SEMblocksurfaceserialimagingfor
phenotypeidentification.PLoSONE12(12): faceserialimagingmode.Wecomparedcalceinstainingoftheskeletonsofthewildtype
e0177731.https://doi.org/10.1371/journal. andnacremutants,whicharetransparentzebrafish,withmicro-CTforthefirst30dayspost
pone.0177731
fertilizationembryos,andidentifiedsignificantdifferences.Wequantifiedthebonevolumes
Editor:DominiqueHeymann,UniversitedeNantes, andmineralcontentsofbones,includingotoliths,duringdevelopment,andshowedthat
FRANCE
suchdevelopmentaldifferences,includingotolithdevelopment,couldbehelpfulinidentify-
Received:September15,2016 ingphenotypes.Inaddition,high-resolutionimagingrevealedthepresenceofmineralized
Accepted:May2,2017 aggregatesinthenotochord,beforetheformationofthefirstboneintheaxialskeleton.
Thesestructuresmightplayaroleinthestorageofthemineral.Ourresultshighlightthe
Published:December8,2017
potentialofthesehigh-resolution3Dapproachestocharacterizethezebrafishskeleton,
Copyright:©2017Silventetal.Thisisanopen
whichinturncouldproveinvaluableinformationforbetterunderstandingthedevelopment
accessarticledistributedunderthetermsofthe
CreativeCommonsAttributionLicense,which andthecharacterizationofskeletalphenotypes.
permitsunrestricteduse,distribution,and
reproductioninanymedium,providedtheoriginal
authorandsourcearecredited.
DataAvailabilityStatement:Allrelevantdataare
withinthepaperanditsSupportingInformation
files.Inaddition,allmoviesandtheirlegendsare Introduction
availableonhttps://figshare.com/s/
Thezebrafish,aspeciesbelongingtotheCyprinidaefamily,isawell-studiedvertebratedevel-
3a0a5945f0d3afda6aed.
opmentalmodelbecauseofitsbasalphylogeneticlocation,becauseofthehighdegreeof
Funding:Thisresearchwassupportedbythe
homologybetweenhumanandzebrafishgenes[1]andorgansystems[2]andalsobecauseof
ISRAELSCIENCEFOUNDATION(grantnumber
875/15).J.S.issupportedbyaClore-Krenter-Katz theopticalclarityofitsembryosandlarvae,allowinginvivoobservationsduringdevelopment
postdoctoralfellowship.Thefundershadnorolein [3–5].Inaddition,theabilitytomanipulatetheembryoenablestheuseofdifferentgenetic
PLOSONE|https://doi.org/10.1371/journal.pone.0177731 December8,2017 1/19
Zebrafishdevelopment:Micro-CT
studydesign,datacollectionandanalysis,decision techniques,suchasreverse-geneticapproachesthatallowthefunctionalstudyofamissing
topublish,orpreparationofthemanuscript. gene,ortheuseoftransgenicapproachesthatenablethecreationofzebrafishexpressingfluo-
Competinginterests:Theauthorshavedeclared rescentproteins[6].Clearlytheinsightsgainedfromgeneticmanipulationsaredirectlyrelated
thatnocompetinginterestsexist. toourabilitytoidentifyandcharacterizetheresultingphenotype.
Skeletondevelopmentisgenerallydocumentedbyusinghistologicalstains,suchascalcein
green,calceinblue,alcianblueoralizarinred[7–15].Thesestainsbindcalciumandcalcium
containingmineral,albeitnotexclusively[16–18].Radiographyhasalsobeenusedtoobtain
2Dprojectionsofonlythemineralizedregions[19,20].Micro-CThasbeenusedformonitor-
ingadultskeletonsatrelativelylowresolution[21],andsynchrotronbasedmicro-CThasbeen
usedforhighresolutionstudiesoftheteeth(0.6microns)[22,23].Hereweshowthatalabora-
torybasedmicro-CTcanprovide3Dhighresolutionimages,aswellasvolumeandmineral
densityquantification.Themicro-CTdataaresubtlydifferentfromthecalceinfluorescence
data,andtheircomparisonprovidesinvaluableinformationforassessingskeletalphenotypes
inlarvalzebrafish.Togofurtherinthecharacterizationoftheskeleton,theserialfocusedion
beam/scanningelectronmicroscopy(FIB-SEM)wasalsousedto3Dvisualizeandquantifythe
bone,asthelacuno-canalicularnetwork[24],thetendon-boneinsertion[25]orthecollagen
network[26–30].witharesolutioninthenanometricscale.
Wereportseveralpreviouslyunidentifiedaspectsofthezebrafishskeleton.Moreover,we
illustratetheeffectivityofthisapproach,bycomparingwildtypetoalbinomutants,asthelat-
terarewidelyusedindevelopmentalstudiesontheassumptionthatintermsofskeletaldevel-
opment,theyaresimilar,ifnotidenticaltothewildtype.
Wefirstdocumentwildtypeskeletaldevelopmentusing,first,awidelyusedfluorescence
dye;calcein,andcomparetheseimagestohigh-resolutionmicro-CT3Dimagestovalidatethe
useofhighresolution(around0.6micron)micro-CTforcharacterizinglarvalskeletalpheno-
types.Calceinisknowntochelatecalciumionsbothinsolutionandinthemineralbulk[10].
Therefore,calceinfluorescencedoesnotonlydetectmineralinthebonesasisoftenassumed
[10],butalsocalciumionconcentrationsatotherlocations.Forexample,theintestinaltract
alsofluorescesstrongly[10],presumablybecausetheintestinaltractcontainshighconcentra-
tionsofcalciumduetothefood.Micro-CTfaithfullymapsthedistributionsofthedensemin-
eralphasesofthebones,theteethandthecalciumcarbonatemineraloftheotoliths,and
thereforemonitorsmineralizationperseinthedevelopmentoftheskeleton.Tocharacterize
furtheraspectsofthedevelopmentofthebones,wealsousedthedualbeamFIB-SEMinthe
“sliceandview”(orserialsurfaceviewSSV)mode,aspreviouslydescribed[26].Thishigh-res-
olutionapproachallowedustoobservenewfeaturesinthetailandthenotochord.
Materialsandmethods
Breedingandcollectingofzebrafish
Zebrafishwerebredandmaintainedinacontrolledenvironmentat28˚Caspreviously
described[4].Thenacremutantischaracterizedbytheabsenceofmelanocytesduetoamuta-
tioninthemiftagene[31].Alltheexperimentswerecarriedoutaccordingtotheguidelines
andapprovedbytheWeizmannInstituteAnimalCareandUseCommittee.Embryoswere
obtainedbyplacing5–6femalesand5–6malesinaspawningtank.Eggswerecollectedand
embryoswereraisedinwaterat28±0.5˚Cinanincubatorfor6days,atwhichtimetheywere
transferredtoanormalwatertankandmaintaineduntilanalysis.
Calceinstaining
Thelarvaewereimmersedina0.2%calceinsolution(Sigma-Aldrich)(pH6.8)for25minand
thenwashedthreetimeswithbluewater.Forinvivoobservations,animalswereanesthetized
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Zebrafishdevelopment:Micro-CT
with0.12%tricaine-metanesulfonate(MS222)inbluewater.Aftermountinginmethylcellu-
lose5%(1.5%)plate,thelarvaewereobservedusinganepifluorescencestereomicroscope
(LeicaM167FC).PicturesweretakenusingLeicaApplicationSuiteimagingsoftwareversion
3.7(Leica,Wetzlar,Germany).
ConfocalimagingwasperformedonaZeissLSM780uprightconfocalmicroscope(Carl
Zeiss,Jena,Germany)withaW-PlanApochromat×20objective,NA1.0.Thecalceinstaining
wasexcitedat488nmandtheemissionwascollectedat492/577nm.Z-stackswereacquired
at1.5μmincrements,every1min.Pictureswereprocessedoff-lineusingImageJ(NIH)and
Avizo(FEI).
4zebrafishlarvaewereobservedforeachconditionandtime.
Micro-CTscan
Weimaged3freshlysacrificedzebrafishusinganon-destructivevolumevisualizationforeach
conditionandat17dpfand30dpf.Thismethodenablesthevisualizationofsofttissueswith-
outneedforchemicalfixationorstaining[32].ThefishwereeuthanizedwithMS222.We
coatedthesurfaceofaplasticsheet3x1x0.2cminsizewithadropofpolylysine(Sigma-
Aldrich)toallowtheinteractionbetweenthepolyanionicsurfacesofthefishandthepolyca-
tioniclayerofadsorbedpolylysine.Thesamplewasplacedinacustom-madesampleholder
thatallowedmaintenanceofhighhumidityaroundthesampleandwasobservedusinganXra-
diaMicro-CT-400(ZeissX-RayMicroscopy,Pleasanton,CA,USA),withanX-raysourceof
30kV,current150μAandmagnification10X.Thepixelsizeforthe17dpfspecimenandfor
thetailwas0.6x0.6x0.6micronsandforthe30dpfspecimenwas1.3x1.3x1.3microns.1,200
projectionimageswererecordedwith20secexposuretime.Inordertocomparealltheinten-
sitiesofalltheacquisitions,ascalewasdesignedusingastandardphantom.Inaddition,a
hydroxyapatiteCTphantom(QRM,Mo¨hrendorf,Germany)wasusedasacalibrationstan-
dardforthequantificationofthedensityofzebrafishbones.Obtaineddataaregivenas
mean±standarddeviation.
FIB-SEMblocksurfaceserialimaging
WeusedthedualbeamFIB-SEM(FEI)intheblocksurfaceserialimagingmodetostudythe
mineralizedanddemineralizedfinstructures.Threesamplesweredemineralizedinasolution
of2%PFA,3%EDTAandcacodylatebufferat0.1Movernightonarotatingtable.Thepre-
servedanddemineralizedsampleswerehigh-pressurefrozen(HPM10;Bal-Tec)indextran
(10%),thenfreeze-substituted(AFS2LeicaMicrosystems,Vienna,Austria)in2%glutaralde-
hydeinabsoluteethanol.Sampleswerestoredat-90˚Cfor42h,beforethetemperaturewas
slowlyincreasedto-30˚Cfor24h(-2˚C/h)tofinallyreach0˚Cin20min(60˚C/h).Thesam-
pleswerestainedbasedontheOTOTOprotocol[26],usingosmiumat1%inethanolfor30
minandthiocarbohydrazideat0.5%inethanolfor15minbothatroomtemperature.The
sampleswerethenembeddedinEpon.Afterpolymerization,thesamplesurfaceswereexposed
usinganUltracutReichertmicrotome(LeicaMicrosystems,Vienna,Austria)withadiamond
knife(DiATOMEAG,Biel,Switzerland).SSVviewsweremadeusingtheHeliosNanolab600
dualbeammicroscope(FEI,TheNetherlands)onsamplessputter-coatedwithgold.The
focusedionbeam(FIB)removesslicesintheXY-planewhereastheSEM,usingamixedsec-
ondaryelectrons/backscatteredelectrons(SE/BSE)detector,scanstheexposedsurfacefrom
theside.Asequenceofserialimagesisrecordedtoformaz-stackinthedirectionperpendicu-
lartothebonecross-section.Theobservationsonthemineralizedsampleswerecarriedout
withaslicethicknessof26nmcovering12μmindepth,overanareaof26x22μm.Isometric
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Zebrafishdevelopment:Micro-CT
voxelsizesweremaintainedduringallexperiments.Asequenceofserialimageswasrecorded
toformaz-stack.
Cryo-SEM
Threetailsweredissectedfromfreshlysacrificedzebrafishandimmediatelyimmersedin10%
dextran(Fluka).Beforebeinghigh-pressurefrozenbyaHPM10(Bal-Tec),thesampleswere
wedgedbetweentwometaldiscs(3mmdiameter,0.05mmcavitieswithaflatcoverabove).
Thefrozensampleswerethenplacedonaholderinliquidnitrogenenvironmentforfreeze
fracture(BAF60;Bal-Tec).Thesampleswerelongitudinallyfracturedat-120˚C,usingavac-
uumbetterthan5x10−7mbar.FracturedsampleswereobservedusinganUltra55SEM
(Zeiss,Germany)withasecondaryelectronin-lensdetectorandabackscatteredelectronin-
lensdetectoroperatingat1kV,witha10μmaperturesizeofanda1.8mmworkingdistance.
Theobservationsweremadeinthefrozen-hydratedstateat-120˚C.
Imageanalysis
ImagesobtainedwiththeCT-scanandtheFIBSEMwereanalyzedusingImageJ(NIH,USA)
andAvizo8(FEIVizualizationSciencesGroup)softwares.Toanalyzethebonedevelopment
inthezebrafishobservedbymicro-CT-scan,weusedvariouscommandsinAvizo.Forthe
LabelFieldcommand,weselectedforeachsamplethebonesineachsliceandalignedthem
oneupontheotherobtaininga3-dimensionalstack.ThentheLabelAnalysiscommandcalcu-
latedthevolumeforeachselectedsample,andtheHistogramcommandprovidedtheintensi-
tiesforeachbone.Afterdatacalibrationwithcalibrationphantomsforhydroxyapatiteand
aragonite,weconvertedintensitiesofeveryvoxelintheimagestomineraldensities.
ToanalyzetheFIBSEMimages,wefirstremovedtheeffectofchargingusingtheFFTsignal
ofeachimagebelongingtothestacks.Thenthecontrastlevelsofalltheimageswereadjusted
usingthepluginEnhanceLocalContrast[33].Thestackswerethenmanuallyalignedusing
theAlignSlicescommandinAvizo.Allthedensematerialsweresurface-renderedusingthe
SurfaceViewcommand.
Thedirectionalityanalysesweresearchedtocharacterizethecollagenstructureofthefin
anddeterminetwoparameters:thedirectionandthedispersion[26,27].Briefly,thefirst
parameterprovidestheazimuthaldirectionangleofthemajorityofthecollagenfibrils,
whereasthesecondparametershowstheangulardispersion,i.e.thestandarddeviationofthis
firstparameter.Theanalysiswasobtainedaspreviouslydescribed[26],withsub-stacksof30
imagesandthemethodofLocalGradientOrientationintheDirectionalityplugin[34].Allthe
imagespresentedinthisarticleinsideviewsareinthesameorientation,namelywiththetail
totheleftandtheheadtotheright.
Statisticalanalysis
Foreachanalysisandcondition,atleast3zebrafishlarvaewereused.Dataaregivenas
mean±standarddeviation.
Results
Developmentoftheskeletonbasedonhighresolutionmicro-CT
Fordirectcomparisonweexaminedthesamecalceinstainedspecimensinthemicro-CTthat
wereimagedusingthefluorescencemicroscope.Wefocusedthecomparisononlyonthehead
andthefirstprecaudalvertebraeat17dpfwhentheheadandaxialskeletonarefullydeveloped
basedoncalceinimaging,andcomparedtheseresultsto30dpf(Fig1).At17dpfthemicro-
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Zebrafishdevelopment:Micro-CT
Fig1.Micro-CT-scanand3D-reconstructions(a,c,e,g)andfluorescencemicroscopyaftercalceinstaining
imaging(b,d,f,h)ofsideviewsofcalcifiedskeletalstructuresinwildtype(a,b,c,d)andnacre(e,f,g,h)zebrafish
larvaeat17dpf(a,b,e,f)and30dpf(c,d,g,h).NovertebraisobservedusingtheCTscanobservationatD17(asterisk).
https://doi.org/10.1371/journal.pone.0177731.g001
CT-scanofwildtypelarvaeshowssomeelementsofthecranium,includingthecleithrum,the
basioccipitalprocess,theexoccipital,theceratobranchial5includingtheteethandthe3pairs
ofotoliths(Tohavemoreinformationonthezebrafishanatomy,seeS1Fig).Thefirst8verte-
braecentrawiththeirneuralspinesandthefirstribsonthefifthvertebraarevisible,aswellas
someelementsfromtheWeberianapparatus.Thefluorescencesignalshowsthepresenceof
thesamebones,butnottheotolithsandtheteeth.At30dpf,weobserveallthebonesinthe
cranium,thevertebraewiththeirneuralspinesandthefirst5ribs.At30dpftheWeberian
apparatusiscompletelyformedwith,forexample,thetripusandtheossuspensorium,theoto-
lithsareclearlyseen,asaretheteeth(Fig1,S1Video).Withthefluorescencemicroscope,the
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Zebrafishdevelopment:Micro-CT
Table1. VolumeofthebonesdeterminedbyCTinthewildtypeat17dpfand30dpfinμm3.
17dpf 30dpf 17dpf 30dpf
Asteriscus 80,000±37,000 560,000±6,000 Orbitosphenoid 14,000±5,000 30,000±3,000
Lapillus 270,000±3,000 830,000±45,000 Ceratobranchial5 280,000±56,000 2,680,000±1,000
Sagitta 340,000±36,000 1,700,000±810,000 Cleithrum 220,000±114,000 1,250,000±4,000
Entopterygoid 140,000±25,000 470,000±8,000 Exoccipital 90,000±5,000 690,000±134,000
Quadrate 60,000±7,000 460,000±45,000 Basioccipitalprocess 280,000±1,000 1,070,000±4,000
Ceratohyalbone 20,000±5,000 250,000±1,000 Vertebra1 30,000±1,000 190,000±4,000
Metapterygoid 20,000±5,000 70,000±9,000 Vertebra2 60,000±1,000 160,000±2,000
Pterosphenoid 95,000±18,000 650,000±117,000 Vertebra3 70,000±5,000 330,000±1,000
Parasphenoid 160,000±3,000 490,000±2,000 Vertebra4 70,000±1,000 210,000±6,000
Branchiostegalray1 10,000±4,000 110,000±21,000 Vertebra5 60,000±5,000 200,000±4,000
Branchiostegalray2 25,000±1,000 100,000±11,000 Vertebra6 60,000±2,000 220,000±2,000
Branchiostegalray3 41,000±2,000 110,000±23,000 Vertebra7 50,000±4,000 210,000±4,000
https://doi.org/10.1371/journal.pone.0177731.t001
completeaxialskeletonat30dpfisobserved,buttheelementsinthecraniumaredifficultto
see.Forexample,thetripusandtheossuspensoriumthatareclearlydetectedbymicro-CT,
arenotvisiblebyfluorescence(Tohavemoreinformationonthedevelopmentoftheskeleton,
seeS2Fig).
Themicro-CTcanprovidequantitativemeasurementsofbonevolumeandmineralden-
sity.Suchquantitativemeasurementscouldprovehelpfulforphenotypeidentification.Table1
showstheincreaseinbonevolumefrom17dpfto30dpfinthewildtype.Inthisperiodof
time,wildtypebonesincreaseinvolumefrom3to10times.The3otolithtypesincreasein
volumefrom3to7times.Thecomparisonofthemineraldensityvaluesforcranialbonesand
vertebraefromthe17and30dpflarvaerespectively(Fig2A)andfortheotoliths(Fig2B)
showsthatthetrendsduringdevelopmentarenotalwaysthesame,duetothecomplexrela-
tionsbetweenrateofvolumeincreaseandrateofmineralizationwithintheformingbone.
Indeed,weobservedforexamplethatthebasioccipitalprocessat17dpfwasmoremineralized
inthewildtypethanat30dpf.
Thevertebraeinthewildtypeat30dpfarealwaysmoremineralizedthanat17dpf,except
forthefirstvertebra.Remarkably,at17dpf,vertebra1isthemostmineralized(930mgHaP/
cm3),comparedtovertebra2(870mgHaP/cm3);vertebrae4,5and3havesimilardensities
((cid:25)850mgHaP/cm3)andtheothervertebraehavelowerdensities((cid:20)800mgHaP/cm3).
Theseresultsshowthat,evenifthemineralizationoftheaxialskeletonbeginswiththeforma-
tionofvertebrae3and4[35],thefirsttwocentraaremoremineralized.At30dpf,vertebrae
1–4allhavethesamemineraldensity,namelymorethan910mgHaP/cm3.Thecomparison
oftherelativemineraldensityfortheotolithsshowsthatthereisnodifferencefrom17dpfto
30dpfforthesagittaortheasteriscus,whereasthelapillusismoredenseat30dpf.
Inthestudiesusingtransparentlines,evenifthequestionwaspreviouslyaskedtoknowif
thelackofpigmentsaffectsthebiologicalprocesses[36],thetransparencywasnotconsidered
todisturbthewildtypephenotypeofthezebrafish.
Weusedourcomplementarymicro-CT–fluorescenceapproachtoalsostudythenacrezeb-
rafish,asitiswidelyusedfordiversestudiesbecauseofitscompletelackofmelanocyte.Our
aimwastodetermineifthemutationthatcausedthetransparencymayalsohaveaffectedskel-
etaldevelopment[31].At17dpfnovertebraeareobservedintheaxialskeletonofnacrefish
usingmicro-CT(Fig1).Thisisinsharpcontrasttothevertebraethatareclearlyseenwithcal-
ceinfluorescence.Presumablythecalceinhighlightsfreeorboundioniccalcium,butnotmin-
eral.Thesame“extra”fluorescenceisobservedintheribs,wherebasedonthemicro-CTno
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Zebrafishdevelopment:Micro-CT
Fig2.Quantificationbymicro-CTofelementsfoundinthewildtypeandnacrezebrafish.A.Absolutemineraldensityquantificationofsome
elementsfoundinthecraniumandthefirst7vertebraeat17dpf(blueandgreen)and30dpf(redandpurple).Allthebonesareexpressedinquantity
ofhydroxyapatitepercm3.B.RelativemineraldensityquantificationbyCTofotolithsat17dpf(blueandgreen)and30dpf(redandpurple).Allthe
bonesareexpressedinpercentageofaragonitecomparedtoapurestandard.Errorbars=standarddeviation.
https://doi.org/10.1371/journal.pone.0177731.g002
mineralcouldbeidentified.At30dpf,nodifferencesbetweenwildtypeandnacrewere
observedusingthemicro-CT(S4Video).Thefluorescenceimagesclearlyshowthevertebrae
andtheribs,butthehighlyfluorescingouterbonesofthecraniumpreventimagingofthe
internalbones.Inconclusionthisdirectcomparisonshowsmajordifferencesbetweenthe
skeletalcomponentsthataremineralizedasrevealedbythemicro-CTandthecalceinfluores-
cencelabellingofthesamespecimens.Thelatterinsomecasesshowsfluorescencewhere
maturemineralizedextracellularmatrixisabsent.
Whenthevolumesofskullbonesarequantifiedat17and30dpf(Table2),theincreasein
volumeofthenacreismuchlargerthaninthewildtype,namelythevolumeincreasesfrom3
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Table2. Volumeofthebonesdeterminedbymicro-CTin17and30dpfnacrefish(μm3).
17dpf 30dpf 17dpf 30dpf
Asteriscus 700±200 390,000±42,000 Orbitosphenoid Absent 20,000±4,000
Lapillus 120,000±60,000 780,000±1,000 Ceratobranchial5 70,000±22,000 1,070,000±5,000
Sagitta 150,000±72,000 970,000±49,000 Cleithrum 60,000±5,000 1,090,000±1,000
Entopterygoid 42,000±10,000 430,000±172,000 Exoccipital 20,000±1,000 600,000±60,000
Quadrate 20,000±2,000 150,000±4,000 Basioccipitalprocess 30,000±1,000 1,820,000±2,000
Ceratohyalbone 20,000±6,000 90,000±28,000 Vertebra1 Absent 300,000±1,000
Metapterygoid Absent 80,000±3,000 Vertebra2 Absent 230,000±1,000
Pterosphenoid Absent 120,000±63,000 Vertebra3 Absent 260,000±2,000
Parasphenoid 60,000±1,000 1,130,000±3,000 Vertebra4 Absent 230,000±3,000
Branchiostegalray1 Absent 90,000±9,000 Vertebra5 Absent 220,000±6,000
Branchiostegalray2 Absent 140,000±45,000 Vertebra6 Absent 220,000±4,000
Branchiostegalray3 Absent 200,000±2,000 Vertebra7 Absent 380,000±4,000
https://doi.org/10.1371/journal.pone.0177731.t002
to58timesforthebonesalreadyformed.Inaddition,twoofthe3nacreotolithpairsincrease
involumearound6times,whereastheasteriscusincreasesmorethan586times.Thisdiffer-
enceisascribedtothefactthattheformationoftheasteriscusislateindevelopment(11–12
dpf)[37]relativetotheotherotoliths(19–22hpf)[38].By30dpfbothwildtypeandnacrefish
aresimilarinsizeandskeletaldevelopmentbasedonfluorescence.Themineraldensities(Fig
2c)arealsosimilarat30dpfwithinexperimentalerror,with4interestingexceptions,namely
theceratohyalbone,thequadrate,theparasphenoidandthepterosphenoid.Inthefirstthree
cases,themineraldensityofthenacreismuchlowerthanthewildtype,whereasthelastoneis
higher.Significantly,allthesebonesarefoundinthesameproximalpartofthecranium.
Comparisonoftherelativemineraldensityfortheotolithsshowsthattheotolithsofnacre
zebrafisharelessmineralizedorasmineralizedasthewildtype,exceptforthesagittaat17dpf
(Fig2d).
Fluorescencemicroscopyrevealsapreviouslyunknowncalcium-richdepositinnacre
mutants(Fig3aand3b).Thisfluorescencewasobservedinthedistalpartofthebodyinthe
sameareaasthelargevacuolatednotochordcells(thenucleuspulposus)(Fig3b),andispres-
entpriortotheformationofthefirstaxialskeletalelement.Usingconfocalmicroscopy,we
observedthatthisfluorescencewaslocalizedinsidethecellsofthe14dpfzebrafish.Micro-CT
showsthatthesefluorescentstructureshavedensitiessimilartothoseofbonemineral.These
structureswerealsoobservedincryo-SEM(Fig3dand3e)underconditionsthatminimizethe
introductionofartifacts.Incryo-SEMtheyappearaslargeaggregates(±2μm)closetothe
notochordsheet,whichhaveapositivebackscatteredelectron(BSE)signal,implyingthatthey
arecomposedofdensematerial.
Newlyidentifiedmineralizedfeaturesinthetailfinbone
Themicro-CTrevealedunknownhighlydensestructuresbetweenthetailfinbonesthatcan-
notbeobservedbyconventionalinvivoconfocalimaging(Fig4aand4b).Theseelongated
denseobjectsarelocatedclosetothelepidotrichiaandarealignedwiththeventralfinbone
hemi-cylinder.WeusedFIB-SEMintheblocksurfaceserialmodetodeterminetherelatedcel-
lularcontentin3Dat10nmresolution.Thesefeaturescannotbeseeninmicro-CT(Fig4d,4e
and4f)(S2Video).Previouslyencounteredproblemsofelectronmicroscopyimagingofan
untreatednon-demineralizedsamplebyFIBSEMaretheabsenceofcontrastinthebiological
tissue[26,27]andthepossibledissolutionofthemineralduringprocessingforspecimen
embedding.Weusedanapproachinvolvingfixation,high-pressurefreezing,freeze-
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Fig3.Tailareashowingtheunknowndepositrevealedbycalceinstainingusingconfocalmicroscopy,micro-CTandcryo-SEM,observed
innacrezebrafishat14dpf.(a,b)Distalregionofthebodyobservedintopviewusingconfocalmicroscopyrevealsapositivestaininglocalizedinthe
nucleuspulposuscells(whitedottedline).(c)Inlateralviewusingmicro-CT,weshowthatthispositivestainingisduetodensematerial.d,e.InLens
secondaryelectronimagesusingcryo-SEMinalongitudinalfractureofthenotochordshowingaggregatesclosetothenotochordsheets
(arrowheads).Thecollagenisfoundinclosevicinity(whiteasterisks).e.Areamagnifiedisdelimitedbytherectangleinpaneld.Inset:Back-scattering
electronimaging(BSE)ofthesameareaobservedine.
https://doi.org/10.1371/journal.pone.0177731.g003
substitution,heavymetalstaining,andpolymerization[39],whichpreservedthemineral
whilesupportingstaining.Themineralizedtailincludingtheupperandlowerlepidotrichia,
i.e.thedermalbonyhemi-segments,appearwhiteduetothehigh-densitycarbonated
hydroxyapatitemineraldeposits.Theactinotrichia,i.e.thelargecollagenousfibers/bundles,
werealsoobservedbetweenthehemi-cylinders.Theactinotrichiaappeargrey.Pigmentlayers
areobservedclosetothebones.ThepigmentlayersarebrightintheBSEmodeduetothepres-
enceofzincandthemetalstaining[40].Weobservedunknownelongatedstructuresinthe
micro-CT.Theyarelocatedclosetothelepidotrichiaandbetweentwoactinotrichia,witha
granularappearance.Heretootheyhavecontrastintensitiessimilartothebones.Thelocaliza-
tionofthisunidentifiedstructureisnotthesamebetweentheCt-scanandtheFIB-SEM,but
wehypothesizedthatthiscanbeduetoadifferenceofthelocationoftheregionofinterestin
thetail.Butthisunidentifiedstructureisalwaysinthevicinityofthebones.
WealsousedFIB-SEMtocharacterizethecollagenfibrilsofthewildtypedemineralized
lepidotrichiaintermsofpreferredorientation(direction)andtheextentofpreferredorienta-
tion(dispersion)[26,27](Fig5)(S3Video).Intheupperpartofamoredeveloppedlepidotri-
chia,closetotheoutersurface(Fig5a),thedirectionalityvaluesplottedagainsttheslice
number(Fig5c)highlight4differentzones.Zone1,whichisthemostdorsalpart,hasthelow-
estdispersion(73.34˚±5.73˚)andthehighestdirectionvalues(94.40˚±2.32˚),characteristic
ofananistropicoralignedstructure.Zone2hasthehighestdispersion(84.17˚±16.48˚)with
theweakestdirectionvalues(80.05˚±8.21˚),implyingadisorderedstructure.Zone3ismore
ordered,withthehighestvariationinthedirectionofthefibrils(84.29˚±9.56˚).Finallyzone
4,whichisthemostventralregion,hasastructureclosertozone1,intermsofdispersion
(80.49˚±10.23˚)anddirection(89.42˚±6.79˚)values.Thepreferredorientationofthecolla-
genfiberscorrespondstothelongitudinalaxisofthelepidotrichia.
PLOSONE|https://doi.org/10.1371/journal.pone.0177731 December8,2017 9/19
Zebrafishdevelopment:Micro-CT
Fig4.NewfeaturesinthetailfinboneobservedbymicroCT-scanandFIB-SEM.a.Confocal
fluorescencemicroscopyobservationsofthetailstainedbyacalceinsolution.b)volumerenderingandc)
cross-sectionofthevolumerenderingusingCT-scanofthetailfinat30dpf,ataresolutionof2.5μm,shows
unknownhighdensitystructures(arrowheads).d,eandfareFIB-SEMscanand3Dreconstructions,
respectively,oftheformingregionofamineralizedtailincrosssection(dande)andreconstructed
longitudinalsection(f)ataresolutionof20nm.Theunidentifiedhighdensitystructures(arrowheads)are
closetothelepidotrichia(bone)andbetweentwoactinotrichia(collagenbundles)(whiteasterisksind).
https://doi.org/10.1371/journal.pone.0177731.g004
Discussion
Weshowherethathighresolution3Dimagingusingconfocalmicroscopy,quantitativeFIB-
SEMintheblocksurfaceserialimagingmodeandquantitativehighresolutionlaboratory
basedmicro-CTofthezebrafishskeletonarepowerfulmethodsforrevealingdetailsofskeletal
development,includingtheotoliths.Highresolutionmicro-CTinparticular,evenwithalabo-
ratorymicro-CT,couldwellprovetobeavaluabletoolforskeletalphenotypeidentificationin
boththeimagingandquantitativemodes.
PLOSONE|https://doi.org/10.1371/journal.pone.0177731 December8,2017 10/19