Epithelial Label-Retaining Cells Are Absent during Tooth Cycling in Salmo salar and Polypterus ...

19 Pages·2016·25.6 MB·English

RESEARCHARTICLE
Epithelial Label-Retaining Cells Are Absent
during Tooth Cycling in Salmo salar and
Polypterus senegalus
SamVandenplas1,MaximeWillems2,P.EckhardWitten1,TomHansen3,Per
GunnarFjelldal3,AnnHuysseune1*
1 EvolutionaryDevelopmentalBiology,GhentUniversity,Ghent,Belgium,2 Pharmaceuticaltechnology,
GhentUniversity,Ghent,Belgium,3 InstituteofMarineResearch(IMR),MatreResearchStation,Matredal,
Norway
*[email protected]
Abstract
a11111
TheAtlanticsalmon(Salmosalar)andAfricanbichir(Polypterussenegalus)arebothacti-
nopterygianfishspeciesthatcontinuouslyreplacetheirteethwithouttheinvolvementofa
successionaldentallamina.Instead,theysharethepresenceofamiddledentalepithelium:
anepithelialtierenclosedbyinnerandouterdentalepithelium.Ithasbeenhypothesized
thatthistiercouldfunctionallysubstituteforasuccessionaldentallaminaandmightbea
OPENACCESS potentialnichetohouseepithelialstemcellsinvolvedintoothcycling.Therefore,inthis
Citation:VandenplasS,WillemsM,WittenPE, studyweperformedaBrdUpulsechaseexperimentonbothspeciesto(1)determinethe
HansenT,FjelldalPG,HuysseuneA(2016)Epithelial localizationandextentofproliferatingcellsinthedentalepitheliallayers,(2)describecell
Label-RetainingCellsAreAbsentduringTooth
dynamicsand(3)investigateiflabel-retainingcellsarepresent,suggestivefortheputative
CyclinginSalmosalarandPolypterussenegalus.
presenceofstemcells.Cellsproliferateinthemiddledentalepithelium,outerdentalepithe-
PLoSONE11(4):e0152870.doi:10.1371/journal.
pone.0152870 liumandcervicalloopatthelingualsideofthedentalorgantoformanewtoothgerm.Using
longchasetimes,bothinS.salar(eightweeks)andP.senegalus(eightweeksandtwelve
Editor:LaurentViriot,Team'EvolutionofVertebrate
Dentition',FRANCE weeks),wecouldnotrevealthepresenceoflabel-retainingcellsinthedentalorgan.Immu-
nostainingofP.senegalusdentalorgansforthetranscriptionfactorSox2,oftenusedasa
Received:October25,2015
stemcellmarker,labelledcellsinthezoneofouterdentalepitheliumwhichgradesintothe
Accepted:March21,2016
oralepithelium(ODEtransitionzone)andtheinnerdentalepitheliumofasuccessoronly.
Published:April6,2016
ThelocationofSox2distributiondoesnotprovideevidenceforepithelialstemcellsinthe
Copyright:©2016Vandenplasetal.Thisisanopen dentalorganand,morespecifically,inthemiddledentalepithelium.ComparisonofS.salar
accessarticledistributedunderthetermsofthe
andP.senegalusrevealssharedtraitsintoothcyclingandthusadvancesourunderstand-
CreativeCommonsAttributionLicense,whichpermits
ingofthedevelopmentalmechanismthatensureslifelongreplacement.
unrestricteduse,distribution,andreproductioninany
medium,providedtheoriginalauthorandsourceare
credited.
DataAvailabilityStatement:Allrelevantdataare
withinthepaper.
Introduction
Funding:Theauthorshavenosupportorfundingto
report. Theenthrallingabilitytocontinuouslyreplaceteeththroughoutlifehasfascinatedscientists
fordecades.Thisabilityismaintainedinalmostallnon-mammalianvertebratesandisatopic
CompetingInterests:Theauthorshavedeclared
thatnocompetinginterestsexist. ofgrowinginterest.Researchhasrecentlyfocusedontheputativeinvolvementofstemcellsin
PLOSONE|DOI:10.1371/journal.pone.0152870 April6,2016 1/19
Label-RetainingCellsandToothReplacement
continuoustoothreplacementinawiderangeofspecies:lesserspottedcatshark(Scyliorhinus
canicula)[1–3];Africanbichir(Polypterussenegalus)[4];rainbowtrout(Oncorhynchusmykiss)
[5–7];Atlanticsalmon(Salmosalar)[8,9];zebrafish(Daniorerio)[10–12];medaka(Oryzias
latipes)[13];leopardgecko(Eublepharismacularius)[14,15]andcornsnake(Pantherophis
guttatus)[16].Thesestudiesrevealconsiderableinterspecificvariationinmorphologicalfea-
tures,organizationandpatterningofthedentition;howeveralmostallspecieshaveteeththat
areorganizedintoothfamilies,i.e.onefunctionaltoothandallofitssuccessors[17].Reif
[17,18]proposedthatthedentitionofalljawbearingvertebratesisformedbyadentallamina,
i.e.adeepepidermalinvaginationinwhichsuccessorteethdevelopbeforetheoldteethare
shedorresorbed.Thisdentallaminacandifferinmorphologicalorspatio-temporalcharacter-
isticsandwascategorizedbyReif[17]eitheras(1)continuousordiscontinuousor(2)asper-
manentornon-permanent.Examplesfor(1)arethecontinuousdentallaminainS.canicula
[17]andthediscontinuousdentallaminainthecichlidHemichromisbimaculatus[19,20].
Examplesfor(2)arethepermanentdentallaminainSyciopterusjaponicus[21]andthenon-
permanentdentallaminainD.rerio[22]).Yet,studiesinO.mykiss[7,23],S.salar[8](both
closelyrelatedbasalprotacanthopterygianteleosts),andmorerecentlyP.senegalus[4](aliving
representativeofabasalcladewithintheactinopterygians),revealedtheabsenceofadental
laminaasdefinedbyReif[17].Inthesespecies,successorteethdevelopdirectlyfromthelin-
gualouterdentalepitheliumcoveringthepredecessorteeth.Here,anepithelialtierisposi-
tionedbetweentheinnerdentalepithelium(IDE)andouterdentalepithelium(ODE)[8].The
latterauthorscoinedthetermmiddledentalepithelium(MDE)forthistier,andhypothesized
thatitcouldfunctionallysubstituteforadentallaminabysupplyingtheouterdentalepithe-
liumwithcellsbeforeitsdifferentiationintoaplacode.
Giventhesuggestedpossibleinvolvementofepithelialstemcellsincontinuoustooth
replacement[10],thedentallamina,ortheMDEforthatmatter,hasbeenconsideredtheobvi-
ouspotentialsourceforsuchstemcells[3,8,22].However,untilnow,littleevidencehasbeen
foundforstemcellinvolvementintoothcyclingofactinopterygians.
Stemcellsaremainlycharacterizedbytheirabilityforself-renewal,i.e.theyhavethecapac-
itytoundergonumerouscellcycles,andproduceprogeny,whilemaintainingtheirundifferen-
tiatedstate,evenafteralonginactiveperiod[24].Dependentonstemcellpotency,their
progenygivesrisetovariousdifferentiatedcellseitherdirectly,orindirectlyviatransient
amplifyingcells.Stemcellsresideinastemcellniche,whichcanbedefinedasastrictlyregu-
latedmicroenvironmentthatmaintainsthestemcellsandtheirfunction[25].Becauseoftheir
undifferentiatedstate,stemcellsaredifficulttoidentify[26].Thereforemanystudieshaveto
relyonindirectevidencetolocateputativestemcells,suchasslowcellcycleortheexpression
ofparticulartranscriptionfactors,e.g.,SRY(sexdeterminingregionY)-box2(sox2).Thistran-
scriptionfactormaintainsthepluripotencyofearlyembryoniccellsandregulatestheforma-
tionofseveralepitheliaduringfoetaldevelopment[27–30].Arnoldandcolleagues[31]showed
sox2expressioninnumerousadultendodermalandectodermalstemcellcompartments.Inthe
mouseincisor,sox2expressionhasbeenobservedinthelabialcervicalloop,asiteknownto
containepithelialstemcells[32].Recently,sox2expressionhasbeenreportedfromthedental
laminagivingrisetosuccessionalteethinmammals(whichdisplaymaximallyonlyoneround
oftoothreplacement),aswellasinreptiles(characterizedbycontinuoustoothreplacement)
[33].Furthermore,GaeteandTucker[16]describedthepresenceofsox2transcriptsintheden-
tallaminaofcornsnake(Pantherophisguttatus)dentalsliceculturesandAbduweliandcol-
leagues[13]demonstratedsox2expressionintheposteriorendofatoothfamilyinthemedaka
(O.latipes),ateleostfish.
Slowcyclingcellscanbedetectedwhenperformingapulse-chaseexperimentwith,e.g.,
BrdU(5-Bromo-2’-deoxyuridine),athymidineanaloguethatisincorporatedinthenucleusof
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Label-RetainingCellsandToothReplacement
cellsintheS-phaseofthecellcycle.Overtime,theBrdUinproliferatingcellsisdiluted(yield-
ingfragmentedlabelintheirnucleus),exceptinthosethatareslow-cycling,andaretherefore
termedlabel-retainingcells,LRCs.Variousstudieshaveusedthismethodtocollectevidence
forthelocationofbothknownandunknownstemcellniches.LRCshavebeenshownindental
systemssuchasinthecontinuouslygrowingrodentincisorsandinthemolars[34–37],inE.
macularius[14]andinO.latipes[13].However,arecentstudyfailedtoshowLRCsinP.sene-
galus[4].Whetherthisfailureisrelatedtotheabsenceofadentallaminaisnotknownbutcan
betestedusinganotherspecieswhereteethderivedirectlyfromthedentalorganoftheprede-
cessor,suchasthesalmonidS.salar.
Thedentitionofsalmonids,inparticularthatofO.mykissandS.salar,hasbeenwellstudied
intermsofpatterningoffirstandlatergenerationteeth[5,6,9,38–41],toothontogenyand
chronologyoftoothdevelopment[23,42]andgeneexpressionpatterns[7,43,44].Inaddition,
BerkovitzandMoore[6]foundthatthelengthofthereplacementcycleinO.mykissvaries
betweeneightandthirteenweeksdependingonthefishlength(suchdataarenotavailablefor
S.salarandP.senegalus).However,whethertheMDEfunctionallysubstitutesforadentallam-
inaandisasourceofepithelialstemcells,asdiscussedbyHuysseuneandWitten[8],hasnot
beeninvestigated.Here,wefocusonthelowerjawdentitionofS.salartotestthishypothesis.
Inparticular,wewantto(1)determinethelocalizationandextentofproliferatingcellsinthe
dentalepitheliallayers,(2)describecelldynamicsthroughaBrdUpulse-chaseexperimentand
(3)investigateiflabel-retainingcellsarepresent,suggestivefortheputativepresenceofstem
cells.Furthermore,(4)wewanttoexpandourdatasetonP.senegalus[4]byusinglongBrdU
chasetimes.Finally,(5)wedeterminethedistributionofthetranscriptionfactorSox2within
thedentalorgan.Comparisonofbothspeciesallowsustoassesswhethertheyshareprolifera-
tioncharacteristics.GiventhephylogeneticpositionofS.salarasabasalprotacanthoperygian,
andP.senegalusasoneofthemostbasalextantactinopterygians,ourresultscanshedlighton
thedevelopmentalmechanismthatensureslifelongreplacementinactinopterygianfishes.
MaterialsandMethods
Animals
Atotalof48S.salar(parrstageofthelifecycle)withanaverageweightof10gandaverage
forklengthof9cm(i.e.lengthofafishmeasuredfromthetipofthesnouttotheposteriorend
ofthemiddlecaudalrays[8])wereusedforBrdUadministration.Theywereobtainedfrom
theHavforskningsinstituttet(InstituteofMarineResearch,IMR,Matre,Norway)andhad
beenrearedinfreshwateratatemperatureof13°C.SixP.senegaluswithanaveragebody
lengthof11.1cmwereusedforBrdUadministrationandhousedandhandledasdescribedina
previousstudy[4].
BrdUadministration
TheS.salarspecimenswereinjectedintraperitoneallywith100μl(10μl/gbodyweight)ofa
solutioncontaining10mg/ml5-Bromo-2’-deoxyuridine(32.6mM)(BrdU,Sigma-Aldrich)in
0.1Mphosphate-bufferedsaline(PBS,pH7.2).Theinjectionwasrepeated5timesevery12
hoursforeachfish,inanattempttoalsolabelcyclingcellswhichwerenotintheS-phaseof
theircellcycleduringthefirstinjection.Priortomanipulation,allfishwereanaesthetized(Fin-
quel,100mg/L).Samplingofthefishoccurredatdifferenttimepoints:4hourspastthelast
BrdUinjectionasapulse,andone(T1),two(T2),four(T3)andeightweeks(T4)afterthelast
BrdUinjectionaschasetime.Thesechasetimeswerechosenbasedonwaximprintexperi-
mentsconductedonacloselyrelatedsalmonidi.e.O.mykiss,wherethetoothlifecycleofa
specimenwithabodylengthof15cm,was16weeks[23].
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Label-RetainingCellsandToothReplacement
BrdUwasadministeredtoP.senegalusexactlyasinS.salaranddescribedinVandenplas
etal.[4].Samplingoccurredatsixweeks(T5),eightweeks(T6)andtwelve(T7)weeksafterthe
finalpulse.AllfishwerehandledwithpermissionoftheethicalcommissionoftheFacultyof
ScienceofGhentUniversity(EC2011-036),whichspecificallyapprovedthewholestudy.
Tissuefixation,decalcificationandsectioning
TheS.salarwereanaesthetized(Finquel,100mg/L)andkilledbydecapitation.Lowerjaws
weredissectedandfixedin4%paraformaldehyde(PFA,pH7.2)at4°Cfor48hrs.Thejaws
wererinsedthreetimesin0.1MPBSanddecalcifiedforatleast3weeksinMorsesolution:
22.5%formicacid,10%sodiumcitrate(Morse,1945)priortoembeddinginparaffin.Blocks
weresectionedsagittallyorfrontally(coronally)at5μm(ProsanMicromHM360).TheP.
senegaluswereanaesthetized,fixed,decalcifiedandsectionedasdescribedinVandenplasetal.
[4]
Immunohistochemistry
ImmunohistologicalstainingforBrdUontheparaffinsectionswasperformedasdescribedin
[45]and[4]:rehydrationthroughadecreasingethanolseries,chromatinprecipitationin
hydrochloricacid,blockin3%BSA/1%milkpowder,exposuretoprimaryantibody(monoclo-
nalanti-BrdUantibodyproducedinRat,ab6326,Abcam)andsecondaryantibody(polyclonal
anti-ratAlexafluor488,a11006,Invitrogen),DAPIcounterstaining(1μl/ml)andmounting
withVectashield(VectorlaboratoriesInc.,Burlingame,USA).ThedetectionofSox2wasdone
byusingaheat-mediatedantigenretrievalstepincitricacid(pH6)for20minutesat95°C,and
astepinblockingsolutioncontaining2%BSAand10%goatserum.Weusedananti-Sox2pri-
maryantibodyproducedinrabbit(ab97959,Abcam)inaconcentrationof1/500at4°Cover-
night,andananti-rabbitsecondaryantibodyDylight488(ab98507,Abcam)ataconcentration
of1/300foronehour.ThesectionswerecounterstainedwithDAPI(1μl/mldistilledwater)
andmountedwithVectashield(Vectorlaboratories).Immunofluorescencewasvisualizedona
NIKONeclipsTE2000-Sconfocallaser-scanningmicroscope.AdobeIllustratorCS5,Adobe
PhotoshopCS5andFijiwereusedtoprocessbrightfieldandfluorescentimagesoftheimmu-
nostainedsections.Thenumberoftoothfamiliesexaminedforimmunohistochemistry(n)is
indicatedthroughouttheresultsandrepresentativesectionswerechosenforthefigureplates.
Three-dimensionalreconstruction
HistologicalsectionsofS.salarstainedwithtoluidinebluewerefrommaterialdescribedin[8].
PhotographsofthesesectionsweretakenonaZeissAxioImager0.Z.1equippedwithaZeiss
AxiocamMRccamera.Inordertounderstandthespatialrelationshipsbetweenthefunctional
tooth(predecessor)andthedevelopingreplacementtooth(successor),andtoidentifythe
potentiallocalizationofastemcellnichewemadethree-dimensionalreconstructionsofthe
dentalorganofasingletoothfamilybymanuallyaligningpicturesfrom28consecutivesec-
tionsandgeneratingsurfacesintheAmira3D5.6software.
Quantification
TheratioofBrdU+toDAPI+cellswasdeterminedindifferentlayers(outerdentalepithelium,
ODE;innerdentalepithelium,IDE,middledentalepithelium,MDE;dentalpapilla,DPand
pulpcavity,PC)ofatoothfamilybycountingBrdUandDAPIlabelledcellsinsuccessivesec-
tionsof5μm(T0:5sections,T2:5sections).Doublecountingofnucleicouldnotbeexcluded;
therefore,theratiocouldbeaslightunderestimate.Sinceonlyonesamplewascountedfor
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Label-RetainingCellsandToothReplacement
eachtimepoint,nostatisticalanalysiswasperformedontheseresults.However,giventhehigh
numberofcellscounted,itcanbeusedasaproxytodetectpreliminarytrendsincelldynamics.
AllcellcountingwasperformedmanuallyandprocessedwithFijisoftware.
Results
ThedentalorganinS.salarisaspatiallycomplexstructurethatenclosestwotoothmembers.
The3Dreconstructionshowsayoungfunctionaltoothassociatedwithasuccessortoothinan
earlycytodifferentiationstageofdevelopment(Fig1Ato1D).Thedentalorganiscomposedof
theinnerdentalepithelium(IDE)ofthefunctionaltooth,theIDEofitssuccessor,amiddle
dentalepithelium(MDE)andtheouterdentalepithelium(ODE).TheIDEisasinglecelllayer,
whichcoversthedentalpapillaofthereplacementtooth,aswellasthefunctionaltooth,except
atitstip.LinguallytheIDEofthesuccessorgradesviathecervicalloopintotheODE(Fig1D).
TheODErepresentstheoutercelllayerofthedentalorganandconnectswithoutclearbound-
arytothebasallayeroftheoralepithelium.TheMDEisenclosedbyIDEandODEatthelin-
gualsideofthefunctionaltooth(Fig1Cand1D,darkgreen).Thistierhasanirregularshape
withthethickestpartsituatedbetweenthefunctionaltoothandthetipofthesuccessor.
Towardsthecervicalloopofthesuccessortooth,theMDEbecomesmorenarrow.TheMDE
extendsasasinglecelllayerbetweentheIDEandODEofthesuccessor.
Cellsproliferateatthelingualsideofthedentalorgantoformanewtooth
germ
Wenextusedthespatialinsightsobtainedthrough3Dreconstructiontoassesstheorientation
ofindividualhistologicalsectionsthroughthedifferenttoothfamiliesinourpulse&chase
experimentinS.salar.Inparticular,wemadesuretostudythelabelinthosesectionsrepre-
sentingthemostadvancedstageofdevelopmentofthetoothgerm,ratherthaninsectionshit-
tingitslessadvancedperiphery.Notethatinallfurtherdescriptions,weuse‘dentalorgan’to
denotetheepithelialstructurecommontopredecessorandsuccessor,andthusincludingthe
IDEandODEofthepredecessor,theMDE,andtheIDEandODEofthesuccessor.
SagittalandfrontalsectionsthroughthelowerjawofS.salarthatreceivedapulse(indicated
asT0,seeMaterialandMethods)showBrdUlabellednucleiinthedentalorgan,theoralepi-
theliumandthemesenchyme(Fig2A,seeFig3foridentificationofthedifferentcelllayers).
ThedistributionofBrdU+cellswithinthedentalorganappearedtobedependentonthedevel-
opmentalstageofthemembersofthetoothfamily(n=5).Thus,atoothfamilycontaininga
replacementtoothinmorphogenesisstageshowsBrdUlabellednucleiintheODEatthelin-
gualandposteriorsideofthedentalorgan(n=3,Fig2A).Labially,thislayermergesintothe
basallayeroftheoralepithelium(OE),hasnoBrdUlabelledcellsandisfurtherreferredtoas
theODEtransitionzone.TheIDEofthereplacementtoothcontainsBrdUlabellednucleiclose
tothecervicalloop;towardsthetoothtiplabelledcellsareonlyoccasionallyseen.IntheIDEof
thepredecessorBrdU+cellsareabsent(Fig2A).Inthecervicalloopofthesuccessor,whichis
thetransitionzonebetweenODEandIDE,allcellsareBrdU-positive.
TheMDEhasmanyBrdU+cells,distributedthroughouttheentiretier.Differentfromthe
ODEandIDE,thecellnucleiareflattened(Fig2A).Afewcellsareenclosedbythecervical
loopattheposteriorsideofthereplacementtoothandarelabelledforBrdU(Fig2A,arrow).
ThesecellsarealsopartoftheMDE.Theseflattenedcellsareorientatedperpendiculartothe
adjacentcellsintheIDEorODE(Fig2A).Givenitsassociationwithasuccessorinmorpho-
genesisstage,thefunctionaltoothisstillyoung.ItspulpcavitycontainsfewBrdU+cells,
restrictedtothecentreregion,andmorefrequentatthelevelofthebaseofthetooththanat
thetip.Almostallmesenchymalcellsthatconstitutethedentalpapillaofthesuccessorare
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Fig1.StructureofthedentalorganinS.salar.(A-D)Three-dimensionalreconstructionsofatoothfamilyonthelowerjawinajuvenileS.salar(forklength:
4.84cm).Tosimplify,theinnerdentalepithelium(IDE)ofthefunctionaltoothandofitssuccessorwerecombinedwiththeouterdentalepithelium(ODE)into
onelayer(Fig1Bto1D,lightgreen).Pleasenotethatthesmoothenedsurfacefacingtheobserverrepresentstheedgeofthe3Dreconstructionanddoesnot
indicatethattheMDEliessuperficially.Instead,theMDEiscoveredbyIDE+ODEoutsidetheareaofreconstruction.(D)Thesamethree-dimensional
reconstructionasin(A-C)butslightlytilted.Thehypothesistobetestedisthatstemcells(asterisk)resideinthemiddledentalepithelium(MDE),andgiverise
totransitamplifyingcells,populatingthecervicallooptoeventuallybecomedifferentiatedameloblasts(dashedarrow).(E)Sagittalhistologicalsection(2μm)
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Label-RetainingCellsandToothReplacement
throughatoothfamilyofajuvenileS.salar(forklength:4.84cm)stainedwithtoluidineblue.Theseserialsectionsareusedtogeneratethethree-dimensional
reconstructionsin(A-D).(F)Samehistologicalsectionasin(E)withdifferentstructuresoverlaidinthecolorsusedinthethree-dimensionalreconstructions
(A-D).(G)Transversehistologicalsection(2μm)throughtwoadjacenttoothfamiliesofjuvenileS.salar(forklength:10cm).Inonetoothfamilythe
replacementtoothisnotvisibleandliesbehindtheplaneofview,whileintheothertoothfamilythefunctionaltoothisnotvisiblebecauseitliesabovethe
planeofview.(H)Samehistologicalsectionasin(G)withdifferentstructuresoverlaidinthecolorsusedinthethree-dimensionalreconstructions(A-D).
Abbreviations:Ab:aboral;Ant:anterior;CV:cervicalloop;FT:functionaltooth;IDE:innerdentalepithelium;Lab:labial;Lin:lingual;MDE:middledental
epithelium;OE:oralepithelium;ODE:outerdentalepithelium;Or:oral;Pos.:posterior;RT:replacementtooth;asterisk:hypotheticalstemcellniche;color
codes:yellow(FT),brown(RT),lightgreen(IDE+ODE),darkgreen(MDE),blue(OE);scalebars:80μm.
doi:10.1371/journal.pone.0152870.g001
BrdU+(Fig2A).SomeofthesecellshaveaslightlyfragmentedBrdUlabelwhencomparedto
theintactlabelincellsoftheIDE,ODEandMDE,indicatingtheydividedalreadyduringthe
pulsetime.Furthermore,thiscondensationofmesenchymalcellsextendsbeyondthebound-
ariesdelimitedbytheIDEofthesuccessor,asitcontinueslingualtothecervicalloopand
alongtheODE.Likewise,mostofthesecellsarelabelled.Elsewhereinthemesenchymesur-
roundingthedentalorgan,BrdUlabelledcellsareonlyoccasionallyfound.Intheoralepithe-
lium,BrdU+cellsarerestrictedtothebasaltwotothreetiers.
Next,wecomparedthepatternofBrdUlabellingoverdifferentchasetimesintoothfamilies
displayinganearlysimilarstateofdevelopmentasdescribedabove,i.e.toothfamilieswitha
youngfunctionaltoothandasuccessorinearlymorphogenesisstage.Afteroneweekofchase
time(T1),theODEcontainsBrdUlabelledcellspredominantlyaroundthefunctionaltooth,
bothonposteriorandanteriorside,andmuchlesssoaroundthereplacementtooth(n=5,Fig
2B).OnlyafewBrdU+cellswithfragmentedlabelarepresentinthecervicalloopofthe
replacementtooth(datanotshown).TheIDEofthefunctionaltoothisBrdU-andtheIDEof
thesuccessorshowsasmallnumberofBrdUlabelledcells,mostlywithafragmentedlabel(Fig
2B).TheMDEdisplaysmanyBrdU+cells,eitherwithintactorfragmentedlabel(Fig2B).The
cellswithintheextensionoftheMDE,enclosedbythecervicalloopofthereplacementtooth,
shownoBrdUlabel.ThepulpcavityofthefunctionaltoothhasonlyafewBrdU+cellslocated
inthecentreandaborally.Thedentalpapillaofthereplacementtooth(includingthemesen-
chymalcondensationextendinglingualtothecervicalloopandODE)displaysafewBrdU+
cells,albeitwithafragmentedBrdUlabel(Fig2B).
Aftertwoweeksofchasetime(T2),onlyafewcellsintheODEaroundthefunctionaltooth
areBrdU-positive,withmostofthenucleihavingafragmentedlabel(n=8,Fig2C).Atits
base,thefunctionaltoothshowsahighnumberofcellswithnon-fragmentedBrdUlabelinthe
IDE.Incontrast,labelledcellsareabsentfromthislayeratthetoothtip.Inthereplacement
tooth,manycellsintheIDEdisplayBrdUlabelinthenucleus,albeitfragmented(Fig2C).In
theMDEBrdU+cellsshowadistinctpatternoflabelling.Cellsincloseproximitytothefunc-
tionaltoothhavefragmentedBrdUlabelwhilecellsclosetotheIDEofthesuccessortoothpos-
sessnon-fragmentedBrdUlabel(Fig2C).ABrdU+cellwithintactlabelisobservedinthe
MDEextensionenclosedbythecervicalloop(Fig2C,arrow).Thepulpcavityofthefunctional
toothshowsBrdU-labelledcellsatitsbasebutnoneatitstip.BrdU+cellsinthedentalpapilla
ofthereplacementtootharerestrictedtosomefaintlylabelledcellswithonlysmallstained
fragmentsintheirnucleus.
Afterfourweeksofchasetime(T3),onlyfewBrdU+cellspersistinthedentalorgan(n=5,
Fig2D).IntheMDEanditsextensionwithinthecervicalloopofthesuccessor,faintandfrag-
mentedBrdUlabelisfoundinalimitednumberofcellnuclei(Fig2D).Thedentalpapillaof
thereplacementtoothlikewisedisplaysfragmentedBrdUlabel.However,non-fragmented
BrdUlabelispresentinmesenchymalcellssurroundingthedentalorganatitsaboral,anterior
andposteriorside.Theoralepitheliumshowsmanynucleiwithfragmentedandnon-frag-
mentedBrdUlabelinitsoutermosttiers.Occasionally,BrdU+cellsarepresentinthebasal
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Fig2.BrdUlabelledtoothfamiliesinS.salarchasedoverdifferentperiods.Immunohistologicalstainingoffrontal(A-C)andsagittal(D)sections
throughatoothfamilyonthelowerjawofS.salarwithBrdUlabelledcellsingreenandDAPIcounterstaininblue.Thetoothfamiliespresentedareinasimilar
stateofdevelopment:ayoungfunctionaltoothanditssuccessorinmorphogenesisstage.(A)Pulsedspecimen,T0;(B)ChasetimeT1,oneweekafterBrdU
administration;(C)ChasetimeT2,twoweeksafterBrdUadministrationand(D)ChasetimeT3,fourweeksafterBrdUadministration.Thewhitearrow
indicatessinglecellswithinthemiddledentalepithelium(MDE)enclosedbytheIDE,cervicalloopandODEofthereplacementtooth.Abbreviations:Ab:
aboral;Ant:anterior;CV:cervicalloop;DP:dentalpapilla;FT:functionaltooth;IDE:innerdentalepithelium;Lab:labial;Lin:lingual;MDE:middledental
epithelium;ME:mesenchyme;OE:oralepithelium;ODE:outerdentalepithelium;Or:oral;PC:pulpcavity;Pos:posterior;yellowasterisk:replacementtooth;
scalebars:100μm.
doi:10.1371/journal.pone.0152870.g002
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Fig3.RatioofBrdU+toDAPI+cellsafterpulseandaftertwoweeksofchasetimeinS.salar.(A)Schematicrepresentationofthedifferentlayersina
toothfamilycontainingtwomembers:ayoungfunctionaltoothandasuccessorinmorphogenesisstage.(B)GraphicalrepresentationoftheratioofBrdU+to
DAPI+cellsinthedifferentlayersasdepictedin(A).(C)Tableshowingresultsfromcellcountinginthedifferentlayers.Thedatawereacquiredfromfive
consecutivesectionsinbothT0(pulsedspecimen)andT2(twoweeksafterBrdUadministration).Abbreviations:Ant:anterior;DP:dentalpapilla;EN:
enameloid;FT:functionaltooth;IDE:innerdentalepithelium;Lab:labial;Lin:lingual;MDE:middledentalepithelium;OE:oralepithelium;ODE:outerdental
epithelium;PC:pulpcavity;Pos:posterior;RT:replacementtooth.
doi:10.1371/journal.pone.0152870.g003
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Label-RetainingCellsandToothReplacement
layeroftheoralepitheliumandintheODEtransitionzonebetweenoralepitheliumandODE
(Fig2D).
WecalculatedtheratioofBrdU+toDAPI+cellsinthedifferentpartsofepitheliumand
mesenchymefromonetoothfamilyinasimilardevelopmentalstagebetweenpulsetimeT0
andchasetimeT2(Fig3Ato3C).Tothisend,BrdU+cellswerecountedthroughfiveconsecu-
tivesections.Nodistinctionwasmadebetweencellswithfragmentedandnon-fragmented
BrdUlabel.ThenumberofBrdU+cellsintheODEandIDEofthereplacementtoothwas
loweraftertwoweeksofchasetime(T2)comparedtothepulse(T0).TheIDEofthefunctional
toothshowedalmostnoBrdU+cellsinT0whilethepercentageofBrdU+cellsinT2wasas
highasaround70%(Fig3Band3C).Theoppositeisobservedinthedentalpapillaofthesuc-
cessor:whilealargefractionofcellswasBrdU+atT0,nearlyallBrdUlabelwaslostaftertwo
weeksofchasetime.TheratioofBrdU+toDAPI+cellsintheMDEwasrathersimilarbetween
T0andT2andfairlyhigh(around38–40%)(Fig3C).Finally,thepercentageofBrdU-labelled
cellsinthepulpcavityofthefunctionaltoothhadslightlydecreasedaftertwoweeksofchase
time(Fig3Band3C).
Tosummarizetheaboveobservations,theMDE,ODEandcervicallooparehighlyprolifer-
ative.Interestingly,theextensionoftheMDE,enclosedbythecervicalloopofthedeveloping
toothgerm,containssomeBrdU-labelledcells.Overtime,theBrdUlabelledcellsshowincreas-
inglyfragmentedlabelinthenucleusleadingtoacompletefailuretofindanybrightlystained
cellswithintactlabelinthedentalorganafterfourweeksofchasetime.Nonetheless,inthe
oralepitheliumcellswithnon-fragmentedlabelpersistuntilthismoment.
CellswithintactBrdUlabelareabsentinthedentalorganofSalmosalar
aftereightweekschasetime
Inordertodetermineiflabel-retainingcells(LRCs)arepresentinthedentalorganofS.salar,
weinvestigatedBrdUlabellinginspecimensthatweresubjectedtoalongerchasetime(eight
weeks,T4).Sevenadjacenttoothfamilieswereanalyzed.ConsistentwiththefindingsofHuys-
seuneetal.(2007)[38],wefindrecurringpatternsofdevelopmentalstateacrossthesefamilies:
(1)toothfamilyone,fourandsevenhaveayoungfunctionaltoothandareplacementtoothin
morphogenesisstage;(2)toothfamilytwoandfivehaveanoldtoothinresorption,ayoung
successor,anddisplayinitiationofathirdtoothgerm,cf.[8];andfinally(3)toothfamilythree
andsixhaveamaturefunctionaltoothwithitssuccessorincytodifferentiationstage(Fig4A).
Thus,similardevelopmentalstagescanbefoundeverythreetoothlocimoreposteriororante-
rioralongthejaw.
Whereanewtoothgermisinitiated,BrdU+cellsarecompletelyabsentfromthedental
organ(Fig4B,toothfamilyfive).However,inareplacementtoothinmorphogenesisstage(Fig
4B,toothfamilyfour),somecellsinthecervicalloopaswellasintheODEclosetothecervical
loopretainsmallfragmentsoflabelintheirnuclei.Afewcells,whichbelongtotheMDEand
areenclosedbythecervicalloop,displayafaintandextremelyfragmentedBrdUlabel.Atthe
baseofthedentalpapillaandtheunderlyingmesenchyme,BrdU+cellsarepresent,however
withfragmentedlabelintheirnucleus.Inareplacementtoothinlatecytodifferentiation,the
dentalorganisagaincompletelydevoidofBrdU+cells(Fig4C).Yet,themesenchymeflanking
theanteriorandposteriorendofthedentalorgancontainsBrdUlabellednucleiwithnon-frag-
mentedlabel.Finally,thepulpcavityofayoungfunctionaltooth(Fig4D,toothfamilyseven)
hasveryfewBrdU+cellswithfragmentedlabel,locatedinthecenter.Intheoralepithelium,
BrdUpositivecellsaredistributedinafashionsimilartowhatisobservedafterfourweeksof
chasetime:bothfragmentedandnon-fragmentedlabelledcellsarefoundintheoutermosttier,
andafewcellsinthebasallayer,albeitfragmented(Fig4Cto4E).
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