Interkinetic Nuclear Migration Is Centrosome Independent and Ensures Apical Cell Division to ...

29 Pages·2015·15.96 MB·English

by  Paulina J. Strzyz

Article
Interkinetic Nuclear Migration Is Centrosome
Independent and Ensures Apical Cell Division to
Maintain Tissue Integrity
Graphical Abstract Authors
PaulinaJ.Strzyz,HyunO.Lee,...,
LouisC.Leung,CarenNorden
Correspondence
[email protected]
In Brief
Strzyzetal.showthat,inzebrafish,
pseudostratifiedretinalneuroepithelia
apicalnuclearmigrationpriortomitosisis
ahighlyreproduciblephenomenon.It
doesnotdependoncentrosomenumber,
location,integrity,orpositionofmitotic
entry.Thisensuresthatproliferativecells
robustlydivideapicallyandsafeguards
tissuearchitectureandmaturation.
Highlights
d ApicalIKNMishighlyrobustandoccursindependentlyofthe
centrosome
d CDK1activityisnecessaryandsufficienttodriveapicalIKNM
d Nonapicaldivisionsperturbintegrityofthepseudostratified
neuroepithelium
d Apicallocalizationofdivisionssafeguardstissuearchitecture
andmaturation
Strzyzetal.,2015,DevelopmentalCell32,203–219
January26,2015ª2015ElsevierInc.
http://dx.doi.org/10.1016/j.devcel.2014.12.001
Developmental Cell
Article
Interkinetic Nuclear Migration
Is Centrosome Independent and Ensures
Apical Cell Division to Maintain Tissue Integrity
PaulinaJ.Strzyz,1HyunO.Lee,1JaydeepSidhaye,1IsabellP.Weber,1LouisC.Leung,1andCarenNorden1,*
1MaxPlanckInstituteofMolecularCellBiologyandGenetics,Pfotenhauerstraße108,01307Dresden,Germany
*Correspondence:[email protected]
http://dx.doi.org/10.1016/j.devcel.2014.12.001
SUMMARY the millimeter scale in the primate neocortex (Rakic, 1972).
Nucleiofthesecellslocateallalongtheapicobasalaxisresult-
Pseudostratified epithelia are widespread during inginthestratifiedappearanceofthetissue(LeeandNorden,
animal development and feature elongated cells 2013).Intheproliferativestate,whenPSEexpandandcellsdo
whose nuclei adopt various positions along the api- not yet differentiate, divisions take place at apical positions
cobasal cell axis. Before mitosis, nuclei migrate with cleavage planes perpendicular to the apical surface (Das
towardtheapicalsurface,andsubsequentdivisions et al., 2003; Morin et al., 2007; Nakajima et al., 2013; Weber
etal.,2014;Xieetal.,2013).Itisproposedthatsuchperpendic-
occur apically. So far, the exact purpose of this nu-
ular division angles are crucial for the maintenance of tissue
clear migration remained elusive. One hypothesis
integrityinthePSEofDrosophilaimaginaldisk(Nakajimaetal.,
was that apical migration ensures that nuclei and
2013) and chick neural tube NE (Morin et al., 2007). In rodent
centrosomesmeetformitosis.Weheredemonstrate
NE,includingretinaandhindbrain,however,anincreaseinnon-
thatinzebrafishneuroepitheliaapicalnuclearmigra-
perpendicular divisions has been observed upon the onset of
tionoccursindependentlyofcentrosomepositionor neurogenesis(CayouetteandRaff,2003;Kosodoetal.,2004).
integrity. It is a highly reproducible phenomenon ApicalmitosesobservedinallPSEresultfromapicalnuclear
linkedtothecellcycleviaCDK1activity.Wepropose migration in G2 (Kosodo et al., 2011; Leung et al., 2011).
that the robustness of bringing nuclei apically for Followingapicaldivision,nucleiaredistributedmorebasallyuntil
mitosis ensures that cells are capable of reintegrat- thecompletionofSphase,afterwhichtheyreturntotheapical
ingintotheepitheliumafterdivision.Nonapicaldivi- sideanddivideagain.Thisbidirectionalmovementofnucleiis
knownasinterkinetic nuclearmigration(IKNM).Since itsinitial
sions lead to cell delamination and formation of cell
observation in 1935(Sauer,1935), IKNMhas beenextensively
clusters that subsequently interfere with neuronal
studied in many model organisms and tissues (Grosse et al.,
layering. Therefore, positioning divisions apically in
2011; Meyer et al., 2011; Norden et al., 2009; Rujano et al.,
pseudostratified neuroepithelia could serve to safe-
2013;Tsaietal.,2010).Thecytoskeletalmachineriesthatdrive
guardepithelialintegrityandenableproperprolifera-
apicalIKNMvarydependingontissuetype.Inshorterepithelia
tionandmaturation. (i.e.,NEofthezebrafishorDrosophilaPSE),forcesformoving
nucleiaregeneratedbyactomyosincontractions(Leungetal.,
2011; Meyer et al., 2011; Norden et al., 2009; Rujano et al.,
INTRODUCTION 2013). Inthe more elongated mammalian neocortex, however,
mainly microtubules (MTs) and their motors are involved (Hu
Pseudostratified epithelia (PSE) are found during the develop- etal.,2013;Kosodoetal.,2011;Tsaietal.,2010).Collectively,
mentofmanyorganisms.Theygiverisetovarioustissuesina thesestudiesprovidedsubstantialmechanisticinsightsintonu-
widerangeofanimals,includinginvertebrateslikeNematostella clear kinetics and the cytoskeletal machineries driving IKNM.
andDrosophila,aswellasvertebratesincludingzebrafish,chick, Whatissofarlessexploredthoughisthebasicfunctionofapical
rodents,andhumans(Grosseetal.,2011;Kosodoetal.,2011; IKNM.Wefurtherlackanexplanationwhydivisionsoccurexclu-
Meyeretal.,2011;Nordenetal.,2009;Rujanoetal.,2013;Spear sively at the apical surface. One study suggests that apical
andErickson, 2012).Pseudostratified neuroepithelia (NE)have mitosisisrelatedtotheexistenceofamitoticzoneattheapical
gatheredspecialattention,astheygeneratemanypartsofthe endfoot (Hu et al., 2013). According to another hypothesis,
nervoussystem. apicalIKNM ismainlya result of theapical position ofcentro-
CellswithinPSEareattachedtobasalandapicallaminaeand somesduringinterphase,astheseorganellesareimportantfor
arehighlyelongated.Theircentrosomesarelocalizedtotheapi- spindleformationbutalsoserveasbasalbodiesforprimarycilia
cal surface during the whole cell cycle (Miyata, 2008; Norden (Miyata,2008;TavernaandHuttner,2010).Additionally,thepo-
et al., 2009; Tsai et al., 2010). Cell lengths range from 40– sitionofthenucleusbeforeapicalIKNMhasbeenlinkedtothe
50 mm in NE of zebrafish or the imaginal disk of Drosophila likelihoodofneurogenesis:Twostudiesprovidedevidencethat
(Meyer et al., 2011; Norden et al., 2009) to 250 mm in rodent the basal depth of nuclei before apical IKNM can be linked to
neocortex (Hu et al., 2013; Kosodo et al., 2011) and up to the probability of a neurogenic division in correlation with an
DevelopmentalCell32,203–219,January26,2015ª2015ElsevierInc. 203
apicobasalNotch/Deltagradient(BayeandLink,2007;DelBene Movie S1 (available online), in the zebrafish retinal NE, the
etal.,2008).Sofar,however,allthesestudiesdealtwiththepro- centrosomebecomesmotileonlyshortlybeforethenucleusrea-
cessofapicalIKNMitselfbutdidnotinvestigatetheimpactof ches the apical endfoot. On average, the centrosome travels
apicaldivisionsontissuedevelopmentandmaturation. 6.7mmtowardthenucleus(Figure1E,n=51cells,fourembryos),
Herewesetouttoexploretheimportanceofapicaldivisions similartoresultsintherodentneocortex(Huetal.,2013).This
for tissue maturation in proliferating pseudostratified zebrafish meansthatcentrosomesmaintaintheirapicallocalizationduring
retinalNE.Weshowthatincontrasttorecentfindingsinrodent thewholecellcycleandthatthemigrationofnucleiresultsinnu-
neocorticalPSE(Huetal.,2013)mitoticentryisnotrestrictedto cleus-centrosomeassociation.
theapicalendfoot.Wefurtherdemonstratethattheapicallocal- IfindeedthemainreasonfortheoccurrenceofapicalIKNM
izationofthecentrosomeisnotaprerequisiteforapicalnuclear wastoensurethatthecentrosomeandthenucleusmeetbefore
migration, as apical IKNM still occurs when centrosomes and division, apical IKNM should not take place when the nucleus
nucleimeetnonapicallyorincaseswhencentrosomeintegrity and centrosome associate nonapically. To test this idea, we
isperturbed.Interestingly,apicalmigrationeventakesplaceaf- introduced additional nonapical ‘‘centrosomes,’’ by interfering
ternonapicalmitoticentry.Thisfindingimpliesthatonceapical with the centriole duplication pathway. To allow for temporal
IKNMistriggered,a‘‘pointofnoreturn’’independentofcentro- control we used heat shock (HS)-inducible constructs (Clark
somepositionispassed.Wefindthatthisupstreamtriggeristhe etal.,2012;Nordenetal.,2009).Wecoexpressedanoverstabi-
activation of CDK1, which is necessary and sufficient for the lized(OS)versionofPlk4—thekeyregulatorofthecentrosome
onsetofapicalIKNM.Wealsorevealthatlocalizingallmitoses duplicationpathway(Hollandetal.,2010)andadominant-nega-
anddivisionsapicallyisofgeneralimportanceforproperprolif- tive(DN)formofCep152,whichperturbscentriolarrecruitment
eration and integrity of pseudostratified NE, as induction of of Plk4 (Cizmecioglu et al., 2010; Coelho et al., 2013) (Fig-
nonapicaldivisionsperturbsretinaldevelopment.Wetherefore ureS1A).Incellsexpressingbothconstructsfusedtothefluo-
suggest that apical migration of nuclei and subsequent apical rescentproteinmKate2,weobservedtheformationofnonapical
divisionsaremechanismswhoserobustnesssafeguardstissue centrosome-like structure appearing as mKate2 positive foci
integrityandthusrepresentsimportantfirststepstoorchestrate thatwereg-tubulinpositive(Figure2A).Thesefocirecruitedcen-
tissuematuration. trin as well as centrosome targeted GFP-PACT (Figures S1B
andS1C).Additionally,liveimagingofEB3-GFPdemonstrated
RESULTS that they can act as microtubule organizing centers (MTOCs)
as nonapical MT nucleation was observed (Figures 2B and
MitoticEntryCanOccurNonapicallyinZebrafish S1D; Movie S2). Together, this shows that these foci indeed
RetinalNE functionallyresemblecentrosomes.
WeusedthedevelopingzebrafishretinalNEtostudytheoccur- We next investigated whether the interaction of nuclei with
renceandreproducibilityofapicalIKNMandapicalmitosesin suchnonapicalcentrosomestriggersnonapicalmitosisandsub-
PSE. We first asked whether in zebrafish NE mitotic entry is sequently impairs apical IKNM. Intriguingly, this was not the
restrictedtotheapicalendfootaspreviouslysuggestedforthe case. While we observed MT nucleation emanating from the
rodentneocortex(Huetal.,2013).Ifthiswerethecase,apical centrosome-likestructures(Figure2B),nucleistilldisplayedapi-
IKNMwouldbeindispensableformitoticentryandsubsequent calmovementwithsimilarkineticsasinthecontrolsituation(Fig-
divisions.Asactomyosincontractionsarethemainforcegener- ures 3C and 3D; Movie S2). Furthermore, all MTOCs of a cell
atorsduringapicalIKNMinzebrafishNE,weblockedactomy- could often be observed to cluster at the apical surface (Fig-
osin contractility using the myosin II inhibitor blebbistatin ure S1D). pH3staining confirmedthatmitotic figures localized
(Nordenetal.,2009).Notably,followingdrugtreatment,rounded apically (n = 28 of 29, seven embryos) (Figure 3A). Altogether
cellsthatassociatedwithcentrosomeswereobservedatnonap- this demonstrates that enabling the mere interaction between
icallocations(Figure1A).Thesecellsstainedpositivelyforthe thenucleusandanMTOCatnonapicalpositionsdoesnotper-
mitoticmarkerphospho-Histone3(pH3),andapicalaswellas turbapicalIKNM.
basalprocessesofcellscouldbeobserved,arguingagainsta Intheconditioninwhichweinducednonapicalcentrosomes,
puredelaminationphenomenon(Figures1Band1C). however, the apically localized centrosome was still present.
WeconcludethatinzebrafishNEmitoticentryisnotrestricted Therefore, we could not exclude the possibility that the apical
to the apical zone. This raises the question why nevertheless position of this centrosome prompted nuclear migration. To
nucleimigrateapicallybeforemitosis. investigate this option, we aimed to induce nonapical centro-
some-nuclear association in cells that featured normal centro-
ApicalIKNMStillOccurswhenNucleusand some number. We noticed that interference with N-cadherin
CentrosomesMeetNonapically via a DN construct (Wong et al., 2012), as well as colcemide
Wenextaimedtotestthepreviouslyraisedhypothesisthatapi- treatment can lead to nonapical association of centrosomes
calIKNMisaresultoftheapicallocationofcentrosomesinPSE andnuclei(Figures2C,2D,andS2A–S2C).Therefore,weused
(Miyata,2008;TavernaandHuttner,2010)andmainlyservesto these approaches to investigate whether nonapical centro-
bring nuclei and centrosomes into close proximity before divi- some-nucleus association influences apical IKNM. In both as-
sion.Wefirstanalyzedthenuclearandcentrosomal dynamics says, cells kept their apical and basal attachments arguing
during the cell cycle using proliferating cell nuclear antigen against mere cell delamination (Figures S2A, S2B, and S2D).
(PCNA),whichunambiguouslylabelsallcellcyclephases(Leung Interestingly, in the DN-N-Cadherin as well as the colcemide
et al., 2011). As seen in the typical example in Figure 1D and condition nuclei that got in contact with centrosomes at basal
204 DevelopmentalCell32,203–219,January26,2015ª2015ElsevierInc.
A B C E
D
Figure1. MitoticEntryIsNotRestrictedtotheApicalSurfaceinZebrafishRetinalNE;CentrosomesAreMaintainedApicallythroughoutthe
CellCycle
(A)ConfocalscanofacellexpressingRas-mKate2(green)andcentrin-GFP(magenta)inanembryotreatedwith200mMblebbistatin1.5hrbeforeimaging.The
cellshowsnonapicalmitoticrounding(filledarrow)andisassociatedwithcentrosomes(openarrows).
(B)ConfocalscanofacellexpressingRas-GFP(green)stainedforpH3(magenta)inanembryotreatedwith200mMblebbistatin1.5hrbeforefixing.Thecell
featuresnonapicalpH3signal(filledarrow)whilemaintainingapicalandbasalattachments(openarrows).
(C)Confocalscansoftheretinaeofanembryotreatedwith200mMblebbistatin1.5hrbeforefixing(lower)andacontrolembryo(upper)stainedforpH3(magenta).
Blebbistatintreatedcellsentermitosisnonapically(lower,arrows).
(D)Time-lapseofthedynamicsofthenucleus-centrosomepairwithrespecttocellcycleprogression.PCNA-RFPlabelsnucleiandmarksthecellcyclestage
(gray).Ras-mKate2(gray)labelscellmembranes.Centrin-GFP(red)labelscentrosomes.Onenucleusislabeledwithayellowdot.Thearrowhighlightsthe
positionofthecentrosome.Timeisinhr:min.TheframesarefromMovieS1.
(E)Centrosomepositionpriortocentrosomesplitting(left)andthemeanvalueofthepositionofcentrosomesatmitosis(middle)withrespecttonuclearlength
(right),n=51cells,4embryos.
Scalebarsrepresent10mm.Thedottedlinerepresentstheapicalsurface,andthesolidlinerepresentsthebasalside.
positions entered mitosis basally, as demonstrated by the mitoticentry,apicalIKNMstilloccurred.Interestingly,whencen-
appearance of cell rounding, chromosome condensation, and trosomes were mispositioned in the DN-N-Cadherin or colce-
nuclearenvelopebreakdown(NEB)(Figures2Cand2D;Movie mide condition, apical IKNM typically started shortly before or
S3).Remarkably,however,despite thepossibilityof nonapical evenaftermitoticentry(Figures2C,2D,3E,and3F).Incontrast,
DevelopmentalCell32,203–219,January26,2015ª2015ElsevierInc. 205
A
B
C D
Figure2. ApicalIKNMPersistsinCellsinwhichCentrosomesandNucleusMeetNonapicallyEvenfollowingNonapicalMitoticEntry
(A)ConfocalscanofaHS-DN-Cep152-mKate2/HS-OS-Plk4-mKate2expressingembryo.PositivecellsfeaturecytosolicmKate2signalaswellasfociofsignal
alongtheapicobasalaxis(magenta).Fociarepositiveforg-tubulinstaining(green).NonapicalfocipositiveformKate2andimmunopositiveforg-tubulinare
markedwithyellowarrows.HSwasperformed8hrpriortofixing.SeealsoFigureS1.
(legendcontinuedonnextpage)
206 DevelopmentalCell32,203–219,January26,2015ª2015ElsevierInc.
incontrolcells,mitoticentryonlytookplaceoncetheapicalside ibility is achieved independently of centrosomes. As IKNM is
was reached (Figure 3C). pH3 staining of embryos expressing tightly linked to the cell cycle, we aimed to elucidate the up-
DN-N-Cadherinshowedthatmitoticfigureswerefoundatapical streamcellcyclecomponentsresponsibleforIKNMrobustness
positions(n=36of36,13embryos)(Figure3B).Thisunderlines andreproducibility.ItisknownthatapicalIKNMinvariablyoc-
thatevenincasesinwhichcentrosomeandnucleicanmeetnon- cursaftercompletionofSphase,whichisinG2(Kosodoetal.,
apically, apical IKNM and apical division still take place. 2011;Leungetal.,2011).Additionally,itisknownthatcellcycle
TogetherthissuggeststhatinitiationofapicalIKNMoccursinde- progression is tightly coordinated by the activity of cyclin-
pendentlyofapicalcentrosomeposition.Additionally,itimplies dependentkinases(CDKs)andtheirassociatedcyclins(Fisher
that the apical position of the centrosome cannot be the sole etal.,2012).EntryintoG2isregulatedbytheactivityofCDK1
reasonfornucleitomigrateapicallypriortocelldivision. in complex with cyclins A or B (Pines and Rieder, 2001). We
therefore hypothesized that CDK1 is the cell cycle-associated
CentrosomeIntegrityIsDispensableforApicalIKNM molecule that controls the timing of apical IKNM. This predic-
SofarwehavedemonstratedthatapicalIKNMisstillreproduc- tionwassupportedbyresultsfromourpreviousworkshowing
ibly initiated when centrosomes and nuclei associate nonapi- thatcellcyclearrestinducedbyCDK1inhibitionleadstostalled
cally.Anindicationthatcentrosomesmightnotbeessentialfor IKNM (Leung et al., 2011). To reproduce these findings, we
apicalnuclearmigrationingeneralcamefromapreviousstudy used the CDK1 inhibitor RO3306 (Vassilev, 2006). Indeed,
in Drosophila. This study showed that flies without centrioles upon CDK1 inhibition, cells completed S phase, as indicated
developrelativelynormally(Bastoetal.,2006)despitefeaturing by the disappearance of nuclear PCNA foci, but stalled in G2
epithelia that display IKNM (Meyer et al., 2011; Rujano et al., without initiating apical IKNM (Figures 4C, 4E, and 4F; Movie
2013). We therefore tested whether centrosomal integrity is a S5). This is in contrast to controls in which apical IKNM
prerequisitefornuclearapicalmigrationinthezebrafishNE.To occurred upon the disappearance of nuclear foci (Figures 1D
achievethis,weperformedlaserablationofcentrosomesinsin- and 4B; Movie S5). This suggested that CDK1 activity is
glecellsusingcentrinlabelingasareadout.Nucleardynamics required to induce apical nuclear movements. Next, we
were followed after ablation, and cells were grouped into two explored whether CDK1 activation is also sufficient to trigger
categories:(1)cellsinwhichsomecentrinsignalremainedbut apicalIKNM.Tothisend,wemadeuseofthefactthattheac-
was much weaker and/or more diffuse; these cells most likely tivity of CDK1 is blocked during S phase due to an inhibitory
featuredonlyremnantsofcentrosomalmaterial(ninecells,eight phosphorylation by Wee1 kinase (Tang et al., 1993). Inhibiting
nucleiperformedapicalmigration)and(2)cellsinwhichnocen- Wee1activityhasbeenshowntoinduceprecociousCDK1ac-
trinsignalwasobservedafterablation(fivecells,fivenucleiper- tivity (McGowan and Russell, 1995). To test whether inducing
formed apical migration). This shows that the vast majority of earlyCDK1activationprematurelytriggersapicalIKNM,wein-
nucleifollowingcentrosomeablationinitiatedmovementtoward hibited Wee1 using PD0166285 (Leijen et al., 2010). Remark-
the apical side. Some cells subsequently did not progress ably, Wee1 inhibition consistently caused nuclear migration to
throughmitosisandremainedroundedatapicalpositions(Fig- occur already during S phase (Figures 4D–4F; Movie S5),
ure4A;MovieS4),whereasothersmanagedtocompleteapical showing that indeed CDK1 activation alone is sufficient to
division(MovieS4).Collectively,thisarguesthateventhepres- trigger apical nuclear movement independent of cell cycle
enceofanintactcentrosomeisnotessentialforapicalmigration phase.
ofnuclei. Together,ourresultsthereforestronglyarguethatCDK1ac-
Takentogether,inallexperimentalsettingsinwhichweinter- tivity is necessary and sufficient to initiate apical IKNM. They
fered withcentrosome position orcentrosome integrity, apical also offer an explanation as to why apical IKNM occurs even
IKNMstilloccurred.ThisstronglyarguesthatapicalIKNMinze- when nuclei and centrosomes meet nonapically or when
brafishNEisaparticularlyrobustphenomenonthatstilloccursin centrosome integrity is impaired. Once cells finish S phase,
challengedconditionsindependentlyofapicalcentrosomes. CDK1 activation most likely leads to a ‘‘point of no return’’
for apical movement. Apical IKNM subsequently occurs
ApicalIKNMIsTriggeredbyCDK1Activity evenincasesinwhichnucleialreadyassociatedwithcentro-
OurexperimentsshowthatapicalIKNMisahighlyrobustphe- somes and initiated NEB or chromosome condensation
nomenon. Next we wanted to understand how this reproduc- nonapically.
(B)Time-lapseofacellexpressingHS-DN-Cep152-mKate2,HS-OS-Plk4-mKate2(green),andthedynamicMTsmarkerHS-EB3-GFP(gray).(Left)Cytosolic
mKate2signalandnonapicalfoci(cyanoutlinedarrows)inthecellofinterestareshown.TheremainingpanelsshowdistributionofdynamicMTs.Clearfociof
nonapicalMTnucleationcanbeobserved(yellowarrows).Intheinsets,magnifiedregionsofnonapicalMTnucleationsitesareshowninfirelookuptable(in
panels4and7,1.53magnification,remainingpanels33magnification).Thereddotmarksthepositionofthenucleus.HSwasperformed12.5hrpriortotime-
lapse.Timeisinhr:min.TheframesarefromMovieS2.
(C)Time-lapseofacellexpressingDN-N-Cadherin.HS-H2B-RFPlabelsnuclei/chromatin(magentaintheupperandgrayinthelower)andcentrin-GFP-RNA
labelscentrosomes(green,upperpanelonly).Thecentrosome(yellowarrow)associateswiththenucleus(cyan,arrow)inanonapicalposition(upper).Thecell
entersmitosisnonapicallyasvisualizedbychromosomecondensation(lower,00:40,cyanarrow).ApicalIKNMandapicaldivisionoccur.HSwaspreformed
17.5hrpriortotime-lapse.Timeisinhr:min.TheframesarefromMovieS2.SeealsoFigureS2.
(D)Time-lapseofacellinanembryotreatedwith100mMcolcemide.ThecellexpressesRas-mKate2(gray)andcentrin-GFP(green).Inthecellofinterestthesplit
centrosome(yellowarrows)travelsbasallyandassociateswiththenucleus(yellowdot)nonapically.Afternonapicalcellrounding(01:50),thecellperformsapical
IKNM.Time-lapsewasstarted5hrafterdrugaddition.Timeisinhr:min.TheframesarefromMovieS3.
Scalebarsrepresent10mm.Thedottedlinerepresentstheapicalsurface.
DevelopmentalCell32,203–219,January26,2015ª2015ElsevierInc. 207
A B
C D
E F
Figure3. ApicalIKNMPersistsDespiteNonapicalMitoticEntry
(AandB)pH3stainings(green)ofembryosexpressingHS-DN-Cep152-mKate2/HS-OS-Plk4-mKate2(A)orDN-N-Cadherin(B)(bothmagenta).
(C–F)Tracksofnuclearpositionaroundmitosisin(C)controlcells,(D)cellsfeaturingnonapicalcentrosomes,(E)cellsexpressingDN-N-Cadherin,(F)cellstreated
with100mMcolcemide.Timeshownintimepoints(tp).1tp=5min.Nuclearpositionwastracked10tp(=50min)priortoand10tpaftermitoticentryoruntil
division.Nuclearpositionhasbeenmeasuredfromthebaseofthenucleustothebasallaminaandwasnormalizedwithrespecttocelllength.Eachtrack
representsasinglenucleus;tpofmitoticentryismarkedwithblueline(alwaysattp=11).
Scalebarsrepresent10mm.
ApicalNonperpendicularDivisionsDoNotPerturb integrityofDrosophilaandchickPSE(Morinetal.,2007;Naka-
RetinalTissueArchitectureandMaturation jima et al., 2013). Therefore, one assumption was that apical
Atthispoint,weunveiledthatapicalIKNMisaparticularlyrepro- IKNM is a prerequisite for successful tissue development of
ducibleeventthatdependsonCDK1activityandtherebyoccurs PSEbyenablingthecontrol ofcleavage planesatdivision.To
eveninchallengedconditions.Wenextaimedtofindoutwhether investigatewhetherthisisthecase,weexaminedhowinterfer-
the reproducibility of bringing all nuclei apically for division is encewithapicalcleavageplanesimpactstheintegrityofretinal
importantfortissuedevelopmentperse.Onepossibleexplana- NE.Weusedpreviouslypublishedmorpholinos(MOs)targeting
tionisthatonlyatapicallocationscancleavageplanesbepre- aPKCl/zasthesehavebeenshowntoinducenonperpendicular
cisely controlled leading to perpendicular divisions. In support divisions(Cuietal.,2007).aPKCl/zMOswereinjectedmosai-
ofthisidea,ithasbeenpreviouslysuggestedthatperpendicular callyinto8-to32-cellstageembryostogetherwithfluorescently
cleavage planes are implicated in the maintenance of tissue tagged RNAs to label affected cells (Norden et al., 2009). We
208 DevelopmentalCell32,203–219,January26,2015ª2015ElsevierInc.
observedthatdivisionstookplaceapicallyat34hourspostferti- aPKCwaslinkedtoaCAAXdomain,thustargetingtheproteinto
lization(hpf)(Figure5A).Thisisincontrasttoapreviousreport themembrane(Ossipovaetal.,2007).Inductionofthisconstruct
usingthesameMOcombination(Cuietal.,2007).Themostlikely led to a gross alteration of aPKC distribution. The proteinwas
reasonsforthisdiscrepancyaredifferencesineffectiveMOcon- observed to localize to cell membranes apically as well as all
centrationorthefactthatweinjectedMOsatthe8-to32-cell alongtheapicobasalcellaxis(Figures6Band6C).Becauseof
stage to avoid early morphological defects as opposed to the the involvement of aPKC in epithelial cell polarity, we investi-
1-cellstageinthepreviousstudy.Inaccordancewiththework gated whether polarity components still localized correctly to
byCuietal.(2007),however,theclearbiasforperpendicularapi- the apical membrane in cells expressing aPKC-CAAX. To this
calcleavageswaslostinaPKCl/zmorphants(Figures5Band end, we first analyzed Par3 localization in these cells. We
5C).Toensurethatmorphantcellsdidnotdisplaymarkedpolar- coinjected a HS-inducible Par3-GFP together with HS-aPKC-
ity defects, we performed staining against the tight junction CAAX-mKate2ormembraneboundHS-Ras-mKate2asacon-
component ZO-1and confirmedthat no differences to control trol.Inbothcases,someresidualnonapicalsignalofPar3was
epitheliawerefound(Figure5D).Weperformedliveimagingto observed(FiguresS3AandS3B).Thisisexplainedbythefact
monitorcentrosomeandPar3behaviorandobservedthatafter thatHSinductionmostlikelyleadstosomeproteinoverexpres-
nonperpendiculardivisionsalsothemorebasaldaughterquickly sion. Nevertheless, the bulk of Par3 signal was localized to a
repositioned its centrosome as well as Par3 toward the apical distinct apical domain both in aPKC-CAAX and control cells
side (Figures 5E and 5F; Movie S6). This confirmed that cells (FiguresS3AandS3B).Additionally,westainedaPKC-CAAX-ex-
wereabletore-establishpolarityandreintegrateintothetissue pressingcellsagainstthetightjunctionmarkerZO-1.ZO-1signal
byrecreatingtheirbipolarmorphology.Subsequently,weinves- wasfoundapicallyandcolocalizedwithapicalPar3(FigureS3C).
tigatedwhetherperturbingapicalcleavageplanesleadstode- We next tested whether aPKC distribution all along the cells’
fects in tissue maturation. To address this issue, we injected apicobasal axis interfered with actomyosin organization. We
aPKCl/z MOs into Tg(Ath5:GAP-GFP) embryos. Ath5-pro- visualizedF-actinbymosaicallyexpressingtheCalponinhomol-
moter-driven membrane-GFP labels the first-born retinal neu- ogydomainofUtrophin,Utr-GFP,whichbindsF-actinwithout
rons—theretinalganglioncells(RGCs).WeexaminedtheRGC stabilizingit(Burkeletal.,2007).Inthecontrolsituation,F-actin
layer to deduce whether early neuronal layer formation was was organized as filaments along the apicobasal axis of cells
impaired. This was not the case, as RGC layer formation was (Figure6A).Thisisinaccordancewithourearlierobservations
similar to control embryos in aPKCl/z morphants (Figure 5G). (Leungetal.,2011;Nordenetal.,2009).Interestingly,whenwe
Additionally,morphantcellscontributedtotheRGClayer(Fig- expressedaPKC-CAAX,actinfilamentorganizationwasdisrup-
ure 5G). This argues that nonperpendicular apical divisions in ted,andthesignalwasmorediffusethaninthecontrolcondition
thezebrafishretinalNEdonotmarkedlyperturbtissuearchitec- (Figure 6B). This argues that aPKC-CAAX interferes with actin
tureandmaturation.Cellsarisingfromsuchdivisionscanreinte- organizationandmighttherebyrepresentavalidtooltoimpair
grateintothedevelopingepitheliumanddifferentiate.Therefore, apicalIKNMandinducenonapicaldivisions.Wenextassessed
controlofcleavageplanepositioningalonedoesnotexplainthe the cell division positions in aPKC-CAAX-expressing cells.
robustnessandreproducibilityofapicalnuclearmigrationbefore Alreadyat30hpf/6hrpostHS(hphs),aboutathirdofalldividing
divisioninPSE. aPKC-CAAX-positive cells (27%, n = 26 cells, 11 embryos)
dividedatectopicbasallocations.Asexpected,thesenonapical
InterferencewithActinDistributionLeadstoImpaired divisionsfeaturedrandomizedcleavageplanes(Figure6D).
ApicalIKNMandNonapicallyDividingCells TheseresultsshowthatinaPKC-CAAX-expressing cellsthe
As we observed that perpendicular cleavage planes are not interferencewithactinorganizationcanindeedleadtoimpaired
absolutelymandatoryforthemaintenanceofPSEarchitecture, apicalIKNM,whichinturnresultsinasignificantamountofnon-
wespeculatedthattheapicalpositionofdivisionsitselfprovides apicaldivisions.
relevant advantages for PSE development. To test this idea,
wewantedtoexplorehownonapicaldivisionsinfluencetissue TheOffspringofNonapically,Nonperpendicularly
developmentandmaturation.Toachievethis,weneededacon- DividingCellsPerturbsRetinalTissueMaturation
ditionthatinterferedwithapicalmotionofnucleiwithoutblocking WenexttestedtheimpactofnonapicaldivisionsofaPKC-CAAX-
cytokinesis (as direct blocking of the actomyosin machinery expressingcellsontissuedevelopmentandretinalmaturation.
would)orcellcyclecontinuation(asthiswouldnotallowcelldi- Interestingly, before and during division, cells showed bipolar
vision).Weandothersrecentlyshowedthatactomyosinaccu- morphology,arguingagainstdelamination(Figures6C,6E,6F,
mulationsbasaltothenucleuscanbelinkedtotheoccurrence andS3D).Followingdivision,theapicaldaughterwasstillinte-
ofapicalIKNM(Leungetal.,2011;Rujanoetal.,2013).Actomy- gratedintheepithelium,whilethebasaldaughterlosttheapical
osindistributioninepithelialcellshasbeenpreviouslyshownto attachmentanddisplayedprotrusiveactivity(Figure6C;Movie
beregulated byaPKC in variouscontexts(Even-Faitelson and S7).Thisimpliedthatfollowingnonapicaldivisiontheabilityof
Ravid,2006;Kishikawaetal.,2008;Uberalletal.,1999).Interest- themorebasaldaughtertoreintegrateintotheepitheliumwas
ingly, in the elongated cells of the pseudostratified epithelium, impaired.Tosubstantiatethisfinding,wemonitoredcentrosome
aPKCisconfinedtotheapicalsideanddoesnotoccuratbaso- behavior and Par3 distribution in nonapically dividing aPKC-
laterallocations(Clarketal.,2012).Therefore,wehypothesized CAAX-positive cells. We demonstrated that while the apical
that altering aPKC distribution in these cells could influence daughtercouldrepositionthecentrosometotheapicalsurface
actomyosinorganizationandmightthusperturbnuclearmigra- following division, this process was inefficient in the basal
tion.Totestthisidea,weusedaHS-inducibleconstructinwhich daughter(Figures6EandS3D;MovieS7).Similarly,theapical
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daughterinheritedapicalPar3signal,maintainedduringdivision, arguing that they could not reintegrate into the epithelium, as
whilethebasaldaughtercreatedanectopicPar3domain(Fig- seen in the aPKC-CAAX condition. We next tested whether
ure6F;MovieS7).Insum,thesedatashowthatapicalprocess also in this condition RGC layer formation can be disturbed.
regrowthandre-establishmentofradialmorphologyarenoteffi- Like in the aPKC-CAAX condition, we used the Tg(Ath5:GAP-
cientinmorebasaldaughtersofnonapical,nonperpendiculardi- GFP)lineasareadout.Herewehadtofixtheembryoswithout
visions,andconsequently,suchcellscannotrobustlyreintegrate knowinginwhichonestheaccesstotheapicalsurfacehadbeen
intotheepithelium. sufficiently blocked earlier in development. Nevertheless, we
Subsequently,wetestedtheeffectofnonapicaldivisionslater found different embryos that showed basal cluster formation.
indevelopment.Notably,at24–30hphs,aPKC-CAAX-express- LikeintheaPKC-CAAXcondition,holesintheAth5layerwere
ingcellswereobservedatectopicbasalpositions,formingclus- observed that contained pH3-positive cells. Additionally, as
ters of rounded cells. These cells continued proliferating, as seen previously, these proliferating cell clusters disturbed
demonstrated by pH3 staining (Figure 7A). We next explored neuronallayerformation(Figures7E,S4B,andS4C).
howsuchbasalcellclusterformationaffectstissuematuration Together,theseresultsstronglyarguethattheoccurrenceof
and architecture. To this end, we injected aPKC-CAAX into nonapicaldivisionscanbelinkedtoperturbationsofPSEinteg-
Tg(Ath5:GAP-RFP) embryos. Remarkably, Ath5 expression rityleadingtodefectsintissuematuration.Therefore,wesug-
was not observed in aPKC-CAAX-positive cell clusters, while gest that the remarkable reproducibility of apical IKNM and
neighboringcontrolcellsdifferentiatednormallyintoRGCs(Fig- subsequentapicaldivisionsservestosafeguardtissueintegrity
ure 7B). These RGCs had to arrange around the aPKC-CAXX andmaturationinproliferativezebrafishNE.
positiveclusters,resultinginholesintheRGClayer(Figure7B,
lower).Thisdemonstratesthatbasalclusterformationofcycling DISCUSSION
aPKC-CAAX-expressing cells negatively impacts retinal devel-
opmentandtissueformation. In this study, we investigated the purpose and significance of
To ensure that what we observed for basal divisions in the localizingdivisionstotheapicalsurfaceinzebrafishNE.Were-
aPKC-CAAX condition was a general phenomenon, we aimed vealedthat(1)mitoticentryisnotrestrictedtoapicalpositions,
to induce such divisions in otherwise nonperturbed cells. To (2) apical IKNM depends on CDK1 and occurs independently
thisend,wemadeuseoftheknownfactthatinhibitionofPlk1 ofcentrosomepositionorintegrity,(3)nonperpendicularapical
leadstoamitoticarrest(Le´na´rtetal.,2007).Therebyanaccumu- divisions are tolerated and do not cause tissue perturbations,
lationofroundedcellsthatarestalledinmitosisformsasteric (4)afternonapical,nonperpendiculardivisionscausedbyinter-
hindrance at the apical surface (Weber et al., 2014). After this ferencewithactomyosindistributionorsterichindranceatapical
boundary has been formed, cells undergoing apical IKNM locations,cellsfailtoefficientlyreintegrateintothetissue;they
cannotreachtheapicalsurfaceleadingtononapicalmitoticen- instead establish proliferative basal cell clusters that perturb
tryofthesecells(Weberetal.,2014).ToinhibitPlk1activity,we overall tissue maturation. Figure 8 presents a schematic sum-
hereusedageneticapproachandexpressedaDNconstructun- maryofthesefindings.
derHSpromoter(Smitsetal.,2000).DN-Plk1wasexpressedina
subsetofcells(markedbyadditionalexpressionofH2B-RFP), ApicalMigrationOccursIndependentlyofCentrosome
leadingtotheformationofroundedapicalcellsstalledinmitosis PositionandIntegrity,aswellasMitoticEntry
thatblockedtheapicalsurface(Figure7C).Wechoseembryosin It was recently suggested that in the highly elongated cells of
whichwedetectedsuchblockingoftheapicalsurfaceforliveim- therodentneocorticalPSE,mitoticentryisrestrictedtotheapi-
aging. In all embryos imaged, we observed that beneath the calterminus(Huetal.,2013).InthezebrafishretinalNE,how-
blockedapicallayer,unperturbedcontrolcellslabeledbyH2B- ever, mitotic entry occurred at nonapical positions when the
GFPandras-GFPmRNAinjectionindeedperformednonapical IKNMmachinerywasinhibitedorwhencentrosomesandnuclei
divisionsshown by pH3staining and live imaging (Figures7C, met basally. Our observation is in accordance with a recent
7D,andS4A;MovieS8).Interestingly,earlyaftertheformation study reporting that nonapical mitotic entry can take place
oftheapicalhindrance,divisionsoccurredclosetothebarrier, also in the PSE of the Drosophila imaginal disk (Liang et al.,
and cells still featured an apical process during division (Fig- 2014).Interestingly,wedocumentedthatapicalmigrationper-
ure7D,upper).Later,however,theoffspringofthesecellsalso sisted despite mitotic entry at basal locations and even upon
divided at very basal positions (Figures 7D, lower, and S4A), perturbing centrosome integrity. We further show that CDK1
Figure4. ApicalIKNMDoesNotDependonCentrosomeIntegrity;CDK1ActivityIsNecessaryandSufficientforApicalIKNM
(A)Time-lapseimagingofacellfollowinglaserablationoftheapicalcentrosome.(Left-mostpanel)Cellpriortoablation.ThenucleusislabeledwithPCNA-GFP
(green).ThemembraneisvisualizedwithRas-GFP(green)andthecentrosomewithcentrin-mKate2(magenta,whitearrow).Afterablation(00:00),nocentrin
signalcanbedetectedintheapicalprocess(whitearrow).Theyellowboxinpanel00:00showsa23magnifiedregionoftheapicalendfoot.Thefollowingpanels
shownuclearbehaviorfollowingablationmarkedbyayellowarrow.Timeisinhr:min.TheframesarefromMovieS4.SeealsoFigureS2.
(B–D)Time-lapseofcellsexpressingPCNA-GFP(gray)upontreatmentwith(B)DMSOonly,(C)CDK1inhibitor(RO3306),(D)Wee1inhibitor(PD166285).Red
arrowsmarkcellsinS-phase(PCNAdots),andgreenarrowsmarkcellsinG2(disappearanceofPCNAdots).Timeisinhr:min.TheframesarefromMovieS5.
(E)TracksofnuclearmovementsinthedifferentdrugconditionsstartingattheonsetofapicalIKNM(PD0166285),theentryintoG2phase(RO3306),orboth
(control).
(F)Averagespeedofapicalnuclearmigrationindifferentconditions.Speedwascalculatedastotaldistancetraveledbynucleiovertime(mm/min).Errorbars
representSEM.
Scalebarsrepresent10mm.Apicalsurfaceisrepresentedbyadottedlinein(A)andasolidlinein(B)–(D).
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