McI-l promotes Neural Precursor Cell Cycle Exit and Differentiation in the Mouse Embryonic Brain

McI-l promotes Neural Precursor Cell CycleExit and Differentiation in the Mouse Embryonic Brain

By

S.M.MahmuduI Hasan

Athcsis submitt cdtothcSchoolofGraduatcStudicsinpartialfullillmcnt ofthc requirementsforadegree of Masterof Science(Medicin e)

FacultyofMedicineC\leuro~cience) MemorialUniversityof Newfoundland

July 2011

Acknow ledg eme nts

Iwouldlike to expre ssmy gratitude and thankmy supervisor, Dr.Jacqueline Vanderluit. for providin gme withallthesupport and best guidance that a studente anask for.

Iwouldalsolike tothank thememb ersof mylab for their support andenco urag ement througho ut

Inaddition.Iwouldlike to thankmy supervisorycommittee,Dr.KarenMearo w and Dr. Miehiru Hirasawa,forprovidingme withthe best advicethroughoutmyprogram

Neura l precurso r cell(NPC) prol iferation andapoptosisarc keyregulato ry aspectsofmamm alian nervous systemdevelopment. Althou gh recentstudiessuggestedthese twoprocessesto be interrelated ,themolecula rmechani smsbehmdthis remain undefin ed .ll ereIshow thatmyeloid cell leukemi a-I (Mel- I),aBeI-2 fami ly memberthat is essentia lfor thesurv ivalof NPCsalso reducesNPCproliferationand promotestheirterminalmitosis.I found that within48 hoursofin uteroelectroporating Mel-Iin EI3.5mouse embryonic brains,the majority ofNPCstransfected with Mel-Ihave migrated into the post mitotic conicalplate.whereascontroltransfected PCs are still within the proliferatingvcntricu lar/subvcntricu lar zo nes. Analysisofproliferation by proli ferating cellnuelea rantigen (PC NA) immunohistoche mistryrevealeda 2-fold red uction in proliferating NPCs in theMel-I treatedbrains.ImmunohistochemistryforTbrl,amark er 1("

newbornneurons,showe da 50%increasein differentratedneu rons111Mel-Itreated brains.Hrd U birthdatingdemonstratedthat McI-lovercxpressionresultsin a greatercohort of' newborn neurons.Furthermore,Mel-ItransfeetedNPCs gaveriseto neurons in the deeper layersofthe cortexthan controltransfected PC seonfirrninganearlierbirthdate.Similarly,transfc ction of Mel- IinPCsillvitropromotes celleyeleexit.Ishowed thatMel-I interacts withkey celleyele regulatorsin NPCs,name lyPC NAand CdkllCyelinHI comp lex. In addition,Ifoundanincrease in Cdk inhibitorp 27"Plprotein, a keypromote rofecllcycleexitwith McI·1overexpress ionand a concomitant decrea seinp27^{Ki p i}in Mel-Icondition alknockout NPCs,suggesting thatMel-!

maymodulatep27' ;P'protein10promoteNPC dilTelentiation.Fina llyIshowed thatp27"'"is required lorMel- I mediatedNPCcellcycleexit, sugges tingthatMel-I regulatesNPCcell eyele through p27"PIactivity.Insummary, theseresultsidentify anovel functionfor Mel-Iin promotingterminalmitosisofIPCs byu.Iluencing the cellcycle regulatorymachinery.

Table of Contents

listof Tables 6

List of Figures 7

Cha p te r 1 9

, ....

Chapter2 40

Cha p le r3 60

Re sul ts 60

3.1 Ilowdoes McI-laffec tem bryo n ic NPCs invivo? 60

3.2McI·lregulates NPCprolifer a tionwith inthe em bryo n ic brain 63 3.3McI·lpromotesNPC differentiat ionwith in theem bry on ic brain 66 3.4 McI· l gain -of- fu ncti o ngenera lesa greatercoh o r tofnewborn ceIIs 69

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~~:~~:.~~~.~:

~~.~~~~.~.~.~

I.~n~~~.~~.~.~~~.~:.~.~.i.~.~

~~.~.~~.~

~:

~~~.~.~.~.~~.1.~~~.~:.~.~~~.~~~72

3.6 McI· !gain· o f·fu nc t ionregu lalesNPC pro lifera lio n;nv ;tro 75 3.7McI·ldire ctlyinte ra cts withcellcycle regula to rsinNPCs 78 3.8 Changcsin McI·lexprcs s io nshow concolllitanl changcsin p2 7KIp1 cxpr ession 81 3.9p27K1p 1affectsNPC p ro liferat io na nd d iffe re n l ia tio nsi m ila r to McI· l;nv ;vo 84 3.10 p2 7K1plan d Mel-t regu la teNPCp r olifer al ion althe same ra teinvit ro 87 3.1 1 McI-l regulates NPCp rolif erat ionth ro ughp 2 7K1P1a cti vily 90

Cha p ler4 93

List of Tables

Bcl-2fami ly membersthatfllnct ionincclla poptosisand proliferat ion

Reaction componentsforMcI-Iand CrePCR

Reaction componentsforpzt'i'"PCR

Bradford AssayStandardCurve

RceipesforPoly-acrylamidescparating andstacking gels

List of Figures

FiJ:urc l. 1

Ncurogc ncsisinthcdevelopingcortex FiJ:urc J. 2

Thechangesin transcripti onfac torexpressionduringneurogenicp rogrcssion . FiJ:ure l .3

rhc "in sidc-ou t"dcvelopm ent of thccortcxandgeneexpression pattcms duringandafter

FiJ:ure l .4

Cellcycleregulation by Cdk s(Cyclin dependent kinascs),cyclins,Cdk inhibitors andthc Rb/E2F pathway.

FiJ:urel .S BcI-2 familyofprote ins FiJ:urc2. 1

Conditional Mel-IKnockoutmediatedbyCrerecombinase.

FiJ:ure2.2

IdentifyingMcl-l,Creand p27^K,p lgenotype byPCR FiJ:ure2 .3

VerificationofMcl-land p27^K,P1ovc rexpressioninEI3NPCs.

FiJ:ure3.1

McI-lgain-of- function promotesmigration ofNPCsinto thccortical pIate.

Figure3.2

Mel-Iregulates NPCproliferation intheembryoni cbrain.

Figure3.3

Mel-I prom otes NPC different iationin theembryonic brain . Figure3.4

Mel-i gain- o f-fun etio n gener ates a greater eohorto fn ewb om eells . Figur e3.5

Mel-!gain-of-f une tion alters thelam inar des tinat ionofNPCs.

Figure3.6

Mel-Iregulates NPCprol iferat ionthrough a cellautono mousmech anism . Figure3. 7

Mel- I directlybindsto celleyel e regulato rs-PC NA andCdkI-C yelin B I.

Figure3 .H

Cha ng esinMel- Iexpress ionshowconcomitantchanges inp27^KiP'proteinin NPCs.

Fig ure 3.9

p27^KiP'affectsNPC differentiationsim ilar toMel- Iinvivo, Figure3.111

p27^KiP'andMeI_1reducesNPCproliferationatsimilarrate sin vitro

Figure3.11

Proliferation isnot affec tedbyMel-Iinp27K'P'nullNPCs.

Figure4.1

Summary&the continuedhypothesis ofhowMel-I regu late cellcyeleexitof NPCs.

Chapter1 Introduction

1.1Dc v ctopment of the l\la m ma lia nCortex

Durio gem bryo nicdevelop me nt,neuralste m cells(NS C s) give rise toalltheneuro ns and macro glia lce lls of themamm al ia ncentralnervoussystem(C NS).The diffe rentiated and funct ion ally speci ali zedcells areder ived e ith erdirectlyfromthe stem c ell s or ind irectly via fatc- re stri ctedprogenitors.Stemcellsarcdeli ned bytheirself-renewalcapability,ideallyforan unlimit cdnumber of celldivisions,and multipot en cy.thcab ilityto giverise tonumeroustyp cso f different iate dcells(Reynoldsand Wei ss.1992).NSC sandthefate-restrictedprogen itorsarc collectivel y calle d neuralprecursorcells(NI'Cs).In themousebra in,cortico gen e sisoccur s betwe enembry o nic da ys11-17(EII-17),whenNI'Csgene rat e the neuro nsto lim n the dist inc t cortica llayers (Ta ka ha shi etal..1996 ).Initiall y,NI'Csmustexpandtheirpool beforea prop o rtion ofthem committo a spe c ificlineageand differe ntiate (NoctorctaI.,200 4,Ih m ncr and Kosod o,2005).As a resu lt,a balancebetwee nprol ifer ati on andcom mitm entto a speci fic lineage regulates the NI'C popul ation.There fore , oneof thecriticalaspec ts ofmammal ia nbrain de velopmentis the regulationof NI'C cell cycle.

1. 1.1Ne uruepithe lj alCells

De vel op me nt of the eNSbegins withthe formationofthe neura ltube.The neura l tubeis formed withthe folding ofa shee t ofneuroe p ithe lialcells,deri vedfrom the ectoder mge rmlayer.

Neuroe p ithe lialcellsarctheprimitiveneural stemcells,lining theneura l tubelumenformingthe ven tr icu larzone(Merk leandAlvarez- Buy lla,2006 ). Thencuro cpithcl ial ce llsarcclongatcdand in contactwith boththe apica l(ventricu lar)andbasal(pia \)surfaces(Figure1.1A).Altho ughthe cellsdivideat the ventric ularzone(VZ) ,theypullthe nuc leito thepial surfaceduring interp hase.

This intcrkineticnuc learmovementma kesthe neuroe pitheli um loo k 'pseudostratificd'orlaye red (Hu ttnerand Kosodo,2005.Zhong and Ch ia,200 8).Be fore the onsetof neurog cnesisin mice, theneuro epithelialcell sdivid e symme tr ica lly.Thistyp e ofcelldivi sion generat es2identic al dau ghte r ste m cells(Figure1.18 )and expandstheneuralste mcell pool(llauben saketal.,200 4, Gotzand l lutt ner,2 005)

With the on set o f neu roge nesis fro m Ell,theneur oepi theli al c ell s swi tch to asymmetricdivisions andgenerate radia lglial cell s.wh ich exhibi t bothresidua lneuroe pithel ia landglia l prop erti es (lla ube nsak etal.,2004,GotzandlIuttne r,2005,Tarui ctal.,2005 ).Like neuroepith elialcells.

radialglialcellsdivid ein the VZ and maint aincontac t with the pialsur faceviaa rad ia lly proje ct ingbasalproc e ss (Fig ure1.11\),Theradialglia lcellsarc the prin cip al progenitorsofthe embryonicbrainand successive ly replace the neuroepithelia lcells,Thus,mostof the ne uronsarc ge nerated either dire c tly fromradialglia l cellsor throughintermediateproge n itors(Anthonyet

aI.,2004,GotzandBard e , 2005) (Figure1.18).Rad ialglia l cellsmaintainsome neuroe pithe lia l

cell propertiesincludingintcrkincticnuclearmigration andexpress ion of theintermediate- filam ent Ncstin (Hartfuss et al.. 200 1).Inaddition,they alsoexhibitseveralglia1characteristics including theastrocytespecificglutamatetransporter (GLAST),thcCa' +-binding proteinS 100fl, glial fibrillary acidic protein (GFAP),vimentin and brain-lipid-bindingprotein(13LBP) (CampbellandGotz,2002,Gotz,2003,Kricgsteinand Gotz, 2003).

Fi!:ure1.1 :Neurog en cst s inthe develupjng corte x

A-An illustration of the developm entalehangesoee urring duringcorticogenesis betweenEIO- 18 (from leftto right). Transition ofneuroepithelial cells(NEPs)to radialgliaoccursafter EIO/11.Thenucleiofneuroepithelial cellsand radial ghacellsremainat theventricularzone (VZ)whilethe basalprogenitors occupythesub-ventricularzone(SVZ).Differentiated neurons make upthecortical plate(Cl')followingncurogenesis,starting afterEl l(Malatesta ctaI., 2008)

B-Thelineagetrees showthe generationofneuronsIN)fromneuroepith elial cells(NE)via the stemcellpopulation-radialglialcells(RG), andfromthe progeni torpopulation thatisthebasal progenitors(BI')(Gotzandlluttner,2005j

A

B

E-18

Lateral ventricle

/~'-.,..Symmetric.

~ / ~'-.,..Asymmetric.dlerentiative dIVision

G'-.,..Asymmetric.differentiativedivision RG ~yrnmetric. derentiativedJVision

/ ' "~~~tnc.neurogenic

1.1 .3Ila salI' ro l:enito rs

Duringmid-neuro gene sis (EI3- 14).anot her neuron alprogenitorpopulation appears, thebasal progenitors orinterm ediateprogenitors.Basalprogenitorsdevelop from thedivisions ofboth neuroepithelialcellsandtheradialglialcells.Duringlater stagesof ncurogenesis.basal progenitor s fonn the subventricularzone(SVZ),which is amitoti c celllayerbasaltothe ventricularzone(llaubensaketa l.,2004.Miyata et al.,2004,Noctor et al.,2004).Characteristic markers for basalprogenitorsincludetranscriptionfactorsTBR2, CUXIand CUX2(Nietoet al., 2004.Englundet al.,2005).

Basalprogenitors contributetoneurogenesisbyundergoi ng symmetriccelldivisionsand generatetwo neuronaldaughter cells(Figure1.1A).Therefore. basalprogenitors amplify the number of cellsproducedbyapreviousprogenitor celldivisionandareanimpo rtantd etenni nant of brainsize (llaub en sak et al.,2004,Noctor etal.,2004.Martinez-Cerdeno etal.. 2006).

ThedifTerentstages ofne uroge nes iscanbe distingu ished bythe seque ntialexpress ionofspec ific transcriptionfactors (Figure 1.2).NPCsat the VZ expressPax6 and divide to generatethe intermediate progenitorcells(lPCs ).which migrateto tbe SVZandexpressTbr2.TheIPCs give rise totheNcurol)"committed ncuroblasts . Finally.differen tiated neuronsaregeneratedfrom neuroblasts and theyexpressTbrl. Tbrl+neuronsmigrate to thecortical plate.andso expression ofTbr lconfirms thecomp letionofncuro genesis(Takaha shiand Liu,2006).

Fig ure 1.2: The chan g e sin transcriptionfactor ex p ress io n duringneuro g en i c progr e s sion,

A-Illustrationof aventricularzone (YZ) NPC (radialglia)progressing toadifferentiated neuron inthecorticalplate(CP),via basal/intermediateprogenitors(IPC)locatedin the subvcntricular zone (SYZ).The sequentialexpression ofthe specific transcription factors(TFs;top row)and differen tphasesofthecell cycle tor radialglia andIPCs(secondrow from bottom) arcalso

B-Immunohistochemi stryon E14.5mousebrain furtherdemonstrate sthesequentia lexpress ion oflhet ran scriptionfactor sduringneurogenieprogressionof NPCs(!Ievneretal., 2006) . (IZ-intenll ediate zone)

1.2 0'1:anizatio na l De vel opm en t of theCo rt ie alLayers

Duringcorticogcne sis(EI I-E I9)in mice,theNPCsgenerate neuronstoform the 6-layered cortex (Dehay andKennedy,2007).Thefirstneurons fo rm thetransient pre-plate,whichisthen split bylater-bom neuron sto formthesuperficial margin al zoneand deeper sub-plate.The cortica l plate (CP)deve lopsbetween thesetwo layers,andeventua llygives riseto the multi- layered neocorte x(Figure 1.3) (Mo lynea uxetal.,2007).The organi zation aldevelopmentof the cortica llayersislargely regulatedbyCajal-Retzius cells, early-bomneurons orlhemargin alzone that expressReelin (Alcantar aetal..1998).Reelinis alargeextracelIularglycoproteinthatplays a role incell migrationand process outgrowth.Mutationsof the reelingene severelydisrupt the normal pattem orcorticallami nat ion.resulling inan invertedorderor cortical layersII-VI (D'Are ange loct aI.,1995,Hirotsune et aI., 1995).The positioningorCajal-Retziuscellsis regulated bythe radialglialcells(Kwon et aI.,20 1I).

Cortica l plate developmentisregulated insucha waythat thelaterbornneuro nsthatarriveat the corticalplatemigratepastthe earlierbomneurons.Thisresultsin the formation or the deeper layersfirst,followedbythe formation of themoresuperfic ial layers.The inside-out patternor corticog cncsis resultsinneuronswithinagivenlayerbeingbomatthe sametimc andsharing commonfunct ional propertiesandconnectivity(Rakic.1988,McConn ell,1995).Neurons in the differe nt layersorthe post-natal cortexcanbe distinguished bythe expressionof'specifi c tran scripti on factors.For example,cut-like transcripti onractor s(Cu xland Cux2) are expresse d in neuronsin LayersII-IV and thezinc-finger transcripti on factors(FezQ andCtip2)are expre ssedinneuronsin deeper LayersV-VI(Figure1.3) (Leone et aI.,2008).

Fi~ u re1.3:The ""i ns id e -o ut"de velupmentof thecortexandgeneex pre s sio n pattern sdurin g and a ftc rc or t ic ogc ncs is.

A-A represent ationof the developmentof the mouseneocortex (Ncx )duringconicogene sis as shown bythe coronalsectionthrough themousebra in at 1010.5(top pa nel ).The embryo nictime- pointscaleshowsthe sequential developm entofdifferen tlayers(bott ompanel ) (Molyneaux ct al.,2(07).Themultilayere d cortexdevelopsinawaythatlaterbo mneu rons arrivingat the corticalplatemigratepastthe earlierbom neurons.

U-Expre ssionpaucms of differenttran scripti on factors duringmid-eo rticogcnesis (embryonic:

left panel ) and afterbirth(rightpanel).Thesemarkers can be usedtoidentif y spec ifi c cortical layer sin thepost-nat albrain (Leone et al.,2008).

(CII.corticalhem;IZ,intermediatezo ne;LG E,lateralganglioniceminen ce;MG E, medial ganglionicemine nce;SYZ.subvcntric ularzone;YZ.ventricularzone;PP.preplatc:MZ.

margin al zone;51',subplate; CP,corticalplate;WM,white matter;I-Vi,thedistinct cortical layersi-VI I

A

tn.s E12.5 rn.s [145 £1,),5

Btrtbdar e of proj e ctt o n neu ro nsu btypes in the VZ/SVZ

Embryonic

To formamatur e nervo ussystemcons isti ngofthe vastnumb er of neurons and glia lcells.Nrcs must be regu latedin abal a ncebe twee n prohferationandtheir commitmentto a specific lineage.

This balanceis largelycontrolledby the cellcycle regulatorymolec u les.which cue the cellto eilherpro lifcrateorto d iffcrcntialew ithsubse que nt matur ati on,

1.3 .1RCl:ulation ofNI'CCe llCycle

Like all somatic cells.sigoa lingpathwaysthatdirect entry,progre ss ioninto and exit from the cell cycle regula tesNPC proli fera tio n.The cellcycleof anactively div idi ngcellis compose dof4 phase s: synthes is (S), mitosi s(M) and two gap(GIandG2)phase s(Fi gurelA A).TheSphaseis responsi b lefor replicationof genomic DNA and theMphase isdedicat edfor physical div isionof cellu larand geno miccontents into two individualcells .The first gapI'ha se (GI)occursbe twe en the end ofMphase and the bcginning of S phase.Gl phasecollsistsofacrilica lrestrictionpoint (Rj.when the ce ll eithe r comm its to thenextround ofcelldivision0rexi ts the cellcycle to ente r GO phas c. The secon dgap pha se (G2 ). betweenthe endofSpha se and bcg innin go f M phase,is dedicat edto repa irany errors inDNA repli eati on an dprep a, e s th e celll'ormi lOsi s (F igur elAA) (Par dee,19 89.Nurse.1994 )

Dur ing corticalneurogenesis.IheGI restrictionpo int Ikrhas beenidentifi ed as the criticalcell cycleregulatio n poi nt.EachsuccessivecyclesofNPCan d the ir restric ted progenit or res ultina greater proportion of post-mitotic cellsthatexit thecellcycleat R (C a vine ss etal.,1999 ).This restriction poi ntRisregul at edby the Retino bl astoma genefami ly an d E2F tra ns cr iptionfactors

1.3 .2Retinohlastom a l:e neandE2F transcriptionfactors

Theretinoblastoma gene (Rb) wasthefirsttumoursuppressor tobeidentified for itsmutations leadi ng topediatric eyetumo ur development(Fung et al., 1987.Lee et al.,1987).Rbfamily proteinsincludepRb,1'130and 1'107.allshari ng the structura l hom ologyofthefunct io nalNB pocket(Cla sson and Dyson.200 1).Thisfunction aldom aincaninteractwith transcription al regulatorslike the E2F transcriptionfactor s,a familyofproteins thatshare a related DNi\- bindingdomain.E2Ftranscription faetorsbindtosetsoftargl'tpromoters and aetiv ateorrepress transcripti on.There fore.Rb/E2F activity plays apivotal role inregul atin g cellcyele progression by controlling tran scripti o n oftarget genes(Polagerand Ginsberg,2008).

During the GIphaseofthece ll cycle,Cycl in dependentkinaseandCycli nactivity hypcrphos phorylatcsRb (Figure1.4).Oncehypcrphosphorylated,RbreleasestheE2F transcriptionfactor s (E2F- 1.2,3),whiehare the aetivato rs o fg ene transeription thatis essential forthe GI toS phasetransiti on andcommitmenttomitosis (Dyson.1998,Nevins.1998) Overexpression ofRbcausescellsto rema in inquiescence orprolon ged GI phase, whereas overexpress ionofE2 Fs inducesquiescentimmortali zed cells to re-entertheeelleyele (l'olager and Ginsberg, 2008)

The functiunufRbandeons equ entlyE 2Fsis eriti ealforneurogen esisand thedevelopmentof the CNS.Germlineknocko utofRbresults in embryoniclethali ty at E15.5 due tohematop oietic and neurologicaldefects.Conditionallyknockingout Rbresultsinectopic mitosis withinthe developingbrain andalthoughNPCscommitto a differenti atedfate .theyfail to exit thecell cycle(Fergusonct al.,2002).Thisisa result of enhancedE2r -1and E2F-3activitythatdelays thetermina l mitosisof NPCsdifferenti at inginabsence ofRb (Callaghan et al., 1999).Rbfamil y member,1'107,has also beenidentifiedtoregulatethe NPC pool. Although1'107 null mice

exhibit inc rea sedprol ife rat ingproge ni to r cells.the pI07-/-progenitors sho wimpairedneuronal commitment (Callaghanet al.,1999,Vanderluitet al.,2004.Vanderluitct aI.. 2007).Therefore, Rband relatedproteins and the ir1021'targetsregula te the NI'Cce llcycleprogre ssionand cell cycleexit.

1.3. 3Cycl i n- depen d e n t kinas e sandCycl i ns

Cyclin-dcpc ndc nt kinasc s(Cdks)regul atethc progre ssionthrougheach of the pha se s of the cell cycle.Cdks arcac tiva ted byphos phorylation/dcphosphorylation eventsas theybind tospec ific Cyclinpartners.thcirrcgolalOry sub units. Distinct Cyclinsarc synthesizedand then destro yedat specific phase s ofthe cellcycle,add ingano ther regulatory ste p for Cdk-Cy cl inact iv ity(Nigg, 1995). The re are 4 Cdks (Cd kl,Cd k2,Cdk4 andCdk6)and10Cyclinsthatbelon gto4 differ ent classe s(CyclinA,Cyclin8,Cycli n DandCyclin10 type),andareresponsibleforcellcycle progression(Ma lu mhr esand llarbaci d,2009).

The initiationand progre ssionthroughGIismediatedby activa tionofmultiple signa ling pathwaysthatconverge on the transcription ofim med iate early ge ncs,D-type Cyc lins,and thei r assemb lywith Cdk4/6kinases(Sherr,1995, Roussel,199 8 ).Once activated,CyclinD-Cdk4/6 complexespreferent iallypho sph o rylatepRb andpRb-re lated pro te ins1'107andpl3 0(Sherr.

1994 .Weinb erg,199 5).Thisisfollo wedby add itio nalphosphorylati onbythe CyclinE-Cdk2 and progression intothecellcycle (Ohtsub o etal.,1995)(Figur eIA ll).During S an dG 2phasc, continuo usCyclinA-Cdk2activ ityis requiredbutthetran sitio ntomitosi srequ ires Cyclin ll- Cdklaetivationbythe phosphata secdc2 5c(KingctaI.. 1994. ursc,1994)(FigureI.4A). Activity ofallCd ks arc regulatedat multiplelevel s including theabundan ce ofCyc lins,

activatingor deactivat ingphosphorylation ofCdksubunitsandthe abundanceof endogenous Cdkinh ibitor prote ins( Figure 1.3)(CunninghamandRousse l.2001. Musgrove et al.,2004).

Recently.theideathat activityo fallCd ksisreq uiredforprogressingthro ughthemamm aliancell cyclehasbeenchallenged.Evenin theabsenceofallinterph ase Cdks(Cdk2, CdkI,Cdk4and Cdk6),themouseembryo can undergoorganogenesiswithcontinueddevelopmentuntil midgestation. Underthesecircumstances,Cdkl binds to allCyclinsandphosphorylatespRb.

resuitingi nt hcex pressionofgenesthata reregulated byE2 F transcriptionfac tors(Santamariaet al..2007)Cdkl can also bring cellsout of quiescencein theabse nceof interphaseCdks by interacti ng withCyclin-Dand/orCyclin-EtophosphorylateRb(Martinctal.,2005).However, Cdk lknockoutmouseembryos failto develop tothemorula orblastocyst stage,sugges tiogthat otherCdks do nothavethesamecompen sato ry capac ityas Cdkl(Santam ariact al.,200 7).

1,3.4Cy cl in-dependen t kin a s e in h i b it ors

Cyclin-dcpcndemkinase inhibitor proteins are importa ntinregulat ing Cdk activity,andhence theprogression of cellcycle or cellquiescence.Two familiesof Cdk inhibitorspromote cell cycleexitby blocking the activityof Cdk-Cy clincomplexes:the Cip/Kipfamily,including p2 ('p,.p27"P' ,and pS7' ;P', andtheINK4family,includingpi S"" ' ,pI6" " ',pIS"""and pI9"" d(Eliedgeandllarp er,1994).

Altho ugh the Cip/Kipfamily ofinhibitors caninteractwithallCdk-Cycl incomplexes.p27' ;P'is themain Cip/Kip inhibito rinNPCs duringdevelopment.Theother family members,p2lc

;p,and p57"'P'. arc onlyexpress ed inpost mitoticcells within the conical plale (Ng uyen ct al.,2006).

P27"P'promotescell cyclearrestof neural progen itorcellsduring em bryogenesis(Fero et al..

1996,Kiyokawaet al.,1996. Nakayamaet al.. 1996.Carruthersetal.,2003).reduces

proliferation oftransitamplifying progeni torsinthe adultsubventr icularzone(Doetschet al., 2002).and. together with pI9'Ok4d.maintainsdilTerentiatedneuronsinanon -mitot ic state (Zindy et al..1999). The p27Ki

"-nullmiceexhibitmulti-organ hype rplasiafrom enhancedcell proliferat ion.lnadditio n.the p27" "-nu llm utan tsd emo nstrateadecreaseinneurona lproduction duringmid-eortieogenesisand an increaseinproductio n oflate-born neurons.This delayin cell cycleexitresults inanenlargementofuppercortica l laye rs (Goto et al.,2004).Similarly.

overexpressingp27KiP'in corticalprogenitorsprom otesprematur e cell cycleexi t andresultsina reduction ofupperlayer neurons (Ta ru ietal.,2005).

Inaddition topromoting cellcycle exit.Cdk inhibitorp27 KiP' alsopromotesdifferentiation and radialmigrationof co rtical projectionneurons.The N-terminusof1'27' ;"isinvolved in stahilizingNeurogenin-2 protein. aproneuralbasicheli x-loop-h eli x (bHLl I)factor,which specificscortical prugenit orstoa neuron al fate.TheCvtcnn inushalfof 1'27' ;' 1inact ivates G'I'PaseRho/v,amodul atorofintracellul aractindynam icsand influencescorticalneuronal migration (Nguy en ct aI.,2006).Thcrel(lre.p27'; PIplays a crucial rolein neuronaldevelopment bypromotingcellcycle exitofNl'Cs,aswellastheirdifferentiation and migration The p27';P'proteinis regulatedviaprimin gphosphorylation .ubiquitination followedby proteasom al degradation(PaganoetaI.,1995,Loda etaI.,1997 ).Cdk-mediatedphosphorylation onTIS7ofp27';P'is requiredforuhiqu itination.This representsa feedback mechanism by whichCdkseao regu late p27';P1turnover.Phosphorylation 01'1'27' ;"on TlS7byCdk-cycJin complexrequiresforma tion of a stahletrimerie complex.However,altho ughCdk l -CycJin 131 phosphorylatesp27"P'on TIS7,itfailsto formastablecomplexwith1'27"" and thus "annot direct itsubiquitinatio n byE3ligase(Momagnoliet aI..1999). Whether the phosph orylation of 1'27" "on T IS7 byCdkl-CycJinBIaffectsitsfunctionalroleISstill unknown.

Apartfromthe 1'187residue,regulatoryphospho rylation ofp27^K,p'protein alsooccurson 510.

Argininedirected serine/threoninekmaseslike Mirk/dyrk lBphosphory latesp27^KiP'on 510.This stabilizes p27^K;P 'proteinand enhancesitsfunctiona l propert iesasa Cdk inhibitor,bindingto Cdk2 (DengetaI.,2004).Interestingly in neuralstem cells,Cdk5canphosphor ylatcpzr'i'"on both 510and 1'187.Phosphorylation onbothsites promotesneuronal differentiationfrom cell cyclearrest followedbyneuriteoutgro wthand migration(Zhengetal..2010). Therefore, phosphorylationofp27^K;Plisa criticalregulatorystep in promotingcellcycle exitofNl' Cs and

Figure\..1: Cellcyc le regul at ionby Cd ks(C ycfin dependent kina s c s), cyc lins, Cd k inbihitorsand theRb/E2 Fpathw a y.

A-Schem aticof the eukaryoticcellcycle show ing the different phases-G I.S.G2.Mand the criticalrestrictionpointR.The specific Cdk-cychncomplexesresponsiblefor progre ssion throu gh each phase are shownwiththei r INK orKWICI!'inh ih itors(Adaptedfrom(Dchayand Kenned y.200?).

B-Toprogressthroughthe GIrestr ictionpoint.GICdk-cyclin complexeshypcrph o spho rylate Rbfree ingE2Ftran scription factors.FreeE2F spromotetranscript ionoftarge t ge nesand progression into theS phase.

A

INKCDKlflh.bltors

(Pls.Plrls.PI9)

CYdnD~

CDK4.CDK6

I

KIP/C1PCDK ,nh,b.tors

" it;;:]'

// i:». ~

Cdk4'6

C>c1in[)~

~

EarlyGl

Targel genetranscription

~r

LateGl

1.4Su rv iva lofN e u ra l l' rec u rs o rCe lls

Thesurviva lofNPCsisimportant to ensure the appropriatenumber ofneu rons withinthe differentlayers ofthecortex.Duringneurogene sis, NPCsand neuron s arc madein excess.This ensures thatifaportionof the cellsexhibitdefectsduringcelldiv ision,differentiation or matu ration,they can be eliminated.Cells exhibiti ngsuchdefectsand the excesscellsarc eliminated by apopt osis (NicholsonctaI.,1995,lIaydar ct al.,1999).AtEIOo nly rare apoptotic cellsarcobserve dwhen the PCs arcundergoi ng symmetricdivis ionsto expandtheirpool.

However, bylate-neu rogenesis at E18,50-70%of NPCsaredyin g (Blaschkeetal.. 1996).This massive celldea th is req uired to reguJatetheNPC po pulationsizeandeventually thebrain size andshape. Alie fncurogcnc si s is complete, there isasecondwaveof apoptos isamon g

cellsandso arc notpartsofthe function al circuitry (de laRosa and dePablo,200 0,Blomgrenct a1..2007)

Studies on pro-apop toticproteins andaspartate-specificcysteineproteas eslcaspases)have providedinsightson NPCsurvival.Specially,studiesonCaspase-Jdeficientmicerevealed hyperp las iaof the NPC popul ationduring CNS developmen t.ThISdemonstra ted thatthe programmedcelldea th of NPCsandneurons during development is apoptotic(Nicho lsonet aI., 1995,llaydar et al.,1999,Roth etal.,2000 1.Apoptosisis an energydependentformofcell death that is characterizedbyDNAfragmentation.nuclearcondensation amimembranechange s withoutinductionof an immuneresponse(Kenctal.,1972).Therearc two main familiesof proteins that regulateapo ptos is:the CaspasefamilyandtheIl-ceillymphoma (llcl-2)family (YouleandStrasser,2008).

104.1Ca spa s efamily

Caspascsarcclassificdaseithcrinitiatorcas pascsorcxccutioncrcas pases . lnitiatorcaspascshavc a caspase recruitmentdoma in (CARD) that allows them tointeract withother apoptosisinitiating moleculeslike apoptoticprotease activating factor -I (Apaf'-l),whichcleaves and activates cxccutioncrcaspases.Once activatcd v executionercaspascs cleave cellularproteinsresulting in thcphysiolo gicalcharacteristicsofapoptoSls.inciudingplasmamcmbraneblcbbingandnu clcar condensation(Fanetal..2005,Wangeta J..2005) .

Caspascfamilymember s are invol ved indevelopmental apo ptos iswithinthemamm al iancortex.

Caspase-Jand casp ase- 9 null-m iceexhibit hypcrccllularity due toimpair edcell death.This results inanexpan ded cortex and ultimatel ylethalityduring the perinatalperiod (Kuidaet al., 199X,llaydaretal.,1999).

104.2 Ilcl-2family

The BcI-2 familyofproteins is characterizedby thepresence of1-41311domains.Itisdivided into ) subtypes based on the functionalhomologyof theBcI-2 familymembers,Theanti- apoptoticBcI-2proteinsareBcI-2,BcI·xL,BcI·W,McI· landAJ,andtheyinhibittheactiv ity of pro-apoptoticIlcl-2 proteins.BcI-2,BeI·xLand BcI· Whaveallfour BIIdomains,whereas McI·1 docs nothave aBII4 domain and AIlacksboth BIBand B1I4domains(Youleand Strasser.

2008).Thesepro-apoptoticproteins arceithereffectorproteinscontainingBII·Ito1lI1-) domains.includingBak and Bax,or the III13domain only.The BII3onlypro-apoptoticprotcins activate thecellintrinsic apoptotic p.ulrvayand include Puma.Noxa. Bim,Bad.Bid.T lrkand Bmf(Figure 1.5) (ChipukandGreen,200X.Youle andStrasser, 2008).Collectively.theBII3

only proteinsfacilitatetheolig ome rization of Hakand Bax,which leadsto mitochondrial membrane permeabilizationand ultimatelyapo ptos is.

Ihcexpress io n of anti-a po ptotic Bel -2protein svariesthroughoutdevelopment. Withinthe de vcl opin g CNS.BcI-2 expression peaksbetween El l-ISand then declinestoan undetectable levelbythe time"I'birth (Krajewskactal.,2002 ).Althou ghBcI-2targeteddeletion inmicedoc s not affec t neuronaldevelopment.postn atallyit resultsina sig nificantloss of sympathetic.motor.

andsens ory neurons (Michaclidis ct al.,1996 ).Bcl-xl rcxpressionisfirstseen in post -mitot ic neuro nsafter EIO.5 (Krajcwska ct al., 2002).Cond itio naldeletionofBcl-xt.in catccholamincrgicneuronsresultsin viablemice witha reduction in thecatccholaminc rgic neuro na l popul at ionby one-thir d (Savitt ctal.,2005).

Incompar ison.germ lineknocko utof anti-upoptotic McI-1 resu lts inperi- impl a ntati o nlethality due tode fect sindifferentia tionof thetrophectod erm (Rinke nberg e retal.,2000).Condit ional knockou t(CKO)ofMcI-1 with intheNI'C po pulation resu ltsinwidespreadapo ptosi s and embryonicleth al ity at E15.McI-1 CKO embryos show apo ptosisamong Nes tin exp re ssing precursor cells.Doub lccortinexpressingmigratin gprecursorcellsami[\111tubulin(T ujl) expressin gnewborn ne urons(Arbour etal., 2008).ThisdemonstratesthatMcI-1 isrequiredfo r thcsurviv al of both proliferating and differentiatin gNPCs.Additionall y.unpubl ished dat afrom our labhasidentifiedMcI-1 asasurvivalfactorof embryonicneuralste mcells(NSCs).To assess se lf-re newa l ofembryonic NSCs. we perfonned a secondary neuro sph e re assay(Vand erluitet al., 2ll04)and demonstratedthat McI-1 CKO re sult sin a-l-foldreductioninseco ndary neurosphe res whenco mpa red to wildtypecontrols.Therefor e.Mci-Iis acritical surv ivalfacto rofbothNSC, and NPCs.Thisisuniqueto McI·:.sinc e all other Bcl-2pro-survivalpro tein s expresse d in the CNS.like BcI-2 and BcI-x L.arc requiredfor thesurv ivalofpost-mitotic neurons.

Figure1.5 :Ilcl-2 familyofprnteins

The Ilcl-2 family isdividedinto the anti-apoptotie. pro-apoptotieand BID-onlypro-apo ptotic subfamily.The1311domainsareillustrated as13111.13112,IlH3 and BII4,while the transmembranedomainisdenotedasTM.ThePESTsequenceisalso shownon Mel-!(Adapted from(You!candStrasser,200S)

Anti-apoptotic

Pro-apoptotic

BidC : '

=========-I::::==:::J

NoxaC : '

===- -====:::J

BmfC : '

====::::::J-===:::::=J

Bim

'C:::====:::::::::J-===~

BadC : '

====::::::J-===:::::=J

BH3-only

_ BHl

_ BH3

_ TM

_ BH4

_ PEST

1.58el - 2 Fami lyandtheCellCycle

Besides apoptos is.so me members o f the BcI-2 famil y o fp roteins regulate cellcycle progre ssion (Table 1.1).Forcedexpressionofsome pro- ap optotic BcI-2p roteins.in thepresenc eofapo ptosis inhibitor s,promot e cellcycle progre ssion andarcprolifcrativc.Tncontrast,forcc dcx prcssionof pro-surv iva l Bcl-2 and BcI-xLproteinsarrestcellcycle progression andare anti-pro liferative (Zinkeletal.,2006).

OverexpressionofBcI- 2 proteinslows cellcycleentryin III3T3 cell linesbypromotin g expressio n ofthe Cdk inhibito r1'27 ", 1and pRb relative1'130.Increase in 1'130resultsinthe forma tion of repressivep130-E2F4complexes and delayin the expre ssion ofE2FI. whichis requir ed for thecellcycleprogr ession (Vairoctal.. 2(00 ).Elevation 01'1'27""protei nalso occurswith Bcl -x Love rexp ression and inducesdel ayed ce ll cycl eprogression.Theeleva ted 1'27';';proteindelays activat ionofCdk2andCdk4 duringprogressio n intoS-p hase(Grc ideret al., 20(2).Therefore.forcedexpressionofsomeanti-apoptoticBcI-2 membersincelllines confersanti-prolifera tiveeffects.

Howev er.the anti-prolifera tive effectsof BcI-xLcanbe rever sedbythe pro- apoptoticBcl- 2 memberBad.Ind uced Bad expressionin fibroblastsresultsin continued proliferation and sustained Cdkz-cyclinEactivity.Bad alsoforms heterodimerswithBcI-xLto overcomethe GO/GIcheckpo intandconti nue cellcycle progr cssionrChauop ad hyaycta1.2( 01) Overexp ressiono f som eotherpro-apopto ticBcl-2membersin thepresen ce ofapopt osis inhibitors alsoincreasesproliferation,Transgen ic ovcrcxprcssion of Bax -alp haincrease sthe numberofproliferating thymocytes,reducesthelevelofp27"ptin mature T-cells andpromotes rapid progre ssiontoSvphase (Bradyctal.,1996).Gcnnlineknockoutof'anothcrpro-apoptotic member.Bid.results in impairedhepaticcell proliferatio nandcarcinogencsis.demollstratingits

role incellcycle regulation(Baietal., 2005).

It hasbeendemonstratedthat the dualrolesofsome Bel-2familymember sincellcycle progressionand cellsurv ivalare functionally separate. Site-speci ficmutationofatyrosine residu e withinth e N-terrninaIB II4regionofBcI-2 abolishesit sfunctionineelleycleprogression but hasno effectoncellsurvival (lluang et al.,199 7).

fable1.1:Bcl-zfamilymcm bcrs thatfun~ti(Jnincellap (Jpthsi salldpr(Jlif('ration( -I-=knocko ut . E3.5=cmhryonic day 3.5)

1. 6 :\Ic l- I Re gul a ti onofCellSurviva l

Mel-Iis ananti-ap optoticmem ber ofthe Bel -2 family.Full lengthMel -Iprotei ncontai ns a transme mbrane dom ain,whichlocalizesittothe outer-mit ochondri almembran e,where it intera ctswith pro-apoptoticBel-2memberslikeBim,Brnf Puma,Noxaand Bakto prevent cell apo plosis (Warrand Shore,2008),Mel-1 proteinalso has 31311domain s and2PESTseqllcn ces (Kozop aset al.,1993. Youlcand Strasser.2008).Pl-S'Lsequcnces areassocia ted with proteins with sho rt half-livesandareabsent inothcrBcl-2pro-surviv almembers(Fuji sc cial.,2000 ) Alternativesplic ing of thcMel-ImRNA result sin asplicevariant containin gonlytheIlH-3 domain.Theshorter Mel-Ipro te in is functio nallyoppositeto fu ll lcngt h Mcl-Ifragmentami prom otescelldeath (Binglect al., 20( 0),Therefo re,processingofMel-ImRNA isimp orta nt in deter minmgitsroleincellsurv iva l.

Md-I waslirst discovcrcdasagclJe lhatisuprcgulatedd uring induccd d iffcrcnt iat iono fhul1lan myelob lastic leukemia cellsML-I(Kozopasctal.,1993).However,stud ieson MeI-1loss-of - functionhavebeen restricted sincegen nlinc knoc koulof1\1c1-1resultsin the peri-implanta tion lethalityofmiceat E3.5.This lethalityisducto defectsin differentiationofth e trophec toderm (Rink cnhcr gc rct al. .2000),ltisthemost sevcrephcnot ypc amon gall of the Bcl -z ant i-apoptotic protein s.SinceMcl-I germline knock o utsarc embryoni clethal,our currentunde rstanding of thc functio ns of'Mel-I comefro mco nd itionalknocko ut mod el susingthe Cre-lox syste m (Sa uer.

1998\.Cond itional knockoutmodelforMel-I wasfirst generated to assessthc funct ionofMel-I inhcm atopoic tic systemdevelopmc m(Opfcnnan etal. ,2005).ltwasdcmOl: stra tcd thatMel-1is esscnti alforthc survivalufhcmatop oietic stcmcclls andthcdeveloplllentandsurv ivalof 13and TIympho cytcs.Thesubseq uelll gencrationofMel-1knockOlll<inhcpalocytes a r.dkcratinocytcs

resultedinwides pread apo ptos is w ith in both popul ati on s.Thisidentifies the survivalrole ofMel - Im hep ati c andepide nna lpro lifera tingprecursor s(Sitailoetal., 2009,Viek et al..2009).Whil e promotin g surv iva lofepidermalkeratinocyte s.Mel-Ialsoinduces expression of kcratinocy te differentiation mar kers,indicatin gthatits role maybecritica latthctimc ofdiffcrcnua tion (Si tailoetaI..2 00 9) .

Condi tiona lkno ck outof Mel-Iin the 'PCpopul ati on causes widespr ead apo ptos isinbo th prolifer atin g anddi ITerenti atin g NI'C s.lnthe ab sence o fl\lel -l,NPCsund er go ap oplosi s as they migr at e away from the ventricular zo neandcommit toaneuronal fate(A rbo uretaI.,2008).In Incl. Mel-I istheonlyBeI-2famil ymember thatisrequ ired for the surv ivalof embryonicNPCs Thus.Mel-Iappears to bea eritiealregulatorduringthetime of differentiationor cellcycleexit

1.7Rc guf utionofMel -I Protein

Regulationof Mel-Iisachievedat multiple levels- transc riptiona l.post -tr an scription aland post- tran slation al(Wa ngetal.,1999.Bingleetal.,2000.Cra ig, 2002,Wan get aI..2 00 3).lJnli ke its othe ranti- apo ptotic BeI-2famil ymemb er s.Mel-Iproteinislabilewitha sho rt hal f-li fe.

Depen din g on thece lltype andcontex t,its half- life ranges fro m minute sto a lew hour s (Craig.

2002.Cucona tiet al.,2003.Adam s andCoo per,2007) .Mel-Iprot einisregu lated by phosp horylati on andubiquitinati onfoll owedbyprot e3Som at degrad ati on.Mel - !ubiq uit inligase E3 (M ule) .an ubiquitinligaseconta iningaBII-3dom ain,intc ractswith Mcl-La ndubiquitin atcs its5 lysincres idues that res ulti n proleaso ma l degra dal io n (W alTe t a I..2 005,Zhonget a l.. 2005) . Glyco gen synt ha se kinase3 (GSK-3)alsophosph orylate sMel-Iand prime s it fo rubiquiunation

by theE3ligase beta-TrCP,prom oti ngitsdegrad ation(Ding et al..2007).Bot hubiqu itinatin g pathways arcopposed hythedeuhiqu itinaseUSPX.whichremovesthelysinelinked polyubiqui tin eh ains andpreventsprotcasornaldcgradation ofMcI-1(Sehwiekartet al.,20 10).

Substituti ngthe lysineresidu esinMcI-1 witharg ininecanexte ndthehalf-life of the protei n (Zhonget al.,2005).Thisdemonstratesthatproteasomaldegrada tion isthe majorregulatorof McI-1protei nand thatpreventi on "fitsn:pidd egradationoffers awaytoeffeciivc lyovcrcxp rcss

McI-1protei nis also regulatedthroughout thecellcycl eandpeaks at mitosis.Duringmito tic arrest,Cdkl-CyclinBI pho spho rylatesMcI-1atSer64and Thr92.Thisphosp horylationinitiat es degradat ionofMcI-1 bypro teasomalactivity of theanaphase -promoungcomplcx/eycloso me V,pC/C)E3ubiqui tinligase(Harleyetal.,2010).Thus.phospho rylat io n ofMcI-1by Cdkl Cyclinllland itsAPCICmed iateddegrad ationinitiatcsapoprosis ofc cllsarrestcd in mito sis

1.8i\lcI-1andtheCellCycle

11/\·itro.McI-1hasbeenshow n to affectcell cycle progression.Forcedexpression ofMel -Iin celllinesleadstodecreasedBrdU(bromodeoxyurid ine)intake, ameasureofcellproliferation.

andaslowerdoublingrate(Fuj isectal.,2000,Jamilctal.,2005).McI-1interact swithpC NA (pro liferatingcellnucl ea r antigen).afactorfor DNAPo lymerase bactivity dur ingDNA replicati on.Thisslowscellcycleprogression into S-phaseinHEK29 31'.IleLa andU20Scell linesIlujise etal.,2000).Furth enn Ol'e.ap roteolyticfrJgmen tofMcI-lhashecndemonSlratedlO bindtoCd klresu ltinginalo werrateofproliferation inamurine myeloidprogenitor cellline.

Cdklregul atesprogre ssion thro ugh Gz andMphasesofthecell cycleby bind ingwithCyclinll

and phosph orylatin gmultipledo wn streamtarget s(Jamil ctal.,2005).There fore, Mel -tmay affect the cellcyclekineticsat differentphases and reduce cell prolifcration.Reeently.

cond itionalknockout of Mel-Iin hepatocytcsresultedincreasedproliferation and hep atocellular carcinoma development(We ber et al.,2010)

1.9Rati un al cand Hypo thesi s

Neural precursorcell prol iferation andapoptos is arccrucial regulato ry aspectsofmamma lia n nervoussystemdevelopment.Althoughrecentevidence suggests that thesetwo processes arc interrclatcdj he molec ular mech anisms behind them arc notwelles tablishe d.Mel-Iisacritical surv ivall1letor !or both proliferatiug andd ifferentiating emb,)' on ie NPCs(Arbo uratal.200S).ln add ition,recentstudiesincelllinesshow tha t Mcl-l canalsoaffect cellcyclekinet icsupon

!'Jrcedexpression(f uj iseeraI2001l,Jam ileral.2005).llowcvcr.theroleofMel-tinregulating cellcycleprogre ssion underphysiological illvrvoconditions hasnotyet bcen dem onstr ared.

Therefore,I put forwardthe followinghypothesis-

Mel -Iregul ate scellcycleprogr es sion and promotes different iat ion ofNPCswith inthe embryon ic brai n.

Objecti ve..:

Themainobj ectivesofthisthesis arc-

I.TodeterminewhetherMcl-lregulate sembryoni c NPC prolif eration anddifferentiation.

2.To dctenninethe mechanismbywhich McI-1 regulatesNrCcycl eprog ression

Chapter 2 Materials& Methods

Micewere keptona12-hourlight/dark cycleandfood/waterwas administeredad libitum.All experi ments were approve d byMemorial University'sAnimalCare EthicsComm ittee,ad her ing

CD- Imicewere providedfromCharles River Laboratories.Forbreeding,mice werehoused in thesame cageforupto3days and theformationof the plugwaschecke devery12 hours.For embryo nic time poin ts.the timeofplugidentification wasconsideredtobeemb ryon icdayO .5(E 0.5) and themalemousewas immediatel y separa tedopon detectionof theplug.Flexed Mel- I (Me l-Ipr)transgenicmice weregeneratedin thelaboratory ofDr.S.Korsmeyc r (Opfenna n etal., 2005) and 'estin Cre (Cre'")transgeni cmiceweregenera ted in thelabo ratory ofDr.R.Slack (Berubeet'II..2005).Both were mainta ined onaFVBN background. Mel-Icon ditio nal knockout (CKO) mice weregenerate d by crossingNestinCre transgenicmicewithMel-Ifle xed (Mel-I'r) adultsasdescn bedby(Arbo uretal.,2008) andillustratedinFigure2.1.Mel-ICKO (Cre' <.Mcl- 1m)micewere compared tolittermatc contro ls(Mcl- ,' I»fora l!experiments studyingMel-I loss-of-function.The p27^K;r^lknockout embryos(p27^KiI' ]' / ')weregenerated bycrossingp27^K'rⁱ hetero zygousmales(p27^{Ki r l}+l.)with p27^K1r1heterozygou sfemales(p27^{Ki r l}+/')(Feroetal.,1'196).

both mainta inedona C57 BL/6 background .Forloss-o f-functionstudies,p27^Kir'knockout embryos (p27^Kirl_'.)werecomparedto wilotype littenna te eonlrols (p27^K'r ''' ').

Figure2.1 : Cood i t io n a l McI-1Knockout mediatedbyCre re cnmhlnus e, McI- 1fifmouse iscrossed withNcstineretransgenicmice.The Ncstinpromotermediates exp ression ofCrerccornbinasc andthe DNAbetwee n the loxP sitesisexcised .Therefore.McI-1 iscondit ionallyknockedoutfrom neu raltissue usingthe neura lspccific Ncstin prom otcr.

~ ~ ~

x

2.2 Gcno ty pi ng Mic e

Todetermi ne thegenotype.DNA wasisolated from ta ilclippingsofadultsand limb buds of embryosat embryon ic day13 (E13)usin g the RED Extra et- N- Amp tissue rCR kit(Sigma.

029K 6262 ).IsolatedDNA was thensubj ectedto Pol ymerase Cha in React ion(PCR )usi ngthe react ioncompo ne nts outlinedin Table 2.1. For p27 ' ;P'rCR .theREDE xt ract -N-AmprCR ReactionMix(Sig ma.R477 5) wasused andthecomponentsarcoutlined in Table 2.2.

IhePCR reaction forMcl -I wasprog rammedto bcin 94"C for6minutes.55"Cfor1minuteand n"cforI minute- rep eat edfor30cycles.The PCRreactionfo r Cre wasprogr amm edtoheIII 94"Cfor 3minutes. 56"CforIminute andnecfor1.5minutes - repea tedfor 30cycles.The PCRreact ionforp27^KiP'waspro gram medto hein94°Cfor6minutes,61"Cfor1minu te and n"cforI minute-rep ea ted for12 cycles.followed by 25 rcpeti tionsof94"Cfor 3minutes,

rCRproducts wererunina 2% agarosegel(Ultra PurcAgarose-Invitrogen.15510-027 ) contai ningEthid ium bromide(155R5-011.Invitrogen)to sta inthe DNAunde rultravioletligh t.

The gel wasrun at120 Volts for 30 minutestodetecttheCre band and for 90minutes for the Mcl-Ihand.Mcl-lf'r aliclehast w0 34bp /nxP sitesflanking exonI(Opfen na nelal..2005 ),asa resultthe wildtype Mcl-I+!'allcle (36 0bp)issmaller thanthe Mcl_l^fIIalle le(400bp ).Usingthe diff erenceinthesizeof the band sunderultravioletlight.thcse twoallele s can be disting uished.

Thewildtypep27^Kip'IP27"P''' +)band canhe iden tifiedat190bpcomparedto mutantp27''' ' (p27^Kip'.!.)handthat can beidentified at280bp .Miceheterozygouslo r p27^{Ki p}'(p27"P!+!')show hothband s. oneat190bpandanot he rat2RObp (Figu re 2.2).

Reaction co m p o ne n ts Mel - II;;~ume^{/S a m Ie} ~re^PCR

- -5-.0 - - 5.0

Table2.1:Rea ction co m p o ne n t s for.Vl cI - 1 andCre PCR

l'uhl c2.2 :Rea ctioncomponentsforp2 7^K;P'I'Cn

Figu re2.2:IdentifyingMel-I, Cre and p27K1pl gen n ty pe byI'CH.

A - The floxedMeI-1(Mcl_If/r)alle lecanhe distin guishedfrom wildtype MeI-1(Mel-I' '')allele bythedifferencein band size.Md-I''''has two 34b p/oxPsitesand is seenasthelarge r band compa red to MeI-1'",Micethat arc heterozygou sto Mel- I(Mel-!'ifl)show hothbands B-Crecan be identifiedbythepresenc eofahandat- 700bp.

C-Wildtypcp27K1p,(p27KiP'>/+)canbe identifiedby aband at 190bp,compa red tomutant p27KiP'(p 27K,p,./.)that canbeidentified byaband at280b p,Mice heterozygousforp27Kip ,

(p27K'P'·")showhothband s.

A

+/+f1/f1 +/fl

McI-1 flax 400 bp

Mcl-1wt 360 bp

B

+/++/- crem 700bP

c

+/++/--/-

p27rnt 280 bp

p27wt 190 bp

2.3Cul turi ng clonallyde ri v edNI'C~

NPCs were harv es tedfrom theneuroepit hel ia ofthe E13 embryos.Pregna ntdam s were euthanize d withalethalintraper iton ealinjecti on of Eutha nyI(250mg/ml,sodi um pentobar bital , Vetoquino l,IEUSOOI),followed bycervical disloca tio n.Theuteru scont ain ingthcembryoswas disscct ed and subme rg ed incoldIxIIBSS pll7.4(madefromlOxHBSS-lla nks'I3alan eed Salt Solut ion: Gibco.14065-056. and Gibcodistilledwater,15230-162 )withphenolred (Sigm a, P0290).lnd ividual embryo s wercrcmo ved from theirembryon icsacandtheirbrains extractedin coldIxlIBSS. TheNPCs were harv estedfrom the ganglioniceminence softheseembryo sby mak ing alongitudi nalincision th roughthe overlying cortex.Theexcised ganglion ic eminence s wereimmediatelytransfe rred intostemcellmed ia(SCM ),prepar edasdescribedinAppe ndi xI.

and manuall ytritur ated tosingle cells.Cellswerecounte d inaI:Imixwith 0.4%Trypa n Blue (Gib co,15250- 61 ) onaHemacytometer (FisherScientific, 0267J10). Neuralprec urso rcells were then plated atclonaldensity(lGcclls/pl.ttogrowncurospheres andincubatedat3'1"Cwith5%

carbon dio xid e and hum idity.

For studying McI-1 gain-of-functio n.a mutant McI-1 (nu Mcl -l )con stru ctfro m(Zho ng etal., 20(5)wasused whe rethelysine resid ues,involvedinubiqu itin-mediate d destructionof the protei n.wereconverted to arginine.ThepCIG2express ion vector (MegasonandMclvlahon.

2002)(Appe nd ixII)wasusedto direct theexpression ofmt McI-1both il/,·il'u a nd il/ ,'itro.T he

mtMcI-lconstruc t wasclonedinto thepCIG2vec tor5'to theinternalribos ome entry sequence (IRES)andenha nc edgree n Iluore sce ntprotein (eGFP) (Appendix1II).

Forstudy ing p27"PIgain-of-funct; on,p27"PIfrompG FP-Ep27 vec tor(DyerandCepko,2001) wasclonedintothepCI G2 vecto r5'tothe internalriboso me entry seq uenc e (IRES ) and enhan ced green Iluorescentp rotein (eGFP ) (Append; xIV).

The expr ess ionofbot hpCIG2tntMcl -l and pClG2p27 "plwere verifiedthroughtransfectionof El3NPCsfollowed byprotein analysisvia Westernl3lot24 hourspost-transfec tion,as shownin Figure2.3. Theoverex pressedtntMeI-lband(human) appearsat 37kDawhilethe endoge nous Mel-Iband(mouse) appea rsat 35kDaa s adoub let.Sincethe endogenousIevelof McI-lis100 low forperformingprotein interactionstudies,allimmunoprccipnationexperiments werehascd onthe overexpressed tntMel-I.

Fig u re 2.3:Veri fi ca t io nof :\lcI-1and p2 7^K;P\uv er expres sio n illE13NI'Cs. A-Western blotana lysisofproteinsamples 24 hoursPOSltransfection withactin used asa loadingcontro l(42kDa).Thc overcxprcsscd Mcl-I band(human)appeare dat 37kDawhilethe endogenous McI-1band (m ouse) app eared at 35 kDa.

Ctl=cellstransfected with pCIG2 controlplasmid .mt McI-1=cells transfcctcd with mt Mel-I.

Il-Westernblotanalysisofproteinsamplcs 24h our sp osttra nsfc ctionwith act inused asloading control (42kDa).Thep27^K;P'band appea redat27 kDa.

Ctl=cellstransfectcdwith pCIG2 control plasmid,pn^KiP''~cellstran sfectcd with p27';'PI

A

B

mt Ctl Mcl-1

Mcl-1 - - _

:~~::~ l~~~:~~ : ~~ ~g:

Actin .Actin

Cli p27Kip1

Actin

3-4days after platin gcneuros phe res werepassagedtosing le cells andthen tra ns fect cd 24hou rs later. Trans fecti on wascarri ed out usi ngthe AmaxaMou se Neura l Ste m CellNuc lcofecto rKit (Lonza,VPG- I0 04)andAmaxaNucleofcctorDevice(Lo nza,AA O-IOOI).accordingto manu factu rer 'sinstru ction.For everymillion (IxI0⁶)NPCs,I0ug of plasm idDAwasused for

Tran s fectcd cellsfor proteinassay were plated instemcell medi a immedia telyfoilow ing transfee tion.Cells for imm unocyt ochemi strywereplatedimme d iately followin gtransfe etionin stemeellmed iaw ilhoutheparin l(Jr ad herent cu lturcs.

2,6/llvitroProliferationAs s a y

NPCswere transfect ed with pCIG2 conu olplasmid.mtMd-Iplasmidorp27 "'PIplasmid and platedinproliferatin gcondition satclona l density, onPol y-ornithine (Sigm a.1'4<)57) coated dishes.Todetermin e the effects of Mcl-lgain-of-functio nonproli feration,transfectcdNPC culturesreceiveda5-Bromo- 2-d cox yur idinc (ImM Brd lJ--Sigm a,B500 2 ) pu lse2 hours immediatelybeforefixation .Cu ltureswere fixed with1:1methano l(Sig ma.179 337-4 L) and acetone(Fishe r Sci entifi c, A949 -4)at24 hours,48hours or 72 hours posttransfectiou Proliferat ionwas assessed tocomparetheeffectsofMd-I or p27^Kipigain-of -func tiononNl'Cs, Th is wasperfo rme dusin gimmu nocytochem istr y forprol iferating cellnuclea rantigen(PC NA)at 24 ho urs,48hoursor72 ho ursposttransfeet ion.

Prote in sampleswere extract edfromNI'Cs.Cells werelysed in complet eimmunop reeipit ation (11')buffercontaining25 mM Tris-llaseI'll7.4,148 mM NaCI,1mMCaC I"1%Triton X-IOO, 0.2mg/mLphcnylmethylsulfonylIluoride(PMSF),lOX proteaseinhib itors(apro tinin and leupeptin ) and 10 mM dithioth reitol(DT T).Sampl eswereIUn in duplicatesusingBio-Rad Protein AssayReagent (Bio Rad.500-0006)todeterm ine theprotein concent rationof each samplebyproduci ng a standar dcurveusingthellrad ford Assay(Table2.3)

Approx I Absorban~e j

0.000 ,

=-~=3

_-~~_ _ _I

The slopeof the standa rdcurve was usedtodetermi neprotein concentrat ion of each sampl e using the followi ngequation'

ul. (Slop e)(Volumeuf sampl e)

2.7lmmunnprcctpttatlunwithI'rotein-GScp h u ro seBe ad s

Individualprotein sam p les cont ainin g 600ugof proteinin250 flLofeompletell'bu ffer,5flgo f spec ific antibodies(C yclindependentkinaseI(Cdkl) -SantaCruz,SC 53219 ; Cycl in B_.Santa Cruz,SC 752 ;McI-1-Santa Cruz,SC 819) were usedlorpullingdown protein co mplexes.

Prot ein swithantibo d ieswere placed onarotatorat 4'Cfor 3hour s,the n40 fl LofProtcin G Seph ar oseBead s (S igma,P3296 -lmL )wereaddedandset back toincubat e ove rn ight The followingday.be ad swere washed incold compl eteII' buffertorem o veany non-spec ificbinding and the n centrifug edat 1000 rpmfor2minute sin 1°C, allowi ng thespec ific prot ei ncompl exes bou nd10thc antib odie s and bead sto sed iment.To thispellet,40 ft Lo f6Xproteinloadi ngbuffer (250mM Tris-IICI.0.5MDTT,10%Sodium dodecyl sulpha te (SDS),0.5%bromophenolblue and50%glyc erol) was adde d. Samp leswere boiledat 100'Cfor 2 minutesto disso cia tethe protein complexes,cooled toroom temperatur e and theneentrif ug edatlO,OOO rpmto sedim e nt thesepharosebeads.The supe rna tan tcontaining theproteinsam p leswa s then loaded illl015%

poly-ae ryl amid e gel a ndwa srun aceord ing tothep roeedurefo . we stembl O!.

2.8 Wesl ernBioi Analysis

Proteinsamp les containing60 ugof'protein weremixed with5flLof6Xproteinload ingbuffer, boiledatI00°Cfor 2minutes and th enl oaded on aI 5%poly-a erylamidege l.

l'ahI0 2. 4:Re cipe sforl' o( y - auyl am id o s op aratin g aod sta ekin g g ol s

1\Mini-PROTEI\ N appara tus(BioRad. 165-8001 )filledwithRunn ing Buffer (0.1 M'Ir is-Base, 0.3Mglycine, 0.01M SDS) wasusedto run the gel.The protein sampleswereloaded intothe stac k mggeland runat 80 volts untilthe dyecleared thestacking gel.Oncethesamplesreached the separatinggel.the gelwasrunat110 voltsfor.)hours .To determine thesit eofva rious proteinband s,10 I,LofHio-RadKaleidoscopepre-sta inedprotein marker (Bio-Rad,:61-0324 ) wasalsoloadedin one of thelanes ineachge l.

Protein sfrom theseparatin g gelwere transferred toanitrocellu losemembrane(A mersharn BioSeie nee s.RPN30320) withthe BioRadMini Tran s-BlotElectrophoreticTransfer Cell (BioRad,170-3930)in WesternTran sferBuffer(0.02M'Iris-B ase .0.15Mglyc ineand4.9M methanol ).Follo win gtransfer.the memb ranewaswashedinIxTween-20phosp hatebuffered saline, TPBS (126mM NaII, POa,62/)mMNaC!,")r.MTwcen-20 ,ddllyO) for15minuteson the shaker,then blocked in 5%blotto(5% skimmilk in TPBS) foran houratroo m temperature.

followedbya washin 0.5%blotto.Blots were incubated overn ight with appropriateprimary

antibody(Append ixV)in0.5%blotto at4°C in ascaledplastic conta inerona shakerat low speed

Thefollowingday,membranes werewashed in 0.5%blotto,followedbyincubat ion with appropriatesecondary antibody(1:2000goat anti-rabbitIgGhorseradishperoxidase (IIRP) conjugate,BioRad,170651 5;1:2000goatanti-mouseIgGIIRPconjugate.Biokad .170651(,) dilutedin 0.5%blottoforIhour inroom temperature.Thenmembranes were washedin IxTPBS and the secondaryantibod iesweredetectedusing a chemiluminescenc e reaction kit(Perkin Elmer LabsInc.-Westem Lightni ng.02118-2512)accordingto themanu facturer' s instructions. Images of thememb raneswere taken1minute after apply ing the che milum inescence reagent.

using GEImagcQu antLAS 4000(GEHealthcare,28-9558-10).To detecttheleve ls ofl>-actin, the loadi ngcontrol,membraneswere stripped usingWesternBlot StrippingBuffer(Sigm a, 21(5 9)at37°Cfor30 minutesandtheprocedurewasrepeated follow inga wash in5%blotto.

2.9/lIl1teroel cctroporalioll

IIIlIteroelectroporationwasperformed on pregnant CD-I fem alemice at EI3 (embryon icday

13).10examine theetTectsofMcI-1 gain-o f..function on neura l precursor cellsill V;l'O .Pregnant fem ales wereanaest hetized withisolluoraneinhalation andwe re clo sel ymonitored daring the entireproccdure.A hypotearopht halmicointment was appliedonthe eyes ofthe mousedunng the surgicalproceduretopre ve nt eyesfromdryingOU:.The entire procedurewasperformed within45minutes.Duringthe surge ry.the mousewas kepton a stcr iIepaddi ng on a heating pad setat30°Ctomaintainbody tcm pe raturc.

Onceanaes thetized,thefur was removedfro m the abdomenof thepregnant mouseusing Nair (Church&DwightCana daCorp.,Mississau ga,ON).The abdo men wasthen clearedwith7l)%

ethanolandan incis ion wasmadedow n themidl ineoftheabdo menandthrough the intraperit onealwall,whichwasthenlined withsteri legauze.Uterine hornswerepulledthrough theincision and placedonsterilegauzeand mo istenedwith pre-warm ed sterile 0.9%saline.

Ind ividu alembryo sreceived an inj ect io noftheplasmid(1~lg/~IL),eitherpClG2 contro l.p27';P' ormt Mel- Iplasm ids,usin g aFernto.let Inj ector(lOOhl'a,1.2 sec,I'C=16) intothe latera i ventric les.The plasmidsolutio nalsocontai nedanon-to xicdyeto visually mon itorinjec tion s into thelateralvcntrieles. Foll o wingtheinje ctio n,elcctroporation pad dles(5ml11,Protech lntcrnational.CUY6501'5) were placedonopposite poles of the embryo'sheadandusing an ECM830Generator (lIarvardApparatus)a series of7pulsesat45volt s,50msec duration with a 500msecinter val,weredeliveredasdescribedpreviously(Lange v inet0/.2(07).Followin g clcctroporation.theuterin e homwasre-inscrtcdbac kintothe abdomen ofthe pregnan tmouse, the musc ulature and overlying skinsuturedandthemo use was allow edtorecover.Atcomp letion oftbesurgery, atopicalgentamicin (Topagen ) wasspray ed on theabdomenof'thepregn ant

Postsurgerymiceweregivensteriliz ed drinkingwater withsulfamethazineantibiotic(O.U5'%

sodiumsulfamethaz inesolution)forthefirst 3 dayspo st-ope rationtopreve ntinfection.The hea lthand wei gh t ofthemicewere monitor edonadall ybasisuntileuthan asia.24 hourspo vt- c1ectropora tion,pregn antmicereceived a singleintraperitonea l Brdl,'inje cti on (lOO,tgl gbody weight)10labelpro liferatingcells.Brains of the electroporatedembryoswere.collected at 48 hour s and 5 daysfollowin gtheilllIfer oelcct ropo ration,or twoweeks postnatall y.

2. 10 Tissue colle c ti o n, fixation, cryo pro tec ti o nandscctioutng

At48hoursand5daysfollo win gtheinuteroclectroporation,pregnantmice werecuthanized with alcthalintraperitone alinjectionofEu thanyl( 250mg/ml.sodium pentob arbital.Vetoquin ol.

IEUSOOI). followe d by cervicaldislocation.Theuteru swas removed bymak ing aninc ision throug htheabdo minalwalland placedinIxPBS.The embryoswere remo ved fromthe embryonicsacsand their brain sdissected .Brains werechecked under the micro scop eforGlP fluorescence,and onlythose with 01'1'expression wer e collected. Pupscollected 2 weeks postnatallywerealsosacrificed withalethalintraper itonea linjectionof Euthanyl.Following euthanasia,pups were perfusedwith icc-coldIxPBSfollowedby4%Para-for maldehyde(PFA~ Fisher Scientifi c,04042-500 ;IxpBS.ddl1,O ,pI17.4\

Brains were postfixedovcrm ghtin 4%pFA.After fixation,thetissue was cryoprotectcd by equilibratingin increasingconcentrationsof suc rosesoiutions(12%,I()%and22%~w/vsucrose in IxPBS).Following cryoprote ctio n.brains were frozenin Tissue-Tek (Sakura Finctck, 0004348-01)o ni sop entane,cooledondryi ce.l3rains wer c sectioned (14fllninthick ness) onthe sameday as freezing.ona cryostat (Microm11M520Cryostat). Tissuesectionswerecollected on Supcrl rostPlus(Fisherb rand ,12-550- i 5) slidesandthenstoredat-XO°Cuntilfurther

2,1 1 Immunohistochemi str yand Immunocytochemist ry

Forimmunohistochemistry,slideswerewarmedto3T=Candahydrophobicmoat(Dakopen)was drawn around the brain sect ions. Fornuclear stains (rC NA.Tbrl,Brd \! andCux lt.slideswere

post-fixed inacetone (Fishe r Scienti fic. A949-4 )for oneminutefollowe dbywashesinlxPBS.

Slideswere nextincubated overnightwithprimaryantibodies(Appen dixV) dilutedin IxPBS at room temp erature. For BrdU and PCNA (proliferating cell nuclear antigen) immu nohi stochemistry.slides werepre-treatedin2NIICIfor30 minutes at37°Cfollowedby0.1 MNa,B,O,(I'"8.0)wash for10 minutesto denatu retheDNA .Slideswere thenwashed inIx PBS be foreincubatin g overnight withprimary antibodyin the humiditychamber atroom

ForPCNA immunocy tochemistry,cultureswerefixedusingcold(-20°C)1:1methanol.acetone (FisherScienti fic.A949-4) for5minutes.Thiswasfollowed bywashes incold (4°C)lx phosphate buffe redsaline(PBS-137mMNaCI.27mM KCI.100mMNa,IIPO, .18mM KII,pO"dd11,0 ,adjusted toI'll7.4).Thenthe cells were firsttreated with2NHydrochloric acid(Fisher Scientific.SA56500)at roomtemp eraturefor15minutesfollowedbywashesillcold IxPBS.Foll owin g this.primaryantibodi es for PCNA(\:300-Vecto rLabs, VP-P980jilllx PBSwas addedto thecells and incubatedovernightat4°C.

ForBrdLJimmunocytochemistry. cultureswerefixedusing cold(4°C)4%PFA 10 1' 10 minutes, followed bywashe sin cold(4°C)IxPAS.Then the cellsweretreatedwithDNase(Iunit/50Il l- Promega,M6101)in DNaseBuffer(40mM Tris-lIc1,III mMNaCI.6111M MgC I,and10mlvl CaCI,)at37°Cfor30 minutes.todenaturethe DNA.Followin gthis.ce llswere washed mix PBSand then incubatedwithprimaryantibod iesfor BrdLJ(1:100-BDBioscicnccs, 347580) ill IxPBS overnig htat4°C.

Thefolluwingday.theslidesorculture dishes were washedill IxPBSandincubatedw:ththe appropriateseco ndaryantibody dilutedillIxPBS(1:200dm:kcy anti-n1'JuS{'lgG (1l-:-LI Akxa Fluor 594-Invitrogen.A2120 "1:200donkey anti-rab bitIgG(11+1..1AlexaFluor 594-

Invitroge n.A21207)for one hour.coveredwith aluminiumfoil. Followingthis.the cellswere stained with the nucleardyeHocchst diluted in IxPBS (1:250BisBenzimide1133258~S igm a.

B1155)for two minutesand thenwas hedagain inIxPBS.Slideswere then coverslippcdwith 1:3 g1ycc rol:lxP B S andth e ed g es we re seal ed w ithn ailp oli sh.

2.1 2 :\lic rosc o py an dSt atis tic s

Cultures were examined on a Zeiss.AxioObserver A.Imicroscopeto confirm tra ns fec tio nand subsequent expression ofplasmids.Immunostained cellsand tissueswere examined on a Zeiss Axiolmager Z.Imicroscope under LEOfluorescenceproduced usin g Colibri. Eachslide conta ined three brainsec tio nsc cach about14 0 )1I11apart. Phot om ic ro graph swe re taken of each embryonic brainsectionat thesame magnification.The samplesize indicatesthenum berof different embryosused for eachtrea tme nt group.Averag enU!11bcrofG FP'cells detectedp"r experiment fo r eachtreatment group islisted in Appendix VI.

Allimages weretakenwithZCi5SAxioC:1nlMRmcame raUSi1gZeissAxio Visto n~. 8software Images were processedand the figureswere compiledusingAdobePhoto-hopCS2 where manipulationswere madeonlytocontrast and brightness.rhefree wareImageJ(1ational Institut e forlIealt h) was usedfor quantilicationof positivecellsfollow ingimmunostainingund the countsweremainta ine dinMicr os oftExcelspreadsheets.All'Statisticswereperformedusrng GraphPadPrism 5 software,includingunpairedTvtcst and One-wayAnalysisofVariance (ANOVA).Tukcy's post hocana lys iswasused to de te rmi nedifferences betweentrea tmen t

Chapter 3 Results

3.1 110'" doc s :\Ie l- I aff e ct em b ry o nicNrC ,in viv n?

Duringneurogene siswithintheembryonic brain.Nl'Csdivid e atthe ventricular zone (VZ) and subve ntricu larzo ne (SVZ ). As Nl'Csexitthe cellcyeleandprogr ess towards aneuronal lineage, theymigrate rad iallyout intothe cortical plate(CP) (Malatestaet'II.,2008).Thcrcforcvthe proliferat ing zonesinthe deve lopingcortexarc theVZand SVZ,while thepost-mitot ic dirrerentiared ee lis mak e up theC P.

Toinvestigatethe effects or Mel-Igain-o f-funct ion onNPCs,IclectroporatcdGFP(control)01 mtMel-Iplasm idsintoEI3mou se embryosillutero,Ico llected the brai ns4&hourspost electroporationandassess ed the locatio nort he G Fp'transrcctcdcelis.

In controlbrains,the distributi o norGFp'cells wasmostlyin theproli ferative zones,VZ (26t2%) and SVZ (52±2%). with lessthanafourthor theGFP'cellsin the post-mitotic CP (21±3%) .In cont rast,therewas ashiftin thelocat ion ortheGFp'cellsto ward,theCP inthemt Mel-!trea ted brai nsLess thanhalfoft he GFp'cell s in mtMel-I trea ted brainswerein the prolifcranvczones.VZ (16±2%)and SVZ (26±2%).andmost or GFp'cells were inthe CP (58±3%)(Fig ure3.1)Th isdistinc tshiftinthelocation or Grp'cellsillthe mtMcl-ltreated bra ins suggestedthatMcI-lgain-o f-func tion induce sipe stoex itthe cellcycleand mig rate to

Fi~ure3.1: :\lcI-1ga i n- o r- f unc ti o n promote smigr ationofNile sinto the co rt i ca l plat e.

A-Representativephotomicrographsofembryo nicbrainsections48hourspost-elcc tro pora tion of contro l (01) ormt Mc1-1plasnuds, showingthclocati on of GFP+cellsinthe VZ (ventricular zone ).SVZ(subvent ricu larzone) andCl'(cor ticalplate).

B·-Quantificationof the perce nt GFP+cellslocated inVZ.SVZ andCPwithin CtlandmtMel-I trcatcdbrains.GFP+ccllswerccountcd in3representative sec tions perembryo(11:::;"5itrea tme ntj.

Mean ce ll countswere analyzedbyt-testwithsta tisticalsignificance assess ed at*p<O.05.Graphs reprc scntmcanseSfrM .

4°fri 60[}j S°b t

30 40 60

20 ^* 20 ^* 40

10 20

o 0 0

Clirnt Mcl-1 Cli mt Mcl-1 ClImt Mcl-1

mtMcl-1 Ctl

B A

3.2 ;\lcI-1regulatesNI'Cproliferation within the em h ry o ni c hrain

To characterizethe effect of Mel-Ion embryn nicNI'Cproliferation,IelectroporatcdGFI' (control)ormt Mcl- Iplasmidsinto 1013mouse embryosin uteroandassessed proliferation with I'CNA immunohis tochemistry4Rhourspostclectroporation.I'C NAis a component ofDNA polym erase-delta andis requiredfor DNA replication.Asaresult,thereis adisrinctincrea sein I'CNAexpre ssionduringthe S·phase (Bacc hiand Gown.1993 )andso itisusedwidely as a markerforcell proliferation. 4Rhourspostelectropora tion,incontro l brains22±3%of transfected cellswere alsoI'CNA'.In contrast,only8±I%oftransfected cells werealsoI'C NA' in themt Mel-I treatedbrains{Figure3.2).Th isgrea ter than two- foldreductioninprol iferating NPCssuggeststhatMel-Igain-of-functio npromotes ceilcycle exitofNPCs.

Fi:: u re3.2::\Icl-Ire::ulat e s NI'Cprutlfcr atlnnin theem bryo n ic hrain . A-Representat ive pho tomicrog raphsofcm bryo nic bra inscclions48hours post clcctroporation show ingGFI"cellsand rC NA+cellsincontro l andmtMel-Ic1cctrop orat cdbrains.Arrows point todoublelabeled cells.

R~.Quantificat ionofthepercentdou blelabeledGFI"andI'CNA+cellsincontro l (Ctl) and mt

McI-1elcctropo ratcdbrains.orr:cellswere countedin3representativesectionsperembryo (n=5/trcat mcn t).Mean cellcoun tswereanalyzed byt-test withstatisticalsigniiicance asscssedat

·p<O.05.GraphsreprcsentmeallsoiSEM.

A

cu

B

b-

3.3 Mcl-Ipromote s NI'C d i ffe re ntia ti o n withinthe emhry oni cbrain

Since Mel-I gain-of- function reduce s NpC proliferation,Iassessed ifMel-IregulatesNpC diff erentia tio n.Thestag es ofneurogenicprogressionofa NPCcan be distingui shedby sequential express ion of spec ific transcriptionfae:tors(Figure1.2).The express ionof theTbrl transcription factorsconfi rmstheneurogeni ctransiti onof NPCsandcanbeusedtolabelnewbornneurons (HevneretaI.,2006 ).Ielectro pora tedGe l'(control)ormt McI-1plasmid sintoEI3mouse embryosin uteroand assesseddifferentia tion with Tbrl immunohistoch emi stry 48hourspost elec troporation. lnc ontrolbra ins18±3%oftransfected celis werealsoTbr !'.lncontrast, 29±3%

oftransfec tedceliswere Tbr l'in the mt Mc l-1treated brains(Figure 3.3).The 50%increase in differenti ated neuronswithin mtMcI-1electropora tcd brains suggeststhat Mcl-L gain- of-function

Figu re3.3: :\lcl-1promote s:"I'Cdifferentia lio n intheem bryo nichrai n.

A-Representative photomicrograp hsof embryon icbrainsections 48 hourspost electro poration showingGFP'cells and Tbrl"cellsincontro land mtMel-Iclcctroporatedbrains.Arrowspoint

B-Quantificationof the percentdnublelabeledGFP'and Tbrl'cellsincontrol(Ctl)andmt Mel-leleetroporated brains.GFP'cellswere counted in3 representativesections per embryo (n=5/treatment).Mean cellcountswere ana lyzedbyt-tcstwithstatisticalsignificunccusscsscdut

·p<O.05.Graphsreprese ntmeans±SEM.

A

B

bt