The role of HDAC2 and Brg1 in adult neural stem cells [Elektronische Ressource] / Melanie Jawerka

Melanie Jawerka

Sujets

• Biologie

Informations

• Publié par: ludwig-maximilians-universitat_munchen
• Publié le: 01 janvier 2009
• Nombre de lectures: 15
• Langue: English
• Poids de l'ouvrage: 34 Mo

Extrait

redTheJarloLudwig-Maximilian-UniversitlofeDr.ofsubmittedHDMunichAtheC2CeandrchBrg1G?tz)inerkadult31.07.2008neyuraPrepalatstemHelmholtz-Zentrum/InstitutecellsStemPhDlThesisReseaSubmitted(Prof.toMagdalenatheMelanieFwacultayatofBiologyT1.Prof.Gutac?ndlichThomaster:derProf.Pr?fung:Dr.Dr.MagdalenaCremerG?tzag2.mGutachenh23.02.2009ter:.Contents.1.Abstract.4.2.Intro28duction.6.2.1.Neural.stem.cells..............4.7.3.administration.lab.analysis...27...........ures.4.6.2...injections...A............6.2.1.1.Cl.assication.ofybridizationadult.neural.stemsignalcells....e.......culture.......30.sso...cultures8.2.1.2.Regulatio32n.and.main.tenance.ofsthesiathe.neural.stem.cellinjectionsfate.......10.2.2.Epigenetic.factors.in.v34olv.ed.in.aBrdUd.ul.t4.9neurogenesis......35...T...........Quan..12.2.2.1.H.istone.deacet.yl.ase4.6s.(HD.A.Cs)..........Cul.primary.neural.....e.............In..14.2.2.2.Chrom.atin.remo.deling.complexes33.a...............4.7.2.adult.........ransplan..17.3.Abb.reviations.19.44.8Materials.and.Metho.ds.23.4.1.Animals..BrdU.............34.el...........situ.....................4.4.1.yramide.amplication............23.4.1.1.M.ouse4.5linestitativ.analysis...........................28.Tissue........................23.4.1.2.Genot.yping.of4.6.1mtutanoftdiandciatedtransgenicprecursorsmice......30.N.urosphere............23.4.2.T.amo.xifen.admi.nistration4.7.vivo...............................4.7.1.n.e..........26.4.3.Histology..............33.Stereotactic.in.mice...............34.T.tations.........................3426BrdU4.3.1.Cry.osections........................4.8.1.pulse.eling.........................4.8.2.lab27retaining4.4.Imm.unostaining..............35.In.h.............................1..Con.tenAtsHD4.9.1.Pl.asmid.preparation.and4.16.3inNvitr.o61transcription..5.2.4.e...solutions...Brg1.....y35.4.9.2.N.on-radioactivAe.in.situPhenoth.ybridization............of...A.expression.....5.1.3...C236.4.10andWunctionalestern.BlottingA...and.def...opmen.5.2.5...n.......Microscop.......Complex.....st.......56.HD...56.HD....37A4.10.1inCell.lysates.and.tissue.preparationC1.....5.1.4.......of.y...A.....71.mice..37714.10.2assaProteindefdetectionH.......brain.A...dev.m...in.C2.2...........45..............37454.10.3buersProtein.analysis......duct...............5.Expression.A.C2.......m.analysis.C1,.and.....5.1.2.of.H37and4.11oViral.v.ect.o.rs........D.m.vivo.........D.in...........Ov.A.imm.....5.2.of.............H.decien39construct4.11.1.Retro.viral.vHectorsactivitandfromretroAvirus.pro74ductionA.-...........bry.e.of.def39.4.12.Fluorescence-activostnatalatedtcellC2sortinge(F.A77CS)c.adult.HD.mi........................40.4.134.16.1RNAyextraction.and.microarra.y......................4.16.2.media,.and...............4541Pro4.13.1liRNA.extraction..........................52.Results.5.1.analysis.HD.C1,.A.and............415.1.14.13.2RMicroarraAyof.A.HD.C2.Brg1.........56.Protein.analysis.HD.C1,.D.C2.Brg1.vitr........................42.4.14.cDNA.preparation.and58RealHTimeA(RimT)unoreactivitPCRin.................58.H.A.expression.vivo43.4.14.1.cDNA.syn.thesis............5.1.5.erlap.HD.C2.Brg1.unoreactivit.........67.F.analysis.HD.C2............43.4.14.2.Real.Time.(R.T)5.2.1PCRD.C2.t.-.design.............5.2.2.D.C.y.y.WT.HD.C2.tissue.....5.2.3.D43C24.15miceHDospringA.C.activit.y.assa.y........74.Em.onic.dev.l.t.HD.C2.mice.......77.P.brain.elopmen.of.A.def.ic........445.2.64.16ypicMaterialshanges.the.brai.of.A.def.c...80...Con.ten.ts.5.2.7.The.adult.neurogenicpregions.in.HD.AAC2C2defPhenotmice:inDG..role.140.6.4.b83.5.2.85.3.4The.adult.neurogenic.regions.in.HDHDA125C2.defAmice:brainSEZ..regions.C2....89.5.2.9.Aneurgliogenicandregion.in149HDadultA.C2Discussiondef.mice:.sub.cortical124whitenmat-.ter.(SCWM)..p.m...of.dev...adult...130.C2.....C2...role.in.prole.......analysis.......Brg1.cells.6.5..92.5.2.10.Analysiswledgmentsof.thedeletionHDculturesA.C2.function120in6.1vitrexpressiono............HD.functional.lysis........97.5.2.11125TdescransplanontationC2ofeWT.cells.inThetoAtheostnatalHDtA.C26.2.3definSEZof.def.....The.HD99mice5.2.12.The.role.of6.2.5HDHDAadultC1.during.brain6.2.6devHDelopmennon-neurogenictadultofBrg1HD.A.C2.def.mice......-.............6.4.1.ation.in.l.....A.-...........A.Curriculum.151.109.Brg1.in.neurosphere......102.5.2.13.W.estern6Blots123ofHDHDC2AproleC1.and.HD.A.C2.of.WT.and.HD.A.C2.def.brain6.2tissueA.-.a.a.........................6.2.1.ypical.ri.ti.of.A.def.ic..........1026.2.25.2.14roleMicroarraHDyC2analysispofbrainWTelopmenand.HD.A.C2128defAbrainoptosisregionsthe.brain.HD.C2104mice5.3.F.unctional.analysis.of6.2.4Brg1adult.of.A.def...............131.The.of.A.in.neurogenic.........132.The.of.A107in5.3.1regionsBrg1theobrainxed6.3miceexpression-.c.onstruct......................142.Brg1.functional............107.5.3.2.Brg1.deletion.during.em.bry143onicAneurogenesisl.of.protein.adult.a.stem.......143.HD109C25.3.3Brg1Brg1comparisondeletion.in.adult.neural.stem.cells..........145.ckno.148.Vitae.Bibliography.3.C2-function.1decienAbstractfromTexaminedoinunderstandexptheandroleereofformepigeneticthemecisolatedhanismspinabsencevinolvsubunitedwinneurogenesis,neuralsulstemalcellsobservdur-Theingfunctiondevmelopmenhe-depttandaadulthotranscriptionoremod,1Icomplexexamis,nephenotdofthetageloBrg1calisationhoandproliferationfunctionunaltered.ofdecitsthestemhistonebdeacneu-etthereforeylaseC2(HDWTAfurtherC)ha2inthattaectse.transcArifunctionptadultiyonalwithregulation,btheythemohro-difyHDingmostthewhetherN-terminalaendslossofthetheahistoneatailslineresultingadultinneuraldecreased4transcedrimaturationpttheiyonalterestinglyactivitneuronalyere.inHDculturesAeC2onicproteindefectswmaturationasstemdeteactedHDattransplanhightslevinelstinoutpyostmitoticadditionalneuronstinAthemicedevtributeelophenotpingus,andfunctionaladultrevbrain.spInadulttematurationrestinglyneurons.,regulatedneuronalmoprecursorsopincthemacdevalelopingrolebraingenedidactsnotTPconthetainremoHDBrg1AC2C2expressedprotein,cwhiletheioftresultincreased-ineneuronalHDprecursorsoinoprg1ostnatallandtoadultvforebrain.aInthetheCretstemwinocellsregionsedofsadultwneurogenesis,defectsHneuronalDwhileAandC2self-renewwcapacitasremainedconIntained,insedoublecortin-pmaturationositivwenotneuroblastsedandneuralincelltransit-amplifyingisolatedprogenitors,thbutemnotryinbrain.theseadultinneuralronalstemfromcells.neuralIncellsorderimplytocell-autonomousunderstandoftheAfunctionandoftationHDerimenAofC2cellsintoneurogenesis,utanImiceexaminedruleantheHDossibilitAtC2tdeciennictendenmousedefectslineHDlacC2kingtthemighcatalyticconac-totivitheyypofThHDtheAofC2.HDWhileC2emealsbryratheronicecicneurogenesisinoneurogenesisccursthenormalofingeneratedtheseAsmice,isthebproliferationhistoneofdicationsadultconeuralerationprogenitorstheihromatinsdelingincreasedhineryandIneuronssoderivtheedoffrombrahma-relatedb(Brg)oththatzonesasofAadultaseneurogenesis,inthecsubmatinedelingpSWI/SNF.endymalandzoneA(SEZ)wandcodenintatebraingyrusell(DG)promptingdiequestionatdeletionaBrg1spouldecicinmasimtuilarratiyponthanstageofinAtheTabsenceexamineofrfunctionalleHDBAinC2.duIntvitrIookcul-dturesanofofneuralmousestemexpressingcellsinduciblefromoftheinadulneuraltcells.HDdeletionAadultC2stemdecienrettSEZinalsob1neuronalAaecbstr,aandcthetemdefectsmaiinThneuronalecicitfateadultacquisitioninandadult,apparenbrain.tstemfateneuronalcononicvofersionrevinwhictoythelateroligomaturation.den-regulationdroincytenotlineage.oniFvurthermore,regulatesthellactenancekfateofemBrg1adultreducestheself-renewingAadultgneuralthestembcellsepigeneticinkvitrectsowith.fateTheseregulatingdataedththeereforeofidenmaturationtifytheforbutthetherstbrytimecaConsperselyecicBrg1roleneuralofcetlwnoandepigeneticcellfactorsininoththebrycenandtralneurogenesis.nervus,ousfunctionssystem,HDHDC2ABrC21andealBrg1,sprevyealingytheirhdistinctregulatorsfunctionstineadultaspneurogenesis.ofHDneurogenesisAtheC2regulatingisandiformernneuronalv5olvnonfermen2neuroIntroneuralductionduceNeuralllstemtralcellshhathatvexpressionemousethenewabilitfoldingywtosuppgenerateanddicomplexstdominanistagncedtandcellwtNeuralypomesecomeforhistoricallytheneuronalformationectivofinthedividenervcenousrebsystemypandwncelltranscriptionalreplacemenbrytbduringteddevfunctionalelopmenforttheirandtheirthroughoutlineageadulthoadultollsd.(CNS)Neuralrstdevneuralelopmene,tTheseandtoplasticitoyprogenitorsareanddeterminedthebferenyepithelialbtootheextrinsichapandvindividetrinsicremofactors(matingthatswitcinandterfaceacttoasregulateator.genefromprogramstoconstrollingproteinsneuralecellllofateyandoffunction.decienTheAmoleculartomecinhanismscellsoftiationcomplextheproconstancessesinsucbrain.hcasmammalianprolifera-oustionaandstructuredierenbtiationnaretonotneuralyhetstemwwellelievunderstoduceoseparated.ofRecgen1889],tgeneraterepa,orts,haOthervaemoshohwneitherthattransformcgliahromatintoremotodelingiaand1897].epigeneticdatageneshoreepithelialgulandation6pladelingySWI/SNFantimpeortanhing/sucrosetting)roleknointodivpre-ersetlyareasasucactivhTheasanalysisneuralemcellonicfateadultspeecicationforandothsynapticwidevbelopmenpresentfoandwfunction.bEpigeneatistudyctransgenicmeclineshanismstincludeHDcC2ellBrg1-texamineyproleeadultspstemecicandtranscriptionaldierenregula-totion,ardhistoneneuronalmogeneratingdicationstlyandneuronscthehromatinmousere2.1mstemoedelingTheenzymes.cenInnervthissystemstudyistcwplexothatepigeneticarisesregulatorsyanditheirtheroleplateibntheneuraltubstewhicmcomprisescellsepithelialwillcells.bcellseereinbtroedduced.proFirst,ttheohistonepdeacetolsylasecommitted(HandDlialA[His,C)whic2,thenaneuronssmallglienzymerespcapableelyofindeacetbrain.ylatingstudieshistonesortasdif-wtelldelaswhicproteinsneuroknocellswnprotooractinasradialtranscriptionalthatrepressors.asymmetricallySecgivond,risebrahma-relatedneuronsgenegl(Brg)[Sc1,er,anRe-AtTPhaaseecatalyticallywnactivneuroecellsisymmetricallyntheaychromatintransform2hoInMcKatroinduction[Williamsgenerateandttowneuronsoenstemsuccellse[Gotztoand2002Huttner,(arro2005]offolloatewateredg