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ZBED6expressionpatternduringembryogenesisandin

thecentralnervoussystem

AxelEricsson

2010

DepartmentofNeuroscience–DevelopmentalgeneticsUppsalaUniversity

Supervisors:KlasKullanderandMartinLarhammar

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Table of contents  

Abstract...................................................................................................................................................3

Abbreviations ......................................................................................................................................3

Aimofstudy ........................................................................................................................................4

Introduction ............................................................................................................................................4

ZBED6..................................................................................................................................................4

Developmentofcentralnervoussystem ............................................................................................4

Materialsandmethods ...........................................................................................................................5

Immunohistochemistry .......................................................................................................................5

Imaging................................................................................................................................................6

Resultsanddiscussion ............................................................................................................................7

Conclusion.............................................................................................................................................12

Acknowledgement ............................................................................................................................12

References ........................................................................................................................................13

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Abstract

ZBED6isarecentlydiscoveredrepressorprotein,whichwasfoundduetoaQuantativetraitlocus(QTL)‐mappingstudycomparingwildboarwithdomesticatedpigs.AsinglenucleotidepolymorphismwhichdisruptedtheZBED6interactionwiththeInsulinlikegrowthfactor‐II(igf‐II)generesultedinanupregulatedgeneexpressionandincreasedmusclemass.ThebindingsiteforZBED6hasbeenfoundinnumerousgrowthfactors,whichindicatesanimportantroleforgeneregulation.InthisstudyweinvestigatedtheZBED6proteinexpressionduringembryonicdevelopmentandinadultCentralnervoussystem(CNS)inmouse.HereweshowthatZBED6isexpressedbydifferentiatedneuronsandstartsapproximatelyatembryonicday10.5,withnoexpressionobservedintheproliferationzone.FromtheexpressionpatternZBED6donotappeartobelinkedtoanyspecificregionsinthespinalcord,ratherageneralexpressionindifferentiatedneurons.TheproteinexpressionwasmappedintheadultbrainshowingthatZBED6iswidelydistributedinmanyregionsandisexpressedinbothastrocytesandneurons,howevertheproportionofZBED6expressingcellsvariesbetweendifferentbrainregions.

Abbreviations BMP ‐BonemorphogeneticproteinbHLH ‐BasichelixloophelixCP ‐CaudateputamenCNS ‐CentralnervoussystemDLHP ‐DorsolateralhingepointsFGF ‐FibroblastgrowthfactorsGFAP ‐GlialfibrillaaryacidicproteinHD ‐HomeodomainIgf ‐InsulinlikegrowthfactorsMHP ‐MediumhingepointsNeuN ‐NeuronnucleispecificantigenNSC ‐NeuralstemcellsQTL ‐QuantativetraitlocusRA ‐Retinoidacid Shh ‐Sonichedgehog

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Fig.1Formationoftheneuraltube.LägginA,B,CIbildenoxåsåmankanfölja.Formationofneuralplate(A)‐bendsandfolds(B)closureoftheneuraltubemigratingcrestcells(C).

AimofstudyTheaimofthisstudywastoinvestigateZBED6expressionpatterninmouseembryonicdevelopmentandadultCNSusingimmunohistochemistrytoassessitspotentialroleasaregulatorofproliferation.

Introduction  Theunderstandingofgeneregulationisimportantinmanyaspectssincethecomplexityofanorganismisnotbasedonthenumberofgenesbutrathertheregulationofthem.Howspecifictranscriptionfactorsinteractandregulategeneexpressionisofinterestforunderstandingandscreeningfordevelopmentaldiseases.Pointmutationsinspecificregionscanhavesevereconsequencesandleadtospecificphenotypes.ApreviouslyunknownrepressorproteinwasdiscoveredusingaQTL‐Mapping,bycomparingEuropeanwildboarwithLargeWhitedomesticpigs1.Itwasfoundthatthedomesticatedpigshadaccumulatedasinglenucleotidesubstitutionandthefavoredallelewaswellconservedduetostrongselectionformeatproductionoverthelast60years1.ThisG‐AtransitionwaslocatedinaCpGislandinintron3oftheinsulinlikegrowthfactor‐II(igf‐II)gene.Themutationinigf‐IIdisruptedtheinteractionwitharepressorprotein,namedZBED6,andledtoanelevatedgeneexpressionofIgf‐IIresultinginanincreasedmusclemass.ZBED6containstwoDNAbindingBEDdomains(namedaftertwochromatinbindingproteinsBEAFandDREAF)whichcanmodifythechromatinstructureandregulatethetranscriptionofgenes,andahATC‐dimerizationdomainwhichisrelatedtothehATCsuperfamilyofDNAtransposons.Thebindingsitehasbeenfoundinover200genesandmanyofthemassociatedwithdevelopmentaldisordersandneurologicaldiseases1.InthisstudyweinvestigatedtheZBED6proteinexpressionduringembryonicdevelopmentusingimmunohistochemistryandcompareditwithneuronalandproliferationmarkers.WehavealsocharacterizedtheexpressionpatterninadultCNStoexaminetheimportanceandpotentialfunctionofthisrepressorprotein. 

Background  

Developmentofcentralnervoussystem

Neurulationistheembryonicprocesswhentheneuraltubeisformed,andstartswiththeformationoftheneuralplate2.Thecellsinthelateralectodermdifferentiateintotheneuralplate,whichthenlengthensandnarrowsandtheneuroepithelialcellsmigratestowardsthemidlineandintercalate.3Theneurulationcanbedividedintoprimaryandsecondaryneurulation.Theprimaryneurulationformthebrainandmostofthespinalcordwhilethesecondarystartsatmorecaudalpartofthespinalcordincludingsacralandcoccygeal

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regions4.Furthermorecantheprimaryneurulationbedividedintofourphases;formation,shaping,bendingandclosureoftheneuraltube(fig.1).Theneuralplatebendattwospecificsites5,attheventralmidlinesonthemedianhingepoints(MHP)andnearthejunctionoftheneuralplate,onthedorsolateralhingepoints(DLHP).Thesetwositesareinvolvedindifferentstagesofthebendingandclosingoftheneuraltube.InmouseneurulationtheneuraltubestartsbendingatMHP(E8.5),proceedsbendingatMHPandDLHP(E9.5)andfinallythelowerneuraltubeonlybendsatDLHP(E10)6.Theclosureoftheneuraltubeisinitiatedinthehindbrain/cervicalpartsandthenproceedsunidirectionaltowardsfuturebrainandspinalregions,additionallytwoclosuresitesoccursatforebrain/midbrainandattherostralendofforebrain.Thespinalcordclosurecontinuestotheposteriorneuroporeinwhichthesecondaryneurulationstarts7.

Arrangementofneuronsinthespinalcord

Fromelevenprogenitordomainsalignalongthemidlineofthedevelopingspinalcordallthespinalcordneuronalsubtypesarise8.Thearrangementoftheseprogenitorcellsandthedifferentiationtodistinctneuronalsubtypesisdependentonalargenumberofintrinsicandextrinsicfactors.Intheventralparttheimportantpatterningproteinsonichedgehog(Shh)isactivewhichisexpressedfromthenotochordan9.AgradientofShhregulatesthehomeodomain(HD)transcriptionfactorsclassIandII,byrepressingclass1andpromotingclass210.Inadditionthebonemorphogenicprotein(BMP)issecretedfromtheroofplateandinhibitsShheffectinthedorsalpartsoftheneuraltube11.Fibroblastgrowthfactors(FGF)andWNTproteinscontributetoproliferationoftheneuralstemcells(NSC)andareactiveintheventricularzoneoftheneuraltube12,13.Thedifferentiationofprogenitorcellsarethendependentonretinoicacid14,basichelixloophelix(bHLH)proteinsandprogenitorspecifictranscriptionsfactors,whichbyasynergiceffectcontributestoeachdistinctsubclassofneuron15.Intheventralhornofthespinalcordfivedistinctsubtypesofneuronsaredeveloped,fourinterneuronsV0‐V3andthemotorneurons(MN)16andinthedorsalpartsixcelltypesareformed,dl1‐3aresomatosensoryrelayinterneuronsanddl4‐6associatedinterneurons8 

Materials and methods  

Immunohistochemistry

Theembryosamplesatembryonicstages9.5‐18,werecollectedfrompregnantfemalesmicesacrificedbycervicaldislocation.Embryosweredissectedandfixedin4%paraformaldehyde(PFA)inPBSfor10–60minonice,followedbycryoprotectionin30%sucrose.TissueTekO.C.Tcompound(A/SChemi‐TEknikk)wasusedforembedding.Thesampleswerecryo‐sectionedinto12‐16µmslicesusingaMicromHM560(MICROMInternationalGmbH,Germany).Sectionswerecollectedonslidesanddried,washedinPBS

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(3x10min)andincubatedinblockingsolution(1xblockingreagent(Roche),0.2%tritonX‐100(Sigma,Germany),0.02%SodiumazidedilutedinPBS)for2h.PrimaryantibodiesagainstZBED6(rabbit,diluted0.2µg/ml)andNeuron‐specificnuclearantigen(NeuN,1:400)dilutedinblockingsolutionwereincubatedat4°Covernight.Thefollowingday,slideswerewashedinPBS(3x5min)andincubatedwithasecondaryantibodyconjugatedtoAlexa594(Invitrogen,USA)andAlexa‐488(Invitrogen,USA),Alexa‐647(Invitrogen,USA)andDAPI(Sigma‐Aldrich,Germany)asacontrolfornuclearstaininginPBSfor1hatroomtemperature.SlideswerewashedinPBSandmountedwithmowiol‐488(ROTH,Germany).Immunolabelingwasanalyzedinafluorescencemicroscope(OlympusBX61WI,Japan),afluorescenceslidescannermicroscope(3DHistechPannoramicmidi,HUNGARY)andaConfocalmicroscope‐(ZeissLSM510META,Germany)

Imaging

ImageswereprocessedinadobePhotoshopCS5(AdobeSystems,USA)andImageJ1.43Uusing;FFTbandpassfilter,desprecklefunctionandarrangedtogetherinadobeIllustratorCS3

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Figure.3.ZBED6expressioninsargitalsectionE12.StainedwithZBED6(red)andNeuN(green)(A‐D).Sargital(A)overviewofembryo.Highermagnificationimagesindicatedin(B‐D)withZBED(B)andNeuN(C)andcolocalizationofZBED6andNeuN(D).Imagestakenwith20xmagnificationimagestakenwith(3DHistechPannoramicmidi)Scalebarindicates100um

 

Results and discussion ThebindingsiteforZBED6hasbeenlocatedinover200genes,manyassociatedtodevelopmentaldisordersandneurologicaldiseases.InthisstudyweinvestigateitsexpressionpatternduringtheembryonicdevelopmentbycomparingZBED6withmarkersforproliferatinganddifferentiatedneurons,furthermoreaZBED6proteinexpressionscreenwasperformedintheadultmousebrain.ImmunohistochemistryonasagittalEmbryoatstageE12.5stainedwithantibodiesagainstneuronalmarkerNeuN17andZBED6canbeseeninfigure3.Weobservedstainingandco‐localizationofZBED6andNeuNinbothsubventricularzoneandinthespinalcordbutlikelynotintheventricularandproliferationzones.ImagesweretakenwithaPannoramicmidiscanner,whichwasusefulinwholeembryoimagingandgivingmultipleimagesinhighmagnificationandresolutionarrangedintoonepicture.ThemajordrawbackwiththePannoramicmidiscanneristhesensitivityforcrumpleswhichresultsinoutoffocusimages.TofurthercharacterizeandestablishwhentheexpressionofZBED6startsweperformedanimmunohistochemistryassayoncoronalsectionsofwholeembryosE9.5‐E18.5,focusingonthedevelopmentofthespinalcordduetoitswell‐knownprogenitordomainsandmanyneuronalmarkersareavailible.Co‐labelingwasperformedwithantibodiesagainstZBED6,NeuNandKi67(ageneralmarkerforproliferatingcells)18.(fig.4),theproteinexpressiononsetwasobservedatE10.5intheventralhorn.ZBED6wasco‐localizedwithNeuNbutnooverlapwasobservedbetweenZBED6andKi67atembryonicstageE10.5(fig.4).ThisindicatesthatZBED6doesnotaffecttheearlyspecificationandisnotexpresseduntiltheprogenitorcellshavedifferentiatedtoneurons(fig.5).TheembryonicexpressionpatternindicatesthatZBED6isnotlinkedtoanyspecificregioninthedorsal‐ventralpatterningofthespinalcordandisalsoexpressedinthedorsalrootganglion(DRG)(fig5M‐P).ThenumberofZBED6expressingcellsincreasedduringdevelopmentinthespinalcordinthesamepatternasNeuN,andintheadultspinalcordZBED6expressionwaswidelydistributedovertheentirespinalcord(fig.6).ZBED6iswidelyexpressedinothertissuesduringembryogenesis,howeveramoreintensestainingwasobservedinCNS.Duetothewidespreadexpressionduringearlydevelopmentandintheadultspinalcordnoco‐stainingwasperformedwithimmuno

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markersforspecificneurons.RegardingtheproliferationmarkerKi67,thisantibodywasproblematictogettobindandseveralantigenretrievalsprotocolsweretriedwithoutsuccess.Themainparameterforki67antibodybindingwasthetissuefixation,oftenshortfixationtimewasnecessarytoreceiveananalyzablesignal.

Figure5.ZBED6expressionpatternduringearlyneuronaldevelopment.ZBED6andNeuNimmunoflourescenceanalysisofcoronalmouseE9.5‐E12.5(A‐P)ImageofSpinalcordE9.5(A‐D),E10.5(E‐H)E11.5,(I‐L),12.5(M‐P),stainedwithDAPI(A,E,I,andM),ZBED6(B,F,J,N),NeuN(C,G,K,O)andtheco‐localizationofZBED6andNeuN(D,H,LandP)indicatedbyarrowheads,doublearrowheadsindicatesDRG.Scalebarindicates100µm.Imagestakenwith20xmagnification(OlympusBX61WI)

Figure4.ZBED6isco‐expressedwithNeuNbutnotKi67inthespinalcordE10.5.A)Ki67asproliferationmarker,(B)ZBED6stainingintheventralhornneuraltube,(C)NeuNstaininginventralparts,(D)YellowstainingshowstheoverlapbetweenZBED6andNeuNwithnooverlapwithki67theproliferationmarker.Scalebarindicates100µm,imagestakenwith20xmagnification(OlympusBX61WI).

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ZBED6expressioniswidelydistributedinthespinalcord(fig6.).ToassessifZBED6expressionwaslinkedtospecificbrainregionswemappedtheexpressionintheadultbrainandmeasuredtherelativeproteinexpressionsignal.CoronalbrainsectionswerestainedwithantibodiesagainstZBED6,NeuNandglialfibrillaryacidicprotein(GFAP),togetherwithDAPI.TheexpressionofZBED6indifferentbrainregionswasanalyzedandmarkedwitheither+(0‐25%ofthecells),++(25‐50%ofthecells),+++(50‐75%ofthecells).ZBED6iswidelydistributedandhasaproteinexpressionrangefrom0‐80%ofthecells(fig.6andtable.1).RegionswithalowexpressionofZBED6appearstobewhitematterareassuchascolossalcommissureandthedorsalhippocampalcommisure,andregionswithhighexpressiondensitywasforexamplemultipleregionsinamygdalaAhighoverlapwithNeuNwasobservedinmostbrainregions.NostatisticalquantificationofZBED6wasdoneduetothewidespreadexpression.TofurtherinvestigateifZBED6expressioninothercelltypesinCNSweusedGFAPamarkerforanintermediatefilamentproteinexpressedinastrocytes19.AsmallpopulationofGFAPpositivecellsco‐localizewithZBED6expressionwasobserved(fig.8)theproportionofGFAPpositivecellsexpressingZBED6wasnotmeasured.

Fig.6ZBED6expressioniswidespreadintheadultspinalcordLumbarspinalcordlabeledwithDAPI(A)andZBED6(B).ZBED6iswidelydistributedovertheentirespinalcord,mainlyinthegreymatter.Imagestakenwith4xmagnification(OlympusBX61WI)

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Figure.6TheZBED6expressionvariesbetweendifferentbrainregionsImmunofluorescenceanalysisofAmygdala,secondaryvisualcortex,caudateputamen,olfactorybulbandDHC.Coronaloverviewofbrain(P,QandR)assembledfrom4xmagnificationimages,boxedareasindicateshighermagnifiedareas:Cortex(A‐D),Caudateandputamen(E‐G),Amygdala(H‐K),Olfactorybulb(M‐O)andDHC(Q‐S)stainedwithDAPI(A‐M)ZBED6(B‐N)co‐localizationofZBED6andDAPI(C‐O).Shortarrowheadsindicatesco‐localizationofZBED6andDAPIwhilelongarrowsindicatesonlyDAPIstaining.Closeupimagesweretakenwith40xmagnification(OlympusBX61WI).Scalebarindicate44µm.

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Conclusion Duringembryogenesis,ZBED6proteinexpressionstartsbeforeE10.5indifferentiatedneurons.ZBED6doesnotappeartobeexpressedinproliferatingprogenitorcells.IntheadultbrainandspinalcordZBED6isexpressedinneuronsandtosomeextentastrocytes.Awidespreadexpressionwasobservedinmostpartsofthebrain,whileafewregionsdidnotexpressZBED6(e.g.dorsalhippocampalcommisureandcolossalcommisure).ThewidespreaddistributionofZBED6andthelargenumberofbindingsitesindicatesafundamentalroleingeneregulation.IthasstillyettobedeterminedwhichgenesandhowZBED6areregulatingthoseintheCSN.HowcrucialZBED6isfornormaldevelopmentanditspotentialroleinneurodevelopmentaldisordershasyettobedetermined.AconditionalZBED6knockoutthatallowsdeletionofZbed6inspecificcelltypesandcellpopulationswillgiveusadeeperunderstandingofthisprotein’sgeneregulatingabilities.

AcknowledgementIwouldliketothankMartinLarhammarwhichhavebeenagreatsupervisorguidingmethroughthisprojectalwaysansweringmyquestions.I’malsogratefultoKlasKullanderandLeifAnderssonwhohasgivenmethechancetodosuchaninterestingproject.

Table.1

Figure7.ZBED6isexpressedbyastrocytesImmunofluorescenceanalysisofHippocampusstainedwithanti‐ZBED6,anti‐NeuNandanti‐GFAP(B‐E)antibodies.Coronaloverviewofbrain(A)assembledof4xmagnificationimages.Boxedareaindicateshighermagnificationregion.Hippocampusstainedwithanti‐GFAP(B),anti‐ZBED6(C)andanti‐NeuN(D).Arrowsindicatesco‐localizationofGFAPandZBED6(E),DoubleheadedarrowsindicatesoverlapbetweenZBED6andNeuN(E)andarrowheadsindicatesGFAPpositivecelllackingZBED6.Imagestakenwith40xmagnification(Confocalmicroscope‐ZeissLSM510META)Scalebar:44µm

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Olfactory bulb Hypothalamus Anterior commissure intrabulbar part +++ latoanterior hypothalamus ++ Anterior olfactory area external part +++ anterior hypothalamus area ++ Anterior olfactory area lateral part +++ paraventriculus,med magnocell ++ Dorsal lateral olfactory tract +++ lateral septal nucleus ++ Ependymal subendymal layer +++ tringualar septal nucleus +++ External plexiform layer of the olfactory bulb +++ septofimbrial nucleus + External plexiform layer of the accessory olfactory bulb +++ ventral hippocampal comm + Gloerular layer of the olfactory bulb +++ Thalamus +

Glomerular layer of the accesory olfactory bulb +++ Anterior commisure intrabulbar part +

Granuel cell layer of the accesory olfactory bulb +++ Agrunular cortex D Granuel cell layer of the olfactory bulb +++ Agrunular insular cortex V Internal plexiform layer of the olfactory bulb +++ Anterior olfactory media lateral olfactory tract +++ Anerior olfactory posterior part ++ mitral cell layer of the olfactory bulb +++ cingulate cortex ++ mitral cell layer of the accessory olfactory bulb +++ dorsal endopiriform claustrum + Hippocampus pyrmidial cell hippocampus +++ Cortical regions polymorph dentat gyrus retrospinal granular cortex ++ dentat gyrus retrosplenial dysgranual cortex ++ granular dentat gyrus +++ primary motorcortex ++ lacunosum moleculare secondary motorcortex ++ PAG + primary somatosens ++ dorsal peduncular cortex ++ secondary somatosensory cortex ++ Superior colliculus ++ granualr insular ++ superficial gray sup coll ++ dysgranular insular ++ optic nerve layer ++ agranular insular ++ intermed gray layer ++ piriform cortex ++ intermediate white layer ++ primary visual cortex ++ dorsal tenia tecta +++ secondary visual cortex: lat,mediolat,mediomed ++ frontal cortex ++

primary primary auditory cortex ++ intermediate endopiriform claustrum +

seconday auditory cortex ++ lateral olfactory tract + temporal association cortex ++ lateral orbital cortex ++ perihinal cortex ++ medial orbital cortex ++ dorsolateral perinal cortex ++ piriform cortex + prelimbic cortex Amygdala rhinal fissura cortex amygdala transition +++ olfactory tubecle + anterior cortical amygdaloid nucleus +++ ventral orbital cortex ++ basomedial amygdala ++ ventral tenia tecta ++ anterior amygdala area ++ caudate putamen + lateral olfactory tract ++ globus pallidus + IPAC lateral ++ internal capsul + IPAC medial ++

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