ZBED6expressionpatternduringembryogenesisandin thecentralnervoussystem AxelEricsson 2010 DepartmentofNeuroscience DevelopmentalgeneticsUppsalaUniversity Supervisors:KlasKullanderandMartinLarhammar 1 P age
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 2 P age
3 P age Abstract ZBED6isarecentlydiscoveredrepressorprotein,whichwasfoundduetoaQuantativetrait locus(qtl) mappingstudycomparingwildboarwithdomesticatedpigs.asinglenucleotide polymorphismwhichdisruptedthezbed6interactionwiththeinsulinlikegrowthfactor II (igf II)generesultedinanupregulatedgeneexpressionandincreasedmusclemass.The bindingsiteforzbed6hasbeenfoundinnumerousgrowthfactors,whichindicatesan importantroleforgeneregulation.inthisstudyweinvestigatedthezbed6protein expressionduringembryonicdevelopmentandinadultcentralnervoussystem(cns)in mouse.hereweshowthatzbed6isexpressedbydifferentiatedneuronsandstarts approximatelyatembryonicday10.5,withnoexpressionobservedintheproliferationzone. FromtheexpressionpatternZBED6donotappeartobelinkedtoanyspecificregionsinthe spinalcord,ratherageneralexpressionindifferentiatedneurons.theproteinexpression wasmappedintheadultbrainshowingthatzbed6iswidelydistributedinmanyregionsand isexpressedinbothastrocytesandneurons,howevertheproportionofzbed6expressing cellsvariesbetweendifferentbrainregions. Abbreviations BMP Bonemorphogeneticprotein bhlh Basichelixloophelix CP Caudateputamen CNS Centralnervoussystem DLHP Dorsolateralhingepoints FGF Fibroblastgrowthfactors GFAP Glialfibrillaaryacidicprotein HD Homeodomain Igf Insulinlikegrowthfactors MHP Mediumhingepoints NeuN Neuronnucleispecificantigen NSC Neuralstemcells QTL Quantativetraitlocus RA Retinoidacid Shh Sonichedgehog
Aimofstudy TheaimofthisstudywastoinvestigateZBED6expressionpatterninmouseembryonic developmentandadultcnsusingimmunohistochemistrytoassessitspotentialroleasa regulatorofproliferation. Introduction Theunderstandingofgeneregulationisimportantinmanyaspectssincethecomplexityof anorganismisnotbasedonthenumberofgenesbutrathertheregulationofthem.how specifictranscriptionfactorsinteractandregulategeneexpressionisofinterestfor understandingandscreeningfordevelopmentaldiseases.pointmutationsinspecificregions canhavesevereconsequencesandleadtospecificphenotypes.apreviouslyunknown repressorproteinwasdiscoveredusingaqtl Mapping,bycomparingEuropeanwildboar withlargewhitedomesticpigs 1.Itwasfoundthatthedomesticatedpigshadaccumulateda singlenucleotidesubstitutionandthefavoredallelewaswellconservedduetostrong selectionformeatproductionoverthelast60years 1.ThisG Atransitionwaslocatedina CpGislandinintron3oftheinsulinlikegrowthfactor II(igf II)gene.Themutationinigf II disruptedtheinteractionwitharepressorprotein,namedzbed6,andledtoanelevated geneexpressionofigf IIresultinginanincreasedmusclemass.ZBED6containstwoDNA bindingbeddomains(namedaftertwochromatinbindingproteinsbeafanddreaf)which canmodifythechromatinstructureandregulatethetranscriptionofgenes,andahatcdimerizationdomainwhichisrelatedtothehatcsuperfamilyofdnatransposons.the bindingsitehasbeenfoundinover200genesandmanyofthemassociatedwith developmentaldisordersandneurologicaldiseases 1.Inthisstudyweinvestigatedthe ZBED6proteinexpressionduringembryonicdevelopmentusingimmunohistochemistryand compareditwithneuronalandproliferationmarkers.wehavealsocharacterizedthe expressionpatterninadultcnstoexaminetheimportanceandpotentialfunctionofthis repressorprotein. Background Developmentofcentralnervoussystem Neurulationistheembryonicprocesswhentheneural tubeisformed,andstartswiththeformationofthe neuralplate 2.Thecellsinthelateralectoderm differentiateintotheneuralplate,whichthenlengthens andnarrowsandtheneuroepithelialcellsmigrates towardsthemidlineandintercalate. 3 Theneurulation canbedividedintoprimaryandsecondaryneurulation. Theprimaryneurulationformthebrainandmostofthe spinalcordwhilethesecondarystartsatmorecaudal partofthespinalcordincludingsacralandcoccygeal 4 P age Fig.1Formationoftheneuraltube. LägginA,B,CIbildenoxåsåmankan följa. Formationofneuralplate(A) bendsand folds(b)closureoftheneuraltube migratingcrestcells(c).
regions 4.Furthermorecantheprimaryneurulationbedividedintofourphases;formation, shaping,bendingandclosureoftheneuraltube(fig.1).theneuralplatebendattwospecific sites 5,attheventralmidlinesonthemedianhingepoints(MHP)andnearthejunctionof theneuralplate,onthedorsolateralhingepoints(dlhp).thesetwositesareinvolvedin differentstagesofthebendingandclosingoftheneuraltube.inmouseneurulationthe neuraltubestartsbendingatmhp(e8.5),proceedsbendingatmhpanddlhp(e9.5)and finallythelowerneuraltubeonlybendsatdlhp(e10) 6.Theclosureoftheneuraltubeis initiatedinthehindbrain/cervicalpartsandthenproceedsunidirectionaltowardsfuture brainandspinalregions,additionallytwoclosuresitesoccursatforebrain/midbrainandat therostralendofforebrain.thespinalcordclosurecontinuestotheposteriorneuroporein whichthesecondaryneurulationstarts 7. Arrangementofneuronsinthespinalcord Fromelevenprogenitordomainsalignalongthemidlineofthedevelopingspinalcordallthe spinalcordneuronalsubtypesarise 8.Thearrangementoftheseprogenitorcellsandthe differentiationtodistinctneuronalsubtypesisdependentonalargenumberofintrinsicand extrinsicfactors.intheventralparttheimportantpatterningproteinsonichedgehog(shh)is activewhichisexpressedfromthenotochordan 9.AgradientofShhregulatesthe homeodomain(hd)transcriptionfactorsclassiandii,byrepressingclass1andpromoting class2 10.Inadditionthebonemorphogenicprotein(BMP)issecretedfromtheroofplate andinhibitsshheffectinthedorsalpartsoftheneuraltube 11.Fibroblastgrowthfactors (FGF)andWNTproteinscontributetoproliferationoftheneuralstemcells(NSC)andare activeintheventricularzoneoftheneuraltube 12,13.Thedifferentiationofprogenitorcells arethendependentonretinoicacid 14,basichelixloophelix(bHLH)proteinsandprogenitor specifictranscriptionsfactors,whichbyasynergiceffectcontributestoeachdistinctsub classofneuron 15.Intheventralhornofthespinalcordfivedistinctsubtypesofneuronsare developed,fourinterneuronsv0 V3andthemotorneurons(MN) 16 andinthedorsalpartsix celltypesareformed,dl1 3aresomatosensoryrelayinterneuronsanddl4 6associated interneurons 8 Materials and methods Immunohistochemistry Theembryosamplesatembryonicstages9.5 18,werecollectedfrompregnantfemales micesacrificedbycervicaldislocation.embryosweredissectedandfixedin4% paraformaldehyde(pfa)inpbsfor10 60minonice,followedbycryoprotectionin30% sucrose.tissueteko.c.tcompound(a/schemi TEknikk)wasusedforembedding.The sampleswerecryo sectionedinto12 16µmslicesusingaMicromHM560(MICROM InternationalGmbH,Germany).Sectionswerecollectedonslidesanddried,washedinPBS 5 P age
(3x10min)andincubatedinblockingsolution(1xblockingreagent(Roche),0.2%tritonX 100(Sigma,Germany),0.02%SodiumazidedilutedinPBS)for2h.Primaryantibodiesagainst ZBED6(rabbit,diluted0.2µg/ml)andNeuron specificnuclearantigen(neun,1:400)diluted inblockingsolutionwereincubatedat4 Covernight.Thefollowingday,slideswerewashed inpbs(3x5min)andincubatedwithasecondaryantibodyconjugatedtoalexa594 (Invitrogen,USA)andAlexa 488(Invitrogen,USA),Alexa 647(Invitrogen,USA)andDAPI (Sigma Aldrich,Germany)asacontrolfornuclearstaininginPBSfor1hatroom temperature.slideswerewashedinpbsandmountedwithmowiol 488(ROTH,Germany). Immunolabelingwasanalyzedinafluorescencemicroscope(OlympusBX61WI,Japan),a fluorescenceslidescannermicroscope(3dhistechpannoramicmidi,hungary)anda Confocalmicroscope (ZeissLSM510META,Germany) Imaging ImageswereprocessedinadobePhotoshopCS5(AdobeSystems,USA)andImageJ1.43U using;fftbandpassfilter,desprecklefunctionandarrangedtogetherinadobeillustrator CS3 6 P age
Results and discussion ThebindingsiteforZBED6hasbeenlocatedinover200 genes,manyassociatedtodevelopmentaldisordersand neurologicaldiseases.inthisstudyweinvestigateits expressionpatternduringtheembryonicdevelopment bycomparingzbed6withmarkersforproliferatingand differentiatedneurons,furthermoreazbed6protein expressionscreenwasperformedintheadultmouse brain.immunohistochemistryonasagittalembryoat stagee12.5stainedwithantibodiesagainstneuronal markerneun 17 andzbed6canbeseeninfigure3.we observedstainingandco localizationofzbed6andneun inbothsubventricularzoneandinthespinalcordbut likelynotintheventricularandproliferationzones. ImagesweretakenwithaPannoramicmidiscanner,whichwasusefulinwholeembryo imagingandgivingmultipleimagesinhighmagnificationandresolutionarrangedintoone picture.themajordrawbackwiththepannoramicmidi scanneristhesensitivityforcrumpleswhichresultsin outoffocusimages.tofurthercharacterizeandestablish whentheexpressionofzbed6startsweperformedan immunohistochemistryassayoncoronalsectionsof wholeembryose9.5 E18.5,focusingonthedevelopment ofthespinalcordduetoitswell knownprogenitor domainsandmanyneuronalmarkersareavailible.colabelingwasperformedwithantibodiesagainstzbed6, NeuNandKi67(ageneralmarkerforproliferatingcells) 18. (fig.4),theproteinexpressiononsetwasobservedat Figure.3.ZBED6expressioninsargital sectione12.stainedwithzbed6(red) andneun(green)(a D). Sargital(A)overviewofembryo.Higher magnificationimagesindicatedin(b D) withzbed(b)andneun(c)andco localizationofzbed6andneun(d). Imagestakenwith20xmagnification imagestakenwith(3dhistech Pannoramicmidi)Scalebarindicates 100um E10.5intheventralhorn.ZBED6wasco localizedwithneunbutnooverlapwasobserved betweenzbed6andki67atembryonicstagee10.5(fig.4).thisindicatesthatzbed6does notaffecttheearlyspecificationandisnotexpresseduntiltheprogenitorcellshave differentiatedtoneurons(fig.5).theembryonicexpressionpatternindicatesthatzbed6is notlinkedtoanyspecificregioninthedorsal ventralpatterningofthespinalcordandisalso expressedinthedorsalrootganglion(drg)(fig5m P).ThenumberofZBED6expressing cellsincreasedduringdevelopmentinthespinalcordinthesamepatternasneun,andin theadultspinalcordzbed6expressionwaswidelydistributedovertheentirespinalcord (fig.6).zbed6iswidelyexpressedinothertissuesduringembryogenesis,howeveramore intensestainingwasobservedincns.duetothewidespreadexpressionduringearly developmentandintheadultspinalcordnoco stainingwasperformedwithimmuno 7 P age
markers for specific neurons. Regarding the proliferation marker Ki67, this antibody was problematic to get to bind and several antigen retrievals protocols were tried without success. The main parameter for ki67 antibody binding was the tissue fixation, often short fixation time was necessary to receive an analyzable signal. Figure 4. ZBED6 is co expressed with NeuN but not Ki67 in the spinal cord E10.5. A) Ki67 as proliferation marker, (B) ZBED6 staining in the ventral horn neuraltube, (C) NeuN staining in ventral parts, (D) Yellow staining shows the overlap between ZBED6 and NeuN with no overlap with ki67 the proliferation marker. Scale bar indicates 100 µm, images taken with 20x magnification (Olympus BX61WI). Figure 5. ZBED6 expression pattern during early neuronal development. ZBED6 and NeuN immunoflourescence analysis of coronal mouse E9.5 E12.5 (A P) Image of Spinal cord E9.5(A D), E10.5(E H) E11.5, (I L), 12.5 (M P), stained with DAPI (A,E,I,and M), ZBED6 (B,F,J,N), NeuN 8 Pand a g the e co localization of ZBED6 and NeuN (D,H,L and P) indicated by arrowheads, double arrowheads (C,G,K,O) indicates DRG. Scale bar indicates 100µm. Images taken with 20x magnification (Olympus BX61WI)
ZBED6expressioniswidelydistributedinthespinalcord(fig6.).ToassessifZBED6 expressionwaslinkedtospecificbrainregionswemappedtheexpressionintheadultbrain andmeasuredtherelativeproteinexpressionsignal.coronalbrainsectionswerestained withantibodiesagainstzbed6,neunandglialfibrillaryacidicprotein(gfap),togetherwith DAPI.TheexpressionofZBED6indifferentbrainregionswasanalyzedandmarkedwith either+(0 25%ofthecells),++(25 50%ofthecells),+++(50 75%ofthecells).ZBED6is widelydistributedandhasaproteinexpressionrangefrom0 80%ofthecells(fig.6and table.1).regionswithalowexpressionofzbed6appearstobewhitematterareassuchas colossalcommissureandthedorsalhippocampalcommisure,andregionswithhigh expressiondensitywasforexamplemultipleregionsinamygdalaahighoverlapwithneun wasobservedinmostbrainregions.nostatisticalquantificationofzbed6wasdonedueto thewidespreadexpression.tofurtherinvestigateifzbed6expressioninothercelltypesin CNSweusedGFAPamarkerforanintermediatefilamentproteinexpressedinastrocytes 19. AsmallpopulationofGFAPpositivecellsco localizewithzbed6expressionwasobserved (fig.8)theproportionofgfappositivecellsexpressingzbed6wasnotmeasured. Fig.6ZBED6expressioniswidespreadintheadultspinalcord LumbarspinalcordlabeledwithDAPI(A)andZBED6(B).ZBED6iswidelydistributedovertheentirespinal cord,mainlyinthegreymatter.imagestakenwith4xmagnification(olympusbx61wi) 9 P age
10 P age
Figure.6TheZBED6expressionvariesbetweendifferentbrainregions ImmunofluorescenceanalysisofAmygdala,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 localizationofzbed6anddapiwhilelongarrows 11 P age indicatesonlydapistaining.closeupimagesweretakenwith40xmagnification(olympusbx61wi).scalebarindicate44µm.
Figure7.ZBED6isexpressedbyastrocytes ImmunofluorescenceanalysisofHippocampusstainedwithanti ZBED6,anti NeuNandanti GFAP(B E)antibodies. Coronaloverviewofbrain(A)assembledof4xmagnificationimages.Boxedareaindicateshighermagnificationregion. Hippocampusstainedwithanti GFAP(B),anti ZBED6(C)andanti NeuN(D).Arrowsindicatesco localizationofgfapand ZBED6(E),DoubleheadedarrowsindicatesoverlapbetweenZBED6andNeuN(E)andarrowheadsindicatesGFAPpositivecell lackingzbed6.imagestakenwith40xmagnification(confocalmicroscope ZeissLSM510META)Scalebar:44µm Conclusion Duringembryogenesis,ZBED6proteinexpressionstartsbeforeE10.5indifferentiated neurons.zbed6doesnotappeartobeexpressedinproliferatingprogenitorcells.inthe adultbrainandspinalcordzbed6isexpressedinneuronsandtosomeextentastrocytes.a widespreadexpressionwasobservedinmostpartsofthebrain,whileafewregionsdidnot expresszbed6(e.g.dorsalhippocampalcommisureandcolossalcommisure).the widespreaddistributionofzbed6andthelargenumberofbindingsitesindicatesa fundamentalroleingeneregulation.ithasstillyettobedeterminedwhichgenesandhow ZBED6areregulatingthoseintheCSN.HowcrucialZBED6isfornormaldevelopmentandits potentialroleinneurodevelopmentaldisordershasyettobedetermined.aconditional ZBED6knockoutthatallowsdeletionofZbed6inspecificcelltypesandcellpopulationswill giveusadeeperunderstandingofthisprotein sgeneregulatingabilities. Acknowledgement IwouldliketothankMartinLarhammarwhichhavebeenagreatsupervisorguidingme throughthisprojectalwaysansweringmyquestions.i malsogratefultoklaskullanderand LeifAnderssonwhohasgivenmethechancetodosuchaninterestingproject. Table.1 12 P age
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 ++ References 13 P age
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