Siew-Lan Ang development Transcriptional controlofmidbraindopaminergicneuron Development 133,3499-3506(2006)doi:10.1242/dev.02501 e-mail: [email protected] Research, TheRidgeway, MillHill,LondonNW71AA,UK. Division ofDevelopmentalNeurobiology, MRCNationalInstituteforMedical transmission inthelimbic system hasbeenlinked tothe Fuxe, 1964;Tzschentke andSchmidt,2000).Dysregulation ofDA in emotionalbehaviour andmechanismsofreward (Dahlstromand striatum, aspartofthemesocorticallimbicsystemthatisinvolved respectively. A10andA8groupsprojecttotheventromedial tegmental area(VTA) andretrorubalfieldofthemidbrain, The medialA10andlateralA8neurongroupsdefinetheventral specific degeneration oftheSNpcneurons(LangandLozano,1998). individuals withParkinson’s disease.Thesesymptomsareduetothe slowing ofvoluntary movement) andgaitdisturbancethatisseenin revealed bytherestingtremor, rigidity, bradykinesia (abnormal of neuronsismainlyinvolved inthecontrolofmovement, as the dorsalstriatumandformnigro-striatalpathway. Thisgroup substantia nigraparscompacta(SNpc),whichmainlyproject into main subgroups.ThelateralA9groupcorrespondstoneuronsof the et al.,2005).Theseneuronscanbeanatomicallydivided intothree (Bjorklund andLindvall, 1984;DahlstromandFuxe, 1964;Marin border andcaudallytothemidbrain/hindbrain(Fig.1) brain thatextends rostrallytotheventral thalamus/hypothalamus called A8-A10)originatefromtheventral partofadomain ofthe enzyme ofdopaminesynthesis.Th of theenzymetyrosinehydroxylase(Th),whichisrate-limiting immunohistochemical studiesthatdeterminetheexpression pattern neurons inthecentralnervous system(CNS)comesfrom drug abuse (ChaoandNestler, 2004). pleasure, andinthedevelopment ofaddictive behaviours suchas midbrain dopaminergic neuronsplaykey rolesinthegenerationof disorders (Hornykiewicz, 1978;Carlsson,2001).Inaddition, because ofitsimplicationindiverse psychiatricandneurological 2003). Themidbraindopaminesystemhasbeenintensively studied dopaminergic neuronsintheadultbrain(Wallen andPerlmann, dopamine asaneurotransmitter)constituteabout75%of The midbraindopaminergic (mDA) neurons(neuronsthatuse Introduction therapeutic useintreatingParkinson’sdisease. into midbraindopaminergicneuronsthatcanbeusedfor will alsosignificantlyadvanceeffortstodifferentiatestemcells midbrain dopaminergicneurons.Importantly,thesediscoveries elucidate thegeneticprogramthatpromotesgenerationof encouragement tothisfield.Thesenewfindingswillhelp these neuronshasbroughtmuchexcitementand transcriptional determinantsregulatingthedevelopmentof this neuronalsubtype.Hence,therecentidentificationofmajor Parkinson’s disease,littleisknownaboutthespecificationof with oneofthemostcommonhumanneurologicaldisorders, Although lossofmidbraindopaminergicneuronsisassociated Our currentunderstandingofthelocationdopaminergic (DA) + mDA neurons(historically expression ofTh,vesicular monoamine transporter2(Vmat2), et al.,1997;Saucedo-Cadenas etal.,1998).Itis also requiredforthe generation, but forthe maintenanceofmDA neurons(Zetterstrom nuclear receptorNurr1was foundtoberequirednotforthe development ofpostmitoticmDA neurons.For example, theorphan have beenidentified anddescribedthatmarkregulate the determinants ofmDA progenitors,several key transcriptionfactors Andersson etal.,2006b). 2003; Puellesetal.,2004;Andersson2006a;Kele etal.,2006; expressed intheseprogenitors(Simonetal.,2001;Puelles Otx2, Lmx1a/b,En1/2,Msx1/2,Ngn2andMash1,are also several groupshave shown thatothertranscriptionfactors, suchas remains tobediscovered. Remarkably, duringthepasttwo years into retinoicacid;however, theroleofRaldh1inmDA progentiors Aldh1a1 (Raldh1).Raldh1catalyzestheoxidationofretinaldehyde progenitors inthemidbrainwas retinaldehyde dehydrogenase recently, theonlymoleculeknown tomarkspecificallyDA specification, earlyandlatedifferentiation (seebelow). Until during successive developmental steps,whicharecalledregional mature neurons(seeFig.2A,B).Thesepopulationsaregenerated populations inthemDA lineage:progenitors,immatureneuronsand Molecular markers allow thedistinctionof three sequentialcell Midbrain dopaminergiclineage next few years. both inthestemcellanddevelopmental biologyfieldsduringthe in thisreview. Thesenew findingswillunderpinexciting research gain-of-function experiments inchickandmice,willbe discussed development ofmDA neurons,whichhave arisenfromloss-and into theroleofthesetranscriptionfactors inregulating the differentiation ofprogenitorsintomDA neurons.Importantinsights candidateregulators forpromotingthespecificationand are address theissueofheterogeneityamongmDA progenitors,and transcription factors provide additionalmarkers withwhichto al., 2006a;Anderssonet2006b;Kele etal.,2006).Thesenew et al.,2001;Puelles2003;2004;Andersson for thefirsttimebyacombinatorialtranscriptionfactor code(Simon in mDA progenitorshasallowed theseprogenitorstobeidentified Msx2 (Msx1/2),Neurogenin2(Ngn2)andMash1,areallexpressed Lmx1b (Lmx1a/b),Engrailed1and2(En1/2),Msx1 that certaintranscriptionfactors, specificallyOtx2,Lmx1aand distinguishable fromotherCNSprogenitors.Therecentdiscovery recently, mDA progenitorswerepoorlydefinedandnoteasily (1) regional specification,(2)earlyand(3)latedifferentiation. Until have beenrecognizedbasedontheexpression ofmolecularmarkers: progenitor cellcanbedivided intodistinctsteps.At leastthreesteps schizoprenia (SesackandCarr, 2002). and isthoughttocontribute tothepsychoticsymptomsof Wightman andRobinson,2002)depression(Daillyetal.,2004), development ofdrugaddition(Kelley andBerridge,2002; Despite thishistoricallackofinformationonmolecular As forallneurons,thegenerationofmDA neuronsfromaneural REVIEW 3499

DEVELOPMENT 3500 2003). Nunes etal.,2003;Smidt2004;van denMunckhofetal., of primarilySNpc,but alsoVTA, neurons(Hwang etal.,2003; subset ofmDA neurons(Maxwelletal.,2005),andforthesurvival transcription factor thatisrequiredfortheexpression ofThina apoptosis (Alberietal.,2004).Pitx3isapairedhomeodomain mDA neuronsforneuronalsurvival throughtheirregulation of have alsobeenshown invitrotoberequiredcellautonomously mDA neuronsandtheirsurvival (Simonetal.,2001).En1/2proteins homeodomain proteinsarerequired,inpart,forthegenerationof 1999; Wallen etal.,2001;Smits2003).Bycontrast,En1/2 dopamine transporter(DAT) andRetinmDA neurons(Wallen etal., Anterior ab Aa C B 1 D rgntr(2)Immature (1) mDAprogenitor Lmx1a/b, Msx1/2, Msx1/2 Lmx1b Mash1 Lmx1a Ngn2, Mash1, En1/2, Raldh1 En1/2 REVIEW Ngn2 boundary Inter-neuromeric boundary Alar-basal Otx2 sP bse ob Otx2, lge mge E7.5 A11d A15 p3 p2 or Dorsal A13

85E. 1. 1. 1. 1. E14.5 E13.5 E12.5 E11.5 E10.5 E9.5 E8.5

p1 A14 mDA neuron Ventral En1/2, Raldh1,

Ngn2, Nurr1, A12 A14-dm A11 β m Lmx1a/b, III-tubulin A9-10 l-r1 A8 Mb (3) MaturemDAneuron Posterior VZ cer Nurr1, En1/2,Pitx3, Th, Aadc,Raldh1, β Lmx1a/b, III-tubulin dorsomedial mDA neuronsexpress ThaheadofPitx3atE12.5.By mDA neuronsexpress Pitx3prior to expressing Th,whereasthe expression ofThandPitx3(Maxwelletal.,2005).Ventrolateral mDA neuronscanbedistinguishedbytheirtemporalprofileof knock-in GFPmouseembryos,two partiallyoverlapping groupsof By usingGFPtotracethelineageofPitx3-expressing cells in prosencephalon. olfactory bulb;or;opticrecess; p,prosomere; sP, secondary mamillary body;Mb,midbrain;mge,medialganglioniceminence;ob, dorsomedial; I-r1,isthmicregion; lge,lateralganglionicemincence;m, and A17groups are notshown.cer, cerebellum; d,dorsal;dm, adapted, withpermission,from Marinetal.(Marinal.,2005).A16 and thealar(dorsal)basal(ventral)platesubdivision.Thisfigure is segments, seePuellesandRubenstein(PuellesRubenstein,2003)] depicted inrelation totheneuromeric map[subdivisionintotranseverse brains rangingfrom E12.5-E14.5.TheDAgroups (lightblueareas) are that distinguishessubpopulationsofmDA neuronsinmiceis that theseneuronsareheterogeneous.Theearliestmolecularmarker with VTA DA neurons,asisevident inParkinson’s disease,indicates vulnerability ofSNpcDA neuronstotoxicinsultswhencompared a homogenousorheterogeneouspoolofprogenitors.Thespecific location ofTh Fig. 1.Dopaminergicneurons intheCNS. Presently, itisstillunknown whethermDA progenitorsrepresent + neurons, corresponding toA8-A15groups, inmouse of sectionshownintheimagetoright.( E12.5 mouseembryo.Theblacklinemarkstheplane are courtesyoftheAngLaboratory. been determined.ImagesinA,parta,andb the precise timingofexpression shutdownhasnot of thelineage)are shown.Dottedlinesindicatethat factors inmDAprogenitors (notinsubsequentsteps cells. ( mDA progenitors. Thecurvedarrow indicatescycling sequential timingoftranscriptionfactoractivationin postmitotic neurons ofthemDAlineage.( mitotic progenitors, immature andmature box inA,parta),illustratingthepositionoccupiedby b in themouseventralmidbrain. Fig. 2.MolecularlydistinctDAcellpopulations ) Anenlargedviewoftheventralmidbrain(square C ) Thetimingofexpression oftranscription Schematic illustratingthe Development 133(18) ( A , part a B ) The A ) An , Pitx3 part Pitx3 .

DEVELOPMENT derived fromShh ventral midlineposition,mDA progenitors arepresumedtobe required fortheexpression ofLmx1aandMsx1/2.Based ontheir suggest thatShhsignallinginducesanothersignalorfactor thatis initiation ofShhexpression (Echelardetal.,1993).These results activated endogenouslyinmouseembryosonedaylaterthanthe (Andersson etal.,2006b).However, thesetranscriptionfactors are and Msx1/2expression inneuraltubeexplants ofchickembryos activation ofthesegenesisnotunderstood.ShhcaninduceLmx1a 2006b). Themolecularmechanismsleadingtothesequential Mash1 expression arenotinitiateduntilE10.75(Andersson etal., Lmx1a andMsx1/2expression turnonaroundE9.5,andNgn2 initiated byE9.0(Angetal.,1994;Smidt2000).Subsequently, (Fig. 2C).Theexpression ofOtx2,Lmx1bandEn1/2genesisalready Lmx1a/b, En1/2,Msx1/2,Ngn2andMash1,inatemporalsequence activation ofacombinationtranscriptionfactors, includingOtx2, anteroposterior (AP)anddorsoventral (DV) identity, andresultinthe patterning ofmidbrainprogenitors.Thesesignalsspecify ␤ addition, Wnt1(Prakashetal.,2006)andtransforminggrowth factor- for theinductionofDA neuronsbeforeE9.5(Ye etal.,1998).In growth factor 8(Fgf8)fromthemid-hindbrainboundaryarerequired that thefloorplate-derived signalsonichedgehog(Shh)andfibroblast Studies usingratandchickneuralplateexplants have demonstrated Regional specificationofmDAprogenitor progenitor stage,or, alternatively, onlylaterinpostmitoticneurons. patterns andresistancetotoxicityarealreadyspecifiedatthe remains tobedeterminedwhethertheirheterogenousconnectivity mDA neuronsareaheterogenouspopulationtransientlyatE12.5,it involve theactivity oftheproneuralgenes neuronal potentialbyfloorplateprogenitors,astepthatislikely to the specificationofmDA progenitorsrequirestheacquistionof E13.5, allTh Development 133(18) postmitotic immaturemDA neuronsbetweenE9.5andE13.5in demonstrate thatmDA progenitorsexit thecellcycle andgenerate Birthdating studiesusingtritiatedthymidineincorporation Early differentiation: generationofimmature mDAneurons Differentiating mDAneurons (Andersson etal.,2006a,Andersson2006b;Kele etal.,2006). N M ur Immature an Nurr1 n/ Pro En1/2 M Lmx1 O P Table 1. A Table 1. T r i g t a t s a (Tgf x x n 2Pro n2 x s 2 3 1 s h c b 1 r i p ␤ t ) (Farkas etal.,2003)probablyalsoplayaroleinthe i o n s

f ummary of the roleof ummary tran + a c cells express Pitx3.Althoughthisstudyindicatesthat t o r + glial-like floorplatecells.Thus,anotherstepin Pro P P P M r r r neurons neurons a o o o t g g g g g g u enitors an enitors, immaturean enitors, immaturean e e e r n n n E e x i i i

t t t p n o o o r e e r r r u s s s s s r i o d o n n mtr ern Require matureneurons d

s i imtr ern Require immatureneurons n

t h e s

m cription factor D A d d

l i mature mature n Ngn2 e a g e and s in neuron development mDA Mash1 Require Require R N R R e e e o markers expression oflate mature mDAneuronsan survival ofmaturemDAneurons mDA neurons for N d spe an for thesurvivalofprimar su q q q t ifferentiation

u u u b r d i e i i c set ofmaturemDAneuronsan r r r alsoVTA,neurons ifi q e e e g u d d d d d d d c n2 fun i forneuronal forthemaintenan forthe formaintenan

ation ofmDApro r f f f F (Table 1). emerge duringthepastfew yearsandwillbethefocusofthisreview differentiation ofmDA progenitorshave onlyrecentlystartedto the transcriptionfactors thatgovern thespecificationandearly Smits etal.,2006;PrakashandWurst, 2006).However, therolesof and Pollock,2003;Wallen andPerlmann,2003;Simeone,2005; extensively reviewed elsewhere (GoridisandRohrer, 2002;Riddle (described brieflyabove, seeFig.2BandTable 1)have been acting duringthelatedifferentiation stepofthemDA lineage The rolesoftranscriptionfactors, suchasNurr1,En1/2andPtx3, development Transcription factorsrequired formDAneuron neurons (Smidtetal.,2004). transcripts arethoughttobeexpressed alreadyinimmaturemDA mDA neurons.Aadcconverts DOPA intodopamine. mDA neurons(Fig.2B).Ngn2,however, isnotexpressed inmature (Aadc), inadditiontotheearliermarkers expressed inimmature neurons express Pitx3,Thandaromaticaminoaciddecarboxylase differentiating intomDA neurons (Kawano etal.,1995).These radially onradialglialfibresintothemantlezone,further From E11.0onwards, immaturemDA neuronscontinuetomigrate Late differentiation: generationofmature mDAneurons postmitotic immatureandmaturemDA neurons(Kele etal.,2006). expression ofgeneralneuronalmarkers, suchas progenitors, downregulate Sox2expression whileinitiatingthe 2B). Inaddition,mDA progenitors,like many otherCNS et al.,2001;Alberi2004)duringthisdifferentiation step(Fig. expression (Zetterstrometal.,1997)andEn1/2expression (Simon mice (Bayeretal.,1995).ImmaturemDA neuronsinitiateNurr1 E12.5 onwards (Mallamacietal.,1996). regions, expression isalsodetectedintherhombencephalonfrom midbrain betweenE8.5andE12.5.Inadditiontotheseanteriorbrain nervous system, after E7.75(Simeoneetal.,1993;Ang1994).Within the progressively restrictedtotheanteriorthirdofmouseembryo widely expressed beforegastrulation,but its expression becomes Otx2 isabicoidclasshomeodomaintranscriptionfactor thatis Otx2 e o o o u d r r r n

c n r T f t e o h e i c o g r u - tion n

e i r n g o b

x o i e n eneration an n ut p n u

a r m d a r e l c ifferentiation

l o D s a

an d d s n A n i c feetainAn ifferentiation i a o

d f c e ofmature c l f n e

ompensate e n d g l

y Otx2 l i r e their s c SNp n enitors e e of u

n a r t o d i a n c expression isrestrictedtotheforebrainand , t a i l o d n Zetterstrom etal.,1997;Sau Simon etal.,2001;Al Smi Kele etal.,2006 Puelles etal.,2003; Hwan A n et al.,2003 Smits al., 1999;Wallenet2001; Car 2004 2004; Verna et al.,2005 2003; Smi 2003; van 2006 d d d ersson etal.,2006a;Kele e t etal.,2000 d r g s enas etal.,1998;Wallen s etal.,2003;Nunes o n

e d d t t etal.,2004;Maxwell Otx2 en Mun

y R a etal.,2005 e l . f , e

2 r e is requiredforthe 0 n 0 REVIEW c c 6 b e ␤ khof etal., b s eri etal., III-tubulin, in Aadc c e mRNA d o- 3501

DEVELOPMENT neuronal development isrecovered in mDA progenitors seemscontradictorytotheobservation that mDA of mDA neurons.Alaterrolefor was deletedinthemidbrainof atdifferent developmental stages.For example, specifically mouse mutantshave beengeneratedinanattempttodelete analyses of and differentiation basedonfindingsobtainedfrom thephenotypical 2003). extent ofShhexpression intheventral midbrain(Puellesetal., midbrain boundary(Brodskietal.,2003),andforlimitingthedorsal required forpositioningtheexpression ofWnt1andFgf8atthe En1cre;Otx2 progenitors atE12.5.Accordingly, expression indirectlyviaitsrepressive effects onNkx2.2inmDA expression ofWnt1,whichindicatesthat E9.5 onwards (Puellesetal.,2004).Inthese by SimeoneandAcampora,2001).Subsequently, fates (Mallamacietal.,1996;Perea-Gomez2001)(reviewed anterior visceralendoderm,whereitfunctionstorestrictposterior formation oftheforebrainandmidbrainasaresultitsrolein 3502 by changesin Shhexpression in the ventral midbrain of mutant embryos,despitetheloss of of results indicatethat embryos, but thelossofmDA neurons isnot.Taken together, these mDA neuronsarerescuedin of ectopicserotonergic neuronsandthereductioninnumberof abnormal expression ofNkx2.2inmDA progenitors,asthepresence mDA neurons.LossofmDA neuronsisdirectlylinked tothe whereas mutant embryos,midbrainexpression of Otx2 Nkx2.2 results inlossofexpression oftheproneuralgenes these conditionalmutants,lossofOtx2proteinfromE10.5onwards studies of nucleus neuronsisfullyrescuedin control oflocomotion(reviewed byKennedy, 1990).Thislossofred normally liedorsaltomDA neurons,and areimplicatedinthe En1cre;Otx2 (Prakash etal.,2006).Theectopicexpression of Nkx2.2 in in regulating reconcile thesetwo findings, Iproposethattherequirementfor to neuronaldifferentiation inthesetriplemutantsatE9.5.Inorderto is requiredfortherepressionof into presumptive DA progenitorsaroundE9.5,indicatingthat expression ofthehomeodomainproteinNkx2.2expands ventrally domain ofmidbraintissuedevelops normally. Within thisdomain, Despite thesechangesinAPandDV patterningmolecules,asmall hindbrain, shiftsanteriorlyintothemidbrain(Puellesetal.,2004). rostral hindbrainof leadstoanupregulation of promoter Cre;Otx2 the rednucleusneuronsthatarereducedinsize expression expands ventrally intotheventricular zoneadjacentto missing attheventral midlineofthemidbrain.Inaddition,Nkx2.2 in ventral mDA progenitors.Subsequently, mDA neuronsare ectopically inthese et al.,2006).Interestingly, serotonergic neuronsaregenerated Otx2 A different rolefor Ngn2 , asectopicexpression of . Itisnoteworthy that REVIEW also hasadditionalrolesinmDA progenitorspecification Fgf8 and flox/flox Nestin-Cre;Otx2 Otx2 Ngn2 Mash1 flox/flox flox/flox expression, whichisnormallyrestrictedtotheanterior embryos (Vernay et al.,2005).Rednucleusneurons mutant mice.Several different conditional expression inmDA progenitorsmaybebypassed ;Nkx2.2 En1 Otx2 embryos isalsoassociatedwithalossof seblw,isalsorequiredforthegeneration (see below), Otx2 Otx2 Wnt1/+ , presumablyviaregulating theexpression conditional mutantsattheexpense Th –/– in mDA progenitorswas identifiedfrom flox/flox Wnt1 En1cre;Otx2 embryos (Prakashetal.,2006). embryos thatlackboth Nkx2.2 Wnt1 En1cre;Otx2 Otx2 embryos (Vernay etal.,2005).In may alsoberequiredupstreamof Nestin-Cre;Otx2 Wnt1 Otx2 under thecontrolof Otx2 in regulating neurogenesisin En1cre;Otx2 in theseprogenitors(Prakash Otx2 expression isrecovered in in mDA progenitors prior flox/flox in thefloorplateof Shh flox/flox might regulate Wnt1 ;Nkx2.2 En1cre;Otx2 expands dorsally, Ngn2 flox/flox flox/flox embryos from Otx2 –/– and ;Nkx2.2 ;Nkx2.2 Otx2 embryos Nestin- is also Mash1 flox/flox Otx2 Otx2 Otx2 Otx2 Otx2 En1 and –/– –/– + in This differentiation defectresultsintheeventual lossofDA neurons paired homeodomaintranscriptionfactor Pitx3(Smidtetal.,2000). contribute tothelossofTh earlier defectsinpatterningofthemid-hindbrainregion may neurons, as demonstratingarolefor mid-hindbrain organizer andfortheexpression of Lmx1b.1 in thesemutants(Prakashetal.,2006). is notcompletelynormalandthismayalsoaffect Ngn2 expression recovered Wnt1expression in via itseffect onLmx1a(seebelow). Alternatively, orinaddition,the This isbecauseShhcaninfluencetheexpression ofNgn2,possibly Th expression ofLmx1ainprogenitorsdirectlyoverlies aregion where expression domainsofthetwo geneslargely coincide.Becausethe the ventral midbrainthandoesLmx1a,but thatbyE10.5the revealed thatatE9.5,Lmx1bexpression encompassesmorecellsin (Andersson etal.,2006b).Doubleantibodylabellingstudieshave E9.5 intheventral midbrainandthenprogressively expands dorsally including thefloorplate.Bycontrast,Lmx1aexpression begins at plate, themid-hindbrainboundaryandventral midbrain, al., 2000),but thisexpression becomesrestrictedbyE9.5totheroof Lmx1b isexpressed inthemidbrainfromE8.0onwards (Smidtet transcription factors, playimportantrolesinthedeveloping brain. Lmx1a andLmx1b,membersofthefamily ofLIMhomeodomain Lmx1a andLmx1b En1cre;Otx2 the maintenanceofTh studies. seems likely but awaits confirmation bylineage-andfate-mapping include progenitorsforothercelltypesaswell.Thisinterpretation 2006b), whereastheinitialexpression ofLmx1bisbelieved to to markthedorsalboundaryofmDA progenitors(Anderssonetal., Lmx1b Wnt10b repression of progenitor cells,asindicated by theactivation ofMsx1andthe neurons inchickembryoswas precededbyare-specificationof neurons over that ofotherneuronalsubtypes.TheinductionDA ventral midbrainofchick embryospromotedthegenerationofDA these researchersshowed thattheoverexpression of neuron fate development (Anderssonetal.,2006b).Inthisstudy, Perlmann hasidentified induce roofplateformation. thus demonstratingthat components ofroofplatesignalling(Chizhikov andMillen,2004b), that caudal roofplate.Inaddition,overexpression studieshave shown point mutationin in thespontaneousmousemutant developmental stages. neurons willbeimportanttoelucidateitsrolesatdifferent detailed analysisof patterning functionsinthemidbrainremainstobeclarified.A embryos isduetoanintrinsicroleinthemDA lineageortoearlier Therefore, whetherthemDA phenotypeof Lmx1b Loss-of-function studieshave shown that A recentbreakthroughstudybythegroupsofEricsonand Positional cloninghasidentified + neurons develop atE11.5,Lmx1aexpression hasbeenproposed Lmx1a –/– , Pax8 Lmx1b –/– and mutants uptoE16.0,althoughthey fail toexpress the can inducetheexpression ofroofplatemarkers andof embryos. Althoughthisphenotypehasbeeninterpreted flox/flox Nkx6.1 Lmx1b.2 and is initiallybroadlyexpressed inthemidbrain;and ;Nkx2.2 Lmx1a Fgf8 expression. Importantly, theactivity of Lmx1b + genes arerequiredformaintenanceofthe in zebrafishembryos(O’Haraetal.,2005). mDA neurons.Th Lmx1a Lmx1a –/– in + mutant embryos(Prakashetal.,2006). dreher functions inmDA progenitorsand En1cre;Otx2 neurons atlaterstages.Inaddition, Lmx1b is bothrequiredandsufficient to as acrucialdeterminantofmDA dreher Lmx1a mice resultsinthelossof in themaintenanceofDA (Millonig etal.,2000).The as beingthegeneaffected flox/flox + Development 133(18) Lmx1b Lmx1b neurons arefoundin ;Nkx2.2 mutant mouse is requiredfor Wnt1 Lmx1a –/– , mutants Wnt3a Lmx1a in the ,

DEVELOPMENT results inthegenerationofectopicTh this context, theoverexpression of dorsal midbraincellstoamoreventral fate. Itisnoteworthy thatin inhibit dorsaldifferentiation programsandthuscannotconvert in ventral midbraincells.Alternatively, suggests aroleforadditionalfactors thatarespecificallyexpressed in ventral, andnotindorsal,midbraincells.Thiscontext dependence (Andersson etal.,2006b).Impressively, into DA neuronsinthepresence,but notintheabsence,ofShh stem (ES)cells.MouseEScellstransfectedwith Lmx1a pathway (Fig.3). functions cooperatively withventral factors inducedbytheShh Lmx1a et al.,1997).Taken together, theseresultsfavour thehypothesisthat chick experiments discussedabove (SasakiandHogan,1994;Hynes mouse embryosatdevelopmental stagesequivalent tothoseofthe dorsal midbrainprogenitorsarecompetenttoacquireamDA fate in mock-transfected EScellsproducedTh neuron markers, suchasPitx3,Lmx1b,En1/2andDAT. Bycontrast, from EScells.Importantly, theseneuronsalsoexpressed othermDA Development 133(18) is context dependent,as 2006b). modified, withpermission,from Anderssonetal.(Anderssonal., indicate hypotheticalfunctionsthatremain tobeproven. Thismodelis suppression ofNkx6.1expression inDAprogenitors. Dottedarrows mDA neurons. Inaddition,Msx1isrequired and issufficient forthe differentiation and,perhaps,alsothesubtypespecificationofimmature activatesMsx1,whichinducesNgn2.Ngn2promotesturn neuronal then actcooperativelytospecifyimmature mDAneurons. Lmx1ain Shh expression, theinductionofLmx1amaybeindirect. Lmx1aandX timing ofinductionendogenousLmx1aexpression compared with X (anunknowntranscriptionfactor)inmDAprogenitors. Basedonthe Fig. 3.ModelofmDAneuron specification. expression of DA neurons, whichwas notcompensatedforbyunperturbed Lmx1a chick embryos(Anderssonet al., 2006b).Intheseexperiments, also supportsarolefor expression inupto80%of dorsal sitesof transgenic embryos,ectopicDA neuronswerefoundadjacentto midbrain (SasakiandHogan,1994;Hynesetal.,1997).Inthese GABA neurons (Smidt etal.,2000); have demonstrated arolefor Shh Additional supportforcooperative interactions between A complementaryloss-of-functionstudybyRNA interference + a comefromstudiesusingthedifferentiation ofembryonic has knockdown bysiRNA electroporationresultedinalossof alone isnotsufficient toinducemDA neurons,andthatit and Pitx3 D rgntrImmaturemDAneuron mDA progenitor Lmx1b Shh Nkx6.1 Lmx1a Msx1 – X . expression. Theseresultsindicatethatsome . Thisresultwas surprising, asstudiesinmice Lmx1a Lmx1a ␤ III-tubulin Lmx1b Lmx1b can onlyinduceectopicDA neurons in thegenerationofDA neuronsin Ngn2 Foxa2 can alsopartially rescueroof in thedevelopment ofmDA + Lmx1a + and neurons thatwerederived neurons thatweremostly + Intrinsic factor Extrinsic factor neurons inthedorsal Shh inducesLmx1aand Lmx1a Gli Neuronal differentiation Subtype specification might notbeableto Lmx1a in transgenicmice promotes Th differentiate Shh and progenitors. leads toagreaterlossofmDA neuronsthanoccursin Mash1 itself isnotrequiredformDA neurondevelopment, the lossofboth Msx1 2006b). Given thatMsxgenesnormallyfunctionasrepressors, expression inventral midbrainprogenitors(Anderssonetal., the timingofmDA neurongenerationpossiblybyinducingNgn2 downregulation ofShhinthefloorplate,indicatingthat the precociousexpression ofNgn2andNurr1, tothe expression of progenitors (Anderssonetal.,2006b)(Fig.3).Premature the lossof mutants (Fig.4),suggestingthat (Andersson etal.,2006a;Kele etal.,2006).Although Mash1 The mouseMsxgenes, Msx1 andMsx2 for (Andersson etal.,2006b).Itisalsopossiblethattherequirements plate formationin Ngn2 nervous system.Intheventral midbrain,theproneuralgenes neurogenesis andofsubtypespecificationinmany areasofthe Proneural basichelix-loop-helixgenesarecrucialregulators of Ngn2 andMash1 Lmx1a the development ofmDA neuronsinmiceawaits furtherstudiesof than consistent withtheobservation that later differentiation events in the DA lineage.Thishypothesisis more efficient atthespecificationstep,while overlapping rolesinmDA development, with way toreconciletheseresultsisproposethatthetwo geneshave required, torepress Msx1;Msx2 Msx2 of mDA neuronsin Ngn2 of mDA neurons,probablyasaresultofthedownregulation of subsequent differentiation intoTh Nurr1 zone attheventral midline. immature mDA neuronsimmediatelyadjacent totheventricular et al.,2006). Mash1 (Shimeld etal.,1996;Wang etal.,1996). the neuraltubeinmouse,caudaltomid-hindbrainboundary Msx3 tooth buds, heart,branchialarchesandincraniofacial processes. mesenchymal inductive interactionsoccur, suchasinthelimbsand tube andneuralcrest,aswellinmany othersiteswhere epithelial- addition tointheroofplateandadjacentcellsdorsalneural progenitors intheventral midbrain(Anderssonetal.,2006b),in by RamosandRobert,2005). homeodomain andfunctionastranscriptionalrepressors(reviewed homeodomain transcriptionfactors thatshare98%homologyinthe conditional mutants. Whether Msx1 Lmx1a Lmx1a , apossibilitythatremainstobeaddressedbytheanalysisof , bycontrast,isexpressed exclusively inthedorsalaspectof and + expression (Anderssonetal.,2006b).Thispartialreduction may regulate theactivity ofarepressor , are expressed inmDA progenitors,whereas are co-localizedintheventricular zonemoredorsally(Kele and –/– immature mDA neurons,andprobably also fortheir Lmx1a;Lmx1b Lmx1a Ngn1 embryos exhibit a40%reductioninthenormalnumber Ngn2 and Ngn2 at promotingmDA neurondifferentiation inEScells double mutants.Inaddition, Msx1 Ngn2 show anintricatepatternofexpression. Lmx1b and function inmDA progenitors. Accordingly, this dreher in in themidbraintransgenicmicealsoleadsto Msx1 is alsoexpressed innewly bornpostmitotic Mash1;Ngn2 Lmx1b Nkx6.1 genes aredifferent forthetwo species. double mutantembryosandof mice (Chizhikov andMillen,2004a).One mutants maybeduetocompensationby Ngn2 have uniqueand/orredundantrolesin Msx1 Msx1 Mash1 expression inventral midbrain is requiredforthegenerationof double mutantmouseembryos , and Lmx1b Msx2 + can partiallycompensatefor Msx2 mature mDA neurons Msx1 is muchlessefficient Lmx1b Lmx1a and are expressed inDA REVIEW Ngn1 Msx3 is able,and Ngn2 is requiredfor being perhaps Ngn2 , Mash1 Msx1 , encode Ngn2 Ngn2 in mDA Mash1 Lmx1b single 3503 sets and and by ,

DEVELOPMENT neurons aregeneratedin partially for (reviewed byBertrandetal.,2002). two markers ofproneuralactivity, beneath mDA progenitors(Kele etal.,2006).Moreover, thelossof Ngn2 permission, from Keleetal.(Keleal.,2006). E14.5 mouseembryos.Asevere reduction inTh neurons intheventralmidbrain of( midbrain. Mash1 partiallycompensatesfor which isnotevidentin(D) as suggestedbythelossof neurons in results inafurtherrescueofTh 3504 Fig. 4. numbers ofTh (B) neurons in classes ofproneuralgenestocompensatefor subtype specificationhasbeendemonstratedbytheinabilityofother (Kele etal.,2006).InotherpartsoftheCNS, roleof with arolefor an effector ofNotchsignalling,intheventral midbrainisconsistent (reviewed byBertrandetal.,2002).ThereducednumberofTh specific, differentiation programsinotherparts oftheCNS the Ngn2 Ngn2 Ngn2 KIMash1/Mash1 Ngn2 REVIEW –/– has aroleinregulating genericneuronal,aswellsubtype- promoter (Kele etal.,2006). Ngn2 Coronal sectionsshowing the expression ofThinDA and in(C) Ngn2 Ngn2 is required forTh + Ngn2 DA neurons. ( KIMash1/KIMash1 mutants isduetoafailure inneuronalproduction, embryos thatexpress function, as60%ofthenormalnumbermDA in regulating thegenericaspectofneurogenesis Ngn2 Mash1 Ngn2 –/– ␤ ;Mash1 ( E III-tubulin Ngn2 ) Partialrescue inthenumber ofTh + KIMash1/KIMash1 –/– DA neuron generation,and A KIM1/KIM1 embryos, whichhavewild-type Ngn2 –/– ) wild-typeand( Dll1 mutant embryosisobserved, Mash1 + , aNotchligand,and Mash1 function intheventral neurons, whichliedirectly + ) embryos.Modified, with neurons (Fig.4)in + is abletocompensate embryos. Thispartial DA neuron numberin under thecontrolof B-D Ngn2 ) mutant Ngn2 activity Hes5 + DA in + , mouse embryoshave demonstratedarolefor studies performedbyelectroporating steps inimmaturemDA neurons.Complementarygain-of-function for thetreatment ofParkinson’s disease includegoodgraft Crucial parametersaffecting thesuccessofatransplantationtherapy developmental historyinadditiontoitsneurotransmitterphenotype. highlight theimportanceof identifying aDA neuronbyits mDA progenitors. (Ohyama etal.,2005).Bycontrast, Six3isnotexpressed inA13and transcription factor Six3inprogenitorsfortheirdevelopment shown todependontheexpression of thehomeodomain express Nkx2.1andMsx1homeodomainproteins,have been tuberal hypothalamicDA neuronsintheforebrain ofchickembryos different CNSpositions.Insupportofthishypothesis, lateral at distinct programsareinvolved inspecifyingDA differentiation theonethatpromotesmDA differentiation, indicatingthat from periglomerular A16DA neurons.These programsalsodiffer the neurogenesisandsubtypedeterminationofolfactory bulb is amoleculardeterminantofA16progenitorsthatrequired for additional anduniquerolefor but notMash1,inpostmitoticDA neuronsisconsistentwithan of themDA neuronalsubtype.Inaddition,theexpression ofNgn2, compensation suggestssomeuniquerolefor Pax6, anddifferentiate intoPax6 progenitors express homeodomaintranscriptionfactors Dlx1/2and of neuromeresegment p3.Recentstudiesdemonstrate thatA13 A13 andA16groups.TheDA neuronsresideinthealarplate about thespecificationofDA fate inthesegroups,except forthe (interplexiform cellsintheretina;seeFig.1).Very littleisknown groups), A16(periglomerularcellsintheolfactory bulb), andA17 They arefoundinareasA11toA15(diencephalicand hypothalamic catecholaminergic neuronsintheforebrainarealsodopaminergic. A10 (VTA) areasofthemidbraindiscussedsofar, some Besides theneuronsinA8(retrorubralarea),A9(SNpc)and factor networks DA neuron developmentviadistincttranscription below. programs thatgovern thedevelopment ofDA neuronsisdiscussed other DA groupsintheCNS,abriefcomparisonofgenetic differentiation. Inlightofrecentstudiesonthedevelopment of progenitors andneurons,but alsoregulate their specificationand new transcriptionfactors thatnotonly better definemDA neurons. targets andcofactors inbothmDA progenitorsandimmaturemDA development willcomefromtheidentificationofitstranscriptional al., 2002).Furtherinsightsintothefunctionof working cooperatively withothertranscriptionfactors (Bertrandet function inotherpartsoftheCNSacontext-dependent manner, 2006). Thisfindingisnotsurprisingbecause markers andthegenerationofectopicDA neurons(Kele etal., is insufficient topromotetheectopicexpression ofDA neuron marker mantle zoneandininducingexpression ofthegeneralneuronal migration ofnewborn neuronsfromtheventricular zonetothe Islet1 andTh.Theseresultsthusidentifyarolefor mice, neuronsgeneratedbyA13progenitorsfail toexpress Pax6, al., 2003;MastickandAndrews, 2001).In A13 progenitorstodifferentiate intoTh specification ofA13DA neurons. The distinctdifferentiation programsforCNSDA neurons In summary, thestudiesdescribed inthissectionhave identified ␤ III-tubulin. However, thesestudiesshow that Ngn2 + Pax6 /Islet in regulating laterdifferentiation Ngn2 , however, isnotrequiredfor + + neurons. Bycontrast,Pax6 /Th Dlx1/Dlx2 in thedorsalmidbrainof Development 133(18) + Ngn2 Ngn2 Ngn2 neurons (Andrews et Ngn2 in promotingthe in specification in mDA neuron double mutant Dlx1/2 is known to Ngn2 in the alone

DEVELOPMENT Lmx1a/b function studiessuggestthatthemajordeterminants,suchas known, theirprecisefunctionsremaintobedeciphered. Gain-of- neural progenitorstowards amDA differentiation programarenow Although someofthekey molecularplayersrequired inspecifying Conclusions transplantation therapies. populations ofDA neuronsfromstemcellsthatwillbeusefulfor progenitor cellsinvivo shouldfacilitate thegenerationofspecific determinants thatregulate mDA neurondifferentiation fromCNS intheseprocedures.Therefore,elucidatingthemolecular used therapies willbestronglyinfluencedbythetypeofDA neurons 1994; Zuddasetal.,1991)indicatethatthesuccessoftransplantation (Thompson etal.,2005).Theseandearlierstudies(Hudson recognition mechanismsregulating reinnervation intheforebrain display distinctresponsestoaxonguidancecuesandtarget forebrain ofadultmice,suggestingthatmDA neuronalsubtypes neurons differ intheiraxonprojectionpatternsthe DA-denervated 2005). RecentfindingsindicatethatgraftedSNpcandVTA DA Isacson, 2002;Lindvall andBjorklund,2004;Snyder andOlanow, integration andfunctionalreinnervation (reviewed byBjorklundand Development 133(18) Andersson, E.,Tryggvason, U.,Deng,Q.,Friling,S.,Alekseenko, Z.,Robert, Andersson, E.,Jensen,J.B.,Parmar, M.,Guillemot,F. andBjorklund, A. Alberi, L.,Sgado,P. andSimon,H. References Parkinson’s DiseaseSociety, UKandtheMedicalResearch Council. for criticalreading ofthismanuscript. Work inmylaboratoryisfundedby Ferri andWai HanYau forhelpwithillustrations,andtomembersofthelab I amextremely gratefulto Stella Uwaborforhelpwithreferences, toAnna Andrews, G.L.,Yun, K., Rubenstein, J.L.andMastick,G.S. based therapiesforParkinson’s disease. of molecularmarkers willleadtosignificantprogressinstemcell- cells oftheSNpcsubtypeandtopurifythembasedonaspecificset The abilitytogenerateDA neuronsfromneuralprogenitor/stem responsible forthesedistinctmigratoryandaxongrowth behaviours. challenge forthefutureistodiscover themoleculesthatare of themidbrainandhave distinctaxonprojection patterns.Amajor DA neuronssettleindifferent positionsalongthemediolateralaxis of SNpcversus VTA DA neuronsremainunknown. SNpcandVTA laboratory. Inaddition,themoleculesresponsibleforgeneration development ofmDA neuronsiscurrentlybeinginvestigated inour mDA progenitors(Puellesetal.,2003),andtheirroleinthe transcription factor family, Foxa1 andFoxa2, arealsoexpressed in transcription factors, membersoftheforkhead/wingedhelix components oftheseregulatory networks. Besidesthese neurons, andwillalsoleadtothediscovery ofdownstream govern thedifferentiation ofmidbrain progenitorsintomDA the specificcombinatorialinteractionsoftranscriptionfactors that et al.,2006;Andersson2006b).Futurework willshedlighton differentiation ofdorsalmidbrainprogenitorsintoDA neurons(Kele expression ofsomethesegenesindividually fails topromotethe combinatorial mannertopromotethemDA fate, astheectopic Ang, S.L.,Conlon,R.A.,Jin,O.and Rossant,J. Bayer, S. A.,Wills,K.V., Triarhou, L. C.andGhetti,B. of midbraindopamineneurons. B., Perlmann,T. andEricson,J. compromised intheabsence ofneurogenin 2. (2006a). 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DEVELOPMENT