Specification of catecholaminergic and serotonergic neurons

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NATURE REVIEWS | NEUROSCIENCE VOLUME 3 | JULY 2002 | 531

The vertebrate nervous system consists of many distinctneuronal cell types, each of which is engaged in multi-ple cellular interactions to generate functional circuits.How the different phenotypic features that underlie thisdiversity are specified during development is an impor-tant issue in developmental neuroscience. The finalphenotype of a neuron comprises pan-neuronal orgeneric neuronal features that are shared by all neurons,features that are common to several neuronal types,and features that are unique to one class of neuron,such as axons that project in a particular direction. So,the question arises of how the expression of specifictraits that are common to several neuronal types is con-trolled during development, and how this is linked tothe expression of generic neuronal properties.

An important aspect of the neuronal phenotype thatis often shared by neurons of different origins is theneurotransmitter that a neuron releases to excite orinhibit its target cells. Chemical neurotransmissionrequires the coordinated expression of a whole set ofspecialized proteins by a given neuron. First, the choiceof neurotransmitter obviously implies the expressionof enzymes that are necessary for its synthesis from

commonly available precursors. Second, neurotrans-mitters are not only taken up, stored and released bysynaptic vesicles that are specific for the type of neuro-transmitter, but the released transmitters or their break-down products are also recaptured by specific membranetransporters. In addition to the coordinated regulationof genes that represent the transmitter phenotype of agiven neuron, the expression of the appropriate recep-tors on the target cells needs to be coordinated duringdevelopment. As neurotransmitter phenotype and target-cell identity are specified in many cases beforesynaptic connections are formed, it is likely that neuro-transmitter choice is regulated in concert with axonalpathfinding and synaptogenesis.

So, the determination of neurotransmitter pheno-type is inextricably linked to other aspects of neuronalfate determination. In general, neurons acquire pheno-types that are stereotypical for their site of origin in thecentral nervous system (CNS) or, in the case of periph-eral neurons, for the site at which the migration of theirprecursors was arrested. If neurons of the same trans-mitter type have similar origins and synaptic connec-tions, it is easy to understand the basis of coordinated

SPECIFICATION OFCATECHOLAMINERGIC AND SEROTONERGIC NEURONSChristo Goridis* and Hermann Rohrer‡

The specification of neurotransmitter phenotype is an important aspect of neuronal fatedetermination. Substantial progress has been made in uncovering key extracellular signals andtranscriptional regulators that control the mode of neurotransmission in several model systems,among which catecholaminergic and serotonergic neurons feature prominently. Here, wereview our current knowledge of the regulatory circuits that direct neurotransmitter choice, anddiscuss the development of well-studied types of catecholaminergic and serotonergic neurons.One emerging concept is that different types of neuron use a similar core programme to controlshared modes of neurotransmission, but recruit different factors that are specific for eachneuronal type. Another is that most factors that specify neurotransmitter identity also controlother features of the neuronal phenotype.

*CNRS UMR 8542,Département de Biologie,École Normale Supérieure,46 rue d’Ulm, 75230 ParisCedex 05, France.‡Max-Planck-Institut für Hirnforschung,Abteilung Neurochemie,Deutschordenstr. 46, 60528Frankfurt/Main, Germany.Correspondence to C.G.e-mail:[email protected]:10.1038/nrn871

© 2002 Nature Publishing Group

SYMPATHETIC NEURONS

The neurons of the sympatheticnervous system, which isresponsible for suchphysiological effects as reductionof digestive secretions,vasoconstriction and increasedheart rate, thereby opposing theeffects of the parasympatheticnervous system.

RAPHE NUCLEI

A series of neuronal groupslocated along the midline of thebrainstem. They constitute themain supply of 5-hydroxy-tryptamine to the rest of thebrain.

532 | JULY 2002 | VOLUME 3 www.nature.com/reviews/neuro

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which catecholamine- and 5-HT-producing cell groupsare numbered in a caudal-to-rostral direction, groupsA1–A7 containing NA, groups A8–A16 producing DA,groups C1 and C2 being adrenergic, and groups B1–B9comprising 5-HT neurons1,2. Catecholaminergic and 5-HT neurons — together known as monoaminergicneurons — produce their neurotransmitters through aseries of enzymatic modifications of the amino-acidprecursors tyrosine and tryptophan, respectively (FIG. 1).Catecholaminergic neurons produce NA, adrenaline or DA. They all share the first two steps of the catechol-amine-synthesis pathway that leads to DA; adrenergicand NA neurons share a third step that leads to NA, andonly adrenergic neurons express the enzyme for adren-aline synthesis1,3. Adrenergic neurons constitute aminor population of ill-defined origin and will not beconsidered here. Catecholaminergic and 5-HT neuronsuse a different amino-acid precursor, but share thebiosynthetic enzyme aromatic amino acid decarboxy-lase. More recent studies of their origin have shown that5-HT and DA neurons both arise from the ventral-most region of the neural tube, albeit at different rostro-caudal levels, perhaps reflecting some phylogeneticrelatedness4.

Neurons that use the same transmitter have manyfeatures in common, but they are operationally definedby the expression of key biosynthetic enzymes or by thepresence of the stored neurotransmitter. So, NA neuronsare generally defined as neurons that express tyrosinehydroxylase (TH) and dopamine β-hydroxylase (DBH),DA neurons by the expression of TH only, and 5-HTneurons by the presence of tryptophan hydroxylaseor 5-HT.

In the vertebrate peripheral nervous system, NA isfound mainly in SYMPATHETIC NEURONS. In the vertebrateCNS, NA neurons form a distinct metencephalicnucleus — the locus coeruleus (LC; groups A4 and A6)(FIG. 2) — and a series of loosely organized cell groupsthat are distributed throughout the hindbrain (A1–A3,A5 and A7). The LC axons irrigate virtually all brainregions, forming one of the most widely distributedprojection systems of the CNS2,3,5. In higher vertebrates,the most prominent groups of DA neurons reside in theventral midbrain (where they are located in the sub-stantia nigra and in the ventral tegmentum; A8–A10)and in the diencephalon (A11–A15); smaller groups arefound in the olfactory bulb and in the retina of somespecies1. In contrast to the diffuse distribution of the NAaxons, DA axons form topographically well-delimitedprojections, the main one being the nigrostriatal pro-jection. 5-HT neurons are found as a series of cellgroups in the hindbrain, which can be separated intorostral (B5–B9) and caudal (B1–B4) divisions, the vastmajority of which are located within the RAPHE NUCLEI.Like NA neurons, 5-HT cells project to most parts ofthe brain2,3,5. So, central NA, DA and 5-HT neuronshave distinct origins and projection patterns, consistentwith the idea that axonal growth is regulated in concertwith neurotransmitter identity.

An interesting aspect of neurotransmitter pheno-type that is not addressed in the present review is its

regulation of different properties. But in other cases, thesame neurotransmitter is used by neurons of very differ-ent origins, and the question arises of whether the sameor different genetic circuits are used for the control oftransmitter phenotype.

We address these questions by reviewing recentprogress in our understanding of the specification anddifferentiation of neurons that have a catecholaminergicor serotonergic (5-hydroxytryptamine (5-HT, sero-tonin)-synthesizing) transmitter phenotype.We will dis-cuss the following questions.What are the key regulatorygenes that specify noradrenergic (NA), dopaminergic(DA) and 5-HT neurons? What extrinsic signals areinvolved in their specification? An important question ishow particular sets of genes, which represent the neuro-transmitter phenotype, come to be expressed in neuronsof different types and origins. This leads to the questionof whether distinct sub-programmes exist that controlspecific components of the mature phenotype and thatare invariably used by all neurons that share this pheno-type, irrespective of their origin and function. Neuronalidentity would therefore be implemented as a collectionof sub-programmes that run in parallel. Alternatively,different classes of neurons might use different modulesto regulate shared features of neuronal identity.

What are catecholaminergic and 5-HT neurons?Historically, the neuronal groups that use catechol-amines or 5-HT as neurotransmitters were the firsttransmitter systems to be described in detail, because ofthe ease of their detection by formaldehyde-inducedfluorescence. This is reflected by a nomenclature in

TyrosineTryptophan

5-Hydroxytryptophan

5-Hyroxytryptamine(serotonin)

Dopamine

Adrenaline

L-DOPA

Tyrosine hydroxylase (TH)

a b

Dopamine β-hydroxylase (DBH)

L-Aromatic amino aciddecarboxylase (L-AADC)

Tryptophanhydroxylase

L-Aromaticamino acid

decarboxylase(L-AADC)

PhenylethanolamineN-methyltransferase Noradrenaline

HO

HO

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NH2+

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NH

NH3+

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OHO

HO

HONH3

+

HONH3

+HO

O–

O

HONH3

+

O–

O

Figure 1 | Enzymatic pathways of catecholamine and 5-HT synthesis. a | Catecholaminesynthesis. b | 5-Hydroxytryptamine (5-HT, serotonin) synthesis.



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related HOMEODOMAIN transcription factors Phox2a andPhox2b. Further downstream in this network of tran-scriptional regulators are the bHLH transcription factordHand (heart and neural-crest derivatives expressed 2)and the ZINC FINGER protein Gata3 (GATA-binding protein 3) (FIGS 4a and 5).

The first indication that BMPs might be extracellularsignals that trigger sympathetic differentiation came fromexperiments showing that BMP7 increases the number ofNA cells that develop in neural crest cultures13. This wassoon substantiated by several observations. First, expo-sure of clonal cultures of neural crest cells to BMP2 leadsto the induction of the AUTONOMIC NEURON markers Mash1and Phox2a14. Second, BMP2, BMP4 and BMP7, as wellas the forced expression of a constitutively active BMPreceptor, massively increase the number of TH-express-ing cells in neural crest cultures15–18. Third, and mostimportantly, overexpression of Bmp4 in the vicinity ofthe developing sympathetic ganglia results in the ectopicdevelopment of TH-positive, DBH-positive cells in vivo15.Fittingly, the dorsal aorta was found to express Bmp2,Bmp4 and Bmp7 at the time neural crest cells arrivethere15,16, identifying it as a likely source of the signal.Final proof that the initiation of sympathetic develop-ment depends on BMPs was provided by experimentsshowing that the implantation of beads releasing theBMP inhibitor noggin in the vicinity of the dorsal aortaprevented the development of sympathetic neurons19.

How are BMP-mediated signals relayed during sym-pathetic-neuron development? Mash1, the expression ofwhich in neural crest cultures is induced by BMPs14, wasthe first transcription factor to be shown to be essentialfor sympathetic development. In Mash1–/– mutants, cellsof the neural crest initially arrive at the dorsal aorta, butmost of them fail to express pan-neuronal markers or theNA biosynthetic enzymes TH and DBH9. Subsequentwork identified the homeodomain transcription factorsPhox2a and Phox2b, which are present in all neurons thatexpress the specific marker of NA neurons DBH20,21. Inthe sympathetic-ganglion primordia, Phox2b precedesPhox2a and is induced by BMPs independently of Mash1,whereas Phox2a expression depends on both Mash1 andPhox2b12,19,21,22. In gain-of-function (GOF) experiments,both Phox2a and Phox2b have the capacity to induce asympathetic-neuron-like phenotype when misexpressedin chick embryos, including the expression of TH andDBH23. However, only Phox2b is required for sympa-thetic-ganglion development. In the absence of Phox2a,sympathetic development is largely normal24. In Phox2b-mutant embryos, by contrast, neural crest cells arrive atthe dorsal aorta and express Mash1, but they do not acti-vate either pan-neuronal markers or the specific markersTH, DBH and Phox2a, and they do not maintain theexpression of Mash1 (REF. 22). Despite the fact that Phox2ais genetically downstream of Phox2b and Mash1 in thislineage, Phox2a misexpression induces Phox2b andMash1 ectopically (REF. 23, and M. Stanke and H.R.,unpublished observations).

Regulation of DBH by Phox2 genes is probably direct,as both Phox2a and Phox2b bind to regulatory elementsin the DBH promoter and stimulate DBH-promoter

plasticity; that is, it can undergo developmental‘switches’. The switch from a NA to a cholinergic phenotype in sympathetic neurons has been studiedmost extensively6–8.

Peripheral noradrenergic neuronsThe peripheral sympathetic nervous system is composedmainly of neurons that use NA as a neurotransmitter.Sympathetic neurons have long been a favoured modelin developmental neurobiology, and knowledge of theirspecification and differentiation has preceded that ofcentral NA neurons. Sympathetic neurons arise from cellsof the NEURAL CREST that arrest their migration once theyhave arrived in the vicinity of the dorsal aorta, aroundembryonic day 10 (E10) in the mouse and E2.5 in thechick (FIG. 3). Soon afterwards, they start to express pan-neuronal markers and the NA biosynthetic enzymes THand DBH9–12. A promising line of research on the deter-mination of the peripheral NA phenotype links togetherBONE MORPHOGENETIC PROTEIN (BMP) signalling molecules,the BASIC HELIX–LOOP–HELIX (bHLH) transcription factorMash1, which is encoded by a vertebrate homologue ofDrosophila PRONEURAL GENES (Mash1 is used here for bothmammalian and avian homologues), and the closely

NEURAL CREST

Groups of cells that migratefrom the neural tube to theperiphery, where they give rise toa wide variety of cell types.

BONE MORPHOGENETIC

PROTEINS

Multifunctional secretedproteins of the transforminggrowth factor-β superfamily.In the early embryo, theyparticipate in dorsoventralpatterning.

BASIC HELIX–LOOP–HELIX

(bHLH). A structural motif thatis present in many transcriptionfactors, which is characterized bytwo α-helices separated by aloop. The helices mediatedimerization, and the adjacentbasic region is required for DNAbinding.

DiencephalonMesencephalon

Metencephalon

Locuscoeruleus

Tectum

Tegmentum

Telencephalon

r2

r1

r3

r4

r5Shh

FGF8

BMP5

Inductiveinfluences

5-HT neurons

Midbrain dopaminergic neurons

Neuronal populations

Locus coeruleus neurons

Figure 2 | Location of 5-HT, locus coeruleus and midbrain dopaminergic neurons withrespect to sources of important signalling molecules. A sagittal view of the neural tube of anembryonic-day-11.5 mouse is shown. 5-Hydroxytryptamine (5-HT, serotonin)-producing neuronsdevelop as two clusters of cells in the ventral hindbrain or rhombencephalon, the locus coeruleusin dorsal rhombomere 1 (r1), and midbrain dopaminergic neurons in the so-called tegmental areaof the midbrain. The arrow indicates the mid–hindbrain border. BMP5, bone morphogeneticprotein 5; FGF8, fibroblast growth factor 8; r2–r5, rhombomeres 2–5; Shh, sonic hedgehog.



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signalling pathway impinges on the Mash1 and Phox2bpromoters, has not yet been elucidated. However, thisinitial description of sympathetic-neuron developmentis not sufficient to explain the specification and differ-entiation of the NA phenotype, as Mash1 and thePhox2 genes are also co-expressed in neuronal lineagesthat do not express NA33. This strongly implies that fur-ther cell-intrinsic factors are required for the NA differ-entiation of sympathetic precursors. Indeed, recentwork indicates a role for the transcription factorsdHand and Gata3 (FIG. 4a).

dHand is expressed in sympathetic-ganglion primor-dia, and promotes sympathetic differentiation both inneural crest cultures and in vivo34,35. The expression ofdHand can be induced by BMPs in neural crest cells, andhas been shown to depend on BMPs in the anlagen ofthe sympathetic ganglia35. The onset of dHand expres-sion in sympathetic precursors — after Mash1 andPhox2b, but before TH and DBH — implied a sequentialaction of Phox2b and dHand on NA differentiation.Indeed, dHand expression in sympathetic gangliadepends on Phox2b (A. Pattyn, personal communica-tion) and Mash1 (REF. 36), indicating that dHand is down-stream of both genes. In spite of this, dHand can inducePhox2b when expressed ectopically35. Recent evidenceimplicates dHand in the maintenance of TH and DBHexpression in sympathetic neurons (F. Müller and H.R.,unpublished observations), an idea that is supported bythe finding that dHand binds to and transactivates theDBH promoter, acting in synergy with Phox2a andcAMP signals (M. Howard, personal communication).Whether dHand is not only sufficient but also necessaryfor sympathetic differentiation has not been investigated,because the available knockout mice die around the timethat sympathetic development is initiated37.

The case of Gata3 is particularly interesting,because in Gata3 mutants, sympathetic ganglia formand express pan-neuronal markers and Phox2 genes,but fail to express TH and possibly also DBH38. So, thistranscription factor seems to be specifically requiredfor the expression of NA traits. Gata3 is geneticallydownstream of Phox2b, as its expression in sympa-thetic precursors is abolished in Phox2b mutants (A. Pattyn, personal communication).

Together, these data establish sympathetic neurons asthe best-understood example of a genetic circuitry thatspecifies a neurotransmitter phenotype in vertebrates.At least five transcription factors seem to collaborate inthe specification of the NA phenotype in this lineage, ofwhich three have been shown to be required for itsexpression (FIG. 5). Although some of these seem to func-tion sequentially (Mash1 before Phox2a and dHand;Phox2b before Phox2a, dHand and Gata3), they form acomplex regulatory network rather than linear cascades.On one hand, dHand and Phox2a, although geneticallydownstream of Phox2b, can induce Phox2b. On theother hand, Phox2b and its downstream targets dHandand Gata3 all seem to act on the TH and DBH genes.For dHand, a synergistic action with Phox2a on theDBH promoter has been shown in cultured cells (M. Howard, personal communication). In other words,

activity in cultured cells, either on their own or in con-junction with the activation of the cyclic-AMP second-messenger pathway25–30. Evidence for the regulation ofTH-promoter activity by Phox2a or Phox2b is lessextensive, but Phox2a has been found to transactivateTH-promoter activity in transient transfection assays bybinding to a previously identified regulatory element31.The negative results that have been reported by others26

might be due to a requirement for enhanced cAMP sig-nalling. In support of this idea, efficient induction of THby Phox2a in neural-crest-cell cultures was found todepend on elevated cAMP levels32. Together, theseresults indicate that extracellular signals other thanBMPs are involved in activating NA traits. The impor-tance of cAMP signalling still needs to be confirmed byin vivo loss-of-function (LOF) approaches.

These results point to a scenario in which, under theinfluence of BMPs that are secreted from the dorsalaorta, cells of the neural crest initiate the expression ofMash1 and Phox2b. The combined input of these tran-scription factors is required to activate NA biosynthesisand pan-neuronal differentiation, as well as the Phox2agene (FIG. 5). Full NA differentiation seems to depend onfurther signals that activate the cAMP second-messengerpathway. The nature of these signals, and how the BMP

PRONEURAL GENES

Genes that encode transcriptionfactors of the basichelix–loop–helix class thatspecify neural progenitor cellsand promote theirdifferentiation.

HOMEODOMAIN

A 60-amino-acid DNA-bindingdomain that comprises three α-helices and is found in manytranscription factors.

ZINC FINGER

A protein module in whichcysteine or cysteine–histidineresidues coordinate a zinc ion.Zinc fingers are often used inDNA recognition and inprotein–protein interactions.

AUTONOMIC NEURON

A neuron that belongs to thepart of the nervous system that isnot under conscious control,and regulates functions such asbreathing, circulation anddigestion.

ROOF PLATE

The point of fusion of the neuralfolds, which forms the dorsal-most part of the neural tube.

Notochord

Dorsal aortaBMPs BMPs

Sympathetic-neuronprecursors

Neural tube

Figure 3 | Migration pathway and location of sympatheticprecursors. Shown is a transverse section through the trunkregion of a vertebrate embryo (embryonic day 10.5 in themouse, or 2.5 in the chick). Sympathetic neurons develop fromneural crest precursor cells that aggregate after their migrationin the vicinity of the dorsal aorta. The dorsal aorta producesand secretes bone morphogenetic proteins (BMPs), which areessential for sympathetic-neuron development.



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expression of the NA phenotype is controlled by thecombinatorial action of several interacting factors.Finally, with the possible exception of Gata3, these factors also control other aspects of the sympathetic-neuronal phenotype and generic neuronal properties.

In addition, some properties of NA neurons seem tobe controlled independently of the Mash1/Phox2b path-way. The plasma-membrane NA transporter (NET/Slc6a2)39, which has an important role in terminatingNA action at the synapse and is part of the NA trans-mitter phenotype, is not co-expressed with TH/DBHduring initial sympathetic-neuron development; inaddition, it is transiently expressed in non-neuronalcells (REF. 40, and M. Stanke and H.R., unpublishedobservations). In agreement with these results, it hasbeen observed that Phox2a cannot transactivate NET-promoter activity41. So, an important property of NAneurons is controlled, at least in part, independently ofTH and DBH during development.

Central noradrenergic neuronsIn the rostral hindbrain or metencephalon, the centralNA neurons form a compact nucleus, the LC, which isthe principal source of NA innervation of the brain3,5.We will not consider here the other groups of NA cells;these are distributed throughout the hindbrain andtheir development has not been studied extensively. TheLC neurons arise in the dorsal isthmic region of thehindbrain (FIG. 2), and are among the earliest-born neu-rons in the brain (E9 to E10.5 in the mouse)42–45. Theirspecification involves, in part, the same players that areencountered during sympathetic differentiation: BMPs,Mash1, Phox2a and Phox2b. Curiously, however, thethree transcription factors function in different sequentialorders during sympathetic and LC development.

The specification of dorsal fates in the early neuraltube depends on BMPs and related signalling factors,which are initially produced by the dorsal ectoderm, andsubsequently by the ROOF PLATE and dorsal-most neuraltube46,47, and the LC is no exception to this rule. Theanalysis of zebrafish mutants yielded the first indicationthat LC development depends on BMPs48. However,impairment of BMP signalling during early zebrafishdevelopment disturbs patterning of the neural plate49,and lack of LC development could be a far-downstreamconsequence of that. In subsequent work, inhibition ofBMP signalling by implanting noggin-releasing beads in ovo showed that the proper development of LC neu-rons, including the expression of Phox2a, requires BMPsignalling at later stages45. Interfering with BMP sig-nalling caused two phenotypes, which were character-ized by a lack of neurons expressing the LC markersPhox2a and DBH, or by their ectopic appearance at amore dorsal location. The latter phenotype could beinterpreted as indicating that the development of LCneurons requires a defined level of BMP signal within aBMP gradient; that is, at lower noggin concentrations, aLC fate is produced nearer to the source of BMP. Insupport of this proposal, LC neurons arise at a moredorsal location in the zebrafish Smad5 mutantsomitabun, which has only weak BMP signalling48.

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Figure 4 | Cell-extrinsic and -intrinsic factors that are known to be involved in thegeneration of noradrenergic, dopaminergic and 5-HT neurons. a | Noradrenergicsympathetic neurons. Their specification is initiated by bone morphogenetic proteins (BMPs)and the transcription factors Mash1, Phox2b, dHand and Gata3, which, in turn, control theexpression of noradrenergic — tyrosine hydroxylase (TH) and dopamine β-hydroxylase (DBH)— and generic neuronal properties. NF160, neurofilament medium polypeptide; SCG10,superior cervical ganglia neural-specific 10. b | Midbrain dopamine (DA)-producing neurons.Their initial specification requires the patterning information that is provided by sonichedgehog (Shh) and fibroblast growth factor 8 (FGF8). The Lmx1b and Nurr1 transcriptionfactors are essential for different aspects of DA differentiation. The Pitx3 transcription factorand the retinoid-synthesizing enzyme Aldh1 are specific markers of developing DA neurons inthe ventral midbrain, but their roles are still unknown. c | 5-Hydroxytryptamine (5-HT,serotonin)-producing neurons. Their initial specification requires the combined input of Shh,FGF4 and FGF8, which provide essential patterning information, and is dependent on theNkx2.2 transcription factor, which probably relays Shh signalling. Pet1, a transcription factorthat is specific for 5-HT neurons, seems to have an essential role in the differentiation ofrostral and caudal cell groups; Gata3 seems to be involved in the differentiation of only caudalgroups of 5-HT cells.



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but Phox2a and Phox2b expression is not affected.However, the precise position of Rnx in the genetic cas-cade that leads from BMP signalling to Phox2b has notbeen elucidated.

Conversely, Phox2a misexpression has been shown toinduce ectopic neurons that express Phox2b, TH andDBH in chick and zebrafish embryos45,48. These putativeLC neurons are generated only at restricted times andlocations, indicating that Phox2 genes must cooperatewith further signals to direct a NA phenotype.

Together, the results show that a similar combina-tion of factors specifies the central and peripheral NAphenotype. In both the peripheral nervous system andthe CNS, NA differentiation depends on BMP signallingand on the Mash1 and Phox2b transcription factors(FIGS 4a and 6). This indicates that the NA transmitterphenotype is controlled by a distinct sub-programmethat is used by neurons of different origins. There are,however, some quirks. First and foremost, Mash1 andthe Phox2 genes act in different sequential orders inthe two lineages (FIGS 5 and 6). One interpretation isthat NA transmitter identity is controlled not by one ortwo master genes, but by a network of genes that cross-regulate each other (although this has not been shownfor all combinations), different sequential orders yield-ing the same final outcome. Second, Mash1, Phox2aand Phox2b, and even Mash1, Phox2a, Phox2b and Rnx,are co-expressed in NA and non-NA lineages, consis-tent with the idea that they must cooperate with fur-ther signals to specify NA identity. Third, dHand hasbeen recruited for determination of the NA phenotypein sympathetic ganglia, but is not expressed in chickLC precursors (A. Vogel-Höpker, personal communi-cation). The status of Gata3 in the LC and of Rnx insympathetic precursors remains unclear; expressionof Gata3 in the LC has not been examined, and Rnx isexpressed in sympathetic ganglia52, although no func-tional data are available. In conclusion, Mash1 andPhox2b are crucial determinants of the NA pheno-type, and can act in a different order and interactwith distinct transcription factors in different types ofNA cell.

Midbrain dopaminergic neuronsMost DA neurons in higher vertebrates are located inthe midbrain within the substantia nigra and the ventraltegmental area1. They are generally defined by theexpression of TH in the absence of DBH. The first DAneurons are detected at around E10 in the mouse, whenTH-expressing cells appear just rostral to the mid–hind-brain boundary (isthmus), close to the FLOOR PLATE at theventral rim of the neuroepithelium53 (FIG. 2). They arisein close proximity to two important organizing centres,the floor plate and the isthmus, which pattern the neuralplate and early neural tube along its dorsoventral andanteroposterior axes, respectively50,54. So, it was not sur-prising that the induction of DA neurons depends onthe correct patterning of the progenitor domain by sig-nalling from these centres. In neural plate explants,sonic hedgehog (Shh), the main signalling molecule tobe secreted by the floor plate, and FGF8, a mediator of

Fibroblast growth factor 8 (FGF8) has been identi-fied as a further factor that is required for LC develop-ment in zebrafish embryos48. FGF8 is an importantmediator of the organizing activity of the ISTHMUS, andhas a key role in maintaining correct patterning of thewhole mes–metencephalic region50. Therefore, the FGF8requirement might reflect this earlier patterning activityrather than a specific function in the specification ofLC precursors.

LOF experiments in the mouse have identified fourtranscription factors that are required for early LC devel-opment: Mash1, Phox2a, Phox2b and Rnx/Tlx312,24,44,51

(FIG. 6). The dependence of the LC on Phox2a, at least, isconserved from zebrafish to mouse48. On the basis oftheir sequential appearance and analysis of the knock-out phenotypes — which show that Phox2a, Phox2band DBH are absent in Mash1 mutants, Phox2b andDBH are absent in Phox2a mutants, and only DBH isabsent in Phox2b mutants — Mash1, Phox2a andPhox2b seem to act in a linear cascade (Mash1 →Phox2a → Phox2b → DBH/TH )12,44. In the simplestscenario, the only function of Mash1 and Phox2awould be to activate Phox2a and Phox2b, respectively,and Phox2b would be responsible for inducing the NAphenotype. The other possibility is that Phox2a andPhox2b act together on the DBH and TH promoters, asthey do in vitro26,28,30. In addition, the function of theRnx homeodomain transcription factor is required forNA differentiation of the LC, except in its rostral-mostpart51. In Rnx mutants, DBH expression is eliminated,

ISTHMUS

A narrow section of the neuraltube that separates the midbrainfrom the hindbrain.

FLOOR PLATE

The ventral cells of the neuraltube that lie along the midline.

BMPs

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TH, DBH

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Figure 5 | Regulatory network controlling sympathetic-neuron development. Bonemorphogenetic proteins BMP2, BMP4 and BMP7 are expressed in the dorsal aorta, and arerequired for the expression of Mash1 and Phox2b. Mash1 and Phox2b precede dHand, Gata3and Phox2a; Mash1 and Phox2b are also genetically upstream of Phox2a and dHand, andPhox2b is genetically upstream of Gata3. Expression of the noradrenaline-synthesizing enzymestyrosine hydroxylase (TH) and dopamine β-hydroxylase (DBH) has been shown to depend onMash1, Phox2b and Gata3, but not on Phox2a. Promoter studies indicate that the regulation ofthe DBH gene by Phox2b, Phox2a and dHand is direct. At present, it is not known whetherMash1 acts on TH and DBH expression only through dHand, or whether Gata3 expressionrequires Mash1 activity. Note that the forced expression of Phox2a and dHand induces theexpression of the upstream genes Mash1 and Phox2b, and that most interactions arereciprocal. Phox2b is also required for the maintenance of Mash1 expression (wavy arrow). Solidarrows indicate that the regulation has been shown by loss-of-function or by both loss- andgain-of-function experiments; stippled arrows show that it has been deduced from gain-of-function experiments and might signify a maintenance rather than an inductive function. Gata3seems to be required for the expression of TH, but not of generic neuronal properties, whereasall the other factors seem to control generic neuronal properties as well.



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Nurr1, an orphan nuclear receptor, was the firsttranscription factor of which inactivation was found toresult in a lack of DA and TH in the midbrain60,62–65. InNurr1–/– mice, no midbrain DA neurons can be detectedat birth using a variety of markers, and it was initiallyconcluded that the lack of Nurr1 causes agenesis of DAneurons62,65. However, further analysis showed that theneurons that normally become DA neurons are bornand express several of their specific markers, includingPitx3 and Lmx1b, but fail to activate the TH gene60,64.Whether these neurons survive up to birth and form anigrostriatal projection is a moot point. Although all theauthors agreed that Pitx3 expression disappeared beforebirth, one group reported the preservation of nigro-striatal innervation and no sign of apoptotic death inNurr1 mutants66. However, it is not clear whether effer-ent and afferent fibres could be distinguished in thisstudy. By contrast, another group reported the absenceof the nigrostriatal projection that is normally formed bymidbrain DA neurons, and extensive cell death60. As DAneurons develop normally in TH–/– mice67, the absenceof TH alone is probably not sufficient to explain thephenotype, and Nurr1 must control other features aswell. In line with this, expression of the neurotrophic-factor receptor Ret has been found to be abrogated inNurr1–/– embryos60. Furthermore, in vitro data indicatethat Nurr1 might be involved in the regulation of theplasma-membrane DA-transporter gene (DAT/Slc6a3)68,but this remains to be validated in vivo. Finally, Nurr1expression is widespread in the embryonic brain, andeven in the ventral midbrain, it is not initially confinedto DA neurons69. Nevertheless, with one exception70, nodefects in other neuronal types have been reported inNurr1-knockout mice.

More recently, the correct specification of midbrainDA neurons has been shown to depend on the Lmx1bgene, which codes for a LIM homeodomain factor thatwas originally described as an essential regulator ofdorsoventral patterning of the limb71. In the midbrainregion of the neural tube, Lmx1b is expressed beforepostmitotic neurons arise61, and before Nurr1 and Pitx3,which are expressed only in postmitotic cells59,60.TH-positive cells are still born on schedule in the mid-brain in Lmx1b –/– embryos, and they express Nurr1, butthey fail to activate the Pitx3 gene and later die61. Thesedata also indicate that Pitx3 expression is not essentialfor induction of the TH gene, and point to a function forPitx3 in neuronal survival, but this possibility remains tobe tested directly by its inactivation. In Lmx1b-mutantembryos, defects in dorsal midbrain structures have alsobeen detected61. Although it seems unlikely, the pheno-type of the DA neurons could be a non-cell-autonomousconsequence of these other deficiencies.

Whereas Nurr1 and Lmx1b seem to control the spec-ification of different aspects of the identity of midbrainDA neurons, the closely related En1 and En2 (engrailed)homeodomain transcription factors seem to have a roleonly in the maintenance of the DA phenotype. The caseof these factors is interesting, because they are impor-tant for patterning the mes–metencephalic region inresponse to signals from the isthmus50. In spite of this,

isthmic activity, can both induce ectopic TH-positivecells, provided that the other factor is present. Therequirement for floor-plate signalling was also shown bythe observation that DA neurons are strikingly reducedin numbers in Gli2-knockout embryos, which lack afloor plate55. Conversely, blocking either Shh or FGF sig-nalling prevents the appearance of DA neurons56. So,DA differentiation seems to depend on the combinatorialaction of both factors (FIG. 4b).

Signalling from the floor plate and the isthmus pat-terns the neuroepithelium well before the first DAneurons are born, and the cell-intrinsic factors thatlink positional information to DA-fate specificationhave remained unknown. The correct specificationand maintenance of midbrain DA neurons has beenfound to depend on the activity of the Nurr1 andLmx1b transcriptional regulators. On the basis of itsexpression, which is highly specific for DA neurons inthe brain, a third transcription factor, Pitx3/Ptx3,might be involved in directing DA fate, but no LOF orGOF data are available at present to prove this point.Mutations in the Pitx3 gene have been found in thespontaneous mouse mutant aphakia, which is charac-terized by small eyes without a lens, but neither the sta-tus of midbrain DA neurons nor any other neuronalphenotype has been reported57,58. None of these tran-scriptional regulators qualifies as a factor that confers aspecific identity to DA-neuronal progenitors inresponse to floor-plate or isthmic signals. Nurr1 andPitx3 are not expressed in the progenitors, appearingonly after cell-cycle exit59,60. Lmx1b is expressed in theprogenitors, but is not confined to the domain inwhich DA neurons arise61.

BMPs

Phox2b

TH, DBH

Dorsal neural tube

Progenitor domain

Locus coeruleusneurons

Postmitotic locuscoeruleus precursorsPhox2a

Mash1

Rnx

?

?

Figure 6 | Transcription-factor cascade controlling locuscoeruleus development. Correct development of the locuscoeruleus depends on bone morphogenetic proteins (BMPs)that are expressed in the dorsal neural tube — BMP5 in thecase of the chick. In this lineage, Mash1 is expressed in theprogenitors and is required for the expression of Phox2a inearly postmitotic neurons, which, in turn, is required for theexpression of Phox2b. Expression of the noradrenaline-synthesizing enzymes tyrosine hydroxylase (TH) and dopamineβ-hydroxylase (DBH) depends on Mash1, Phox2a, Phox2band Rnx. The precise position of Rnx within the regulatorycascade has not been clarified.



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preserved in the respective mutants59,61,63,65. So, DA fatemight be regulated differently at different locations,although forebrain DA neurons also depend on previ-ous patterning by FGF signalling, and develop close toa source of Shh4,56. Finally, TH expression is a sharedfeature of DA and NA neurons, but seems to be con-trolled in completely different ways in the two classesof neurons.

Interest in the development of DA neurons has beenheightened by the implication of this neurotransmitterin psychiatric and neurological disorders, and by thebeneficial effect of DA-neuron transplantation inParkinson’s disease. Much attention has been focused ongenerating DA cells from embryonic stem cells.Substantial progress towards this goal has been maderecently using different approaches. One protocolmade use of FGF8 and Shh, which afforded only a mod-est (twofold) increase in the yield of DA neurons81.Another group identified an activity that was secreted bya bone-marrow-derived stromal-cell line as a potentinducer of the DA phenotype82. So far, studies on devel-opmental DA-neuron specification have contributedonly little to this line of research, which has proceededlargely by trial and error. This situation might change aswe learn more about the cell-extrinsic and -intrinsicpathways that control the fate of DA neurons. Nurr1might turn out to be interesting in this respect, becauseas a member of the nuclear hormone-receptor family, itmight respond directly to extracellular signals that remainto be identified. In this context, it is worth mentioningthat unknown signals that are released from ventralmesencephalic astrocytes strongly increase DA differen-tiation of a Nurr1-overexpressing neural stem-cell line,resulting in a population of cells that is composedmainly of DA neurons83.

5-HT neuronsLike DA neurons, 5-HT neurons arise from ventralneuroepithelial progenitors in proximity to the floorplate, albeit at different rostrocaudal levels. They developas two separate clusters within the hindbrain, where theyform rostral cell groups just caudal to the isthmus, andcaudal cell groups in the MYELENCEPHALON4 (FIG. 2). Rostral5-HT neurons have ascending projections that innervatevirtually all areas of the brain, whereas the caudal cellgroups project to the spinal cord1,2,5. In the mouse, 5-HTneurons can first be detected with anti-5-HT antibodiesat E11.5 (REF. 84), although birthdating studies indicatethat the first 5-HT neurons become postmitotic one-dayearlier42. The recent discovery of the Pet1 (plasmacytoma-expressed transcript 1) gene, which encodes an ETS DOMAIN

transcription factor and is specifically expressed in the 5-HT lineage before the appearance of 5-HT85,86, allowsthe detection of immature 5-HT neurons from aroundE10.75 in the mouse (A. Pattyn, personal communica-tion). Using this marker, it could be shown that the rostral5-HT cells arise in rhombomeres 1–3, and that rhombo-mere 4 corresponds to the gap between rostral and caudalcells, relating their origin in a straightforward manner tohindbrain compartmentalization (J. Ericson, personalcommunication; see also REF. 87).

TH-positive cells initially arise in the ventral midbrainin En1/En2 double mutants, but they are no longerdetected at birth72. At present, it is not known whetherthe cells just shut off TH synthesis or whether they die.Although postmitotic DA neurons express En1 andEn2, it remains possible that their phenotype is a conse-quence of the defects in dorsal midbrain structures inthese mutant mice.

Another gene that, on the basis of its expression pattern, could have a role in DA specification is Aldh1(also called AHD2 or Raldh1). It codes for an aldehydedehydrogenase that is involved in the synthesis of retinoicacid from vitamin A. In the brain, it is specificallyexpressed in TH-positive mesencephalic neurons73,74.Originally reported to be activated after TH73, Aldh1 waslater shown to be expressed in the progenitor domain,possibly restricted to DA progenitors60. Retinoic acid,which is well known for its role as a signalling moleculein many developmental processes, could have autocrineor paracrine functions during DA development. So, itmight be worth exploring the status of midbrain DAneurons in animals with defective retinoic-acid sig-nalling, such as retinoic-acid-receptor-knockout mice orvitamin-A-deficient animals. Finally, Nurr1 has beenreported to heterodimerize with the retinoid X receptor(RXR) and to bind to retinoic-acid-responsive promoterelements75 (but see REF. 76), indicating a possible linkbetween Aldh1 function and DA differentiation.

A new kind of regulation of the DA phenotype hasbeen uncovered in zebrafish. In this species, the develop-ment of DA neurons in the diencephalon has beenfound to require the activity of the regulator of tran-scriptional elongation Foggy77. Interestingly, in foggymutants, 5-HT neurons develop in the diencephalon atthe expense of DA neurons (in contrast to higher verte-brates, the zebrafish contains 5-HT neurons in the diencephalon), indicating that both neurotransmitterphenotypes can arise from similarly specified precur-sors, and that the DA and 5-HT phenotypes are, to someextent, interchangeable. A similar situation is observedin the chick, in which FGF4 has been reported to induce5-HT neurons ectopically in the midbrain at the expenseof DA neurons56.

Although the picture is still fragmented, the availabledata point to the existence of independent regulatorycircuits that control the DA transmitter phenotype(exemplified by Nurr1) and other aspects of DA-neuronidentity (represented by Lmx1b and Pitx3) (FIG. 4b). Theidea that Nurr1, but not Pitx3, participates in the induc-tion of TH is supported by the observations that forcedexpression of Nurr1, but not of Pitx3, in a hippocampalprogenitor-cell line induces TH, and that Nurr1 binds toand transactivates the TH promoter78. However, twoother groups have reported the presence of a Pitx3-responsive element in the TH promoter79,80. So far, tran-scription factors that coordinately regulate transmitterchoice and generic neuronal differentiation (as doMash1 and Phox2b during NA differentiation) have notbeen identified in this lineage. Furthermore, othergroups of DA neurons in the brain express neitherNurr1 nor Lmx1b (nor Pitx3), and these neurons are

MYELENCEPHALON

The caudal subdivision of theembryonic hindbrain, whichgives rise to the medullaoblongata.

ETS DOMAIN

The DNA-binding domain ofthe transcription factors of theEts family, so-called after theavian ets1 proto-oncogene.



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Pet1-binding sites in their promoter regions85. However,the widespread expression of SERT in non-5-HT neu-rons during early development92,93 does not correlatewith the restricted Pet1 expression. This, together withthe data on NET expression, implies that genes thatencode plasma-membrane transmitter transportersand neurotransmitter-synthesizing enzymes might becontrolled independently.

An interesting aspect of 5-HT-neuron developmentthat deserves further study is that the progenitordomains that give rise to 5-HT neurons also producecranial BRANCHIOMOTOR and visceromotor neurons at ear-lier stages of development33. This is a striking example ofa temporal mechanism that operates in the progenitordomains to generate different cell types over time. As theformation of 5-HT neurons coincides in time with the extinction of Phox2b expression in the progenitors(REF. 33, and A. Pattyn, personal communication), onepossibility is that Phox2b, which is essential for thedevelopment of branchiomotor neurons, in addition to specifying the NA phenotype, represses the 5-HT-neuronal fate. This situation is reminiscent of the mech-anisms that are involved in the successive generation ofmotor neurons and oligodendrocytes in the ventralspinal cord94.

ConclusionsOver the past few years, great strides have been taken inseveral model systems towards defining the transcrip-tional regulations that result in the generation of specificneuronal types at defined positions in the central andperipheral nervous systems7,36,95,96. However, our knowl-edge of the mechanisms by which distinct componentsof the mature neuronal phenotype are regulated in acoordinated fashion within any neuronal lineage remainssketchy at best. One of the better-documented aspects ofneuronal identity is the choice of neurotransmitter, andsome of the key regulatory genes and crucial extrinsicfactors that are involved have been identified. Still, ourunderstanding of the programmes that specify neuro-transmitter phenotype and link neurotransmitter controlto other aspects of neuronal identity is fragmentary. Insome cases — for example, in the case of 5-HT cells —the neurons that use a given transmitter have a similarorigin and similar phenotypic properties, and are there-fore expected to use common regulatory programmes.However, even in this apparently simple situation, differ-ent molecules seem to be involved in the specification ofrostral and caudal 5-HT neurons. In other cases —exemplified by NA neurons — the same transmitter isused by very different types of neuron. Here, a conservednetwork of interacting factors seems to direct transmitterphenotype in central and peripheral neurons, but also inthis case, cell-type-specific variations and modificationsof this network are evident. It remains to be seen whetherother transmitter phenotypes are also controlled byshared transcription factors, or whether the mode of NA-neuron specification is the exception rather than the rule.Finally, the expression of TH, which is common to bothDA and NA neurons, seems to depend on a completelydifferent set of regulators in each type of neuron.

The development of 5-HT neurons, like that of DAneurons, depends on previous patterning of the neuro-epithelium by Shh signalling and, for the rostral cells,on FGF signalling56. Ectopic 5-HT neurons can beinduced in the dorsal hindbrain, but not in other loca-tions, by the floor plate (as a source of Shh) and by Shhitself, and their development in explant cultures isblocked by anti-Shh antibodies. Consistent with therequirement for Shh signalling, fewer 5-HT neuronsdevelop in embryos that lack the Shh downstreameffector Gli255. Blocking FGF signalling prevented theappearance of 5-HT neurons in rostral, but not in cau-dal, hindbrain explants. This surprising result leads tothe general question of why Shh and FGF8 confer com-petence to the neuroepithelium to develop DA neuronsrostral to the mid–hindbrain border and to generate 5-HT neurons caudal to it. One factor that could bemissing in the midbrain is FGF4, because ventral mid-brain explants that are treated with FGF4 develop 5-HT at the expense of TH-positive neurons56 (FIG. 4c).Patterning signals other than Shh that are responsiblefor 5-HT-neuron development in the caudal hindbrainstill await identification.

Subsequent work has identified three transcriptionalregulators that are required for 5-HT-neuron develop-ment. These are Nkx2.2, a homeodomain factor that isexpressed in the ventral-most neuroepithelium inresponse to Shh signalling84; Gata3, already mentionedas a regulator of the NA phenotype; and Pet1, which isselectively expressed in 5-HT-neuron precursors and isrequired for their correct specification (FIG. 4c).

Nkx2.2 is a good candidate for a regulator that relaysearly Shh patterning by imposing a ventral progenitoridentity88, thereby conferring competence to become 5-HT neurons. In the absence of Nkx2.2 activity, few, ifany, 5-HT neurons develop in the rostral hindbrain,although 5-HT neurons of the dorsal raphe nucleus aresaid to develop in normal numbers84. Nkx2.2 isexpressed throughout the ventral hindbrain and spinalcord89, and must cooperate with other factors to direct a5-HT phenotype. Gata3 is expressed in the embryonicCNS in several populations of presumably postmitoticneurons90, among which the rostral and caudal 5-HTneurons feature prominently91. In the adult hindbrain,Gata3 expression becomes specific for the raphe nuclei,where the main neurotransmitter is 5-HT 91. LOF analy-sis of Gata3 shows that this factor is required for the cor-rect development of caudal 5-HT neurons. In Gata3–/–

CHIMERIC MICE, the caudal raphe nuclei appear disorga-nized and few mutant cells express 5-HT. Rostral groupsof 5-HT cells appear to be normal, although they alsoexpress Gata3 (REF. 91).

On the basis of its highly specific expression pattern,Pet1 has been proposed to have a role in 5-HT-neuronspecification85, a hypothesis that has been confirmed bythe recent analysis of Pet1-knockout mice, which showsthat Pet1 is required for the initial specification of the 5-HT phenotype (E. Deneris, personal communication).In addition, the genes that encode the 5-HT biosyn-thetic enzyme tryptophan hydroxylase and the plasma-membrane 5-HT transporter (SERT/Slc6a4) contain

CHIMERIC MICE

Mice that are genetically mosaic.They are produced mainly byinjecting embryonic stem (ES)cells of one genotype intoblastocysts of another genotype,resulting in mice whose cells arepartly derived from the ES cellsand partly from the hostblastocyst.

BRANCHIOMOTOR NEURONS

The neurons of the cranialnerves that control musclesderived from the branchialarches. In humans, they are thetrigeminal, the facial, theglossopharyngeal, the vagus andthe spinal accessory nerves.



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combinations of transcription factors to control not onlythe expression of shared properties, such as transmitter-synthesizing enzymes, but also the expression of genericneuronal properties. But there are obvious gaps in our knowledge: apart from transmitter-synthesizingenzymes, the downstream targets of transcription factorsthat are involved in transmitter choice are poorlydefined. The complexity and, in particular, the lineageselectivity of the programmes that are involved in neu-ron-subtype generation are significant challenges forfuture work. Beyond expanding our knowledge of neu-ronal fate determination, a better understanding of thepathways that control neurotransmitter choice will opennew ways of manipulating them. This, in turn, mightultimately find application in cell-replacement therapiesfor neurological diseases.

In invertebrates, several transcription factors seemto control both neurotransmitter identity and the ori-entation of axonal projections (for review, see REF. 97), asatisfying finding in view of the fact that the mode ofneurotransmission has to match receptor expression onthe target cells. In vertebrates, however, there is still littleevidence for this. For example, whether or not Nurr1 isrequired in DA neurons to generate the correct axonalprojection is a matter for debate60,66, and other types ofevidence are circumstantial. Both Phox2b 21,98 and Rnx51

are expressed in neuronal groups that are synapticallyinterconnected, and as they are expressed well beforesuch connections form, it is tempting to speculate thatthey have a role in the choice of synaptic partner.

Taken together, the available data are compatible witha model in which different neuronal types use distinct

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AcknowledgementsWe thank E. Deneris, J. Ericson, M. Howard and A. Pattyn for disclosing unpublished results, and J.-F. Brunet, F. Müller and M. Stanke for helpful comments on the manuscript. The work carried out in the authors’ laboratories was supported by grants toH.R. from the Deutsche Forschungsgemeinschaft, the Fonds derchemischen Industrie and the European Community (QLG3-CT-2000-0072), and by grants to C.G. from the European Community(QLGT-CT-2001-01467), the Association Française contre lesMyopathies and the Ministère de la Recherche.

Online links

DATABASESThe following terms in this article are linked online to:GenBank: http://www.ncbi.nlm.nih.gov/Genbank/BMP5 | FGF4 | FGF8 | Foggy | Smad5LocusLink: http://www.ncbi.nlm.nih.gov/LocusLink/Aldh1 | BMP2 | BMP4 | BMP7 | DAT | DBH | dHand | En1 | En2 |Gata3 | Gli2 | Lmx1b | Mash1 | NET | NF160 | Nkx2.2 | noggin |Nurr1 | Pet1 | Phox2a | Phox2b | Pitx3 | Ret | Rnx | RXR | SCG10 |SERT | Shh | TH | tryptophan hydroxylaseOMIM: http://www.ncbi.nlm.nih.gov/Omim/Parkinson’s disease

FURTHER INFORMATIONEncyclopedia of Life Sciences: http://www.els.net/adrenaline and noradrenaline | amine neurotransmitters | aminetransporters | dopamine | serotoninAccess to this interactive links box is free online.


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