DOI: 10.1007/s12210-008-0019-3Rendiconti Lincei 19, 271 – 290 (2008)

Carla Perrone-Capano · Floriana Volpicelli ·Umberto di Porzio

The Molecular Code Involved in Midbrain DopaminergicNeuron Development and Maintenance

Received: 15 February 2008 / Accepted: 19 May 2008 – © Springer-Verlag 2008

Abstract Midbrain dopaminergic neurons comprise a small group of cellsthat regulate important functions in the mammalian brain. Their degenerationor dysfunction is involved in many common neurological disorders such asParkinson’s disease, depression, schizophrenia, attention deficit hyperactivedisorder and drug addiction. Here we review recent studies that unravel themolecular events involved in various steps required for the embryonic de-velopment and maturation of midbrain dopaminergic neurons, with emphasison the role of epigenetic cues and transcription factors acting during the latedifferentiation steps.

Further knowledge on the extrinsic and intrinsic signals governing fate,development and survival of midbrain dopaminergic cells will increase theability to generate these neurons from stem cells and neural progenitors, andwill lead to significant progress in cell replacement therapies for Parkinson’sdisease.

Keywords Dopamine, Drug addiction, Parkinson’s disease, Stem cells, Tran-scription factors

Subject codes B18006, L18000, L25066

C. Perrone-Capano (B)Institute of Genetics and Biophysics “Adriano Buzzati Traverso”, CNR, Via Pietro Castellino 111,80131 Naples, Italy; Dept. of Biological Sciences, University of Naples “Federico II”, ItalyTel.: +39 081/6132362, Fax: +39 081/6132350, E-mail: perrone@igb.cnr.it

F. VolpicelliInstitute of Genetics and Biophysics “A. Buzzati Traverso”, CNR, Naples, Italy

U. di PorzioInstutute of Genetics and Biophysics “A Buzzati Traverso”, CRN, Naples, Italy

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1 Introduction

Dopamine (DA) is one of the most extensively studied neurotransmitters inthe brain because of its involvement in numerous physiological processes andin diverse psychiatric and neurological disorders. In the mammalian brain,DA systems play a central role in the control of movement, neuroendocrinehormone release, cognition, emotional balance and reward.

The first 50 years of DA research culminated in the award of the Nobel Prizein Physiology and Medicine 2000 to Arvid Carlsson and Paul Greengard, twoof the pioneers in this field. Carlsson showed that DA is a neurotransmitter inthe brain and mapped its regional distribution, and Greengard provided insightinto the cellular signaling mechanisms triggered by the activation of DA re-ceptors. Progress in biochemical and molecular pharmacology research duringthese years, coupled with the analysis of genetically modified mice lacking DAreceptors, has lead to important advances in our knowledge of dopamine neu-rotransmission and signal transduction pathways (Carlsson 2001; Greengard2001). At the biochemical level, DA is synthesized from tyrosine by tyrosinehydroxylase (TH), the rate-limiting enzyme in catecholamine biosynthesis,and is stored in the specific nerve-ending organelles called “dense core vesi-cles”. Following the arrival of the nerve impulse to the synaptic terminal, theneurotransmitter is released into the synaptic cleft. This process takes placefollowing a complex cascade of events that require the specific docking andfusion of the vesicles with the presynaptic membrane. The released DA canact on receptors that are distant from its release site (more than the width ofthe synaptic cleft), a modality of communication called volume or paracrinetransmission. The activation of postsynaptic dopaminergic receptors triggers acascade of intracellular signaling pathways that lead to the neuronal response.These receptors are classified in the D1R (D1 and D5) and D2R (D2, D3,D4) subfamilies and belong to the G-protein-coupled receptor superfamily.Neurotransmission is terminated mainly by a process named “high affinity up-take” by which the released DA is actively captured back into the presynapticDA endings through the plasma membrane glycoprotein dopamine transporter(DAT; Bannon 2005), Fig. 1.

DA neurons in the mammalian central nervous system(CNS) are an anatom-ically and functionally heterogeneous group of cells, localized in areas A8-A10of the ventral midbrain (Mb), areas A11–A15 of the diencephalon, area A16of the olfactory bulbs and area A17 of the retina (Bjorklund and Dunnet 2007),Fig. 2.

In this review we will focus on the most prominent dopaminergic cellgroups, localized in the Mb, given their importance in animal homeostasis andhuman pathology. In rodents, the total number of midbrain DA (mDA) neuronsis 20,000–40,000; this number increases to 160,000–300,000 in monkeys and400,000–600,000 in humans. mDA neurons can be anatomically divided into

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Fig. 1 Dopamine metabolism. Tyrosine is converted to L-3, 4-dihydroxyphenylalanine(DOPA) by tyrosine hydroxylase (TH) and DOPA is converted to dopamine (DA, star) byaromatic L- amino acid decarboxylase (AADC). DA is transported into the dense core vesiclesby the synaptic vesicle transporter VMAT2, a secondary active transporter driven by the pro-tonic pump H(+)-ATPase. When the vesicles fuse with the presynaptic plasma membrane, DAis released into the synaptic cleft and binds to the postsynaptic D1-type or D2-type receptors(which modulate cAMP level). DA action at the synapse is terminated predominantly by highaffinity uptake into the presynaptic terminal through the Na+/Cl−coupled dopamine transporter,DAT. Once back into the DA terminals, the neurotransmitter can be repackaged into vesicles orcatabolized by mitochondrial glial monoamine oxidase MAO into 3,4 dihydroxylphenylaceticacid (DOPAC) or by the catechol-O-methyl transferase enzyme (not shown).

three distinctive cell clusters: the retrorubral area (area A8), the substantianigra (SN; A9) and the ventral tegmental area (VTA; A10), Fig. 3. In rodents,the A8 neurons project to the SN, VTA and to striatal, limbic and corticalareas. Neurons originating in the SN project mainly to the dorsal striatum(corresponding to the caudate-putamen in primates, Fig. 4) and receive inner-vations from multiple structures in the diencephalon and telencephalon. Theyform the ascending nigrostriatal pathway, the first relay of the exptrapyrami-dal tract, which controls non-volitional muscular tone, balance and initiationof movement (Lynd-Balta and Haber 1994). The neurons of the VTA mainlyproject to the limbic system (nucleus accumbens, olfactory tubercle) and cor-

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Fig. 2 Anatomical localization of dopaminergic neuron cell groups in the developing ro-dent brain. The dopamine neurons in the mammalian brain are distributed in ten cell groups,from A8 to A17. The A17 dopaminergic neurons, represented by the retina amacrine interneu-rons, are not depicted in the figure. Abbreviations:Cx, cortex; Ob, olfactory bulb; Mb, midbrain;MHJ, mid-hindbrain junction; Hb, hindbrain.

Fig. 3 Tyrosine hydroxylase-immunostaining of mDA neurons and fibers in the adultmouse brain. (a) The ventral tegmental area (VTA) and the substantia nigra (SN) in the adultmouse ventral midbrain are visualized by anti-tyrosine hydroxylase antibodies (bar, 222 µm).A magnification of the substantia nigra is displayed in (b) (bar, 64 µm).

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Fig. 4 Diagram of the main midbrain dopaminergic circuits in the human brain. Midbraindopaminergic (mDA) neurons localized in the substantia nigra (SN) project to the dorsal stria-tum (corresponding to the caudate-putamen in primates), forming the nigrostriatal pathway.The neurons of the ventral tegmental area (VTA) mainly project to the Nucleus Accumbens(ventral striatum) and frontal cortex, forming the mesocorticolimbic pathway.

tical areas (prefrontal, cingulate and perirhinal cortex), and also innervate theseptum, amygdala and hippocampus. This mesocorticolimbic pathway regu-lates emotional behavior (attention and reward processes; Oades and Halliday1987; Wise 2004). Degeneration of nigrostriatal pathway in humans is associ-ated with Parkinson’s disease (PD; Thomas and Beal 2007), the syndrome de-scribed by the English neurologist in his “Essay on the shaking palsy” in 1817.By the use of specific toxins (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine,MPTP, or 6-hydroxydopamine, 6-OHDA) or lesions, as well as mutations in PDassociated genes (Orth and Tabrizi 2003), experimental PD has been elicited inanimal models, and used to assess mDA differentiation and cell-replacementstrategies, as described below. Dysregulation of the mesocorticolimbic path-way has been linked to mood disorders (Dailly et al. 2004), schizophrenia(Lang et al. 2007), attention deficit hyperactive disorder (ADHD; Swanson etal. 2007) and drug addiction (Nestler 2005), altering function of mDA neuronsin the reward circuits, especially during adolescence (Adriani et al. 2006).

2 Embryonic development

The establishment of functional mDA neurons from multipotent progenitorsis orchestrated by cell-intrinsic factors and environmental cues and occursthrough a complex multi-step process with a number of stages: early Mb pat-terning, induction of precursors, differentiation of postmitotic neurons, and

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Fig. 5 Diagram model of midbrain dopaminergic neuron development. The diagram sum-marizes the sequential stages and the molecules involved in rodent midbrain DA neuron em-bryonic development. The expression of transcription factors and secreted molecules involvedin the induction, specification, differentiation and maturation of the mDA phenotype are indi-cated, at various embryonic (E) ages. Possible interactions between this network of moleculesare indicated by arrows (see text).

functional maturation (Abeliovich and Hammond 2007; Ang 2006). Key reg-ulators of this developmental time course are the activation of transcriptionfactors (TFs) and the action of secreted molecules. Their role and subsequentaction in the various stages of rodent DA neuron development are summarizedin Fig. 5.

2.1 Early midbrain patterning and induction of DA precursors

The specification of the permissive region for DA neuron generation is a fun-damental event that initially occurs through the formation and positioning ofmolecular borders, such as the mid-hindbrain junction (MHJ, also known asisthmus), Fig. 2. Together with the floor plate, an embryonic structure liningthe ventral midline of the neural tube, the MHJ is a key signaling center inthe developing Mb. Genetic loss of function studies have identified a networkof TFs and signaling molecules that underlie early patterning events of theMb, Fig. 5. Initially, expression of the two homeodomain TFs, Otx2 in theMb and Gbx2 in the hindbrain, are required for positioning the MHJ, whichis a source of inductive molecules required for the specification of the mDAneuronal field. Otx2, as its family member Otx1, is the murine homolog of

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the Drosophila orthodenticle gene. Both Otxs play a remarkable role in spec-ification and regionalization of the forebrain and midbrain (Simeone et al.2002). Gbx2 regulates Mb and cerebellar development primarily through thesecreted fibroblast growth factor-8 (FGF8, see below). The identity of earlyproliferating DA progenitor cells in the mammalian brain is achieved throughthe action and gradient disposition of various diffusible factors. The secretedfactors FGF8, together with the other morphogen Sonic hedgehog (SHH), de-rived from the floor plate of the ventral midline, cooperate in the induction ofDA neurons early during gestation (before day E9.5 in mouse). In vitro studiessupport the notion that these secreted signals establish a functional epigeneticCartesian grid of positional information that specifies Mb progenitors (Hynesand Rosenthal 1999).

Gene expression studies and in vitro culture assays have implicated addi-tional secreted factors, such as Wnt1 (Schulte et al. 2005) and transforminggrowth factor-beta (TGFβ; Farkas et al. 2003), in the correct positioning of theMHJ, Fig. 5.

The morphogenetic signals activate cascades of recently identified TFsexpressed in Mb neuroblasts that are early markers of these progenitors still inthe cell division stage (Ang 2006). For instance, a morphogen is effective onlyin those cells that are competent to receive and respond to its signal. Thus, SHHact on those neural precursors that express its receptor system, composed oftwo transmembrane proteins, Patched (PTC) and Smoothened (SMO), whichelicit an intracellular response that involves the activation of other molecules,such as the zinc-finger transcription factor Gli1. The latter, thus, represents amarker of early mDA precursors (Zervas et al. 2004), Fig. 5.

A second marker characterizing early mDA precursors (E8.5 in the mouse)is the enzyme aldhehyde dehydrogenase-1 (AHD2) involved in retinoic acidmetabolism (Wallen et al. 1999). Indeed, retinoic acid is an additional moleculethat is required for the correct positioning of the MHJ (Avantaggiato et al.1996). Besides its role in CNS patterning, Otx2 is also an early marker of mDAprecursors, and is required for their generation. Interestingly, mDA neuronsare replaced by serotonergic neurons in the absence of Otx2 (Puelles et al.2004). This occurs because Otx2 normally represses the homeodomain proteinNkx2.2 that is in turn a negative regulator of mDA differentiation (Prakashand Wurst 2006; Puelles et al. 2004). Otx2 also regulates the expression of“proneural” genes that promote neuronal cell fate (proneural), such as Mash1and Neurogenin 2 (Ngn2), in dividing Mb progenitors (Vernay et al. 2005).

Lmx1a is a member of the LIM homeodomain family, named from threemembers, Lin-11, Isl-1 and Mec-3; LIM domain is defined by the presence ofone to three repeats of a 52-residue segment containing two adjacent zinc fin-gers separated by a two residue linker. Lmx1a expression is induced by SHH individing Mb precursors (E9 in the mouse). Interestingly, forced misexpression

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of Lmx1a promotes ectopic DA neuron generation in the anterior ventral Mb,but not in the dorsal Mb, while its elimination by RNA interference results inloss of DA neurons (Andersson et al. 2006a). Lmx1a in turn activates Msx1,which induces Ngn2, Fig. 5. The latter is required for the normal generationand maturation of mDA neurons, since their number is reduced in Ngn2-nullmutant mice (Andersson et al. 2006b). Importantly, Msx1 is neither neces-sary nor sufficient for mDA generation, indicating that other still unknownmolecules must function downstream of Lmx1a.

Lmx1b, an other member of the LIM homeodomain family, is expressedbroadly in early development at the MHJ, and is subsequently expressed specif-ically in mDA neurons. Studies in Lmx1b-null mice show that it is an essentialfactor for mDA postmitotic differentiation (Smidt et al. 2000). Lmx1b is notable to compensate for the loss of Lmx1a in the specification of DA neurons andis much less efficient than Lmx1a in promoting mDA neuron differentiationfrom embryonic stem cells (ES; Andersson et al. 2006a). These observationssupport the notion that Lmx1a may be involved in the early specification steps,while Lmx1b is required for later differentiation events in the DA lineage.

2.2 Differentiation of postmitotic DA neurons

Recent studies indicate that mDA neuroblasts, generated near the MHJ, arederived from the medial floor plate region (Kittappa et al. 2007). As theseprecursors exit from the cell cycle, they radially migrate from the ventricu-lar surface to their final position in the ventral Mb. Birthdating studies using3H-thymidine incorporation show that DA progenitors exit the cell cycle andgenerate postmitotic immature mDA neurons between E9.5 and E13.5 in ro-dents (Bayer et al. 1995). Immature mDA neurons then further differentiateinto mature DA neurons that express TH, in addition to the other markers men-tioned above. The first TH+ cells and fibers have been detected close to theventricular ependymal layer, suggesting that DA differentiation can occur inearly radially migrating postmitotic mDA precursors (di Porzio et al. 1990),Fig. 6.

Several key transcription factors, such as Engrailed (En) 1 and 2, Nurr1,Pitx3 and Lmx1b are involved in the development of postmitotic mDA neu-rons, Fig. 5. However, none of these TFs appears by themselves sufficient tospecify all aspects of mature mDA phenotype, suggesting that they must actin a combinatorial manner.

The homeodomain TFs En1 and 2 are expressed in mDA neurons from earlydevelopment to adulthood (Simon et al. 2001). They are cell-autonomouslyrequired to prevent apoptosis of these neurons towards the end of embryonicdevelopment, suggesting a key role for both genes in the regulation of mDA

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Fig. 6 Radial migration of TH-positive cells during embryonic midbrain development. (a)TH+ cells in the ventral midbrain of E13 mouse embryos (bar, 100 µm). (b) A magnificationof the inset in (a) shows many TH+ cells with putative neurites that appear to be still migrating(bar, 40 µm).

neuron survival and death, a function that may endure also during postnataldevelopment and maintenance (Alberi et al. 2004).

The TF Nurr1 is a nuclear receptor of the steroid/thyroid hormone receptorsuperfamily expressed in postmitotic mDA precursors at approximately E10.5in rodents, just one day before TH expression (Jankovic et al. 2005; Volpicelliet al. 2004a). It is essential for the development of mDA neurons since Nurr1-deficient animals show agenesis of TH+ cells in the Mb; however, other earlymarkers of mDA precursors (Pitx3, Lmx1b, En1 and 2, Aldh2) are normallyexpressed in Nurr1 null mice, suggesting that mDA cell fate is not completelyabolished in the absence of Nurr1, although the surviving precursors die atlater stages (E15-16; Jankovic et al. 2005).

Nurr1 regulates the expression of a number of key DA genes, includingTH, L-aromatic amino acid decarboxylase (AADC), DAT, VMAT-2, cyclin-

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dependent kinase inhibitor p57Kip2, neuropilin-1, as well as the genes encod-ing the glial derived neurotrophic factor (GDNF) receptors Ret and GFRα1(Jankovick et al. 2005; Hermanson et al. 2006). The latter finding suggeststhat Nurr1 might be involved in the responsivity of mDA neurons to trophicfactors. This hypothesis received further support by recent data obtained inour laboratory showing that the gene encoding the neurotrophin brain derivedneurotrophic factor (BDNF) is a novel Nurr1 target in rat Mb neurons in vitro(Volpicelli et al. 2007). Interestingly, BDNF exerts its trophic effects on theseneurons in vivo and in vitro (Hyman et al. 1991) and has a neuroprotectiveeffect against neurotoxicity or axotomy (Spina et al. 1992; Frim et al. 1994;Hagg 1998). Nurr1 plays an important role in mDA neuron function also inadult life. It appears to be an important regulator of DA levels in the adult(Zetterstrom et al. 1997) and its down-regulation can increase the vulnerabil-ity of mDA neurons to the selective dopaminergic neurotoxin MPTP (Le et al.1999). Defects in the Nurr1 gene or its altered expression have been found inmDA pathologies: Nurr1 is down-regulated in the Mb after chronic cocainetreatment in humans and rats (Bannon et al. 2004; Leo et al. 2007); geneticalterations at the Nurr1 locus have been associated with ADHD (Smith et al.2005) and with PD (Jankovic et al. 2005), although Nurr1 variability is un-likely to play a major role in PD patients (Nichols et al. 2004). In addition,missense mutations have been found in cases of schizophrenia and bipolardisorder (Buervenich et al. 2000).

A second TF that plays an important role in the development and functionof vertebrate mDA neurons is the bicoid-related homeodomain Pitx3 expressedalmost exclusively in mDA cells, starting one day after Nurr1 expression. Lossof Pitx3 has been observed in Lmx1b mutant mice (Smidt et al. 2000). Nat-urally occurring loss of Pitx3 expression has been observed in aphakia mice.These mutants show loss of the SN subset of mDA neurons and an overallreduced locomotor activity, but not alteration in VTA neurons (Smidt and Bur-bach, 2007). Strikingly, a similar pattern of mDA deficiency is observed in PDpatients. Data from Pitx3 null mice strongly suggest that distinct developmen-tal programs for SN and VTA DA neurons exist. In the absence of Pitx3, theSN precursors, which normally express Pitx3 prior to TH, fail to induce TH; incontrast the VTA cells, which normally express Pitx3 and TH at the same time,express TH even in the absence of Pitx3 (Maxwell et al. 2005). From thesedata emerge a new concept that mDA neurons are not equal, and therefore thatthey may be also functionally different (see below).

In vitro overexpression studies of single TFs involved in mDA developmentsupport the notion that none of these genes are self-sufficient to promote theappropriate maturation of the DA phenotype. Although Nurr1 transfection inneuronal cell lines, hippocampal or midbrain neurons and embryonic stemcells (Wagner et al. 1999; Sakurada et al. 1999; Kim et al. 2003; Sonntag

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et al. 2004) promotes the differentiation of the DA phenotype, only a subsetof mDA markers is induced by Nurr1 alone (Kim et al. 2003). On the otherhand, overexpression of Pitx3 in neuronal progenitors or in embryonic stemcells appears to have no effect or to induce only a limited number of mDAmarkers (Sakurada et al. 1999; Chung et al. 2005). Thus, Nurr1 and Pitx3seem to control different aspects and pathways of mDA neuron differentiationand, probably, survival (Simeone 2005; Chung et al. 2005). Consistent withthe notion that these TFs can function in a combinatorial mode to promotemDA maturation, it has been shown that co-overexpression of Nurr1 and Pitx3induces late mDA neuron maturation markers such as DAT. In contrast, theearlier phenotypic markers TH and AHD2 appear induced by Nurr1 or Pitx3alone (Martinat et al. 2006).

Recent data demonstrate novel roles for the forkhead/winged helix tran-scription factors Foxa1 and Foxa2 in the specification and differentiation ofmDA neurons. By analyzing the phenotype of Foxa1 and Foxa2 single- anddouble-mutant mouse embryos it has been shown that these TFs regulate multi-ple phases of mDA neuron development in a dosage-dependent manner. Duringspecification, Foxa1 and Foxa2 regulate the extent of neurogenesis in mDAprogenitors by positively regulating Ngn2 expression. Subsequently, Foxa1and Foxa2 regulate the expression of Nurr1 and En1 in immature neurons andthe expression of AADC and TH in more mature neurons during the differ-entiation of mDA neurons (Ferri et al. 2007). Interestingly, late in life, Foxa2heterozygous mice spontaneously develop significant motor problems associ-ated with a significant loss of mDA neurons preferentially in the SN, similarto the events observed in PD patients (Kittappa et al. 2007).

2.3 Maturation and survival of the dopaminergic phenotype

Following the early commitment of DA neuroblasts, the activation of genesinvolved in DA neurotransmission takes place in a precise temporal sequenceduring embryonic development under the control of various environmentalcues.

By monitoring the expression of a number of “dopaminergic” genes andtheir function during embryonic development of the Mb, we have shownthat DA synthesis, storage and high-affinity uptake develop asynchronously(Perrone-Capano and di Porzio 1996, 2000). Following TH gene expression,the gene codifying for VMAT-2 is readily expressed. These events take placeseveral days before the establishment of nigrostriatal connections (which inrodents occurs at E15–16). They suggest that VMAT-2 could play a protectiverole before the establishment of functional DA neurotransmission, since itsactivity is necessary to accumulate DA into presynaptic vesicles and thus clear

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it from the cytoplasm. Thus, VMAT-2 limits DA oxidation (since dense corevesicle pH is acidic) and its neurotoxic effects.

Interactions of presynaptic mDA neurons with striatal target neurons playan important role, at least in vitro, in promoting key aspects of DA neuro-transmission, namely DA synthesis and uptake, mediated by TH and DAT,respectively (Prochiantz et al. 1979; di Porzio et al. 1980; Perrone-Capano anddi Porzio 1996).

In contrast to the early appearance of endogenous DA levels in the Mb,specific high-affinity DA uptake and DAT transcripts are found in rodents onlyat E15–16, concomitantly with the arrival of DA fibers to the striatum (Fiszmanet al. 1991). Accordingly, in mDA primary cultures the levels of DAT genetranscripts and the number of uptake sites are selectively increased when co-cultured with striatal cells. Thus, functional maturation of mDA neurons isconditioned by a specific cellular environment and promoted by specific anddirect cell interactions with striatal target tissue (Perrone-Capano and di Porzio2000).

Interestingly, recent data support the hypothesis that correct target recog-nition may improve maintenance and survival of DA neurons. It has beenproposed that ephrin signaling (involved in axonal guidance) can regulate thecorrect targeting of nigrostriatal neurons (Yue et al. 1999). Indeed applicationof ephrin-B2 (expressed in vivo by the developing striatum) to Mb culturesresults in Nurr1 upregulation (Calò et al. 2005). Other guidance cues can con-tribute to regulate correct mDA projections, such as netrin-1 and slit-2, whichact in concert to regulate and direct mDA neurite outgrowth (reviewed by Smidtand Burbach 2007).

It is not clear how higher TH+ neuron survival in vitro is elicited by the tar-get striatal neurons (Prochiantz et al. 1979; di Porzio et al. 1980). It is possiblethat target-derived trophic factors, together with autocrine and paracrine se-creted molecules promote arborization, maturation and survival/maintenanceof postmitotic DA neurons. Putative “dopaminotrophic” factors include TGF-β, GDNF, BDNF, and the recently identified conserved dopamine neurotrophicfactor (CDNF; Krieglstein 2004; Lindholm et al. 2007). GDNF and BDNFhave been shown to exert a protective role on mDA neurons following a num-ber of experimental lesions. They also promote survival and differentiation ofmDA neurons in culture (Hyman et al. 1991; Feng et al. 1999, Consales etal. 2007). That Bdnf expression in mDA neurons is regulated by Nurr1 sug-gests the existence of a Nurr1-BDNF loop protecting mDA neurons duringdevelopment and, possibly, in the adult (Volpicelli et al. 2007). Bdnf null miceare born with the normal asset of mDA neurons but have fewer dopaminergicdendrites in the substantia nigra, a defect evident only in the postnatal period,suggesting that BDNF plays a role in phenotypic maturation (Baquet et al.2005).

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Fig. 7 Hypothetical scheme of cues involved in the development and maintenance of mDAneurons. Specific transcription factors are required for mDA neuron development and through-out maturation and adult life. Among other agents, neuronal activity can modulate the expressionof transcription factors, such as Nurr1. The latter can in turn stimulate Bdnf gene expression indopaminergic neurons (Volpicelli et al. 2007), thus establishing a trophic loop. Other trophicfactors are also involved in mDA neuron maintenance and function, such as GDNF, TGFβ andothers. This equilibrium can be altered by the actions of toxic agents (such as MPTP, 6-OHDAand oxidative stress), addictive drugs (such as psychostimulants like cocaine or amphetamines),genes (such as those involved in PD) and developmental disorders (schizophrenia, ADHD) thataffect dopaminergic neurons.

Lack or limited production of “dopaminotrophic” factors may play an im-portant role in the pathogenesis, as well as in the potential treatment, of DA-associated neurological diseases, Fig. 7.

3 In vitro generation of dopaminergic neurons

In the last decade, the use of a number of cytokines and growth factors, and thediscovery of stem cells in the embryonic and adult CNS has been exploited togenerate DA neurons, expand DA neuron precursor populations and increaseDA phenotype in vitro. These new approaches offer a simplified model for thein vitro analysis of intrinsic and extrinsic cues involved in mDA cell fate.

The totipotent ES cells are clonal cell lines derived from the inner cell massof the pre-implantation embryonic blastula. If injected into the blastocyst of ahost embryo, ES cells can give rise to all cell lineages, including germ cells.In vitro ES cells are capable of unlimited self-renewal and can differentiate

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into all cell types of an organism. When correctly directed or engineered, theycan produce neural stem cells and give rise to differentiated neurons. Neuralstem cells can also be obtained from “neurospheres”, specific neural precur-sor aggregates that are formed in defined culture conditions in suspension inthe presence of epidermal and fibroblast growth factors 2 (EGF and FGF2),generated from embryonic or adult CNS. When dissociated and attached toa substrate, the neurospheres can differentiate and, under appropriate stimuli,can give rise to all CNS cellular phenotypes (neurons, astrocytes, oligodendro-cytes) or to specific neural cell populations.Several studies have shown that EScultures and neurosphere-derived neural progenitors can generate TH+ cellsexpressing mDA phenotypic markers and that the developmental program invitro appears to recapitulate the temporal course of normal mDA development(Andersson et al. 2006a; Barberi et al. 2003; Kim et al. 2002; Martinat et al.2004; Sonntag et al. 2004). mDA neurons have also been generated in vitrofrom embryonic tissues and neuroblasts expanded through the mitotic activityof FGF2. Whatever the origin of the precursors, soluble factors implicated inthe specification of mDA neurons in vivo (that is morphogens, trophic factorsas well as ascorbic acid) further enhance differentiation and survival of theDA phenotype in vitro (Krieglstein 2004; Volpicelli et al. 2004b). Similarly,overexpression of cell-intrinsic transcription factors such as Nurr1 (Kim et al.2002; Chung et al. 2002; Sonntag et al. 2004) and Pitx3 (Chung et al. 2005)appear to drive mDA phenotype (see above). Overexpression of Lmx1a in EScells also promotes mDA differentiation up to 80%, but only in the presenceof SHH (Andersson et al. 2006a).

Increased dopaminergic differentiation from ES cells can also be achievedby loss of function. For instance, DA neurons were obtained in a large numberfrom ES mouse mutant lacking the EGF-CFC protein Cripto, a key player inthe signaling pathways controlling neural induction (Parish et al. 2005).

These strategies have raised the possibility of generating unlimited numbersof DA neurons to be grafted in animal models for functional reconstitution ofthe nigrostriatal pathway with the aim of learning how to transplant theseneurons into patients and increase their survival (Lindvall et al. 2004; Winkleret al. 2005).

Regarding this, an open question remains unanswered, whether DA neuronscan be functionally used in nigrostriatal grafts independently of their anatom-ical and functional origin. For instance, only embryonic DA neurons obtainedfrom Mb, but not from other CNS regions, are able to restore function whengrafted into mouse hosts with a MPTP lesioned nigrostriatal system (Zuddaset al. 1991). These results can be explained as a consequence of distinct DAdifferentiation programs at different CNS positions. In support of this hypoth-esis, the fate of DA forebrain neurons appears to be governed by the expressionof TFs different from those involved in the specification of mDA neurons (An-

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drews et al. 2003; Ohyama et al. 2005). Limited survival of transplanted cells,observed in many studies, may also reflect the existence of different molecularcodes for various DA neuron subtypes which determine limited or ineffectivedevelopment of the grafted cells. Similarly, it has been shown that mDA neu-rons grafted into non-target brain areas do not develop and show little survival,indicating that grafted mDA neuron, in vivo may need, as in vitro, still uniden-tified extrinsic support by the host adult brain (Zuddas et al. 1991; Martinatet al. 2006). Recent data also show that grafted SN and VTA neurons differin their axonal projections in the host adult forebrain, suggesting that mDAneuronal subtypes display distinct responses to axon guidance cues and targetrecognition mechanisms (Thompson et al. 2005).

These findings suggest that in the nigrostriatal system, proper matchingbetween target and presynaptic elements is required not only during embryo-genesis for the maturation of developing DA neurons, but also when DA neu-rons are grafted into hosts with a lesioned nigrostriatal system, where they canachieve functional restoration.

To date, the hope for a cell replacement strategy for PD patients needs betterunderstanding of the molecular and celluar biological cues directing the differ-entiation program of uncommitted stem cells or precursors into functionallymature mDA neurons.

4 Concluding remarks

In the last years, the development of the mDA system has been studied exten-sively. It shows how complex and varied neural functions subserved by a smallgroup of neurons can be achieved during embryonic development throughthe correct interplay of genetic and epigenetic cues. New tools such as genetargeted-disruption (knock-out mice, mRNA silencing approaches), cell linesas well as primary cell cultures obtained from CNS and neural stem cellsoverexpressing selected genes are currently being used to unravel the molec-ular mechanisms underlying DA functions. Studies analyzing the functions ofdopaminergic TFs with a key role in various steps of DA neuron developmenthave not only increased our knowledge on how DA neurons are generated invivo, but also allows the development of new strategies for engineering DAneurons in vitro.

Nevertheless, many questions related to the transcriptional regulation ofmDA differentiation remain to be answered. Namely, what is the interaction,if any, of the distinct mDA TFs described above? What are their downstreamtarget and upstream activators? Further knowledge of the key regulators andhierarchical network involved in mDA neuron differentiation will be highlyuseful for pharmacological manipulations of DA neurons that can be used in

286 C. Perrone-Capano, F. Volpicelli, U. di Porzio

future clinical application and in regenerative medicine to aid or solve one ofthe most widespread neurodegenerative conditions that affects man.

Acknowledgements. This work was supported by COFIN MURST/MIUR 2007, Italy. We aregrateful to Dr. Gian Carlo Bellenchi for critical reading of the manuscript.

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