Molecular mechanisms of dopaminergic subset specification: fundamental aspects and clinical perspectives

Molecular mechanisms of dopaminergic subset specification: fundamental

aspects and clinical perspectives

Veenvliet JV and Smidt MP*

Swammerdam Institute for Life Sciences, University of Amsterdam, Amsterdam, The Netherlands

*, author for correspondence ([email protected])

as published in:

Cellular and Molecular Life Sciences

The original publication is available at www.springerlink.com

Publisher's version/PDF is available at:

http://link.springer.com/article/10.1007%2Fs00018-014-1681-5

Please cite as:

Veenvliet, J. V. and Smidt, M. P. (2014). Molecular mechanisms of dopaminergic subset

specification: fundamental aspects and clinical perspectives. Cell. Mol. Life Sci. 2014 Jul 27. [Epub

ahead of print]

Abstract

Dopaminergic (DA) neurons in the ventral mesodiencephalon control locomotion and emotion and

are affected in psychiatric and neurodegenerative diseases, such as Parkinson's Disease (PD). A

clinical hallmark of PD is the specific degeneration of DA neurons located within the Substantia

Nigra (SNc), whereas neurons in the Ventral Tegmental Area (VTA) remain unaffected. Recent

advances have highlighted that the selective vulnerability of the SNc may originate in subset

specific molecular programming during DA neuron development, and significantly increased our

understanding of the molecular code that drives specific SNc development. We here present an up-

to-date overview of molecular mechanisms that direct DA subset specification, integrating our

current knowledge about subset specific roles of transcription factors, signaling pathways and

morphogenes. We discuss strategies to further unravel subset specific gene regulatory networks, and

the clinical promise of fundamental knowledge about subset specification of DA neurons, with

regards to cell replacement therapy and cell-type specific vulnerability in PD.

Introduction

Mesodiencephalic dopaminergic (mdDA) neurons in the ventral mesodiencephalon are involved in

the control of voluntary movement and regulation of emotion, and dysfunction is associated with

several psychiatric and neurodegenerative diseases, such as obesity, addiction, schizophrenia and

Parkinson's Disease (PD). A pathological hallmark of PD is the specific degeneration of neurons in

the Substantia Nigra pars compacta (SNc), whereas neurons in the Ventral Tegmental Area (VTA)

remain unaffected [1, 2]. Despite the realization that this selective vulnerability of a specific DA

cell group implies the existence of different subpopulations of mdDA neurons, it was only recently

postulated that this specific vulnerability may be explained by specific molecular programming of

the SNc. This increasingly popular hypothesis has lead to extensive research into the molecular

code of developing mdDA subsets in the last decade and was initially reviewed by Smidt et al. [3,

4]. Ever since, major progress has been made in our understanding of developing mdDA subsets.

Here, we review the molecular circuitry that defines mdDA subsets and present an up-to-date model

of the molecular mechanisms that direct mdDA subset specification, integrating our current

knowledge about subset specific roles of transcription factors, signaling molecules and

morphogenes.

Early specification and patterning of the mdDA domain

In the developing brain, mdDA neurons are located in the neural tube area that later becomes the

mesencephalon (midbrain) and diencephalon (forebrain). The early specification of the permissive

region for mdDA neuron generation requires concerted action of signaling factors such as Shh

(sonic hedgehog), Fgf8 (fibroblast growth factor 8), Tgf-b (transforming growth factor beta), Wnt1

(wingless-type MMTV integration site 1) and (Wnt5a wingless-type MMTV integration site 1) on the

one hand, and several key transcription factors (TFs) including Nurr1 (nuclear receptor related-1

protein), Pitx3 (paired-like homeodomain transcription factor 1), En1 (engrailed-1), En2

(engrailed-2), Otx2 (orthodenticle homeobox 2), Lmx1a (lim homeobox transcription factor 1a),

Lmx1b (lim homeobox transcription factor 1b), Foxa1 (forkhead box protein a1) and Foxa2

(forkhead box protein a2) on the other hand [3–8] (Figure 1).

The mid-hindbrain border (MHB), or isthmus, is a critical signaling center in the establishment of

the mdDA neuronal field, since it produces Fgf8, that acts together with the ventralizing factor Shh

to specify the initial mdDA neuronal field [9]. Rostral to the isthmus, in the mdDA neuronal field,

Otx2 is expressed, whereas caudal to the isthmus, in the presumptive serotonergic field,

gastrulation brain homeobox 2 (Gbx2) is expressed, and manipulation of these factors affects the

size of the early mdDA domain [3, 10–13]. Multiple other signaling molecules are involved in the

early establishment of the mdDA neuronal field. Transforming growth factor beta (Tgf-b) induces

early Shh signaling, and is therefore required for the induction of the ventral mdDA neuronal field

[14, 15]. In human pluripotent stem cells, Tgf-b pathway mediators have critical upstream roles as

regulators of Wnt1, Lmx1a and Foxa2, and in complex interplay with Shh and Fgf8, promote mdDA

neuron differentiation [16]. Wnt1, as well as Wnt5a, are critical factors in the establishment of the

mid-hindbrain region. Among other functions, Wnt1 induces expression of engrailed genes,

important in multiple stages of mdDA development [6, 17, 18]. The expression of Wnt1 and Fgf8 in

the isthmic organizer (IsO) is in turn controlled by Lmx1b [19, 20]. Recently, an Lmx1b-miR135a2

regulatory circuit was identified, that modulates Wnt signaling and determines the size of the mdDA

progenitor pool [21]. Also, the first wave of retinoic acid (RA) signaling in the midbrain is

important for positioning of the MHB [22, 23].

Once the permissive mdDA region has been established, multiple TFs are involved in the

establishment of the size and specification of the mdDA progenitor domain. Neurogenin 2 (Ngn2) is

important for the proper specification of mdDA precursors. Ngn2 activates Sox2+ progenitors that

later develop into Nurr1+ mdDA neurons [24] and suppresses Nurr1-induced gene transcription in

mdDA neurons. Achaete-schute homologue 1 (Ascl1/Mash1) is involved in the maintenance of

progenitor populations in the developing mdDA area, and Mash1-null mice display a reduced

neurogenesis in the mdDA system [25, 26]. The role of Mash1 in mdDA neuron development may

however be rather permissive than instructive, and it is well possible that there is functional

redundancy between Mash1 and Ngn2 [24] (Figure 1).

Transcription Factors involved in mdDA subset specification

After the early establishment of the permissive mdDA region and expansion of the mdDA

progenitor domain, multiple TFs and signaling molecules act together to provide cellular diversity

and subset specification in the developing mdDA system. From rostral to caudal, transverse

domains of the Central Nervous System (CNS) are designated telencephalon, rostral diencephalon,

prosomere 3-1, the midbrain and hindbrain, of which prosomere 1/2/3 and the midbrain comprises

the mdDA neuronal field [3, 27–30].

The roles of key mdDA TFs in mdDA neuron development and maintenance have been extensively

reviewed in two excellent recent reviews [7, 31]. In this review, we focus on subset specific roles of

TFs most prominently associated with mdDA subset specification: Pitx3, En1, Otx2 and Lmx1a/b

(Figure 1-3).

Pitx3

Pitx3 is expressed in the mouse ventral mdDA area from E11.5 onwards and a terminal selector

gene for mdDA neurons [32]. The spatiotemperal expression pattern of Pitx3 suggests that Pitx3 is

critical for terminal differentiation of mdDA neurons, and not involved in earlier DA neuron

development [33]. Indeed, many studies have demonstrated critical involvement of Pitx3 in the

terminal differentiation and survival of an mdDA subset. In the absence of Pitx3, specific loss of

mdDA neurons in the SNc is observed with concomitant loss of nigrostriatal projections to the

dorsal striatum, whereas the neighbouring neurons of the VTA are relatively unaffected. This was

initially demonstrated in aphakia mice, a natural Pitx3-mutant that lacks Pitx3 transcript due to

mutations in its promoter [34, 35], and later confirmed in the transgenic Pitx3 knockout, gfp knock-

in mouse model [33, 36–38]. These early observations indicated a paradoxical role for Pitx3 in the

mdDA system: whereas Pitx3 is expressed in most if not all mdDA neurons during late

differentiation, in the absence of Pitx3 only a small subset is affected, and this subset comprises

neurons destined to form the SNc. In Pitx3-mutant embryos, a rostrolateral subpopulation of mdDA

neurons, that ultimately forms the SNc, is halted in its terminal differentiation, indicated by the lack

of Th expression (Figure 2D,F; Figure 3) [33, 39–41]. In this rostrolateral subset, Pitx3 induces the

expression of Ahd2/Aldh1a1 (aldehyde dehydrogenase 1 family, member A1), most likely by direct

activation, since Pitx3 binds the Ahd2 promoter [40]. Ahd2 is selectively expressed in the mdDA

subset that is critically dependent on Pitx3 (Figure 2E,K, 3A, 4A-D). Ahd2 encodes an aldehyde

dehydrogenase enzyme, that, in addtion to the detoxification of DOPAL

(dihydroxyphenylacetaldehyde) through the conversion to DOPAC (dihydroxyphenylacetic acid)

[42], converts retinaldehyde to Retinoic Acid (RA), and therefore serves as a potent generator of RA

in the developing mdDA system [40, 42]. Functional relevance of Pitx3/Ahd2-mediated RA

signaling in a developmental mdDA subset has been demonstrated in vivo and ex vivo (discussed

below), and RA-dependent and -independent gene-regulatory pathways of Pitx3 in mdDA neurons

have been identified [40, 41]. At developmental stage E14.5, rostrolateral mdDA neurons display a

specific dependence on RA to induce Th (tyrosine hydroxylase), and most likely D2R (Drd2;

dopamine 2 receptor) expression (mainly expressed in the rostrolateral area at this stage).

Expression of other key genes in DA metabolism, Dat (Slc6a3; dopamine transporter) and Vmat2

(Slc18a2; vesicular monoamine transporter 2), is not affected by RA (Figure 3A, 4A-D).

In addition to the subset specific induction of Ahd2, other Pitx3-induced molecular programs may

explain the subset specific vulnerability observed in Pitx3-mutant mice. Pitx3 was recently

demonstrated to activate the expression of brain-derived neurotrophic factor (BDNF) in

rostrolateral SNc mdDA neurons specifically. In these experiments, it was elegantly shown that loss

of BDNF expression correlates with the loss of SNc neurons in Pitx3-deficient mice [43]. Treatment

of primary cultures of Pitx3-deficient mdDA neurons with BDNF augmented their survival,

suggesting functional relevance for Pitx3-induced BDNF signaling in the development and

maintenance of mdDA neurons of the SNc subset (Figure 3A) [43]. Notably, specific dependency

of SNc neurons on BDNF for their survival has been demonstrated in many studies (as discussed

below). The same study reported that Pitx3 expression is induced by glial derived neurotrophic

factor (GDNF), another growth-factor implicated in subset specific vulnerability of mdDA neurons

(see below) (Figure 3).

Pitx3 regulates the conserved micro-RNA miR-133b, that in turn suppresses Pitx3 expression [44].

Although the exact role of this Pitx3-miR-133b negative feedback loop is difficult to interpret, and

mdDA development appears to be normal in miR-133b null mice [45], this miRNA may have more

subtle roles in subset-specification of mdDA neurons, especially given its recently established role

in adipocyte specification [46, 47].

Regarding the function of Pitx3 in mdDA neurons, at least three important questions have remained

unanswered: 1) is Pitx3-itself directly responsible for survival of mdDA neurons (for example, by

activation of pro-survival and/or anti-apoptotic pathways), or is SNc neurodegeneration in Pitx3-

deficient mice solely the result of mis-specification of SNc neurons? 2) at what stage do SNc

neurons exactly start degenerating in Pitx3-deficient mice?, and 3) does dependency of SNc neurons

on Pitx3 extend into adulthood? Regarding the latter, it would be of utmost interest to conditionally

remove Pitx3 at postnatal and/or adult stages in future studies.

Interplay of Pitx3 and Nurr1

Nurr1 regulates Pitx3 expression in vitro [48], but Pitx3 expression appears unaffected in Nurr1-

null mutants. Recent studies have unambiguously demonstrated that Pitx3 and Nurr1 coordinately

regulate mdDA neuron specification. Pitx3 potentiates Nurr1 in terminal differentiation of mdDA

neurons, most likely by releasing SMRT/HDAC-mediated repression of Nurr1 target genes [49–53].

Whereas most studied target genes were shown to be activated by both Pitx3 and Nurr1 (such as

Vmat2 and Dat), at least two Nurr1 target genes have been described that are downregulated in the

absence of Nurr1, but upregulated and ectopically expressed in the absence of Pitx3:

cholecystokinin (Cck) and delta-like homologue 1 (Dlk1). In contrast to the genes activated by

Nurr1 and Pitx3 in concert, Cck and Dlk1 expression is confined to the caudal mdDA area in wild-

type embryos at the terminal differentiation stage. The molecular mechanisms behind such subset

specific interplay of Nurr1 and Pitx3 remain to be determined (Figure 2E, 3A,B).

En1

During early development, En1 and its paralogue En2 are critical for the proper patterning of the

mdDA area. Initially, En1 and En2 are expressed at the MHB, and crucial for the formation of the

isthmus [54, 55]. Although En1 was initially described to be broadly expressed in mdDA neurons

starting at their early development and continuously into adulthood [56, 57], recent in-depth spatial

analysis performed in our lab justifies a refinement of this model. In situ hybridization analysis of

the E14.5 mdDA area clearly indicated substantially lower En1 expression in rostrolateral mdDA

neurons (as compared to caudal mdDA neurons) (Figure 2A,B). These findings have been

confirmed by qPCR in purified mdDA neurons, that indicate 2-3 fold higher levels of En1 in caudal

mdDA neurons (JV Veenvliet, unpublished results). In line with subset specific levels and roles of

En1 is the recent observation that expression of En1 in more rostral diencephalic DA progenitors is

lost early, and then only weakly reactivated in precursors [58]. Moreover, this study suggested that a

small diencephalic Th+ population is generated independent of En1. However, conclusions were

drawn from analyses at early timepoints (E10.5-E12.5) and not based on analysis of En1-null

embryos, but on phenotypical consequences of En1 ablation in Fgfr1/2 conditional compound

mutants, and should therefore be treated with caution.

Whereas En1 and En2 were initially reported to be critical for the development and survival of all

mdDA neurons in a dose-dependent manner [56, 59], recent research has indicated more refined and

subset specific roles of En1 (Figure 2C,G, 3A,B). Initial studies were performed in En1 and En2

double mutants, and indicated massive loss of mdDA neurons by E14, due to caspase-dependent

apoptosis [56, 60]. Simon et al. [60] analyzed various allelic combinations (wild-type,

En1-/-;En2-/-, En1-/-, and En1-/-;En2+/-) and concluded that mice mull for either En1 or En2 had

no apparent reductions in mdDA neurons of the SNc and VTA subtype and may therefore

compensate for each others loss partially or even entirely. It was reported that, at P0, distribution

and density of mdDA neurons of the SNc were similar in En1-null mice as compared to wild-type,

and that the only detectable difference was a more loose arrangement of VTA neurons. In contrast,

later research indicated that En1 is critical for the maintenance of mdDA neurons, since En1-

heterozygous mice displayed progressive DA cell loss in both an En2-null and a wild-type

background [61, 62]. However, the exact fate of mdDA neurons in the absence of En1 remained

unclear in these studies, hampered by the perinatal lethality in En1-null mutant mice related to

cerebellar deletions [59]. We and others have circumvented this impediment by transferring the

original En1-null allele from the 129/Sv strain to the C57BL/6J background, thereby suppressing

the cerebellar phenotype and facilitating the analysis of En1 knock-out adult mice [63, 64]. Six-

week old En1-null mice have severe loss of both SNc and VTA neurons as assessed by the number

of Th+ neurons, whereas En1 heterozygous mice display an intermediate phenotype, indicating a

dose-dependent effect of En1 on the development and survival of mdDA neurons. Concomitantly,

Th+ fibers are lost in the both SNc projection areas (dorsal striatum), as well as VTA projection

areas (nucleus accumbens). In the same study, Nissl staining on adjacent sections revealed a clear

reduction of DA cell density in both SNc and VTA of En1-null mice, indicating that these neurons

were lost in the absence of En1 [64]. Similar to the Pitx3-knockout, where loss of SNc neurons in

adult is preceded by developmental programming defects in a rostrolateral subset, the absence of

En1 also leads to programming defects during mdDA neuron development. At the terminal

differentiation stage, expression of all genes of the DA gene battery (Th, Dat, Aadc (aromatic L-

amino acid decarboxylase), Vmat2, D2R) are affected in En1-null embryos (Figure 2G). Especially

Th and Dat displayed subset specific dependence on En1, since expression is severely

downregulated in rostrolateral mdDA neurons, but less affected in the caudal mdDA subpopulation

[64]. Importantly, unaffected expression of early postmitotic mdDA precursor marker Nurr1 and

mdDA progenitor marker Lmx1a was reported in En1-null embryos in the same study, suggesting

that the loss of DA neuron marker expression is a direct consequence of En1-driven programming

deficits, and not the result of cell death at this stage [64]. Notably, we have reported ectopic

expression of multiple mdDA markers in rhombomere 1 of En1-deficient embryos (Figure 2G).

Interplay of En1 and Pitx3

We have recently reported how extensive and subset specific crosstalk of En1 and Pitx3 may be

critically involved in DA subset specification (Figure 3). Initial evidence for functional crosstalk of

En1 and Pitx3 came from the observation that En1 expression is highly upregulated in Pitx3-null

embryos [41]. In turn, the expression of Pitx3 is ablated in a rostrolateral subset in En1-null

embryos [64], and the rostrolateral phenotype in En1-null embryos resembles that of Pitx3-deficient

embryos of the same stage (Figure 2C,D,F,G), suggesting that both En1 and Pitx3 are essential for

the induction of the rostrolateral phenotype. Thus, En1 and Pitx3 clearly influence each others

expression level, although it remains to be studied if this is mediated by direct or indirect effects.

The ablation of Ahd2 in both Pitx3- and En1-deficient embryos, and the downregulation of multiple

other factors involved in RA metabolism in En1-null embryos (Figure 2K) [64] point to RA

signaling as a convergence point of En1 and Pitx3 in rostrolateral DA subset specification.

Although En1 and Pitx3 have similar effects in the rostrolateral mdDA domain (i.e., induction of the

mdDA phenotype), reciprocal regulation of target genes has been observed in the caudal mdDA

domain using both genome-wide and in-depth expression analysis tools. The most striking example

of such a reciprocally regulated gene is Cck (Figure 3). In wild-type embryos, Cck expression is

restricted to the caudal mdDA subpopulation. In Pitx3-deficient embryos, Cck is upregulated and

expression is expanded into the rostrolateral region that, as a consequence of Pitx3 ablation, is

devoid of Th expression, suggesting that Pitx3 antagonizes Cck in a rostrolateral mdDA subset. In

sharp contrast, En1-null embryos completely lose Cck expression in the caudal mdDA domain,

suggesting a critical role for En1 in the induction of Cck expression [41, 64]. Based on these data,

and the evidence that birth of rostrolateral SNc neurons precedes that of caudal VTA neurons [65],

we have proposed a model for dopaminergic subset specification based on functional and subset

specific interplay of Nurr1, En1 and Pitx3 [64]. In short, the induction of Nurr1 would generate a

default DA neuron, that could acquire the rostrolateral or caudal mdDA neuron phenotype based on

differential interplay of En1 and Pitx3. A rather complicated negative feedback system, where En1

initially induces Pitx3 expression and is subsequently repressed by Pitx3 was proposed to program

the rostrolateral mdDA phenotype (Figure 2H,I, 3A) [64]. If such transcriptional regulation is

executed directly (via promoter binding) or indirectly is unknown. Although the lower levels of En1

in rostrolateral mdDA neurons, and their upregulation in Pitx3-deficient embryos suggest that Pitx3

represses En1 at the transcriptional level, modulation of En1 protein function cannot be excluded,

and may also be involved (Figure 2J). Expression of multiple genes encoding En1-modulatory

proteins (by binding to the En1 homology domain) of the Tle- and Pbx-family (Pbx1, Pbx3 and

Tle3) is downregulated in Pitx3-deficient embryos and in wild-type these genes display subset

specific expression, that is maintained in adulthood [30, 64, 66]. After establishment of the

rostrolateral phenotype, the molecular signature of the remaining caudal subpopulation could be

established by a relatively simple and default program in which En1 and Pitx3 can (partially)

compensate for each others loss (Figure 3A,B).

Although subset specific En1/Pitx3 crosstalk is insufficient to fully explain the differential

molecular programming underlying DA subset specification, comparative analysis of Pitx3- and

En1-deficient embryos lead to the identification of two mdDA subpopulations differentially

dependent on En1 and Pitx3: the rostrolateral Ahd2+ population, and the caudal Cck+ population.

At E14.5, it was demonstrated that both appear to be mutually exclusively expressed, and this is

most likely maintained into adulthood, where Ahd2 expression is enriched in SNc neurons, whereas

Cck expression is enriched in VTA neurons [30]. The subset specific expression of Ahd2 and Cck

may give rise to crucial physiological and functional differences. Although this remains to be

assessed in future studies, Cck is associated with VTA functions, whereas Ahd2 is associated with

SNc functions, and related to PD pathogenesis [67, 68]. Intriguingly, it has been described that Cck

inhibits DA neurotransmission [69], but if increased levels of Cck in Pitx3-deficient mice affect DA

neurotransmission remains to be established. Moreover, functional blockade of Cck may relieve PD

symptoms, since Cck-B receptor antagonists potentiate L-DOPA effects in MPTP-lesioned monkeys

[70].

Otx2

Otx2 belongs to the bicoid family of homeodomain TFs. During early mdDA neuron development,

Otx2 has been suggested to activate the expression of Lmx1a, Msx1, Ngn2 and Mash1, either

directly or indirectly, and thereby trigger proliferation and differentiation of mdDA progenitors [71,

72]. Pioneering work of Antonio Simeone and colleagues established Otx2 as one of the critical

factors involved in mdDA neuron subset specification. Although Otx2 is expressed in all mdDA

progenitors, it is selectively expressed in a subset of mdDA neurons of the VTA subtype at later

developmental and adult stages, and required for the differentiation of this subset [73–76]. This

pattern is conserved in primate and human, and maintained into adulthood, where Otx2 is

selectively expressed in a subgroup of VTA neurons, that expresses low levels of glycosylated Dat

(glyco-Dat) and Girk2. Otx2 specificies this subset of VTA neurons by antagonizing molecular

features of the dorso-lateral VTA, including Girk2 and Dat expression [74]. In the same study, it

was demonstrated that Otx2 limits the number of neurons with efficient DA uptake, and that

neurons with low levels of Otx2, and therefore high levels of glyco-Dat, are most vulnerable to

MPTP toxicity. Moreover, ectopic expression of Otx2 in SNc neurons protected these neurons from

MPTP induced degeneration [74]. Otx2 gain-of-function experiments have been demonstrated to

elevate expression of known VTA-enriched genes in vitro in both the MN9D cell line model and

ventral midbrain primary cultures [76], including many factors involved in the formation of DA

neuronal projections. Indeed, Otx2 conditional knock-out mice (En1-cre/+;Otx2-flox/flox) loose

VTA DA neuronal projections, whereas SNc neuronal projections (to the dorsolateral striatum) are

not affected. In the same study, it was elegantly demonstrated that Otx2 knockdown reduces the

number of mdDA neurons, and increases the vulnerability of mdDA neurons to MPTP [76]. Mild

overexpression of Otx2 in SNc progenitors and neurons rescued the defects observed in case of En1

haploinsufficiency (i.e. progressive loss and increased MPTP sensitivity of SNc neurons, as

discussed above). Based on these observations the author suggested that Otx2 and En1 share similar

properties, controlling mdDA neuron development and maintenance in the VTA and SNc

respectively [73]. Although our observations that En1-heterozygous and En1-null adult mice display

both SNc and VTA defects contradicts this theory to some degree, this is an intriguing possibility,

and could be investigated by in-depth analysis of the downstream molecular pathways of En1 and

Otx2 on a genome-wide scale. This is especially important, because the En1-cre driver is also

heterozygous for endogenous En1, and therefore it is critical to establish to what extent defects

observed in the Otx2 conditional knock-out model are the result of En1 and Otx2 interplay.

Recently, the functional consequences of over- and ectopic expression of Otx2 were reported. When

Otx2 was expressed throughout the En1 expression domain, increased numbers of glyco-Dat

positive mdDA neurons and DA innervation were observed [72, 77]. This observation is

contradictory to the Otx2 overexpression driven decrease of glyco-Dat levels reported in an earlier

study [74]. An explanation for this discrepancy may be found in the use of different Cre-drivers. In

the study by Tripathi et al., En1-cre was used to drive Otx2 overexpression [77], a model previously

shown to enhance mdDA neuron progenitor proliferation [72]. In contrast, Di Salvio et al. used Dat-

cre as a driver, and Otx2 overexpression was therefore only induced after the mdDA progenitor

stage. Thus, the effect of Otx2 overexpression on glyco-Dat levels in mdDA neurons appears highly

timing-dependent.

In the En1-cre driven Otx2 overexpression model, the number of glyco-Dat positive mesocortical

DA fibers is increased, and correlated with increased density of parvalbumin-positive interneurons

and hypolocomotion behaviour [77]. Since hypolocomotion is observed in multiple murine PD-

models [78–80], it may therefore be questioned if Otx2 overexpression, despite its clear protective

role against MPTP-toxicity, can truly serve as a future treatment option in PD, since it might induce

transdifferentiation of mdDA neurons from the SNc to the VTA subtype. It would therefore be

critical to assess the true identity of neurons rendered less vulnerable to MPTP toxicity by Otx2

overexpression, for example, by analyzing their relative Ahd2 and Cck transcript levels.

Additionally, the decrease of MPTP toxicity upon Otx2 overexpression, may well be related to

lower uptake of MPTP in cells that express lower levels of Dat (expression of Dat is decreased upon

Otx2 overexpression, as discussed above), since there is a quantitative relationship between the

expression of Dat and the extent of cytotoxic effects of MPTP [81, 82].

Lmx1a and Lmx1b

LIM homeodomain transcription factors Lmx1a and Lmx1b are both involved in mdDA neuron

development. Initial studies in chick suggested a critical role for Lmx1a in establishment of the

mdDA neuron phenotype [83], supported by the observation that overexpression of Lmx1a in mouse

embyronic stem cells (ESCs) induces the DA phenotype [83] which probably involves a Wnt1-

Lmx1a regulatory loop [84]. However, results obtained in recent studies in mouse mutants for

Lmx1a indicate that the initial observations in chick and ESCs must be treated with caution and

suggest that Lmx1a may be less critical for mdDA neuron development than previously thought, at

least in mouse. Several studies have described only mild mdDA phenotypes in Lmx1a mutant mice

[85–88]. These mild phenotypes can be explained by functional redundancy of Lmx1a and Lmx1b

in the developing mdDA system, but although indeed severe DA cell loss is observed in

Lmx1a/Lmx1b double mutants, this hypothesis remains controversial [85, 87].

Intriguingly, some studies have described subset specific roles of Lmx1a and Lmx1b in developing

mdDA neurons. Perlmann and colleagues [87] have reported that Lmx1a and Lmx1b are most

important for the specification of mdDA neurons in the medial and lateral progenitor domain

respectively. In Lmx1a-deficient embryos, generation of post-mitotic mdDA neurons was limited to

lateral positions at E11.5, suggesting that Lmx1a critically controls neurogenesis in the medial part

of the mdDA progenitor domain. In contrast, lateral markers D2R (at E13.5) and Wnt1 (E11.5) are

severely affected in Lmx1b-null embryos, but almost unaffected in Lmx1a-deficient embryos.

However, Lmx1b may also be critical for differentiation of medial derived mdDA neurons, since

Pitx3 and Th co-expression is not induced at terminal differentiation stages in the absence of Lmx1b

[87, 89]. Since Lmx1b regulates Fgf8, Wnt1, and several isthmus-related TFs, and may therefore

critically induce isthmic organizer (IsO) activity and specification of the mdDA progenitor domain

[19, 90] it is also possible that the effect observed in Lmx1b-deficient embryos is secondary to its

essential role for IsO activity. In support of this, specific inactivation of Lmx1b in mdDA

progenitors does not affect mdDA neuron development [85].

More evidence for a role of Lmx1a in mdDA subset specification has come from a recent in-depth

study of Lmx1a-deficient mice at embryonic and adult stages. This study indicated an esssential role

for Lmx1a in the induction of the rostrolateral mdDA neuronal phenotype and using microarray

analysis the molecular pathways underlying this phenotype were partially elucidated [86].

Downstream targets of Lmx1a included Nurr1 and Wnt/b-catenin signaling activator Rspo2. It was

suggested that, within the rostrolateral mdDA domain, Lmx1a indirectly regulates the expression of

Th, Ahd2, Aadc and Vmat2 via the induction of Nurr1. Possibly in parrallel, Lmx1a induces Rspo2

expression, that is in turn involved in the induction of Th, Ahd2 and Pitx3 , since expression of these

genes is downregulated in the rostrolateral mdDA domain in Rspo2-deficient mice [86]. However, it

remains to be established if the observed defects reflect programming deficits, rather than migration

defects or cell-loss, although in Lmx1a-deficient embryos Th fiber outgrowth to the striatum

appears unaltered, and in Rspo2-deficient embryos normal DAPI- and BGAL-staining (the Rspo2-

mutant is an Rspo2-knockout;Lac-Z knockin model) suggests that there is no immediate neuronal

loss in the mdDA region of these mice [86].

Other critical transcription factors

Nurr1 (Nr4a2) is an orphan nuclear receptor, for which no endogenous ligand has been identified to

date. Nurr1 may function as a 'master' regulator of mdDA neuron development, since Nurr1 is

critical for the development and maintenance of all mdDA neurons [91–93]. Although its role as a

'master' regulator strongly suggests that Nurr1 is not involved in mdDA subset specification, Nurr1

inactivation in already mature mdDA neurons induces a Parkinson-like phenotype, with SNc

neurons being more severely affected than VTA neurons [94]. However, this may simply reflect the

increased sensitivity of SNc neurons to environmental stress, especially since nuclear-encoded

mitochondrial genes are massively affected upon conditional inactivation of Nurr1 [95].

Additionally, since Dat-cre was used as a driver in these studies, and Dat expression levels are

typically higher in SNc than VTA neurons, lower Cre levels in the VTA could have lead to partial

inactivation of Nurr1 in the VTA, thus explaining the 'resilient' phenotype of VTA neurons.

Therefore, the most likely model, is that Nurr1 functions as a 'master switch' to induce mdDA

neuron specification, and that subset specificity is determined by other TFs that modulate Nurr1

transcriptional activity by association with the Nurr1 transcriptional complex, such as En1 and Pitx3

[50, 64, 96].

Foxa1 and Foxa2 are broadly expressed in the mdDA neuron progenitor domain and critically

involved in mdDA neuron development and maintenance at various stages [5, 97]. Regulatory

mechanisms include their activation of Lmx1a and Lmx1b in mdDA neuron progenitors [98],

induction of Nurr1 and En1 expression in immature mdDA neurons, and regulation of Pitx3, Th,

Dat, Vmat2 and Aadc during terminal differentiation [5, 99, 100]. Foxa1 and Foxa2 appear to

regulate mdDA neuron development in a dose-dependent manner, since failure to induce the mdDA

phenotype is only observed when all four copies of Foxa1/2 are deleted [99]. As in the case of

Nurr1, the only evidence for subtype specific roles of Foxa1/2 comes from studies in adult mice.

Foxa2 haploinsufficiency results in progressive degeneration of mdDA neurons of the SNc subtype,

whereas VTA neurons remain unaffected, and is associated with Parkinson-like motor problems.

Interestingly, Ahd2-positive neurons are specifically affected [101]. Moreover, conditional deletion

of Foxa1 and Foxa2 in postmitotic mdDA neurons results in a severe decrease of Ahd2+ mdDA

neurons of the SNc, whereas the number of Otx2+ mdDA neurons in the VTA is only mildly

affected. In line with these observations, projections to the dorsolateral striatum are severely

affected, whereas those to the nucleus accumbens are unaffected [97]. Although specific

degeneration of SNc neurons in the absence of Foxa1/2 could again simply reflect the increased

vulnerability of these neurons to environmental stress, this may not necessarily be the case,

especially since Stott et al. have shown that mdDA neurons fail to maintain the expression of genes

involved in DA metabolism, but continue to express Lmx1a, Lmx1b and Nurr1 at E18.5 [97]. This

suggests that mdDA neurons are not lost, but rather lose their mdDA neuronal phenotype. It is

therefore possible that SNc and VTA neurons are truly differentially dependent on Foxa1 and

Foxa2, and it should be investigated in more detail how specific allelic combinations of Foxa1 and

Foxa2 loss-of-function mutants affect the development of mdDA neuronal subsets.

Engrailed-2 (En2) is expressed in a specific subset of the mdDA neuronal domain from early

development into adulthood. Since the replacement of the En1 coding sequence with En2 in mouse

has been described to completely rescue brain and skeletal muscle defects in the En1-null mutant, it

has been suggested that the two paralogs are redundant, and that functional differences are the

consequence of differences in spatiotemporal expression rather than different molecular properties

[60, 102]. However, En2-null mice in an En1-heterozygous background have a less severe

phenotype than En1-null mice in an En2-heterozygous background, and En2-null mice in an

otherwise wild-type background have a less severe phenotype than En2-null mice in an En1-

heterozygous background [103], suggesting that En2 is less critical for mdDA neuron development

and/or maintenance than En1 [60]. Despite this interesting observation, in-depth analysis of mdDA

neuron development and maintenance in single En2-null embryos and adult mice has not been

performed to date, probably since initial studies reported no difference in distribution and number

of mdDA neurons in adult En2-null mice [61, 103]. However, as discussed in the previous section,

the same was initially reported for En1-null mice and recently contradicted based on more in-depth

analysis [64]. It would therefore be of utmost interest to assess if the subset specific expression of

En2 is reflected by specific vulnerability or programming defects in case of its deletion. Moreover,

not only En1, but also En2, is upregulated in Pitx3-deficient mice, and En2 is one of the most

downregulated genes in En1-null embryos [41, 64]. Therefore, it is critical to investigate both

convergence and divergence points of the molecular pathways of En1 and En2 during mdDA

neuron subset specification in the near future.

Signaling mechanisms involved in mdDA subset specification

The roles of various signaling mechanisms involved in general DA neuron development and

maintenance have been addressed in multiple excellent reviews [6, 104–106]. In this section, we

discuss the possible roles of such signaling mechanisms in mdDA subset specification, focussing on

neurotrophic factors that may have protective, restorative and/or stimulatory effects in mdDA

neurons [107].

Neurotrophins

Critical roles for many neurotrophins in mdDA neuron development and maintenance have been

shown [3, 104, 107]. However, only for few neurotrophins subset specific dependency in the mdDA

system has been investigated, and the few studies that have addressed this issue have mainly

focused on the adult system.

Brain-derived neurotropic factor (BDNF) levels are reduced in the Parkinsonian SNc [108]. During

mouse embryogenesis, BDNF expression is activated by Pitx3 in the rostrolateral mdDA neuronal

subset that ultimately forms the SNc [43]. Further evidence for Pitx3-driven activation of BDNF

comes from in vitro studies [109, 110]. In turn, in BDNF-deprived E13 mouse ventral midbrain

primary cultures, a ~50% reduction of Pitx3-expressing neurons was observed with concomitant

loss of Th+ neurons, possibly due to death receptor and caspase activation. Similar effects were

observed in GDNF-deprived cultures [111]. Interestingly, GDNF signaling may be upstream of

Pitx3, since it has been demonstrated that expression of GDNF in the ventral midbrain induces

Pitx3 transcription via NF-kB signaling [43]. The Pitx3/BDNF pathway may be controlled by PGC-

1a (peroxisome proliferator-activated receptor gamma co-activator-1 alpha), a positive regulator of

genes required for mitochondrial biogenesis and antioxidant responses, since AAV-mediated

overexpression of PGC-1a in the adult SNc results in downregulation of Pitx3, increased

susceptibility to MPTP, and reduced BDNF levels [112]. However, it should be noted that this

particular finding is in contrast with other studies, that have mainly suggested protective effects of

PGC-1a against MPTP toxicity in SNc neurons [113, 114].

The conditional removal of GDNF receptor Ret by means of the Dat-Cre driver induces slow and

progressive degeneration of SNc, but not VTA neurons, with concomitant loss of dorsolateral

striatal innervation [115]. Although these findings not necessarily imply that GDNF is exclusively

important for the maintenance of SNc neurons, especially given the profound degeneration of both

SNc and VTA neurons upon tamoxifen-induced GDNF removal in adult mice [116], it suggests that

the nigrostriatal pathway from SNc to dorsal striatum is more dependent on GDNF/Ret signaling

than the mesolimbic pathway from VTA to ventromedial striatum. A similar specific dependency of

SNc neurons on a neurotrophin has been described In Tgf-a knockout mice, where a 50% loss of

DA neurons in the SNc is observed, with a concomitant 20% reduction of the dorsal striatum

volume, whereas VTA neuron numbers remain unchanged [117].

Despite these critical roles in mdDA neuron development and, especially, maintenance, little is

known about the roles of neurotrophins in mdDA neuron subset specifciation. Detailed analysis of

developing mouse embryos in which neurotrophins have been (conditionally) removed is a

prerequisite to understand the roles of these factors in mdDA subset specification and specific

neuron vulnerability. Although such mechanisms have not yet been studied in mdDA subset

specification, evidence for an instructive role of neurotrophin signaling in neuron subtype

specification comes from two elegant studies in dorsal root ganglia (DRG). Expression of TrkC (the

receptor for NT-3) from the TrkA (the receptor for nerve growth factor) locus causes a subset of

DRG neurons to switch fate [118], and Ret is involved in differentiation and diversification of Low-

Treshold mechanoreceptor neurons of the DRG [119].

FGF and Wnt family members

The roles of Wnts and FGFs in mdDA neuron development and maintenance are complex, and for

in-depth coverage of Wnt- and FGF-regulated networks in mdDA neuron development and

maintenance we refer to some excellent recent reviews [6, 20, 105, 120]. In this section, we mainly

restrict ourselves to mechanisms that may confer subset specification of mdDA neurons.

Wnt1 activates the expression of engrailed genes, that are critical for mdDA neuron development,

maintenance and subset specification, as discussed in previous sections [6, 17, 18]. Additionally, a

Wnt1-Lmx1a autoregulatory loop directly activates Otx2 expression and influences Nurr1 and Pitx3

expression through direct activation of Lmx1a [84]. In their review, Wurst and Prakash have

recently proposed the existence of two different Wnt1-regulated genetic networks that may infer

mdDA subset specification [20]. In a gene regulatory network (GRN) specifying caudal mdDA

neurons, Otx2 would initially induce Wnt1 expression. Subsequent activation of Wnt1 target gene

Ccnd1 (cyclin D1) [121] then gives rise to mdDA progenitors that eventually differentiate into VTA

mdDA neurons of the Otx2+, but glyco-Dat low subtype, a view supported by the increased and

ectopic expression of Wnt1 and Ccnd1 in En1-cre induced Otx2 overexpressing mice in the

posterior mdDA area [72], although the possibility that the apparent subset specificity is in fact the

result of differential rostral versus caudal expression of En1 (and therefore cre) should not be

excluded [64]. In the Wnt1 GRN in the rostrolateral mdDA area, Wnt1 would induce Lmx1b and

Lmx1a in an autoregulatory loop. Subsequent activation of Pitx3 and Nurr1 by Lmx1a/b would then

lead to the activation of Pitx3/Nurr1 gene-regulatory pathways, including the activation of Ahd2

and BDNF, and thus specification of mdDA neurons of the SNc subtype [20]. However, as

acknowledged by the authors, also from this model it remains unclear how Wnt1 could function in a

subset specific manner in the generation of the SNc and VTA mdDA neuronal subsets. In this light,

the recent discovery of an Lmx1b-miR135a2 regulatory circuit that modulates Wnt1 signaling is

intriguing [21]. Although the data suggest that this regulatory circuit is involved in determining the

size of the mdDA neuron progenitor pool rather then conferring subset specificity, it would be

interesting to study this possibility in more detail. Recently, it was discovered that the Wnt/b-

catenin signaling activator Rspo2 (r-spondin 2) is a marker for the rostral set of mdDA neurons that

is encoded by Lmx1a and affected in Lmx1a knock-out mice (discussed above). Although no

mechanism was shown, Rspo2-deficient mice partly phenocopied the Lmx1a null mutant, since

expression of Ahd2, Pitx3 and Th was affected in the rostrolateral mdDA domain, suggesting that

modulation of Wnt signaling specifically within this subpopulation partially defines the specific

molecular profile of this mdDA neuronal subset [86].

Several fibroblast growth factors (FGFs), as well as their receptors may be involved in mdDA

neuron subset specification. Among these is Fgf2, one of the few factors of which deletion results in

increased numbers of mdDA neurons in the SNc specifically, possibly due to increased or prolonged

neurogenic production of Lmx1a in mdDA progenitor cells or decreased apoptosis at P0. In

agreement with these observations, overexpression of Fgf2 in mdDA neurons during development

results in decreased numbers of Th+ mdDA neurons [122, 123], and loss of Fgf2 results in increased

mdDA fiber outgrowth during nigrostriatal wiring [124]. It should, however, be noted, that the same

group reported that in 6-OHDA lesioned mice significantly less mdDA neurons survive in Fgf2-

deficient mice, indicating that endogenous Fgf2 may actually protect mdDA neurons in case of

neurotoxicity and that maintaining Fgf2 levels within certain constraints may be critical. The same

study reported that mice heterozygous for Fgf receptor Fgfr3 display reduced mdDA neuron

numbers in the SNc [123]. Although another study described that Fgfr3, as well as Fgfr2, are not

required for early patterning of the mid-hindbrain region, and maintenance of mdDA neurons [125],

this study was less detailed, and no quantification was performed, indicating that subtle differences

may have been overlooked.

The knock-out of another Fgf receptor, Fgfr1, has been studied extensively and shows an interesting

phenotype. Upon conditional deletion of Fgfr1 (using En1-cre as a driver), a rhombomere 1-to-

midbrain transition is observed, resulting in a posterior shift of the mdDA neuron population, with

concomitant caudal expansion of Pitx3, Ahd2 and Otx2 expression at E11.5. At E15.5, the total

number of mdDA neurons is not affected, but significantly more mdDA neurons are located in a

caudal position, and consequently less in a rostral position [126, 127]. An excellent study by the

same group described the contributions of the different Fgfrs to mdDA neuron development and

maintenance using compound conditional mutant embryos for Fgfr1/2 (En1-cre driver) and a full

Fgfr3 knock-out. In single Fgfr1 mutants, mdDA neuron number was not affected, but SNc and

VTA appeared disorganized. However, upon combined deletion of Fgfr1 and Fgfr2 mdDA neuron

cell number in both SNc and VTA were severely reduced by E12.5, and deleted by E18.5. In

Fgfr1/2 compound mutants Ahd2 and Pitx3 expression was ablated at E10.5 and E12.5 respectively

[128]. Later, the molecular mechanisms behind this phenotype were studied in more detail. It was

unveiled that the diencephalic mdDA domain contains not only DA neurons, but also non-DA

Pou4f1+ cells, and suggested that in the absence of Fgfr1/2 postmitotic mdDA precursors in the

mdDA area acquire a phenotype similar to these neurons [58].

Although these data together suggest that Fgf signaling via Fgfr1/2 is primarily important for

anterior-posterior patterning and possibly programming of early mdDA neuron progenitors, an

elegant study where a dominant negative form of Fgfr1 was expressed from the Th-promoter, found

that the roles of Fgfr1 in the SNc and VTA may not be similar. In these mice the density of Th+

neurons is equally reduced in the SNc and VTA at birth, but in adult mice this decrease is only

observed in the SNc. Concomitant loss of Dat expression in the striatum was observed, whereas Dat

expression in the nucleus accumbens was even increased, supporting specific ablation of the

nigrostriatal projections [129]. Moreover, these animals displayed a schizophrenia-like phenotype,

possibly due to increased activity of VTA DA neurons. Altogether, these findings justify the study

of Fgf receptors, in particular Fgfr1, in more detail at later developmental stages (i.e. after initial

patterning and expansion of the progenitor pool) to assess possible roles in mdDA subset

specification.

Fgf20 is a fibroblast growth factor that may be involved in mdDA subset specification. SNPs in

Fgf20 have been associated with PD susceptibility [130]. Moreover, multiple experiments have

indicated critical roles of Fgf20 in the development and maintenance of neurons of the SNc subtype

[130, 131], but a clear role in mdDA subset specification has not yet been established.

Retinoic Acid

A direct transcriptional target of Pitx3 is the retinoic acid (RA) synthesizing enzyme Ahd2 [40], that

converts retinaldehyde into RA and is highly expressed in the developing mesodiencephalon [42].

In addition to Pitx3, Ahd2 is also transcriptionally regulated by Nurr1 (Figure 3A) [132]. Although

Ahd2 is expressed in a small ventral subset of VTA neurons [42], we [40, 41] and others [101, 133]

have shown that Ahd2 is mainly expressed in the Substantia Nigra pars compacta (SNc) area at both

developmental and adult stages. The finding that cultured embryonic midbrains selectively activate

RAR-lacZ but not RXR-lacZ constructs, indicates the presence of endogenous atRA in the

developing mesodiencephalon [134] and the functional relevance of RA signaling in mdDA neurons

has been demonstrated in vitro and in vivo, in Pitx3-deficient stem cell cultures and embryos, that

lack Ahd2 expression and are therefore deprived of RA signaling in post-mitotic mdDA neurons,

respectively. RA increases the number of Th+ mdDA neurons in Pitx3-deficient stem cell cultures

in vitro [135], and in vivo supplementation of RA during the window of embryonic development

where Pitx3 activates Ahd2, bypassed the requirement for Pitx3 and Ahd2, and restored Th

expression in the SNc [40]. In addition, RA restored expression of D2R [41] (Figure 4A-C).

Moreover, multiple in vitro studies support a role of retinoids in mdDA neuron development. RA

critically induces cellular differentation of the embryonic ventral midbrain derived DA cell line

MN9D [136], retinoid receptor ligand DHA facilitates iPSC differentiation into Th-positive neurons

[137], and RA activates Th expression in human neuroblastoma SK-N-BE(2)C cells, most likely

through activation of RAR [138]. Although these results suggest a critical role for Nurr1/Pitx3

mediated RA signaling during final mdDA neuron differentiation and subset specification, little is

known about the mechanisms by which RA directs subset specification in the developing

mesodiencephalon. In vitro evidence, as discussed above, suggests that RA may act via binding to

Retinoic Acid Receptors (RARs) (Figure 3A). Indeed, treatment of Pitx3-deficient explant cultures

with the pan-RAR agonist TTNPB increases Th transcript levels [41]. However, despite the

detection of RARα and RARβ protein in the adult midbrain [139], the presence of RAR proteins

(and their subset specificity) in the developing mdDA area remains to be demonstrated in future

studies.

Another outstanding question regarding RA signaling relates to its spatial constriction (Figure 4D-

F). Although Ahd2, and therefore RA production, is spatially constricted to the rostrolateral mdDA

subset, free diffusion of RA would result in a RA-gradient into the caudal mdDA area, resulting in a

'transition zone'. Such local fluctuations in RA concentration would make it difficult to induce a

sharp boundary between rostrolateral and caudal expression domains, since some cells could adapt

an RA-induced molecular profile, whereas others would not, depending on whether or not

exceeding an RA treshold [140]. Using the developing hindbrain as a model, where much more is

known about the roles of RA in rhombomere specification, Zhang et al. have used an elegant

combination of in situ experiments and computational modeling to address this issue. In the

zebrafish hindbrain, RA initially induces expression of hoxb1a in rhombomere 4 (r4) and krox20 in

r3 and r5. However, somewhat in analogy with Pitx3/En1 (as discussed above), hoxb1a/krox20 gene

expression boundaries are not sharp, and such noise in hoxb1a/krox20 expression is required in an

elegant feedback system, that eventually results in the sharpening of rhombomere boundaries

initially created by RA [141]. If a similar mechanism exists in the developing mesodiencephalon,

and if this could be dependent on interplay of Pitx3, En1 and possibly other homeodomain TFs (e.g.

Pbx family members that are RA-inducible [142, 143]), remains to be investigated. Another

possible mechanism to assure spatial confinement of RA signaling could involve the combined

spatial expression of RA-synthesizing enzymes (e.g. Ahd2) and RA-degrading enzymes. In the

developing hindbrain, such feedback is mediated by Dhrs3 (dehydrogenase/reductase member 3),

that attenuates RA signaling by reducing retinal levels [144] and Cyp26a1 (cytochrome P450,

family 26, subfamily A, polypeptide 1), that actively degrades RA [140, 145] (Figure 4D-F).

Indeed, Dhrs3 and Cyp26b1 (cytochrome P450, family 26, subfamily B, polypeptide 1), an RA

hydroxylase qualitatively similar to Cyp26a1 [146], display enriched expression in caudal mdDA

neurons, as well as surrounding non-mdDA cells at E14.5 (JV Veenvliet, unpublished results) and

Cyp26b1 is expressed in a subset of the mdDA area in the adult stage (Allen Brain Atlas,

http://mouse.brain-map.org/experiment/show/72081548).

Importantly, multiple lines of evidence suggest that the functional role of RA signaling in the SNc is

not limited to developmental stages. Lower mRNA levels of the human Ahd2 homologue RALDH1

have been detected in the SNc of PD patients [147–149] and RA receptor stimulation using two

different RAR agonists protected midbrain dopaminergic neurons from inflammatory degeneration

in adult mice induced by lipopolysaccharide-activated microglia [139]. This rescue was

accompanied by increased tissue levels of BDNF mRNA, and the neuroprotective effect of a RAR

agonist was suppressed by TrkB inhibition and anti-BDNF neutralizing antibodies, suggesting that

the neuroprotective effect of RAR activation is mediated via enhancement of BDNF expression. A

later study by the same group further unraveled the RAR-BDNF molecular cascade and

demonstrated an essential role for nNOS-derived nitric oxide (NO) in RAR signaling, by recruiting

cyclic GMP and PKG, leading to ERK-dependent BDNF up-regulation in mesodiencephalic DA

neurons [150]. If similar molecular pathways downstream of RA are involved in mdDA subset

specification in developing mdDA neurons remains an open question, although the confinement of

both RA and BDNF signaling (as discussed above) to the rostrolateral subpopulation are suggestive

of such a mechanism.

Dlk1

A recent study by our lab described how in vivo treatment of pregnant mother mice with RA

suppresses the ectopic expression of Dlk1 mRNA and protein expression in the rostrolateral part of

developing mdDA neurons in Pitx3-deficient embryos, the exact region that harbours the neurons

that are devoid of Th in Pitx3 ablated mice. Dlk1 is a transmembrane protein that has mainly been

described for its role in adipocyte and osteoblast differentiation [151, 152]. In adipocytes, Dlk1

inhibits differentiation [153–155]. At adult stages, Dlk1 has been reported to be expressed in Th-

positive neurons of both SNc and VTA [156, 157]. However, at the terminal differentiation stage of

mdDA development (E14.5), Dlk1 expression is limited to a caudal subpopulation of mdDA

neurons [41, 49]. Initial in vitro studies indicated that treatment of VM-derived DA neuron

precursors with Dlk1 protein increased DA precursor proliferation during primary culture

expansion, whereas treatment during DA neuronal differentiation did not affect the number of Th-

expressing neurons. However, interfering with Dlk1 expression during differentiation decreased the

expression of some mdDA markers, suggesting a permissive role for Dlk1 during terminal

differentiation [158]. In striking contrast with these data, analysis of the mdDA area in Dlk1-null

mice in our laboratory suggested an inhibitory role for Dlk1 in the expression of Dat, since in the

absence of Dlk1 Dat was ectopically and/or prematurely expressed. Notably, expression of other

mdDA markers appeared unaffected in Dlk1-null mice [49]. In vivo, Dlk1 is therefore likely to play

a suppressive role in terminal mdDA differentiation, in line with its function in many non-neuronal

tissues [152–155]. The suppression of ectopic Dlk1 transcript and protein expression in the mdDA

area of Pitx3-deficient embryos that have received embryonic RA treatment, suggests that the

increased Dlk1 expression in Pitx3-deficient embryos is the consequence of RA signaling

deprivation in the absence of Pitx3. Subset specific Pitx3/Ahd2-mediated RA signaling may thus

play an active role in the local suppression of Dlk1, allowing terminal differentiation of mdDA

neurons in a specific subset (Figure 3A,B). Notably, such interplay between RA signaling and Dlk1

may not be confined to mdDA neurons, since in neuroblastoma cells, both various doses of RA and

Dlk1 knockdown induce differentiation [159]. However, although in multiple, both neural and non-

neural tissues, critical roles for Dlk1 have been demonstrated in inferring molecular and functional

subset specific cellular characteristics [160–163], the exact role for, and downstream pathways of,

Dlk1 in mdDA neuron development and subset specification remain poorly understood and should

be studied in more detail. One important question is, if Dlk1 functions as a Notch ligand in DA

cells. Studies in drosophila have shown roles for delta/notch singaling in early DA specification

events [164, 165], and Dlk1 may act as a non-canonical Notch ligand [166], but if this is the case in

vertebrate DA neurons remains to be revealed.

Translational value of fundamental knowledge about subset specific molecular

programming

Obtaining knowledge about molecular programs that regulate mdDA neuron subset specification is

not only interesting from a fundamental point-of-view. It also harbors great translational value,

since it may (1) increase our understanding of molecular mechanisms of neuronal vulnerability in

PD and (2) offer novel leads for the generation of SNc subtype specific neurons from stem cells

(SCs).

Translational value for the understanding of PD pathogenesis

The potential translational value of developmental genetics for the understanding of PD

pathogenesis, is exemplified by the many studies that have found associations between PD and

SNPs in genes that are critically involved in mdDA neuron development. Polymorphisms in EN1,

PITX3, NURR1, LMX1A and LMX1B are associated with PD [167–171]. Also, AHD2 expression is

downregulated in the SNc of PD patients and may serve as a peripheral biomarkers for diagnosing

PD [147, 172]. Likewise, polymorphisms in, and/or downregulated expression of growth factors

critically involved in mdDA neuron development and maintenance, such as BDNF, TGF-B and

FGF20 has been observed in PD patients [130, 173–175].

An outstanding question is if these polymorphisms affect mdDA development and subset-

specification, and if developmental deficits in the specification of mdDA sub-populations are

observed in PD patients and related to these SNPs. This is impossible to study in vivo because this

would require studying human SNc and VTA ante mortem. However, an interesting alternative

would be the generation of iPSCs from patients with such polymorphisms, differentiate them

towards the mdDA neuronal lineage, and investigate if these polymorphisms bias iPSC-derived

mdDA neurons towards an SNc or VTA phenotype, which would suggests that these

polymorphisms result in developmental defects in mdDA subset specification that may precede SNc

degeneration in PD.

Do developmental TFs influence the terminal phenotypes of SNc neurons that put them at risk

to neurodegeneration?

Could polymorphisms in critical TFs and signaling factors affect later survival of mdDA neurons

without affecting subset specification and/or terminal differention of mdDA neurons? An excellent

recent review by Doucet-Beaupré and Lévesque has suggested that developmental TFs contribute to

mdDA neuron survival and maintenance in the adult system by transcriptional and/or translation

regulation of nuclear-encoded mitochondrial genes and genes involved in mitochondrial metabolism

[31]. Mitochondrial dysfunction has long been suggested to be at the heart of PD pathogenesis

[176]. Indeed, several TFs that are implicated in mdDA neuron development and subset-

specification have recently been shown to regulate mitochondrial function. En1/2 protects adult

mdDA neurons from mitochondrial complex I insults by regulating the translation of subunits of

this complex, Ndufs1 and Ndufs3 [177]. In fetal muscle progenitors, Pitx2/3 regulates Nrf1, a TF

that regulates mitochondrial biogenesis, and in the absence of Pitx2/3 excessive levels of Reactive

Oxygen Species (ROS) are observed [178]. In the case of Otx2, more than half of Otx2 target genes

are nuclear-encoded mitochondrial mRNAs [179].

Mitochondrial dysfunction in the pathogenesis of PD is closely intertwined with another terminal

phenotype of SN neurons that makes them more susceptible to degeneration in PD: the reliance of

SN neurons on Ca2+ for their autonomous pacemaking. SNc neurons use L-type Ca2+ channels

[180], as opposed to VTA neurons that rely on sodium with minimal Ca2+ channel contribution

[181]. The resulting continuous influx of Ca2+ puts a strain on mitochondria, that produce ATP to

keep intracellular levels of Ca2+ within limits, and are involved in Ca2+ buffering themselves [182].

Thus, metabolic stress as a result of sustained Ca2+ entry in SN, but not VTA neurons, may underly

the specific vulnerability of these neurons in PD. In addition, it has been suggested that Ca2+ boosts

DA synthesis from L-DOPA in SNc, but not VTA neurons, which would imply Ca 2+ driven increase

of toxic DA metabolites in SNc neurons specifically [183], further increasing metabolic and

proteostatic stress in these neurons. In addition to its role in RA synthesis, Ahd2 is also involved in

the detoxification of DA metabolites, suggesting that misregulation of Ahd2 as a result of reduced

(functional) levels of these TFs can further increase oxidative stress in SNc neurons. In line with the

Ca2+ hypothesis for SNc specific vulnerability, mdDA neurons that express the Ca2+ buffer,

calbindin (most VTA neurons, but only a small subset of SN neurons), are relatively spared from

neurodegeneration in PD [184].

Another channel that appears to be involved in selective vulnerability of SN neurons is the K-ATP

channel. This channel is selectively activated in SN neurons upon toxic challenges and this prevents

action potential generation. Studies in K-ATP knock-out mice confirmed the instrumental role of

these channels in SNc neuron selective vulnerability, since these mice were restistant to MPTP

induced neurodegeneration [185]. Quantitative single-cell analysis has revealed that mRNAs coding

for these channels are enriched in SN neurons as compared to VTA neurons. It would be highly

interesting to study if genes affecting mitochondrial function and channel composition are

differentially expressed in SN as compared to VTA neurons, if such a distinction is already visible

during development, and if these possible differences are encoded by subset specific roles of critical

TFs and signaling molecules.

Translational value for cell replacement strategies

In the last decade, many excellent neurodevelopmental studies have focused on the generation of a

good cell replacement model by reprogramming of inducible pluripotent SCs (iPSCs) and/or

embryonic SCs (ESCs) [186–191]. In most of these studies resemblance of SC-derived neurons

with their in vivo counterpart was assessed based on mainly morphological and functional

properties, as well as mdDA specific gene sets [189–193]. Such scarce characterization of

iPSC/ESC-derived DA neurons is problematic, since classical differentiation protocols for mouse

and human iPSCs and ESCs typically yield a heterogeneous population of DA neurons [194, 195],

that is associated with problems that limit the clinical application of cell replacement therapy in PD,

such as poor graft survival and tumour formation [195]. These are critical issues that need to be

overcome before iPSCs and ESCs can be considered as a safe and successful treatment for PD.

Recently, some studies have therefore focused on the resemblance of SC-derived DA neurons with

their in vivo counterpart. Roessler et al. performed a detailed analysis of the transcriptomic and

epigenetic signature of iPSC-derived DA neurons [196]. Using microarray and Reduced

Representation Bisulfite Sequencing (RRBS) technology, the genetic and epigenetic profile of

iPSC-derived FACS (Fluorescence Activated Cell Sorting)-purified Pitx3(gfp/+) DA neurons was

compared with their in vivo counterpart at various developmental stages. Although the iPSC-derived

DA neurons had largely adopted characteristics of their in vivo counterparts that are generally used

to assess the resemblance of SC-derived mdDA neurons with true mdDA neurons (e.g. morphology,

DA production, functionality in behavior models, expression of mdDA markers), major relevant

deviations in genome-wide gene expression and CpG island methylation profiles were observed.

Many genes involved in neurodevelopment were hypermethylated and consequently showed

reduced expression levels in iPSC-derived DA neurons as compared to their in vivo counterparts.

Moreover, residual expression and a permissive methylation state of fibroblast markers and

enriched expression of genesets involved in mesodermal lineage specification was reported,

indicative of epigenetic memory of the cells of origin, in line with previous reports that iPSCs are

prone to differentiate along their lineage of origin [197, 198]. To what extend the observed

epigenetic memory and expression profile deviations interfere with in vivo functionality of SC-

derived DA neurons after grafting remains to be investigated in future studies. However, such

findings stress the importance of genome-wide screens of SC-derived DA neurons to evaluate their

true potential for stem cell therapy in PD.

More studies have focused on global transcriptome analysis of iPSC- and ESC-derived DA neurons.

Momcilovic et al. [199] described changes in gene expression during mdDA differentiation of ESCs

and iPSCs using bead microarrays at four stages during ESC- and iPSC –differentiation towards

mdDA neurons (undifferentiated, neural stem cells, DA precursors, DA neurons). An excellent study

by Studer and colleagues [190] described global transcriptome analysis of progeny from Hes5:GFP,

Nurr1:GFP and Pitx3:YFP ESC reporter lines and identified expression of many known and novel

DA genes in these ESC-derived DA neurons. Unfortunately, these studies lack a comparison of SC-

derived mdDA neurons with their in vivo counterpart. In this regard, Salti et al. [194] took an

interesting, although not genome-wide, approach. They performed quantitative comparison of gene

expession profiles during DA differentiation in vitro and in vivo (E11.5-E13.5) at five stages of

differentiation (ES cells, Embryoid Bodies (EBs), Neural Selection, Neural Patterning and Neural

Differentiation), and used such profiles to relate gene expression milestones at various stages of

differentiation to the efficiency of DA differentiation at a protocol's endpoint. In this particular

study, levels of DA metabolism genes (Th, Vmat2) and critical mdDA TFs (Otx2, En1, Foxa2) were

comparable with E11.5 dissected midbrain tissue. Such in-depth characterization studies, that

compare the molecular signature of SC-derived mdDA neurons with that of their in vivo counterpart

are of critical importance to reveal the true potential of protocols that generate SC-derived mdDA

neurons to use in cell replacement strategies. However, all studies that have used cross-comparison

approaches, have not taken the existence of multiple mdDA neuronal subsets into account, and this

should be addressed in future studies.

Generation of specific mdDA neuron subtypes from stem cells

Given the specific degeneration of SNc neurons in PD, generating neurons of the correct (SNc)

subtype from iPSCs / ESCs is an important focus of SC research, and has been proposed as a

critical criterium for assuring pluripotent SC quality for cell therapy [200]. In support of this, it was

shown that in intrastriatal grafts the SNc component is of critical importance for recovery in a

rodent model of PD [201]. Also, it has been demonstrated that, in intrastriatal grafts, distinctive

features are retained after transplantation, and can be used to distinguish SNc and VTA

subpopulations, that innervate distinct projection areas, providing evidence that axonal outgrowth

from these subpopulations is differentially regulated in the grafts. Indeed, fiber outgrowth upon

grafting in PD models appears to reflect, at least to some extent, normal innervation patterns, as

SNc neurons extend their fibers towards the dorsolateral striatum after transplantation, whereas

VTA neurons project to the frontal cortex and other forebrain areas [201–203]. Since the axon

terminal network is critical for the release of DA in the dorsolateral striatum, grafted SC-derived

mdDA neurons need to have the capacity to innervate the host dorsolateral striatum for functional

improvement in PD patients, and this appears to be a unique feature of SNc neurons. Distinct

molecular features of SNc and VTA neurons with respect to axon guidance factors, as observed in

normal developing mdDA neurons, are likely to form the basis for this different dorsostriatal

innervation capacity [8]. Notably, even if this innervation issue could be circumvented, it is not sure

whether grafting SC-derived neurons of the VTA subtype would be functionally beneficial, since

their distinct molecular make-up is, at least to some extend, reflected by functional diversity, and it

is not sure to what degree SC-derived VTA neurons would be flexible enough to adapt SNc neuron

functionality upon grafting (for detailed reviews about molecular versus functional diversity in the

adult mdDA system we refer to [204, 205]).

Two different strategies can be applied to obtain a homogeneous population of SNc-subtype mdDA

neurons (Figure 5). The first possibility is using a protocol that allows for the specific generation of

SNc DA neurons from SCs. Multiple studies have claimed enriched and/or specific generation of

SNc-subtype DA neurons using a variety of differentiation strategies [133, 206–210]. Some of these

approaches involve over-expression of critical TFs, such as Pitx3 [133], Lmx1a [207] and Lmx1b

[208]. However, in virtually all of these studies the succesful conversion to the SNc-subtype is

solely based on the observation that SC-derived DA neurons express the G protein-gated inwardly

rectifying K+ channel Girk2. Although Girk2 has indeed been reported to exclusively mark SNc

neurons [202, 211], later studies have reported that Girk2 is expressed in both SNc neurons, as well

as 50-60% of VTA neurons in humans and mice [74, 212, 213], and therefore the true potential of

all reported approaches in the specific generation of SNc-subtype DA neurons is questionable.

Although more in-depth knowledge of the transcriptional programs that direct mdDA subset-

specification may eventually lead to a protocol to drive mdDA subset specification in a dish (for

example by careful titration of TFs such as Pitx3, En1, and Otx2, and/or application of retinoids), it

is conceivable that such an approach is treacherous, since the process of mdDA subset specification

appears to be extremely complex, and requires not only detailed knowledge of the transcriptional

and signaling machinery, but also detailed knowledge about timing and the critical timeframe

during which induction of SNc and VTA subsets can be achieved in vitro.

A second, more straightforward approach is the generation of a heterogeneous pool of SC-derived

DA neurons, followed by FACS-purification of mdDA neurons of the SNc-subtype. Such an

approach requires the identification of SNc-specific cell-surface markers that can be used in flow-

cytometry. Although SNc-specific cell-surface markers have not been identified yet, such a strategy

harbors great promise, since it circumvents the necessity for laboreous optimization of the current

protocols that generate heterogeneous populations of DA neurons. Moreover, if an SNc-specific

cell-surface marker is combined with an mdDA-specific surface marker, this additionally

circumvents the need for mdDA-specific fluorescent reporters in SC-lines (such as Pitx3-GFP) that

are relatively easily implemented in vitro and in murine models, but problematic when translated to

human cells. A list of such markers has been suggested based on the global expression profiling of

Nurr1:GFP and Pitx3:YFP ESC reporter lines by Studer and colleagues [190], including Chrna6

and Chrnb3, that are expressed in developing mdDA neurons in vivo [51]. Several strategies can be

applied to identify SNc- and VTA-subtype specific cell-surface markers. One possibility is genome-

wide expression profiling of SNc versus VTA neurons after purification by Laser Capture

Microdissection (after staining for a DA-specific gene), and subsequent mining of these data for

subtype-specific surface markers. Such data have been obtained in adult mouse and rat [66, 214,

215], and these data could potentially be used. However, given the typical similarity of SC-derived

DA neurons with embryonic DA neurons [196], genome-wide expression data of developing SNc

versus VTA neurons may be favorable. Unfortunately, such a resource is currently lacking.

Conclusions and future directions

Although the specific vulnerability and degeneration of SNc neurons in PD was acknowledged long

ago [1, 216, 217], the molecular mechanisms that underly such specific vulnerability are still largely

unclear. In their recent perspective, Fishell and Heintz argue that, although studying the immense

diversity of cell types in the mouse CNS provides many harsh challenges for researchers, the

ultimate identification and molecular characterization of neuron subtypes will offer a great potential

for therapeutic intervention in neurodegenerative diseases that affect a certain neuronal subtype,

such as PD [218]. They stated that “It seems apparent that detailed molecular profiling of the

affected cell types during development and disease progression is a necessary step in understanding

the molecular consequences of destructive genetic or environmental events”, but that “these studies

cannot be pursued without comprehensive information regarding CNS cell types” [218].

The last decade has brought enormous advances in our understanding of the molecular

programming underlying the development of 'default' mdDA neurons, but relatively few in-depth

understanding has been acquired about mechanisms regulating mdDA subset specification in the

developing DA system, although important progress has been made, as reviewed here. However, to

truly understand the molecular cascades that underly mdDA subset specification, a mixture of clever

fate-mapping designs, -omics approaches, single-cell expression analysis, and in-depth

spatiotemporal analysis of the expression of critical factors, should be used to identify subset

specific marker genes and pathways.

In this respect, some pioneering work has come from the laboratories of Roeper and Stuber [219,

220]. Lammel et al. first mapped the anatomical distribution of retrogadely labeled Th+ neurons in

the mdDA area for different projection areas of the mesocorticolimbic mdDA system, and then used

a combination of electrophysiological and molecular techniques to unravel the functional and

molecular diversity of these subpopulations. They demonstrated that in the adult system at least two

functionally and molecularly distinct types of mdDA neurons exist. A particular unique subset

consisted of the mdDA neurons that project to the medial prefrontal cortex, and was shown to lack

functional D2R receptors. Also, Dat expression levels were not equal among mdDA subsets, with

low absolute Dat and relative Dat/Th and Dat/Vmat2 expression ratios observed in

mesocorticolimbic mdDA neurons (with more than half of these neurons not expressing Dat

protein), but high levels in mesostriatal mdDA neurons and neurons projecting to the mesolimbic

lateral shell [220]. This is suggestive of differential DA metabolism in distinct subsets of mdDA

neurons. Moreover, at the level of DA synthesis, Aadc and Th may be differentially post-

translationally modified as a result of Ca2+ influx in SNc as compared to VTA neurons [183]. The

study of Lammel et al. also clearly demonstrated that the sole distinction of the mdDA system in

SNc and VTA neurons is not exhaustive. Also within the SNc and VTA, multiple subsets exist with

distinct molecular profiles and electrophysiological properties [212, 220], that reflect functional

diversity. For example, Stamatakis et al. recently described a subset of VTA mdDA neurons that co-

release GABA (gammma-aminobutyric acid) from their terminals, and express GABA neuron

markers [219]. Further form-to-function profiling, by relating molecular diversity with functional

diversity is therefore critical in future research, and it will be of utmost interest to determine to what

extend this further segretation of the mdDA area in distinct subsets is reflected in the developing

mdDA system.

As a final remark, we note that unveiling the degree of conservation of subset specific programming

is critical to select for those mechanisms that hold translational value. Eventually, increased

understanding of mdDA subset specification may find its way to the clinic, given the potential

importance of such knowledge to generate SC-derived DA neurons that can safe and soundly

replace those neurons lost in PD.

Acknowledgements

This work was supported by a VICI-ALW grant [865.09.002 to M.P.S.] from the Dutch

Organisation for Scientific Research (NWO).

Figure 1: Timescale of mdDA neuron development

Key factors in three subsequent steps of mdDA development are displayed, and regulatory roles are

indicated for factors most clearly associated with mdDA subset-specification (i.e., BDNF, RA,

Dlk1, and En1, Otx2, Pitx3 (yellow boxes)). During the regional specification phase, several TFs

and signaling molecules interact to establish the mdDA neuronal field. In the mdDA progenitor

expansion phase, Otx2 controls proliferation of mdDA progenitors by regulating the expression of

Lmx1a, Msx1, Ngn2 and Mash1, and during this phase these precursors expresss a large set of genes

from early mdDA progenitor specification (En1/2, Lmx1a/b, Foxa1/2). Around E10.5 neurons start

to express Nurr1 that acts in concert with Pitx3 and En1 to activate the mdDA phenotype during

mdDA differentiation (i.e., transition of postmitotic mdDA precursors to fully differentiated mdDA

neurons). During this phase, complex interplay between Otx2, En1, Pitx3 and other factors defines

mdDA subsets, as specified in more detail in Figure 2.

Figure 2: Transcription Factors in mdDA subset specification

(A) Expression domains of critical mdDA Transcription Factors at E14.5 in wild-type. (B)

Expression domains of Pitx3 and En1 in E14.5 saggital medial and lateral sections. (C,D)

Expression domains of En1, Pitx3 and Nurr1 in En1- and Pitx3-deficient embryos at E14.5. (E-G)

Expression domains of DA metabolism genes in E14.5 wild-type (E), Pitx3-deficient (F), and En1-

deficient (G) embryos. (H) During mdDA neuron development, En1 may initially activate Pitx3

expression (1) in a rostrolateral subset; subsequently, Pitx3 downregulates En1 in turn (2). (I) One

option to infer subset specific Pitx3-mediated repression of En1, is that active Pitx3 protein exceeds

a certain repression treshold (Trepr). (J) Another option is that Pitx3 functionally modulates En1

protein activity, by subset specific activation of genes encoding En1 modulatory proteins. (K) Both

En1 and Pitx3 are critical for the induction of the RA-synthesizing enzyme Ahd2 in mdDA neurons,

Pitx3 activates Ahd2 directly, whereas En1 may activate Ahd2 either directly, or indirectly, via

regulation of Pitx3. In panel (A,C,E-G) a color gradient indicates expression in a midbrain and/or

p1/2/3 subset, and reduced color intensity (opacity) indicates lower expression. A, activator; R,

repressor; C, caudal; R, rostral; ko, knock-out; mdDA, mesodiencephalic dopaminergic; p1,

prosomere 1; p2, prosomere 2; p3, prosomere 3.

Figure 3: Gene Regulatory Networks in mdDA subset specification

(A) In the rostrolateral mdDA neuronal subset that harbours neurons destined to form the SNc in

adult, Pitx3 expression is initially induced by GDNF and En1 (1). Pitx3 then potentiates Nurr1 to

drive the expression of Vmat2 and Dat [50]. Expression of Ahd2 is also under the combined

transcriptional control of Nurr1 and Pitx3, and expression is restricted to the rostrolateral mdDA

neuronal subset. Ahd2 synthesizes Retinoic Acid (RA) from its precursor retinaldehyde, and

activates the expression of Th, D2R, and possibly BDNF, presumambly through binding to Retinoic

Acid Receptors (RARs). Moreover, RA represses Nurr1-mediated expression of Dlk1 in the

developing SNc. In rostrolateral mdDA neurons, Pitx3 is possibly engaged in a positive feedback

loop with BDNF, augmenting survival of SNc neurons. Also, within this subset, Pitx3 antagonizes

the caudal mdDA neuronal phenotype (as illustrated by the inhibition of Cck expression) through an

RA-independent mechanism, possibly via direct or indirect inhibition of En1 expression within this

subset. (B) In the developing caudal mdDA neuronal subset that eventually gives rise to the adult

VTA, Pitx3 and En1 can both induce the expression of Dat and Vmat2, in an RA-independent

manner, since Ahd2 is not expressed in this subset. Pitx3 is not required for Th expression within

this subset, but partially depends on En1 expression. In caudal mdDA neurons, Pitx3 can not

antagonize Cck expression, possibly due to the higher levels of En1. In the absence of RA, Nurr1-

mediated expression of Dlk1 is not repressed, and this may suppress SNc properties in VTA

neurons, although this remains to be established. In a subset of VTA neurons, Otx2 antagonizes the

expression of Dat. These subset specific GRNs illustrate the importance of RA-dependent and

-independent aspects of the Pitx3-gene regulatory networks, as well as the critical roles of Otx2, and

subset specific interplay of En1 and Pitx3 in mdDA subset specification.

Figure 4: Retinoic Acid-dependent gene regulatory pathways in mdDA

development

(A) In wild-type mdDA neurons of the SNc subtype, Pitx3 cooperates with Nurr1 to induce the

expression of the RA-synthesizing enzyme Ahd2. RA then induces the expression of Th and D2R.

(C) In wild-type mdDA neurons of the VTA subtype, the expression of Th is RA-independent. In

addition, expression of Vmat2 and Dat in these neurons is Pitx3-dependent, but RA-independent, as

is the case in SNc neurons (A). (B) Supplementation of RA to pregnant mice during the critical

period for mdDA neuron differentiation restores the expression of Th and D2R in neurons of the

SNc subtype, whereas expression of Vmat2 and Dat is not rescued. (D) In a passive diffusion

model, retinal would be omnipresent in the mdDA system, only locally converted to RA by Ahd2 in

the p1/p2 area, but diffusing freely into the caudal midbrain. (E) In an active degradation model,

Cyp26 enzymes in the caudal midbrain could degrade RA, thus lowering the local RA levels. (F)

When active degradation by Cyp26 enzymes is combined with caudal-specific expression of Dhrs3,

that catalyzes the reversal of retinal into vitamin A, local retinal levels would be low in the caudal

midbrain, which, if combined with local RA synthesis by Ahd2, could induce a relatively strict

border of RA levels (high in the rostrolateral p1/2, low in the caudal midbrain). SNc, Substantia

Nigra pars compacta; VTA, Ventral Tegmental Area; RA, Retinoic Acid; m, midbrain; p1,

prosomere 1; p2, prosomere 2.

Figure 5: Strategies for the generation of SNc-subtype specific SC-derived DA

neurons

(A) One option to obtain a pure population of SC-derived mdDA neurons of the SNc subtype is to

use a differentiation protocol that specifically generates SNc neurons ('Directed Differentiation').

However, this requires detailed knowledge about the Gene Regulatory Network that underlies the

specific programming of these neurons, and may involve a complicated strategy, using a

combination of transduction of many TFs, and multiple signaling molecules. (B-D'') A much easier

approach to obtain a purified population of SC-derived SNc neurons, is to use a Molecular Sort

strategy. In vivo, neurons of the SNc and VTA can be roughly separated based on their anatomical

position (C). However, after in vitro differentiation of SC towards mdDA neurons a heterogeneous

pool of both SNc and VTA subtype mdDA neurons is obtained, and it is not possible to distinguish

between both subtypes (C', D'). However, if a SNc specific surface marker can be identified, live

cells can be stained, and SC-derived DA neurons of the SNc subtype can then be purified using

FACS (C'', D''). Such an approach is greatly facilitated if it is combined with a fluorescent reporter

that marks all DA neurons (e.g. Pitx3-gfp) (C-C''). However, in the absence of such a reporter, a

combination of a DA surface marker that stains all SC-derived DA neurons, and a SNc subtype

specific surface marker would suffice to obtain a purified population of SC-derived SNc DA

neurons (D-D''). SC, Stem Cell; TF, Transcription Factor; DA, dopaminergic.

References

1. Hirsch E, Graybiel AM, Agid YA (1988) Melanized dopaminergic neurons are differentially susceptible to degeneration in Parkinson’s disease. Nature 334:345–348. doi: 10.1038/334345a0

2. Lees AJ, Hardy J, Revesz T (2009) Parkinson’s disease. Lancet 373:2055–2066. doi: 10.1016/S0140-6736(09)60492-X

3. Smidt MP, Burbach JPH (2007) How to make a mesodiencephalic dopaminergic neuron. Nat Rev Neurosci 8:21–32. doi: 10.1038/nrn2039

4. Smits SM, Burbach JPH, Smidt MP (2006) Developmental origin and fate of meso-diencephalic dopamine neurons. Prog Neurobiol 78:1–16. doi: 10.1016/j.pneurobio.2005.12.003

5. Ang S-L (2009) Foxa1 and Foxa2 transcription factors regulate differentiation of midbrain dopaminergic neurons. Adv Exp Med Biol 651:58–65.

6. Alves dos Santos MTM, Smidt MP (2011) En1 and Wnt signaling in midbrain dopaminergic neuronal development. Neural Dev 6:23. doi: 10.1186/1749-8104-6-23

7. Hegarty SV, Sullivan AM, O’Keeffe GW (2013) Midbrain dopaminergic neurons: a review of the molecular circuitry that regulates their development. Dev Biol 379:123–138. doi: 10.1016/j.ydbio.2013.04.014

8. Van den Heuvel DMA, Pasterkamp RJ (2008) Getting connected in the dopamine system. Prog Neurobiol 85:75–93. doi: 10.1016/j.pneurobio.2008.01.003

9. Hynes M, Rosenthal A (1999) Specification of dopaminergic and serotonergic neurons in the vertebrate CNS. Curr Opin Neurobiol 9:26–36.

10. Wassarman KM, Lewandoski M, Campbell K, et al. (1997) Specification of the anterior hindbrain and establishment of a normal mid/hindbrain organizer is dependent on Gbx2 gene function. Development 124:2923–2934.

11. Rhinn M, Brand M (2001) The midbrain--hindbrain boundary organizer. Curr Opin Neurobiol 11:34–42.

12. Acampora D, Gulisano M, Broccoli V, Simeone A (2001) Otx genes in brain morphogenesis. Prog Neurobiol 64:69–95.

13. Acampora D, Gulisano M, Simeone A (1999) Otx genes and the genetic control of brain morphogenesis. Mol Cell Neurosci 13:1–8. doi: 10.1006/mcne.1998.0730

14. Farkas LM, Dünker N, Roussa E, et al. (2003) Transforming growth factor-beta(s) are essential for the development of midbrain dopaminergic neurons in vitro and in vivo. J Neurosci 23:5178–5186.

15. Roussa E, Krieglstein K (2004) Induction and specification of midbrain dopaminergic cells: focus on SHH, FGF8, and TGF-beta. Cell Tissue Res 318:23–33. doi: 10.1007/s00441-004-0916-4

16. Cai J, Schleidt S, Pelta-Heller J, et al. (2013) BMP and TGF-β pathway mediators are critical upstream regulators of Wnt signaling during midbrain dopamine differentiation in human

pluripotent stem cells. Dev Biol 376:62–73. doi: 10.1016/j.ydbio.2013.01.012

17. Danielian PS, McMahon AP (1996) Engrailed-1 as a target of the Wnt-1 signalling pathway in vertebrate midbrain development. Nature 383:332–334. doi: 10.1038/383332a0

18. Castelo-Branco G, Wagner J, Rodriguez FJ, et al. (2003) Differential regulation of midbrain dopaminergic neuron development by Wnt-1, Wnt-3a, and Wnt-5a. Proc Natl Acad Sci USA 100:12747–12752. doi: 10.1073/pnas.1534900100

19. Guo C, Qiu H-Y, Huang Y, et al. (2007) Lmx1b is essential for Fgf8 and Wnt1 expression in the isthmic organizer during tectum and cerebellum development in mice. Development 134:317–325. doi: 10.1242/dev.02745

20. Wurst W, Prakash N (2014) Wnt1-regulated genetic networks in midbrain dopaminergic neuron development. J Mol Cell Biol 6:34–41. doi: 10.1093/jmcb/mjt046

21. Anderegg A, Lin H-P, Chen J-A, et al. (2013) An Lmx1b-miR135a2 Regulatory Circuit Modulates Wnt1/Wnt Signaling and Determines the Size of the Midbrain Dopaminergic Progenitor Pool. PLoS Genet 9:e1003973. doi: 10.1371/journal.pgen.1003973

22. Holder N, Hill J (1991) Retinoic acid modifies development of the midbrain-hindbrain border and affects cranial ganglion formation in zebrafish embryos. Development 113:1159–1170.

23. Avantaggiato V, Acampora D, Tuorto F, Simeone A (1996) Retinoic acid induces stage-specific repatterning of the rostral central nervous system. Dev Biol 175:347–357. doi: 10.1006/dbio.1996.0120

24. Kele J, Simplicio N, Ferri ALM, et al. (2006) Neurogenin 2 is required for the development of ventral midbrain dopaminergic neurons. Development 133:495–505. doi: 10.1242/dev.02223

25. Tomita K, Moriyoshi K, Nakanishi S, et al. (2000) Mammalian achaete-scute and atonal homologs regulate neuronal versus glial fate determination in the central nervous system. EMBO J 19:5460–5472. doi: 10.1093/emboj/19.20.5460

26. Kim H-J, Sugimori M, Nakafuku M, Svendsen CN (2007) Control of neurogenesis and tyrosine hydroxylase expression in neural progenitor cells through bHLH proteins and Nurr1. Exp Neurol 203:394–405. doi: 10.1016/j.expneurol.2006.08.029

27. Puelles L, Rubenstein JLR (2003) Forebrain gene expression domains and the evolving prosomeric model. Trends Neurosci 26:469–476. doi: 10.1016/S0166-2236(03)00234-0

28. Rubenstein JL, Martinez S, Shimamura K, Puelles L (1994) The embryonic vertebrate forebrain: the prosomeric model. Science 266:578–580.

29. Puelles L, Harrison M, Paxinos G, Watson C (2013) A developmental ontology for the mammalian brain based on the prosomeric model. Trends Neurosci 36:570–578. doi: 10.1016/j.tins.2013.06.004

30. Smits SM, Von Oerthel L, Hoekstra EJ, et al. (2013) Molecular marker differences relate to developmental position and subsets of mesodiencephalic dopaminergic neurons. PLoS ONE 8:e76037. doi: 10.1371/journal.pone.0076037

31. Doucet-Beaupré H, Lévesque M (2013) The role of developmental transcription factors in adult

midbrain dopaminergic neurons. OA Neurosciences Oct 01;1(1):3.

32. Smidt MP, Van Schaick HS, Lanctôt C, et al. (1997) A homeodomain gene Ptx3 has highly restricted brain expression in mesencephalic dopaminergic neurons. Proc Natl Acad Sci USA 94:13305–13310.

33. Smidt MP, Smits SM, Bouwmeester H, et al. (2004) Early developmental failure of substantia nigra dopamine neurons in mice lacking the homeodomain gene Pitx3. Development 131:1145–1155. doi: 10.1242/dev.01022

34. Rieger DK, Reichenberger E, McLean W, et al. (2001) A double-deletion mutation in the Pitx3 gene causes arrested lens development in aphakia mice. Genomics 72:61–72. doi: 10.1006/geno.2000.6464

35. Semina EV, Murray JC, Reiter R, et al. (2000) Deletion in the promoter region and altered expression of Pitx3 homeobox gene in aphakia mice. Hum Mol Genet 9:1575–1585.

36. Hwang D-Y, Ardayfio P, Kang UJ, et al. (2003) Selective loss of dopaminergic neurons in the substantia nigra of Pitx3-deficient aphakia mice. Brain Res Mol Brain Res 114:123–131.

37. Nunes I, Tovmasian LT, Silva RM, et al. (2003) Pitx3 is required for development of substantia nigra dopaminergic neurons. Proc Natl Acad Sci USA 100:4245–4250. doi: 10.1073/pnas.0230529100

38. Van den Munckhof P, Luk KC, Ste-Marie L, et al. (2003) Pitx3 is required for motor activity and for survival of a subset of midbrain dopaminergic neurons. Development 130:2535–2542.

39. Maxwell SL, Ho H-Y, Kuehner E, et al. (2005) Pitx3 regulates tyrosine hydroxylase expression in the substantia nigra and identifies a subgroup of mesencephalic dopaminergic progenitor neurons during mouse development. Dev Biol 282:467–479. doi: 10.1016/j.ydbio.2005.03.028

40. Jacobs FMJ, Smits SM, Noorlander CW, et al. (2007) Retinoic acid counteracts developmental defects in the substantia nigra caused by Pitx3 deficiency. Development 134:2673–2684. doi: 10.1242/dev.02865

41. Jacobs FMJ, Veenvliet JV, Almirza WH, et al. (2011) Retinoic acid-dependent and -independent gene-regulatory pathways of Pitx3 in meso-diencephalic dopaminergic neurons. Development 138:5213–5222. doi: 10.1242/dev.071704

42. McCaffery P, Dräger UC (1994) High levels of a retinoic acid-generating dehydrogenase in the meso-telencephalic dopamine system. PNAS 91:7772–7776.

43. Peng C, Aron L, Klein R, et al. (2011) Pitx3 is a critical mediator of GDNF-induced BDNF expression in nigrostriatal dopaminergic neurons. J Neurosci 31:12802–12815. doi: 10.1523/JNEUROSCI.0898-11.2011

44. Kim J, Inoue K, Ishii J, et al. (2007) A MicroRNA feedback circuit in midbrain dopamine neurons. Science 317:1220–1224. doi: 10.1126/science.1140481

45. Heyer MP, Pani AK, Smeyne RJ, et al. (2012) Normal midbrain dopaminergic neuron development and function in miR-133b mutant mice. J Neurosci 32:10887–10894. doi: 10.1523/JNEUROSCI.1732-12.2012

46. Trajkovski M, Ahmed K, Esau CC, Stoffel M (2012) MyomiR-133 regulates brown fat differentiation through Prdm16. Nat Cell Biol 14:1330–1335. doi: 10.1038/ncb2612

47. Liu W, Bi P, Shan T, et al. (2013) miR-133a regulates adipocyte browning in vivo. PLoS Genet 9:e1003626. doi: 10.1371/journal.pgen.1003626

48. Volpicelli F, De Gregorio R, Pulcrano S, et al. (2012) Direct Regulation of Pitx3 Expression by Nurr1 in Culture and in Developing Mouse Midbrain. PLoS ONE 7:e30661. doi: 10.1371/journal.pone.0030661

49. Jacobs FMJ, Van der Linden AJA, Wang Y, et al. (2009) Identification of Dlk1, Ptpru and Klhl1 as novel Nurr1 target genes in meso-diencephalic dopamine neurons. Development 136:2363–2373. doi: 10.1242/dev.037556

50. Jacobs FMJ, Van Erp S, Van der Linden AJA, et al. (2009) Pitx3 potentiates Nurr1 in dopamine neuron terminal differentiation through release of SMRT-mediated repression. Development 136:531–540. doi: 10.1242/dev.029769

51. Chakrabarty K, Von Oerthel L, Hellemons A, et al. (2012) Genome Wide Expression Profiling of the Mesodiencephalic Region Identifies Novel Factors Involved in Early and Late Dopaminergic Development. Biology Open. doi: 10.1242/bio.20121230

52. Martinat C, Bacci J-J, Leete T, et al. (2006) Cooperative transcription activation by Nurr1 and Pitx3 induces embryonic stem cell maturation to the midbrain dopamine neuron phenotype. Proc Natl Acad Sci USA 103:2874–2879. doi: 10.1073/pnas.0511153103

53. Hwang D-Y, Hong S, Jeong J-W, et al. (2009) Vesicular monoamine transporter 2 and dopamine transporter are molecular targets of Pitx3 in the ventral midbrain dopamine neurons. J Neurochem 111:1202–1212. doi: 10.1111/j.1471-4159.2009.06404.x

54. Davis CA, Joyner AL (1988) Expression patterns of the homeo box-containing genes En-1 and En-2 and the proto-oncogene int-1 diverge during mouse development. Genes Dev 2:1736–1744.

55. Nakamura H, Katahira T, Matsunaga E, Sato T (2005) Isthmus organizer for midbrain and hindbrain development. Brain Res Brain Res Rev 49:120–126. doi: 10.1016/j.brainresrev.2004.10.005

56. Albéri L, Sgadò P, Simon HH (2004) Engrailed genes are cell-autonomously required to prevent apoptosis in mesencephalic dopaminergic neurons. Development 131:3229–3236. doi: 10.1242/dev.01128

57. Simon HH, Thuret S, Alberi L (2004) Midbrain dopaminergic neurons: control of their cell fate by the engrailed transcription factors. Cell Tissue Res 318:53–61. doi: 10.1007/s00441-004-0973-8

58. Lahti L, Peltopuro P, Piepponen TP, Partanen J (2012) Cell-autonomous FGF signaling regulates anteroposterior patterning and neuronal differentiation in the mesodiencephalic dopaminergic progenitor domain. Development 139:894–905. doi: 10.1242/dev.071936

59. Wurst W, Auerbach AB, Joyner AL (1994) Multiple developmental defects in Engrailed-1 mutant mice: an early mid-hindbrain deletion and patterning defects in forelimbs and sternum. Development 120:2065–2075.

60. Simon HH, Saueressig H, Wurst W, et al. (2001) Fate of midbrain dopaminergic neurons controlled by the engrailed genes. J Neurosci 21:3126–3134.

61. Sgadò P, Albéri L, Gherbassi D, et al. (2006) Slow progressive degeneration of nigral dopaminergic neurons in postnatal Engrailed mutant mice. Proc Natl Acad Sci USA 103:15242–15247. doi: 10.1073/pnas.0602116103

62. Sonnier L, Le Pen G, Hartmann A, et al. (2007) Progressive loss of dopaminergic neurons in the ventral midbrain of adult mice heterozygote for Engrailed1. J Neurosci 27:1063–1071. doi: 10.1523/JNEUROSCI.4583-06.2007

63. Bilovocky NA, Romito-DiGiacomo RR, Murcia CL, et al. (2003) Factors in the Genetic Background Suppress the Engrailed-1 Cerebellar Phenotype. J Neurosci 23:5105–5112.

64. Veenvliet JV, Dos Santos MTMA, Kouwenhoven WM, et al. (2013) Specification of dopaminergic subsets involves interplay of En1 and Pitx3. Development 140:3373–3384. doi: 10.1242/dev.094565

65. Bye CR, Thompson LH, Parish CL (2012) Birth dating of midbrain dopamine neurons identifies A9 enriched tissue for transplantation into Parkinsonian mice. Experimental Neurology. doi: 10.1016/j.expneurol.2012.04.002

66. Chung CY, Seo H, Sonntag KC, et al. (2005) Cell type-specific gene expression of midbrain dopaminergic neurons reveals molecules involved in their vulnerability and protection. Hum Mol Genet 14:1709–1725. doi: 10.1093/hmg/ddi178

67. Rotzinger S, Vaccarino FJ (2003) Cholecystokinin receptor subtypes: role in the modulation of anxiety-related and reward-related behaviours in animal models. J Psychiatry Neurosci 28:171–181.

68. Fitzmaurice AG, Rhodes SL, Lulla A, et al. (2013) Aldehyde dehydrogenase inhibition as a pathogenic mechanism in Parkinson disease. Proc Natl Acad Sci USA 110:636–641. doi: 10.1073/pnas.1220399110

69. Lane RF, Blaha CD, Phillips AG (1987) Cholecystokinin-induced inhibition of dopamine neurotransmission: comparison with chronic haloperidol treatment. Prog Neuropsychopharmacol Biol Psychiatry 11:291–299.

70. Boyce S, Rupniak NM, Tye S, et al. (1990) Modulatory role for CCK-B antagonists in Parkinson’s disease. Clin Neuropharmacol 13:339–347.

71. Simeone A, Puelles E, Omodei D, et al. (2011) Otx genes in neurogenesis of mesencephalic dopaminergic neurons. Dev Neurobiol 71:665–679. doi: 10.1002/dneu.20877

72. Omodei D, Acampora D, Mancuso P, et al. (2008) Anterior-posterior graded response to Otx2 controls proliferation and differentiation of dopaminergic progenitors in the ventral mesencephalon. Development 135:3459–3470. doi: 10.1242/dev.027003

73. Di Giovannantonio LG, Di Salvio M, Acampora D, et al. (2013) Otx2 selectively controls the neurogenesis of specific neuronal subtypes of the ventral tegmental area and compensates En1-dependent neuronal loss and MPTP vulnerability. Dev Biol 373:176–183. doi: 10.1016/j.ydbio.2012.10.022

74. Di Salvio M, Di Giovannantonio LG, Acampora D, et al. (2010) Otx2 controls neuron subtype

identity in ventral tegmental area and antagonizes vulnerability to MPTP. Nat Neurosci 13:1481–1488. doi: 10.1038/nn.2661

75. Di Salvio M, Di Giovannantonio LG, Omodei D, et al. (2010) Otx2 expression is restricted to dopaminergic neurons of the ventral tegmental area in the adult brain. Int J Dev Biol 54:939–945. doi: 10.1387/ijdb.092974ms

76. Chung CY, Licznerski P, Alavian KN, et al. (2010) The transcription factor orthodenticle homeobox 2 influences axonal projections and vulnerability of midbrain dopaminergic neurons. Brain 133:2022–2031. doi: 10.1093/brain/awq142

77. Tripathi PP, Di Giovannantonio LG, Sanguinetti E, et al. (2014) Increased dopaminergic innervation in the brain of conditional mutant mice overexpressing Otx2: Effects on locomotor behavior and seizure susceptibility. Neuroscience 261:173–183. doi: 10.1016/j.neuroscience.2013.12.045

78. Kim RH, Smith PD, Aleyasin H, et al. (2005) Hypersensitivity of DJ-1-deficient mice to 1-methyl-4-phenyl-1,2,3,6-tetrahydropyrindine (MPTP) and oxidative stress. Proc Natl Acad Sci USA 102:5215–5220. doi: 10.1073/pnas.0501282102

79. Smith PD, Crocker SJ, Jackson-Lewis V, et al. (2003) Cyclin-dependent kinase 5 is a mediator of dopaminergic neuron loss in a mouse model of Parkinson’s disease. Proc Natl Acad Sci USA 100:13650–13655. doi: 10.1073/pnas.2232515100

80. Wang S, Hu L-F, Yang Y, et al. (2005) Studies of ATP-sensitive potassium channels on 6-hydroxydopamine and haloperidol rat models of Parkinson’s disease: implications for treating Parkinson’s disease? Neuropharmacology 48:984–992. doi: 10.1016/j.neuropharm.2005.01.009

81. Pifl C, Giros B, Caron MG (1993) Dopamine transporter expression confers cytotoxicity to low doses of the parkinsonism-inducing neurotoxin 1-methyl-4-phenylpyridinium. J Neurosci 13:4246–4253.

82. Bezard E, Gross CE, Fournier MC, et al. (1999) Absence of MPTP-induced neuronal death in mice lacking the dopamine transporter. Exp Neurol 155:268–273. doi: 10.1006/exnr.1998.6995

83. Andersson E, Tryggvason U, Deng Q, et al. (2006) Identification of intrinsic determinants of midbrain dopamine neurons. Cell 124:393–405. doi: 10.1016/j.cell.2005.10.037

84. Chung S, Leung A, Han B-S, et al. (2009) Wnt1-lmx1a forms a novel autoregulatory loop and controls midbrain dopaminergic differentiation synergistically with the SHH-FoxA2 pathway. Cell Stem Cell 5:646–658. doi: 10.1016/j.stem.2009.09.015

85. Yan CH, Levesque M, Claxton S, et al. (2011) Lmx1a and lmx1b function cooperatively to regulate proliferation, specification, and differentiation of midbrain dopaminergic progenitors. J Neurosci 31:12413–12425. doi: 10.1523/JNEUROSCI.1077-11.2011

86. Hoekstra EJ, Von Oerthel L, Van der Heide LP, et al. (2013) Lmx1a Encodes a Rostral Set of Mesodiencephalic Dopaminergic Neurons Marked by the Wnt/B-Catenin Signaling Activator R-spondin 2. PLoS ONE 8:e74049. doi: 10.1371/journal.pone.0074049

87. Deng Q, Andersson E, Hedlund E, et al. (2011) Specific and integrated roles of Lmx1a, Lmx1b and Phox2a in ventral midbrain development. Development 138:3399–3408. doi:

10.1242/dev.065482

88. Ono Y, Nakatani T, Sakamoto Y, et al. (2007) Differences in neurogenic potential in floor plate cells along an anteroposterior location: midbrain dopaminergic neurons originate from mesencephalic floor plate cells. Development 134:3213–3225. doi: 10.1242/dev.02879

89. Smidt MP, Asbreuk CH, Cox JJ, et al. (2000) A second independent pathway for development of mesencephalic dopaminergic neurons requires Lmx1b. Nat Neurosci 3:337–341. doi: 10.1038/73902

90. Adams KA, Maida JM, Golden JA, Riddle RD (2000) The transcription factor Lmx1b maintains Wnt1 expression within the isthmic organizer. Development 127:1857–1867.

91. Smits SM, Ponnio T, Conneely OM, et al. (2003) Involvement of Nurr1 in specifying the neurotransmitter identity of ventral midbrain dopaminergic neurons. Eur J Neurosci 18:1731–1738.

92. Zetterström RH, Solomin L, Jansson L, et al. (1997) Dopamine neuron agenesis in Nurr1-deficient mice. Science 276:248–250.

93. Saucedo-Cardenas O, Quintana-Hau JD, Le WD, et al. (1998) Nurr1 is essential for the induction of the dopaminergic phenotype and the survival of ventral mesencephalic late dopaminergic precursor neurons. Proc Natl Acad Sci USA 95:4013–4018.

94. Kadkhodaei B, Ito T, Joodmardi E, et al. (2009) Nurr1 is required for maintenance of maturing and adult midbrain dopamine neurons. J Neurosci 29:15923–15932. doi: 10.1523/JNEUROSCI.3910-09.2009

95. Kadkhodaei B, Alvarsson A, Schintu N, et al. (2013) Transcription factor Nurr1 maintains fiber integrity and nuclear-encoded mitochondrial gene expression in dopamine neurons. Proc Natl Acad Sci USA 110:2360–2365. doi: 10.1073/pnas.1221077110

96. Van Heesbeen HJ, Mesman S, Veenvliet JV, Smidt MP (2013) Epigenetic mechanisms in the development and maintenance of dopaminergic neurons. Development 140:1159–1169. doi: 10.1242/dev.089359

97. Stott SRW, Metzakopian E, Lin W, et al. (2013) Foxa1 and foxa2 are required for the maintenance of dopaminergic properties in ventral midbrain neurons at late embryonic stages. J Neurosci 33:8022–8034. doi: 10.1523/JNEUROSCI.4774-12.2013

98. Metzakopian E, Lin W, Salmon-Divon M, et al. (2012) Genome-wide characterization of Foxa2 targets reveals upregulation of floor plate genes and repression of ventrolateral genes in midbrain dopaminergic progenitors. Development 139:2625–2634. doi: 10.1242/dev.081034

99. Ferri ALM, Lin W, Mavromatakis YE, et al. (2007) Foxa1 and Foxa2 regulate multiple phases of midbrain dopaminergic neuron development in a dosage-dependent manner. Development 134:2761–2769. doi: 10.1242/dev.000141

100. Lin W, Metzakopian E, Mavromatakis YE, et al. (2009) Foxa1 and Foxa2 function both upstream of and cooperatively with Lmx1a and Lmx1b in a feedforward loop promoting mesodiencephalic dopaminergic neuron development. Dev Biol 333:386–396. doi: 10.1016/j.ydbio.2009.07.006

101. Kittappa R, Chang WW, Awatramani RB, McKay RDG (2007) The foxa2 Gene Controls the

Birth and Spontaneous Degeneration of Dopamine Neurons in Old Age. PLoS Biol 5:e325. doi: 10.1371/journal.pbio.0050325

102. Hanks M, Wurst W, Anson-Cartwright L, et al. (1995) Rescue of the En-1 mutant phenotype by replacement of En-1 with En-2. Science 269:679–682.

103. Sgadò P, Viaggi C, Pinna A, et al. (2011) Behavioral, Neurochemical, and Electrophysiological Changes in an Early Spontaneous Mouse Model of Nigrostriatal Degeneration. Neurotox Res 20:170–181. doi: 10.1007/s12640-010-9232-9

104. Andressoo J-O, Saarma M (2008) Signalling mechanisms underlying development and maintenance of dopamine neurons. Curr Opin Neurobiol 18:297–306. doi: 10.1016/j.conb.2008.07.005

105. Joksimovic M, Awatramani R (2014) Wnt/β-catenin signaling in midbrain dopaminergic neuron specification and neurogenesis. J Mol Cell Biol 6:27–33. doi: 10.1093/jmcb/mjt043

106. Kipp M, Karakaya S, Pawlak J, et al. (2006) Estrogen and the development and protection of nigrostriatal dopaminergic neurons: concerted action of a multitude of signals, protective molecules, and growth factors. Front Neuroendocrinol 27:376–390. doi: 10.1016/j.yfrne.2006.07.001

107. Aron L, Klein R (2011) Repairing the parkinsonian brain with neurotrophic factors. Trends in Neurosciences 34:88–100. doi: 10.1016/j.tins.2010.11.001

108. Howells DW, Porritt MJ, Wong JY, et al. (2000) Reduced BDNF mRNA expression in the Parkinson’s disease substantia nigra. Exp Neurol 166:127–135. doi: 10.1006/exnr.2000.7483

109. Peng C, Fan S, Li X, et al. (2007) Overexpression of pitx3 upregulates expression of BDNF and GDNF in SH-SY5Y cells and primary ventral mesencephalic cultures. FEBS Lett 581:1357–1361. doi: 10.1016/j.febslet.2007.02.054

110. Yang D, Peng C, Li X, et al. (2008) Pitx3-transfected astrocytes secrete brain-derived neurotrophic factor and glial cell line-derived neurotrophic factor and protect dopamine neurons in mesencephalon cultures. J Neurosci Res 86:3393–3400. doi: 10.1002/jnr.21774

111. Yu L, Saarma M, Arumäe U (2008) Death receptors and caspases but not mitochondria are activated in the GDNF- or BDNF-deprived dopaminergic neurons. J Neurosci 28:7467–7475. doi: 10.1523/JNEUROSCI.1877-08.2008

112. Clark J, Silvaggi JM, Kiselak T, et al. (2012) Pgc-1α overexpression downregulates Pitx3 and increases susceptibility to MPTP toxicity associated with decreased Bdnf. PLoS ONE 7:e48925. doi: 10.1371/journal.pone.0048925

113. St-Pierre J, Drori S, Uldry M, et al. (2006) Suppression of reactive oxygen species and neurodegeneration by the PGC-1 transcriptional coactivators. Cell 127:397–408. doi: 10.1016/j.cell.2006.09.024

114. Zheng B, Liao Z, Locascio JJ, et al. (2010) PGC-1?, A Potential Therapeutic Target for Early Intervention in Parkinson’s Disease. Sci Transl Med 2:52ra73. doi: 10.1126/scitranslmed.3001059

115. Kramer ER, Aron L, Ramakers GMJ, et al. (2007) Absence of Ret signaling in mice causes progressive and late degeneration of the nigrostriatal system. PLoS Biol 5:e39. doi:

10.1371/journal.pbio.0050039

116. Pascual A, Hidalgo-Figueroa M, Piruat JI, et al. (2008) Absolute requirement of GDNF for adult catecholaminergic neuron survival. Nat Neurosci 11:755–761. doi: 10.1038/nn.2136

117. Blum M (1998) A null mutation in TGF-alpha leads to a reduction in midbrain dopaminergic neurons in the substantia nigra. Nat Neurosci 1:374–377. doi: 10.1038/1584

118. Moqrich A, Earley TJ, Watson J, et al. (2004) Expressing TrkC from the TrkA locus causes a subset of dorsal root ganglia neurons to switch fate. Nat Neurosci 7:812–818. doi: 10.1038/nn1283

119. Bourane S, Garces A, Venteo S, et al. (2009) Low-threshold mechanoreceptor subtypes selectively express MafA and are specified by Ret signaling. Neuron 64:857–870. doi: 10.1016/j.neuron.2009.12.004

120. Partanen J (2007) FGF signalling pathways in development of the midbrain and anterior hindbrain. J Neurochem 101:1185–1193. doi: 10.1111/j.1471-4159.2007.04463.x

121. Shtutman M, Zhurinsky J, Simcha I, et al. (1999) The cyclin D1 gene is a target of the beta-catenin/LEF-1 pathway. Proc Natl Acad Sci USA 96:5522–5527.

122. Ratzka A, Baron O, Stachowiak MK, Grothe C (2012) Fibroblast growth factor 2 regulates dopaminergic neuron development in vivo. J Neurochem 122:94–105. doi: 10.1111/j.1471-4159.2012.07768.x

123. Timmer M, Cesnulevicius K, Winkler C, et al. (2007) Fibroblast growth factor (FGF)-2 and FGF receptor 3 are required for the development of the substantia nigra, and FGF-2 plays a crucial role for the rescue of dopaminergic neurons after 6-hydroxydopamine lesion. J Neurosci 27:459–471. doi: 10.1523/JNEUROSCI.4493-06.2007

124. Baron O, Ratzka A, Grothe C (2012) Fibroblast growth factor 2 regulates adequate nigrostriatal pathway formation in mice. J Comp Neurol 520:3949–3961. doi: 10.1002/cne.23138

125. Blak AA, Naserke T, Saarimäki-Vire J, et al. (2007) Fgfr2 and Fgfr3 are not required for patterning and maintenance of the midbrain and anterior hindbrain. Developmental Biology 303:231–243. doi: 10.1016/j.ydbio.2006.11.008

126. Trokovic R, Trokovic N, Hernesniemi S, et al. (2003) FGFR1 is independently required in both developing mid- and hindbrain for sustained response to isthmic signals. EMBO J 22:1811–1823. doi: 10.1093/emboj/cdg169

127. Jukkola T, Lahti L, Naserke T, et al. (2006) FGF regulated gene-expression and neuronal differentiation in the developing midbrain-hindbrain region. Dev Biol 297:141–157. doi: 10.1016/j.ydbio.2006.05.002

128. Saarimäki-Vire J, Peltopuro P, Lahti L, et al. (2007) Fibroblast growth factor receptors cooperate to regulate neural progenitor properties in the developing midbrain and hindbrain. J Neurosci 27:8581–8592. doi: 10.1523/JNEUROSCI.0192-07.2007

129. Klejbor I, Myers JM, Hausknecht K, et al. (2006) Fibroblast growth factor receptor signaling affects development and function of dopamine neurons - inhibition results in a schizophrenia-like syndrome in transgenic mice. J Neurochem 97:1243–1258. doi:

10.1111/j.1471-4159.2006.03754.x

130. Itoh N, Ohta H (2013) Roles of FGF20 in dopaminergic neurons and Parkinson’s disease. Front Mol Neurosci 6:15. doi: 10.3389/fnmol.2013.00015

131. Murase S, McKay RD (2006) A specific survival response in dopamine neurons at most risk in Parkinson’s disease. J Neurosci 26:9750–9760. doi: 10.1523/JNEUROSCI.2745-06.2006

132. Wallén A, Zetterström RH, Solomin L, et al. (1999) Fate of mesencephalic AHD2-expressing dopamine progenitor cells in NURR1 mutant mice. Exp Cell Res 253:737–746. doi: 10.1006/excr.1999.4691

133. Chung S, Hedlund E, Hwang M, et al. (2005) The homeodomain transcription factor Pitx3 facilitates differentiation of mouse embryonic stem cells into AHD2-expressing dopaminergic neurons. Mol Cell Neurosci 28:241–252. doi: 10.1016/j.mcn.2004.09.008

134. De Urquiza AM, Liu S, Sjöberg M, et al. (2000) Docosahexaenoic acid, a ligand for the retinoid X receptor in mouse brain. Science 290:2140–2144.

135. Papanikolaou T, Amano T, Lennington J, et al. (2009) In-vitro analysis of Pitx3 in mesodiencephalic dopaminergic neuron maturation. Eur J Neurosci 29:2264–2275. doi: 10.1111/j.1460-9568.2009.06784.x

136. Castro DS, Hermanson E, Joseph B, et al. (2001) Induction of cell cycle arrest and morphological differentiation by Nurr1 and retinoids in dopamine MN9D cells. J Biol Chem 276:43277–43284. doi: 10.1074/jbc.M107013200

137. Chang Y-L, Chen S-J, Kao C-L, et al. (2012) Docosahexaenoic acid promotes dopaminergic differentiation in induced pluripotent stem cells and inhibits teratoma formation in rats with Parkinson-like pathology. Cell Transplant 21:313–332. doi: 10.3727/096368911X580572

138. Jeong H, Kim M-S, Kim S-W, et al. (2006) Regulation of tyrosine hydroxylase gene expression by retinoic acid receptor. J Neurochem 98:386–394. doi: 10.1111/j.1471-4159.2006.03866.x

139. Katsuki H, Kurimoto E, Takemori S, et al. (2009) Retinoic acid receptor stimulation protects midbrain dopaminergic neurons from inflammatory degeneration via BDNF-mediated signaling. J Neurochem 110:707–718. doi: 10.1111/j.1471-4159.2009.06171.x

140. Schilling TF, Nie Q, Lander AD (2012) Dynamics and precision in retinoic acid morphogen gradients. Curr Opin Genet Dev 22:562–569. doi: 10.1016/j.gde.2012.11.012

141. Zhang L, Radtke K, Zheng L, et al. (2012) Noise drives sharpening of gene expression boundaries in the zebrafish hindbrain. Mol Syst Biol 8:613. doi: 10.1038/msb.2012.45

142. Qin P, Haberbusch JM, Soprano KJ, Soprano DR (2004) Retinoic acid regulates the expression of PBX1, PBX2, and PBX3 in P19 cells both transcriptionally and post-translationally. J Cell Biochem 92:147–163. doi: 10.1002/jcb.20057

143. Vitobello A, Ferretti E, Lampe X, et al. (2011) Hox and Pbx Factors Control Retinoic Acid Synthesis during Hindbrain Segmentation. Developmental Cell 20:469–482. doi: 10.1016/j.devcel.2011.03.011

144. Kam RKT, Shi W, Chan SO, et al. (2013) Dhrs3 protein attenuates retinoic acid signaling and

is required for early embryonic patterning. J Biol Chem 288:31477–31487. doi: 10.1074/jbc.M113.514984

145. White RJ, Nie Q, Lander AD, Schilling TF (2007) Complex regulation of cyp26a1 creates a robust retinoic acid gradient in the zebrafish embryo. PLoS Biol 5:e304. doi: 10.1371/journal.pbio.0050304

146. Topletz AR, Thatcher JE, Zelter A, et al. (2012) Comparison of the function and expression of CYP26A1 and CYP26B1, the two retinoic acid hydroxylases. Biochem Pharmacol 83:149–163. doi: 10.1016/j.bcp.2011.10.007

147. Galter D, Buervenich S, Carmine A, et al. (2003) ALDH1 mRNA: presence in human dopamine neurons and decreases in substantia nigra in Parkinson’s disease and in the ventral tegmental area in schizophrenia. Neurobiology of Disease 14:637–647. doi: 10.1016/j.nbd.2003.09.001

148. Bossers K, Meerhoff G, Balesar R, et al. (2009) Analysis of Gene Expression in Parkinson’s Disease: Possible Involvement of Neurotrophic Support and Axon Guidance in Dopaminergic Cell Death. Brain Pathology 19:91–107. doi: 10.1111/j.1750-3639.2008.00171.x

149. Moran LB, Duke DC, Deprez M, et al. (2006) Whole genome expression profiling of the medial and lateral substantia nigra in Parkinson’s disease. Neurogenetics 7:1–11. doi: 10.1007/s10048-005-0020-2

150. Kurauchi Y, Hisatsune A, Isohama Y, et al. (2011) Midbrain dopaminergic neurons utilize nitric oxide/cyclic GMP signaling to recruit ERK that links retinoic acid receptor stimulation to up-regulation of BDNF. J Neurochem 116:323–333. doi: 10.1111/j.1471-4159.2010.06916.x

151. Moon YS, Smas CM, Lee K, et al. (2002) Mice lacking paternally expressed Pref-1/Dlk1 display growth retardation and accelerated adiposity. Mol Cell Biol 22:5585–5592.

152. Abdallah BM, Jensen CH, Gutierrez G, et al. (2004) Regulation of human skeletal stem cells differentiation by Dlk1/Pref-1. J Bone Miner Res 19:841–852. doi: 10.1359/JBMR.040118

153. Enomoto H, Furuichi T, Zanma A, et al. (2004) Runx2 deficiency in chondrocytes causes adipogenic changes in vitro. J Cell Sci 117:417–425. doi: 10.1242/jcs.00866

154. Hansen LH, Madsen B, Teisner B, et al. (1998) Characterization of the inhibitory effect of growth hormone on primary preadipocyte differentiation. Mol Endocrinol 12:1140–1149.

155. Smas CM, Chen L, Zhao L, et al. (1999) Transcriptional repression of pref-1 by glucocorticoids promotes 3T3-L1 adipocyte differentiation. J Biol Chem 274:12632–12641.

156. Christophersen NS, Grønborg M, Petersen TN, et al. (2007) Midbrain expression of Delta-like 1 homologue is regulated by GDNF and is associated with dopaminergic differentiation. Exp Neurol 204:791–801. doi: 10.1016/j.expneurol.2007.01.014

157. Jensen CH, Meyer M, Schroder HD, et al. (2001) Neurons in the monoaminergic nuclei of the rat and human central nervous system express FA1/dlk. Neuroreport 12:3959–3963.

158. Bauer M, Szulc J, Meyer M, et al. (2008) Delta-like 1 participates in the specification of ventral midbrain progenitor derived dopaminergic neurons. J Neurochem 104:1101–1115. doi: 10.1111/j.1471-4159.2007.05037.x

159. Kim Y (2010) Effect of retinoic acid and delta-like 1 homologue (DLK1) on differentiation in neuroblastoma. Nutr Res Pract 4:276–282. doi: 10.4162/nrp.2010.4.4.276

160. Armengol J, Villena JA, Hondares E, et al. (2012) Pref-1 in brown adipose tissue: specific involvement in brown adipocyte differentiation and regulatory role of C/EBPδ. The Biochemical Journal. doi: 10.1042/BJ20111714

161. Hudak CS, Sul HS (2013) Pref-1, a gatekeeper of adipogenesis. Front Endocrinol (Lausanne) 4:79. doi: 10.3389/fendo.2013.00079

162. Müller D, Cherukuri P, Henningfeld K, et al. (2014) Dlk1 promotes a fast motor neuron biophysical signature required for peak force execution. Science 343:1264–1266. doi: 10.1126/science.1246448

163. Raghunandan R, Ruiz-Hidalgo M, Jia Y, et al. (2008) Dlk1 influences differentiation and function of B lymphocytes. Stem Cells Dev 17:495–507. doi: 10.1089/scd.2007.0102

164. Wheeler SR, Stagg SB, Crews ST (2008) Multiple Notch signaling events control Drosophila CNS midline neurogenesis, gliogenesis and neuronal identity. Development 135:3071–3079. doi: 10.1242/dev.022343

165. Tio M, Toh J, Fang W, et al. (2011) Asymmetric Cell Division and Notch Signaling Specify Dopaminergic Neurons in Drosophila. PLoS ONE 6:e26879. doi: 10.1371/journal.pone.0026879

166. Falix FA, Aronson DC, Lamers WH, Gaemers IC (2012) Possible roles of DLK1 in the Notch pathway during development and disease. Biochimica Et Biophysica Acta. doi: 10.1016/j.bbadis.2012.02.003

167. Haubenberger D, Reinthaler E, Mueller JC, et al. (2011) Association of transcription factor polymorphisms PITX3 and EN1 with Parkinson’s disease. Neurobiol Aging 32:302–307. doi: 10.1016/j.neurobiolaging.2009.02.015

168. Bergman O, Håkansson A, Westberg L, et al. (2010) PITX3 polymorphism is associated with early onset Parkinson’s disease. Neurobiol Aging 31:114–117. doi: 10.1016/j.neurobiolaging.2008.03.008

169. Fuchs J, Mueller JC, Lichtner P, et al. (2009) The transcription factor PITX3 is associated with sporadic Parkinson’s disease. Neurobiol Aging 30:731–738. doi: 10.1016/j.neurobiolaging.2007.08.014

170. Zheng K, Heydari B, Simon DK (2003) A common NURR1 polymorphism associated with Parkinson disease and diffuse Lewy body disease. Arch Neurol 60:722–725. doi: 10.1001/archneur.60.5.722

171. Bergman O, Håkansson A, Westberg L, et al. (2009) Do polymorphisms in transcription factors LMX1A and LMX1B influence the risk for Parkinson’s disease? J Neural Transm 116:333–338. doi: 10.1007/s00702-009-0187-z

172. Grünblatt E, Zehetmayer S, Jacob CP, et al. (2010) Pilot study: peripheral biomarkers for diagnosing sporadic Parkinson’s disease. J Neural Transm 117:1387–1393. doi: 10.1007/s00702-010-0509-1

173. Karamohamed S, Latourelle JC, Racette BA, et al. (2005) BDNF genetic variants are

associated with onset age of familial Parkinson disease: GenePD Study. Neurology 65:1823–1825. doi: 10.1212/01.wnl.0000187075.81589.fd

174. Pihlstrøm L, Axelsson G, Bjørnarå KA, et al. (2013) Supportive evidence for 11 loci from genome-wide association studies in Parkinson’s disease. Neurobiol Aging 34:1708.e7–13. doi: 10.1016/j.neurobiolaging.2012.10.019

175. Goris A, Williams-Gray CH, Foltynie T, et al. (2007) Investigation of TGFB2 as a candidate gene in multiple sclerosis and Parkinson’s disease. J Neurol 254:846–848. doi: 10.1007/s00415-006-0414-6

176. Sulzer D (2007) Multiple hit hypotheses for dopamine neuron loss in Parkinson’s disease. Trends Neurosci 30:244–250. doi: 10.1016/j.tins.2007.03.009

177. Alvarez-Fischer D, Fuchs J, Castagner F, et al. (2011) Engrailed protects mouse midbrain dopaminergic neurons against mitochondrial complex I insults. Nat Neurosci 14:1260–1266. doi: 10.1038/nn.2916

178. L’honoré A, Commère P-H, Ouimette J-F, et al. (2014) Redox regulation by pitx2 and pitx3 is critical for fetal myogenesis. Dev Cell 29:392–405. doi: 10.1016/j.devcel.2014.04.006

179. Spatazza J, Di Lullo E, Joliot A, et al. (2013) Homeoprotein signaling in development, health, and disease: a shaking of dogmas offers challenges and promises from bench to bed. Pharmacol Rev 65:90–104. doi: 10.1124/pr.112.006577

180. Chan CS, Guzman JN, Ilijic E, et al. (2007) “Rejuvenation” protects neurons in mouse models of Parkinson’s disease. Nature 447:1081–1086. doi: 10.1038/nature05865

181. Khaliq ZM, Bean BP (2010) Pacemaking in dopaminergic ventral tegmental area neurons: depolarizing drive from background and voltage-dependent sodium conductances. J Neurosci 30:7401–7413. doi: 10.1523/JNEUROSCI.0143-10.2010

182. Chan CS, Gertler TS, Surmeier DJ (2009) Calcium homeostasis, selective vulnerability and Parkinson’s disease. Trends in Neurosciences 32:249–256. doi: 10.1016/j.tins.2009.01.006

183. Mosharov EV, Larsen KE, Kanter E, et al. (2009) Interplay between cytosolic dopamine, calcium, and alpha-synuclein causes selective death of substantia nigra neurons. Neuron 62:218–229. doi: 10.1016/j.neuron.2009.01.033

184. Yamada T, McGeer PL, Baimbridge KG, McGeer EG (1990) Relative sparing in Parkinson’s disease of substantia nigra dopamine neurons containing calbindin-D28K. Brain Res 526:303–307.

185. Liss B, Haeckel O, Wildmann J, et al. (2005) K-ATP channels promote the differential degeneration of dopaminergic midbrain neurons. Nat Neurosci 8:1742–1751. doi: 10.1038/nn1570

186. Arenas E (2010) Towards stem cell replacement therapies for Parkinson’s disease. Biochem Biophys Res Commun 396:152–156. doi: 10.1016/j.bbrc.2010.04.037

187. Gaillard A, Jaber M (2011) Rewiring the brain with cell transplantation in Parkinson’s disease. Trends Neurosci 34:124–133. doi: 10.1016/j.tins.2011.01.003

188. Toulouse A, Sullivan AM (2008) Progress in Parkinson’s disease-where do we stand? Prog

Neurobiol 85:376–392. doi: 10.1016/j.pneurobio.2008.05.003

189. Kriks S, Shim J-W, Piao J, et al. (2011) Dopamine neurons derived from human ES cells efficiently engraft in animal models of Parkinson’s disease. Nature 480:547–551. doi: 10.1038/nature10648

190. Ganat YM, Calder EL, Kriks S, et al. (2012) Identification of embryonic stem cell-derived midbrain dopaminergic neurons for engraftment. J Clin Invest 122:2928–2939. doi: 10.1172/JCI58767

191. Caiazzo M, Dell’Anno MT, Dvoretskova E, et al. (2011) Direct generation of functional dopaminergic neurons from mouse and human fibroblasts. Nature 476:224–227. doi: 10.1038/nature10284

192. Wernig M, Zhao J-P, Pruszak J, et al. (2008) Neurons derived from reprogrammed fibroblasts functionally integrate into the fetal brain and improve symptoms of rats with Parkinson’s disease. Proc Natl Acad Sci USA 105:5856–5861. doi: 10.1073/pnas.0801677105

193. Hedlund E, Pruszak J, Lardaro T, et al. (2008) Embryonic stem cell-derived Pitx3-enhanced green fluorescent protein midbrain dopamine neurons survive enrichment by fluorescence-activated cell sorting and function in an animal model of Parkinson’s disease. Stem Cells 26:1526–1536. doi: 10.1634/stemcells.2007-0996

194. Salti A, Nat R, Neto S, et al. (2013) Expression of early developmental markers predicts the efficiency of embryonic stem cell differentiation into midbrain dopaminergic neurons. Stem Cells Dev 22:397–411. doi: 10.1089/scd.2012.0238

195. Hwang D-Y, Kim D-S, Kim D-W (2010) Human ES and iPS cells as cell sources for the treatment of Parkinson’s disease: current state and problems. J Cell Biochem 109:292–301. doi: 10.1002/jcb.22411

196. Roessler R, Smallwood S, Veenvliet J, et al. (2014) Detailed analysis of the genetic and epigenetic signature of iPS cell-derived mesodiencephalic dopaminergic neurons. Stem Cell Reports

197. Bar-Nur O, Russ HA, Efrat S, Benvenisty N (2011) Epigenetic memory and preferential lineage-specific differentiation in induced pluripotent stem cells derived from human pancreatic islet beta cells. Cell Stem Cell 9:17–23. doi: 10.1016/j.stem.2011.06.007

198. Kim K, Doi A, Wen B, et al. (2010) Epigenetic memory in induced pluripotent stem cells. Nature 467:285–290. doi: 10.1038/nature09342

199. Momčilović O, Liu Q, Swistowski A, et al. (2013) Genome Wide Profiling of Dopaminergic Neurons Derived from Human Embryonic and Induced Pluripotent Stem Cells. Stem Cells Dev. doi: 10.1089/scd.2013.0412

200. Cooper O, Parmar M, Isacson O (2012) Chapter 13 - Characterization and criteria of embryonic stem and induced pluripotent stem cells for a dopamine replacement therapy. In: Stephen B. Dunnett and Anders Björklund (ed) Progress in Brain Research. Elsevier, pp 265–276

201. Grealish S, Jönsson ME, Li M, et al. (2010) The A9 dopamine neuron component in grafts of ventral mesencephalon is an important determinant for recovery of motor function in a rat model of Parkinson’s disease. Brain 133:482–495. doi: 10.1093/brain/awp328

202. Thompson L, Barraud P, Andersson E, et al. (2005) Identification of dopaminergic neurons of nigral and ventral tegmental area subtypes in grafts of fetal ventral mesencephalon based on cell morphology, protein expression, and efferent projections. J Neurosci 25:6467–6477. doi: 10.1523/JNEUROSCI.1676-05.2005

203. Allodi I, Hedlund E (2014) Directed midbrain and spinal cord neurogenesis from pluripotent stem cells to model development and disease in a dish. Front Neurosci 8:109. doi: 10.3389/fnins.2014.00109

204. Roeper J (2013) Dissecting the diversity of midbrain dopamine neurons. Trends Neurosci 36:336–342. doi: 10.1016/j.tins.2013.03.003

205. Liss B, Roeper J (2008) Individual dopamine midbrain neurons: functional diversity and flexibility in health and disease. Brain Res Rev 58:314–321. doi: 10.1016/j.brainresrev.2007.10.004

206. Kirkeby A, Grealish S, Wolf DA, et al. (2012) Generation of regionally specified neural progenitors and functional neurons from human embryonic stem cells under defined conditions. Cell Rep 1:703–714. doi: 10.1016/j.celrep.2012.04.009

207. Sánchez-Danés A, Consiglio A, Richaud Y, et al. (2012) Efficient generation of A9 midbrain dopaminergic neurons by lentiviral delivery of LMX1A in human embryonic stem cells and induced pluripotent stem cells. Hum Gene Ther 23:56–69. doi: 10.1089/hum.2011.054

208. Tian L-P, Zhang S, Zhang Y-J, et al. (2012) Lmx1b can promote the differentiation of embryonic stem cells to dopaminergic neurons associated with Parkinson’s disease. Biotechnol Lett 34:1167–1174. doi: 10.1007/s10529-012-0888-5

209. Roy NS, Cleren C, Singh SK, et al. (2006) Functional engraftment of human ES cell-derived dopaminergic neurons enriched by coculture with telomerase-immortalized midbrain astrocytes. Nat Med 12:1259–1268. doi: 10.1038/nm1495

210. Narytnyk A, Verdon B, Loughney A, et al. (2014) Differentiation of Human Epidermal Neural Crest Stem Cells (hEPI-NCSC) into Virtually Homogenous Populations of Dopaminergic Neurons. Stem Cell Rev. doi: 10.1007/s12015-013-9493-9

211. Mendez I, Sanchez-Pernaute R, Cooper O, et al. (2005) Cell type analysis of functional fetal dopamine cell suspension transplants in the striatum and substantia nigra of patients with Parkinson’s disease. Brain 128:1498–1510. doi: 10.1093/brain/awh510

212. Fu Y, Yuan Y, Halliday G, et al. (2012) A cytoarchitectonic and chemoarchitectonic analysis of the dopamine cell groups in the substantia nigra, ventral tegmental area, and retrorubral field in the mouse. Brain Struct Funct 217:591–612. doi: 10.1007/s00429-011-0349-2

213. Reyes S, Fu Y, Double K, et al. (2012) GIRK2 expression in dopamine neurons of the substantia nigra and ventral tegmental area. J Comp Neurol 520:2591–2607. doi: 10.1002/cne.23051

214. Greene JG, Dingledine R, Greenamyre JT (2005) Gene expression profiling of rat midbrain dopamine neurons: implications for selective vulnerability in parkinsonism. Neurobiol Dis 18:19–31. doi: 10.1016/j.nbd.2004.10.003

215. Grimm J, Mueller A, Hefti F, Rosenthal A (2004) Molecular basis for catecholaminergic neuron diversity. PNAS 101:13891–13896. doi: 10.1073/pnas.0405340101

216. D’Amato RJ, Lipman ZP, Snyder SH (1986) Selectivity of the parkinsonian neurotoxin MPTP: toxic metabolite MPP+ binds to neuromelanin. Science 231:987–989.

217. D’Amato RJ, Alexander GM, Schwartzman RJ, et al. (1987) Evidence for neuromelanin involvement in MPTP-induced neurotoxicity. Nature 327:324–326. doi: 10.1038/327324a0

218. Fishell G, Heintz N (2013) The neuron identity problem: form meets function. Neuron 80:602–612. doi: 10.1016/j.neuron.2013.10.035

219. Stamatakis AM, Jennings JH, Ung RL, et al. (2013) A unique population of ventral tegmental area neurons inhibits the lateral habenula to promote reward. Neuron 80:1039–1053. doi: 10.1016/j.neuron.2013.08.023

220. Lammel S, Hetzel A, Häckel O, et al. (2008) Unique properties of mesoprefrontal neurons within a dual mesocorticolimbic dopamine system. Neuron 57:760–773. doi: 10.1016/j.neuron.2008.