The role of developmental transcription factors in adult midbrain dopaminergic neurons

Abstract Introduction Mesodiencephalic dopamine neurons play crucial roles in the control of a variety of brain functions, including voluntary movement and behavioural processes such as mood, reward, and attention. Degeneration of mesodiencephalic dopaminergic neurons also represents one of the principal pathological features of Parkinson’s disease. Recent progress led to the identification of transcription factors that are expressed in dopaminergic progenitors and are required for their differentiation. The expression of some of these transcription factors persists into adulthood but their exact functions in postnatal and adult dopaminergic neurons remain puzzling. The objective of this review is to gather recent findings on the potential roles of Foxa1, Foxa2, Nurr1, Pitx3, Otx2, Lmx1a, Lmx1b, En1, and En2 in the maintenance of dopaminergic circuitry throughout adulthood. This maintenance appears to be underpinned by the continued action of developmental transcription factors. The loss of functional alleles of Foxa1/2, Nurr1, Pitx3, or En1/2 seriously impairs the survival dopaminergic neurons, affecting preferentially the subtantia nigra pars compacta. Moreover, for almost all transcription factors reviewed here, a genetic association to Parkinson’s disease has been established. Currently, it is unclear how exactly these developmental transcription factors contribute to the maintenance and survival of the dopaminergic system, but one possible mechanism could be related to transcriptional/translational regulation of mitochondrial bioenergetics and biogenesis. Conclusion This review suggests that developmental transcriptional networks are essential for maturity maintenance of mesodiencephalic dopaminergic neurons. Much remains to be done to dissect the effects of each developmental transcription factor on the functional properties of mDA neurons in adults.


Introduction
Over the last few decades, extensive efforts have been deployed to identify and understand the role of key transcription factors (TFs) mediating mesodiencephalic dopaminergic (mDA) neurons development (Figure 1A-H).The multiple roles of TFs at different embryonic stages have become increasingly clear 1,2 .Several TFs promote or repress the activity of thousands of genes that trigger the morphological and metabolic transformations.During early development, the isthmus organizer (a neuroepithelial signalling centre localized at the midbrain-hindbrain boundary) and the floor plate (a ventral signalling centre) drive pattern formation and the generation of mDA neurons in the developing embryos [3][4][5][6] .During the late stages of development, mDA neurons are organized in three main groups called the substantia nigra pars compacta (SNpc), the ventral tegmental area (VTA), and the retrorubral field (RRF).Dopaminergic neurons from the SNpc form the nigrostriatal pathway while neurons from the VTA and RRF are associated with the mesolimbic/mesocortical pathways.Degeneration or dysfunctions of these neurons (or subpopulations of these neurons) can lead to severe neurological disorders such as Parkinson's disease (PD), schizophrenia, and addiction [7][8][9] .
In addition to their fundamental role during mDA neuron development, a large number of TFs remain expressed in adulthood while dopaminergic neuronal maturation is completed (Figure 1I-L).Gaining knowledge with regard to this sustained expression of developmental TF into later life could provide precious mechanistic insights into the maintenance of mDA neurons.This review summarizes the current data on the transcriptional control of dopaminergic neurons in the adult midbrain.By surveying recent studies on Foxa1, Foxa2, Nurr1, Pitx3, Otx2, Lmx1a, Lmx1b, and En1/2, the purpose is to gain information about the functional significance of each one in the adult midbrain but more importantly is to begin to develop a general understanding of how these developmental TFs contribute to maintenance and survival of the dopaminergic system.We also discuss recent insights into the role of these TFs in mitochondrial transcriptional regulation and their potential implications in PD.

Discussion
The authors have referenced some of their own studies in this review.The protocols of these studies have been approved by the relevant ethics and Lmx1b 21 and subsequently continue to function cooperatively with them 22 .Loss-of-function studies revealed that Foxa1 and Foxa2 are also important for maturation of mDA neurons by regulating Nurr1 and En1 in immature mDA neurons.During early and late differentiation stages of mDA neurons, they regulate the expression of Pitx3, tyrosine hydroxylase (TH), dopamine transporter (DAT), vesicular monoamine transporter 2 (VMAT2), and aromatic L-amino acid decarboxylase (AADC) 18,[22][23][24][25] .and Foxa2 are expressed in a wide domain in ventral mesodiencephalic progenitors [15][16][17] .Foxa1 and Foxa2 are known to play a central role at various steps of mDA neuron development.Initially, they are required, in a gene dosage-dependent manner, for specification of mDA progenitors 18,19 .They regulate expression of sonic hedgehog (Shh) signalling components 20,21 and neurogenesis by controlling neurogenin 2 (Ngn2) expression 18 .They participate in mDA neurons differentiation by controlling the expression of Lmx1a committees related to the institution in which they were performed.

Foxa1 and Foxa2
The forkhead box protein A (Foxa) family of transcription factors participate in transcriptional programmes governing embryonic development and organogenesis of a number of metabolic and endocrine-related tissues 10,11 including the brain 12,13 .In mammals, the Foxa subfamily has three members: Foxa1, Foxa2, and Foxa3 (previously termed, HNF3α, HNF3β, and HNF3γ) 14 but only Foxa1 A, Representation of a mouse embryo at E12.5 and a section at midbrain level (dashed red line).Ventral midbrain region is boxed and magnified in B. The generation of mDA neurons from neural progenitors follows three sequential steps defining mDA lineage into mDA progenitors, immature neurons, and mature neurons (B).Foxa1/2, Otx2, Lmx1a/b, and En1/2 genes are already expressed in mDA progenitors by E9.5 and their expression persists in both immature and mature mDA neurons (C-F).At E12.5, only a fraction of postmitotic mDA neurons express Otx2 (D) 128 Nurr1 expression is initiated in immature postmitotic mDA neurons (G) whereas Pitx3 appears only in mature mDA neurons (H).Note that Otx2, En1/2, Foxa1/2 are present in other non-DA cell populations in ventral midbrain 18,20,128,154 .Some cells outside mDA domain also express Nurr1.A midbrain section from adult mouse is illustrated in I and the dashed box is magnified in J-L to show expression of the different transcription factors in VTA and SNpc.Nurr1, En1/2, Lmxa/b, and Foxa1/2 genes remain expressed in adult mDA neurons of both VTA and SNpc (J) while Otx2 is only present in central and ventral portion of the VTA (K).Pitx3 gene is expressed in both VTA and SNpc (L) 61,155 , but protein levels appear lower (or absent) in dorsal SNpc and in scattered mDA neurons in VTA 71 .postmitotic and mature mDA neurons 42,48 .Conditional inactivation of Nurr1 in postmitotic mDA neurons during late development resulted in rapid loss of striatal dopamine, loss of mDA markers, and neuron degeneration.A similar but slower progressing loss of striatal dopamine and mDA axonal markers was observed following ablation of Nurr1 in adult brain 42 .Nurr1 deletion in mature mDA neurons produced a more severe loss of dopamine neurons in the SNpc than in the VTA and consequently engendered a Parkinson'slike phenotype 42 .Moreover, Nurr1 ablation specifically in adult mice results in a distinct fibre pathology that affects both dendrites and axons 49 .
Nurr1 heterozygous mutant mice are more vulnerable to injury induced by MPTP, the classic toxin-induced animal models of PD 50 and to toxicity of methamphetamine exposure 51 .The role of Nurr1 in the maintenance of mDA neurons is also highlighted by the identification of several mutations in the genes associated with PD [52][53][54][55][56] .To identify Nurr1-regulated genes in mature DA neurons, Kadkhodaei et al. 49 performed gene expression profiling on conditional Nurr1 gene-targeted mice.Nuclear-encoded mitochondrial genes were identified as the major functional category of Nurr1-regulated target genes 49 .Nuclear-encoded mitochondrial genes and mRNA levels of each oxidative phosphorylation component are relevant for mitochondrial regulation and energy production.Long-and short-term axon survival depends on mitochondria 57 and since Nurr1 targets several nuclear-encoded mitochondrial genes, deregulation of axonal mitochondrial functions might contribute to the progressive retrograde degeneration of mDA neurons in Nurr1-ablated adult mice.Further studies are required to elucidate this issue.

Pitx3
The Pitx3 gene encodes a homeobox bicoid-like transcription factor Pitx3 mDA neurons but rather to the loss of their dopaminergic properties 25 .Further studies looking at specific inactivation of Foxa1/2 in adult mDA neurons would be necessary to fully elucidate the role of these TFs in the maintenance of mDA neurons.

Nurr1
The Nuclear receptor related 1 (Nurr1 also known as NR4A2) gene encodes an orphan member of the steroid/thyroid hormone receptor super family.These orphan receptors are a group for which endogenous ligands do not exist or have not been identified yet.Nurr1 is an immediate early gene activated in response to extracellular stresses 31,32 , which is predominantly, but not exclusively expressed in the brain 33 .In the developing central nervous system, Nurr1 is primarily known for its association with mDA neuron differentiation [33][34][35][36] .Nurr1-defficient mice result in the absence of mDA neurons [37][38] .Nurr1 directly transactivates the promoter activity of the TH gene in a cell type-dependent manner and is, for that reason, necessary for the region-specific expression of TH in mDA neurons 39 .Nurr1 could be defined as a master regulator in the induction of the neurotransmitter phenotypic identity of mDA neurons 1 .In addition to TH regulation, Nurr1 also controls the expression of several key proteins that are necessary for the synthesis and regulation of dopamine including DAT, VMAT2, and AADC [40][41][42][43][44][45] .A cross-regulatory model, between Nurr1 and the well-known Wnt/β-catenin signalling components have been described in the development of the mDA system 46 .Similarly, nuclear fibroblast growth factor receptor 1 (FGFR1) signals have been reported to partner with Nurr1 in developing postmitotic and mature mDA neurons 47 .
Expression of Nurr1 in mDA neurons of the SNpc and VTA is maintained in adult life and appears to be critical for the maintenance of a dopaminergic phenotype in both Foxa1 and Foxa2 remain expressed into adulthood 12,13 but their exact roles in the adult brain remain largely unknown.Loss-of-function studies in mice have, however, demonstrated the requirement of Foxa2 in the maintenance of mDA neurons.Foxa2 heterozygous mutant mice spontaneously develop significant motor problems and an associated late-onset degeneration of mDA neurons.The initial deficit is asymmetric and preferentially affects dopamine neurons of the SNpc 23 while leaving the VTA intact 23 .The loss of a single functional allele does not compromise the development of mDA neurons but impairs their survival in the postnatal and adult brain.It is possible that Foxa1 and Foxa2, while redundant during development, have evolved divergent roles in the mature brain.A comparable hypothesis has recently been confirmed in the liver for these two TFs 26 .If this is the case in mDA neurons, it might explain the deficient compensation observed in adulthood.
Similar to what it is observed in human patient with PD, nigral dopaminergic neurons are the first to degenerate in Foxa2 heterozygous mutant mice.Based on anatomical reports showing an impressive difference in axonal arborisation between mDA neurons from SNpc and VTA [27][28][29] , it is reasonable to speculate that SNpc neurons have a higher energetic demand and are likely more vulnerable to metabolic changes.Loss of a single Foxa allele could result in a reduced capacity to withstand endogenous cell stress, which could further cause a progressive cell loss 30 .
The contribution of Foxa1 and Foxa2 to the maintenance of mDA neurons is also stressed by a recent in vivo study in which Foxa1 and Foxa2 were specifically deleted in postmitotic mDA neurons.In these animals, a significant reduction of TH-positive neurons in the SNpc was observed.Surprisingly, this diminution is not attributed to the death of by Pgc-1α 84 .Over-expression of Pgc-1α in the SNpc unexpectedly results in dopamine depletion associated with lower levels of Pitx3 and enhances susceptibility to MPTP 82 .While the authors were exploring the efficacy of Pgc-1α as a therapeutic target, the forced expression of Pgc-1α probably disrupts the finetune regulation of metabolic activity in the mitochondrial expression/ energy balance network.These metabolic changes induced by Pgc-1α could also be explained by an indirect regulation of Pitx3.
An important negative regulatory relationship has also been uncovered in which Pitx3 regulates miR-133b transcription, a conserved micro-RNA.This latter, in turn, was found to suppress Pitx3 expression post-transcriptionally to terminate the developmental signals 85 .It has also been proposed that neurite outgrowth can be regulated by exosome release of miR-133b 86 .MicroRNAs have been implicated in age-associated decline of organ functions and miR-133b is specifically enriched in the midbrain and deficient in PD patients 85 .
Several studies have explored the association between polymorphism in PITX3 gene and the manifestation of PD [87][88][89][90][91][92][93][94] but this led to inconsistent results.A recent meta-analysis of these studies, however, reporting on more than 5000 PD patients and controls, confirmed a significant association of three single nucleotide polymorphisms (SNPs) in the Pitx3 gene and the risk of PD 95 .Thus far, the functional significance of these SNPs remains unknown.All SNPs associated with an increased risk of PD are located in intronic sequences (except for one SNP located in the PITX3 promoter 96 ; and should not be confused with disease-causing amino acid substitutions.Alternatively, intronic SNPs could act as genetic regulators, being associated with exon skipping events, or may impair transcription, alter splicing, and reduce mRNA stability [96][97][98] .vulnerable to degeneration induced by MPTP treatment 71 . Molecular targets of Pitx3 include TH, DAT, and VMAT2 [72][73][74] .Interestingly, these genes are also targets of Nurr1 and Foxa1/2 (see previous sections).Functional cooperation between Nurr1 and Pitx3 75,76 , between En1 and Pitx3 77 , and between Foxa1/2 and Nurr1 25 have also been reported.Alternatively, both Nurr1 and Foxa1/2 have been shown to regulate Pitx3 25,78 .Two neurotrophic factors, BDNF (brain derived neurotrophic factor) and GDNF (glial cell line derived neurotrophic factor), are also regulated by Pitx3 79 .Moreover, GDNF acts as an upstream stimulatory factor of Pitx3 80 .In cultured cells, over-expression of Pitx3 increased the mRNA and protein levels of BDNF and GDNF 79 .The interplay between Pitx3, GDNF, and BDNF appears different during preand postnatal stages 80 .In the adult brain, striatal uptake and retrograde axonal transport of GDNF from axon terminals to the soma, maintains the proper levels of Pitx3 and BDNF expression in SNpc mDA neurons 80 .As the main known function of BDNF throughout adulthood is to enhance synaptic transmission, facilitate synaptic plasticity, and promote synaptic growth 81 , it might contribute to the synaptic maintenance of nigrostriatal mDA neurons over the course of aging.
Interestingly, Clark et al. 82 found that over-expression of Peroxisome proliferator-activated receptor gamma co-activator-1 alpha (Pgc-1α) down-regulates Pitx3 expression in the SNpc 82 .Pgc-1α has been shown to positively regulate the expression of genes required for mitochondrial biogenesis and the cellular antioxidant responses, and also to have a newly discovered role in the formation and maintenance of dendritic spines and synapses 83 .Nigral dopaminergic neurons are critically sensitive to the modifications in mitochondrial homeostasis induced (previously named Ptx3).During embryogenesis, Pitx3 is transiently present in the eye lens and in skeletal muscles, but constantly expressed from E11.5 in mouse mDA neurons where it plays an important role in dopaminergic differentiation 58,59 .The expression pattern of Pitx3 is close to the expression pattern of TH 60 .After birth, Pitx3 expression is confined to mDA neurons in mammals 61 .
The Pitx3-deficient aphakia mouse strain, discovered in the 1960s, has facilitated the analysis of the function of the Pitx3 TF during development 62 .Although Pitx3 is selectively expressed in mDA neurons of both SNpc and VTA, it is the neurons from the SNpc that are mainly affected in mutant mice lacking Pitx3, while the VTA appears much less affected [58][59][60][63][64][65] . The diferential dependence of mDA neurons on Pitx3 has been associated to the downstream target gene Aldehyde dehydrogenase 2 (Ahd2, also known as Raldh1) which is crucial for the enzymatic production of retinoic acid 66 .Retinoic acid is a pleiotropic activation factor that has several essential functions during brain development that are likely to persist into postnatal life 67 .The main Ahd2 expression site in the brain is a subpopulation of dopaminergic cells within the SNpc and adjoining VTA 68,69 .Retinoic acid also exerts anti-apoptotic and antioxidant activities 70 and may explain, in part, the role of Pitx3 in the survival of the SNpc mDA neurons.The dependence on Pitx3 for the survival of dopaminergic neurons is however not uniform between all mDA neurons.A recent study using mice with hypomorphic alleles of Pitx3 (aphakia) supports the existence of distinctive dopaminergic neuronal population with different degrees of susceptibility to neurodegeneration 71 .A subset of dopaminergic neurons within SNpc is preserved in Pitx3-deficient mice, indicating that not all mDA neurons require Pitx3 for their survival.These neurons also seem less were identified, including the glucocorticoid receptor DNA binding factor (GRLF1) and Myosin 1c (MY-O1C), two proteins link to neurite outgrowth, and a stress marker, the heat-shock protein 70 (HSP70) 124 . Lm1b has been found in the Nurr1 transcriptional complex suggesting that Lmx1b may act directly in the transcriptional activation of Nurr1 target genes 124 .
Interestingly, in central serotonergic (5-HTergic) neurons, utilization of an inducible Cre-LoxP system to selectively inactivate Lmx1b expression in the raphe nuclei of adult mice, revealed that Lmx1b was required for the biosynthesis of 5-HTergic receptor, and it may be involved in maintaining normal functions of central 5-HTergic neurons by regulating the expression of Tph2, Sert, and Vmat2 125 .There is no comparable data for the mDA neurons.
Polymorphisms in LMX1A and LMX1B have previously been linked to PD 126 as well as with schizophrenia 127 .In search of a possible association between schizophrenia and SNPs in three dopamine-related transcription factors, LMX1A, LMX1B and PITX3, Bergman et al. 127 found five SNPs that were more common in subjects with schizophrenia which were the same as those previously shown to be over represented in subjects with PD 87,91,92,96,126 .Studies aiming to elucidate the role of Lmx1a and Lmx1b in adult mDA neurons as well as to identify the molecular mechanisms regulated by these factors in the maintenance of mDA neurons are still lacking.

Otx2
Otx2 is a member of the bicoid subfamily of the homeodomain transcription factor.It has been demonstrated that Otx2 regulates mDA neurogenesis and subtype identity in VTA 128,129 .Otx2 might directly or indirectly control the activation of Lmx1a and succeeding steps of proliferation and differentiation of mDA progenitors 130,131 .tion and maintenance of midbrainhindbrain boundary, or isthmic organizer 115 .Lmx1a and Lmx1b show similar roles in mDA neuron development in gain-of-function studies in mouse embryos 22,24 and in ES cells 116 .Lmx1b can, partially rescue roof plate development in dreher (Lmx1a) mice, indicating that Lmx1b has some functional redundancy to Lmx1a during development 105 .More recently, by studying the phenotype of double mutants for Lmx1a and Lmx1b, functional cooperation has been demonstrated between Lmx1a and Lmx1b in regulating proliferation, specification, and differentiation of mDA pro genitors 109,112 .
The co-expression of Lmx1a and Lmx1b persists in mature dopaminergic neurons of the SNpc and VTA throughout birth and into adulthood 117,118 .Their independent and/ or combinatory functions in the adult midbrain remain unknown.Gene profiling analysis of LMX1A overexpressing MN9D dopaminergic cells allowed for the identification of putative downstream targets of Lmx1a, including Grb10, Rgs4, and Vmat2, and also 13 mitochondrial nuclearencoded structural subunits of the mitochondrial respiratory chain 119 .
Developmental studies of knockout and conditional mutant mice revealed that Lmx1b regulate Fgf8 and Wnt1, which are essential for the inductive action of the isthmic organizer 109,117,[120][121][122] .Lmx1b expression also precedes the expression of Nurr1, TH, and Pitx3.Consequently, directly targeted genes by Lmx1b during development are difficult to circumscribe.Genes under the control of Lmx1b during adulthood are not confirmed yet.A microarray analysis using a tetracycline-inducible LMX1B expression system in HeLa cells has revealed that Lmx1b binds to the proximal promoter of IL-6 and IL-8 and activate the nuclear factor-κB (NF-κB) 123 .In an open search for binding partners of Lmx1b in mDA neurons, new possible interactors

Lmx1a and Lmx1b
Lmx1a and Lmx1b genes encode LIM-homeodomain (LIM-HD) proteins, a subfamily of transcription factors that have been well-preserved throughout evolution.The characteristic features of LIM-HD proteins are two specialized zinc fingers, called LIM domains, which are recognized by a number of co-factors.According to the co-factors, complex formation could either lead to transcriptional activation or repression 99 .The mouse Lmx1a and Lmx1b proteins share an overall 64% identity in amino acid sequences (100% identity in their HD and 67% and 83% identity in each LIM domain) 99 .Lmx1a is widely expressed in the developing embryo, including the roof plate of the neural tube, the cerebellum, ventral midbrain, otic vesicles, inner ear, the notochord, and the developing pancreas [100][101][102] .Lmx1a spontaneous mutantsdreher (Lmx-1a dr/dr ) exhibit a complex phenotype, including circling behaviour, sterility, pigmentation, and tail abnormalities [103][104][105][106] .Lmx1b is required for the normal development of dorsal limb structures, the glomerular basement membrane in the kidney, the anterior segment of the eye, skull development as well as dopaminergic and serotonergic neurons 107 .Mutations in the human LMX1B gene have been demonstrated to be responsible for the nail-patella syndrome, an autosomal dominant disorder characterized by dysplasia of the patellae, nails, and elbows and focal segmental glomerulosclerosis 108 .
During midbrain development, Lmx1a and Lmx1b are among the first markers that identify the mDA precursors, and are key regulators of their differentiation [109][110][111][112][113] .Lmx1a is an efficient inducer of mDA neurons from embryonic stem cells 114 .Loss-and gain-of-function studies in chick embryos demonstrated that Lmx1a is an essential determinant of mDA neuron development 110,113 .Lmx1b is associated with the forma-phenotype at birth but leads a massive mDA neuronal death in SNpc of young adult mice 144 .Furthermore, a single mutated allele of En1 in an En2 wild-type context, results in a progressive reduction of adult mDA neurons affecting both SNpc and VTA 145 .En1 and En2 are thus cell-dependant -autonomously needed in a gene-dosage -for the maintenance of mDA neurons in the adult brain.Polymorphism in EN1 has also been correlated with PD 91 .This genetic association suggests a role for EN1 in long-term maintenance of human mDA neurons.
In an elegant study, Alvarez-Fisher et al. 146 shed light into the putative mechanism by which En1/2 operate in adult neurons.En1/2 might participate in the local energetic metabolism of adult mDA neurons by regulating the translation of nuclear-encoded subunits of mitochondrial complex I (NDUFS1 and NDUFS3) and the enzymatic activity of this complex.In vitro and in vivo experiments of this study, demonstrated that exogenous En1/2 could protect nigrostriatal mDA neurons.Infusion of En1/En2 into the SNpc and VTA protected mDA neurons against MPTP and rotenone toxicity 146 .Ndufs1 translation is part of this En1/2-mediated neuroprotective pathway.En1/2 were also tested in other PD models in vitro and similarly, confirmed their protective action against 6-hydroxydopamine (6-OHDA) and mutated α-synuclein-A30P 2 .In En1 mutant mice, expression of Ndufs1 and Ndufs3 was also 30% lower in dopaminergic SNpc neurons compared to other mDA neurons.
Of interest, Engrailed extracellular stimulation of isolated axons in cell culture, unexpectedly results in translation of intermediate filament protein, Lamin B2, a protein normally known as a major constituent of the nuclear envelope.Detailed analysis confirmed Lamin B2 translation in axon and its mitochondrial of adult mDA neurons as well as in the difference in vulnerability of VTA versus SNpc neurons remain to be studied.
Interestingly, the observed degeneration in Otx2 deficient tissues might take its origin from acute perturbation of energy metabolism since 60% of Otx2 target genes are nuclear-encoded mitochondrial mRNAs 137 .Recent findings put forward that coordinate regulation of the expression of multiple nuclear-encoded mitochondrial mRNAs modulate local energy metabolism in the axon 138,139 .In this regard, one interesting hypothesis is that, developmentally acting transcription factors are required throughout adulthood as they are key regulators of the axonal energy balance.Although some alternative explanations cannot be eliminated, this possibility accords well with the fact that Nurr1, Lmx1a, Otx2, and En1/2 (see next section) target mR-NAs of nuclear-encoded subunits of mitochondrial complexes.

En1 and En2
The role of the homeoprotein Engrailed has been intensively studied in insect and invertebrate development.Expression of Engrailed allows a cell to interpret morphogen gradients, which spread from a compartment boundary and specify a posterior pattern 140 .In vertebrates, there are two Engrailed proteins, Engrailed-1 (En1) and Engrailed-2 (En2).These two paralogues play critical roles in patterning and neurogenesis during central nervous system development 141 .
En1/2 are expressed by SNpc and VTA neurons from early development and continuously into adulthood 142 .Loss-of-function studies revealed the importance of these TFs for mDA neurons maintenance.For example, when all four alleles of En are absent, mutant mice die at birth and mDA neurons are completely absent 142,143 .

Deletion of one allele of En1 and two alleles of En2 results in a normal
Early studies in adult mice showed that elimination of Otx2 expression in the ventral midbrain resulted in a selective loss of axonal projection from dopaminergic neurons in the VTA 132 .It is now known that Otx2 is expressed in the adult mouse brain but only in a specific, functionally distinct, subgroup of neurons of the VTA, i.e., the central and medial-ventral area of the VTA (Figure 1K) 128 .Studies, in which Otx2 has selectively been ablated from VTA neurons suggest that Otx2 negatively regulates the expression of the dopamine transporter DAT and an inverse correlation between Otx2 expression and the glycosylated active form of DAT, glyco-DAT, is observed 128 .When ectopically expressed in the SNpc, Otx2 suppresses the expression of the glycol-DAT 128 .It also confers efficient neuroprotection to MPTP toxicity by suppressing dopamine recapture and accordingly the uptake of the neurotoxic cation MPP+ 133 .A recent study showed that a mild over-expression of Otx2 in neurons significantly compensates vulnerability to MPTP and the progressive SNpc neuronal loss caused by En1 haploin sufficiency.Therefore, it is thought that Otx2 and En1 may share similar molecular basis of actions (see next section about En1/2 and the translation of nuclearencoded subunits of mitochondrial complex I) 134 .
In other highly metabolically active cells in the adult retina, loss of Otx2 elicits photoreceptor degeneration and could be rescued by constitutive Otx2 expression in Otx2-ablated retinas 135 .In this context, Otx2 ablation results in inflammation and expression of stress genes that lead to apoptosis.It has been suggested that Otx2 regulates a critical period of plasticity in the developing mouse visual cortex and infusion of Otx2 can reopen a window of plasticity in adult retina 136 .In the mature visual cortex, the persistent internalization of Otx2 in a specific group of cells maintained the critical period of plasticity closure 136 .The role of Otx2 in plasticity the changes during senescence are transcriptional regulation through microRNA and TFs, and represent reversals or extensions of developmental patterns 153 .

Conclusion
Studies of TF acting during development of the mDA neurons revealed that many factors like Foxa1/Foxa2, Nurr1, Pitx3, Otx2, Lmx1a/b, and En1/2 remain expressed in adults.The fundamental roles of these TFs in different tissues in maturity maintenance of cell-specific secretory and metabolic pathways are being more frequently acknowledged and one should expect that mDA-related TFs engage in similar roles.
Reviewing knowledge on the role of Foxa1/2, Nurr1, Pitx3, Otx2, Lmx1a/b, and En1/2 in adult midbrain supports the idea that: (1) the loss of a functional allele of Foxa2, Nurr1, and En1/2 does not compromise the development of mDA neurons but seriously impairs their survival in the postnatal and adult brain.Data for gene dosage effect of Lmx1a/b, Pitx3, and Otx2 are, up till now, not available; (2) Loss-offunction studies for Foxa1/2, Nurr1, Pitx3, and En1/2 show a massive dopaminergic cell death preferentially in SNpc while leaving the VTA more or less intact; (3) Cell death in SNpc is concomitant to loss of striatal innervations but line of evidences from Nurr1, Pitx3, and En1/2 experiments point towards a specific role of these TFs in axon maintenance throughout adulthood.Alternatively, axon degeneration might be the earliest feature of metabolic vulnerability; (4) Each developmentally expressed TF targets thousands of genes and initiates downstream cascades leading to profound physiological changes.Aside from the midbrain-specific well-known genes that induce the DA phenotype, nuclear-encoded mitochondrial genes (and mitochondrial bioenergetics related genes) have just begun to come into sight energy-transducing mitochondrial electron transport chain depends upon the interaction of both nuclear and mitochondrial-encoded gene products.Coordinated regulation of the two genomes in distal cellular compartments poses a singular challenge for neurons.Mitochondria distant from the cell body are therefore more vulnerable than their somatic counterparts 57 .
The action of En1, through the synthesis of lamin B2, on axonal mitochondrial size and activity is evidence of a local and non-traditional function exertion by TFs in axons 147 .Local axonal translation of TFs is thought to be involved in long-range retrograde signalling, in maintenance of connectivity with target cells, and in mediating neuronal cell survival.It has been proposed that stimulating the local synthesis of proteins involved in mitochondrial function might be a common mechanism by which target-derived cues support axon maintenance 152 .In this context, exploring the role of developmental TFs in mature mDA axon, including their implication in local regulation of mitochondrial protein synthesis, would be especially insightful.

Perspectives
A combination of several transcription factors orchestrates the development of mDA neurons.These TFs promote the differentiation of subpopulations and specify neural fate.Each neuronal subtype displays a specific gene expression profile and physiological properties that direct the formation of selective connections with appropriate target cells.Neurons of each subtype have different size, neurite topology, and plasticity and present differential vulnerability to cell death when exposed to moderate or acute stress.Although the development of mDA neurons is tightly regulated, the transition between pre-and postnatal development and the aging process remains unclear.Many of localization where it regulates mitochondrial size and mitochondrial membrane potential, and supports axon survival 147 .These observations have been done in Xenopus axon, a model far from mammalian midbrain model, but inspire further exploration of the role of En1/2 in mitochondrial activity in axon and axon maintenance throughout adulthood.

Mitochondrial regulation by TFs and potential significance in PD
The TFs involved in the development of mDA neurons that remain expressed in postnatal stages target a wide variety of genes involved in the dopaminergic phenotype.Nurr1, Lmx1a, Otx2, Pitx3, and En1 and En2 also mediate the expression of several nuclear-encoded mitochondrial genes as well as key genes implicated in the mitochondrial metabolism.Conditional gene inactivation of these TFs in the mDA neurons results in cell death.A preferential loss of neurons in the SNpc is the pathological hallmark of PD.Neurons critically depend on mitochondrial energy for their maintenance and mitochondrial dysfunction has long been implicated in the etiopathogenesis of PD.Functional characterization of genes associated with familial variants of PD revealed that several aspects of mitochondrial biology are perturbated in PD including mitochondrial biogenesis, bioenergetics, dynamics, transport, and quality control [148][149][150] .
as the major functional category of TF-regulated target genes for Nurr1, Lmx1a, Otx2, Pitx3, and En1/2.Neuronal-specific transcriptional regulation of mitochondrial bioenergetics and biogenesis has not been the subject of extensive studies and therefore molecular mechanisms remain to be elucidated.( 5) All TFs reviewed here, except Foxa1/2, have a genetic association to PD. Accordingly, this strongly suggests that mDA neuron functional preservation throughout aging is under the influence of developmental TFs.
This review suggests that developmental transcriptional networks are essential for maturity maintenance of mDA neurons.Much remains to be done to dissect apart the effects of each developmental TFs on functional properties of mDA neurons in adults.Efforts to understand the molecular basis of their actions could provide precious insights into specific mechanisms underlying axon degeneration in neurodegenerative diseases and normal survival of neurons through the late stages of life.

Figure 1 :
Figure 1: Schematic overview showing the expression pattern of key transcription factors during mDA neurons development and in ventral midbrain of adult mouse.A, Representation of a mouse embryo at E12.5 and a section at midbrain level (dashed red line).Ventral midbrain region is boxed and magnified in B. The generation of mDA neurons from neural progenitors follows three sequential steps defining mDA lineage into mDA progenitors, immature neurons, and mature neurons (B).Foxa1/2, Otx2, Lmx1a/b, and En1/2 genes are already expressed in mDA progenitors by E9.5 and their expression persists in both immature and mature mDA neurons (C-F).At E12.5, only a fraction of postmitotic mDA neurons express Otx2 (D)128 Nurr1 expression is initiated in immature postmitotic mDA neurons (G) whereas Pitx3 appears only in mature mDA neurons (H).Note that Otx2, En1/2, Foxa1/2 are present in other non-DA cell populations in ventral midbrain18,20,128,154 .Some cells outside mDA domain also express Nurr1.A midbrain section from adult mouse is illustrated in I and the dashed box is magnified in J-L to show expression of the different transcription factors in VTA and SNpc.Nurr1, En1/2, Lmxa/b, and Foxa1/2 genes remain expressed in adult mDA neurons of both VTA and SNpc (J) while Otx2 is only present in central and ventral portion of the VTA (K).Pitx3 gene is expressed in both VTA and SNpc (L)61,155 , but protein levels appear lower (or absent) in dorsal SNpc and in scattered mDA neurons in VTA71 .