Transcriptional Regulation of Mesencephalic Dopaminergic
Neurons: The Full Circle of Life and Death
Kambiz N. Alavian, PhD, Christian Scholz, MS Biology, and Horst H. Simon, PhD*
Interdisciplinary Centre for Neuroscience, Department of Neuroanatomy - Ruprecht-Karls,
Universita ¨t Heidelberg, Heidelberg, Germany
Abstract: Since mesencephalic dopaminergic neurons are as-
sociated to one of the most prominent human neurodegenera-
tive ailments, Parkinson’s disease, the molecular mechanism
underlying their development and adult cellular properties has
been the subject of intense investigations. Throughout life,
transcription factors determine the fate of this neuronal popu-
lation and control essential processes such as localization in the
ventral midbrain, their neurotransmitter phenotype, their target
innervations and synapse formation. Studies of transcription
factors, such as Nurr1, Pitx3, Engrailed-1/2, and Lmx1a/b,
have not only revealed importance of these genes during de-
velopment, but also roles in the long-term survival and main-
tenance of these neurons. In this review, we will discuss the
function of these transcription factors throughout the life of
mesencephalic dopaminergic neurons and their value in the
study of the disease mechanism. © 2007 Movement Disorder
Key words: mesencephalic dopaminergic neurons; mid-
brain; neurodegenerative disease; Parkinson’s disease; substan-
tia nigra; transcription factors
(mesDA) neurons are located in three distinct nuclei:
substantia nigra pars compacta (SNpc), ventral teg-
mental area (VTA), and retrorubral field (RRF).1His-
torically, these nuclei were designated as A8 (RRF),
A9 (SNpc), and A10 (VTA). The number of DA cells
in this region strongly differs among mammalian spe-
cies. Humans possess hundreds of thousands of
them2,3whereas mice have 10–30 thousands depend-
ing on the strain.4,5Three major axonal projections
originate from this neuronal population: the nigrostri-
atal pathway, a projection form the SNpc to the dorsal
striatum (caudate putamen), and the mesolimbic and
mesocortical pathways, which connect the DA neurons
in the VTA with the ventral striatum (nucleus accum-
bens and olfactory tubercle) and the frontal cortex,
respectively. Changes in these three pathways are as-
sociated to common human neurological disorders,
such as Schizophrenia, drug addiction6and, most
prominently, Parkinson’s disease (PD). The main
characteristic of PD is the progressive degeneration of
DA neurons in the SNpc,7,8resulting in diminished
release of dopamine in the caudate putamen and in
motor deficits like resting tremor, muscular rigidity,
difficulty to initiate movement, akinesia, and loss of
postural reflexes.9Studies of postmortem human
brains of different ages (up to 110 years) and old
non-human primates10,11have demonstrated that de-
spite late onset of the disease, significant biochemical
alterations and loss of axonal terminals in the stria-
tum,12,13mesDA neurons do not show an age-related
decline in number,10,11,14suggesting that while age
may be a risk factor for PD, it is not the primary cause
for the pathological degeneration.
The neurons, which degenerate during the course of
PD, are characterized by a particular vulnerability to cell
*Correspondence to: Horst Simon, Interdisciplinary Centre for Neu-
roscience (IZN), Department of Neuroanatomy, University of Heidel-
berg, Im Neuenheimer Feld 307, 69120 Heidelberg, Germany.
Received 15 March 2007; Revised 14 May 2007; Accepted 23 May
Published online 28 November 2007 in Wiley InterScience (www.
interscience.wiley.com). DOI: 10.1002/mds.21640
Vol. 23, No. 3, 2008, pp. 319–328
© 2007 Movement Disorder Society
death. This is supported by experimental models for
the disease and by human genetic evidence. Firstly,
rather unspecific toxins like the proteasome inhibitor,
epoxomicin,15or the inhibitor of the mitochondrial
respiratory chain rotenone16can cause, at distinct con-
centrations, a specific demise of nigral DA neurons
without affecting the VTA (for contradicting results
see also Refs. 17-19). Secondly, the genes with PD-
associated mutations20are, with the exception of
?-synuclein, widely expressed throughout the CNS
(UCHL1, DJ-1, and PINK1) or barely detectable (Par-
kin and LRRK2) (for the expression pattern see the
Allen Brain Atlas at http://www.brain-map.org). The
particular sensitivity to toxins and to the mutations is
likely an innate cellular property of nigral DA neu-
rons. However, the focus of most basic research into
PD has been the molecular mechanisms, which are
active when cell death is induced in these neurons and
the molecular foundation of the underlying selective
vulnerability has been only occasionally the tar-
get.21-24Unfortunately, the obtained data sets of the
latter experiments are still too large and a more de-
tailed analysis is still required. Since all cellular prop-
erties result from a cascade of developmental events,
which is ultimately controlled on the transcriptional
level, we suggest observation of the vulnerable cell
populations during development as an alternative ap-
proach. In this review we will, therefore, discuss the
transcription factors, essential for generation, survival,
and maintenance of mesDA neurons and will address,
their possible connections to the molecular etiology of
EARLY PATTERNING EVENTS LEADING TO
All neurons in the central nervous system and most in
the periphery, mediated by neural crest migration, are
generated in a region-specific manner from the neuroep-
ithelium by a series of inductive events. During gastru-
lation, neural tissue is generated from the embryonic
ectodermal layer by planar and vertical signals, arising
from Spemann Organizer and the underlying mesoder-
mal tissue.25After its induction, the neuroectoderm be-
gins to thicken, starts to roll up along its anterior–poste-
rior axis, and closes eventually to form the neural tube.26
At this time, the neural tissue becomes specified along its
rostrocaudal axis and slightly later along its dorsoventral
axis by graded signals.27The first sign of this polariza-
tion is the appearance of transcription factor expression
domains28and emergence of the morphogenetic subdi-
visions; forebrain, midbrain, hindbrain, and spinal
cord.29-31Secondary centers of organization appear as a
next step32and neuronal populations are generated at
distinct rostrocaudal and dorsoventral positions in anal-
ogy to a Cartesian grid of coordinates.29The precursor
cells of mesDA neurons are induced in the ventral mid-
brain by an interaction of two diffusible factors, sonic
hedgehog (Shh) and the fibroblast growth factor 8
(Fgf8).33The former is secreted by the floor plate34and
the latter is released by the border region between mid/
hindbrain, called isthmus or mid-hindbrain organizer
(MHO).32While the floor plate is present almost
throughout the entire length of the neural tube, the MHO
is a unique center of organization, playing a key role in
controlling the size and relative location of mesDA neu-
rons.35The MHO is established very early during devel-
opment by the mutual repression of two opposing
transcription factors, the homolog of drosophila ortho-
denticle, Otx2, and the gastrulation brain homeobox 2
(Gbx2), thereby defining a sharp border. Otx2 is already
expressed in the epiblast and in the anterior-visceral
endoderm before gastrulation.36However, during further
development, its expression becomes progressively lim-
ited to the anterior neuroectoderm and then defines the
region of forebrain and midbrain.37The opposing factor,
Gbx2, is expressed early on by all three germ layers of
the posterior embryo but later becomes limited to the
anterior hindbrain.38Otx2 homologous-recombinant null
mutant mice exhibit defects during gastrulation and fail
to develop fore-, mid-, and anterior hindbrain,39,40
whereas in null mutants for Gbx2, the Otx2 expression
domain is shifted posteriorly, leading to the caudal ex-
pansion of anterior brain structures.38Although Otx2 and
Gbx2 are not required for the induction of MHO-genes
(like Fgf8), however, the two transcription factors are
essential for the correct positioning of their expression
At the time point when the caudal border of the Otx2
expression domain begins to sharpen and before the
induction of mesDA neurons, a second group of tran-
scription factors appears which participates in the region-
alization of mid- and hindbrain. This second wave in-
cludes the pair-ruled gene Pax2,44the lim-homeodomain
factor Lmx1b45and also the diffusible glycoprotein
Wnt1.46Shortly thereafter, Engrailed-1 (En1) (at the
1-somite stage), Engrailed-2 (En2) (at the 3–5 somite
stage), and Pax5 are expressed around the forming
MHO. The homozygous mutant mice null for these genes
(in case of En1/2 or Pax2/5, the double mutant is re-
quired), all have one feature in common: a large deletion
of midbrain tissue combined with a complete or partial
loss of mesDA neurons (Fig. 1B).39,40,46,48-53
320 K.N. ALAVIAN
Movement Disorders, Vol. 23, No. 3, 2008
LMX1A (LIM HOMEOBOX TRANSCRIPTION
FACTOR 1, ?) AND MSX1 (HOMEOBOX,
After induction by Fgf8 and Shh in the ventral mid-
brain, the precursor cells and later the postmitotic neu-
rons go through several steps of specification. The first
indications of the cellular phenotype of mesDA neurons
are the appearance of Lmx1a and Msx1 in the still pro-
liferating precursor cells. The two genes seem to function
as key determinants of the midbrain DA neuronal cell
fate, with Lmx1a being the more important of the two.
Silencing of Lmx1a by RNAi (RNA interference) in
chick embryos results in the loss of DA neurons in the
midbrain and the gain of function experiment in embry-
onic stem cells leads to a robust generation of mesDA
neurons with correct molecular identity.54
After this initial round of specification, the precursor
cells gradually become postmitotic (in mice from about
E10–E14)29and the first signs of the neurotransmitter
phenotype appear, like the expression of the rate-limiting
enzyme for dopamine synthesis, tyrosine hydroxylase
(TH). At the same stage, several transcription factors
begin to be expressed, which are essential for their dif-
ferentiation and long-term survival. Deficiencies in
Lmx1b, Nurr1, Pitx3, and the Engrailed genes all result
in the loss of mesDA neurons before birth.52,55-57These
genes represent at least three independent molecular
pathways and are discussed below (Fig. 1B).
NURR1 (ALSO CALLED NR4A2, NOT, RNR-1,
Nurr1 is a member of the steroid–thyroid hormone
receptor family of transcription factors,58-61that atypi-
cally lacks a ligand cavity and a classical canonical
binding site for coactivators.62,63The expression of
Nurr1 starts briefly after induction of the neurons in the
ventral midbrain64at the time when they become post-
mitotic,65and remains high in almost all of them
throughout life of a mammal.66One of the main func-
tions of Nurr1 is the control over synthesis, vesicle
packaging, axonal transport, and reuptake of dopamine,
thereby critically determining the neurotransmitter phe-
notype of mesDA neurons. Surprisingly, this control is
exclusive to this group of DA neurons and is not required
in any other TH-expressing cell population. As a result,
mesDA neurons deficient of Nurr1, do not express key
components of the DA phenotype, including TH, the
vesicle monoamine transporter 2 (VMAT2) and the do-
pamine transporter (DAT).67,68The regulation of TH is
FIG. 1. Early and late development, regional identity and induction of
mesDA neurons. (A) Interaction of sonic hedgehog (Shh), secreted by
the floor plate, and the fibroblast growth factor 8 (Fgf8), released from
the mid-hindbrain organizer (MHO), induces mesDA neurons in the
ventral midbrain. These neurons will later give rise to three distinct
nuclei, retrorubral field (RRF), ventral tegmental area (VTA) and
substantia nigra pars compacta (SNpc). The location of mesDA neurons
is dependent upon the MHO, which is defined by the expression of two
opposing factors, Otx2 and Gbx2, and also by Pax2, Pax5, and Wnt1,
which participate in the regional specification of midbrain tissue. (B)
Genes involved in the late development of mesDA neurons. After the
early midbrain development and induction of mesDA neurons, the
development of postmitotic neurons is controlled by at least three
distinct pathways under the influence of Nurr1, the Engrailed genes,
Lmx1b, and Pitx3. These factors start to be expressed in mesDA
neurons around the same time (E10.5-13) and are important for their
neurotransmitter phenotype, their survival and maintenance throughout
the lifetime of the mouse. Expression of several vital genes is con-
trolled by these transcription factors. In the mice lacking Engrailed
genes, the expression of ?-synuclein, is significantly reduced. The
expression of tyrosine hydroxylase (TH), the rate-limiting enzyme in
dopamine synthesis, and of the GDNF receptor component, Ret, which
is required for long-term survival of nigral DA neurons,47is regulated
by Nurr1. The Engrailed genes and Lmx1b are expressed in the region
before induction of mesDA neurons (dotted lines); yet, their expression
becomes relatively specific to postmitotic mesDA neurons during later
embryogenesis (continuous lines).
DIFFERENTIATION OF MIDBRAIN DA NEURONS321
Movement Disorders, Vol. 23, No. 3, 2008
due to a direct interaction of Nurr1 with a NBRE se-
quence in the 5? untranslated region.69,70Such a binding
site was also found in the DAT gene, suggesting that it is
regulated in the same manner.71,72
Although Nurr1 is considered a postmitotic differen-
tiation factor, its expression in embryonic neuronal stem
cells is potent enough to induce a general DA pheno-
type.73-75Because of these properties, Nurr1 deficient
mesDA neurons lack key markers, by which they are
normally identified. This led originally to the erroneous
conclusion that deficiency in Nurr1 results in the failure
to generate mesDA neurons during embryogenesis.76
Later, the observation of agenesis was revised to a mere
lack of DA phenotype, since the cells adopt a normal
ventral position in the midbrain and express other cell-
type specific markers like Pitx3, Engrailed1/2, Lmx1b,
Gfra1, and Ahd2.77,78In later development, Nurr1 ob-
tains a role in survival, migration, and target innervation.
The reports regarding this latter function are, however,
controversial. Whereas, one group found an increase in
apoptotic figures in the ventral midbrain and lack of
axons, traceable by retrograde labeling from the basal
ganglia to the SN and VTA,78another group, using the
same techniques, concluded that there is no evidence that
Nurr1 has a survival function or participates in terminal
differentiation processes.79The two studies used differ-
ent constructs when they had originally generated the
homologous recombinant mouse mutant strains, which
may explain their different observations.
Nurr1 provides the most compelling evidence for our
original hypothesis that investigation of regulators of
mesDA neuronal cell fate may provide a link to the
molecular mechanism underlying PD. A recent study
identified two mutations, mapping to the first exon of the
Nurr1 gene, which were associated with familial PD
(?291Tdel and ?245T3G).80These mutations in the 5?
untranslated region lead, in vitro, to a marked decrease in
Nurr1 mRNA levels and to a lowered expression of TH.
Two other studies identified mutations in exon 6 with
higher frequency in familial and sporadic forms of the
disease.81,82Mouse mutant studies suggest that a lowered
Nurr1 expression is correlated with an increased vulner-
ability of nigral DA neurons. Heterozygous Nurr1 mu-
tant animals are characterized by a higher sensitivity to
MPTP-induced toxicity,83,84and by an age-dependent
(?15 months) loss of nigral DA neurons, leading to a
lowered striatal dopamine content and to motor dysfunc-
tions analogous to PD.85Taken together, the human and
mouse mutant genetic studies show that beside its devel-
opmental functions, Nurr1 is also crucial for long-term
survival of a cell population which is intimately associ-
ated to this prevalent human age-related degenerative
PITX3 (PAIRED-LIKE HOMEODOMAIN
TRANSCRIPTION FACTOR 3 OR PITUARY
Pitx3, also called Ptx3 in earlier publications (due to
an error in gene family association), is a homeodomain-
containing transcription factor with binding activity to
DNA, similar to the drosophila bicoid.86It is transiently
expressed in the developing eye lens and skeletal muscle
during embryogenesis, but its most striking feature is the
exclusivity of its expression in the central nervous sys-
tem to mesDA neurons.87Slightly after Nurr1, Pitx3
starts to be expressed in the ventral midbrain. This ex-
pression continues into adulthood. Initially, a role of
Pitx3 in the lens was established by the discovery of a
human mutation in the gene, associated with cataracts
and anterior segment dysgenesis.88Later a spontaneously
occurring blind mouse mutant, Aphakia, which had been
first described in 1968,89was linked to a 652 bp deletion
in the promoter of Pitx3,90which leads to loss of Pitx3
expression.91The subsequent analysis of the mutant with
respect to mesDA neurons revealed a specific absence of
nigral DA neurons and a loss of axonal projection to the
dorsal striatum.86,91-93Surprisingly, despite the strong
reduction of dopamine levels (?90%) in the striatum, the
behavioral consequences were limited to lower motor
activity.91,93These motor deficiencies could be rescued
by administration of levodopa.94,95The nigral deficiency
in Aphakia mice was associated to an early developmen-
tal failure of nigral DA neurons to migrate.93The role of
Pitx3 in nigral DA neurons is also evident from stem
cells studies.96These investigations showed an up-regu-
lation of genes, which are highly expressed by nigral DA
neurons, like aldehyde dehydrogenase 2 (AHD2), sug-
gesting a role of retinoids during the development of
these neurons97,98regulated in part by Pitx3. Further-
more, Pitx3 also seems to regulate the expression of TH,
since there is an active high-affinity binding site for the
transcription factor in the promoter.99-101The role of
Pitx3 in the adult is still unknown and may be explored
by generation of conditional knockout animals or in vivo
knockdown by RNAi.
The Engrailed homeobox genes are one of the most
widely studied group of transcription factors, described
and investigated in a variety of species from annelids,102
mollusks,103insects, and crustaceans,104-106to echino-
derms107and chordates, such as amphioxus108and mul-
tiple vertebrate species109-113including humans.114,115
Movement Disorders, Vol. 23, No. 3, 2008
Engrailed was first mentioned as a spontaneous, non-
lethal mutation in Drosophila melanogaster leading,
among others, to scetullar modification and wing mal-
formation.116Insects and other invertebrates normally
possess one copy of the gene, whereas vertebrates mostly
have two (En1 and En2). Functional homology between
paralogs and orthologs is very high, demonstrated by
replacement experiments in mouse. Substitution of
mouse En1 with En2 or the drosophila homolog leads to
the rescue of the otherwise lethal En1 null phenotype that
exhibits a large mid/hindbrain deletion and severe skel-
Functionally, in all the investigated species, the En-
grailed genes participate in two events during develop-
ment. Early on when the basic body plan is laid
down,120,121they take part in regionalization of the em-
bryo, and, later they have a role in neuronal specifica-
tion.53,122-124Regarding the mesDA neurons, the two
mammalian paralogs, En1 and En2, determine first the
size of the Fgf8 expression domain in the mid/hind-
brain,50which influences the amount of mesDA neurons
that are generated.53En1 and En2 appear at E8 in the
anterior mouse neuroectoderm as patches which subse-
quently fuse to form a band of cells which will later give
rise to the border region between midbrain and hind-
brain, the isthmic organizer. The early onset of Fgf8 is
independent of the two transcription factors; however, its
later expression in the isthmus seems to be under their
direct regulatory control. A highly conserved region in
human, mouse, rat, and chicken in the large intron of the
Fgf8 gene contains an active En1 binding site125(our
unpublished promoter analysis), and ectopic En expres-
sion induced by a retroviral construct in chicken or by
mRNA injection in medaka and xenopus 1–2 cell em-
bryos leads to ectopic Fgf8 expression.126,127
During later development and throughout life, the two
genes are required for the survival and maintenance of
mesDA neurons in a cell-autonomous and gene dose-
dependent manner. After their initial expression in the
mid/hindbrain, mesDA neurons start to express the two
En genes between E11.5 and E14 in mice and the ex-
pression continues throughout the life of the animals.128
Their survival function for mesDA neurons became ap-
parent from mouse mutant studies. In animals homozy-
gote null for both genes (En1?/?;En2?/?), mesDA
neurons are initially generated, become postmitotic, and
begin to express their neurotransmitter phenotype.
Briefly after En expression starts in the wild type, the
mutant neurons die by caspase-dependent apoptosis and
the entire population is lost by E14.53,128By using clas-
sical cell mixing in vitro experiments, RNAi on primary
midbrain cell cultures and by the analysis of chimeric
animals,128,129we concluded that despite a large mid/
hindbrain deletion in the En double mutant embryos, the
requirement of the En genes in postmitotic mesDA neu-
rons is cell-autonomous.128
The intermediate genotypes between the double null
mutant and wild type show various degrees of deficiency
in the mesDA system. En2?/? mutants and En2?/? are
viable and fertile and show no significant deficit. Mutants
for En1 (En1?/?) die at birth and exhibit a small alter-
ation in distribution of DA neurons in the VTA. In the
En1?/?;En2?/? mice, the SNpc and VT are reduced to
a small cluster of TH-positive cells (?90%), close to the
ventral surface of the midbrain.53In neonatal En1?/?;
En2?/? mice, mesDA neurons are normal in respect to
cell number, cell body distribution and target innerva-
tions; however, during the first two months of postnatal
maturation, 70% of nigral DA neurons are gradually lost
(VTA is not affected), resulting in diminished storage
and release of dopamine in the caudate putamen, motor
deficits similar to akinesia and bradykinesia, and a re-
duction in weight, resembling pathogenesis and symp-
toms of PD.57We could not find any significant abnor-
malities, with regard to mesDA systems, in En1?/?;
En2?/? animals; however, there is a report that the
overall number of mesDA neurons decreases in En1?/?
mutants between 8th and 48th postnatal weeks.130
Interestingly, during the embryonic stage when
mesDA neuron disappear in the En double mutant em-
bryos, the expression of ?-synuclein (and the Engrailed
genes) starts in the wild type. Despite the presence of
mesDA neurons in E12/13 double mutants (En1?/?;
En2?/?), the ?-synuclein expression is not observable.
At the same age, in the single mutants for En1 (En1?/
?;En2?/?), which show no significant major deficien-
cies in development of the mesDA system until birth, the
expression of ?-synuclein is significantly reduced.53This
could be a sign of delayed differentiation in the mutants
or a result of the direct interaction of En1 and En2 with
the promoter of ?-synuclein. The existence of two high
affinity binding sites for En1 several kb upstream of the
coding region of ?-synuclein, conserved among human,
rat, and mouse (our in silico analysis), suggests that the
latter may be the case and the dose of Engrailed deter-
mines the level of ?-synuclein expression. The first
known genetic link to familial forms of PD, ?-synuclein
(SNCA, PARK1),131,132is important in normal synaptic,
membrane-associated processes and seems to be an ac-
tivity-dependent negative regulator of dopamine neuro-
transmission.133Its accumulation in Lewy body aggre-
gates is one of the main pathological characteristics of
sporadic PD,134and the association of a triplication of its
locus to familiar PD135suggests that the survival of
DIFFERENTIATION OF MIDBRAIN DA NEURONS 323
Movement Disorders, Vol. 23, No. 3, 2008
nigral DA neurons depends on the level of its expression.
The putative positive regulation of ?-synuclein by the
Engrailed genes and the gene-dose effect in Engrailed
mutant mice collectively suggest that a very tight regu-
lation of the En1/2 expression may be necessary for
survival of DA neurons in the SNpc. This may be ex-
ploitable for therapeutic purposes to stabilize nigral DA
neurons in two manners; either by lowering the En-
grailed expression with an RNAi approach or by eleva-
tion of its activity by direct application of the protein.
Intriguingly, despite being transcriptional regulators,
which are localized in the nucleus, a small proportion
(?5%) of the intracellular En protein is found associated
to membrane vesicles,136becomes secreted and is then
internalized by other cells.137-139The internalized protein
is functional and acts as a signaling molecule.140
LMX1B (LIM HOMEOBOX TRANSCRIPTION
Lmx1b is best investigated as a determinant of dorsal–
ventral patterning in the developing limb buds141but is
also known as a regulator of neuronal cell fate.142-144In
humans, mutations in the gene can cause abnormal skel-
etal development and renal dysplasia as seen in nail
patella syndrome.145Lmx1b was first associated to
mesDA neurons in a screen which aimed to identify
homeobox genes with unique neuronal lineages in ver-
tebrates.87Its expression in the midbrain starts before
neural tube closure,52where it is co-expressed with
Lmx1a and Msx1 in mesDA neuronal precursors.54While
the expression of Lmx1a and Msx1 is restricted to the
ventral midbrain at this age, the Lmx1b expression is
found in a broad band, reaching from the ventral to the
dorsal surface of the mesencephalon. Later this primary
expression is down-regulated and disappears at around
E11 in order to reappear later in the postmitotic neurons.
From at least E16 continuing into adulthood, it is colo-
calized with TH and Pitx3 in the ventral midbrain.
Although Lmx1b is structurally related to Lmx1a, its
function is distinctively different from its ortholog. It is
not able to induce a DA cell fate in the midbrain when
ectopically expressed, but, instead, controls the onset and
maintenance of Wnt1 around the isthmic organizer, dem-
onstrated by a gain-of-function experiment using a rep-
lication-competent retroviral vector in chicken.46Fur-
thermore, the Fgf8 expression in the midbrain also
requires the presence of Lmx1b. The deletion of the gene
in homologous recombinant mutant mice causes the ma-
jority of the TH-positive cells in the ventral midbrain to
disappear by E12.5. The remaining cells entirely vanish
a few days later. Transient expression in the mesDA
precursors cells and the late onset in the postmitotic
neurons deem it unlikely that the deficiency in mutant
mice is due to a cell-autonomous requirement for Lmx1b.
More likely, the deficit is linked to the requirement of
Lmx1b for the expression of Wnt1 in the midbrain during
early embryogenesis. Both Lmx1b and Wnt1 null mutants
have very similar phenotypes in respect to mesDA neu-
rons, a small residue of TH-positive neurons in the
ventral midbrain, which do not express Pitx3 and totally
disappear during further development.47,52,56This simi-
larity in mutant phenotypes also suggests that the lack of
Pitx3 expression in the midbrain of Lmx1b-deficient em-
bryos is not due to a regulation of Pitx3 by Lmx1b, as
previously proposed,52but due to an arrest in differenti-
ation of mesDA neurons.
Up to date, most of the research on the cause of nigral
cell loss during PD has been focused on the analysis of
neurotoxin-based models. Although these classical ap-
slow progressive degeneration of the nigrostriatal system in
the course of PD and the late appearance of symptoms are
mimicked by an acute or a relatively fast progressive loss in
species with short life expectancy. Therefore, the observa-
tion of pathological features does not guarantee that the
underlying molecular mechanism is the same as during the
progression of PD. The susceptibility of nigral DA neurons
to degeneration and cell death must lie, as any other cellular
property, within the transcriptional profile of these neurons,
which is ultimately a result of a cascade of developmental
events. With this premise in mind, the transcription factors
essential for survival of mesDA neurons during develop-
ment may be the molecular key to the vulnerability of those
cell populations, which degenerate during PD. The success-
ful association of three nucleotide variations in NR4A2
(Nurr1) to PD and the identification of several genetic
mouse models with a slow progressive degeneration of the
mesDA system, like En1?/?;En2?/? mice, are the first
fruits of these efforts. Furthermore, a better understand-
ing of the molecular mechanisms essential for the gen-
eration of mesDA neurons during embryogenesis may
lead to improved or novel protocols for differentiation of
stem cells into mesDA neurons, as evident in two exam-
ples, the terminal differentiation of mesDA neurons from
stem cells by over-expression of Lmx1a or a combination
of Nurr1 and Pitx3.54,73
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