RESEARCH ARTICLE
531
Development 136, 531-540 (2009) doi:10.1242/dev.029769
Pitx3 potentiates Nurr1 in dopamine neuron terminal
differentiation through release of SMRT-mediated repression
Frank M. J. Jacobs, Susan van Erp, Annemarie J. A. van der Linden, Lars von Oerthel, J. Peter H. Burbach and
Marten P. Smidt*
In recent years, the meso-diencephalic dopaminergic (mdDA) neurons have been extensively studied for their association with
Parkinson’s disease. Thus far, specification of the dopaminergic phenotype of mdDA neurons is largely attributed to the orphan
nuclear receptor Nurr1. In this study, we provide evidence for extensive interplay between Nurr1 and the homeobox transcription
factor Pitx3 in vivo. Both Nurr1 and Pitx3 interact with the co-repressor PSF and occupy the promoters of Nurr1 target genes in
concert. Moreover, in vivo expression analysis reveals that Nurr1 alone is not sufficient to drive the dopaminergic phenotype in mdDA
neurons but requires Pitx3 for full activation of target gene expression. In the absence of Pitx3, Nurr1 is kept in a repressed state
through interaction with the co-repressor SMRT. Highly resembling the effect of ligand activation of nuclear receptors, recruitment of
Pitx3 modulates the Nurr1 transcriptional complex by decreasing the interaction with SMRT, which acts through HDACs to keep
promoters in a repressed deacetylated state. Indeed, interference with HDAC-mediated repression in Pitx3–/– embryos efficiently
reactivates the expression of Nurr1 target genes, bypassing the necessity for Pitx3. These data position Pitx3 as an essential
potentiator of Nurr1 in specifying the dopaminergic phenotype, providing novel insights into mechanisms underlying development
of mdDA neurons in vivo, and the programming of stem cells as a future cell replacement therapy for Parkinson’s disease.
INTRODUCTION
The dopaminergic neurons of the meso-diencephalic dopaminergic
system (mdDA system) have been extensively studied in relation to
Parkinson’s disease, and pioneering studies have explored the
possibility of using cell replacement therapy with stem cells as a
future treatment (Freed et al., 2001; Chung et al., 2002; Kim et al.,
2002; Olanow et al., 2003; Andersson et al., 2006; Hedlund et al.,
2008). Ultimately, stem cells could be exploited as an unlimited
source of transplantable DA neurons. However, in order to engineer
stem cells with mdDA characteristics, the need to obtain the
appropriate dopaminergic phenotype through correct molecular
coding has been underlined (Chung et al., 2005; Martinat et al.,
2006; Smidt et al., 2007). Therefore, much effort has been put into
unravelling the multi-step process that produces a genuine mdDA
neuronal population in vivo, as this is believed to hold the key to the
successful engineering of stem cells in vitro.
One of the key regulators for development of mdDA neurons in
vivo is the orphan nuclear receptor Nurr1 (Nr4a2), which is crucial
for expression of a set of genes involved in DA metabolism, such as
tyrosine hydroxylase (Th), vesicular monoamine transporter
(Vmat2), dopamine transporter (Dat) and aromatic L-amino acid
decarboxylase (Aadc) (Zetterström et al., 1997; Saucedo-Cardenas
et al., 1998; Baffi et al., 1999; Le et al., 1999; Wallén et al., 1999;
Smits et al., 2003). Another key factor for the development of
mdDA neurons is the paired-like homeobox gene Pitx3. Because of
its highly exclusive expression in mdDA neurons within the brain,
Pitx3 is a selective marker for the generation of transplantable ES
cells (Zhao et al., 2004; Chung et al., 2005; Hedlund et al., 2008).
Rudolf Magnus Institute of Neuroscience, Department of Neuroscience &
Pharmacology, University Medical Center Utrecht, Universiteitsweg 100, 3584 CG,
Utrecht, The Netherlands.
*Author for correspondence (e-mail: [email protected])
Accepted 12 November 2008
Pitx3 serves a unique function in mdDA neurons, highlighted by the
dramatic loss of neurons of the substantia nigra in Pitx3-deficient
mice (Hwang et al., 2003; Nunes et al., 2003; Van der Munckhof et
al., 2003; Smidt et al., 2004), which is preceded by deficient Th
expression during embryonic development (Smidt et al., 2004;
Maxwell et al., 2005; Jacobs et al., 2007). Strikingly, although Pitx3
is expressed in all mdDA neurons, only mdDA neurons of the
substantia nigra are affected by the loss of Pitx3, emphasizing the
complicated role of Pitx3 in mdDA neurons and the diverse
properties of subsets within the mdDA neuronal population.
Despite the fact that both Pitx3 and Nurr1 are crucial for proper
expression of Th in mdDA neurons, it has long been assumed that
Pitx3 and Nurr1 are components of independent developmental
pathways (Smidt et al., 2000; Chung et al., 2005). However, there is
growing evidence to suggest that these pathways are interconnected
at a functional level. Studies aimed at constructing functional DA
neurons from ES cells, have shown that combined transduction of
Nurr1 and Pitx3 promotes the maturation of ES cells into a
dopaminergic phenotype (Martinat et al., 2006). Importantly, the
recent discovery of aldehyde dehydrogenase 2 (Ahd2; Aldh1a1) as
transcriptional target of Pitx3 in mdDA neurons has highlighted a
significant similarity between the phenotype of Pitx3- and Nurr1-null
mice (Jacobs et al., 2007). Similar to what was observed in Pitx3–/–
embryos, Ahd2 expression is not detected in the midbrain area of
Nurr1-deficient embryos during final differentiation of mdDA
neurons (Wallén et al., 1999). These observations strongly suggest a
functional relationship between the homeodomain transcription factor
Pitx3 and the orphan nuclear receptor Nurr1 in the development of
mdDA neurons. In this study, we deduce the molecular mechanism of
this functional interplay between Pitx3 and Nurr1 in vivo, with
extensive consequences for mdDA development. We establish Pitx3
as an essential regulator of Nurr1-mediated transcription in mdDA
neurons, which positions Pitx3 as a crucial factor for the specification
of the dopaminergic phenotype both in vivo and in stem cell
engineering for future treatment of Parkinson’s disease.
DEVELOPMENT
KEY WORDS: DA phenotype, Dat, Dopamine, Parkinson’s disease, VMAT2, mdDA
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RESEARCH ARTICLE
MATERIALS AND METHODS
Cell culture
MN9D-Nurr1Tet On13N (MN9D) cells were cultured and transfected as
described previously (Jacobs et al., 2007).
Identification of Pitx3 interacting proteins
MN9D cells were transfected with 0.5 μg Pitx3-pcDNA3.1(–)myc-His or
an equivalent molar amount of pcDNA3.1(–)myc-His expression vector and
His-tagged proteins were purified using Ni-NTA magnetic agarose beads
(Qiagen) according to manufacturer’s protocol. Purified proteins were
separated by SDS PAGE and visualized by protein gel silver-staining.
Subsequently, the gel was fixed in 50% methanol (2⫻15 minutes) and 5%
methanol (10 minutes), rinsed with water (3⫻10 seconds), soaked in 10 μM
DTT (20 minutes), incubated in 0.1% AgNO3 (20 minutes), rinsed with
water and incubated in developer solution (3% NaCO3, 0.05%
formaldehyde) until protein bands were visible. The reaction was stopped
by adding citric acid and the gel was washed in water. Protein bands of
interest were excised from the gel and subjected to nanoLC-ESI-MS Mass
Spectrometry analysis (Proteome Factory).
Development 136 (4)
CTTG-3⬘. The amount of amplified DNA was determined by densitometry
on a FLA5000 multi imaging system (Fuji) and relative enrichment by
Pitx3- and Nurr1-ChIP over pre-immune serum (Ctrl)-ChIP was calculated.
Statistical analysis was performed using Student’s t-test, comparing the
relative values of enrichment of promoters of Vip and Vmat2 to the relative
values of a non-enriched region (control) in the Vmat2 promoter, as
determined by ChIP-on-chip.
Animals
Pitx3–/– and Nurr1–/– embryos were obtained as described previously (Smits
et al., 2003; Jacobs et al., 2007). Pitx3-deficient Aphakia (Pitx3–/–) or
C57Bl6-Jico wild-type mice were crossed with Pitx3gfp/gfp mice to obtain
Pitx3gfp/+ and Pitx3gfp/– embryos. Pitx3gfp/– embryos contain both the
classical Ak allele and an allele in which green fluorescent protein (GFP) is
knocked-in the Pitx3 locus (Zhao et al., 2004; Maxwell et al., 2005) and are
therefore Pitx3 deficient. Pitx3gfp/+embryos are heterozygous for wild-type
Pitx3 and GFP, and are described to have a normal development of the
mdDA system (Maxwell et al., 2005).
Genotyping
MN9D cells or tissue was homogenized in lysis buffer [50 mM Tris HCl
(pH 8), 150 mM NaCl, 5 mM MgCl2, 0.5 mM EDTA, 0.2% NP-40, 5%
glycerol and 0.5 mM DTT + Complete Protease Inhibitor Cocktail
(Roche)], and subjected to IP. Two dishes (10 cm in diameter) of MN9D
cells or five ventral midbrains of E14.5 C57Bl6-Jico embryos were used
for each IP. Magnetic dynal beads (Invitrogen) were blocked in 0.5%
BSA and incubated with antibodies overnight. Lysates were incubated
with antibody-bound beads for 3 hours at 4°C. Beads were washed in lysis
buffer (five times) and captured using a magnetic separator. Elution was
performed in elution buffer [50 mM Tris (pH 8.0), 10 mM EDTA, 1%
(v/v) SDS and Complete protease inhibitors] at 65°C with frequent
vortexing. Proteins were separated on Nupage 4-12% gradient gels
(Invitrogen), transferred to Hybond C extra (Amersham) and after
overnight blocking in 5% milk powder in PBS at 4°C the membranes
were incubated with antibodies in PBS-T [PBS + 0.05% (v/v) Tween-20].
Antibodies used: anti-Nurr1 [E-20; Santa Cruz (SC)], 1:100; anti-Pitx3
(Smidt et al., 2004), 1:10.000; anti-PSF (B92; Sigma), 1:2500; anti-Sin3a
(K-20; SC), 1:1000; anti-Ncor (C-20; SC), 1:1000; anti-SMRT (H300;
SC), 1:200; anti-RXR (sc-774; SC), 1:500; anti-HDAC1 (sc7872; SC),
1:300. Blots were incubated with SuperSignal West Dura Extended
Duration Substrate (Pierce) and exposed to ECL films (Pierce). When
applicable, films were digitized on a FLA5000 multi imaging system
(Fuji), and the amount of co-immunoprecipitated proteins was determined
by densitometry. Ratios were calculated as relative increase in amount of
co-immunoprecipitated proteins in Pitx3–/– embryos compared with
C57BL6 embryos.
Chip-on-chip analysis
Chromatin immunoprecipitation was performed according to the protocol
supplied by Nimblegen. For the in vitro ChIP-on-chip analysis, MN9DNurr1Tet On13N cells were transfected with 0.5 μg Pitx3-pcDNA3.1(–).
Nurr1 expression was induced by 3 μg/ml doxycyclin and cells were
cultured for 48 hours. Two 10 cm plates were used for each antibody. For the
in vivo ChIP-on-chip analysis, five ventral midbrains from C57Bl6-Jico
E14.5 embryos were used for each ChIP. Chromatin was sonicated to an
average length of 500 bp on a Soniprep 150 sonicator through 15 pulses of
10 seconds at one-third of maximum power. ChIP was performed with either
Nurr1 antibodies, Pitx3 antibodies or pre-immune serum (Ctrl). ChIP-DNA
was amplified using a Whole Genome Amplification kit (Sigma), according
to the manufacturer’s protocol and shipped for ChIP-on-chip analysis
(Nimblegen) where Cy5-labelled Pitx3- and Nurr1-ChIP samples were
hybridized to Cy3-labelled input chromatin on a mouse promoter array set
(MM8). Microarray data was analysed using Signalmap software (v1.9).
PCR-based validation was performed on 10 ng of amplified ChIP-DNA
using the following primers: Vip, 5⬘-AGCGACTGAGTTGGAGATTC-3⬘
and 5⬘-GTAAGCCATGGCACTAGCAC-3⬘; Vmat2, 5⬘-GTGTCTACTGTCTATCACAG-3⬘ and 5⬘-GTCAAAGTGTCCATGAAGCC-3⬘; control, 5⬘GACCCGTGTCACTGACCTAC-3⬘ and 5⬘-GCCTGCTAGGAGCAGC-
Genotypes were determined by PCR analysis as described previously
(Saucedo-Cardenas et al., 1998; Jacobs et al., 2007).
In situ hybridization
In situ hybridization was performed as described previously (Smits et al.,
2003; Smidt et al., 2004). The following digoxigenin (DIG)-labelled probes
were used: Aadc; bp 22-488 of the mouse coding sequence (cds) (Smits et
al., 2003), Vmat2; bp 290-799 of mouse cds (Smits et al., 2003), Dat; bp
789-1153 of rat cds, Nurr1; 3⬘ region of rat Nurr1, En1; 5⬘ region of mouse
transcript, D2R; bp 345-1263 of mouse cds.
Tissue culture
Ventral midbrains of Pitx3gfp/+ and Pitx3gfp/- embryos at stage E13.5 were
dissected in L15 medium (Gibco) and cultured in Neurobasal Medium
(Gibco) supplemented with: 2% (v/v) B-27 supplement (Gibco), 18 mM
HEPES-KOH (pH 7.5), 0.5 mM L-glutamine, 26 μM beta-mercapto-ethanol
and 100 units/ml penicillin/streptomycin. Midbrains were cultured and
treated with either 0 mM, 0.3 mM or 0.6 mM of sodium butyrate (Sigma)
for 48 hours.
FACS sorting
Cultures were dissociated using a Papain dissociation system (Worthington)
and cells were sorted on a Cytopeia Influx Cell sorter. Sort gates were set on
forward scatter versus side scatter (life cell gate), on forward scatter versus
pulse width (elimination of clumps) and on forward scatter versus
fluorescence channel 1 (528/38) filter; GFP fluorescence). Cells were sorted
using a 100 μm nozzle at a pressure of 15 PSI with an average speed of 7000
cells/second.
Semi-quantitative RT-PCR
RNA was isolated in Trizol (Invitrogen), according to manufacturer’s
protocol. Semi-quantitative RT-PCR was performed on equal amounts of
RNA (0.1 ng) using a one-step RT-PCR kit (Qiagen), according to the
supplied protocol. The following primers were used: Vmat2, 5⬘-GCAGTCACACAAGGCTACCA-3⬘ and 5⬘-TGAATAGCCCCATCCAAGAG-3⬘;
D2R, 5⬘-GCCGAGTTACTGTCATGATC-3⬘ and 5⬘-ACGGTGCAGAGTTTCATGTC-3⬘; Th, 5⬘-GCCACGTGGAATACACAAAG-3⬘ and 5⬘GAGGCATGACGGATGTACTG-3⬘; Tbp, 5⬘-GAGAATAAGAGAGCCACGGAC-3⬘ and 5⬘-TCACATCACAGCTCCCCAC-3⬘. The amount of
amplified DNA was determined by densitometry as described above.
Statistical analysis was performed by two-way unpaired Student’s t-test,
comparing the relative transcript levels of sodium butyrate-treated cultures
with transcript levels of untreated cultures.
Statistical analysis
The quantified results represent the average values of experiments
performed in triplicate and presented data indicate means with standard
errors (s.e.m.). Statistical analysis was performed using Student’s t-test (twoway unpaired). P≤0.05 is considered significant and are indicated by
asterisks, P<0.01 is indicated with double asterisks.
DEVELOPMENT
Immunoprecipitation (IP) and western blotting
RESULTS
Both Pitx3 and Nurr1 form a complex with the corepressor PSF
Based on the discussed similarities between Nurr1- and Pitx3-null
mice, we hypothesised that Pitx3 and Nurr1 are interconnected at the
level of transcriptional regulation. Notably, functional interactions
between homeobox transcription factors and nuclear receptors have
been proposed previously (Tremblay et al., 1999; Shan et al., 2005).
In the pituitary gland, Pitx1 interacts with the orphan nuclear
receptor ‘steroidogenic factor 1’ (SF1; Nr5a1) and multiple coregulators to activate SF1-mediated transcription (Tremblay et al.,
1999; Quirk et al., 2001). For mdDA neurons, the identification of
proteins that physically interact with Pitx3 might provide clues to
the relationship between Nurr1 and Pitx3 at the level of gene
transcription.
Through affinity purification of Pitx3-His proteins followed by
mass spectrometry, we identified the transcriptional co-repressors
PSF (SFPQ; PTB-associated splicing factor) and Nono [non-POUdomain-containing, octamer binding protein/p54(nrb)] as proteins
that specifically interact with Pitx3 in dopaminergic MN9D cells
(Choi et al., 1991) (Fig. 1A). Interestingly, PSF and Nono are
reported to form a heterodimeric complex involved in repression of
nuclear receptor-mediated transcription (Mathur et al., 2001; ShavTal and Zipori, 2002; Sewer et al., 2002; Dong et al., 2007). PSF has
been most extensively studied in relation to nuclear receptor
repression and therefore we focussed on PSF. The interaction of
Pitx3 with endogenous PSF in MN9D cells was confirmed by means
of co-immunoprecipitation (Co-IP) (Fig. 1B). To investigate
whether the interaction of Pitx3 with PSF also exists in vivo, we
performed immunoprecipitation (IP) on E14.5 mdDA neurons.
Indeed, endogenous PSF clearly co-immunoprecipitated with Pitx3
(Fig. 1C), demonstrating the interaction of Pitx3 and the nuclear
receptor co-repressor PSF in developing mdDA neurons.
Most compelling, PSF has been shown to interact with the
retinoic acid receptor RXR (Mathur et al., 2001), a well-described
heterodimerization partner for Nurr1 in mdDA neurons (Perlmann
and Jansson, 1995; Wallen-Mackenzie et al., 2003). Therefore, we
hypothesised that, like Pitx3, Nurr1 might also interact with PSF.
Immunoprecipitation with Nurr1 antibodies was performed in
RESEARCH ARTICLE
533
MN9D cells, and endogenous PSF was indeed detected in Nurr1
immunoprecipitates (Fig. 1D). Moreover, PSF was also detected in
Nurr1 and RXR-immunoprecipitates of E14.5 mdDA neurons,
suggesting that besides Pitx3, the nuclear receptors Nurr1 and RXR
also interact with PSF in vivo (Fig. 1E). These observations, together
with the previously described involvement of PSF in repression of
nuclear receptors, makes PSF an appealing candidate for bridging
Pitx3 to Nurr1 in mdDA neurons.
Pitx3 and Nurr1 target the same promoter regions
in the genome
The identification of PSF as interacting protein of both Pitx3 and
Nurr1 provided novel leads to the molecular background of the
functional interplay between Pitx3 and Nurr1. In their role as
developmental regulators, both Pitx3 and Nurr1 are crucial for the
induction of Th in mdDA neurons of the substantia nigra (Smidt et
al., 2004; Maxwell et al., 2005; Jacobs et al., 2007). In addition, PSF
has been shown to repress the TH-promoter in human CHP-212 cells
(Zhong et al., 2006). Although the involvement of Pitx3 or Nurr1
has not been investigated in this context, other studies have
independently shown that both Nurr1 and Pitx3 can bind and
activate the Th promoter (Cazorla et al., 2000; Iwawaki et al., 2000;
Lebel et al., 2001; Kim et al., 2003). The finding that both Pitx3 and
Nurr1 interact with PSF could indicate that Nurr1 and Pitx3 are part
of the same transcriptional complex on the Th promoter and possibly
other target genes as part of a general mechanism for transcription
regulation of mdDA-associated genes.
In order to investigate the occupancy of Pitx3 and Nurr1 on
promoters of a large and unbiased set of genes, we performed ChIPon-chip analysis. We chose two different approaches to obtain DA
neurons. For the first approach, we expressed Pitx3 and Nurr1 in
dopaminergic MN9D cells and for the second approach we used in
vivo mdDA neurons derived from E14.5 ventral midbrain.
Chromatin immunoprecipitation (ChIP) was performed with either
Pitx3, Nurr1 or control (pre-immune serum) antibodies, and the IPefficiency was determined by western blot analysis (Fig. 2A,B).
Amplified ChIP-DNA fragments were labelled with Cy3 and
hybridized to Cy5-labelled input chromatin on a MM8 mouse
promoter array (Nimblegen). The ChIP-on-chip analysis in MN9D
Fig. 1. Both Pitx3 and Nurr1 interact with the co-repressor protein PSF. (A) Pitx3-His interacts with the co-repressor complex PSF/Nono. Histag based affinity purification of Pitx3-His proteins from MN9D cells, followed by protein gel silverstaining, revealed four protein bands that
specifically interacted with Pitx3-His. Through mass spectrometry analysis, the protein bands of 100, 95 and 75 kDa were identified as PSF and the
55 kDa band was identified as Nono. (B,C) Pitx3 interacts with PSF in MN9D cells and in vivo. Lysates of Pitx3 transfected MN9D cells (B) or E14.5
mdDA neurons (C) were subjected to IP for Pitx3 or pre-immune serum, and immunoblotted for Pitx3 and PSF. (D,E) Nurr1 interacts with PSF in
MN9D cells and in vivo. Lysates of MN9D cells expressing Nurr1 (D) and E14.5 mdDA neurons (E) were subjected to IP for Nurr1, RXR or preimmune serum, and immunoblotted for Nurr1 and PSF. I, input; IP, immunoprecipitation; Ctrl, pre-immune serum control.
DEVELOPMENT
Pitx3 outSMRTs Nurr1 in dopamine neurons
534
RESEARCH ARTICLE
Development 136 (4)
cells revealed that 1652 and 541 promoter regions were enriched by
ChIP for Nurr1 or Pitx3, respectively (Fig. 2C). In the in vivo ChIP,
a smaller number of promoter regions was enriched: 208 by Nurr1
and 140 by Pitx3 (Fig. 2D). This lower number may reflect the more
stringent conditions of promoter occupancy in the in vivo situation
compared with in a cell line. Strikingly, a substantial amount of
promoter regions that were enriched by Nurr1, were also enriched
by Pitx3 (see Tables S1 and S2 in the supplementary material). This
was observed for ~22% and 28% of the Nurr1-enriched promoter
regions in MN9D cells and in vivo, respectively. Of special interest
was the enrichment of promoter regions near genes that have
previously been described to be regulated by Nurr1. The ChIP-onchip analysis of MN9D cells revealed that the promoter of the Nurr1
target gene Vip (vasoactive intestinal peptide) (Luo et al., 2007) was
highly enriched by Nurr1-ChIP (Fig. 2E). Remarkably, the same Vip
promoter region was clearly also enriched by Pitx3-ChIP, which
suggests that Pitx3 and Nurr1 bind to the same promoter region of
the Nurr1 transcriptional target Vip. The enrichment of the Vip
promoter was validated by PCR on ChIP-DNA, confirming a
significant enrichment of the Vip promoter region by both Pitx3- and
Nurr1-ChIP compared with Control-ChIP in MN9D cells [Fig.
2K,L; n=3; P<0.01 (Pitx3-ChIP)/P<0.05 (Nurr1-ChIP)].
The promoter of Vmat2, a well described target of Nurr1 in vivo,
was clearly also enriched by ChIP for both Nurr1 and Pitx3 (Fig.
2F). The enrichment was observed in both MN9D cells and E14.5
mdDA neurons, which is in line with the described regulatory effect
of Nurr1 on Vmat2 expression in MN9D cells and in vivo
(Hermanson et al., 2003; Smits et al., 2003). PCR-based validation
confirmed the significant enrichment of the selected Vmat2
promoter region by ChIP for both Pitx3 and Nurr1 (Fig. 2K,L; n=3;
P<0.01). Within the tiled regions of the promoters of Ahd2, Dat, Th
and D2R, which are established as Nurr1-regulated genes in mdDA
neurons (Zetterström et al., 1997; Le et al., 1999; Castillo et al.,
1998; Wallén et al., 1999; Smits et al., 2003), we did not observe
DEVELOPMENT
Fig. 2. Pitx3 and Nurr1 target the
same regions on promoters of Nurr1
target genes.
(A,B) Immunoprecipitation of Pitx3 and
Nurr1 from crosslinked chromatin. ChIPEluates from E14.5 mdDA neurons were
immunoblotted for Nurr1 (A) or Pitx3
(B). (C,D) Representation of uniquely
and mutually enriched promoter regions
by ChIP for Nurr1 and Pitx3 in MN9D
cells (C) and E14.5 mdDA neurons (D)
[false discovery rate (FDR)<0.01].
(E-J) Pitx3 and Nurr1 interact with the
same regions within promoters of a set
of Nurr1-regulated genes. Note that
high confidence binding sites of
transcription factors are identified as a
positive signal for multiple neighbouring
probes (visualised as peaks). Relative
enrichment of the promoters of Vip (E),
Vmat2 (F), Ahd2 (G), Dat (H), Th (I) and
D2R (J) by ChIP for Pitx3 and Nurr1 in
MN9D cells and in vivo E14.5 mdDA
neurons. Regions enriched by both Pitx3
and Nurr1 are indicated as series of red
peaks. (K,L) ChIP-PCR validation.
Significant enrichment of the regions
indicated in red in E and F within the
promoters of Vip (Pitx3-ChIP; n=3;
P=0.002, Nurr1-ChIP; n=3; P=0.012)
and Vmat2 (Pitx3-ChIP; n=3; P=0.006,
Nurr1-ChIP; n=3; P=0.009) by ChIP for
Pitx3 and Nurr1, but not pre-immune
serum. No enrichment was observed for
a region (Control) in the Vmat2
promoter that was not enriched by
ChIP-on-chip. Data are represented as
mean±s.e.m., *P<0.05, **P<0.01; IP,
immunoprecipitation; Ctrl, Pre-immune
serum; TSS, transcription start site; AB,
antibody; E14.5, dissected E14.5 mdDA
area.
Pitx3 outSMRTs Nurr1 in dopamine neurons
RESEARCH ARTICLE
535
mutually enriched regions (Fig. 2G-J). However, because on average
5 kb of each promoter is represented on the array, we cannot exclude
the possibility that Nurr1 and Pitx3 targeted sites are outside the tiled
regions. Altogether, these data demonstrate the concerted occupancy
of Pitx3 and Nurr1 on a set of promoters, including those of the
Nurr1-regulated genes Vip and Vmat2.
Loss of Pitx3 expression in vivo increases the
interaction of the co-repressor SMRT with Nurr1
Our data demonstrate the concurrent involvement of Pitx3 and
Nurr1 in the regulation of the same set of genes within mdDA
neurons. However, the relative severity of the analyzed molecular
phenotypes suggests that, whereas Nurr1 can be considered a master
switch on DA-gene transcription, Pitx3 appears to regulate the
transcriptional activity of Nurr1. Generally, the activity of nuclear
receptors is highly regulated by co-regulating proteins. Upon
Fig. 3. Pitx3 is crucial for expression of a set of Nurr1 target
genes. (A) Overview of sagittal sections of 14.5 embryos (middle panel
was adapted from www.genepaint.org). In the right panel, the location
of the mdDA neurons in the medial part of the mdDA area is indicated
in red, the broken line represents the area shown in B-M. (B-M) In situ
hybridization using DIG-labelled probes for a set of mdDA expressed
genes in Pitx3+/+/Pitx3–/– (B-G) and Nurr1+/+/Nurr1–/– (H-M) littermate
embryos at stage E14.5. Homozygous mutant embryos are indicated
with an asterisk (*). (B,C,H,I) Normal presence of mdDA neurons in
Pitx3–/– and Nurr1–/– embryos. (B,H) In situ hybridization for Nurr1. Note
that truncated Nurr1 transcripts were still detected in Nurr1–/– embryos
(H*). (C,I) In situ hybridization for En1. (D-F,J-L) Expression of a set of
Nurr1-regulated genes was affected in Pitx3–/– embryos. In situ
hybridization for Dat (D,J), Vmat2 (E,K) and D2R (F,L). Note the mdDAspecific defect of Vmat2 expression in Pitx3–/– (E*) and Nurr1–/– (K*)
embryos. The red line indicates the location of the mid-hindbrain
border. (G,M) In situ hybridization for Aadc. h, hindbrain; m, midbrain;
f, forebrain.
activation of nuclear receptors, co-repressors are released and coactivators are recruited (Fowler and Alarid, 2004; Wolf et al., 2008).
Possibly, recruitment of Pitx3 affects the composition of the Nurr1
transcriptional complex in relation to the interaction with corepressor proteins such as PSF. As we have demonstrated that the
co-repressor PSF interacts with both Pitx3 and Nurr1, the interaction
of Nurr1 with PSF might depend on the presence of Pitx3. Another
plausible candidate in this respect is the co-repressor Sin3a, which
is recruited by PSF to mediate nuclear receptor repression (Mathur
et al., 2001). In order to test any alterations of the composition of the
Nurr1 transcriptional complex upon Pitx3 ablation in vivo, we
performed IP for Nurr1 from E14.5 wild-type and Pitx3–/– mdDA
DEVELOPMENT
Pitx3 regulates a set of genes involved in
dopamine metabolism, previously described as
Nurr1 target genes
Thus far, we have shown that Pitx3 and Nurr1 form a complex with a
common co-repressor, and bind to regions within the promoters of
Nurr1 target genes. These observations make it tempting to
hypothesize that these physical associations have functional
consequences in mdDA neurons. Two lines of evidence are in
agreement with this hypothesis. First, combined transduction of Pitx3
and Nurr1 in ES cells leads to increased transcript levels of DA genes,
such as Vmat2, Dat, Th and Aadc (Martinat et al., 2006). Second, the
Pitx3 transcriptional target gene Ahd2 is not detected in the midbrain
area of Nurr1-deficient embryos at E15.5 (Wallén et al., 1999) or
E14.5 (F.M.J.J. and M.P.S., unpublished). In order to elucidate the
gene-regulatory function of Pitx3 in relation to Nurr1 in the
development of mdDA neurons, we performed in situ hybridization
on sagittal sections of E14.5 Pitx3–/– embryos, and compared them
with E14.5 Nurr1–/– embryos (Fig. 3A). The normal expression of En1
and Nurr1 in Pitx3–/– embryos and the conserved expression of En1
and truncated Nurr1 transcripts in Nurr1–/– embryos is indicative of a
normal presence of mdDA neurons in medial sagittal sections (Fig.
3B,C,H,I). As described previously, the embryonic expression of Dat
and Vmat2 was completely lost in Nurr1–/– mdDA neurons (Fig.
3J,K), whereas the expression of D2R and Aadc was strongly
decreased (Fig. 3L,M). Interestingly, Dat was not detected in Pitx3–/–
mdDA neurons, whereas it was clearly expressed in Pitx3+/+ littermate
controls (Fig. 3D). Furthermore, the expression of Vmat2, which is
completely absent in Nurr1–/– mdDA neurons, was drastically reduced
in Pitx3–/– mdDA neurons (Fig. 3E). Notably, Vmat2 was abundantly
expressed in the hindbrain of Nurr1- and Pitx3-deficient embryos,
clearly demonstrating a mdDA neuron-specific deficiency of Vmat2
expression. Similarly, D2R was expressed at barely detectable levels
in Nurr1–/– mdDA neurons (Fig. 3L) and was strongly reduced in
Pitx3–/– mdDA neurons compared with littermate controls (Fig. 3F).
Whereas Aadc expression was clearly decreased in Nurr1–/– mdDA
neurons (Fig. 3M), we observed no difference of Aadc expression
between Pitx3–/– and Pitx3+/+ embryos in these medial sections (Fig.
3G). Altogether, besides the previously described effects on the
expression of Th and Ahd2, loss of Pitx3 clearly affects the expression
of Vmat2, Dat and D2R in mdDA neurons during development.
Interestingly, next to Nurr1 as essential factor for the dopaminergic
phenotype, Pitx3 is crucial for canonical expression of genes
previously described as Nurr1 downstream targets, strengthening our
hypothesis that Pitx3 and Nurr1 constitute a physical and functional
complex in transcription regulation.
RESEARCH ARTICLE
Fig. 4. Pitx3 decreases the interaction of Nurr1 with the nuclear
receptor co-repressor SMRT. (A-E) Co-IP of Nurr1 with a set of
nuclear receptor co-repressors in wild-type and Pitx3–/– E14.5 mdDA
neurons. Lysates were subjected to IP for Nurr1 or pre-immune serum,
and immunoblotted for PSF (A), Sin3a (B), Ncor (C), SMRT (D) or Nurr1
(E). (F) The interaction of Nurr1 with SMRT was increased in the
absence of Pitx3. Relative increase of interaction with Nurr1 in Pitx3–/–
mdDA neurons over wild type was calculated for PSF (n=3; P=0.12),
Sin3a (n=3; P=0.35) and SMRT (n=4; P=0.002). Data are represented as
mean±s.e.m., *P<0.01; I, input; IP, immunoprecipitation; Ctrl, preimmune serum; WT, wild type.
neurons. Importantly, in accordance with what was observed
by in situ hybridization, similar amounts of Nurr1 were
immunoprecipitated from wild-type and Pitx3–/– mdDA neurons
(Fig. 4E). Besides PSF, the PSF-interacting co-repressor Sin3a also
interacted with Nurr1 in vivo. However, the level of interaction of
Nurr1 with either PSF or Sin3a was not affected by the presence of
Pitx3 (Fig. 4A,B; n=3). These data indicate that the physical
interaction of PSF and Sin3a with Nurr1 is unrelated to the potency
of Nurr1 to activate its target genes. This is in line with other studies,
reporting that activation of nuclear receptors by ligand binding does
not result in dissociation of Sin3a or PSF (Mathur et al., 2001).
Although no endogenous ligand has been identified for Nurr1, its
ligand-binding domain interacts with the co-repressors Ncor
(nuclear receptor co-repressor 1) and SMRT (silencing mediator of
retinoic acid and thyroid hormone receptor) (Codina et al., 2004;
Lammi et al., 2008). In general, Ncor and SMRT bind to unliganded
nuclear receptors, and are believed to keep these complexes in a
repressed state (Nishihara et al., 2004). Ligand binding to the nuclear
receptor complex results in dissociation of Ncor and SMRT,
allowing activation of gene transcription. As a possible regulatory
mechanism of Nurr1-mediated transcription in mdDA neurons, the
recruitment of Pitx3 to the Nurr1 transcriptional complex might
mimic the effect of ligand activation of Nurr1, by inducing
the dissociation of SMRT or Ncor. To test this, we examined
whether the level of interaction between Nurr1 and the co-repressors
Ncor and SMRT was altered upon ablation of Pitx3 in mdDA
neurons. Although we did not detect an interaction between Ncor
and Nurr1 (Fig. 4C), the co-repressor SMRT was clearly coimmunoprecipitated with Nurr1. Strikingly, we observed a
significant increase in SMRT interaction with Nurr1 in the absence
of Pitx3 (Fig. 4D). The SMRT interaction with Nurr1 was increased
approximately threefold in Pitx3–/– mdDA neurons compared with
wild type, as determined by densitometry (Fig. 4F; n=4; P<0.01).
This suggests that in the absence of Pitx3, the transcriptional activity
of Nurr1 is kept in a repressed state by SMRT, and recruitment of
Pitx3 leads to activation of Nurr1, through release of the SMRTmediated repression.
Development 136 (4)
Release of HDAC-mediated repression in Pitx3deficient embryos restores Nurr1 target gene
expression in vivo
SMRT exerts its repressive actions through recruitment of a set of
class I and class II histone deacetylases (HDACs) (Huang et al.,
2000; Guenther et al., 2001; Fischle et al., 2002). The de-acetylated
state of histones near the transcription start site is responsible for
the repression of gene transcription. The potential involvement of
HDACs in the regulation of Nurr1 was further strengthened by the
detection of endogenous HDAC1 in Nurr1 immunoprecipitates
from E14.5 dissected ventral midbrains (see Fig. S1 in the
supplementary material). Our data suggest that Nurr1 occupies the
promoters of target genes in Pitx3–/– embryos but is kept in a
repressed state owing to the interaction with SMRT/HDAC
complexes. Hypothetically, interference with HDAC activity in
Pitx3–/– embryos would result in re-activation of Nurr1-mediated
transcription, bypassing the necessity for Pitx3. To test this
hypothesis, we focussed on the expression of Vmat2 and D2R
because of the reduced transcript level throughout the mdDA area
in Pitx3–/– embryos (Fig. 5B,C) and on Th because of the mdDA
subset-specific loss of Th expression in Pitx3–/– embryos (Fig. 5D).
In order to isolate mdDA neurons for gene expression analysis by
means of FACS sorting, we set up a breeding scheme to obtain
Pitx3-deficient Pitx3gfp/– and heterozygous Pitx3gfp/+ embryos
(Zhao et al., 2004; Maxwell et al., 2005). Importantly, the
expression of GFP was highly similar in Pitx3gfp/– and Pitx3gfp/+
embryos at stage E14.5 (Fig. 5E), showing the normal presence of
mdDA neurons in Pitx3-deficient mdDA neurons during embryonic
development. Ventral midbrains of Pitx3gfp/+ and Pitx3gfp/– embryos
were dissected at stage E13.5 and cultured for 2 additional days. To
interfere with HDAC activity, we treated the ventral midbrains with
different concentrations of the a-specific HDAC inhibitor sodium
butyrate, after which GFP-positive mdDA neurons were selected
by FACS sorting (Fig. 5F-H). Importantly, similar numbers of GFPpositive neurons were collected from all treated and untreated
Pitx3gfp/– and Pitx3gfp/+ cultures. Equal amounts of RNA from GFPpositive mdDA neurons were subjected to semi-quantitative RTPCR to determine the relative transcript levels of Vmat2, D2R and
Th. Tbp (TATA-box binding protein) was taken along as control,
which showed no difference in transcript levels in mdDA neurons
of both genotypes and upon treatment with sodium butyrate (Fig.
5I,M; n=3). In heterozygous Pitx3gfp/+ mdDA neurons, no
significant increase in relative amounts of Vmat2, D2R and Th
transcripts was observed upon sodium butyrate treatment (Fig. 5IL; n=3). In agreement with what was observed for Vmat2
expression by in situ hybridization, a significant lower level of
Vmat2 transcripts was observed in untreated Pitx3gfp/– mdDA
neurons, at ~7% compared with the level of Vmat2 in untreated
Pitx3gfp/+ mdDA neurons (Fig. 5I,J; n=3). Strikingly, treatment with
0.3 mM sodium butyrate resulted in a significant increase in Vmat2
transcripts, restoring levels to 56% (n=3; P<0.01). Vmat2 levels
were even restored to 84% in cultures treated with 0.6 mM sodium
butyrate (n=3; P<0.01). Similarly, D2R levels were restored from
53% in untreated Pitx3gfp/– mdDA neurons to 98% in 0.6 mMtreated cultures (Fig. 5I,K; n=4; P<0.01) and levels of Th were
increased from 60% in untreated Pitx3gfp/– mdDA neurons to 94%
in cultures treated with 0.6 mM sodium butyrate (Fig. 5I,L; n=3;
P=0.05). These data clearly demonstrate that inhibition of HDACmediated repression in Pitx3–/– embryos efficiently restores the
transcriptional activation of a set of Nurr1 target genes, bypassing
the necessity for Pitx3. These observations signify our proposed
mechanism that in mdDA neurons, full activation of Nurr1 target
DEVELOPMENT
536
Pitx3 outSMRTs Nurr1 in dopamine neurons
RESEARCH ARTICLE
537
genes depends on the release of the SMRT/HDAC-mediated
repression of the Nurr1 transcriptional complex through the
regulatory action of Pitx3.
DISCUSSION
The regulatory effect of Pitx3 on the association of Nurr1 with
SMRT and the finding that Nurr1-mediated transcription of Vmat2,
D2R and Th is repressed in a HDAC-dependent manner in the
absence of Pitx3, enabled us to propose a model in which Pitx3 is
a key regulator of Nurr1-mediated transcription (Fig. 6).
Accordingly, the recruitment of Pitx3 to PSF within the Nurr1
transcriptional complex leads to (full) activation of Nurr1 target
genes, by inducing the release of SMRT/HDAC-mediated
repression from Nurr1. Ablation of Nurr1, acting as a master
switch on gene transcription, results in complete loss of target gene
expression. However, ablation of Pitx3 mainly affects Nurr1
transcriptional activity, which would still allow Nurr1-mediated
transcription at a lower level (Vmat2/D2R), or in a certain mdDA
subpopulation (Th). This model also explains why expression of
the Nurr1 target genes Dat, Vmat2 and D2R is not detected until
after Pitx3 is expressed at E11.5, although Nurr1 is already
expressed at E10 (Simon et al., 2003; Wallen-Mackenzie et al.,
2003; Smits and Smidt, 2006; Alavian et al., 2008).
Interestingly, another member of the paired like homeobox
transcription factors has been described to functionally and
physically interact with nuclear receptors in the pituitary. Pitx1
enhances the transcriptional activity of the orphan nuclear receptor
SF1 (Tremblay et al., 1999; Quirk et al., 2001; Melamed et al., 2002),
and Pitx1, together with the estrogen receptor, modify the repressive
androgen receptor/SF1 complex on the LHβ promoter (Jorgensen
and Nilson, 2001). Moreover, SF1 has also been shown to interact
with PSF (Sewer et al., 2002). The apparent analogy between the
regulation of an orphan nuclear receptor by Pitx1 and our data are
indicative of a more general mechanism involving PSF, in which
homeodomain transcription factors modulate nuclear receptormediated transcription. Most likely, more components are involved
in the proposed mechanism. The release of the co-repressor SMRT
from Nurr1 could be accompanied by the recruitment of activating
proteins, although co-activators of Nurr1 have yet to be identified
(Castro et al., 1999; Wang et al., 2003; Volakakis et al., 2006; Lammi
DEVELOPMENT
Fig. 5. Interference with HDAC-mediated
repression in Pitx3–/– embryos, restores Nurr1
target gene expression. (A) Overview of coronal
sections of an E14.5 embryonic brain. Adapted, with
permission, from Kaufman (Kaufman, 1992). The
location of the mdDA neurons is indicated in red, the
broken line represents the area shown in B-D. (B-D) In
situ hybridization on coronal sections of E14.5 Pitx3+/+
and Pitx3–/– littermate embryos. Homozygous mutant
embryos are indicated with an asterisk. (B,C) Expression
of Vmat2 and D2R was decreased throughout the
mdDA area in E14.5 Pitx3–/– embryos. (D) Expression of
Th was deficient in a subpopulation of the mdDA
neurons in E14.5 Pitx3–/– embryos. (E) A similar pattern
of GFP-positive neurons in E14.5 Pitx3gfp/+ (E) and
Pitx3gfp/– (E*) reveals the apparent normal presence of
mdDA neurons in Pitx3-deficient Pitx3gfp/– embryos.
(F-H) The FACS sorting gate was set, using a E14.5
C57Bl6-Jico (Bl6) reference sample (F) to select GFPpositive mdDA neurons from Pitx3gfp/+ (G) and Pitx3gfp/–
(H) midbrain cultures with a purity of 98% (data not
shown). (I-M) Treatment with the HDAC inhibitor
sodium butyrate restores expression of Nurr1 target
genes in Pitx3gfp/– mdDA neurons. (I) RNA from FACSsorted mdDA neurons derived from cultures treated
with 0 mM, 0.3 mM or 0.6 mM of sodium butyrate
(n=3) were subjected to semi-quantitative RT-PCR.
Relative transcript levels were determined by
densitometry and compared with transcript levels in
untreated Pitx3gfp/+ mdDA neurons. Relative values
were calculated for Vmat2 (J; Pitx3gfp/– 0.3 mM P=0.006,
Pitx3gfp/– 0.6 mM; P=0.007), D2R (K; Pitx3gfp/– 0.3 mM;
P=0.0006, Pitx3gfp/– 0.6 mM; P=0.001), Th (L;
Pitx3gfp/– 0.3 mM; P=0.19, Pitx3gfp/– 0.6 mM; P=0.05) and
Tbp (M; Pitx3gfp/– 0.3 mM P=0.5, Pitx3gfp/– 0.6 mM; P=0.5).
Data are represented as mean±s.e.m., *P=0.05,
**P<0.01.
538
RESEARCH ARTICLE
Development 136 (4)
et al., 2008). Notably, β-catenin has been reported to affect the
transcriptional activity of both Pitx factors and SF1 (Vadlamudi et al.,
2005; Hossain and Saunders., 2003) which makes β-catenin an
interesting potential component of the Nurr1/PSF/Pitx3 complex.
In zebrafish, PSF is involved in neuronal differentiation, and loss
of PSF results in developmental defects in the mid- and hindbrain
(Lowery et al., 2007). Although PSF is expressed in mdDA neurons
(data not shown) and forms a complex with Pitx3 and Nurr1, the role
of PSF in the Nurr1 transcriptional complex is unclear. The
interaction of Nurr1 with PSF is unchanged in Pitx3–/– embryos,
which makes it unlikely that Pitx3 is involved in the recruitment or
dissociation of PSF to the Nurr1 transcriptional complex.
Importantly, besides PSF, we showed that Nurr1 also interacts with
Sin3a, which is involved in transcriptional repression by recruiting
SMRT and HDACs (Wolffe, 1997). This may indicate that the effect
of Pitx3 on dissociation of SMRT from the Nurr1 transcriptional
complex also involves PSF and Sin3a.
The shared interaction of Pitx3 and Nurr1 with PSF suggests that
Pitx3 and Nurr1 are part of the same transcriptional complex. The
results from the ChIP-on-chip analysis further strengthen this
hypothesis, as we identified a number of promoter regions that were
enriched by ChIP for both Pitx3 and Nurr1. Most intriguingly, both
Pitx3 and Nurr1 interact with the promoter of Vmat2, which is
crucial for DA metabolism and is regulated by Nurr1 in vivo (Smits
et al., 2003). These observations strongly suggest that Pitx3 and
Nurr1 are recruited in concert with a number of promoters, including
some that have been previously associated with Nurr1.
The shared physical interaction of Pitx3 and Nurr1 with the
promoter of Vmat2 is translated to function in vivo, as expression
analysis of E14.5 Pitx3–/– embryos revealed an essential role for
Pitx3 for the proper expression of a set of described Nurr1 target
genes, including Vmat2. These observations shed new light on the
complicated role of Pitx3 in mdDA neurons, as it has been puzzling
for a long time that although Pitx3 is expressed in all mdDA neurons,
only the subpopulation of the substantia nigra is dependent on Pitx3
for induction of Th and subsequential maintenance into adulthood.
This discrepancy indeed holds true for the expression of Th,
underlining the existence of mdDA subsets with differential reliance
on Pitx3. However, based on analysis of the expression patterns of
Vmat2, Dat and D2R, the entire mdDA population is affected by
Pitx3 deficiency. Our observations clearly indicate that in addition
to the previously described subset-specific role for Pitx3 in the
substantia nigra, Pitx3 is crucial for the correct molecular phenotype
of mdDA neurons in general. This finding could be an explanation
for the aberrant electrophysiological properties of surviving Thpositive neurons in Pitx3-deficient mice, which has remained
unexplained until now (Smits et al., 2005).
Interestingly, in vivo expression analysis revealed a striking
difference between the relative severity of the Nurr1 target gene
deficit between Nurr–/– and Pitx3–/– embryos, which is most evident
for the expression of Vmat2. It appears that although Nurr1 is crucial
for the expression of its target genes, Pitx3 is essential for the level
of Nurr1-mediated transcription. The increased interaction of Nurr1
with the co-repressor SMRT in the absence of Pitx3 is a suitable
molecular explanation for this observation. The regulatory effect of
Pitx3 on the physical interaction of Nurr1 with the co-repressor
SMRT strongly suggests that Pitx3 has the potency to induce the
release of SMRT-mediated repression from the Nurr1 transcriptional
complex. Indeed, interference with the repressive activity of
HDACs, which are recruited to unliganded nuclear receptors by
SMRT, efficiently restores the transcript levels of the Nurr1 target
genes Vmat2, D2R and Th in cultured Pitx3gfp/– midbrains. This
clearly indicates that, in the absence of Pitx3, the Nurr1
transcriptional complex is kept in a repressed state by SMRT/
HDAC, which signifies the regulatory role of Pitx3 on the
expression of Nurr1 target genes as we proposed in our model.
Altogether, as part of a proposed general modulatory effect of Pitx
factors on nuclear receptor activity, our data provide evidence for
the existence of a transcriptional complex involving Pitx3 and
Nurr1, positioning Pitx3 as an essential activator of the potency of
DEVELOPMENT
Fig. 6. Pitx3 is a crucial regulator of Nurr1-mediated transcription. In the absence of Pitx3, the Nurr1 transcriptional complex is kept in a
repressed state by SMRT through recruitment of HDACs, which keep the target gene promoter in a de-acetylated (repressed) state, resulting in
deficiency of Nurr1 target gene expression. Interference with HDAC-mediated repression by sodium butyrate restores Nurr1-mediated transcription
of Nurr1 target genes in the absence of Pitx3. Recruitment of Pitx3 to Nurr1 target gene promoters strongly resembles the effect of ligand
activation of nuclear receptors by inducing the dissociation of SMRT from the Nurr1 transcriptional complex, which favours (full) activation of
transcription of Nurr1 target genes.
Nurr1 to drive expression of genes essential for mdDA neurons. In
conclusion, our data significantly contribute to a better
understanding of mdDA neuron development, as we have shown
that Nurr1 and Pitx3 act in concert to induce the mdDA
characteristics of neurons, which has large consequences for stemcell engineering as a future treatment for Parkinson’s disease.
We thank Thomas Perlmann for the generous gift of the MN9D cells, Orla
Conneely for providing us with Nurr1 knockout mice, Meng Li for providing us
with the Pitx3-GFP knock-in mice, Koen Dreijerink for providing us with the
HDAC antibodies, Simone Smits for many helpful discussions, Ger Arkestein
for FACS sorting and excellent technical assistance, Jeroen Pasterkamp for
helpful comments on the manuscript, and Roger Koot for his excellent support
in the data analysis. This work was supported by a HIPO-grant (University of
Utrecht) to M.P.S.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/136/4/531/DC1
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