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First published online June 25, 2007
doi: 10.1242/10.1242/dev.02865
Rudolf Magnus Institute of Neuroscience, Department of Pharmacology and Anatomy, University Medical Center Utrecht, Universiteitsweg 100, 3584 CG Utrecht, The Netherlands.
Author for correspondence (e-mail:
m.p.smidt-2{at}umcutrecht.nl)
Accepted 3 May 2007
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SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: Pitx3, Retinoic acid, Aldehyde dehydrogenase, Development, Midbrain, Dopamine, Parkinson's disease, Ahd2, aldh1a1, Mouse
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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).
Analysis of animal models for PD demonstrates a similar susceptibility to
degeneration of SNc DA neurons, indicating a conserved phenomenon among
species (Blum et al., 2001).
Although several genes have now been associated with PD
(Moore et al., 2005;
Abou-Sleiman, 2006), the exact
mechanism underlying the specific degeneration of DA neurons located in the
SNc is not fully understood.
The homeodomain transcription factor Pitx3, exclusively expressed in
meso-diencephalic DA (mdDA) neurons in the brain
(Smidt et al., 1997), is
essential for the proper development of the mdDA system. Analysis of
Pitx3-deficient mice has revealed that the loss of Pitx3 expression
leads to the selective loss of a neuronal subset located primarily in the SNc
(Hwang et al., 2003;
Nunes et al., 2003;
van den Munckhof et al., 2003;
Smidt et al., 2004a;
Smidt et al., 2004b). The
mechanism by which Pitx3 influences the development of this specific mdDA
subpopulation is still unknown. Although Pitx3 deficiency results in
early developmental defects of mdDA neurons, whereas PD is a progressive
neurodegenerative disease with late onset, both affect the same mdDA neuronal
population. Therefore, knowledge of molecular pathways controlled by Pitx3
might provide new insights into mdDA pathology, as in PD. However, until now,
no target genes of Pitx3 have been identified, although some genes are
associated with Pitx3 function (Smits et
al., 2006). One of these genes encodes the enzyme aldehyde
dehydrogenase family 1, subfamily A1 (Aldh1a1; also known as Raldh1 or Ahd2).
In embryonic stem (ES) cells, transgenic expression of Pitx3 leads to an
increased ratio of ES cells that are positive for both Ahd2 and tyrosine
hydroxylase (Ahd2+/TH+) (Chung et al.,
2005). Although this suggests a relationship between Pitx3 and
Ahd2 expression, a relatively large portion of cells (approximately 70%) that
were positive for both Pitx3 and TH still showed no induction of Ahd2.
In mice, Ahd2 is first detected in the mesencephalic flexure as early as
embryonic day (E)9 (Haselbeck et al.,
1999; Westerlund et al.,
2005). There is, however, a marked change in Ahd2 distribution
during different stages of development within this area. From E9 until E11.5,
Ahd2 is found in a broad area ranging from the ventricular zone to the most
ventral neuronal population within the mantle layer
(Wallen et al., 1999). By
contrast, from E12.5 onwards, Ahd2 distribution is largely confined to a
selective subpopulation of mdDA neurons in a rostro-ventral portion of the
mdDA area (McCaffery and Drager,
1994; Wallen et al.,
1999; Chung et al.,
2005). These data suggest that Ahd2 has a dual role in the
presumptive mdDA area throughout development, functioning both in the
proliferative and migratory stages, and during the final differentiation and
maintenance of a selective mdDA neuronal subpopulation. Ahd2 is involved in
the production of retinoic acid (RA) from retinol, which is crucial for
neuronal patterning and differentiation
(Duester, 2000;
McCaffery et al., 2003). Via
binding to the RA receptor (RAR) and retinoid X receptor (RXR), RA has been
shown to induce tissue-specific gene transcription leading to cellular
differentiation (Chambon,
1996). RA is detected in the midbrain area during early embryonic
stages (Niederreither et al.,
2002a), and in the postnatal and adult brain (McGaffery and
Drager, 1994). Interestingly, Ahd2 is the only aldehyde dehydrogenase present
in the mdDA area during the developmental and adult stage
(Niederreither et al., 2002b),
suggesting an important role for Ahd2-mediated RA production in the mdDA
system.
Based on the restricted expression pattern of Ahd2 within the mdDA neuronal population, together with the implicated relationship between Pitx3 and Ahd2 in ES cells, we studied the relationship between Pitx3 and Ahd2 in more detail in mdDA neurons. Our results show that Pitx3 regulates Ahd2 expression both in vivo and in vitro. Furthermore, Pitx3 interacts with a highly conserved region in the proximity of the transcriptional start site of the Ahd2 gene, suggesting that Pitx3 activates Ahd2 directly at the transcriptional level. Most intriguingly, we show that maternal supplementation of RA counteracts the developmental defects caused by Pitx3 deficiency in a specific mdDA neuronal subpopulation. In this study, we provide evidence for the existence of a developmental cascade in which Pitx3 is positioned centrally in RA-dependent final differentiation of mdDA neurons.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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; Rieger et al.,
2001; Smidt et al.,
2004a) were used in homozygous breeding. In heterozygous breeding,
male ak mice were crossed to female C57BL/6-Jola mice to generate heterozygous
F1 hybrids. F1 hybrids were intercrossed to generate
Pitx3+/+, Pitx3+/- and
Pitx3-/- progeny. Genotypes were determined by PCR
analysis of tail DNA. Pregnant mice were decapitated or euthanized by
CO2 asphyxiation and embryos were collected at E13.5, E14.5 or
E18.5 (the day on which the copulatory plug was detected was considered E0).
Adult mice were euthanized by CO2 asphyxiation and brains were
collected. Mice were bred in our laboratory under standard conditions
(21.0±1.0°C and 50% humidity) with ad libitum access to standard
food (Hope Farms) and tap water, on a 12:12 hour light:dark cycle. All
experimental procedures were approved by the ethical committee for animal
experimentation of the Utrecht University, the Netherlands.
In situ hybridization
Embryos and adult brains were collected and immediately frozen on dry ice.
Sections (16 µm) were cut and collected on SuperFrost Plus slides (Menzel
Gläser). In situ hybridization (ISH) with digoxigenin (DIG)-labeled and
[33P]-labeled cRNA probes was performed as described previously
(Smidt et al., 2004a;
Smits et al., 2005).
[33P]-labeled sections were dehydrated, air-dried and exposed to
BAS-MS 2340 imaging plates (Fuji) for 3-5 days or to Kodak Biomax MR films
(Kodak) for 3 weeks. Autoradiographic BAS-MS 2340 imaging plates containing
the hybridization signals were scanned using the FLA-5000 imaging system
(Fuji), and quantitative analysis was performed using the AIDA Image Analyzer
Software (Raytest). Gene expression was analyzed in both the left and right
SNc (Pitx3+/+: n=8; Pitx3+/-:
n=5). Per animal, two adjacent sections in the SNc were analyzed. To
evaluate statistical significance, data were subjected to the Student's
t-test (two tailed). The following probes were used: a 1142 bp
fragment of the rat Th cDNA
(Grima et al., 1985), an
Aadc (also known as Ddc - Mouse Genome Informatics) fragment
containing bp 22-488 of the mouse coding sequence
(Smits et al., 2003), a
fragment containing bp 290-799 of the coding region from the mouse
Vmat2 (also known as Slc18a2 - Mouse Genome Informatics)
gene (Smits et al., 2003), an
alpha-synuclein (a-synuclein; Snca) fragment containing bp 20-420 of
the coding region from the mouse cDNA
(Smidt et al., 2004a), and an
Ahd2 fragment containing bp 568-1392 of the mouse coding
sequence.
Immunohistochemistry
Embryos and adult brains were collected, incubated overnight in 4%
paraformaldehyde (PFA) at 4°C, and embedded in paraffin. Sections (7
µm) were cut on a microtome, collected on SuperFrost Plus slides (Menzel
Gläser), de-paraffinated through xylene, rehydrated through an ethanol
series and incubated in 0.3% H2O2 in Tris-buffered
saline (TBS) for 30 minutes. Next, sections were boiled in citrate buffer (pH
6) for antigen retrieval, blocked with 4% fetal calf serum in TBS for 30
minutes and incubated overnight with rabbit anti-TH antibody (Pel-Freez,
Arkansas, USA; 1:1000) in TBS/0.5% Triton at 4°C. The next day, sections
were incubated for 1 hour with biotinylated goat anti-rabbit immunoglobulin in
TBS (1:1000), followed by incubation with avidin-biotin-peroxidase reagents
(ABC elite kit, Vector Laboratories, 1:1000) for 1 hour in TBS. The slides
were stained with DAB (3,3'-diamino-benzidine) for a maximum of 10
minutes, until the background was lightly stained. Slides were dehydrated with
ethanol and mounted using Entellan. Quantification of neurons was performed
using a microscope (Zeiss Axiovert 405M) attached to a camera system
(MicroPublisher 5.0 RTV) and imaging software (Openlab 5.0.1, Improvision).
The number of TH-immunoreactive (TH-IR) neurons was counted unilaterally in
anatomically matched adjacent coronal sections. For each E14.5 embryo, four
sections were counted in the caudal and rostral domain of the mdDA system. For
each E18.5 embryo, sections containing the SNc were analyzed (27-40 sections)
and the average number of TH-IR neurons per section was calculated. Only
neurons in which cell nuclei could be recognized were counted. Quantification
was performed by an independent observer in a blind design. To evaluate
statistical significance, data were subjected to the Student's t-test
(two tailed).
For double-immunofluorescence staining, sections were incubated overnight with rabbit anti-Ahd2 (Abcam, diluted 1:100) in combination with sheep anti-TH (Chemicon, diluted 1:500) in PBS/0.5% Triton at 4°C. The next day, sections were incubated with fluorophore-conjugated secondary antibodies in PBS (Alexa-Fluor-488-conjugated goat anti-rabbit and Alexa-Fluor-555-conjugated donkey anti-sheep, diluted 1:400; Molecular Probes) for 1 hour at room temperature and embedded with 90% glycerol.
Nissl staining
TH-IR sections were counterstained for 5 minutes in 0.5% cresyl violet and
briefly rinsed in an acetate buffer (pH 4). The sections were then
differentiated in 96% ethanol for 60 seconds, dehydrated in 100% ethanol,
cleared in xylene and mounted with Entellan. Neuronal number per section in
the rostral mdDA area was determined in cresyl violet-stained sections,
delineated according to TH-IR area. Quantification (n=3) was
performed by an independent observer in a blind design. To evaluate
statistical significance, data were subjected to the Student's t-test
(two tailed).
Combined ISH-immunohistochemistry
Adult brains were collected, incubated overnight in 4% PFA at 4°C, and
embedded in paraffin. Sections (7 µm) were cut and collected on SuperFrost
Plus slides (Menzel Gläser). ISH on paraffin wax sections was performed
as described for frozen sections with the following modifications: sections
were first deparaffinated through xylene, rehydrated through an ethanol
series, boiled in citrate buffer (pH 6), and incubated in 0.2 M HCl for 15
minutes. Sections were further treated as described above for DIG-ISH on
frozen sections, except that, after termination of the alkaline phosphatase
reaction, sections were immunostained for TH with the avidin-biotin-peroxidase
complex (ABC) method, as described above.
RNA isolation, PCR and cloning
RNA from E18 mouse whole brain or MN9D cells was isolated using Trizol
(Invitrogen) according the manufacturer's guidelines. A sample of
Pitx3+/GFP FACS-sorted mdDA cells from E16 embryos was isolated via
the guanidine thiocyanate method.
Full-length Pitx3 cDNA was amplified from cDNA originating from E18 whole-brain RNA, with the following primers: forward, 5'-CCCTGCCTGCGCTCCAGAAC-3'; reversed: 5'-CCCTGTTCCTGGCCTTAGTC-3'. Pitx3 was ligated in pGEM-T easy vector (Promega) for sequence analysis, and subsequently cloned into pcDNA3.1(-) vector (Invitrogen) using the ApaI and BstxI restriction sites.
To distinguish between the Ahd2 and Aldh1a7 genes, primers with 100% homology to both genes were designed: forward, 5'-GACTGATGAGATGCGCATTG-3'; reversed 5'-GTCTTGAGCTCAGTGTATTC-3'. For in vivo determination, RNA originating from E16 ventral midbrain Pitx3+/GFP cells was subjected to OneStep reverse transcriptase (RT)-PCR (Qiagen). For in vitro determination, RNA from MN9D cells transfected with Pitx3-pcDNA3.1 was used. The obtained PCR fragments were cloned into pGEM-T easy vectors and eight different clones of each cloning were sequence-analyzed (Baseclear, The Netherlands).
MN9D cell culture and cell transfections
MN9D (MN9D-13N) cells were cultured in Dulbecco's Modified Eagle Medium
(DMEM) supplemented with 10% (v/v) hiFCS, 100 units/ml penicillin, 100
units/ml streptomycin and 2 mM L-glutamine in a humidified atmosphere with 5%
CO2 at 37°C. Cells were grown on 10-cm-diameter dishes coated
with poly L-lysine. At 2 hours prior to transfection, culture medium was
replaced with medium without antibiotics, and transfection was performed using
Lipofectamine 2000 (Invitrogen), according to the manufacturer's guidelines.
Cells were transfected with 5 µg of Pitx3-pcDNA3.1 or an equal
molar amount of empty pcDNA3.1 vector together with carrier DNA and 1 µg of
EGFP-N1 vector (Clontech). At 6 hours after transfection, cells were divided
and allowed to grow for an additional 30 hours. Transfection efficiency was
determined based on GFP fluorescence using a fluorescent microscope, and
plates were matched based on their relative transfection efficiency. For MN9D
cells, a typical transfection efficiency with Lipofectamine is approximately
80-90%. Finally, cells were harvested for RNA isolation or used for a
chromatin immunoprecipitation assay.
Semi-quantitative RT-PCR
Relative gene expression levels were determined using a OneStep RT-PCR kit
(Qiagen). We used 50 ng total RNA per reaction, and for each transcript the
linear phase was determined and reactions were stopped during that phase for
analysis (for primer sequences, see Table
1). Samples were loaded on 2% agarose gels and, after gel
electroforesis, gels were scanned using a FLA-5000 imaging system (Fuji) and
relative amounts of DNA were measured with a densitometer. Each analysis was
replicated at least three times with independent RNA samples to confirm the
obtained results. To determine whether RA regulates the expression of TH, we
cultured MN9D cells in the presence of 1 µM all-trans RA for 36 hours as
described previously (Castro et al.,
2001).
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), with
some adjustments. Cells were resuspended in lysis buffer [50 mM Tris-HCl (pH
8.0), 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% Na-deoxycholate]
containing protease inhibitors (Complete Protease Inhibitor Cocktail Tablets,
Roche) and were incubated on ice for 20 minutes. Subsequently, the lysate was
put through a 27/3-4 G syringe three times. Chromatin was sonicated using a
Soniprep 150 sonicator to obtain DNA of an average length of 400-500 bp.
Chromatin was pre-cleared with 60 µl of a 50% slurry of Protein A
agarose/Salmon Sperm DNA (Upstate) for 1.5 hours at 4°C. Pre-cleared
chromatin was incubated overnight with approximately 1 µg of Pitx3 antibody
(Smidt et al., 2000), or with
1 µg of rabbit whole serum immunoglobulins (Sigma) as a negative control.
Antibody-DNA complexes were captured by adding 20 µl of a 50% slurry of
Protein A agarose/Salmon Sperm DNA for 1 hour at 4°C. Immunoprecipitated
complexes were washed for 10 minutes with the following wash buffers: twice
with 1.5 ml of buffer 1 [20 mM Tris-HCl (pH 8.0), 150 mM NaCl, 2 mM EDTA, 1%
Triton X-100, 0.1% sodium dodecyl sulfate (SDS)], once with 1.5 ml of buffer 2
[20 mM Tris-HCl (pH 8.0), 500 mM NaCl, 2 mM EDTA, 1% Triton X-100, 0.1% SDS],
once with 1.5 ml of LiCl buffer [20 mM Tris-HCl (pH 8.0), 250 mM LiCl, 1 mM
EDTA, 1% NP-40, 1% Na-deoxycholate], and twice with 1.5 ml of TE [10 mM
Tris-HCl (pH 8.0), 1 mM EDTA]. For elution of the immunocomplexes, 150 µl
of elution buffer (TE, 1% SDS) was added and beads were shaken on a vortex for
30 minutes at 4°C. This was performed twice and samples were pooled.
Crosslinks were reversed overnight at 67°C and complexes were precipitated
and dissolved in TE. Proteinase K was added and samples were incubated for 2
hours at 45°C. Samples were extracted once with phenol/chloroform (1:1)
and twice with chloroform. DNA was precipitated and dissolved in 100 µl of
PCR grade MilliQ. For each PCR reaction, 2 µl of this DNA sample was used
(for primer sequences, see Table
1).
Retinoic acid treatment of pregnant Pitx3+/- mice
Following timed matings of Pitx3+/- parents, pregnant
mice were supplemented with all-trans-RA (Sigma), which will be referred to as
RA in this article, twice daily from E10.75 to E13.75. RA was dissolved in
corn-oil and mixed with powdered food to a final concentration of 0.25 mg/g
food, which was supplied ad libitum as previously described
(Niederreither et al., 2002c;
Mic et al., 2003). RA was
dissolved freshly each time and supplemented food was supplied at 12-hour
intervals. The control Pitx3+/- animals were treated
according to the same protocol, but were supplemented with corn oil without
RA. Embryos were isolated at E14.5 and E18.5, and weight-matched
Pitx3+/+ and Pitx3-/- littermates were
analyzed by immunohistochemistry.
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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;
Montplaisir et al., 2002).
Most previous analyses did not clearly distinguish between these two
transcripts. To determine which of these genes is actually expressed in mdDA
neurons, reverse transcriptase (RT)-PCR was performed on RNA of FACS-sorted
Pitx3+/GFP mdDA neurons from E16 embryos
(Zhao et al., 2004). Sequence
analysis of cloned PCR products identified all clones (100%) as Ahd2,
indicating that Ahd2 is the predominantly expressed gene in mdDA
neurons (data not shown).
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;
Chung et al., 2005). To study
the expression pattern of Ahd2 in more detail, the distribution of Ahd2 was
examined in adult mdDA neurons of wild-type mice
(Fig. 1). High levels of
Ahd2 mRNA were observed in cells located in the SNc, as revealed by
the expression pattern of Th on adjacent sections
(Fig.
1AA-AF). In addition, a large number of
Ahd2+ cells was observed in the VTA. These cells were predominantly
located in the ventral and lateral part of the VTA, with some scattered
Ahd2+ cells located more dorsally
(Fig.
1AE-AL). Although Ahd2 is expressed
in a large number of cells in the SNc and VTA, the expression patterns of
Ahd2 and Th did not overlap completely. In particular,
Th+ cells in the medial part of the SNc and dorso-medial part of the
VTA appeared to lack Ahd2 expression. Ahd2 in situ
hybridization (ISH) combined with an immunohistochemical staining for TH
clearly confirmed that not all TH+ neurons in the adult SNc and VTA
co-expressed Ahd2 (Fig.
1B). In the lateral tip of the SNc, TH+ cells that did not express
Ahd2 were intermingled with TH+ cells that did express Ahd2
(Fig. 1BB). In the
more medial part of the SNc, TH+ cells that lacked Ahd2 expression
were predominantly located dorsally (Fig.
1BC). In addition, in the VTA, TH+ cells that lacked
Ahd2 expression were mainly observed in the dorsal VTA
(Fig.
1BD-BF). Finally, antibody double-labeling
experiments assured the subpopulation-specific distribution of Ahd2 protein in
the SNc (Fig.
1CA-CC,CG-CI) and VTA
(Fig.
1CD-CF,CJ-CL). Not all
TH+ cells in the SNc and VTA expressed Ahd2 protein, but those cells that did
express Ahd2 protein were all TH+, indicating that Ahd2 and TH are
co-expressed in a specific mdDA subpopulation. Taken together, based on both
transcript and protein distribution, Ahd2 marks a specific mdDA subpopulation
in the adult brain.
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; Wallen et al.,
1999). To study the developmental expression of Ahd2
during late differentiation in more detail, the distribution of Ahd2
mRNA was examined in E13.5 wild-type embryos
(Fig. 2). Ahd2
expression was confined to neurons located in the more lateral parts of the
mdDA area, as identified by the expression of Th mRNA on adjacent
sections (Fig. 2).
Aadc-positive young migrating neurons, which are located more dorsally
(Smidt et al., 2004a), and
proliferating cells in the ventricular zone did not express Ahd2 at this time
point. These data indicate that, at E13.5, Ahd2 expression is
restricted to a specific subpopulation of mdDA neurons located in the lateral
part of the mdDA area. By contrast, the medial part of the mdDA area was
devoid of Ahd2 expression. Clearly, Ahd2 shows mdDA
subpopulation-specific expression, indicating that mdDA subpopulations are
already specified during development.
Ahd2 expression in Pitx3-deficient mdDA neurons
The expression pattern of Ahd2 described above, appears to identify the
neuronal subset that is lost in Pitx3-deficient animals. To study
that possibility in detail, we analyzed the expression of Ahd2 in adult brain
and during the late differentiation phase in E13.5 mdDA neurons of
Pitx3-deficient mice (Fig.
3). The expression pattern of Th revealed the previously
reported malformation of the adult mdDA system in Pitx3-deficient
mice (Smidt et al., 2004b),
characterized by neuronal loss predominantly in the SNc
(Fig. 3A). As expected,
Ahd2 expression was completely absent in the rostral part of the mdDA
area of adult Pitx3-deficient mice, which includes the SNc and
lateral part of the VTA, as identified by the expression of Th mRNA
on adjacent sections (Fig.
3AA-AD). By contrast, in the caudal part of
the mdDA area, some Ahd2+ neurons could still be observed in the VTA
(Fig.
3AE,AF). Antibody double-labeling
experiments demonstrated that the Ahd2+ neurons co-expressed TH protein,
indicating that these cells are DA neurons (see Fig. S1 in the supplementary
material). At E13.5, Ahd2 expression was completely lost in the
lateral part of the Pitx3-deficient mdDA area, which is normally
positive for Ahd2. By contrast, Th expression in adjacent
sections was still observed, indicating that there were still mdDA neurons
present in this area (Fig. 3B).
Taken together, these data clearly demonstrate that Ahd2 expression
is highly affected in Pitx3-deficient mice, already early in
development.
Decreased Ahd2 expression in mdDA neurons of Pitx3+/- mice
The apparent loss of Ahd2 expression in Pitx3-deficient
mdDA neurons during development suggests that Pitx3 is involved in
Ahd2 gene activation. To study this in more detail, we analyzed the
Ahd2 expression level in Pitx3+/- mice, which
display reduced Pitx3 expression
(Rieger et al., 2001). We
performed quantitative radio-active ISH studies on coronal adult mdDA sections
of Pitx3+/- mice containing the SNc
(Fig. 4). Ahd2 mRNA
levels in the SNc of Pitx3+/- mice
(Fig. 4, black bar;
n=5) were significantly decreased compared with
Pitx3+/+ mice (Fig.
4, white bar; n=8; P<0.05). Expression levels
of other mdDA markers, including Th, Vmat2 and alpha-synuclein were
not altered in Pitx3+/- mice. The fact that Ahd2
expression was specifically decreased in Pitx3+/- mice
strongly indicates a dose-dependent effect of Pitx3 on the expression level of
Ahd2.
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). In
addition, Nurr1 induces morphological differentiation and cell cycle arrest
(Castro et al., 2001).
Therefore, we argued that this cell line is an appropriate cell model in which
to analyze Pitx3 function. Under normal conditions, Pitx3 and
Ahd2 were both expressed endogenously in MN9D cells, albeit at a very
low level (Fig. 5A). However
Pitx3 overexpression led to a drastic increase in endogenous Ahd2
transcript levels, whereas the control gene, TATA box binding protein
(Tbp), showed no alteration in expression levels
(Fig. 5A,B). To confirm that
the upregulated gene was Ahd2 and not Aldh1a7, as was the
case in vivo (see above), we performed sequence analysis on cloned PCR
fragments, revealing that the majority of transcripts (88%) were encoded by
the Ahd2 gene (data not shown). In addition, Pitx3 overexpression
showed no effect on the transcript levels of other DA markers, including
Th, Aadc, Vmat2 and alpha-synuclein
(Fig. 5A,B). In total, three
independent RNA samples were analyzed, which all gave similar results. These
data demonstrate that endogenous Ahd2 expression largely relies on
Pitx3 function, whereas Pitx3 has no effect on the expression of genes
involved in neurotransmitter phenotype in these cells.
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|
Pitx3 antibodies or non-specific rabbit
immunoglobulins (IgG). The immunoprecipitated DNA fragments were subjected to
PCR, including IgG immunoprecipitated DNA as a negative control and input
chromatin as a positive control. Region 2
(Fig. 6A) contains a putative
Pitx3-binding site that has been shown to be bound by Pitx3 in an electro
mobility shift assay (EMSA) (Chung et al.,
2005). In our assay, however, we observed no amplification of this
region, indicating that, under the conditions we used, Pitx3 is not bound to
that region. This difference can be explained by the use of different
approaches to determine functional interactions of a transcription factor to a
presumed binding site. It should be noted that, in contrast to a ChIP assay,
the chromatin structure in living cells, which can strongly reduce
accessibility for transcription factors, is disregarded in an EMSA approach.
Interestingly, we successfully amplified a highly conserved genomic region
(region 5) specifically from Pitx3 immunoprecipitated DNA
(Fig. 6A,B), whereas no
amplification of any of the other selected conserved regions was observed
(data not shown). The genomic region 4, which lies further upstream than
region 5 (Fig. 6A) and shows
moderate sequence conservation between rat and mouse, but contains the
consensus Pitx3-binding site TAATCC
(Wilson et al., 1996), was not
amplified (Fig. 6B). This
implies selective binding of Pitx3 to the highly conserved region 5.
Confirmation of the observed results in a second independent ChIP assay
assured selective binding of Pitx3 to region 5. Furthermore, closer analysis
of this highly conserved region revealed that, although sequence conservation
between mouse/rat and human is generally poor in introns of Ahd2
(approximately 60%), the homology in this specific intronic region is 91% over
a stretch of 60 bp (Fig. 6C).
In addition, a non-consensus putative Pitx3-binding site, AAATCT
(Wilson et al., 1996;
Yuan et al., 1999), which is
fully conserved between mouse, rat and human, is found in this region.
Altogether, these data indicate that Pitx3 interacts with a highly conserved
region 2.1 kb downstream of the TSS of the Ahd2 gene, which may
account for the observed regulation of Ahd2 gene transcription in
MN9D cells.
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;
Mic et al., 2004;
Molotkov et al., 2006). Ahd2
is a potent generator of RA and is present in the mdDA area throughout
embryonic development (Hsu et al.,
1999; Haselbeck et al.,
1999; Wallen et al.,
1999). This suggests a possible role for Ahd2 at this stage of
mdDA neuronal development in the synthesis of RA. With Ahd2 under the
transcriptional control of Pitx3, it is appealing to hypothesize that the
developmental defect in Pitx3-deficient mice is due to a loss of RA
synthesis. If RA is crucial for proper development of Ahd2+ neurons in the
mdDA system, we could bypass the necessity of Pitx3-mediated Ahd2
expression in these neurons by maternal dietary RA administration. Because
Ahd2 is expressed in a subset of mdDA neurons, we expected a compensating
effect of RA exclusively in that subpopulation. To identify the Ahd2+ mdDA
subpopulation at E14.5, when the mdDA phenotype is clearly visible in
Pitx3-deficient mice (Smidt et
al., 2004a; Smidt et al.,
2004b; Maxwell et al.,
2005), we analyzed Ahd2 expression in wild-type brain at this
stage. Ahd2 expression was not detected in caudal sections of the mdDA area,
where, ultimately, the VTA is formed (Fig.
7BA-BC). In rostral sections, Ahd2 fully
co-localized with TH in a specific subset of TH-immunoreactive (TH-IR) neurons
(Fig.
7BD-BF). Ahd2 was predominantly present in
mdDA neurons in lateral and ventral positions. The majority of TH-IR neurons
in dorso-medial positions did not co-express Ahd2. In addition, TH-IR neurons
in a distinct population in most lateral positions were largely negative for
Ahd2.
To determine whether RA synthesis by Ahd2, mediated via Pitx3, has a role
in the development of these Ahd2+ neurons, we aimed to compensate for
Pitx3 deficiency by maternal dietary RA administration. Pregnant
Pitx3+/- mice were supplemented with RA from E10.75 to
E13.75, and Pitx3+/+ and Pitx3-/-
littermates were analyzed at E14.5. Detailed analysis showed that, in caudal
regions of the mdDA area, no effect of RA treatment is observed in either the
Pitx3+/+ or Pitx3-/- embryos
(Fig.
7CA-CD;
Fig. 7D, left panel). In
Pitx3+/+ embryos, RA treatment had no effect on the
occurrence of mdDA neurons in the rostral regions
(Fig.
7CG,CH;
Fig. 7D, right panel). As
previously reported (Smidt et al.,
2004a), untreated Pitx3-/- embryos displayed a
significant loss (61%; n=3, P<0.01) of TH-IR neurons in
the ventro-lateral regions (Fig.
7CE,CG;
Fig. 7D, right panel).
Strikingly, in rostral sections of Pitx3-/- embryos, RA
treatment led to a significant increase (70%; n=3,
P<0.01) in number of TH-IR neurons
(Fig.
7CE,CF;
Fig. 7D, right panel). As a
result, the loss in number of TH-IR neurons caused by Pitx3
deficiency was drastically reduced after RA treatment. This demonstrates that
RA treatment has an exclusive effect in Pitx3-/- embryos,
affecting specifically the rostral mdDA subpopulation, which is most affected
by Pitx3 deficiency.
|
To determine whether the effect of temporary RA treatment is maintained at later developmental stages, we treated pregnant Pitx3+/- mice with RA from E10.75 until E13.75 and analyzed Pitx3+/+ and Pitx3-/- littermate embryos at E18.5 (Fig. 8). RA-treated Pitx3-/- embryos showed a significant increase in number of TH-IR neurons (59%; n=3, P=0.01) within the SNc. These data indicate that temporary RA treatment has a sustained effect on the mdDA neuronal population. In agreement with the E14.5 data, the effect was highly restricted to Pitx3-/- embryos (Fig. 8A,B).
Pitx3-/- mice are characterized by impaired innervation of the dorsal striatum. To determine whether the increased number of TH-IR neurons in RA-treated E18.5 Pitx3-/- embryos was accompanied by increased innervation, we analyzed TH-immunoreactivity in dorsal regions of the striatum at multiple levels (Fig. 8C,D). Interestingly, increased TH-immunoreactivity was observed in dorsal regions of the striatum in RA treated Pitx3-/- embryos, whereas this area was largely devoid of TH-immunoreactivity in untreated Pitx3-/- embryos. These observations indicate that the TH-IR neurons that are maintained in RA-treated Pitx3-/- embryos are those neurons that normally innervate striatal regions located more dorsally. Altogether, these data strongly suggest that RA-dependent signaling is essential for the proper development of a specific subset of mdDA neurons initially affected in Pitx3-deficient mice.
|
DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). An early
determinant of subset specification is Pitx3 and, based on its temporal
expression pattern, two distinct mdDA neuronal populations exist during early
development. A ventro-lateral population, which expresses Pitx3 before TH, can
be identified, as well as a dorso-medial population, which expresses TH before
Pitx3 (Maxwell et al., 2005).
The existence of mdDA subpopulations is highlighted by the expression pattern
of Ahd2. In the adult brain, we observed Ahd2 expression in a ventrally
located subset of TH-IR neurons in the SNc and VTA. During embryonic
development, this mdDA subpopulation-specific expression was observed as early
as E13.5, showing Ahd2 expression exclusively in the lateral parts of
the mdDA area. The restricted expression of Ahd2 is also clearly visible in
the major projection area of the mdDA system, where Ahd2 protein is detected
in dorso-lateral parts of the adult striatum (McGaffery and Drager, 1994).
Interestingly, innervation to these specific regions of the striatum is lost
in Pitx3-deficient mice, caused by a selective loss of mdDA neurons
predominantly in the SNc. In order to investigate the fate of Ahd2-expressing
mdDA neurons in Pitx3-deficient mice, we determined the distribution
of Ahd2 transcripts in both the adult and the embryonic brain.
Although we observed a small population of mdDA neurons in the ventro-medial
part of the VTA that still expressed Ahd2, most of the expression of Ahd2 was
completely lost. This indicates that the majority of Ahd2+ mdDA neurons,
except for a small distinct population, are fully dependent on Pitx3
expression for proper development. In E13.5 Pitx3-deficient embryos,
no expression of Ahd2 was observed in the lateral region of the mdDA
area, where the gene is normally expressed. However, Th is still
expressed in that area, which indicates that a considerable amount of mdDA
neurons is still present. Considering the amount of TH+/Ahd2+ cells in
wild-type E13.5 embryos, and the amount of remaining TH+ cells in the
Pitx3-deficient brain, this suggests that loss of Ahd2 expression is
due to the ablation of Pitx3 itself rather than to neuronal loss.
The fact that Pitx3 expression is reduced in
Pitx3+/- animals
(Rieger et al., 2001) provided
a tool by which to analyze the in vivo consequence of Pitx3 gene
dosage. Quantitative analysis of Ahd2 expression in the SNc of
Pitx3+/- mice revealed a significant reduction in the
expression level of Ahd2. Expression of other DA marker genes was
unaffected in the SNc of Pitx3+/- animals, which points to
a gene-dosage effect of Pitx3 on Ahd2 expression specifically,
unrelated to neuronal loss. In perfect agreement with these data, we observed
a drastic increase in endogenous Ahd2 transcript levels when Pitx3
was overexpressed in the DA cell line MN9D. This effect was specific for
Ahd2, because the levels of Th, Aadc, Vmat2, Tbp and
alpha-synuclein were unaffected. Further evidence for the
Pitx3-mediated regulation of Ahd2 transcription was provided via a
ChIP assay, which was performed to analyze the binding of Pitx3 to a selection
of highly conserved regions in the proximity of the Ahd2 TSS. We
showed that Pitx3 immunoprecipitation specifically selected for a genomic
region in intron 1 of the Ahd2 gene. This region displays a
surprisingly high sequence conservation in mouse, rat and human, in contrast
to the surrounding intronic sequence of the Ahd2 gene. A
non-consensus putative Pitx3-binding site, AAATCT, is contained within this
region, which was also found to be fully conserved.
Because different lines of evidence suggest that Ahd2 is under the
transcriptional control of Pitx3, we hypothesized that Ahd2, mediated through
Pitx3, might have a role in the development of mdDA neurons. Ahd2 is a potent
generator of RA (Hsu et al.,
1999; Fan et al.,
2003), which is essential for the proper development of many
structures in the embryo (Duester et al.,
2003), and is involved in neuronal patterning, survival and
neurite outgrowth (Clagett-Dame et al.,
2006). Although Ahd2-deficient animals are viable and
exhibit no gross abnormalities, the brain, and, in particular, the mdDA
system, have not been analyzed yet (Fan et
al., 2003). Although the first step in RA synthesis by alcohol
dehydrogenases is an ubiquitous process, tissue specificity of RA synthesis is
achieved by restricted expression of aldehyde dehydrogenases, such as Ahd2
(Molotkov et al., 2002). In
agreement with the Ahd2 expression pattern, RA is synthesized in the mdDA area
during early development, and during the postnatal and adult stages (McGaffery
and Drager, 1994; Niederreither et al.,
2002a). In this study, we show that maternal supplementation of RA
counteracts the developmental defect in the mdDA area in
Pitx3-/- mice. After RA treatment, a significant increase
in the number of TH-IR mdDA neurons was observed in the rostral part of the
mdDA system of E14.5 Pitx3-/- embryos. This effect was
specific to the rostral part of the mdDA system which is the region that is
most affected by Pitx3 deficiency. Importantly, no effect of
RA-treatment was observed in Pitx3+/+ embryos, which rules
out the possibility that the observed increase in number of TH-IR neurons is
due to increased proliferation. In addition, RA is unable to induce TH
expression in MN9D cells, which suggests that the observed increase in TH-IR
neurons is not due to a general induction of TH expression. Rather, a possible
explanation for the increase in TH-IR neurons is that RA induces the
establishment of the proper mdDA identity of immature
Pitx3-/- neurons. Indeed, previous studies show that, at
E14.5, a large population of immature Pitx3-/- neurons do
not achieve their proper mdDA identity, but are still present in the area as
TH-negative neurons (Maxwell et al.,
2005). In agreement with these findings, our data indicate that
the total number of neurons in the rostral region of the mdDA system is
unaffected by either Pitx3 deficiency or RA treatment. This strongly
suggests that the observed decrease of TH-immunoreactivity in E14.5
Pitx3-/- embryos is caused by the fact that immature
neurons have not established their proper mdDA identity, rather than being
lost. Therefore, the increase in the number of TH-IR neurons in RA-treated
Pitx3-/- embryos appears to be the result of restored mdDA
neuronal differentiation.
The effect of the temporary RA treatment was preserved to a comparable level at E18.5, as the number of TH-IR neurons was still significantly increased in RA-treated Pitx3-/- embryos. This implies that, for a specific mdDA subset, RA is crucial between E10.75 and E13.75 to induce proper mdDA neuronal identity. Once the correct neuronal identity is established, these neurons appear to be maintained without further RA treatment. Strikingly, the increase in TH-IR number in the mdDA area is accompanied by increased dorsal innervation of the striatum. This strengthens the idea that RA induces the occurrence of a distinct functional mdDA subset in Pitx3-/- embryos, which is normally severely affected by Pitx3-deficiency.
Altogether, these data indicate that RA can compensate for the loss of Ahd2
expression as a consequence of Pitx3 deficiency. This positions Ahd2
and its RA-generating potency central in a pathway for mdDA neuronal
development, which might be linked to the final differentiation of a specific
mdDA neuronal subpopulation. Interestingly, Ahd2 is downregulated in the
Parkinsonian SNc, and other components of the RA-synthesis pathway have been
correlated to PD (Buervenich et al.,
2000; Buervenich et al.,
2005; Galter et al.,
2003; Grünblatt et al.,
2004). Most appealing, by linking local RA synthesis to mdDA
neuronal development and maintenance, a novel mechanism is proposed, with
essential implications for clinical pathology as seen in PD.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/134/14/2673/DC1
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ACKNOWLEDGMENTS |
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Footnotes |
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0.01). (C)
Schematic representation of an E18.5 sagittal section showing the position of
the striatal regions (I and II) demonstrated in D. (D)
TH-immunoreactivity at two levels of the striatum of E18.5 embryos. The arrows
mark the dorsal striatal areas in which an increase of TH-immunoreactivity is
observed.