1 Rudolf Magnus Institute of Neuroscience, Department of Pharmacology and
Anatomy, University Medical Center, Universiteitsweg 100, 3584 CG Utrecht, The
Netherlands
2 GSF, Institute of Developmental Genetics, Ingolstaedter Landstrasse 1, D-85764
München, Germany
* Author for correspondence (e-mail: m.p.smidt{at}med.uu.nl)
Accepted 28 November 2003
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SUMMARY |
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Key words: Mesencephalic, Dopaminergic, Pitx3, Substantia nigra
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Introduction |
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Two key molecules on the anterior side of the isthmus play an essential
role in the commitment of progenitors to mesDA neuron development. The
signaling molecule sonic hedgehog (Shh), which is generated in the neural
plate, diffuses into the ventral mesencephalon and, together with FGF8,
induces cells at a specific location in the ventral midbrain from which mesDA
neurons later arise (Hynes et al.,
1995; Hynes et al.,
1997
; Ye et al.,
1998
; Shamim et al.,
1999
; Hynes and Rosenthal.,
1999
). MesDA neurons, which express tyrosine hydroxylase (Th) and
Pitx3 (Ptx3) (Smidt et al.,
1997
), first appear at the most ventral rim of the
neuroepithelium, lining up along the mesencephalic flexure of the ventral
mesencephalon. Ventral midbrain markers are present in this area prior to
Pitx3 and Th. Among the earliest markers of the region are En1
(Danielian et al., 1996; Wurst et al.,
1994
), En2 (Hanks et
al., 1995
), Wnt1 (Danielian et al., 1996), Pax2
(Favor et al., 1996
) and
Pax5 (Urbanek et al.,
1997
). Th expression is induced at E11.5, 1 day after the
induction of the orphan nuclear hormone receptor Nurr1
(Nr4a2 - Mouse Genome Informatics)
(Law et al., 1992
). The
expression of Nurr1 is, however, not restricted to the ventral
midbrain but is also expressed in the dorsal mesencephalon and diencephalon,
where Th is not expressed at this developmental stage
(Smidt et al., 2000
). MesDA
neurons in Nurr1 knockout mice do not express Th, Dat
(Slc6a3 - Mouse Genome Informatics) and Vmat2
(Slc18a2 - Mouse Genome Informatics)
(Smits et al., 2003
) and are
lost after initial generation
(Zetterström et al.,
1997
; Saucedo-Cardenas et al.,
1998
). Thus, Nurr1 is part of a molecular cascade
required for neurotransmitter synthesis and survival.
Additional cascades have been discovered that specify other properties of
the mesDA neuronal phenotype. Recently, the homeobox genes En1, En2
(Davidson et al., 1988;
Simon et al., 2001
) and
Lmx1b (Johnson and Tabin,
1997
; Dreyer et al.,
1998a
; Chen et al.,
1998a
; Chen et al.,
1998b
; Tucker et al.,
1999
) have been implicated in the development of the mesDA system
(Smidt et al., 2000
).
En1 and En2 are not required for the induction of
Th, but control the survival of mesDA neurons in a gene
dose-dependent manner (Simon et al.,
2001
). Lmx1b is present in the premordial ventral mesencephalon
and diencephalon at E7.5, and continues to be expressed in the adult in a
limited set of brain structures derived from these areas, including mesDA
neurons (Smidt et al., 2000
).
Null mutation of Lmx1b showed that this gene is not required for the
expression of Nurr1 and is not essential for Th induction.
Moreover, Lmx1b-null mutation impairs the proper development of mesDA
neurons, such that the mesDA-specific homeodomain gene Pitx3 is not
expressed in Th-positive cells in the ventral midbrain
(Smidt et al., 2000
).
The Pitx3 gene belongs to the Pitx (Ptx)
subfamily of paired-like homeodomain proteins, of which two other members are
known, Pitx1 and Pitx2, which are involved in hindlimb
patterning, mandible development (Lanctot
et al., 1999), left-right asymmetry of the body
(Lin et al., 1999
), pituitary
genesis (Suh et al., 2002
) and
pituitary hormone regulation (Quirk et
al., 2001
; Quentien et al.,
2002
). Pitx3 is expressed in the eye and the brain
(Semina et al., 1997
;
Smidt et al., 1997
), where its
expression is restricted to mesDA neurons in rodents and humans
(Smidt et al., 1997
). The
Pitx3 gene is induced at the same developmental stage as Th
and continues to be expressed in adulthood
(Smidt et al., 1997
). Although
in vitro studies have suggested that Pitx3 is important for the
induction of the Th gene (Lebel
et al., 2001
; Cazorla et al.,
2000
), analysis of the Lmx1b-null mutant indicated that
Pitx3 is not essential for Th expression
(Smidt et al., 2000
). Recent
reports have shown that the Pitx3 gene is inactivated in the aphakia
(ak) mouse mutant (Semina et al.,
2000
; Rieger et al.,
2001
) and that the architecture of the midbrain DA system is
compromised (Nunes et al.,
2003
; van den Munckhof et al.,
2003
; Hwang et al.,
2003
; Burbach et al.,
2003
). However, the early developmental fate of mesDA progenitors,
and the role of Pitx3 in this, are uncertain. We show that SNc DA
neurons are absent in the ak mice from E12.5 onwards. Pitx3
appears to be an essential part of a transcription factor cascade required for
the developmental specification of the SNc subpopulation of mesDA neurons.
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Materials and methods |
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Surgery
Six- or 7-day-old C57Bl/6-Jico mice underwent surgery to remove the eyes.
The mice were anesthetized with isoflurane (florane®) 2% and
N2O (carrier-gas) and O2 (75:65% volume, respectively),
and the eyelids were treated with lidocaine as local anesthetic. The animals
were kept at 37°C with a heating mat. The eyelids were opened with a pair
of scissors and the membrane covering the eye was removed with a micro hook.
The eyeball was removed by cutting the nerve and blood vessel with a scalpel
after they had been forced together by the use of a forceps. The eyelids were
glued together with tissue glue (Histoacryl® (Braun)) applied with a
micropipette. The animals recovered from surgery and did not appear to be
affected by the procedure.
In situ hybridization
The brains of adult animals and E12.5 embryos of wild-type and ak
mice were collected and immediately frozen on dry ice. Sagittal and coronal
sections (16 µm) were cut on a cryostat and collected on SuperFrost Plus
slides (Menzel Gläser). In situ hybridization with digoxigenin
(DIG)-labeled probes was performed essentially according to Schaeren-Wiemers
and Gerfin-Moser (Schaeren-Wiemers and
Gerfin-Moser, 1993). Briefly, sections were fixed in 4%
paraformaldehyde (PFA) for 10 minutes and acetylated with 0.25% acetic
anhydride in 0.1 M triethanolamine for 10 minutes. Hybridization was carried
out at 72°C in a hybridization solution containing 50% deionized
formamide, 5xSSC, 5xDenhardt's solution, 250 µg/ml tRNA Baker's
yeast and 500 µg/ml sonificated salmon sperm DNA. Post-hybridization washes
were carried out in 0.2xSSC for 2 hours at 72°C. DIG was detected
with an alkaline phosphatase-labeled antibody (Roche, Mannheim) using NBT/BCIP
as a substrate. After DIG in situ hybridization, slides were dehydrated in
ethanol, cleared in xylene and mounted using Entellan. DIG in situ
hybridization was performed with the following probes: a
BalI/EcoRI (bp 915-1137) fragment of the rat Th
cDNA (Grima et al., 1985
), an
EcoRI/PstI fragment containing bp 1 to 285 of the rat
Pitx3 cDNA (Smidt et al.,
1997
), a Nurr1 cRNA containing bp 1022 to the 3'
end of the full-length cDNA (U72354), an En1 fragment containing bp
1-1842 of the full-length mouse cDNA sequence (L12703), an En2
fragment (BglII/XbaI) containing bp 1351-2101 of the mouse
cDNA sequence (L12705), a Lmx1b fragment (EcoRI) containing
the full-length mouse cDNA sequence, a Ret fragment containing bp
1733-1281 of the mouse cDNA sequence (X67812), a dopamine transporter
(Dat) fragment (ApaI/HindIII) containing bp
762-1127 of the rat cDNA sequence (m80570), an aromatic-L-amino acid
decarboxylase (Aadc) fragment containing bp 22-488 of the coding
region from the mouse cDNA, a vesicular monoamine transporter 2
(Vmat2) fragment containing bp 290-799 of the coding region from the
mouse cDNA, a Nli fragment containing bp 651-1778 of the mouse cDNA
(U69270), a dopamine receptor 2 (D2r) fragment containing bp 342-1263
of the coding region from the mouse cDNA, a cholecystokinin (Cck)
fragment containing the full cDNA of the rat (nm_012829), a neurotensin
receptor 1 (Ntr1) fragment containing bp 426-931 of the coding region
from the mouse cDNA, and an alpha-synuclein fragment containing bp 20-420 of
the coding region from the mouse cDNA.
Combined in situ hybridization-immunohistochemistry
Sections were treated as described above for DIG hybridization, except that
after termination of the alkaline phosphatase reaction, the sections were
washed twice for 5 minutes in TBS. Then they were incubated in 0.3%
H2O2 in TBS for 30 minutes to reduce endogenous
peroxidase activity, washed twice for 5 minutes in TBS, blocked with 4% fetal
calf serum in TBS for 30 minutes, washed twice for 5 minutes in TBS, and
incubated overnight at room temperature with either anti Th (Pel-Freez,
Arkansas, USA; 1:1000) in TBST (0.5 M Tris-HCl at pH 7.4, 9% NaCl, 0.5%
Triton) or anti Pitx3 (1:500) (Smidt et
al., 2000) in TBST. The next day, sections were washed three times
with TBS for 5 minutes, incubated for 1 hour with biotinylated goat
anti-rabbit immunoglobulin in TBST (1:1000), washed three times with TBS for 5
minutes, incubated for 1 hour with avidin-biotin-peroxidase reagents (ABC
elite kit, Vector Laboratories, 1:1000) in TBST and washed with TBS three
times for 5 minutes. The slides were stained with DAB
(3,3'-diamino-benzidine) until background was lightly stained. Slides
were washed twice with demineralized water for 5 minutes, dehydrated with
ethanol and mounted using Entellan.
Immunohistochemistry
PFA (4%)-fixed vibratome sections were used for direct
immunohistochemistry, as described earlier
(Smidt et al., 2000).
Immunohistochemistry with paraffin wax sections was performed as described
earlier for frozen sections (Smidt et al.,
2000
) with the following modifications. Sections were
deparaffiniated through xylene and rehydrated through an ethanol series.
Sections were boiled in 0.06 M sodium citrate (pH 6) for 9 minutes and then
allowed to cool down to room temperature. The following antibodies were used:
polyclonal rabbit anti-Th (1:1000; PelFreez) and a polyclonal rabbit
anti-Pitx3 (1:500) (Smidt et al.,
2000
).
Nissl staining
Paraffin-embedded coronal midbrain sections (7 µm) obtained from adult
wild-type and ak mice were mounted on SuperFrost plus slides (Menzel
Gläser). Sections were deparaffinated, rinsed in water, stained for 10
minutes in 0.5% Cresyl Violet and briefly rinsed in an acetate buffer, pH 4.
The sections were then differentiated in 96% ethanol for 30 seconds,
dehydrated in 100% ethanol, cleared in xylene and mounted with Entellan.
Fluorogold retrograde tracing
A `David Kopf' stereotact apparatus with mouse adaptor (Stoelting, USA) was
used for injections in wild-type and ak mice at the following
coordinates for the dorsal striatum: (bregma=0), 1.1 to anterior, 1.5 lateral.
The skull surface was set at vertical position 0, and the injection depth was
3 mm. The animals were anesthetized with 2.5 µl/g Hypnorm (Janssen; 0.315
mg/ml fentanyl citrate, 10 mg/ml fluanisone, IP), additionally 0.8 µl/g
dormicum (Roche; 5 mg/ml midazolam, IP) was given 5 minutes after the Hypnorm
injection. During the operation `CAF-zalf' (Apharmo) was applied to the eyes
of the wild-type mice to prevent drying of the eyes. Body temperature was kept
at 37°C by placing the mice on heating mats. The retrograde tracer
Fluorogold (Fluorochrome, USA) was iontophoretically delivered through a glass
micropipette (internal diameter 10-15 µm) filled with 2% Fluorogold in 0.1
M cacodylate buffer (pH 7.3). The tracer was applied using a midgard 51413
precision current source instrument [polarity -, 5 µA for 10 minutes (pulse
setting 7 seconds on, 7 seconds off) red pen(+) at the ear of the mice, black
pen(-) in Fluorogold solution]. The glass capillaries (1.5 mm OD, 0.86 mm ID,
Borosilicate glass, standard wall with inner filament, Clark electromedical
instruments, Reading, UK) were prepared on a micropipette-puller (Getra,
Munchen, Germany) with the following settings: Heating: 7.5-8, Magnet: 3. The
animals were killed 48 hours after the infusions, and the brains were isolated
and fixed for 24 hours in 4% PFA (fresh) at 4°C. Sections (50 µm) were
cut with a vibratome (Leica) and examined under a fluorescence microscope or
used for additional immunohistochemical experiments.
Climbing test
The test was performed as described
(Costall et al., 1978a;
Costall et al., 1978b
) except
that climbing was scored every 5 minutes for 90 minutes (0=all paws on the
floor, 1=2 paws on the cage, 2=all paws on the cage). None of the animals was
injected with drugs.
Medium open field test
The open field consisted of a Plexiglas open cylinder (diameter 20 cm,
height 30 cm) placed on a white plastic board. Locomotor activity was
monitored for 15 minutes using a fully automated observation system (1.6
frames per second, Ethovision, Noldus Information Technology, The
Netherlands). The animals were used at 3 months of age. The experiment was
performed under normal lighting conditions between 10 am and 3 pm.
Automated quantitative gait analysis
The gait of wild-type and ak mice was analyzed as described
previously (Hamers et al.,
2001).
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Results and discussion |
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As mesDA progenitor neurons start to express Th after they have
migrated to a ventral position in the midbrain, we sought to determine their
fate by analyzing Aadc (Ddc - Mouse Genome Informatics)
expression. Aadc is expressed solely by catecholaminergic and
serotonergic neurons (Cooper et al.,
1977) and is first expressed 2 days earlier than Th
(Teitelman et al., 1983
).
Analysis of sagittal sections of E12.5 wild-type mice showed that
Aadc was expressed in serotonergic neurons located caudal to the
mid-hindbrain border (MHB) and in midbrain dopaminergic neurons located
rostral to the MHB (Fig. 3A),
being expressed over a larger area than Th
(Fig. 3B). In coronal sections
from three different levels containing dopaminergic neurons
(Fig. 3C),
Aadc-positive neurons were seen immediately below the neuroepithelium
(Fig. 3D-F). This suggests that
the Aadc gene is activated early in the differentiation of
neuroepithelial cells.
|
Architecture of mesDA neurons of adult ak mice
The anatomical architecture of mesDA neurons was analyzed by comparing the
distribution of Th-immunoreactive neurons in the midbrain of ak and
wild-type mice (Fig. 4). The
morphology of the mesDA system of ak mice was dramatically altered in
two aspects: the field of dopaminergic bodies in the ventral midbrain was
smaller (Fig. 4A) and there
were fewer projections to the striatum.
(Fig. 4B,C). These findings are
consistent with other findings (Nunes et
al., 2003; van den Munckhof et
al., 2003
; Hwang et al.,
2003
). In ak mice, Th-positive neurons were not detected
in the SNc. The region of the VTA was less affected, although in the region
where the SNc and VTA are overlapping, Th positive neurons were also absent
(Fig. 4A). The
non-Pitx3 expressing dopamine systems including the periglomerular
dopamine neurons (A16) located in the olfactory bulb and the
tuberohypophycial/incertohypothalamic dopamine neurons (A11-A14) located in
the hypothalamus were not affected, as analyzed by Th staining in coronal
sections through the brain (data not shown). This was expected as
Pitx3, in contrast to Lmx1b and Nurr1, is expressed
only in mesDA neurons in the brain (Smidt
et al., 1997
). Furthermore, the defect of Pitx3 seems to be
restricted to a subset of mesDA neurons. To address the reportedly limited
expression of Pitx3 in a subset of DA neurons and the compromised survival of
these neurons (van den Munckhof et al.,
2003
), the overlap in expression of Th and Pitx3 was investigated
in detail by combined in situ hybridization/immunohistochemistry for
transcripts and protein of both Th and Pitx3
(Fig. 5).
|
|
Restricted connectivity of mesDA neurons and forebrain regions in ak mice
Analysis of sagittal sections immunostained for Th revealed dramatic
changes in the connectivity of the mesDA cell bodies with main target areas,
specifically the caudate putamen (Fig.
4B). In the striatal projection area, Th-positive fibers were
mainly lost in the dorsal caudate putamen. Parts of the medial forebrain
bundle were still visible. These fibers innervated ventral striatal areas,
particularly the nucleus accumbens area and the olfactory tubercle. The
caudate putamen was devoid of innervating Th-positive axons. This loss of
mesDA projections was seen throughout the caudate putamen
(Fig. 4C).
In order to determine whether mesDA neurons in the SNc were absent in
ak mice or no longer expressed Th, Nissl staining was performed
(Fig. 6A). Sections were
compared to adjacent sections stained for Th
(Fig. 6B). In ak mice,
particularly DA neurons in the lateral part of the SNc were absent
(Fig. 6A, parts
a,b,a',b') and the density of DA cells in the medial part of the
SNc (Fig 6A, parts
c,d,c',d') was reduced. Taken together, the data indicate that a
null mutation of the Pitx3 gene results in the absence of mesDA
neurons in the SNc. Moreover, when present the mesDA neurons in the VTA of
ak mice had an altered morphology
(Fig. 6A,B, parts e-h;
indicated by arrows in g,h), consistent with data from a recenlty published
study (Hwang et al., 2003).
This indicates that mesDA neurons of the SNc and the VTA, differ in their
dependence on Pitx3 expression. The loss of neurons in the
ak brain results in altered mesDA projections to the caudate
putamen.
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ACKNOWLEDGMENTS |
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