 |
 Previous Article | Next Article 
The Journal of Neuroscience, May 1, 2001, 21(9):3126-3134
Fate of Midbrain Dopaminergic Neurons Controlled by the Engrailed
Genes
Horst H.
Simon1,
Harald
Saueressig1,
Wolfgang
Wurst2, 3,
Martyn D.
Goulding1, and
Dennis D. M.
O'Leary1
1 Molecular Neurobiology Laboratory, The Salk
Institute, La Jolla, California 92037, 2 Max Planck
Institute for Psychiatry, D-80804 Munich, Germany, and
3 GSF-Research Center, Institute for Mammalian Genetics,
D-85758 Oberschleißheim, Germany
 |
ABSTRACT |
Deficiencies in neurotransmitter-specific cell groups in the
midbrain result in prominent neural disorders, including Parkinson's disease, which is caused by the loss of dopaminergic neurons of the
substantia nigra. We have investigated in mice the role of the
engrailed homeodomain transcription factors, En-1 and En-2, in
controlling the developmental fate of midbrain dopaminergic neurons.
En-1 is highly expressed by essentially all dopaminergic neurons in the substantia nigra and ventral tegmentum, whereas En-2 is highly expressed by a subset of them. These
neurons are generated and differentiate their dopaminergic phenotype in
En-1/En-2 double null mutants, but
disappear soon thereafter. Use of an En-1/tau-LacZ
knock-in mouse as an autonomous marker for these neurons indicates that
they are lost, rather than that they change their neurotransmitter
phenotype. A single allele of En-1 on an En-2 null background is sufficient to produce a wild
type-like substantia nigra and ventral tegmentum, whereas in contrast a single allele of En-2 on an En-1 null
background results in the survival of only a small proportion of these
dopaminergic neurons, a finding that relates to the differential
expression of En-1 and En-2. Additional
findings indicate that En-1 and En-2 regulate expression of
 -synuclein, a gene that is genetically linked to Parkinson's disease. These findings show that the engrailed genes are
expressed by midbrain dopaminergic neurons from their generation to
adulthood but are not required for their specification. However, the engrailed genes control the survival of midbrain dopaminergic neurons in a gene dose-dependent manner. Our findings also suggest a
link between engrailed and Parkinson's disease.
Key words:
-synuclein; En-1; En-2; neuronal death; neuronal specification; dopamine; Parkinson's disease; substantia nigra; mouse; transcription factors; tyrosine hydroxylase; ventral tegmentum; tau-LacZ
| |
INTRODUCTION |
The enormous variety of neuronal
types in the nervous system becomes specified during development in a
stepwise process (Tanabe and Jessell, 1996 ) from an originally uniform
pool of neuroepithelial cells (Sechrist and Bronner-Fraser, 1991).
Overlapping signals lay down the rostrocaudal and dorsoventral neuraxes
of the embryo in a chronological order and determine where specific
neuronal types will form (Simon et al., 1995). During this process,
unique sets of transcription factors are activated in subsets of
neuroepithelial cells and their progeny, and they specify neuronal
phenotype. For example, the distinct subsets of spinal motor neurons
and their progenitors are determined by their expression of unique combinations of Lim homeodomain transcription factors (Pfaff et al.,
1996).
Engrailed, a homeodomain transcription factor, has well defined roles
in insect development. During early development, engrailed establishes
the body plan, defining for example the posterior half of each
parasegment (Kornberg, 1981a,b; Poole and Kornberg, 1988). Later,
engrailed determines neuronal identity. For example, engrailed controls
the glial-neuronal fate decision of certain multipotent neuroblasts in
the grasshopper CNS (Condron et al., 1994) and of midline serotonergic
neurons in Drosophila (Lundell et al., 1996).
Vertebrates have two engrailed homologs, En-1 and
En-2, that are expressed during development in a domain
encompassing the posterior midbrain and anterior hindbrain (Davidson et
al., 1988; Gardner et al., 1988). This domain coincides with parts of
the neural tube that generate dorsal structures, including the
cerebellum and colliculi, as well as ventral midbrain nuclei. Targeted
deletion of En-1 and En-2 in mice reveals their
role in regulating the development of dorsal structures originating
within their expression domain. En-1 mutants, which die on
the day of birth, lack the cerebellum and inferior colliculus (Wurst et
al., 1994). En-2 mutants are viable and fertile and have
minor defects in cerebellar foliation (Joyner et al., 1991; Millen et
al., 1994). Overexpression of En-1 and En-2 in
chick midbrain indicates that engrailed regulates the
anterior-posterior polarity of the optic tectum, the homolog of the
mammalian superior colliculus (Friedman and O'Leary, 1996; Itasaki and
Nakamura, 1996; Logan et al., 1996). Although these studies show that
engrailed has a prominent role in vertebrate neural development, a
function for engrailed in vertebrate neuronal specification has only
recently been suggested (Saueressig et al., 1999).
The ventral midbrain nuclei, substantia nigra (SN) and ventral
tegmentum (VT), are the most prominent sources of dopaminergic neurons
in the CNS (German and Manaye, 1993). Degeneration of these neurons and
their projections to the basal ganglia and frontal cortex is implicated
in several CNS disorders, including Parkinson's disease (PD), which is
characterized by a debilitating loss of motor control (Polymeropoulos
et al., 1997). The expression domains of En-1 and
En-2 approximate the location of these neurons (Davis and
Joyner, 1988; Davis et al., 1988), suggesting that engrailed may
regulate their specification and differentiation. To investigate this
issue, we related the expression of En-1 and En-2
to dopaminergic neurons and addressed the requirement of engrailed for
their developmental fate in En-1 and En-2 mutant
mice using cell autonomous and neurotransmitter-specific markers. Our
findings led us to show that expression of -synuclein, a
gene genetically linked to PD (Polymeropoulos et al., 1997; Kruger et
al., 1998), is dependent on engrailed.
| |
MATERIALS AND METHODS |
Animals. Generation of the En-1- and
En-2-deficient mice by targeted gene deletion has been
described previously (Joyner et al., 1991; Wurst et al., 1994). The
En-1/tau-LacZ mice were generated by a
"knock-in" strategy in which the first 71 codons, including the
start codon, were replaced by a tau-LacZ sequence (Callahan and Thomas, 1994) and resulted in an En-1 null allele. The
construct and procedures are described in detail elsewhere (Saueressig
et al., 1999). Parental lines for producing the mutant mice deficient for both En-1 and En-2 were kept as
En-1+/ /En-2/
or
En-1/tau-LacZ+//En-2/.
A minimum of four mice of a given genotype, but usually more, were
analyzed for each finding described. Each set of findings described for
a given genotype and methodology was observed in each animal analyzed.
Immunohistochemistry on sections. The animals were perfused
with 4% paraformaldehyde in 100 mM phosphate
buffer (PB), pH 7.4, and immersion-fixed overnight at 4°C. The brains
were cryoprotected with 30% sucrose in 100 mM PB
and cut at 50 µm with a sledge microtome. Alternatively, the brains
were embedded in an albumin-gelatin mixture as follows: 3% gelatin
bloom 100 was heated until dissolved and cooled to <40°C, chicken
egg albumin was added to a concentration of 30%, and then the mixture
was filtered through a 100 µm nylon mesh. The tissue was put
into a plastic mold containing the albumin-gelatin mixture and exposed
to formaldehyde vapor overnight at 4°C (formalin in a glass tray).
When the top was firm, a 4% paraformaldehyde solution was poured into
the mold and left overnight at 4°C. The next day, the block was
trimmed and immersed overnight in 4% paraformaldehyde. Then the
specimen was sectioned with a vibratome at 70 µm.
The sections were immunostained in a plastic chamber; blocked with 10%
heat-inactivated newborn calf serum (NCS), 1%
H2O2, 1% Triton X-100 in
PBS for 1 hr at room temperature; washed with PBT (PBS, 1%
Triton X-100) three times for 10 min each; and incubated with
primary antibody in PBT, 10% NCS [rabbit anti-tyrosine hydroxylase (TH) 1:2000 (Chemicon, Temecula, CA); rabbit anti- -galactosidase (-gal) 1:10,000 (Organon Tecknika-Cappel, Durham, NC); or goat anti--galactosidase 1:10,000 (Arnel Products Co., New York, NY)] at
4°C overnight. They were washed three times with PBT for 10 min, incubated with secondary antibody at 1:500 in PBT, 10% NCS [biotinylated goat anti-rabbit, all secondary antibodies from Jackson
ImmunoResearch (West Grove, PA)] for 2 hr, washed three times with
PBT, and incubated with streptavidin-peroxidase (Jackson ImmunoResearch) at 1:1000. The sections were washed three times with PBT and twice with PBS and developed (0.05% DAB, 0.01%
H2O2 in PBS or with 0.7%
nickel ammonium sulfate added for nickel intensification). The sections
were mounted on slides, dehydrated with a series of alcohols, xylene,
and embedded in DPX. TH-labeled sections were counterstained
with neutral red before dehydration and embedding. For immunostaining
with the Enhb antibody, brains were fixed in Zamboni's solution
(4% paraformaldehyde, 100 mM PB, 15% saturated picric
acid) and then further treated as described above.
The fluorescent double labeling using Enhb and anti-TH (both
antibodies raised in rabbits) was performed by labeling first one,
using a biotinylated anti-rabbit antibody and streptavidin-FITC, and
then the other using an anti-rabbit TRITC. Labeling of the first
primary antibody by the second secondary antibody was negligible and
easy to differentiate because the Enhb antigens, En-1 and En-2, are
localized to the nucleus, whereas TH is localized to the cytoplasm. In
the case of double labeling for the -gal reporter and TH, a goat
anti--gal, a rabbit anti-TH, and species-specific secondary
antibodies were used. After the staining procedure, the sections were
mounted on slides and embedded in
glycerol-1,4-diazabicyclo-[2.2.2]octane.
Immunohistochemistry on whole mounts. Embryos were fixed in
4% paraformaldehyde and stained as described (Simon et al., 1994) using the same antibody concentrations as above. Brains were isolated after fixation but before staining to facilitate penetration of the
antibodies. After staining, the ventral brainstem was dissected and
flat mounted in glycerol/PBS (9:1).
RNA in situ hybridization. In situ
detection of En-1 and En-2 on 20 µm
fresh-frozen sections of mouse brain was performed according to a
previously described method (Goulding et al., 1994). The
S35 antisense probes corresponded to
regions described by Davis et al. (1988). After hybridization, sections
were dipped in NTB 2 emulsion (Eastman Kodak, Rochester, NY),
developed after 3-5 weeks, and counterstained.
Terminal deoxynucleotidyl transferase-mediated biotinylated UTP
nick end labeling. Apoptotic cell nuclei were detected
with the In Situ Cell Death Detection Kit (catalog #1 684 809; Boehringer Mannheim, Indianapolis, IN) on either section or on
isolated whole-mount embryo brains. Embryos were fixed in 4%
paraformaldehyde, cryoprotected with sucrose, and cut with a freezing
microtome. Sections were treated for 10 min with proteinase K and then
stained according to the kit. The whole-mount brains were obtained by
isolating the neural tubes of living embryos, which then were fixed in
4% paraformaldehyde. The specimens were dehydrated briefly and
rehydrated with a series of methanol/PBS, 0.1% Tween 20 (PBT), and
incubated for 10 min in 10 µg/ml proteinase K in PBT. After a brief
wash with PBT, the specimens were incubated in the reaction mixture of
the kit overnight at 4°C. The reaction buffer was exchanged, and specimens were incubated again for 3 hr at 37°C. The specimens were then washed several times with PBT, 1 hr each time, and incubated with alkaline phosphatase-coupled anti-fluorescein antibody in PBT (1:5000) overnight at 4°C. The specimens were then washed several
times with PBT, 1 hr each time, followed by two washes with 100 mM Tris, pH 9.5, 100 mM
NaCl, 50 mM MgCl2, and
developed with NBT/BCIP (Boehringer Mannheim).
Image processing. All images were photographed on a Nikon
Microphot-FX microscope. The film was digitized using a Nikon LS1000 scanner, and figures were assembled using Adobe Photoshop. Some images
were photographed with a 2.5× lens and montaged digitally.
| |
RESULTS |
En-1 and En-2 are expressed in the
substantia nigra and ventral tegmentum in neonatal mice
In situ hybridizations were done on sagittal sections
of postnatal day (P)0 mouse brains to determine the expression of the murine engrailed genes in the midbrain and hindbrain, focusing on the
dopaminergic nuclei in the ventral midbrain (German and Manaye, 1993).
In the ventral midbrain, En-1 and En-2 have
largely overlapping patterns of expression (Fig.
1A,B)
and are coincidentally expressed in the SN, the VT, and the
periaqueductal central gray. Within the SN and VT, En-1 is
expressed more or less throughout the nuclei at relatively high levels,
whereas En-2 appears to be expressed highly in a small
proportion of cells and at lower levels in the remainder. In the
anterior hindbrain, En-1 expression is largely limited to
the superior olive, whereas En-2 is not expressed at
detectable levels. Dorsally, we found that En-1 and En-2 have overlapping patterns of expression, most notably
the inferior colliculus and the cerebellum (Fig.
1A,B), as reported previously
(Davis and Joyner, 1988; Davis et al., 1988).
View larger version (54K):
[in this window]
[in a new window]
|
Figure 1.
Dopaminergic neurons in the ventral midbrain of
neonatal mice express En-1 and En-2.
In situ hybridization (A-C) and double
immunolabeling (D-G) of sagittal (A-C,
F, G) and coronal (D,
E) sections of P0 mouse brains. A,
B, Adjacent sections of a P0 wild-type mouse hybridized
with riboprobes against En-1 (A)
and En-2 (B) reveal the
distribution of engrailed transcripts in the substantia nigra
(SN), periaqueductal central gray
(CG), inferior colliculus (IC), and
cerebellum (Cb). En-1 is expressed at
high levels throughout the ventral tegmentum (VT)
and SN, whereas En-2 is expressed at relatively high
levels by only a small subset of cells and at much lower levels in most
cells. En-1 is also expressed in a subpopulation of
cells in the superior olive (SO), a hindbrain nucleus.
C, A P0 En-1/
mutant hybridized with riboprobes against En-2. In
En-1/ mutants,
En-2 expression appears to be upregulated and expressed
at relatively high levels throughout the SN and VT. D,
E, Double immunohistochemistry on a coronal section of a
P0 wild-type mouse brain using the Enhb antibody, which recognizes
both En-1 and En-2 proteins (D, red), and
an antibody against TH (E,
green). Engrailed and TH proteins are coexpressed in
dopaminergic neurons of the SN and VT. Engrailed protein is located in
the nuclei, whereas TH is located in the cell somata and their axonal
processes. F, G, A lateral section of P0
mouse brain heterozygous for En-1/tau-LacZ
(En-1+/tLZ), double-labeled with
antibodies against -gal, the protein product of
LacZ (F, red) and TH
(G, green). TH and the -gal reporter
for En-1/tau-LacZ are coexpressed in the somata and
axons of the midbrain dopaminergic neurons. For the sagittal sections,
rostral is to the left, and dorsal to the
top. For the coronal sections, dorsal is to the
top, and the midline is in the middle of
the image. Scale bars: A-C, 300 µm;
D-G, 100 µm.
|
|
To determine whether SN and VT dopaminergic neurons express engrailed,
as suggested by our findings using in situ hybridization, we
performed antibody double labeling experiments. Engrailed expression was revealed with either the Enhb antibody, which recognizes both
En-1 and En-2 proteins (Davis and Joyner, 1988) or an antibody against
-gal to detect the tau--gal product of the
tau-LacZ reporter in the En-1/tau-LacZ
mice (Saueressig et al., 1999). Because the tau--gal protein
integrates into the microtubule network, it effectively labels the cell
body and axonal processes of En-1-expressing neurons
(Callahan and Thomas, 1994; Mombaerts et al., 1996). Dopaminergic
neurons were detected immunohistochemically using an antibody against
TH, a key enzyme in the synthetic pathway of dopamine.
Immunostaining of P0 wild type with Enhb reveals the nuclear
distribution of engrailed protein in the ventral midbrain (Fig.
1D). Immunostaining the same section with the TH
antibody labels the corresponding cell bodies (Fig.
1E), confirming that dopaminergic neurons of the SN
and VT express engrailed. Because the Enhb antibody recognizes both
En-1 and En-2 proteins, we performed additional double-labeling studies
to determine whether midbrain dopaminergic neurons express both
proteins, as suggested by our in situ data (Fig.
1A,B). Double-immunolabeling of
sections from P0 heterozygous En-1/tau-LacZ
mutant brains with antibodies against -gal and TH reveals an
identical distribution (Fig. 1F,G), demonstrating that the dopaminergic neurons express En-1. In
addition, double-immunolabeling of sections from P0
En-1/ mice with the
Enhb and TH antibodies shows that En-2 protein is also expressed by
the midbrain dopaminergic neurons (data not shown) (Fig.
1C).
En-1 and En-2 are required for the
development of midbrain dopaminergic neurons
To determine whether the engrailed genes are required for the
development of the dopaminergic neurons of the SN and VT, we analyzed
En-1 (Wurst et al., 1994) and En-2 (Joyner et
al., 1991; Millen et al., 1994) mutant mice. In situ
hybridization analysis of P0 En-1 mutants shows that the
basic expression pattern of En-2 in the ventral midbrain,
including expression in the SN and VT, is similar to that described
above in wild-type mice, with the exception that En-2
appears to be highly expressed throughout the SN (Fig. 1C),
in contrast to wild type where it is highly expressed in only a
proportion of SN neurons (Fig. 1B). This finding suggests that the normal coincident expression patterns of
En-1 and En-2 in these nuclei do not depend on
one another, although the absolute level of expression may. Therefore,
because En-2 can functionally replace En-1 (Hanks
et al., 1995), and likely vice versa, we first focused our analyses on
mice deficient for both En-1 and En-2.
Dense clusters of dopaminergic neurons comprising the SN and VT
are readily detected in sections of wild-type P0 mouse brains using the
TH antibody (Fig.
2A,A').
In contrast, in engrailed double mutants
(En-1//En-2/),
dopaminergic neurons of the SN and VT are completely absent (Fig.
2B,B'). Dopaminergic cell
groups located outside of the expression domains of En-1 and
En-2, for example the dorsomedial hypothalamic nucleus,
remain detectable and appear normal in engrailed double mutants. These
findings indicate that targeted deletion of both En-1 and
En-2 results in the selective loss of dopaminergic neurons
in the SN and VT.
View larger version (97K):
[in this window]
[in a new window]
|
Figure 2.
Midbrain dopaminergic neurons require
En-1 and En-2 for their survival in a
gene dose-dependent manner. Immunohistochemistry on sagittal sections
of P0 mouse brains using antibodies against tyrosine hydroxylase
(TH) to identify dopaminergic neurons
(A, A', B,
B', C, C',
D, D'), or against -gal (A", B", C",
D") to identify En-1/tau-LacZ
(En-1/tLZ)-expressing neurons in the ventral midbrain of
wild-type (WT) mice
(A-A"), engrailed double mutants
(En-1//En-2/)
(B-B"),
En-1/ mutants
(C--C"), and
En-1//En-2+/
mutants (D--D"). In
A"-D", mice with the
En-1/tau-LacZ (En-1/tLZ) reporter
construct were used. Therefore, in
A"-D", wild type is actually
En-1+/tLZ, and
En-1/ is actually
En-1tLZ/tLZ.
A-A", Wild type. TH immunostaining
reveals the normal distribution of the midbrain dopaminergic neurons.
In the midline (A), the neurons of the ventral
tegmentum (VT) are labeled, and in a more lateral
section (A'), those of the substantia nigra
(SN) are labeled. The -gal
immunostaining of a section from an
En-1+/tLZ reveals a similar labeling
pattern as TH for both the VT and SN. In addition, SN axons projecting
rostrally to the forebrain (A", arrowhead) and
En-1-expressing radial glia (white arrow)
are labeled. DMH, dorsomedial hypothalamic nucleus.
B-B", Engrailed double mutant.
Dopaminergic neurons of the SN and VT are not detected by
immunostaining with either TH (B, B') or
-gal (B"). However, dopaminergic neurons of the
DMH (a diencephalic nucleus) remain TH-positive
(B-B"), indicating that their dopaminergic phenotype
does not depend on En-1 or En-2. In
addition, neurons in the superior olive (SO) that
normally express En-1 are marked by the
En-1/tau-LacZ reporter in engrailed double mutants
(B"), as well as in the other mutant genotypes examined
(C", D"), indicating that
En-1 expression is not auto-regulated.
C-C", En-1/
mutant. Distribution of the dopaminergic neurons appears normal with
the exception of those located in the VT that appear more loosely
clustered (compare C with A).
D-D",
En-1//En-2+/
mice. The dopaminergic neurons of the SN and VT are reduced to a small
cluster of cells in the ventral midbrain, demonstrating an engrailed
gene dose-dependent effect on the development of these neurons. Rostral
to the left, dorsal to the top. Scale
bars, 200 µm.
|
|
En-1 and En-2 compensate for each
other in the development of dopaminergic neurons
To assess whether En-1 and En-2 can
compensate for the targeted deletion of one another, we analyzed mutant
mice null for either En-1 or En-2. The SN and VT
in the En-2/ mice resemble wild
type (data not shown), as expected on the basis of the integrity of
dorsal structures in En-2/ mice
(Joyner et al., 1991; Millen et al., 1994). In contrast, because
several major dorsal structures are absent or substantially diminished
in En-1/ mice (Wurst et al.,
1994), we anticipated a comparable reduction in the SN and VT. However,
the distribution and packing density of the dopaminergic cells of the
SN in En-1/ mice (Fig.
2C') appears similar to that in wild-type mice (Fig. 2A'). The only difference that we observed is that
the dopaminergic cells of the VT appear more loosely arranged in
En-1 mutants than in wild-type mice (Fig. 2, compare
A, C). The relatively normal appearance of the SN
and VT in engrailed single mutants, compared with their absence in
engrailed double mutants, indicates that En-1 and
En-2 compensate substantially, if not entirely, for the loss
of one another in regulating the proper development of these nuclei.
En-1 and En-2 act in a gene
dose-dependent manner
To determine whether En-1 and En-2 act in a
gene dose-dependent manner, we analyzed mutant mice null for one
engrailed gene and heterozygous for the other. In
En-1//En-2+/
mice, the SN and VT are substantially diminished, compared with engrailed single mutants or wild-type mice, being reduced to a small
cluster of dopaminergic neurons close to the ventral surface of the
midbrain (Fig. 2D,D'). In contrast
to these substantial defects in
En-1//En-2+/
mice, in
En-1+//En-2/
mice the SN and VT are essentially identical to the wild type (data not
shown). Thus, with respect to the dopaminergic neurons of the SN and
VT, in mice in which only one of the four engrailed allelesis present,
one En-1 allele is sufficient to produce a phenotype that
resembles wild type, but one En-2 allele is not.
Midbrain dopaminergic neurons are absent rather than phenotypically
defective in engrailed double mutants
One of three possible explanations could account for our inability
to detect in engrailed double mutants the dopaminergic neurons that
would normally form the SN and VT: they are present but defective in
expressing their dopaminergic phenotype, they are not generated, or
they are generated but disappear later. To address this issue, we used
the En-1/tau-LacZ mice in which En-1
is replaced with tau-LacZ, creating a null En-1
allele (termed En-1tLZ) (see
Materials and Methods) (Saueressig et al., 1999). In these mice, the
tau--gal protein encoded by tau-LacZ is a cell autonomous marker specific for En-1-expressing neurons, and it labels
their cell bodies and axonal processes. With respect to the SN and VT, the distribution of tau--gal protein is the same as the distribution of TH protein in each of the six En-1/En-2 genotype
combinations analyzed in this study (Fig. 2; data not shown) (for
determining six genotypes, the
En-1tLZ allele is considered
equivalent to the En-1/ allele),
including the engrailed double mutants
(En-1tLZ/tLZ/En-2/)
in which no tau--gal-positive or TH-positive neurons are detected in
the ventral midbrain (Fig. 2B--B").
However, En-1-expressing cells in the superior olive are
present in all genotypes analyzed, including the engrailed double
mutant, indicating that En-1 is not positively autoregulated
(Saueressig et al., 1999). These findings strongly suggest that the
lack of detection of the midbrain dopaminergic neurons is attributable
to their absence rather than their failure to express a dopaminergic phenotype.
Midbrain dopaminergic neurons are generated in engrailed
double mutants
The absence of dopaminergic neurons of the SN and VT in engrailed
double mutants could be caused by an early event such as the failure of
these cells to be generated or to a later event such as the loss of
these cells after they have differentiated and started to express TH.
At embryonic day (E)8, En-1 and En-2 are broadly
expressed in the neuroepithelium of the posterior midbrain and anterior
hindbrain and appear to be involved in specifying these fields (Wurst
et al., 1994). If the deletion of En-1 and En-2
results in a respecification or diminished proliferation of the
neuroepithelial cells that normally would generate the dopaminergic
neurons or their precursors, the dopaminergic neurons would not be
detectable at any stage of development. If the defect observed in
engrailed double mutants is caused by a later event, we would expect
that the dopaminergic neurons could be detected over at least a brief
embryonic period. To address these alternatives, we first determined
whether any cells that normally express En-1 remain in the
midbrain after the full extent of the morphological deletion becomes
apparent around E9 (Wurst et al., 1994). This approach is made possible
by using the En-1/tau-LacZ mice.
In hetero-homo E9 embryos
(En-1+/tLZ/En-2/),
the tau-LacZ domain detected by the -gal antibody
coincides exactly with the normal En-1 expression domain in
the wild type (Fig. 3A). In
engrailed double mutant embryos at the same age, this domain is
substantially reduced to a small group of tau-LacZ positive
cells in the ventral neural tube. This zone corresponds with the area
in which the dopaminergic neurons are induced (Ye et al., 1998)
slightly later. Because the precursor cells giving rise to
midbrain dopaminergic neurons are present in engrailed double mutants,
we used TH immunostaining to determine whether neurons of the SN and VT
are generated and express their dopaminergic phenotype. At E11, in
wild-type and hetero-homo
(En-1+//En-2/)
mutants, a distinct ventral domain of TH-positive cells is present in
the midbrain (Fig. 3C). These cells will later form the SN and VT (Voorn et al., 1988; Lieb et al., 1996). In engrailed double mutants
(En-1//En-2/),
this ventral domain of TH-positive cells is present at this age (Fig.
3D), although it is smaller than that observed in
En-1+//En-2/
littermates or wild-type mice. By E14, these TH-positive cells are no
longer detectable in engrailed double mutants (data not shown; for P0,
see Fig. 3B). These findings indicate that neurons of the
dopaminergic phenotype are generated in the ventral midbrain of
engrailed double mutants, but they disappear soon thereafter.
View larger version (132K):
[in this window]
[in a new window]
|
Figure 3.
Dopaminergic neurons of the ventral midbrain are
generated in engrailed double mutants. Whole-mount immunohistochemistry
of E9 (A, B) and E11 (C, D) embryos using
antibodies against -gal and TH, respectively. A,
Heterozygous E9 embryo (En-1+/tLZ).
-gal immunostaining reveals the normal distribution of En-1. En-1 is
expressed rostral and caudal to the isthmus (arrow)
spanning the entire neuraxis from dorsal to ventral. B,
In the E9 engrailed double mutant embryo
(En-1tLZ/tLZ/En-2/),
a large deletion of the posterior midbrain and anterior hindbrain is
apparent. -gal immunostaining is only detected in a small domain in
the ventral neural tube (arrow), likely corresponding to
the precursor cells of the dopaminergic neurons. C, E11
wild-type flat mount of ventral midbrain immunostained for TH. The
immunostaining reveals the bilateral distribution of dopaminergic
neurons adjacent to the floor plate
(asterisk). D, E11 engrailed double
mutants
(En-1//En-2/).
A cluster of dopaminergic neurons is detected by TH immunostaining,
although it is smaller than that observed in wild-type or heterozygous
mice. These neurons disappear in engrailed double mutants over the next
few days of development. Rostral is to the top. Scale
bars, 100 µm.
|
|
Our findings suggest that in engrailed double mutants, midbrain
dopaminergic neurons are generated but die over a brief time window of
embryonic development. Because most embryonic cell death is apoptotic
and apoptotic cell death can often be recognized by methods detecting
chromosomal fragmentation (Compton, 1992), we used a terminal
deoxynucleotidyl transferase-mediated biotinylated UTP nick end
labeling (TUNEL) technique to identify apoptotic cell nuclei in the
ventral midbrain in wild-type mice and engrailed double mutants at E12
and E13, ages when the midbrain dopaminergic neurons can still be
identified in the mutants but are reduced in number compared with wild
type. We found no TUNEL-positive cells in either wild-type or engrailed
double mutants in the region of the ventral midbrain in which the
dopaminergic neurons are located, whereas unrelated neuronal
populations known to undergo apoptotic cell death at these ages, such
as the trigeminal ganglia (Davies and Lumsden, 1984), show large
numbers of TUNEL-positive cells (data not shown). These findings
suggest that if the midbrain dopaminergic neurons die via apoptosis,
their turnover rate or some other feature precludes their detection
with the TUNEL method.
Engrailed expression in midbrain dopaminergic neurons is maintained
in the adult
Because the degeneration of dopaminergic neurons in the SN and VT
is implicated in PD, we investigated whether engrailed expression observed during embryogenesis and in neonates is maintained into the
adult. We determined whether En-1 and En-2 are
expressed by the dopaminergic neurons of the SN and VT of adult mice by
immunostaining for -gal in mice heterozygous for
En-1/tau-LacZ or by in situ hybridization. The SN and VT are strongly positive for tau--gal protein in adult En-1+/tLZ mice
(Fig. 4A). In addition,
the axonal projections of the SN and VT to their major targets, such as
the striatum, nucleus accumbens, and the olfactory tubercle (Voorn et
al., 1988; Paxinos et al., 1994), are also immunostained for
tau--gal protein. This overall pattern of -gal immunostaining is
identical to that for TH immunostaining (Fig. 4B),
demonstrating that the dopaminergic neurons of the SN and VT continue
to express En-1 in the adult. In situ
hybridization confirms the En-1 expression data obtained
with the En-1/tau-LacZ mice and shows that En-2
is also expressed in the SN and VT (Fig. 4C,D).
However, as during development (see above), En-1 is
expressed at high levels throughout the SN and VT, whereas
En-2 appears to be expressed highly in only a subset of
cells in these nuclei and at considerably lower levels in the
remainder. Thus En-1 and En-2 are expressed in
the dopaminergic neurons of the SN and VT from early embryogenesis into
adult, although their relative levels of expression appear to
differ.
View larger version (77K):
[in this window]
[in a new window]
|
Figure 4.
Dopaminergic neurons of the substantia nigra
express En-1 and En-2 in adult mice.
Immunohistochemistry (A, B) and in situ
hybridization (C, D) on sagittal sections of adult mouse
brain. A, Immunostaining for -gal on an
En-1+/tLZ brain section reveals
En-1 expression domains in the midbrain: the substantia
nigra (SN), inferior colliculus
(IC), parts of the periaqueductal central
gray, and a subset of the Bergmann glia in the cerebellum
(Cb). Because of the cytoplasmic localization of the
tau--gal reporter protein, the axonal projection of the SN-VT
dopaminergic neurons to the striatum (St), the nucleus
accumbens (Ac), and the olfactory tubercle
(OT) is also labeled by the -gal antibody.
B, Immunostaining for TH on a section adjacent to the
one shown in A, revealing the distribution of
dopaminergic neurons in the SN and their axonal terminations
(St, Ac, and OT).
The similar distribution of -gal and TH in A and
B demonstrates that dopaminergic neurons of the SN
express En-1 in the adult. C, D, Adjacent
sections hybridized with S35-labeled
riboprobes against En-1 (C) or
En-2 (D). En-1 is
expressed at high levels throughout the SN. In contrast,
En-2 is expressed at high levels in only a small subset
of cells in the SN and at much lower level in the majority of SN cells.
Rostral is to the left, dorsal to the
top. Scale bars: A, B, 1 mm; C, D, 500 µm.
|
|
-Synuclein may be regulated by engrailed
Our findings in mice that En-1 and En-2 are
required for the survival of dopaminergic neurons of the SN and VT and
are expressed by these neurons in the adult suggest a link between
these genes and PD. Thus, we investigated whether the engrailed genes
influence the expression of -synuclein, which
is expressed by the midbrain dopaminergic neurons (Maroteaux et al.,
1988; Maroteaux and Scheller, 1991) and has been genetically linked to
PD in humans (Polymeropoulos et al., 1997). We isolated mouse
-synuclein cDNA and performed an in
situ hybridization analysis of its expression at late E12, an age
when midbrain dopaminergic neurons have been generated and can be
defined by TH expression but have yet to be lost in engrailed double
mutants. We found that in wild-type mice, -synuclein expression (Fig. 5A)
colocalizes with midbrain dopaminergic neurons identified by in
situ hybridization for TH (Fig. 5B). Expression of
-synuclein by midbrain dopaminergic neurons is diminished in En-1 null mice (Fig. 5, compare C,
D) and absent in engrailed double mutants (Fig. 5, compare
C, E), although the TH-positive neurons are still
present (Fig. 3). These findings indicate that En-1 and
En-2 regulate the expression of
-synuclein in ventral midbrain dopaminergic
neurons.
View larger version (96K):
[in this window]
[in a new window]
|
Figure 5.
The engrailed genes influence the
expression of -synuclein. Whole-mount in
situ hybridization using riboprobes against mouse
-synuclein (A, C,
D, E) and mouse TH
(B) on E12 brains from wild-type mice
(A-C),
En-1/ mutants
(D), and engrailed double mutants
(En-1//En-2/)
(E). A, B, Whole-mount in
situ of a wild-type brain showing a view of the ventral
midbrain (dorsal midbrain is removed). -synuclein
(A) and TH
(B) transcripts are largely colocalized in
bilateral domains (arrows) adjacent to the midline
(asterisks) in the anterior ventral midbrain. The
TH expression domain is the larger of the two and is
composed of the dopaminergic neurons that give rise to both the SN and
VT, whereas the -synuclein expression domain likely
only marks developing SN neurons at this age. C,
Wild-type flat mount of the ventral midbrain shows the bilateral
-synuclein expression domain (arrow)
adjacent to the midline. D,
En-1/ flat mount of the ventral
midbrain shows a domain of -synuclein expression
(arrow) at the same location as in the wild type, but
the expression level is greatly reduced. E, Engrailed
double mutant, -synuclein is not detected in its
normal expression domain adjacent to the midline, although TH and
engrailed positive dopaminergic neurons are still present at this age
(Fig. 3D). Note that the more anterior and lateral
domains of -synuclein expression in the diencephalon
(C-E, arrowheads) is
unaffected in all three genotypes, indicating that the diminished
-synuclein expression in the engrailed mutants is
attributable to the lack of engrailed expression in the SN and not to a
developmental delay.
|
|
| |
DISCUSSION |
We show that En-1 and En-2 are required in
mice for the survival of dopaminergic neurons of the SN and VT in a
gene dose-dependent manner. These neurons are generated and
differentiate their dopaminergic phenotype independent of engrailed
expression but are lost soon thereafter if both En-1 and
En-2 are absent. The proportion that survives depends on the
En-1/En-2 genotype. Furthermore, we show that the
expression of -synuclein is dependent on engrailed. These
findings raise issues regarding the mechanisms of action of
En-1 and En-2 in controlling the developmental
fate and survival of these neurons and a potential link between
engrailed and PD.
Mechanisms underlying the loss of midbrain
dopaminergic neurons
Our analyses using two independent markers, TH and
En-1/tau-LacZ, show that dopaminergic neurons of
the SN and VT are generated in engrailed double mutants but cannot be
detected a few days later. This finding indicates that engrailed is not
required for TH expression and therefore is the dopaminergic phenotype
of SN and VT neurons, which is consistent with the fact that the
expression domains of neither En-1 nor En-2
coincide elsewhere in the CNS with the location of dopaminergic neurons
(Davis and Joyner, 1988; Davis et al., 1988). It is likely that
the ventral midbrain dopaminergic neurons die shortly after they
are generated in engrailed double mutants, because the alternative,
that they are present but no longer detectable, would demand that the
initiation of expression of TH and En-1/tau-LacZ
does not require either En-1 or En-2, but their
maintained expression does. Such a late onset of regulation of
En-1 by engrailed is rendered further unlikely by our
finding that En-1-expressing neurons in the superior olive
appear unaffected and are marked by the tau--gal reporter in
engrailed double mutants. At least in these cells, the maintained
expression of En-1/tau-LacZ is engrailed independent.
Thus, the evidence indicates that the midbrain dopaminergic cells
require engrailed for their survival. Their survival may depend on
signals or trophic support provided by other cells that are normally
present in the ventral midbrain of wild-type mice and single null
mutants for either En-1 or En-2 but are missing in engrailed double mutants. This explanation is consistent with the
more substantial truncation of the midbrain-anterior hindbrain in
engrailed double mutants than in either single mutant (Millen et al.,
1994; Wurst et al., 1994). Alternatively, the loss of the midbrain
dopaminergic neurons in engrailed double mutants could be caused by a
defect autonomous to these neurons. For example, essential housekeeping
genes or components of essential signaling pathways, including
receptors for required trophic factors, might be controlled by
engrailed and are improperly regulated in midbrain dopaminergic neurons
in engrailed double mutants. Candidate receptors are Nurr1, an orphan
member of the steroid-thyroid hormone receptor family, and receptors
for glial cell line-derived neurotrophic factor (GDNF), a member of the
TGF- superfamily.
TH-positive cells are not detected at embryonic or postnatal ages in
the ventral midbrain of Nurr1 mutant mice (Zetterström et al., 1997). However, use of markers for afferents to the SN, substance P, and glutamic acid decarboxylase, and the
neuron-specific nuclear marker NeuN, suggests that SN neurons are
present in Nurr1 mutants but do not express TH (Castillo et
al., 1998), consistent with the in vitro demonstration that
Nurr1 can induce TH expression in cells resembling midbrain
dopaminergic neurons (Sakurada et al., 1999). It is unlikely that
En-1 and En-2 regulate Nurr1 because, in engrailed double mutants, ventral midbrain TH-positive neurons are
detected during early embryogenesis but later disappear.
GDNF, which has potent trophic effects on SN and VT dopaminergic
neurons, increases their survival and differentiation in vitro (Lin et al., 1993) and prevents their loss in the adult after axotomy (Beck et al., 1995) or treatment with the
neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (Tomac
et al., 1995), which induces a PD-like syndrome. The receptor complex
for GDNF is composed of GDNFR (GDNF receptor ) and Ret (Jing et
al., 1996; Baloh et al., 1997), which are expressed in the SN and VT of
mice from at least E13.5 into adulthood (Avantaggiato et al., 1994;
Nosrat et al., 1997). Regulation of GDNFR and
Ret by En-1 and En-2 is possible, but
unlikely to be the cause for the loss of midbrain dopaminergic neurons
in engrailed double mutants, because a reduction in this population is
not apparent in mice with a targeted deletion of either GDNF
or Ret (Schuchardt et al., 1994; Moore et al., 1996).
Compensatory mechanisms of En-1
and En-2
In contrast to the complete loss of SN and VT dopaminergic neurons
in engrailed double mutants, mice null for either En-1 or
En-2 have no apparent reductions in these populations. This finding indicates that En-1 and En-2 compensate
for the deletion of each other in maintaining the differentiation and
survival of these neurons. Although En-2 can functionally
replace En-1 when knocked-in to the En-1 locus
(Hanks et al., 1998), they differ substantially in their compensatory
abilities to maintain ventral midbrain dopaminergic neurons; whereas a
single allele of En-1 is sufficient to maintain a wild
type-like phenotype, a single allele of En-2 is not. This
difference may be attributable to En-1 expression beginning
before En-2 (Davidson et al., 1988; Davis and Joyner, 1988;
Davis et al., 1988; McMahon et al., 1992). More likely though, it is
attributable to our finding that during development and in the adult,
En-2 is expressed at high levels in only a small proportion
of SN and VT neurons, whereas En-1 is expressed at high
levels in most or all of them. We suggest that the small proportion of
SN and VT neurons that normally express high levels of En-2
are those that survive in the
En-1//En-2+/
mice, whereas no loss is apparent in
En-1+//En-2/
mice because essentially all of these neurons highly express En-1.
Potential effects on induction of midbrain
dopaminergic neurons
Progenitor cells in the neuroepithelium of the ventral midbrain
are induced to produce dopaminergic neurons by the combined action of
sonic hedgehog (SHH) released by the floorplate at the ventral midline
and FGF8 produced by anterior forebrain and the isthmus, a strip of
tissue at the midbrain-hindbrain junction (Ye et al., 1998). The
population of ventral midbrain dopaminergic neurons observed at E12 was
reduced in every engrailed double mutant analyzed, compared with their
littermates: both
En-1+/+/En-2/
embryos, which have a wild-type-like phenotype, and
En-1/+/En-2/
embryos, which have an intermediate phenotype. One potential explanation for the reduced population at E12 in the engrailed double
mutants is a temporal overlap in the loss and generation of TH-positive
neurons. Alternatively, levels of SHH or FGF8 are reduced in engrailed
double mutants, and fewer progenitor cells respond to these lower
concentrations to produce dopaminergic neurons. Because the isthmus is
absent in engrailed double mutants (present study), a lower level of
FGF8 might be expected. However, the isthmus is also absent in
En-1 mutants (Wurst et al., 1994), but the number of
midbrain dopaminergic neurons appears unaffected. A third possibility
is that engrailed double mutants have a reduced number of progenitor
cells capable of producing dopaminergic neurons. Consistent with this
possibility is our finding that the En-1/tau-LacZ expression
domain, which includes the ventral midbrain neuroepithelium in which
these progenitors are located, is substantially reduced in engrailed
double mutants (Fig. 3A,B).
Engrailed and Parkinson's disease
Degeneration of SN and VT neurons and the loss of their
dopaminergic innervation of the forebrain is the primary cause of the
severe movement disorders associated with PD (Jenner et al., 1992;
Temlett, 1996). PD, which affects 1-2% of humans during their
lifetime (Polymeropoulos et al., 1996), can be caused by genetic and
epigenetic factors (Tomac et al., 1995). A link between engrailed and
PD is suggested by our findings that SN and VT dopaminergic neurons
require En-1 and En-2 for their survival, that
En-1 and En-2 continue to be expressed by these
neurons in the adult, and that expression of -synuclein,
a gene on human chromosome 4 genetically linked to PD in some families
(Polymeropoulos et al., 1997), is dependent on engrailed. It is
unlikely, though, that both En-1 and En-2 are
mutated in a given individual, especially because they are located on
different chromosomes (human chromosomes 2 and 7, respectively) (Logan
et al., 1989). However, because En-1 is expressed at high
levels throughout the SN and VT in adult mice, whereas En-2
is expressed at high levels in only a small proportion of SN and VT
neurons, a mutation in En-1 may be sufficient to promote the
onset of PD. A mutation of En-1 would not necessarily affect
the development of midbrain dopaminergic neurons, because as shown
here, they appear unaffected even in En-1 null mice, indicating that during development En-2 compensates for the
lack of En-1. However, an age-related decrease in the
ability of En-2 to compensate for either the loss of
En-1 or a change in its amount or efficacy caused by a
mutation in the regulatory or coding sequences for En-1 may
lead to a gradual degeneration of dopaminergic neurons in the SN and VT
as occurs in PD. This scenario is consistent with our finding of an
En-2 gene dose effect, which indicates that the number of
dopaminergic neurons that survive in the absence of En-1
appears to depend on the level of En-2 expression.
Another potential link with PD is our finding suggesting that engrailed
regulates -synuclein expression. Transgenic mice engineered to overproduce -synuclein, a presynaptic and nuclear protein believed to be involved in synaptic plasticity, exhibit a loss
of midbrain dopaminergic neurons (Masliah et al., 2000). It is
conceivable that an alteration in the binding sequence of the
-synuclein gene for En-1 or En-2 or a mutation in either engrailed gene could lead to an increase of -synuclein, which may in
turn lead to the degeneration of dopaminergic neurons in the ventral midbrain.
| |
FOOTNOTES |
Received Dec. 8, 2000; revised Feb. 12, 2001; accepted Feb. 23, 2001.
This work was supported by National Institutes of Health Grants R01
NS31558 (D.D.M.O.) and R01 NS37075 (M.D.G.), Eu Biotech Grant PL960146
(W.W.), Human Frontier Science Program (HFSP) Grant RG-83/96
(W.W.), and fellowships NIH F32 NS10284 (H.H.S.), HFSP LT-526/94
(H.H.S.), and Deutsche Forschungsgemeinschaft SA652/2-1 (H.S.). We are grateful to A. Joyner for providing breeding
pairs of En-1 and En-2 deficient mice,
En-1 and En-2 cDNA, and the Enhb antibody. We thank J.-P. Pinaud for technical assistance and R. Dyck
and V. Blanquet for helpful discussions.
Correspondence should be addressed to Dennis D. M. O'Leary,
Molecular Neurobiology Laboratory, The Salk Institute, 10010 N. Torrey
Pines Road, La Jolla, CA 92037. E-mail:
doleary{at}salk.edu.
| |
REFERENCES |
-
Avantaggiato V,
Dathan NA,
Grieco M,
Fabien N,
Lazzaro D,
Fusco A,
Simeone A,
Santoro M
(1994)
Developmental expression of the RET protooncogene.
Cell Growth Differ
5:305-311[Abstract].
-
Baloh RH,
Tansey MG,
Golden JP,
Creedon DJ,
Heuckeroth RO,
Keck CL,
Zimonjic DB,
Popescu NC,
Johnson Jr EM,
Milbrandt J
(1997)
TrnR2, a novel receptor that mediates neurturin and GDNF signaling through Ret.
Neuron
18:793-802[Web of Science][Medline].
-
Beck KD,
Valverde J,
Alexi T,
Poulsen K,
Moffat B,
Vandlen RA,
Rosenthal A,
Hefti F
(1995)
Mesencephalic dopaminergic neurons protected by GDNF from axotomy-induced degeneration in the adult brain.
Nature
373:339-341[Medline].
-
Callahan CA,
Thomas JB
(1994)
Tau-beta-galactosidase, an axon-targeted fusion protein.
Proc Natl Acad Sci USA
91:5972-5976[Abstract/Free Full Text].
-
Castillo SO,
Baffi JD,
Palkovits M,
Goldstein DS,
Kopin IJ,
Witta J,
Magnuson MA,
Nikodem VM
(1998)
Dopamine biosynthesis is selectively abolished in substantia nigra/ventral tegmental area but not in hypothalamic neurons in mice with targeted disruption of the Nurr1 gene.
Mol Cell Neurosci
11:36-46[Web of Science][Medline].
-
Compton MM
(1992)
A biochemical hallmark of apoptosis: internucleosomal degradation of the genome.
Cancer Metastasis Rev
11:105-119[Web of Science][Medline].
-
Condron BG,
Patel NH,
Zinn K
(1994)
Engrailed controls glial/neuronal cell fate decisions at the midline of the central nervous system.
Neuron
13:541-554[Web of Science][Medline].
-
Davidson D,
Graham E,
Sime C,
Hill R
(1988)
A gene with sequence similarity to Drosophila engrailed is expressed during the development of the neural tube and vertebrae in the mouse.
Development
104:305-316[Abstract].
-
Davies A,
Lumsden A
(1984)
Relation of target encounter and neuronal death to nerve growth factor responsiveness in the developing mouse trigeminal ganglion.
J Comp Neurol
223:124-137[Web of Science][Medline].
-
Davis CA,
Joyner AL
(1988)
Expression patterns of the homeo box-containing genes En-1 and En-2 and the proto-oncogene int-1 diverge during mouse development.
Genes Dev
2:1736-1744[Abstract/Free Full Text].
-
Davis CA,
Noble-Topham SE,
Rossant J,
Joyner AL
(1988)
Expression of the homeo box-containing gene En-2 delineates a specific region of the developing mouse brain.
Genes Dev
2:361-371[Abstract/Free Full Text].
-
Friedman GC,
O'Leary DDM
(1996)
Retroviral misexpression of engrailed genes in the chick optic tectum perturbs the topographic targeting of retinal axons.
J Neurosci
16:5498-5509[Abstract/Free Full Text].
-
Gardner CA,
Darnell DK,
Poole SJ,
Ordahl CP,
Barald KF
(1988)
Expression of an engrailed-like gene during development of the early embryonic chick nervous system.
J Neurosci Res
21:426-437[Web of Science][Medline].
-
German DC,
Manaye KF
(1993)
Midbrain dopaminergic neurons (nuclei A8, A9, and A10): three-dimensional reconstruction in the rat.
J Comp Neurol
331:297-309[Web of Science][Medline].
-
Goulding M,
Lumsden A,
Paquette AJ
(1994)
Regulation of Pax-3 expression in the dermomyotome and its role in muscle development.
Development
120:957-971[Abstract].
-
Hanks M,
Wurst W,
Anson-Cartwright L,
Auerbach AB,
Joyner AL
(1995)
Rescue of the En-1 mutant phenotype by replacement of En-1 with En-2.
Science
269:679-682[Abstract/Free Full Text].
-
Hanks MC,
Loomis CA,
Harris E,
Tong CX,
Anson-Cartwright L,
Auerbach A,
Joyner A
(1998)
Drosophila engrailed can substitute for mouse Engrailed1 function in mid-hindbrain, but not limb development.
Development
125:4521-4530[Abstract].
-
Itasaki N,
Nakamura H
(1996)
A role for gradient en expression in positional specification on the optic tectum.
Neuron
16:55-62[Web of Science][Medline].
-
Jenner P,
Schapira AH,
Marsden CD
(1992)
New insights into the cause of Parkinson's disease.
Neurology
42:2241-2250[Abstract/Free Full Text].
-
Jing S,
Wen D,
Yu Y,
Holst PL,
Luo Y,
Fang M,
Tamir R,
Antonio L,
Hu Z,
Cupples R,
Louis JC,
Hu S,
Altrock BW,
Fox GM
(1996)
GDNF-induced activation of the ret protein tyrosine kinase is mediated by GDNFR-alpha, a novel receptor for GDNF.
Cell
85:1113-1124[Web of Science][Medline].
-
Joyner AL,
Herrup K,
Auerbach BA,
Davis CA,
Rossant J
(1991)
Subtle cerebellar phenotype in mice homozygous for a targeted deletion of the En-2 homeobox.
Science
251:1239-1243[Abstract/Free Full Text].
-
Kornberg T
(1981a)
Engrailed: a gene controlling compartment and segment formation in Drosophila.
Proc Natl Acad Sci USA
78:1095-1099[Abstract/Free Full Text].
-
Kornberg T
(1981b)
Compartments in the abdomen of Drosophila and the role of the engrailed locus.
Dev Biol
86:363-372[Web of Science][Medline].
-
Kruger R,
Kuhn W,
Muller T,
Woitalla D,
Graeber M,
Kosel S,
Przuntek H,
Epplen JT,
Schols L,
Riess O
(1998)
Ala30Pro mutation in the gene encoding alpha-synuclein in Parkinson's disease.
Nat Genet
18:106-108[Web of Science][Medline].
-
Lieb K,
Andersen C,
Lazarov N,
Zienecker R,
Urban I,
Reisert I,
Pilgrim C
(1996)
Pre- and postnatal development of dopaminergic neuron numbers in the male and female mouse midbrain.
Dev Brain Res
84:37-43.
-
Lin LF,
Doherty DH,
Lile JD,
Bektesh S,
Collins F
(1993)
GDNF: a glial cell line-derived neurotrophic factor for midbrain dopaminergic neurons.
Science
260:1130-1132[Abstract/Free Full Text].
-
Logan C,
Willard HF,
Rommens JM,
Joyner AL
(1989)
Chromosomal localization of the human homeo box-containing genes, EN1 and EN2.
Genomics
4:206-209[Web of Science][Medline].
-
Logan C,
Wizenmann A,
Drescher U,
Monschau B,
Bonhoeffer F,
Lumsden A
(1996)
Rostral optic tectum acquires caudal characteristics following ectopic engrailed expression.
Curr Biol
6:1006-1014[Web of Science][Medline].
-
Lundell MJ,
Chu-LaGraff Q,
Doe CQ,
Hirsh J
(1996)
The engrailed and huckebein genes are essential for development of serotonin neurons in the Drosophila CNS.
Mol Cell Neurosci
7:46-61[Medline].
-
Maroteaux L,
Scheller RH
(1991)
The rat brain synucleins; family of proteins transiently associated with neuronal membrane.
Brain Res Mol Brain Res
11:335-343[Medline].
-
Maroteaux L,
Campanelli JT,
Scheller RH
(1988)
Synuclein: a neuron-specific protein localized to the nucleus and presynaptic nerve terminal.
J Neurosci
8:2804-2815[Abstract].
-
Masliah E,
Rockenstein E,
Veinbergs I,
Mallory M,
Hashimoto M,
Takeda A,
Sagara Y,
Sisk A,
Mucke L
(2000)
Dopaminergic loss and inclusion body formation in alpha-synuclein mice: implications for neurodegenerative disorders.
Science
287:1265-1269[Abstract/Free Full Text].
-
McMahon AP,
Joyner AL,
Bradley A,
McMahon JA
(1992)
The midbrain-hindbrain phenotype of Wnt-1-/Wnt-1- mice results from stepwise deletion of engrailed-expressing cells by 9.5 days postcoitum.
Cell
69:581-595[Web of Science][Medline].
-
Millen KJ,
Wurst W,
Herrup K,
Joyner AL
(1994)
Abnormal embryonic cerebellar development and patterning of postnatal foliation in two mouse Engrailed-2 mutants.
Development
120:695-706[Abstract].
-
Mombaerts P,
Wang F,
Dulac C,
Chao SK,
Nemes A,
Mendelsohn M,
Edmondson J,
Axel R
(1996)
Visualizing an olfactory sensory map.
Cell
87:675-686[Web of Science][Medline].
-
Moore MW,
Klein RD,
Farinas I,
Sauer H,
Armanini M,
Phillips H,
Reichardt LF,
Ryan AM,
Carver-Moore K,
Rosenthal A
(1996)
Renal and neuronal abnormalities in mice lacking GDNF.
Nature
382:76-79[Medline].
-
Nosrat CA,
Tomac A,
Hoffer BJ,
Olson L
(1997)
Cellular and developmental patterns of expression of Ret and glial cell line-derived neurotrophic factor receptor alpha mRNAs.
Exp Brain Res
115:410-422[Web of Science][Medline].
-
Paxinos G,
Ashwell KWS,
Törk I
(1994)
In: Atlas of the developing rat nervous system, Ed 2. London: Academic.
-
Pfaff SL,
Mendelsohn M,
Stewart CL,
Edlund T,
Jessell TM
(1996)
Requirement for LIM homeobox gene Isl1 in motor neuron generation reveals a motor neuron-dependent step in interneuron differentiation.
Cell
84:309-320[Web of Science][Medline].
-
Polymeropoulos MH,
Higgins JJ,
Golbe LI,
Johnson WG,
Ide SE,
Di Iorio G,
Sanges G,
Stenroos ES,
Pho LT,
Schaffer AA,
Lazzarini AM,
Nussbaum RL,
Duvoisin RC
(1996)
Mapping of a gene for Parkinson's disease to chromosome 4q21-q23.
Science
274:1197-1199[Abstract/Free Full Text].
-
Polymeropoulos MH,
Lavedan C,
Leroy E,
Ide SE,
Dehejia A,
Dutra A,
Pike B,
Root H,
Rubenstein J,
Boyer R,
Stenroos ES,
Chandrasekharappa S,
Athanassiadou A,
Papapetropoulos T,
Johnson WG,
Lazzarini AM,
Duvoisin RC,
Di Iorio G,
Golbe LI,
Nussbaum RL
(1997)
Mutation in the alpha-synuclein gene identified in families with Parkinson's disease.
Science
276:2045-2047[Abstract/Free Full Text].
-
Poole SJ,
Kornberg TB
(1988)
Modifying expression of the engrailed gene of Drosophila melanogaster.
Development [Suppl]
104:85-93.
-
Sakurada K,
Ohshima-Sakurada M,
Palmer TD,
Gage FH
(1999)
Nurr1, an orphan nuclear receptor, is a transcriptional activator of endogenous tyrosine hydroxylase in neural progenitor cells derived from the adult brain.
Development
126:4017-4026[Abstract].
-
Saueressig H,
Burrill J,
Goulding M
(1999)
Engrailed-1 and netrin-1 regulate axon pathfinding by association interneurons that project to motor neurons.
Development
126:4201-4212[Abstract].
-
Schuchardt A,
D'Agati V,
Larsson-Blomberg L,
Costantini F,
Pachnis V
(1994)
Defects in the kidney and enteric nervous system of mice lacking the tyrosine kinase receptor Ret.
Nature
367:380-383[Medline].
-
Sechrist J,
Bronner-Fraser M
(1991)
Birth and differentiation of reticular neurons in the chick hindbrain: ontogeny of the first neuronal population.
Neuron
7:947-963[Medline].
-
Simon H,
Guthrie S,
Lumsden A
(1994)
Regulation of SC1/DM-GRASP during the migration of motor neurons in the chick embryo brain stem.
J Neurobiol
25:1129-1143[Web of Science][Medline].
-
Simon H,
Hornbruch A,
Lumsden A
(1995)
Independent assignment of antero-posterior and dorso-ventral positional values in the developing chick hindbrain.
Curr Biol
5:205-214[Web of Science][Medline].
-
Tanabe Y,
Jessell TM
(1996)
Diversity and pattern in the developing spinal cord.
Science
274:1115-1123[Abstract/Free Full Text].
-
Temlett JA
(1996)
Parkinson's disease: biology and aetiology.
Curr Opin Neurol
9:303-307[Medline].
-
Tomac A,
Lindqvist E,
Lin LF,
Ogren SO,
Young D,
Hoffer BJ,
Olson L
(1995)
Protection and repair of the nigrostriatal dopaminergic system by GDNF in vivo.
Nature
373:335-339[Medline].
-
Voorn P,
Kalsbeek A,
Jorritsma-Byham B,
Groenewegen HJ
(1988)
The pre- and postnatal development of the dopaminergic cell groups in the ventral mesencephalon and the dopaminergic innervation of the striatum of the rat.
Neuroscience
25:857-887[Web of Science][Medline].
-
Wurst W,
Auerbach AB,
Joyner AL
(1994)
Multiple developmental defects in Engrailed-1 mutant mice: an early mid-hindbrain deletion and patterning defects in forelimbs and sternum.
Development
120:2065-2075[Abstract].
-
Ye W,
Shimamura K,
Rubenstein JL,
Hynes MA,
Rosenthal A
(1998)
FGF and Shh signals control dopaminergic and serotonergic cell fate in the anterior neural plate.
Cell
93:755-766[Web of Science][Medline].
-
Zetterström RH,
Solomin L,
Jansson L,
Hoffer BJ,
Olson L,
Perlmann T
(1997)
Dopamine neuron agenesis in Nurr1-deficient mice.
Science
276:248-250[Abstract/Free Full Text].
Copyright © 2001 Society for Neuroscience 0270-6474/01/2193126-09$05.00/0
This article has been cited by other articles:
|
|
|
|
 
S. Friling, E. Andersson, L. H. Thompson, M. E. Jonsson, J. B. Hebsgaard, E. Nanou, Z. Alekseenko, U. Marklund, S. Kjellander, N. Volakakis, et al.
Efficient production of mesencephalic dopamine neurons by Lmx1a expression in embryonic stem cells
PNAS,
May 5, 2009;
106(18):
7613 - 7618.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
M. Yin, S. Liu, Y. Yin, S. Li, Z. Li, X. Wu, B. Zhang, S.-L. Ang, Y. Ding, and J. Zhou
Ventral Mesencephalon-Enriched Genes That Regulate the Development of Dopaminergic Neurons In Vivo
J. Neurosci.,
April 22, 2009;
29(16):
5170 - 5182.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
R. V. Sillitoe, D. Stephen, Z. Lao, and A. L. Joyner
Engrailed Homeobox Genes Determine the Organization of Purkinje Cell Sagittal Stripe Gene Expression in the Adult Cerebellum
J. Neurosci.,
November 19, 2008;
28(47):
12150 - 12162.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
B. Mugat, M.-L. Parmentier, N. Bonneaud, H. Y. E. Chan, and F. Maschat
Protective role of Engrailed in a Drosophila model of Huntington's disease
Hum. Mol. Genet.,
November 15, 2008;
17(22):
3601 - 3616.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
S. Chiba, Y. M. Lee, W. Zhou, and C. R. Freed
Noggin Enhances Dopamine Neuron Production from Human Embryonic Stem Cells and Improves Behavioral Outcome After Transplantation into Parkinsonian Rats
Stem Cells,
November 1, 2008;
26(11):
2810 - 2820.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
D. Omodei, D. Acampora, P. Mancuso, N. Prakash, L. G. Di Giovannantonio, W. Wurst, and A. Simeone
Anterior-posterior graded response to Otx2 controls proliferation and differentiation of dopaminergic progenitors in the ventral mesencephalon
Development,
October 15, 2008;
135(20):
3459 - 3470.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
R. V. Sillitoe and M. W. Vogel
Desire, Disease, and the Origins of the Dopaminergic System
Schizophr Bull,
March 1, 2008;
34(2):
212 - 219.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
A. L. M. Ferri, W. Lin, Y. E. Mavromatakis, J. C. Wang, H. Sasaki, J. A. Whitsett, and S.-L. Ang
Foxa1 and Foxa2 regulate multiple phases of midbrain dopaminergic neuron development in a dosage-dependent manner
Development,
August 1, 2007;
134(15):
2761 - 2769.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
D. Gherbassi, L. Bhatt, S. Thuret, and H. H. Simon
Merging Mouse Transcriptome Analyses with Parkinson's Disease Linkage Studies
DNA Res,
May 23, 2007;
(2007)
dsm007v1.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
J. A. Rodriguez-Gomez, J.-Q. Lu, I. Velasco, S. Rivera, S. S. Zoghbi, J.-S. Liow, J. L. Musachio, F. T. Chin, H. Toyama, J. Seidel, et al.
Persistent Dopamine Functions of Neurons Derived from Embryonic Stem Cells in a Rodent Model of Parkinson Disease
Stem Cells,
April 1, 2007;
25(4):
918 - 928.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
F. S. Goes, P. P. Zandi, K. Miao, F. J. McMahon, J. Steele, V. L. Willour, D. F. MacKinnon, F. M. Mondimore, B. Schweizer, J. I. Nurnberger Jr., et al.
Mood-Incongruent Psychotic Features in Bipolar Disorder: Familial Aggregation and Suggestive Linkage to 2p11-q14 and 13q21-33
Am J Psychiatry,
February 1, 2007;
164(2):
236 - 247.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
M. Geraerts, O. Krylyshkina, Z. Debyser, and V. Baekelandt
Concise Review: Therapeutic Strategies for Parkinson Disease Based on the Modulation of Adult Neurogenesis
Stem Cells,
February 1, 2007;
25(2):
263 - 270.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
L. Sonnier, G. Le Pen, A. Hartmann, J.-C. Bizot, F. Trovero, M.-O. Krebs, and A. Prochiantz
Progressive Loss of Dopaminergic Neurons in the Ventral Midbrain of Adult Mice Heterozygote for Engrailed1
J. Neurosci.,
January 31, 2007;
27(5):
1063 - 1071.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
P. Sgado, L. Alberi, D. Gherbassi, S. L. Galasso, G. M. J. Ramakers, K. N. Alavian, M. P. Smidt, R. H. Dyck, and H. H. Simon
Slow progressive degeneration of nigral dopaminergic neurons in postnatal Engrailed mutant mice
PNAS,
October 10, 2006;
103(41):
15242 - 15247.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
S.-L. Ang
Transcriptional control of midbrain dopaminergic neuron development
Development,
September 15, 2006;
133(18):
3499 - 3506.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
N. Prakash and W. Wurst
Genetic networks controlling the development of midbrain dopaminergic neurons
J. Physiol.,
September 1, 2006;
575(2):
403 - 410.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
Y. Luo, C. Schwartz, S. Shin, X. Zeng, N. Chen, Y. Wang, X. Yu, and M. S. Rao
A Focused Microarray to Assess Dopaminergic and Glial Cell Differentiation from Fetal Tissue or Embryonic Stem Cells
Stem Cells,
April 1, 2006;
24(4):
865 - 875.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
H. Fukuda, J. Takahashi, K. Watanabe, H. Hayashi, A. Morizane, M. Koyanagi, Y. Sasai, and N. Hashimoto
Fluorescence-Activated Cell Sorting-Based Purification of Embryonic Stem Cell-Derived Neural Precursors Averts Tumor Formation after Transplantation
Stem Cells,
March 1, 2006;
24(3):
763 - 771.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
C. Martinat, J.-J. Bacci, T. Leete, J. Kim, W. B. Vanti, A. H. Newman, J. H. Cha, U. Gether, H. Wang, and A. Abeliovich
Cooperative transcription activation by Nurr1 and Pitx3 induces embryonic stem cell maturation to the midbrain dopamine neuron phenotype
PNAS,
February 21, 2006;
103(8):
2874 - 2879.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
E. Andersson, J. B. Jensen, M. Parmar, F. Guillemot, and A. Bjorklund
Development of the mesencephalic dopaminergic neuron system is compromised in the absence of neurogenin 2
Development,
February 1, 2006;
133(3):
507 - 516.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
N. Prakash, C. Brodski, T. Naserke, E. Puelles, R. Gogoi, A. Hall, M. Panhuysen, D. Echevarria, L. Sussel, D. M. V. Weisenhorn, et al.
A Wnt1-regulated genetic network controls the identity and fate of midbrain-dopaminergic progenitors in vivo
Development,
January 1, 2006;
133(1):
89 - 98.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
R. M. Miller and H. J. Federoff
Altered Gene Expression Profiles Reveal Similarities and Differences Between Parkinson Disease and Model Systems
Neuroscientist,
December 1, 2005;
11(6):
539 - 549.
[Abstract]
[PDF]
|
|
|
|
|
|
|
Y. Yan, D. Yang, E. D. Zarnowska, Z. Du, B. Werbel, C. Valliere, R. A. Pearce, J. A. Thomson, and S.-C. Zhang
Directed Differentiation of Dopaminergic Neuronal Subtypes from Human Embryonic Stem Cells
Stem Cells,
June 1, 2005;
23(6):
781 - 790.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
T. C. Schulz, S. A. Noggle, G. M. Palmarini, D. A. Weiler, I. G. Lyons, K. A. Pensa, A. C.B. Meedeniya, B. P. Davidson, N. A. Lambert, and B. G. Condie
Differentiation of Human Embryonic Stem Cells to Dopaminergic Neurons in Serum-Free Suspension Culture
Stem Cells,
December 1, 2004;
22(7):
1218 - 1238.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
A. L. Perrier, V. Tabar, T. Barberi, M. E. Rubio, J. Bruses, N. Topf, N. L. Harrison, and L. Studer
From the Cover: Derivation of midbrain dopamine neurons from human embryonic stem cells
PNAS,
August 24, 2004;
101(34):
12543 - 12548.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
L. Alberi, P. Sgado, and H. H. Simon
Engrailed genes are cell-autonomously required to prevent apoptosis in mesencephalic dopaminergic neurons
Development,
July 1, 2004;
131(13):
3229 - 3236.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
M. P. Smidt, S. M. Smits, H. Bouwmeester, F. P. T. Hamers, A. J. A. van der Linden, A. J. C. G. M. Hellemons, J. Graw, and J. P. H. Burbach
Early developmental failure of substantia nigra dopamine neurons in mice lacking the homeodomain gene Pitx3
Development,
March 1, 2004;
131(5):
1145 - 1155.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
J.-W. Shim, H.-C. Koh, M.-Y. Chang, E. Roh, C.-Y. Choi, Y. J. Oh, H. Son, Y.-S. Lee, L. Studer, and S.-H. Lee
Enhanced In Vitro Midbrain Dopamine Neuron Differentiation, Dopaminergic Function, Neurite Outgrowth, and 1-Methyl-4-Phenylpyridium Resistance in Mouse Embryonic Stem Cells Overexpressing Bcl-XL
J. Neurosci.,
January 28, 2004;
24(4):
843 - 852.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
B. Marie and J. M. Blagburn
Differential Roles of Engrailed Paralogs in Determining Sensory Axon Guidance and Synaptic Target Recognition
J. Neurosci.,
August 27, 2003;
23(21):
7854 - 7862.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
J. Holzschuh, G. Hauptmann, and W. Driever
Genetic Analysis of the Roles of Hh, FGF8, and Nodal Signaling during Catecholaminergic System Development in the Zebrafish Brain
J. Neurosci.,
July 2, 2003;
23(13):
5507 - 5519.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
C. Brodski, D. M. V. Weisenhorn, M. Signore, I. Sillaber, M. Oesterheld, V. Broccoli, D. Acampora, A. Simeone, and W. Wurst
Location and Size of Dopaminergic and Serotonergic Cell Populations Are Controlled by the Position of the Midbrain-Hindbrain Organizer
J. Neurosci.,
May 15, 2003;
23(10):
4199 - 4207.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
P. J. Solano, B. Mugat, D. Martin, F. Girard, J.-M. Huibant, C. Ferraz, B. Jacq, J. Demaille, and F. Maschat
Genome-wide identification of in vivo Drosophila Engrailed-binding DNA fragments and related target genes
Development,
April 1, 2003;
130(7):
1243 - 1254.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
S. Agarwala and C. W. Ragsdale
A role for midbrain arcs in nucleogenesis
Development,
March 14, 2003;
129(24):
5779 - 5788.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
B. Marie, L. Cruz-Orengo, and J. M. Blagburn
Persistent engrailed Expression Is Required to Determine Sensory Axon Trajectory, Branching, and Target Choice
J. Neurosci.,
February 1, 2002;
22(3):
832 - 841.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
TOP
ABSTRACT
INTRODUCTION
