1 Department of Biotechnology and Biosciences, University of Milano-Bicocca,
Piazza della Scienza 2, 20126 Milano, Italy
2 Department of Pharmacology, Chemotherapy and Medical Toxicology, University of
Milano, via Vanvitelli 32, 20129 Milano, Italy
3 Cancer Biology and Genetics Program, and Department of Pathology, Memorial
Sloan-Kettering Cancer Center, 1275 York Avenue, Box 110, New York, NY 10021,
USA
4 Department of Neuroscience, `Mario Negri' Institute of Pharmacological
Research, via Eritrea 62, 20157 Milano, Italy
5 Department of Biomolecular Sciences and Biotechnology, University of Milano,
via Celoria 26, 20133 Milano, Italy
* Author for correspondence (e-mail: silvia.nicolis{at}unimib.it)
Accepted 2 April 2004
![]() |
SUMMARY |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: Neural stem cells, Nervous system, Mouse, Sox2, Transcription factors, Neurogenesis, Hippocampal precursors, Neurodegeneration, Neuronal inclusions
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
To investigate the role of Sox2 in the nervous system, we
generated different mouse mutant alleles: a null allele
(Sox2ß-geo `knock-in')
(Zappone et al., 2000;
Avilion et al., 2003
), and a
regulatory mutant allele (Sox2
ENH), in which a
neural cell-specific enhancer (Zappone et
al., 2000
) is deleted. Compound
Sox2ß-geo/
ENH heterozygotes are born with
important cerebral malformations and neural cell pathology. In addition to
proliferative defects of adult stem/progenitor cells, our results unexpectedly
reveal a crucial role for Sox2 in neuronal maintenance in selected
brain areas.
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
ß-Geo expression in tissues
X-gal staining was performed as described
(Zappone et al., 2000).
Fresh-frozen embryos or dissected brains were sectioned at 20-40 µm, air
dried, postfixed for 30 minutes in 1% glutaraldehyde in PBS, washed in PBS and
stained according to Zappone et al.
(Zappone et al., 2000
) for 2-4
hours. Sections were then dehydrated through an ethanol series and
mounted.
In vitro neurosphere cultures, immunocytochemistry and RT-PCR
Neurosphere cultures were grown in vitro from adult brain lateral
ventricles in the presence of G418 for 5-12 passages (about 5-12 weeks), as
previously described (Zappone et al.,
2000). Individual G418-resistant neurospheres were picked from
low-density cultures (to ensure clonal origin), dissociated to single cells,
and allowed to grow secondary neurospheres (in G418). Individual neurospheres
were again picked, briefly expanded as clonal cultures, allowed to
differentiate, then probed with antibodies against ß-tubulin 3 (neurones)
(BABCO mouse monoclonal, 1:500) and GFAP (astroglia) (Diasorin, rabbit
polyclonal, 1:5), or GalC (oligodendroglia) (Chemicon mouse monoclonal,
1:100), followed by detection with FITC or TRITC-conjugated Chemicon secondary
antibodies (1:100). For X-gal staining, neurospheres were fixed
(Zappone et al., 2000
) for 10
minutes and stained for 2 hours.
RT-PCR for Sox2 (and HPRT control) was performed according to
Zappone et al. (Zappone et al.,
2000), on DNAse-treated total RNA. Samples obtained after 20
cycles were analyzed by Southern blotting with radioactive probes to obtain a
quantitative estimate in the exponential phase of amplification. Images were
recorded with a Typhoon 8600 (Molecular Dynamics-Amersham Biosciences)
apparatus and analyzed with ImageQuant (Molecular Dynamics) software.
Histology, immunohistochemistry and electron microscopy
For morphological/histological adult brain studies, animals were perfused
with 4% paraformaldehyde (PFA) in PBS. Brains were dissected, postfixed
overnight in the same fixative, cryoprotected in 30% sucrose, frozen and
sectioned at 40 µm. Sections were stained with thionine, quickly dehydrated
through an ethanol series and mounted. Analysis (coronal sections) was
performed by complete coronal sectioning of 10 mutant (8
Sox2ß-geo/ENHneo and 2
Sox2ß-geo/
ENH
neo) and four wild-type
littermates brains. The abnormalities reported were consistently observed in
all mutants. For comparisons at E14.5, a total of six mutant and four
wild-type embryos were analyzed. The same analyses were carried out on
Sox2ß-geo/+ heterozygotes and on
Sox2
ENH/
ENH homozygotes, and no significant
difference with respect to wild type was observed, except for a mild ventricle
enlargement in the occasional Sox2ß-geo/+ mouse.
For immunohistochemistry of intraneuronal aggregates in adult brain, mice (6-months old) were perfused with 4% PFA in 0.1 M phosphate buffer (PB; pH 7.4), brains were postfixed for 12 hours to 2 days, dehydrated in ethanols and embedded in paraffin wax. Dewaxed and rehydrated 10 µm sections were blocked with 1% BSA in PBS for 30 minutes, incubated for 48 hours at 4°C in primary antibodies, followed by the appropriate biotinylated secondary antibodies and developed with a standard Vector ABC kit. Four mutant and four wild-type littermates brains were analyzed; all mutants (and no wild-type) showed the alterations described. Primary antibodies were: SMI 32 (non-phosphorylated neurofilaments) and SMI 31 (phosphorylated neurofilaments) (Sterberger monoclonals, both 1:1000), anti-ubiquitin (Chemicon monoclonal, 1:1000). Immunolabelled sections were lightly counterstained with thionine.
For histology on semithin sections and electron microscopy, mice were perfused with 2.5% glutaraldehyde and 0.5% paraformaldehyde, and brains were postfixed in the same fixative overnight. Coronal vibratome sections (80 µm) were osmicated and flat-embedded in Epon-Spurr. Semithin (1 µm) sections were stained with Toluidine Blue for light microscopy, and adjacent thin sections collected on grids were counterstained with uranyl acetate and lead citrate and viewed in a Jeol electron microscope.
For SOX2 immunodetection, paraffin sections were prepared as above and pre-treated for antigen unmasking by bringing to the boil (in a microwave oven) in citrate buffer, then incubated with a rabbit polyclonal anti-SOX2 antibody (Chemicon, 1:500) for 24-48 hours at room temperature. Incubation with a biotinylated secondary antibody (VECTOR, 1:200) was then performed, followed by revelation with the VECTASTAIN ABC system (VECTOR) using DAB as the chromogen. For SOX2/BrdU double immunofluorescence, 30 µm cryosections (prepared as above, morphological studies) were microwaved for antigen unmasking (see above), then pre-treated for BrdU immunodetection as described below, and then incubated with a mixture of rabbit anti-SOX2 (see above) and rat anti-BrdU (see below) primary antibodies (24-36 hours), followed by fluorescent Rhodamine-X anti-rat (see below) and FITC anti-rabbit (Jackson, 1:200) secondary antibodies. For SOX2/GFAP and SOX2/PSA-NCAM double immunofluorescence, cryosections (as for SOX2/BrdU) were pre-treated for antigen unmasking as above, and incubated with a mixture of rabbit anti-SOX2 and either mouse monoclonal anti-GFAP antibody (Boehringer, 1:200) or mouse anti-PSA-NCAM monoclonal antibody (AbCys S.A., Paris, 1:800). For SOX2/GFAP detection, this was followed by FITC anti-rabbit and Rhodamine-X anti-mouse (both Jackson, 1:200) secondary antibodies. For SOX2/PSA-NCAM detection, secondary antibodies were Rhodamine-X anti-rabbit (Jackson, 1:200) and Cy2 anti-mouse IgM (Jackson, 1:200).
For nestin and nestin/GFAP double immunofluorescence, cryosections (30 µm) were prepared as above (no unmasking) and incubated with an anti-nestin antiserum (nestin 130, a gift from R. McKay, 1:50) and (for the double immunofluorescence) a monoclonal anti-GFAP antibody (Boehringer, 1:200). For the single GFAP staining using DAB reaction, the same anti-GFAP antibody was used, at a dilution of 1:1000.
Appropriate controls for all antibodies used ruled out non-specific reactions by secondary antibodies, or cross-reactions between antibodies used simultaneously. SOX2 immunoreactivity was quantitated in DAB-stained sections by densitometry of equivalent areas of the periventricular region (embryo); for adult sections, the average staining of the populations of Sox2-positive cells was obtained by individually scanning all visible cells in a field and subtracting the average background staining. Densitometric analysis was carried out using ImageQuant (Molecular Dynamics) software.
In situ hybridization
A Sox2 probe was transcribed from an AccI-XbaI
750 nucleotide fragment (Avilion et al.,
2003), and hybridized to 80 µm vibratome sections essentially
as described by Wilkinson (Wilkinson,
1998
). Briefly, sections were treated in 2%
H2O2 in PBT (0.1% Triton X-100 in PBS) for 1 hour,
washed in PBT, treated with proteinase K (10 µg/ml in PBT) for 8 minutes,
incubated with 2 mg/ml glycine in PBS for 10 minutes, prehybridized and
hybridized at 60°C overnight with 1 µg/ml probe in 50% formamide,
5xSSC (pH 4.5), 1% SDS and 50 µg/ml yeast RNA. Sections were then
successively washed with 50% formamide, 5xSSC (pH 4.5), 1% SDS (for 1
hour at 60°C), 0.5 M NaCl, 10 mM TrisCl (pH 7.5), 0.1% Tween-20 (for 1
hour at room temperature), and then with 50% formamide, 2xSSC (pH 4.5)
(for 1 hour at room temperature). Washing was followed by preincubation in
TBST [0.5 M NaCl, 20 mM Tris (pH 7.5), 0.1% Tween-20] with 10% normal goat
serum (NGS) (for 90 minutes at room temperature), and incubation with anti-DIG
antibody (Boehringer, 1:2000) in TBST with 1% NGS overnight at 4°C. This
was followed by several washes in TBST, and then in NTMT [100 mM NaCl, 100 mM
TrisCl (pH 9.5), 50 mM MgCl2, 0.1% Tween-20], and incubation in
BM-purple DIG substrate (SIGMA). Sections were then postfixed briefly and
mounted in 80% glycerol in PBS.
Quantitation of the hybridization signal was carried out on equivalent areas of the periventricular region by densitometric scanning using ImageQuant software.
BrdU labelling and immunohistochemistry
BrdU was administered to adult mice (75 µg/g intraperitoneally) with a
daily injection for 6 days (or 9 days for BrdU/calbindin double labelling, see
below), and it was also supplied in the drinking water at 1 mg/ml. Mice were
perfused on the seventh day (or twelfth day for BrdU/calbindin double
labelling) with 4% paraformaldehyde and treated for cryosectioning as above.
For a total count of BrdU-positive cells, 10-20 µm sections were cut, air
dried, and treated with 50% formamide, 50% 2xSSC at 65°C for 1 hour.
This was followed by washes in 2xSSC at room temperature, denaturation
with 2N HCl in H2O at 37°C for 1 hour, neutralization with 0.1
M borate buffer (pH 8.5) for 10 minutes and treatment with 0.6%
H2O2 in PBS for 10 minutes at room temperature. Sections
were then blocked in 1% BSA in PBS for 1 hour at room temperature, and probed
with a SIGMA anti-BrdU mouse monoclonal antibody (1:150, in 0.1% BSA in PBS)
overnight at 4°C. Secondary fluorescent or biotinylated antibodies were
subsequently used, and sections were dehydrated and mounted. BrdU-positive
nuclei surrounding the lateral ventricles, or in the germinative layer of the
dentate gyrus, were counted on one out of every four coronal sections along
the entire extension of the forebrain.
For neurogenesis studies by BrdU/calbindin double labelling, 40 µm
floating cryosections were cut, treated as above for BrdU immunostaining
(except for omission of the formamide/SSC pre-treatment), and incubated
overnight at room temperature with rat anti-BrdU (Harlan, 1:500) and rabbit
anti-calbindin (SWANT, 1:200) antibodies, followed by rhodamine-X fluorescent
anti-rat (Jackson, 1:200), biotinylated anti-rabbit (VECTOR, 1:200 followed by
FITC-streptavidin, Jackson, 1:200) secondary antibodies. For quantification,
BrdU/calbindin double-positive and BrdU single-positive cells were counted on
one out of every three coronal sections throughout the length of the
hippocampus. For lateral ventricle BrdU/ß-tubulin double
immunofluorescence studies, the method described by Capela and Temple
(Capela and Temple, 2002) was
used. Briefly, periventricular cells were dissociated and allowed to adhere to
polylysine-coated plastic chambered slides for 2-3 hours, then fixed with 4%
paraformaldehyde, pre-treated for BrdU immunostaining (as above) and reacted
with a mixture of rat anti-BrdU and rabbit polyclonal anti-ß-tubulin
antibodies. A minimum of 500 cells (corresponding to an average of 200
BrdU-positive cells for the wild type) were counted for each mouse.
Behavioral tests
Animals were individually housed in laboratory cages, with a 12-hour
light-dark cycle, with free access to food and water throughout the study.
Spontaneous motor activity
Mice from each genotype were placed in an activity cage (Ugo Basile,
Varese, Italy) as previously described
(Braida et al., 2000).
Cumulative horizontal counts (measuring horizontal motility) were recorded for
30 minutes.
Circling behavior
Rotations of each animal were recorded by an observer, blind of treatments,
during motor activity test. Cumulative turns were evaluated for 30 minutes, by
counting for 1 minute every 5 minutes. Only complete (360°) left or right
rotations were counted for quantification of circling behavior. All
experiments were performed during the light period between 9 am and 1 pm. Each
animal was subjected to all forms of treatments in a random order with at
least one week elapsing between experiments.
Evaluation of EEG activity
Mice were surgically implanted under deep Equithesin anesthesia (1%
phenobarbital/4% chloral hydrate, 3 ml/kg, ip, Sigma) with a unilateral
bipolar hippocampal electrode and a screw electrode positioned onto the
contralateral overlying cortex under stereotaxic guidance, as previously
described (Vezzani et al.,
2000). The coordinates from bregma for the implantation of the
electrodes were (mm): anteroposterior, -1.9; lateral±1.5; horizontal,
1.5 below dura. The electrodes were connected to a multipin socket and secured
to the skull with acrylic dental cement. Animals were allowed 3-5 days to
recover from surgical procedures before the start of the experiment.
EEG recordings were carried out in freely-moving mice, as previously
described (Vezzani et al.,
2000), to assess the spontaneous EEG pattern. The EEG recordings
were made twice a day (between 9 am and 12 pm, and 4 and 6 pm) continuously
for at least 30 minutes. Mice were recorded three times per week for two
consecutive weeks. The EEG of each mouse was visually analyzed by two
independent investigators who were unaware of the identity of the experimental
groups. Particular attention was paid to the occurrence of ictal episodes
(high frequency and/or multispike complexes, and/or high voltage synchronized
spikes, simultaneously occurring in both leads of recordings) and/or spiking
activity (Vezzani et al.,
2000
).
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
|
|
In the ventricular zone, ependymal cells and choroid plexi are strongly positive (Fig. 3C,D). Furthermore, the lateral ventricle adjacent to the rostral migratory stream, and the stream itself, include numerous cells strongly positive for SOX2 expression (Fig. 3A,B), again matching ß-galactosidase distribution (Fig. 2H,K,L). In the hippocampus dentate gyrus, many neurones are weakly positive (using DAB staining), whereas a minority of the cells show stronger expression (data not shown; see also below, Fig. 4).
To better characterize the Sox2-expressing cells in the neurogenic regions,
double-immunofluorescence experiments were performed. Among neural cells, only
stem cells and their early descendants (such as precursors, transit amplifying
progenitors and neuroblasts) divide in the adult brain
(Temple and Alvarez-Buylla,
1999). We thus labelled proliferating cells by BrdU
administration, and investigated whether BrdU-positive cells express SOX2.
Most, if not all, BrdU-positive cells in the neurogenic region of the lateral
ventricle are also SOX2 positive (Fig.
4A,B, and data not shown). Notably, some of the double
BrdU/SOX2-positive cells are found within subependymal cell clusters, as is
expected for stem cells or their immediate progeny
(Doetsch et al., 1999
;
Capela and Temple, 2002
),
whereas the overlying ependyma is BrdU negative
(Fig. 4A,B). In addition, many
positive cells are also visible in the rostral migratory stream
(Fig. 4A).
Neural stem cells have been proposed to be part of a GFAP-positive `glial'
population residing in the subependymal region
(Doetsch et al., 1999;
Doetsch, 2003
). Several cells
within the subependyma are labelled with both SOX2 and GFAP antibodies
(Fig. 4C,D), and might
represent early neural stem cell/precursor cell types. In addition, strongly
GFAP-positive cells ensheath newly born
(Doetsch et al., 1997
;
Doetsch et al., 1999
;
Capela and Temple, 2002
)
neuroblasts migrating anteriorly along the ventricle to form the rostral
migratory stream; these cells appear as `empty' GFAP-negative areas. Several
of these GFAP-negative cells are also SOX2 positive
(Fig. 4), indicating that the
SOX2-positive/GFAP-negative population may include migrating neuroblasts.
Migrating neuroblasts express PSA-NCAM, a membrane antigen, and the proportion
of PSA-NCAM-positive cells progressively and strongly increases from posterior
to anterior along the migratory route
(Doetsch et al., 1997
).
Strongly SOX2-positive cells were in general PSA-NCAM weakly positive or
negative; however, weakly SOX2-positive cells often showed greater expression
of PSA-NCAM (see Fig. S3 at
http://dev.biologists.org/supplemental/).
At more anterior levels, few if any SOX2/PSA-NCAM double-positive cells were
seen.
Thus, SOX2 appears to be expressed in most proliferating neural precursors. The observation of rare GFAP/SOX2-positive cells in the periventricular neurogenic region, together with the expression of SOX2 in essentially all of the BrdU-positive cells in the subventricular zone, suggests that SOX2/GFAP-positive cells may include GFAP-positive neural stem cells.
In the second adult neurogenic region, the hippocampus, SOX2 is expressed
along the entire dentate gyrus (Fig.
4E), similar to as observed with
Sox2ß-geo (Fig.
2N). However, prominent expression is detected only in a minority
of the cells, at the base of the granule cell layer
(Fig. 4F-H). To better
characterize these cells, we labelled proliferating precursors with BrdU. Most
BrdU-positive cells were located at the base of the granule cell layer, and
the large majority of these cells were also strongly SOX2 positive
(Fig. 4G). BrdU-positive cells
located more deeply inside the granule cell layer showed a much lower level of
SOX2 expression, or no expression at all (not shown). As in the subventricular
region, `stem cells' in the hippocampus have been reported to be part of a
GFAP-positive population (Seri et al.,
2001; Fukuda et al.,
2003
). Double SOX2/GFAP-positive cells are detected almost
exclusively at the base of the granule cell layer, in a location corresponding
to that of double SOX2/BrdU-positive cells, with GFAP-labelled processes
characteristically extending radially into the granule cell layer
(Fig. 4H).
Sox2 is expressed in neural stem cells grown in vitro from adult brain
Neural stem cells have been grown in vitro from the neurogenic regions of
adult mice (McKay, 1997;
Gage, 2000
;
Temple, 2001
). To investigate
whether Sox2ß-geo is expressed in adult neural stem
cells, we exploited the ability of the ß-geo fusion protein to confer
resistance to G418. Neural stem cells from the periventricular region of adult
Sox2ß-geo mice were tested in clonal in vitro assays
(Zappone et al., 2000
). Cells
from these cultures extensively self-renew, forming neurospheres, for at least
12 passages (i.e. about 12 weeks), even in the presence of G418, indicating
that neurosphere-forming cells continuously express the
Sox2ß-geo knock-in reporter
(Fig. 2O). Moreover, individual
G418-resistant neurosphere-forming cells retain multipotency, that is the
ability to give rise to progeny able to differentiate into all three main
neural lineages, neurones, astroglia and oligodendroglia
(Fig. 2O-Q). These results
indicate that adult neural stem cells grown in vitro continuously express
Sox2ß-geo.
Sox2 deficiency causes reduced viability and neurological defects
Homozygosity for the Sox2ß-geo null allele results
in early embryonic lethality (Avilion et
al., 2003). Compound
Sox2ß-geo/
ENH heterozygotes,
irrespective of whether the PGKneo cassette has been retained or not, show
severe abnormalities, as described below; the two genotypes will be discussed
together.
Sox2 expression was evaluated in compound heterozygotes for the null and
the knockdown alleles (Sox2ß-geo/ENHneo) by
antibody staining, in situ hybridization and RT-PCR on neural precursors,
grown in vitro as neurospheres (see Figs S1 and S2 at
http://dev.biologists.org/supplemental/;
data not shown). As anticipated for a regulatory mutation, Sox2 was still
detected in the expected brain regions in compound
Sox2ß-geo/
ENHneo heterozygotes, but at a
reduced level (approximately 25-30% relative to the wild-type genotype). As
this level of expression is due only to the Sox2
ENH
allele, the transcriptional output of this allele is about half that of a
wild-type Sox2 allele.
Neither simple heterozygotes (Sox2ß-geo/+ or
Sox2ENH/+) nor
Sox2
ENH/
ENH homozygotes show the
abnormalities described for compound
Sox2ß-geo/
ENHneo heterozygotes. The fact that
Sox2
ENH/
ENH homozygotes are not affected is
consistent with the
50% reduction of expression of the
Sox2
ENH allele, which would give a level of
Sox2 expression in Sox2
ENH/
ENH
homozygotes close to that expected for the
Sox2ß-geo/+ null heterozygotes.
The viability of the Sox2ENH/+,
Sox2ß-geo/+ and
Sox2
ENH/
ENH genotypes was identical to that
of the wild type. By contrast, compound
Sox2ß-geo/
ENH heterozygotes were born in
reduced numbers compared with the expected frequency, and their number further
declined in postnatal life (Fig.
5A). They showed growth retardation, normally compensated by six
weeks of age, and slowed reactivity. When held by their tail, 40% of the
mutants retracted their limbs toward their trunk in a dystonic fashion, rather
that extending them as did control littermates. This feet-clasping phenotype
is observed in several mutants with neurological impairment due to
neurodegeneration (Mangiarini et al.,
1996
; Mantamadiotis et al.,
2002
; Wang et al.,
2002
). Forty percent of the mutants showed epileptic spikes in the
cortex and hippocampus in electroencephalographic recordings
(Fig. 5B). Finally, `circling'
behaviour was observed in Sox2ß-geo/
ENH
mutants at 3-4 weeks of age, or later; this phenotype was transmitted with
incomplete penetrance (25%).
|
These findings indicate that a dysfunction in the dopaminergic system is involved in generating circling in the mutants, working through reduced D1 and particularly D2 receptor signalling.
Neurological abnormality is accompanied by brain morphological defects
In adult mutant brains, the cortex is reduced, in particular posteriorly
and medially (Fig. 6A,F,
sections). In addition, there is progressive anteroposterior reduction of the
corpus callosum [that is prematurely interrupted
(Fig. 6C) in most mutants, and
is completely absent in 15% of the mice), marked decrease of anterior
thalamus, dorsal striatum and septum (Fig.
6B-D,I), and lateral and third ventricle enlargement
(Fig. 6B-E,I). Reduction of the
posterior cerebral cortex is consistently observed already at E14.5
(Fig. 6G,H); of note, no
ventricle enlargement is yet present at this stage
(Fig. 6G,H), indicating that
ventricle enlargement does not, per se, initiate the reduction of the cortical
parenchyma. Thalamic-striatal reduction and ventricle enlargement are already
developed at E17.5 (not shown) and at birth
(Fig. 6I). With the exception
of a slight ventricle enlargement in occasional mice, none of these changes is
seen in Sox2ß-geo/+ heterozygotes.
|
In the mutant brain, several neurones show typical features of degeneration
(Hodgson et al., 1999;
Turmaine et al., 2000
;
Capsoni et al., 2000
;
Tanemura et al., 2002
), such
as a severely shrunken and hyperchromatic cell body, nuclear and cytoplasmic
condensation, and irregular, `scalloped' plasma and nuclear membranes
(Fig. 7A-D,L), whereas others
exhibit characteristic perinuclear inclusions. Neither abnormality was
observed in control littermates. The degenerating cells are located in
selected regions, such as striatum, and, particularly, septum and thalamus
(centromedian and paraventricular nuclei)
(Fig. 7A-D), whereas inclusions
are observed in the thalamus, in paraventricular nuclei, and (abundantly) in
basolateral and lateral geniculate nuclei
(Fig. 7E-L), and (less
frequently) in striatum (not shown). Significantly, these regions correspond
to those where parenchymal shrinkage is observed
(Fig. 6B-E), and match areas of
persistent Sox2 expression in late embryonic and adult stages
(Fig. 2F,I,J,M;
Fig. 3G,H). Inclusions were
rarely detected at birth, were abundant at six months of age, and were
extremely abundant in mice aged 18-24 months. Neurodegeneration was not
observed at birth, was already apparent at three months, and was abundant
(Fig. 7) at six months, with
little apparent progression at later stages (we cannot rule out a selective
effect, as more severely affected mice may be lost prematurely). In several
hereditary and multifactorial neuropathies in man and mouse, inclusions are a
hallmark of the disease; they result from aggregation of a variety of
proteins, such as neurofilaments, tau protein and alpha-synuclein, and are
often ubiquitinated (Taylor et al.,
2002
; Zoghbi and Botas,
2002
). In Sox2 mutant mice, the perinuclear aggregates
contain neurofilaments (Fig.
7J) and, less frequently, ubiquitinated protein
(Fig. 7K,L), though not
alpha-synuclein (not shown). They are found predominantly in otherwise
apparently healthy neurones (Fig.
7E,I-K), but occasional `hyperchromatic'/degenerating cells can
also exhibit them (Fig.
7L).
|
Impairment of neural cells proliferation and of neurogenesis in adult neurogenic regions
In the ependymal/subependymal region of the lateral ventricles, as well as
in the hippocampus dentate gyrus, some neurogenesis is maintained in the
adult, through the proliferation and differentiation of neural stem and
precursor cells (McKay, 1997;
Gage, 2000
;
Temple, 2001
). Adult neural
stem cells (a site of Sox2 expression;
Fig. 2O-Q) have been reported
to be a subset of ependymal cells themselves
(Johansson et al., 1999
), or
to lie in the subependyma (Doetsch et al.,
1999
; Capela and Temple,
2002
). As Sox2 is expressed in essentially all dividing
progenitors (see above and Fig.
4), we investigated cell proliferation in these regions by BrdU
incorporation. In Sox2ß-geo/
ENH mutants, the
number of BrdU-positive cells was markedly reduced in the hippocampus
germinative layer (an
65% reduction)
(Fig. 8A,B) and in the lateral
ventricle (an
55% reduction) (Fig.
8A,B). In the hippocampus, some sparse cells were found in the
hilus, where labelling is only rarely seen in the wild type.
|
GFAP-positive radial glia cells in the hippocampus germinative layer
generate new neurones in dentate gyrus of adult rats
(Seri et al., 2001); in mouse,
double GFAP/nestin-positive progenitors share many morphological properties
with these cells, and have been identified by BrdU pulse-chase analyses as the
earliest progenitors to dentate gyrus adult-born neurones
(Doetsch, 2003
;
Fukuda et al., 2003
). In
Sox2ß-geo/
ENH mutants, nestin-positive cells
in the dentate gyrus are greatly decreased
(Fig. 9A,C), and GFAP-positive
radial glia are also significantly (although less) decreased
(Fig. 9B,D). Strikingly, double
GFAP/nestin-positive cells are almost absent from the dentate gyrus of mutant
mice (Fig. 9E,F). These data
suggest that neural stem cells and/or precursors in the adult hippocampal
neurogenic region of Sox2 mutants are depleted.
|
In contrast to the BrdU incorporation defects observed postnatally, no significant differences between normal and mutant were observed when labelling was done in the embryo for a single day (between E12.5 and E16.5) and analysis was performed 24 hours later (to detect newly born neural cells) or a month later (to detect neurones labelled during their terminal division). It is conceivable that embryonic cells might be less dependent than adult cells on Sox2 levels, possibly because of Sox family gene redundancy, or because of higher normal expression of Sox2 in these cells.
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Reduction of Sox2 expression in Sox2ß-geo/ENH compound heterozygotes is sufficient to cause brain defects
We have combined a null mutation of Sox2
(Sox2ß-geo) with a regulatory (`knockdown') mutation
(Sox2ENH), obtained by deleting a neural-specific
enhancer (Zappone et al.,
2000
) from the Sox2 locus. Although the latter deletion
decreases Sox2 expression, it does not abolish it
(Zappone et al., 2000
) (see
also Figs S1, S2 at
http://dev.biologists.org/supplemental/).
Neither the heterozygous null mutant
(Sox2ß-geo/+), nor the homozygous knockdown mutants
(Sox2ENH/
ENH) show any pathology, with the
exception of some mild ventricle enlargement in occasional heterozygous null
(Sox2ß-geo/+) mice. Significantly, this suggests that
the observed phenotype is critically sensitive to thresholds effects; in fact,
a pathological phenotype is observed in
Sox2ß-geo/
ENH compound heterozygotes, at a
level of expression of Sox2 of 25-30% (relative to wild type),
moderately below the level expected for the unaffected
Sox2ß-geo/+ null heterozygote. This effect resembles
that observed with combinations of different knockdown alleles of the
Fgf8 gene (Meyers et al.,
1998
). Importantly, it is unlikely that the regulatory
Sox2
ENH mutation causes its effects through
interference with adjacent genes, as no pathological effects are observed in
mice homozygous for this mutation.
Thalamo-striatal defects and neurodegeneration in adult Sox2 mutants
A major defect of Sox2ß-geo/ENH
mutant mice is the loss of thalamo-striatal parenchyma with ventricle
enlargement. In murine models of Huntington's and Alzheimer's disease,
parenchymal loss with secondary ventricle enlargement represents the
consequence of primary neurodegeneration
(Capsoni et al., 2000
;
Yamamoto et al., 2000
;
Mantamadiotis et al., 2002
).
The correlation between the expression of Sox2 (in the late foetal
and in the adult periods) in the differentiated parenchyma of thalamus and
striatum (Figs 2,
3), and the tissue loss
(Fig. 6) with abundant
neurodegeneration (Fig. 7) in
these same areas in compound Sox2ß-geo/
ENH
mutants, implies an important functional role of Sox2 for adult
neurones.
An additional unexpected observation is the presence of intracellular
inclusions in neurones, close to (and occasionally within) the degenerated
cells (Fig. 7). Protein
inclusions in neurodegenerative diseases are caused by the aggregation of
potentially toxic misfolded proteins, and may represent an `attempt' to clear
the cell of harmful proteins through, for example, ubiquitination
(Bucciantini et al., 2002;
Taylor et al., 2002
;
Zoghbi and Botas, 2002
). In
inherited human neurodegenerative disorders, aggregates are often the result
of mutations in a gene encoding a structural protein found in the aggregates
themselves (e.g. alpha-synuclein, huntingtin), or of an enzyme (e.g.
presenilin) involved in protein processing
(Taylor et al., 2002
;
Zoghbi and Botas, 2002
).
However, in the majority of `common' multifactorial degenerations,
aggregates may represent a progressive, age-related response to
`environmental' (i.e. metabolic, toxic) factors. Few genetic modifiers of this
response have been identified (Zoghbi and
Botas, 2002). Neurofilament-rich aggregates ultrastructurally very
similar to those described here were reported in the thalamus of mice with
thiamine-deficient encephalopathy (Aikawa
et al., 1983
), and of a small proportion of aging mice
(Fraser, 1969
). The
development after birth of large numbers of inclusion-containing neurones in
mutant but not control mice, at a relatively young age, indicates that
Sox2 deficiency can represent a major genetic contribution to this
pathology. In conclusion, the presence, in mutants, of inclusion-containing
neurones and, in particular, of degenerated cells (see above), shows that
Sox2 is required for the maintenance of vital functions of neurones.
Sox2, being a transcription factor, might, directly or indirectly,
affect the activity of genes encoding chaperons, proteolytic enzymes, and so
on, involved in general mechanisms of neurone protection. It will be
interesting to evaluate whether Sox2 deficiency increases the
severity of other mutations causing neurodegeneration
(Zoghbi and Botas, 2002
), by
breeding to appropriate strains.
The expression of Sox2 in the ependyma, together with the lipidic
inclusions and ciliary loss, raises the possibility that malfunction of these
cells may also contribute to the development of hydrocephalic features.
Hereditary hydrocephalus is observed in humans with Karthagener's syndrome, a
primary ciliary dyskinesia. It is also observed in the mouse knock-out model
of the dynein gene Mdnah5 (Dnahc5 - Mouse Genome
Informatics), which is essential for ciliary mobility
(Ibanez-Tallon et al., 2002),
or of the Isg15 (Glp2 - Mouse Genome Informatics) gene,
which encodes an ependymal ubiquitin ligase
(Ritchie et al., 2002
).
However, we note that in Sox2 mutants the cortex, which in primary
hydrocephalus is often very thin because of cerebrospinal fluid pressure
(Lindeman et al., 1998
;
Ibanez-Tallon et al., 2002
;
Ritchie et al., 2002
), is
comparatively unaffected and does not show neuronal pathology. Conversely,
thalamo-striatal neuronal pathology with intraneuronal inclusions is not
described in primary hydrocephalus with ventricle enlargement that is even
more pronounced than that seen in Sox2 mutants
(Lindeman et al., 1998
;
Ibanez-Tallon et al., 2002
;
Ritchie et al., 2002
;
Blackshear et al., 2003
).
The regions affected by neuropathology are part of the
nigrostriatal-cortico-thalamic circuitry, which controls motor behaviour and
requires dopamine as a crucial neurotransmitter
(Carlsson, 2001). This makes
their lesion a plausible reason for the functional defects observed. Some of
these defects, such as circling, have been likened to those observed in humans
with Parkinson's disease (Kim et al.,
2002
). Dopaminergic neurones of the substantia nigra, whose lesion
causes Parkinson's disease in humans, do not show gross abnormalities or
important reduction of TH immunoreactivity in our mutants (not shown). It
should be considered that the vulnerability of these neurones to damage (and
aggregate formation) appears to be extremely reduced in mice versus humans
(Lee et al., 2002
). Thus,
motor dysfunction in mouse models, including the Sox2 mutant, might
be due to defects located elsewhere in the basal ganglia-thalamocortical
circuitry (Carlsson, 2001
;
Wang et al., 2002
).
Interestingly, in preliminary experiments we found altered GABA
immunoreactivity in the same thalamic nuclei that the neuropathology is
observed in (S.D.B. and S.K.N., unpublished).
Sox2 is expressed in neural precursor cells
In the adult brain, neural stem cells divide at a low rate, both renewing
themselves and generating neural precursors. Virtually all newly divided cells
(i.e. BrdU+ cells) express Sox2 in both the subventricular
region and hippocampus subgranular zone
(Fig. 4). These results suggest
that Sox2 is expressed in the majority, if not all, of the neural
precursor cells. A number of observations suggest that Sox2 might be
expressed in stem cells as well. (1) Within the subventricular region, a
proportion of Sox2-positive cells also express GFAP; GFAP-positive
cells (B cells) within this location have been proposed to include stem cells
(Doetsch et al., 1999), and
their morphology (Doetsch et al.,
1999
; Doetsch,
2003
) strikingly resembles that observed by us for SOX2/GFAP
double-positive cells (see Fig.
4D). (2) In the hippocampus subgranular zone, GFAP/SOX2
double-positive cells, characteristically extending radial processes into the
dentate gyrus, are detected (Fig.
4H); again, the location and morphology of these cells are
strongly reminiscent of GFAP-positive
(Seri et al., 2001
) and
GFAP/nestin-positive (Fukuda et al.,
2003
) cells thought to include the in vivo hippocampal primary
precursors (Seri et al., 2001
;
Fukuda et al., 2003
;
Doetsch, 2003
). (3) In in
vitro culture, Sox2ß-geo-expressing cells show
functional properties of neural stem cells (self renewal, multipotency)
throughout extensive propagation in the presence of G418 (implying continued
Sox2 expression in these cells).
In Sox2 mutants, neural precursor proliferation and neurogenesis are affected
In adult Sox2 mutants, BrdU labelling in the subventricular zone
and hippocampus is strongly decreased (Fig.
8A,B). This might be due either to loss of stem cells/precursors,
or to a decreased ability to proliferate in response to physiological stimuli.
Our results suggest that, in the hippocampus, both the number of
stem/precursor cells, and the ability of their progeny to differentiate into
neurones, is decreased (Figs 8,
9). In fact, GFAP/nestin
double-positive cells, which represent the earliest precursors to neurones in
the dentate gyrus (Fukuda et al.,
2003), are dramatically decreased in Sox2 mutants
(Fig. 9). This is in agreement
with the presence of SOX2 in GFAP-positive subventricular cells (see above),
and suggests that Sox2 is cell-autonomously requested for the
maintenance of these cells. In addition, the proportion of calbindin-positive
cells (i.e. neurones) within BrdU-positive cells is decreased
(Fig. 8E), indicating that
newly generated precursors may either die or fail to differentiate properly. A
similar result is obtained in the subventricular zone; here, the number of
BrdU/ß-tubulin III double-positive (i.e. recently generated early
neuronal) cells is strongly decreased (Fig.
8F). Thus, both in the hippocampus and subventricular zone,
neurogenesis is strongly affected.
It has been speculated that neuronal cell replacement by endogenous
precursors in adults, following chronic neurodegeneration, might slow down the
course of the disease (Kruger and
Morrison, 2002). If this were the case, the decreased neurogenesis
in Sox2 mutants might contribute to the severity of the
phenotype.
Recent experiments in chicken using electroporation of a Sox-dominant
negative construct (affecting Sox1, Sox2 and Sox3
activities) suggest that at least one member of the SOX family is necessary to
prevent premature commitment to differentiation; alternatively, a single Sox
gene expressed ectopically prevents neural differentiation
(Graham et al., 2003;
Bylund et al., 2003
). Our
experiments are consistent with a role of Sox family members in the
maintenance of functional properties of neural precursor cells
(Graham et al., 2003
;
Bylund et al., 2003
). However,
the situation may be more complex. We show that adult neural precursors, in
mouse, are specifically sensitive to Sox2 gene dosage, even in the
presence of normal Sox1 and Sox3 genes. It is possible that
the subset of neural precursors affected by Sox2 deficiency do not
co-express Sox1 and/or Sox3 together with Sox2;
alternatively, Sox2 might play specific functions within these cells
that cannot be complemented by Sox1 and Sox3. Furthermore,
Sox2 may also affect cell survival or differentiation even downstream
of the precursor stages, as shown by the requirement for Sox2 in
neurone generation (see above, and Fig.
8), and in preventing the death or degeneration of some
differentiated neurones (Fig.
7). As important neural cell and brain alterations are detected in
Sox2ß-geo/
ENH compound heterozygotes retaining
significant amounts of Sox2, it will be important to assess the
effect of the complete ablation of Sox2 expression in different types
of neural cells using a conditional Sox2 knock-out strategy.
Recently, it was shown (Fantes et al.,
2003) that heterozygous Sox2 truncating-point mutations
in humans are associated with anophtalmia, and with variable extraocular
defects, including seizures, microcephaly and motor abnormalities. The ocular
phenotype of our mutant mice is presently being investigated. However, these
observations in humans, taken together with our findings, suggest that it
might be worth looking for Sox2 defects in a wider range of inherited
human neurological diseases.
![]() |
ACKNOWLEDGMENTS |
---|
![]() |
Footnotes |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Avilion, A. A., Nicolis, S. K., Pevny, L. H., Perez, L., Vivian,
N. and Lovell-Badge, R. (2003). Multipotent cell lineages in
early mouse development depend on SOX2 function. Genes
Dev. 17,126
-140.
Aikawa, H., Suzuki, K. and Iwasaki, Y. (1983). Ultrastructural observation on the thalamic neuronal inclusions in young mice. Acta Neuropathol. 59,316 -318.[Medline]
Blackshear, P. J., Graves, J. P., Stumpo, D. J., Cobos, I.,
Rubenstein, J. L. and Zeldin, D. C. (2003). Graded phenotypic
response to partial and complete deficiency of a brain-specific transcript
variant of the winged helix transcription factor RFX4.
Development 130,4539
-4552.
Braida, D., Pozzi, M. and Sala, M. (2000). CP 55,940 protects against ischemia-induced electroencephalographic flattening and hyperlocomotion in Mongolian gerbils. Neurosci. Lett. 296,69 -72.[CrossRef][Medline]
Bucciantini, M., Giannoni, E., Chiti, F., Baroni, F., Formigli, L., Zurdo, J., Taddei, N., Ramponi, G., Dobson, C. M. and Stefani, M. (2002). Inherent toxicity of aggregates implies a common mechanism for protein misfolding diseases. Nature 416,507 -511.[CrossRef][Medline]
Bylund, M., Andersson, E., Novitch, B. G. and Miuhr, J. (2003). Vertebrate neurogenesis is counteracted by Sox1-3 activity. Nat. Neurosci. 11,1162 -1168.[CrossRef]
Capela, A. and Temple, S. (2002). LeX/ssea-1 is expressed by adult mouse CNS stem cells, identifying them as nonependymal. Neuron 35,865 -875.[Medline]
Capsoni, S., Ugolini, G., Comparini, A., Ruberti, F., Berardi,
N. and Cattaneo, A. (2000). Alzheimer-like neurodegeneration
in aged antinerve growth factor transgenic mice. Proc. Natl. Acad.
Sci. USA 97,6826
-6831.
Carlsson, A. (2001). A paradigm shift in brain
research. Science 294,1021
-1024.
Doetsch, F., Garcìa-Verduga, J. M. and Alvarez-Buylla
A. (1997). Cellular composition and three-dimensional
organization of the subventricular germinal zone in the adult mammalian brain.
J. Neurosci. 17,5046
-5061.
Doetsch, F., Caille, I., Lim, D. A., Garcia-Verdugo, J. M. and Alvarez-Buylla, A. (1999). Subventricular zone astrocytes are neural stem cells in the adult mammalian brain. Cell 97,703 -716.[Medline]
Doetsch, F. (2003). The glial identity of neural stem cells. Nat. Neurosci. 6,1127 -1134.[CrossRef][Medline]
Fantes, J., Ragge, N. K., Lynch, S. A., McGill, N. I., Collin, J. R., Howard-Peebles, P. N., Hayward, C., Vivian, A. J., Williamson, K., van Heyningen, V. and FitzPatrick, D. R. (2003). Mutations in SOX2 cause anophthalmia. Nat. Genet. 33,461 -463.[CrossRef][Medline]
Fraser, H. (1969). Eosinophilic bodies in some neurones in the thalamus of ageing mice. J. Pathol. 98,201 -204.[Medline]
Fukuda, S., Kato, F., Tozuka, Y., Yamaguchi, M., Miyamoto, Y.
and Hisatune, T. (2003). Two distinct subpopulations of
nestin-positive cells in adult mouse dentate gyrus. J.
Neurosci. 23,9357
-9366.
Gage, F. H. (2000). Mammalian neural stem
cells. Science 287,1433
-1438.
Gould, E., Beylin, A., Tanapat, P., Reeves, A. and Shors, T. J. (1999). Learning enhances adult neurogenesis in the hippocampal formation. Nat. Neurosci. 2, 260-265.[CrossRef][Medline]
Graham, V., Khudyakov, J., Ellis, P. and Pevny, L. (2003). SOX2 functions to maintain neural progenitor identity. Neuron 39,749 -765.[Medline]
Gubbay, J., Collignon, J., Koopman, P., Capel, B., Economou, A., Münsterberg, A., Vivian, N., Goodfellow, P. and Lovell-Badge, R. (1990). A gene mapping to the sex-determining region of the mouse Y chromosome is a member of a novel family of embryonically expressed genes. Nature 346,245 -250.[CrossRef][Medline]
Hodgson, J. G., Agopyan, N., Gutekunst, C. A., Leavitt, B. R., LePiane, F., Singaraja, R., Smith, D. J., Bissada, N., McCutcheon, K., Nasir, J., Jamot, L., Li, X. J., Stevens, M. E., Rosemond, E., Roder, J. C., Phillips, A. G., Rubin, E. M., Hersch, S. M. and Hayden, M. R. (1999). A YAC mouse model for Huntington's disease with full-length mutant huntingtin, cytoplasmic toxicity, and selective striatal neurodegeneration. Neuron 23,181 -192.[Medline]
Ibanez-Tallon, I., Gorokhova, S. and Heintz, N.
(2002). Loss of function of axonemal dynein Mdnah5 causes primary
ciliary dyskinesia and hydrocephalus. Hum. Mol. Genet.
11,715
-721.
Johansson, C. B., Momma, S., Clarke, D. L., Risling, M., Lendahl, U. and Frisen, J. (1999). Identification of a neural stem cell in the adult mammalian central nervous system. Cell 96,25 -34.[Medline]
Kamachi, Y., Uchikawa, M. and Kondoh, H. (2000). Pairing SOX off, with partners in the regulation of embryonic development. Trends Genet. 16,182 -187.[CrossRef][Medline]
Kempermann, G., Kuhn, H. G. and Gage, F. H. (1997). More hippocampal neurons in adult mice leaving in an enriched environment. Nature 386,493 -495.[CrossRef][Medline]
Kim, J. H., Auerbach, J. M., Rodriguez-Gomez, J. A., Velasco, I., Gavin, D., Lumelsky, N., Lee, S. H., Nguyen, J., Sanchez-Pernaute, R., Bankiewicz, K. and McKay, R. (2002). Dopamine neurons derived from embryonic stem cells function in an animal model of Parkinson's disease. Nature 418,50 -56.[CrossRef][Medline]
Kishi, M., Mizuseki, K., Sasai, N., Yamazaki, H., Shiota, K.,
Nakanishi, S. and Sasai, Y. (2000). Requirement of
Sox2-mediated signaling for differentiation of early Xenopus neuroectoderm.
Development 127,791
-800.
Kruger, G. M. and Morrison, S. J. (2002). Brain repair by endogenous progenitors. Cell 110,399 -402.[CrossRef][Medline]
Lee, M. K., Stirling, W., Xu, Y., Xu, X., Qui, D., Mandir, A.
S., Dawson, T. M., Copeland, N. G., Jenkins, N. A. and Price, D. L.
(2002). Human alpha-synuclein-harboring familial Parkinson's
disease-linked Ala-53 Thr mutation causes neurodegenerative disease
with alpha-synuclein aggregation in transgenic mice. Proc. Natl.
Acad. Sci. USA 99,8968
-8973.
Lindeman, G. J., Dagnino, L., Gaubatz, S., Xu, Y., Bronson, R.
T., Warren, H. B. and Livingston, D. M. (1998). A specific,
nonproliferative role for E2F-5 in choroid plexus function revealed by gene
targeting. Genes Dev.
12,1092
-1098.
McDevitt, M. A., Shivdasani, R. A., Fujiwara, Y., Yang, H. and
Orkin, S. H. (1997). A `knockdown' mutation created by
cis-element gene targeting reveals the dependence of erythroid cell maturation
on the level of transcription factor GATA-1. Proc. Natl. Acad. Sci.
USA 94,6781
-6785.
McKay, R. (1997). Stem cells in the central
nervous system. Science
276, 66-71.
Mangiarini, L., Sathasivam, K., Seller, M., Cozens, B., Harper, A., Hetherington, C., Lawton, M., Trottier, Y., Lehrach, H., Davies, S. W. and Bates, G. P. (1996). Exon 1 of the HD gene with an expanded CAG repeat is sufficient to cause a progressive neurological phenotype in transgenic mice. Cell 87,493 -506.[Medline]
Mantamadiotis, T., Lemberger, T., Bleckmann, S. C., Kern, H., Kretz, O., Martin, V. A., Tronche, F., Kellendonk, C., Gau, D., Kapfhammer, J., Otto, C., Schmid, W. and Schutz, G. (2002). Disruption of CREB function in brain leads to neurodegeneration. Nat. Genet. 31,47 -54.[CrossRef][Medline]
Meyers, E. N., Lewandoski, M. and Martin, G. R. (1998). An Fgf8 mutant allelic series generated by Cre- and Flp-mediated recombination. Nat. Genet. 18,136 -141.[CrossRef][Medline]
Ritchie, K. J., Malakhov, M. P., Hetherington, C. J., Zhou, L.,
Little, M. T., Malakhova, O. A., Sipe, J. C., Orkin, S. H. and Zhang, D.
E. (2002). Dysregulation of protein modification by ISG15
results in brain cell injury. Genes Dev.
16,2207
-2212.
Schwenk, F., Baron, U. and Rajewsky, K. (1995). A cre-transgenic mouse strain for the ubiquitous deletion of loxP-flanked gene segments including deletion in germ cells. Nucleic Acids Res. 23,5080 -5081.[Medline]
Seri, B., Garcia-Verdugo, J. M., McEwen, B. S. and
Alvarez-Buylla, A. (2001). Astrocytes give rise to new
neurons in the adult mammalian hippocampus. J.
Neurosci. 21,7153
-7160.
Tanemura, K., Murayama, M., Akagi, T., Hashikawa, T., Tominaga,
T., Ichikawa, M., Yamaguchi, H. and Takashima, A. (2002).
Neurodegeneration with tau accumulation in a transgenic mouse expressing V337M
human tau. J. Neurosci.
22,133
-141.
Taylor, J. P., Hardy, J. and Fischbeck, K. H.
(2002). Toxic proteins in neurodegenerative disease.
Science 296,1991
-1995.
Temple, S. (2001). The development of neural stem cells. Nature 414,112 -117.[CrossRef][Medline]
Temple, S. and Alvarez-Buylla, A. (1999). Stem cells in the adult mammalian central nervous system. Curr. Opin. Neurobiol. 9,135 -141.[CrossRef][Medline]
Turmaine, M., Raza, A., Mahal, A., Mangiarini, L., Bates, G. P.
and Davies, S. W. (2000). Nonapoptotic neurodegeneration in a
transgenic mouse model of Huntington's disease. Proc. Natl. Acad.
Sci. USA 97,8093
-8097.
Uwanogho, D., Rex, M., Cartwright, E. J., Pearl, G., Healy, C., Scotting, P. J. and Sharpe, P. T. (1995). Embryonic expression of the chicken Sox2, Sox3 and Sox11 genes suggests an interactive role in neuronal development. Mech. Dev. 49, 23-36.[CrossRef][Medline]
Vezzani, A., Moneta, D., Conti, M., Richichi, C., Ravizza, T.,
De Luigi, A., De Simoni, M. G., Sperk, Andell-Jonsson, S., Lundkvist, J.,
Iverfeldt, K. and Bartfai, T. (2000). Powerful anticonvulsant
action of IL-1 receptor antagonist upon intracerebral injection and astrocytic
overexpression in mice. Proc. Natl. Acad. Sci. USA,
97,115
-134.
Wang, Q., Bardgett, M. E., Wong, M., Wozniak, D. F., Lou, J., McNeil, B. D., Chen, C., Nardi, A., Reid, D. C., Yamada, K. and Ornitz, D. M. (2002). Ataxia and paroxysmal dyskinesia in mice lacking axonally transported FGF14. Neuron 35, 25-38.[Medline]
Wilkinson, D. G. (1998). In Situ Hybridization, A Practical Approach. Oxford, Oxford University Press.
Wood, H. B. and Episkopou, V. (1999). Comparative expression of the mouse Sox1, Sox2 and Sox3 genes from pre- gastrulation to early somite stages. Mech. Dev. 86,197 -201.[CrossRef][Medline]
Yamamoto, A., Lucas, J. J. and Hen, R. (2000). Reversal of neuropathology and motor dysfunction in a conditional model of Huntington's disease. Cell 101, 57-66.[Medline]
Zappone, M. V., Galli, R., Catena, R., Meani, N., De Biasi, S.,
Mattei, E., Tiveron, C., Vescovi, A. L., Lovell-Badge, R., Ottolenghi, S. and
Nicolis, S. K. (2000). Sox2 regulatory sequences direct
expression of a ß-geo transgene to telencephalic neural stem cells and
precursors of the mouse embryo, revealing regionalization of gene expression
in CNS stem cells. Development
127,2367
-2382.
Zoghbi, H. Y. and Botas, J. (2002). Mouse and fly models of neurodegeneration. Trends Genet. 18,463 -471.[CrossRef][Medline]