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The Journal of Neuroscience, March 15, 2002, 22(6):2255-2264
Lack of the Cell-Cycle Inhibitor p27Kip1 Results in Selective
Increase of Transit-Amplifying Cells for Adult Neurogenesis
Fiona
Doetsch1, 2,
José Manuel-Garcia
Verdugo3,
Isabelle
Caille2,
Arturo
Alvarez-Buylla2,
Moses V.
Chao4, and
Patrizia
Casaccia-Bonnefil5
1 Department of Molecular and Cellular Biology, Harvard
University, Cambridge, Massachusetts 02138, 2 Rockefeller
University, New York, New York 10021, 3 Department of
Cellular Biology, University of Valencia, Valencia 46100, Spain,
4 Molecular Neurobiology Program, Skirball Institute, New
York University, New York, New York 10016, and
5 Department of Neuroscience and Cell Biology, University
of Medicine and Dentistry of New Jersey, Piscataway, New Jersey
08854
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ABSTRACT |
The subventricular zone (SVZ) is the largest germinal layer in the
adult mammalian brain and comprises stem cells, transit-amplifying progenitors, and committed neuroblasts. Although the SVZ contains the
highest concentration of dividing cells in the adult brain, the
intracellular mechanisms controlling their proliferation have not been
elucidated. We show here that loss of the cyclin-dependent kinase inhibitor p27Kip1 has very specific effects on a
population of CNS progenitors responsible for adult neurogenesis. Using
bromodeoxyuridine and [3H]thymidine incorporation
to label cells in S phase and cell-specific markers and electron
microscopy to identify distinct cell types, we compared the SVZ
structure and proliferation characteristics of wild-type and
p27Kip1-null mice. Loss of p27Kip1 had no effect on the number of stem
cells but selectively increased the number of the transit-amplifying
progenitors concomitantly with a reduction in the number of
neuroblasts. We conclude that cell-cycle regulation of SVZ adult
progenitors is remarkably cell-type specific, with p27Kip1 being a key
regulator of the cell division of the transit-amplifying progenitors.
Key words:
cell division; differentiation; neural progenitors; stem
cells; CNS; proliferation
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INTRODUCTION |
The adult mouse subventricular zone
(SVZ) is the richest source of stem cells in the adult brain and
constantly generates new neurons destined for the olfactory bulb. The
SVZ is composed of a thin layer of dividing cells extending along the
length of the lateral wall of the lateral ventricle (Steindler et al.,
1996 ; Temple and Alvarez-Buylla, 1999). On the basis of cell-cycle
length and ultrastructural characteristics, four distinct cell types can be identified in this region: slowly dividing SVZ astrocytes (type
B cells), rapidly dividing progenitors (type C cells), proliferating migratory neuroblasts (type A cells), and ependymal cells, which line
the lateral ventricle (Lois and Alvarez-Buylla, 1994; Doetsch et al.,
1997; Peretto et al., 1999). Recently a lineage progression from
B CA cell has been proposed (Doetsch et al., 1999a,b), with type B
cells being the primary precursors (stem cells) that generate migratory
neuroblasts via the highly proliferative transit-amplifying progenitor
(type C cell). Cells within this lineage possess the capability of
undergoing proliferation, differentiation, or apoptosis (Morshead and
van der Kooy, 1992; Lois and Alvarez-Buylla, 1994; Biebl et al., 2000).
Recent studies have suggested that cell death may be one of the
potential mechanisms for lineage restriction in the postnatal SVZ
(Levison et al., 2000).
The SVZ of adult mice has a characteristic architecture. The migrating
neuroblasts form elongated aggregates called "chains" that are
ensheathed by GFAP-positive B cells. Type C cells are found as clusters
interspersed among the chains (Doetsch et al., 1997). The chains of
neuroblasts form an extensive network of interconnected pathways that
converge at the anterior tip of the SVZ to join the rostral migratory
stream (RMS), which terminates in the olfactory bulb (Lois and
Alvarez-Buylla, 1994; Doetsch and Alvarez-Buylla, 1996).
Several studies on cell-cycle length in the neonatal rat brain
(Schultze and Korr, 1981; Menezes et al., 1995, 1998; Thomaidou et al.,
1997; Smith and Luskin, 1998) and adult mouse (Schultze and Korr, 1981;
Morshead and van der Kooy, 1992) have indicated the existence of
distinct populations with different cell-cycle times within the SVZ and
RMS. It has been reported, for instance, that cells in the anterior SVZ
have a faster cell-cycle time than the migratory cells in the RMS
(Smith and Luskin, 1998).
The length of the cell-cycle transit time during neurogenesis depends
primarily on the duration of the G1 transition
(Caviness et al., 1999) and is modulated by the enzymatic activity of
cyclin-dependent kinases (CDKs). Two main enzymatic activities have
been described: CDK4, acting in early to mid-G1,
and CDK2, acting in late G1, very close to the
entry into the S replicative phase. These two activities differ in
terms of substrate specificity and modality of regulation. CDK4, for
instance, is positively regulated by cyclin D and can be inhibited by
binding to members of the inhibitors of CDK4 (INK4) family, which act
by preventing association of the CDK4 catalytic subunit to the positive
regulator cyclin D1. Inhibitors of the kinase inhibitory protein (Kip)
family can also bind CDK4/cyclin D complexes, although with lower
affinity than the INKs, but this event does not result in efficient
functional inhibition of enzymatic activity (Sherr and Roberts, 1995).
CDK2, in contrast, is positively regulated by cyclin E and negatively regulated by the Kips. Three members of the Kip family have been identified: p21Waf1, p27Kip1, and p57Kip2. It has been shown that p27Kip1 effectively binds to preformed CDK2/cyclin E complexes and
stoichiometrically inhibits their enzymatic activity, thus affecting
the duration of the G1 phase of the cell cycle
(Sherr and Roberts, 1995). Interestingly, only two
G1 regulators are expressed in the SVZ and in the
RMS: p27Kip1 and p19Ink4d. P27Kip1 is expressed in the proximity of the
ventricle (van Lookeren Campagne and Gill, 1998), whereas p19Ink4d
expression is low close to the ventricle and highly enriched in the RMS
(Coskun and Luskin, 2001).
We have demonstrated previously that loss of p27Kip1 function in mice
results in a twofold increase in glial cell number as a result of
impaired exit from the cell cycle of glial progenitors in the cortex
and in the cerebellum (Casaccia-Bonnefil et al., 1997, 1999). The
defective growth arrest was observed for both oligodendrocytes and
astrocytes and resulted in expanded pools of glial cells
(Casaccia-Bonnefil et al., 1997, 1999). The pattern of p27Kip1
expression in the SVZ (van Lookeren Campagne and Gill, 1998) and the
consideration of differences in cell-cycle transit time in distinct
neonatal SVZ subpopulations (Menezes et al., 1995, 1998) led us to
hypothesize a role for p27Kip1 in growth arrest of SVZ progenitor
cells. The detailed information available for the identification of the
primary precursors for adult neurogenesis and their lineage progression
makes the SVZ an ideal experimental system in which to investigate the
cell types that are affected by lack of p27Kip1 function. We now
characterize distinct cell populations in the SVZ of p27Kip1-null mice
(Kiyokawa et al., 1996; Casaccia-Bonnefil et al., 1997, 1999) and show
that p27Kip1 plays an important role in the regulation of type C cell
number. The data presented indicate that CDK2 regulators may have a
critical role in cell-cycle control of transit-amplifying progenitors
and suggest that distinct cell-cycle inhibitors are used by the
different SVZ cell types.
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MATERIALS AND METHODS |
Mouse genotyping. Null and wild-type mice were
obtained from heterozygous p27Kip1 breeding pairs (in a mixed genetic
background of C57BL/6 and B6SJL) (Kiyokawa et al., 1996). The genotype
was determined by PCR analysis of genomic tail DNA. Briefly, tails were
digested in lysis buffer (100 mM NaCl, 10 mM Tris, pH 8.0, 0.5% SDS, 25 mM EDTA, 148 µg/ml proteinase K stock) for
14-18 hr at 55°C. Genomic DNA was isolated and amplified by PCR
using the following primers: 5'-CGCCCCGACTGCATCTGCGTGTTCGAA-3' and
5'-TCAAACGTGAGAGTGTCTAACGG-3' directed against the
p27Kip1 gene and 5'-AGGGCTTATGATTCTGAAAGTCG-3' corresponding to the neo sequence. After a 35 cycle reaction
(denaturation at 94°C for 45 sec, annealing at 62°C for 1 min,
extension at 72°C for 1 min), the wild-type allele yielded a 210 bp
PCR product and the null allele yielded a longer 290 bp product because
of neo insertion in the first exon.
Whole-mount bromodeoxyuridine incorporation.
Bromodeoxyuridine (BrdU, Sigma, St. Louis, MO) was injected
intraperitoneally (100 µg/gm body weight) 1 hr before whole-mount
dissection. Whole mounts were fixed overnight in 3% paraformaldehyde;
washed in PBS; sequentially incubated in 100% methanol (30 min at
 20°C) and 100% acetone (30 min at 20°C); washed three times in
PBS/0.5% Triton X-100; incubated in 2N HCl at 37°C for 10 min;
washed; blocked in 10% horse serum in PBS/0.5% Triton X-100 for 2 hr; incubated for 48-72 hr with 1:200 anti-BrdU antibodies (Dako, Carpinteria, CA) in blocking solution; and revealed with secondary antibodies, avidin-biotin complex (ABC) reaction (Vector
Laboratories, Burlingame, CA), and 0.02% DAB (Sigma) and 0.01%
H2O2 (Fisher, Houston, TX).
The distribution and number of BrdU-positive cells were analyzed in
whole-mount preparations of the lateral wall of the lateral ventricle
using a computerized mapping system (Alvarez-Buylla and Vicario, 1988).
Counts were also analyzed in three subregions of the SVZ, as defined by
the anatomy of the ventricle: the anterior horn from +1.4 mm to 0 mm
relative to bregma, the intermediate bridge from 0 mm to 1.5 mm, and
the inferior horn from 1.5 mm to 2 mm (Mitro and Palkovits, 1981).
Statistical analysis of the samples was performed using a two-tailed
t test distribution for samples of unequal variance.
Whole-mount terminal deoxynucleotidyl transferase-mediated
biotinylated UTP nick end labeling staining. Adult mice (2-3
months of age) were intracardially perfused with 0.9% saline, and the brains were removed. The lateral walls of the lateral ventricle were
dissected, and the resulting whole mounts were fixed overnight in 3%
paraformaldehyde. Whole mounts were immersed for several hours in PBS,
followed by methanol and acetone, as described above, and then washed
three times in PBS. For terminal deoxynucleotidyl transferase
(TdT)-mediated biotinylated UTP nick end labeling (TUNEL) processing,
whole mounts were treated with proteinase K (20 µg/ml in 10 mM Tris/HCl, pH 7.4-7.8) for 30 min at room temperature; washed three times in PBS; and immersed in TdT buffer (Boehringer Mannheim, Indianapolis, IN), 1.25 mM
cobalt chloride, biotinylated dUTPs, and 0.25 U/µl TdT (Boehringer
Mannheim) for 1.5 hr at 37 C. After washing, positive cells were
revealed with Elite ABC (Vector Laboratories) and DAB as substrate. The
distribution and number of TUNEL-positive cells were analyzed in
whole-mount preparations of the lateral wall of the lateral ventricle
using a computerized mapping system (Alvarez-Buylla and Vicario, 1988). For immunostaining in combination with TUNEL, the whole mounts were
first stained with antibodies against polysialic acid
(PSA)-neural cell adhesion molecule (NCAM) (1:1000; a gift from Dr.
Rougon, University de Luminy, Marseille, France) and Dll (1:50;
a gift from Dr. Panganiban, University of Wisconsin, Madison,
WI), GFAP (1:200; Boehringer Mannheim), or Mac-1 (1:50;
Boehringer Mannheim), followed by incubation in Cy-3- or
Cy-5-conjugated secondary antibodies (1:500; Jackson ImmunoResearch
Laboratories, West Grove, PA). The whole mounts were then processed for
TUNEL, with the omission of the proteinase K step, using biotinylated
dUTPs followed by avidin Cy-2 (1:1000; Jackson ImmunoResearch
Laboratories).
Frozen sections and immunohistochemistry on sections.
Wild-type and p27/ animals were anesthetized with avertin and
perfused with 4% paraformaldehyde in PBS. After overnight
post-fixation at 4°C in the same fixative, the tissue was incubated
for 24 hr in 30% sucrose in 0.1 M phosphate
buffer, pH 7.5, embedded in optimal cutting temperature (OCT)
compound, and frozen in cryomolds in an 8-methylbutane bath on
dry ice. Cryosections (10-15 µm thick) were collected on Superfrost
glass slides (Fischer Scientific, Swanee, GA) and processed for
immunofluorescence. Frozen sections were thawed and incubated for 1 hr
in blocking solution (PGBA: 0.1 M PBS, 0.1%
gelatin, 1% BSA, 0.002% sodium azide) with 10% NGS (Vector
Laboratories). Primary antibodies were typically incubated overnight
[Dll 1:50, PSA-NCAM 1:1000, GFAP (Dako, Carpinteria, CA) 1:500,
or TuJ-1 (Sigma) 1:1000 in PGBA containing 10% NGS and 0.3% Triton
X-100], followed by biotinylated secondary antibodies (1:200; Southern
Biotechnology, Birmingham, AL) and avidin conjugated to distinct
fluorophores (1:500; Amersham Biosciences, Arlington Heights, IL). For
immunolabeling with anti-BrdU antibodies, sections were then treated
with 2N HCl for 10 min at 37°C to denature DNA, followed by
equilibration in 0.1 M sodium borate, pH 8.6, for 10 min. Primary antibody anti-BrdU was used at a 1:100 dilution in 0.1 M PBS, 0.1% gelatin, 1% BSA, 0.002% sodium
azide, and 0.5% Triton X-100 for at least 3 hr at room temperature,
followed by Texas Red-conjugated secondary antiserum. Sections were
counterstained with 4',6'-diamidino-2-phenylindole (DAPI) (Molecular
Probes, Eugene, OR) to visualize cell nuclei. Photomicrographs were
obtained with a Hamamatsu (Bridgewater, NJ) CCD camera interfaced to a Leica (Nussloch, Germany) TMC fluorescence microscope. Double labeling was confirmed by scanning individual cells along the z-axis with a Leica TCS confocal microscope.
Electron microscopy, [3H]thymidine
autoradiography, and cell counts. Four adult male wild-type mice
and four p27Kip1-null littermates were injected 1 hr before they were
killed with 50 µl of 1 mCi [3H]thymidine. Animals were deeply
anesthetized with Nembutal and transcardially perfused with 0.9%
saline followed by 100 ml of Karnovsky's fixative (2%
paraformaldehyde and 2.5% glutaraldehyde). The heads were removed and
post-fixed in the same fixative overnight. The brains were then removed
from the skull and washed in 0.1 M PBS for 2 hr.
Transverse 200 µm sections were cut on a Vibratome, post-fixed in 2%
osmium for 2 hr, rinsed, dehydrated, and embedded in araldite
(Durcupan; Fluka, Buchs, Switzerland). Tritiated thymidine autoradiography was performed as described in detail by Doetsch et al.
(1997). For the identification of individual cell types, ultrathin
(0.05 µm) sections were cut with a diamond knife, stained with lead
citrate, and examined under a Jeol (Peabody, MA) 100CX electron
microscope. The number of profiles corresponding to the different cell
types along the ventricular wall of the anterior horn was quantified in
a fixed region in six to eight ultrathin sections for each genotype
under the electron microscope (Doetsch et al., 1997). The dorsolateral
corner of the SVZ was not included in the analysis, because the high
density of cells precludes an accurate quantification in this area.
Cells with only small fragments of cytoplasm or nucleus in a given
section were classified as unidentified. All quantifications were
performed blind as to the genotype. Total cell counts obtained by
single-section analysis are comparable with those in three-dimensional
reconstructions, indicating that our counts are not biased by cell size
(Doetsch et al., 1997). For
[3H]thymidine counts, the dorsoventral
extent of four semithin sections each from wild-type and p27Kip1-null
mice were photographed in their entirety, then re-embedded for electron
microscopy. Labeled and unlabeled cells were then identified and
quantified with an electron microscope.
Neurosphere cultures, BrdU labeling, and staining. Adult
wild-type and knock-out mice were killed, and the SVZ was dissected and
collected in Petri dishes containing PIPES buffer (in
mM: 20 PIPES, 25 glucose, 120 NaCl, 0.5 KCl), pH
7.4. After digestion with papain and DNase as described previously
(Doetsch et al., 1999), cells were dissociated by trituration, using a
fire-polished Pasteur pipette and resuspended in DMEM-F12 supplemented
with N2, 2 mM glutamine, 20 µg/ml insulin, and 15 mM HEPES. The cells were
counted, and equal numbers of cells were plated for wild-type and
knock-out mice in the medium described above supplemented with 20 ng/ml
epidermal growth factor (EGF). After 5 d in culture, the number of
neurospheres from wild-type and knock-out mice was counted. For
differentiation of neurospheres, cells were dissociated by gentle
trituration and then plated onto laminin-coated LabTek chambers
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RESULTS |
Increased incorporation of BrdU in the SVZ of
p27Kip1-null mice
Because p27Kip1 is the only member of the Kip family to be
expressed in the SVZ (van Lookeren Campagne and Gill, 1998), we asked
whether loss of p27Kip1 affects proliferation in this area of postnatal
neurogenesis in the rodent brain. To address this question, 9-week-old
wild-type and p27Kip1-null littermates were pulsed with BrdU for 1 hr
in vivo. The incorporation of BrdU into SVZ cells was then
evaluated by immunohistochemistry in whole-mount preparations of the
walls of the lateral ventricles. Labeled nuclei were counted, and each
BrdU-positive cell was recorded and its location displayed within the
outline of the entire ventricular wall as shown in Figure
1. In p27Kip1-null mice
(n = 8), we observed a statistically significant
increase (p = 0.0028) in the number of
proliferating cells compared with wild-type mice (n = 5). The total number of BrdU-positive cells counted in p27Kip1-null
mice was 4499.5 ± 579 (mean ± SEM; n = 8);
the total number in wild-type littermates was 3756 ± 185 (mean ± SEM; n = 5). Interestingly, the increased
number of BrdU-positive cells was not evenly distributed throughout the
SVZ but rather was localized in the anterior horn, with no differences
in the intermediate bridge or in the posterior horn (Fig. 1). This
pattern of increased BrdU labeling in the mutant mice parallels the
distribution of type A and C cells, whose incidence is higher in the
anterior horn of the lateral ventricles (Doetsch et al., 1997).
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Figure 1.
Increased BrdU incorporation in the SVZ of adult
p27Kip1-null mice. A computer-generated map of BrdU-labeled cells in
the SVZ of adult wild-type (A) and knock-out
(B) mice is shown. Nine-week-old mice received a
single pulse of BrdU and were killed after 1 hr. The number of
proliferating cells was determined by staining SVZ whole mounts with
anti-BrdU antibodies. Each dot represents the nucleus of
a BrdU-positive cell. The black lines indicate arbitrary
anatomical subdivisions of the SVZ, as described in Materials and
Methods. Note that the increased number of BrdU-positive cells in the
p27Kip1-null mice is localized predominantly to the anterior horn
(ah) rather than the intermediate bridge
(ib) or the posterior horn (ph).
The topographic location of the BrdU-positive cells parallels the
distribution of type A and C cells in the adult SVZ.
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Loss of p27Kip1 results in selective increase of type C cells in
the adult SVZ
To identify the cell types that were affected by loss of p27Kip1
function, we performed electron microscopic analysis of the anterior
horn of the SVZ. Six to eight ultrathin transverse sections of the
ventricular lateral wall were obtained from wild-type
(n = 4) and p27Kip1-null (n = 4) mice,
and distinct cell types were identified according to established
ultrastructural criteria using an electron microscope (Doetsch et al.,
1997). Briefly, type A cells (neuroblasts) were identified on the basis
of their elongated morphology, scant dark cytoplasm, abundant free
ribosomes, and microtubules oriented along the longitudinal axis of the
cells. Type B cells (SVZ astrocytes) were characterized by the presence of multiple processes intercalating among other cells, thick bundles of
intermediate filaments, glycogen granules, and dense bodies in the
cytoplasm and gap junctions. Type C cells (transit-amplifying progenitors) were more electron-dense than type B cells and more electron-lucent than type A neuroblasts. They were identified on the
basis of their large size, deeply invaginated nuclei, the presence of
free ribosomes in the cytoplasm, and lack of the intermediate filaments
typical of type B cells.
The total number of cells counted with the electron microscope was 1751 for p27Kip1-null mice and 1536 for wild-type littermates (Table
1). The most striking difference between
the two genotypes was the increased number of type C cells in the
p27Kip1-null mice (Table 1). On average, 19 ± 4 type C cells were
counted in a wild-type mouse SVZ per 1000 µm length, whereas 38 ± 7 C cells were counted in p27Kip1-null mice. Interestingly, no
difference was observed either in the number of ependymal cells
(wild-type, 80 ± 8; p27Kip1-null, 74±18) or in the number of
type B stem cells (wild-type, 63 ± 11; p27Kip1-null, 58 ±16).
The mutant mice also showed a twofold reduction in the number of type A
cells (p27Kip1-null, 29 ± 6) compared with wild-type mice
(56 ± 12).
The increased number of C cells combined with the decrease in type A
cells resulted in the disruption of the characteristic architecture of
the SVZ in p27Kip1-null mice (Figs. 2,
3). In the wild-type mice, B cells were
organized in gliotubes surrounding chains of migratory type A
neuroblasts (Fig. 2). Type C cells, the precursors of type A, cells
were distributed in clusters interspersed among the chains, as
described previously (Doetsch et al., 1997). In contrast, in the
p27Kip1-null mice, type A neuroblasts were sometimes observed at
unusual locations (Figs. 3,
4B,F), whereas the more abundant type C cells were closer to the ventricle and scattered in distribution, primarily interspersed among type B cells
(Figs. 3, 4D,F). Microglia were also more
common in the p27Kip1-null mice than in wild-type mice (see Fig. 6).
These data indicate that loss of p27Kip1 function selectively increases
the number of type C cells and that modification of the relative
proportion of each cell type disrupts the orderly structure of the
SVZ.
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Figure 2.
Loss of p27Kip1 function results in increased
C-cell number and disorganization of the SVZ. A,
Photomicrograph of the typical structure of the adult mouse SVZ as
observed in wild-type animals. The migrating neuroblasts (A cells),
identified by scant dark cytoplasm and dense heterochromatin, are
surrounded by type B cells (B), characterized by
light cytoplasm with several inclusions and irregular contours. Type C
cells (C) are interspersed in clusters, whereas
ependymal cells (E) line the ventricular wall
(4812× magnification). B, Photomicrograph of the SVZ
structure in p27Kip1-null littermates. There are significantly more
type C cells, whereas type B cells and ependymal cells are constant and
type A cells are reduced in number (4156× magnification).
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Figure 3.
Displaced A cells in the p27Kip1-null mice.
Electron micrograph of the anterior horn of the SVZ of mutant mice
showing the ependymal cells (E) and the transit amplifying
progenitors (C) close to the ventricular lumen
(left). Note the unusual location of the migratory A
cells (arrow) away from the lumen of the lateral
ventricle.
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Figure 4.
Identification of proliferating type C cells in
p27Kip1-null mice. Triple immunofluorescence of frozen coronal sections
obtained from the SVZ of wild-type (+/+, A, C, E) and
p27Kip1-null (/, B, D, F) mice and stained
for BrdU (green nuclei), PSA-NCAM
(blue immunofluorescence), and Dll (red
immunofluorescence). The arrowhead indicates a
cell that is BrdU+/PSA-NCAM (B, F) but Dll+
(D, F) and therefore identified as a
proliferating type C cell. Note that in the mutant, the accumulation of
proliferating C cells close to the ventricular lumen (on the
left of each panel) displaces the
PSA-NCAM+ type A cells laterally. Scale bar, 100 µm.
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Proliferation in p27Kip1-null mice
To determine the effect of loss of p27Kip1 on cell proliferation
in the SVZ, we used [3H]thymidine and
BrdU incorporation to label cells in S phase and electron microscopic
analysis and immunohistochemistry to identify labeled cells in the SVZ.
Electron microscopic analysis of brain sections labeled for 1 hr with
[3H]thymidine revealed that the relative
contribution of each population to the total number of proliferating
cells was completely altered in p27Kip1-null mice compared with
controls (Table 2). Although the
proportion of labeled type B cells did not change significantly between
the two genotypes (Table 2), there was an increase in proliferating
transit-amplifying type C cells and a decrease in proliferating type A
neuroblasts in the anterior horn of the SVZ. Type A cells, for
instance, which account for 26% of the total number of proliferating
cells in wild-type mice (8 of 31 cells), represent only 7% of
the total number of cells in S phase in p27Kip1-null mice (2 of 28 cells). Conversely, type C cells accounted for 64% (20 of 31 cells) of
the total number of
[3H]thymidine-labeled cells in wild-type
animals and 86% (24 of 28 cells) of the proliferating population in
the p27Kip1-null mice (Table 2). Proliferation of type B cells did not
change (Table 2). From these data we conclude that the loss of
p27Kip1 does not equally affect the cell-cycle regulation of distinct subpopulations in the SVZ. Loss of this cell-cycle inhibitor increases the number of type C cells and decreases the number of type A cells in
S phase.
To confirm the data obtained from the electron microscopic analysis, we
performed immunohistochemistry on frozen sections, using BrdU to label
cells in S phase and cell-specific markers to identify distinct cell
types. Type A and C cells can be distinguished by double-immunostaining
for Dlx-2 and PSA-NCAM. Type A cells express both PSA-NCAM and Dlx-2,
whereas type C cells express only Dlx-2 (F. Doetsch and A. Alvarez-Buylla, unpublished results). Thus, triple immunostaining for
Dll (which recognizes all forms of the Dlx family, including Dlx-2)
(Panganiban et al., 1995), PSA-NCAM, and BrdU allowed us to distinguish
BrdU-positive type C cells (Dll+/PSA-NCAM) from BrdU-positive type A
cells (Dll+/PSA-NCAM+). Consistent with the results of the
[3H]thymidine labeling,
immunohistochemical analysis of the SVZ of wild-type and p27Kip1-null
mice injected with BrdU revealed that the majority of the BrdU-labeled
cells in p27Kip1-null mice were PSA-NCAM and Dll+ type C cells (Fig.
4).
Loss of p27Kip1 function results in increased apoptosis in
the SVZ
Because type C cells are increased in number and represent the
transit-amplifying population of progenitors for type A neuroblasts, we
investigated why the number of A cells was significantly reduced in the
mutant animals. We therefore performed TUNEL and analyzed the
distribution and number of TUNEL-positive cells in whole-mount preparations of the SVZ to evaluate cell death in the mutant mice. In
p27Kip1-null mice, we counted a total of 568 ± 64 (mean ± SEM, n = 6) TUNEL-positive cells, compared with the
290 ± 18 (mean ± SEM, n = 5) apoptotic
cells in wild-type animals (Fig. 5).
Consistent with an increase in apoptosis in the mutant mice, a large
number of microglial cells (which are typically involved in
phagocytosis of dead cell fragments) were observed in the SVZ of mutant
mice (Fig. 6). To
identify the dying cells, we performed
TUNEL in combination with immunohistochemistry for cell-specific
antigens (Figs. 7, 8). When triple
immunostaining was performed for PSA-NCAM, Dll, and TUNEL, we very
rarely observed labeled A cells (TUNEL+/PSA-NCAM+/Dlx2+) and
occasionally found labeled type C cells (TUNEL+/PSA-NCAM/Dlx2+) (Fig.
7). Colabeling for TUNEL and specific antibodies for type B cells
(i.e., GFAP) or microglia (i.e., Mac-1) also revealed few
double-labeled cells. The majority of TUNEL-positive cells did not
stain with any cellular markers, suggesting that apoptotic cells may
downregulate expression of markers before death.
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Figure 5.
Increased apoptosis in whole mounts of SVZ from
p27Kip1-null mice. A computer-generated map of the SVZ of adult
wild-type (A) and knock-out
(B) mice whole mounts stained with TUNEL. Each
dot represents the nucleus of a TUNEL-positive cell, and
the black lines indicate arbitrary subdivisions of the SVZ
based on anatomical landmarks, as defined in Materials and Methods.
Note that the increased number of TUNEL-positive cells in the
p27Kip1-null mice is localized predominantly to the anterior horn
(ah) of the SVZ rather than the intermediate bridge
(ib) or the posterior horn (ph),
which parallels the distribution of type A and C cells in the adult
SVZ.
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Figure 6.
Microglial cells in the p27Kip1-null mouse SVZ.
A, Photomicrograph of the adult mouse SVZ, as observed
in wild-type animals. Occasional microglial cells (M,
arrows) can be observed (4812× magnification).
B, A typical section from p27Kip1-null mice reveals an
increased number of microglial cells (M,
arrows) (6453× magnification). A, A
cells; B, B cells; E, ependymal
cells.
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Figure 7.
Identification of apoptotic cells in the SVZ of
wild-type and p27Kip1-null mice using type A- and type C-specific cell
markers. Triple immunofluorescence of whole mounts from wild-type mice
(+/+, left column) and p27Kip1-null mice (/,
right column) stained for TUNEL
(green nuclei), PSA-NCAM (blue
immunofluorescence), and Dll (red immunofluorescence).
The arrow indicates the apoptotic nucleus (B)
of a cell that is Dll-positive (F) and PSA-NCAM-negative
(D) and therefore identified as a type C apoptotic cell in
the mutant mice. Scale bar, 50 µm.
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Figure 8.
Identification of apoptotic cells in the SVZ of
wild-type and p27Kip1-null mice using type B-specific (gfap)
and microglial-specific (mac-1) cell markers. Double
immunofluorescence of whole mounts from wild-type mice (+/+) and
p27Kip1-null mice (/) stained for TUNEL
(green nuclei), GFAP (red
immunofluorescence; A-F), or Mac-1
(red immunofluorescence; G-L). The
arrow indicates an apoptotic nucleus of a microglial
cell in wild-type mice. The arrowheads indicate
apoptotic cells that do not stain with Mac-1 in the mutant mice. Scale
bar, 150 µm.
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Loss of p27Kip1 function results in increased number
of neurospheres
Single precursor cells isolated from the adult brain and cultured
in suspension divide to generate clusters of cells called neurospheres
in the presence of appropriate growth factors (Reynolds and Weiss,
1992; Morshead et al., 1994). The SVZ is the richest source of
EGF-responsive neurospheres in the adult brain. We therefore dissociated the SVZ from wild-type and p27Kip1 littermates (four mice
per experiment) and cultured the same number of cells in medium
containing EGF (10 ng/ml) to determine the ability of p27Kip1-null mice
to generate neurospheres. After 5 d, the total number of neurospheres was counted in wild-type and knock-out dishes.
Interestingly, the average of three independent experiments revealed a
significant increase in the number of neurospheres generated from
p27Kip1-null mice (mean, 2077 ± 91) compared with wild-type mice
(mean, 1461 ± 165). No difference was observed between wild-type
and p27Kip1-null neurospheres in their ability to generate O4+
oligodendrocytes, GFAP+ astrocytes, and TuJ-1+ neurons (Fig.
9). We conclude that loss of p27Kip1
results in increased numbers of neurospheres, although it does not
affect their ability to generate neurons and glia.
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Figure 9.
Neurospheres isolated from p27Kip1-null mice
maintain their ability to differentiate in vitro.
Pluripotent cells were isolated from the SVZ of p27Kip1-null mice and
grown in vitro to generate neurospheres. After
dissociation and plating on laminin-coated dishes, these cells
differentiate into glial cells (A) or into
neurons (B). A, p27Kip1-null
GFAP-positive astrocyte (green
immunofluorescence). B, p27Kip1-null A cell stained with
the neural marker TuJ-1 (red immunofluorescence).
Individual nuclei are labeled by DAPI (blue
immunofluorescence). Scale bar, 25 µm.
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DISCUSSION |
The number of stem cells, transit-amplifying progenitors, and
neuroblasts at any given stage in the SVZ lineage is the result of a
dynamic equilibrium between self-renewal, lineage progression, and
death (Fig. 10). In this report, we
have examined the consequences of eliminating one of the crucial
G1 regulators of the cell cycle, the CDK
inhibitor p27Kip1, on the cell types in the SVZ.
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Figure 10.
Schematic model of population dynamics in the
anterior horn of the SVZ. A model of SVZ lineage progression is shown.
Type B cells are the GFAP-positive primary precursors that give rise to
type C transit-amplifying progenitors, which can further differentiate
into type A migratory neuroblasts. The number of cells in each stage of
the lineage is determined by self-renewal, differentiation, or
death.
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The main conclusion of this study is that loss of the
G1 regulator p27Kip1 selectively increases the
number of the transit-amplifying type C cells, concomitant with a
decrease in the number of type A neuroblasts. This clearly indicates
that cell-cycle regulation is not equivalent in the different cell
populations of the SVZ. If the role of p27Kip1 was equivalent in
distinct SVZ cell types, then its loss would equally affect cell
division in the three cell populations and proportionally increase the
number of cells in each population. In contrast, loss of p27Kip1
enhanced proliferation of type C cells and not B cells or A
neuroblasts, as evidenced by the increased number of
[3H]thymidine-positive and BrdU-positive
C cells and by the increase in the total number of type C cells. Our
results suggest that the different SVZ cell types rely on distinct
molecules regulating the G1/S transition, which
represents, for the majority of eukaryotic cells, the decisional point
between exit or re-entry into the cell cycle. More specifically, we
show here that the number of type C cells is regulated primarily by the
p27Kip1 inhibitor acting at the G1/S checkpoint.
P27Kip1 is expressed in the SVZ (van Lookeren Campagne and Gill, 1998)
and has been shown to regulate the length of the
G1 phase of the cell cycle in CNS progenitors
(Mitsuhashi et al., 2001). It is likely that in the absence of p27Kip1,
type C cells lose their inhibitory control and that this results in an
untimely exit from the cell cycle, as reported in other cellular systems (Durand et al., 1998). Although our data do not directly rule
out the possibility that loss of p27Kip1 may selectively increase the
duration of the S phase in C cells and therefore result in an increased
number of [3H]thymidine-positive and
BrdU-positive cells, this is an unlikely scenario. P27Kip1 was
originally identified as a regulator of the G1
phase of the cell cycle (Polyak et al., 1994; Toyoshima and Hunter,
1994). Consistent with this role, transgenic mice overexpressing
p27Kip1 in CNS progenitor cells show lengthening of the
G1 phase of the cell cycle (Mitsuhashi et al.,
2001). In addition, loss of p27Kip1 function in rodents
(Casaccia-Bonnefil et al., 1997; Durand et al., 1998), in
Drosophila (de Nooij et al., 1996; Lane et al., 1996), or in
Caenorhabditis elegans (Hong et al., 1998) has been shown
not to affect the length of the S phase but rather the
G1 checkpoint of progenitor cells, which is
crucial for determining the timing of cell-cycle exit before differentiation. We propose that the increased number of type C cells
in the adult SVZ of p27Kip1-null mice is the consequence of additional
rounds of cell division caused by loss of one of the crucial
regulators of the G1 checkpoint.
The selectivity of the effect of p27Kip1 in regulating the
G1 phase of type C cells is also supported by our
finding that the number of type B cells is not changed and type A cells
are actually decreased in number. The lower number of type A cells in S
phase in the p27Kip1-null mice may be attributable to compensatory mechanisms of homeostasis. Recently, type A neuroblasts have been shown
to express the G1 regulator p19Ink4d (Zindy et
al., 1997; Coskun and Luskin, 2001). The increased number of type C
cells in p27Kip1-null mice may elicit a feedback signal onto type A cells, which, through a p19Ink4d-dependent mechanism, results in type A
cells undergoing fewer rounds of cell division. Distinct cell-cycle
molecules, therefore, may have cell type-selective and stage-specific
effects during the SVZ lineage progression. A second mechanism that
could underlie the decreased number of type A cells is increased
apoptosis (Fig. 10). In p27Kip1-null mice, increased numbers of
TUNEL-positive cells were present. Triple immunohistochemistry for
TUNEL, PSA-NCAM, and Dll revealed that some TUNEL-positive cells were
type C cells, and very occasionally A cells, but that the majority of
TUNEL-positive cells did not express cellular markers. This could be
attributable to the downregulation of markers by dying cells. It is
likely that increased apoptosis is an additional compensatory mechanism
activated in the p27Kip1-null mice to maintain cell number in the SVZ.
The most likely explanation for the decreased number of type A cells is
therefore a delay in cell-cycle exit by type C cells. Loss of the
G1 checkpoint in p27Kip1-null mice likely causes
type C cells to undergo extra rounds of cell division at the expense of
lineage progression. The fact that C cells do eventually progress into
A cells suggests that other cell-cycle inhibitors are involved in this
decision. Because of the increased number of dividing C cells close to
the ventricle and their delayed progression into the lineage, type A
cells appear "dislocated" away from the ventricular lumen. As these
C cells progress into type A neuroblasts, however, the cell-cycle
control is taken over by the G1 inhibitor
p19Ink4d (Coskun and Luskin, 2001). This possible compensatory
mechanism results in decreased A cell proliferation, exit from the cell cycle, and aggregation of type A neuroblasts in chains that converge to
form the RMS.
In conclusion, by taking advantage of the known lineage relationships
in the adult SVZ, we have identified p27Kip1 as one of the main
cell-cycle inhibitors regulating proliferation of the
transit-amplifying progenitors, type C cells. In the absence of p27Kip1
function, control of proliferation is lost in a cell-specific manner,
and this results in increased division of type C cells and in a
consequent decrease of type A neuroblasts and altered histogenesis of
the SVZ.
| |
FOOTNOTES |
Received March 19, 2001; revised Dec. 10, 2001; accepted Dec. 18, 2001.
This paper was supported by National Multiple Sclerosis Society Grant
RG 3154-A-2 (P.C.B.) and National Institutes of Health Grants HD23315
(M.V.C.) and HD32116 (A.A.-B.). We are extremely grateful to Dr.
Pangagiban for the generous gift of the anti-Dll antibody; to Parawmen
Navidad, and Michela Muggironi for technical help; and to Drs.
Nowakowski and Di Cicco-Bloom for critical reading of this manuscript.
Correspondence should be addressed to Patrizia Casaccia-Bonnefil,
Department of Neuroscience and Cell Biology, University of Medicine and
Dentistry of New Jersey, 675 Hoes Lane, Piscataway, NJ 08854. E-mail: casaccpa{at}umdnj.edu.
| |
REFERENCES |
-
Alvarez-Buylla A,
Vicario DS
(1988)
Simple microcomputer system for mapping tissue sections with the light microscope.
J Neurosci Methods
25:165-173[Web of Science][Medline].
-
Biebl M,
Cooper CM,
Winkler J,
Kuhn HG
(2000)
Analysis of neurogenesis and programmed cell death reveals a self-renewing capacity in the adult rat brain.
Neurosci Lett
291:17-20[Web of Science][Medline].
-
Casaccia-Bonnefil P,
Kiyokawa H,
Tikoo R,
Friedrich V,
Koff A,
Chao MVC
(1997)
Oligodendrocyte precursor differentiation is perturbed in the absence of the cyclin-dependent kinase inhibitor p27Kip1.
Genes Dev
11:2335-2346[Abstract/Free Full Text].
-
Casaccia-Bonnefil P,
Hardy RJ,
Teng KK,
Levine JM,
Koff A,
Chao MV
(1999)
Loss of p27Kip1 function results in increased proliferative capacity of oligodendrocyte progenitors but unaltered timing of differentiation.
Development
126:4027-4037[Abstract].
-
Caviness Jr VS,
Takahashi T,
Nowakowki RS
(1999)
The G1 restriction point as critical regulator of cortical neuronogenesis.
Neurochem Res
24:497-506[Web of Science][Medline].
-
Coskun V,
Luskin MB
(2001)
The expression pattern of cell cycle inhibitor p19 ink4d by progenitor cells of the rat embryonic telencephalon and the neonatal anterior subventricular zone.
J Neurosci
21:3092-3103[Abstract/Free Full Text].
-
de Nooij J,
Letendre M,
Hariharan I
(1996)
A cyclin-dependent kinase inhibitor, Dacapo, is necessary for timely exit from the cell cycle during Drosophila embryogenesis.
Cell
87:1237-1247[Web of Science][Medline].
-
Doetsch F,
Alvarez-Buylla A
(1996)
Network of tangential pathway for neural migration in adult mammalian brain.
Proc Natl Acad Sci USA
93:14895-14900[Abstract/Free Full Text].
-
Doetsch F,
Garcia-Verdugo JM,
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[Abstract/Free Full Text].
-
Doetsch F,
Caille I,
Lim DA,
Garcia-Verdugo JM,
Alvarez-Buylla A
(1999a)
Subventricular zone astrocytes are neural stem cells in the adult mammalian brain.
Cell
97:703-716[Web of Science][Medline].
-
Doetsch F,
Garcia-Verdugo JM,
Alvarez-Buylla A
(1999b)
Regeneration of a germinal layer in the adult mammalian brain.
Proc Natl Acad Sci USA
96:11619-11624[Abstract/Free Full Text].
-
Durand B,
Fero ML,
Roberts JM,
Raff MC
(1998)
p27Kip1 alters the response of cells to mitogen and is part of a cell-intrinsic timer that arrests the cell cycle and initiates differentiation.
Curr Biol
8:431-440[Web of Science][Medline].
-
Hong Y,
Roy R,
Ambros V
(1998)
Developmental regulation of a cyclin-dependent kinase inhibitor controls cell cycle progression in Caenorhabditis elegans.
Development
125:3585-3597[Abstract].
-
Kiyokawa H,
Kineman RD,
Manova-Todorova KO,
Soares VC,
Hoffman ES,
Ono M,
Khanam D,
Hayday AC,
Frohman LA,
Koff A
(1996)
Enhanced growth of mice lacking the cyclin-dependent kinase inhibitor function of p27(Kip1).
Cell
85:721-732[Web of Science][Medline].
-
Lane M,
Sauer K,
Wallace K,
Jan Y,
Lehner C,
Vaessin H
(1996)
Dacapo, a cyclin-dependent kinase inhibitor, stops cell proliferation during Drosophila development.
Cell
87:1225-1235[Web of Science][Medline].
-
Levison SW,
Rothstein RP,
Brazel CY,
Young GM,
Albrecht PJ
(2000)
Selective apoptosis within the rat subependymal zone: a plausible mechanism for determining which lineages develop from neural stem cells.
Dev Neurosci
22:106-115[Medline].
-
Lois C,
Alvarez-Buylla A
(1994)
Long distance neuronal migration in the adult mammalian brain.
Science
264:1145-1148[Abstract/Free Full Text].
-
Menezes JR,
Smith CM,
Nelson KC,
Luskin MB
(1995)
The division of neuronal progenitor cells during migration in the neonatal mammalian forebrain.
Mol Cell Neurosci
6:496-508[Web of Science][Medline].
-
Menezes JR,
Dias F,
Garson AV,
Lent R
(1998)
Restricted distribution of S-phase cells in the anterior subventricular zone of the postnatal mouse forebrain.
Anat Embryol
198:205-211[Medline].
-
Mitro M,
Palkovits M
(1981)
Morphology of the rat brain ventricles, ependymal and periventricular structures.
In: Bibliotecha anatomica, No. 21 (Lierse W,
ed). Basel: Karger.
-
Mitsuhashi T,
Aoki Y,
Eksaioglu YZ,
Takahashi T,
Bhide PG,
Reeves SA,
Caviness VSJ
(2001)
Overexpression of p27kip1 lengthens the G1 phase in a mouse model that targets inducible gene expression to central nervous system progenitor cells.
Proc Natl Acad Sci USA
98:6435-6440[Abstract/Free Full Text].
-
Morshead CM,
van der Kooy D
(1992)
Postmitotic death is the fate of constitutively proliferating cells in the subependymal layer of the adult mouse brain.
J Neurosci
12:249-256[Abstract].
-
Morshead C,
Reynolds BA,
Craig CG,
McBurney MW,
Staines WA,
Morassutti D,
Weiss S,
van der Kooy D
(1994)
Neural stem cells in the adult mammalian forebrain: a relatively quiescent subpopulation of subependymal cells.
Neuron
13:1071-1082[Web of Science][Medline].
-
Panganiban G,
Sebring A,
Nagy L,
Carroll S
(1995)
The development of crustacean limbs and the evolution of arthropods.
Science
270:1363-1366[Abstract/Free Full Text].
-
Peretto P,
Merighi A,
Fasolo A,
Bonfanti L
(1999)
The subependymal layer in rodents: a site of structural plasticity and cell migration in the adult mammalian brain.
Brain Res Bull
49:221-243[Web of Science][Medline].
-
Polyak K,
Lee MH,
Erdjument-Bromage H,
Koff A,
Roberts JM,
Tempst P,
Massague J
(1994)
Cloning of p27Kip1, a cyclin-dependent kinase inhibitor and a potential mediator of extracellular antimitogenic signals.
Cell
78:59-66[Web of Science][Medline].
-
Reynolds BA,
Weiss S
(1992)
Generation of neurons and astrocytes from isolated cells of the adult mammalian central nervous system.
Science
255:1707-1710[Abstract/Free Full Text].
-
Schultze B,
Korr H
(1981)
Cell kinetic studies of different cell types in the developing and adult brain of the rat and mouse: a review.
Cell Tissue Kinet
14:309-325[Web of Science][Medline].
-
Sherr CJ,
Roberts JM
(1995)
Inhibitors of mammalian G1 cyclin-dependent kinases.
Genes Dev
9:1149-1163[Free Full Text].
-
Smith CM,
Luskin MB
(1998)
Cell cycle length of olfactory bulb neuronal progenitors in the rostral migratory stream.
Dev Dyn
213:220-227[Web of Science][Medline].
-
Steindler DA,
Kadrie T,
Fillmore H,
Thomaas LB
(1996)
The subependymal zone: "brain marrow."
Prog Brain Res
108:349-363[Web of Science][Medline].
-
Temple S,
Alvarez-Buylla A
(1999)
Stem cells in the adult mammalian central nervous system.
Curr Opin Neurobiol
9:135-141[Web of Science][Medline].
-
Thomaidou D,
Mione MC,
Cavanagh JFR,
Parnavelas JG
(1997)
Apoptosis and its relation to the cell cycle in the developing cerebral cortex.
J Neurosci
17:1075-1085[Abstract/Free Full Text].
-
Toyoshima H,
Hunter T
(1994)
p27, a novel inhibitor of G1 cyclin-Cdk protein kinase activity, is related to p21.
Cell
78:67-74[Web of Science][Medline].
-
van Lookeren Campagne M,
Gill R
(1998)
Tumor-suppressor p53 is expressed in proliferating and newly formed neurons of the embryonic and postnatal rat brain: comparison with expression of the cell cycle regulators p21waf1, p27Kip1, p57Kip2, p16Ink4a, cyclin G1, and the protooncogene Bax.
J Comp Neurol
397:181-198[Web of Science][Medline].
-
Zindy F,
Soares H,
Herzog K-H,
Morgan J,
Sherr CJ,
Roussel MF
(1997)
Expression of INK4 inhibitors of cyclin D-dependent kinases during mouse brain development.
Cell Growth Differ
8:1139-1150[Abstract].
Copyright © 2002 Society for Neuroscience 0270-6474/02/2262255-10$05.00/0
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|
|
|
|
|
|
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An inhibition of cyclin-dependent kinases that lengthens, but does not arrest, neuroepithelial cell cycle induces premature neurogenesis
J. Cell Sci.,
December 15, 2003;
116(24):
4947 - 4955.
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D. Kaushal, J. J. A. Contos, K. Treuner, A. H. Yang, M. A. Kingsbury, S. K. Rehen, M. J. McConnell, M. Okabe, C. Barlow, and J. Chun
Alteration of Gene Expression by Chromosome Loss in the Postnatal Mouse Brain
J. Neurosci.,
July 2, 2003;
23(13):
5599 - 5606.
[Abstract]
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ABSTRACT
INTRODUCTION
