1 Institute of Molecular and Cellular Biosciences, University of Tokyo, 1-1-1
Yayoi, Bunkyo-ku, Tokyo 113-0032, Japan
2 Department of Anatomy, Keio University School of Medicine, Tokyo 160-8582,
Japan
3 Department of Molecular Neurobiology, Institute of DNA Medicine, Jikei
University School of Medicine, 3-25-8 Nishi-shinbashi, Minato-ku, Tokyo
105-8461, Japan
4 National Institute for Physiological Sciences, Myodaiji, Okazaki 444-8585,
Japan
* Author for correspondence (e-mail: ygotoh{at}iam.u-tokyo.ac.jp)
Accepted 27 February 2004
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SUMMARY |
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Key words: ß-catenin, Wnt, Neurogenesis, Neocortex, Neural precursor cell, Mouse
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Introduction |
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Wnt genes encode secreted factors that regulate various cell fate decisions
depending on the cellular context. In mammals, 19 Wnt genes have been
identified to date, and several of these (such as Wnt7a, Wnt7b, Wnt2b and
Wnt8b) are expressed during cortical development in complex spatiotemporal
patterns (Fougerousse et al.,
2000; Grove et al.,
1998
; Kim et al.,
2001a
; Lee et al.,
2000
). Wnt proteins signal through a receptor complex composed of
members of the Frizzled (Fz) and low-density lipoprotein receptor-related
protein (Lrp) families, and activate a number of intracellular signaling
pathways including the ß-catenin/TCF pathway (known as the canonical Wnt
pathway) (Brantjes et al.,
2002
; Wodarz and Nusse,
1998
) and non-canonical pathways, including those mediated by Jun
N-terminal kinase (Jnk) or Ca2+
(Habas et al., 2003
;
Huelsken and Birchmeier, 2001
;
Kuhl et al., 2000
). Recent
reports have shown that the canonical Wnt pathway promotes the self-renewal
capacity of some tissue stem cells such as hematopoietic stem cells
(Reya et al., 2003
;
Willert et al., 2003
). The Wnt
signals have also been implicated in promoting self-renewal during neural
development; ectopic expression of Wnt1 or stabilized ß-catenin in the
early stages of chick spinal cord or mouse forebrain development,
respectively, has been shown to result in an increased number of NPCs and
suppression of neuronal differentiation
(Chenn and Walsh, 2002
;
Megason and McMahon, 2002
).
Infection of mouse forebrain explants with a retrovirus expressing HA
epitope-tagged Wnt7a also promoted proliferation and suppressed neuronal
differentiation of NPCs (Viti et al.,
2003
). However, activation of a TCF-dependent reporter gene
construct was detected in differentiating neurons of mouse cortical
development (Maretto et al.,
2003
), implying a potential role of the canonical Wnt pathway in
the differentiation of cortical neurons. In addition, Wnt1 promotes neuronal
differentiation of the embryonic carcinoma cell line P19
(Lyu et al., 2003
;
Smolich and Papkoff, 1994
;
Tang et al., 2002
). It has
remained unclear, however, whether Wnt is important for promoting neuronal
differentiation in the developing neocortex.
In contrast to the recent reports mentioned above that indicate a role for Wnts in the expansion of NPCs, we now show that Wnt7a promotes neuronal differentiation of NPCs in the developing mouse neocortex, at the expense of NPCs. Inhibition of the canonical Wnt pathway suppressed neuronal differentiation in vitro and in the developing neocortex. This effect appears to be mediated at least in part by direct regulation of Ngn1 promoter by the ß-catenin-TCF complex. Importantly, Wnt signaling promotes neuronal differentiation only at late stages of cortical development. These results demonstrate that Wnt7a or related Wnt proteins may play an essential role in triggering neurogenesis during cortical development in a stage-specific manner and imply that the timing of differentiation can be determined not only by the expression of differentiation-inducing factors, but also by the responsiveness of the cells to these factors.
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Materials and methods |
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The pMX-IRES-EGFP vector was used for the production of recombinant retroviruses. Ecotropic virus-packaging (PLAT-E) cells were transfected with the desired plasmids with the use of FuGENE 6 (Roche) and then cultured for 3 days at 32°C. Retroviral particles were collected from the culture supernatant by centrifugation at 6000 g for 16 hours at 4°C and were resuspended in serum-free Dulbecco's modified Eagle's medium (DMEM)-F12 (1:1) (Invitrogen).
Primary antibodies used in this study included mouse monoclonal antibodies to ßIII-tubulin (TuJ1, Babco), to GFAP (Chemicon), to Nestin (Becton Dickinson) and to HuC/D (Molecular Probes), as well as rabbit polyclonal antibodies to the 150 kDa neurofilament protein (Chemicon) and to GFP (MBL). Alexa-labeled secondary antibodies were from Molecular Probes.
Primary NPC culture and immunostaining
Primary NPCs were prepared from the dorsal cerebral cortex of ICR mouse
embryos at E11.5 (E1 was defined as 12 hours after detection of the vaginal
plug). Dissected cortices were transferred to artificial cerebrospinal fluid
(aCSF: 124 mM NaCl, 5 mM KCl, 2 mM CaCl2, 26 mM NaHCO3,
1.3 mM MgCl2, 10 mM glucose) containing 0.05% trypsin (Sigma) and
incubated for 10 minutes on ice to remove overlying epidermal ectoderm. The
cortices were then transferred to aCSF containing 0.1% trypsin, DNase I (0.1
mg/ml) (Roche) and hyaluronidase (0.67 mg/ml) (Sigma), and incubated at
37°C for 10 minutes. After the addition of an equal volume of aCSF
containing trypsin inhibitor (0.7 mg/ml) (Sigma), the neuroepithelium was
transferred to DMEM-F12 (1:1) and mechanically dissociated into single cells.
The dissociated cells were cultured in DMEM-F12 (1:1) supplemented with B27
(Invitrogen), Fgf2 (20 ng/ml) (Roche) and Egf (20 ng/ml) (Upstate
Biotechnology). For retroviral infection, cells were mixed with recombinant
viruses for 24 hours, washed with phosphate-buffered saline (PBS) and then
incubated in the absence or presence of Fgf2. For immunostaining, cells were
fixed with 4% paraformaldehyde in PBS, permeabilized with 0.5% Triton X-100
for 30 minutes, incubated with primary antibodies for 2 hours and then with
secondary antibodies for 30 minutes, and mounted in Mowiol (Calbiochem).
Clonal analysis
NPCs (7.9x102 cells/mm2) were plated on dishes
coated with poly-D-lysine and infected with retroviruses encoding either GFP
alone (control) or both GFP and S33Y ß-catenin at a low titer (0.21
infected cells/mm2). To confirm each colony was a `clone' generated
from a single cell, we infected NPCs with a mixture of retroviruses encoding
GFP and those encoding DsRed2 at the same titer. After incubation for 3 days,
no colonies in the culture contained both GFP+ cells and
DsRed2+ cells, showing that each colony was derived from a single
cell under this condition.
In utero electroporation
Introduction of plasmid DNA into the neuroepithelial cells of mouse embryos
in utero was performed as described
(Tabata and Nakajima, 2001).
Plasmid DNA for histone H2B-GFP (pH2B-EGFP) (0.1 mg/ml) and the test plasmid
(0.4 mg/ml) were injected into the lateral ventricle of each littermate at
E13.5; in other experiments, the pMX-based vectors were injected at a
concentration of 5 µg/µl. Electrodes were placed flanking the equivalent
ventricular region of each embryo, covered with a drop of PBS and pulsed 8
times at 40 V for 50 ms separated by intervals of 950 ms with an
electroporator (CUY21E; Tokiwa Science). The uterine horn was placed back into
the abdominal cavity to allow the embryos to continue development. Two days
after electroporation, the embryos were harvested, and the brains were removed
and fixed with 4% paraformaldehyde in PBS at 4°C overnight. After
equilibration with 30% (w/v) sucrose in PBS, the fixed tissue was embedded in
OCT compound (Sakura) and frozen. Coronal sections were prepared by cutting
the frozen brain with a cryostat (CM1850, Leica) at a thickness of 6 or 10
µm and mounted on glass slides coated with MAS (Matsunami). Sections with a
thickness of 6 µm were used for quantitative analysis.
Chromatin immunoprecipitation assay
Primary NPCs were cultured in suspension for 3 days, after which
neurospheres were collected and dissociated. The dissociated cells were plated
on dishes coated with poly-D-lysine and harvested after incubation for 2 days.
The cells were suspended in lysis solution [1% SDS, 10 mM EDTA, 50 mM Tris-HCl
(pH 8.1)] and sonicated to shear genomic chromatin into DNA fragments of
0.5 to 1.0 kb. The lysate was incubated for 2 hours with protein
A-conjugated beads, after which the beads were removed and the lysate was
incubated overnight at 4°C with antibodies to ß-catenin or control
IgG (Santa Cruz Biotechnology). After the addition of protein A beads, the
mixture was incubated with rotation for 1 hour. The beads were then isolated
and washed consecutively with a low-salt solution [0.1% SDS, 1% Triton X-100,
2 mM EDTA, 20 mM Tris-HCl (pH 8.1), 150 mM NaCl], with a high-salt solution
[0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl (pH 8.1), 500 mM NaCl],
with a LiCl solution [0.25 M LiCl, 1% NP-40, 1% sodium deoxycholate, 1 mM
EDTA, 10 mM Tris-HCl (pH 8.1)] and twice with a Tris-EDTA solution [10 mM
Tris-HCl (pH 8.0), 1 mM EDTA]. Immune complexes were then eluted from the
beads with a solution containing 10 mM dithiothreitol, 1% SDS and 0.1 M
NaHCO3, after which NaCl was added to a final concentration of 0.2
M and the elute was incubated at 65°C overnight to induce the dissociation
of proteins from DNA. The proteins were eliminated by digestion with
proteinase K at 45°C for 1 hour and the DNA was purified with a QIAquick
spin column (Qiagen). PCR was performed with the eluted DNA and primers
specific for the TCF binding element within the promoter region of the mouse
Ngn1 gene (forward, 5'-CTGCCCAAGAGCTGCTACAGAGGG-3'; reverse,
5'-GCCGGACAGCAATAGAGGCTCAGG-3'). The PCR products were labeled by
incorporation of [
-32P]dCTP and were detected by
electrophoresis on a 5% polyacrylamide gel and autoradiography. The PCR
reaction was performed within the range that the PCR products increased
approximately twofold every cycle.
Luciferase assay
NPCs (8.0 x 105 cells/ml) were plated on dishes coated
with poly-D-lysine and transfected with expression plasmids and the pRL-TK
plasmid encoding Renilla luciferase (Promega) with the use of
Lipofectamine 2000 (Invitrogen). Cell extracts were subsequently prepared and
assayed for luciferase activity (Toyo Ink). Firefly luciferase activity was
normalized relative to the activity of Renilla luciferase.
Quantitative analysis of immunohistochemistry
Immunohistochemistry of the brain sections was carried out as described
(Shen et al., 2002).
Quantitative analysis of marker expression was performed by Laser Scanning
Cytometry (LSC, Olympus) as follows. The area of the nucleus of each
electroporated cell was outlined automatically from the fluorescence of
histone H2B-GFP with the use of a CCD camera and LSC software. Within each
outlined area, the integral of the fluorescence intensity of the
Alexa633-conjugated secondary antibodies was measured. This analysis was
performed doubly blind with several sections, and a total of at least 200
electroporated cells were examined per sample. As LSC software can show the
location of each electroporated cell in the neocortex and its fluorescence
intensity, we determined the approximate expression level of the marker
proteins at the border between the ventricular zone (VZ) and the intermediate
zone (IMZ). We confirmed that the cells exhibiting lower and higher expression
levels of the neuronal marker proteins relative to the expression level at
this border were predominantly localized in the VZ or in the IMZ and cortical
plate, respectively (data not shown). Other fluorescence images were obtained
with a confocal laser microscope (LSM510, Zeiss).
RT-PCR
Total RNA was obtained from infected NPCs using TRIzol (Invitrogen)
following the instructions of the manufacturer. Reverse transcription (RT) was
performed with 10 µg of total RNA, oligo d(T)12-18 (Invitrogen)
primers and ReverTra Ace (TOYOBO). cDNA was amplified by PCR using ex Taq
(TaKaRa). The sense and antisense primers used were as follows: neurogenin 1,
sense 5'-ATGCCTGCCCCTTTGGAGAC-3' and antisense
5'-TGCATGCGGTTGCGCTCGC-3'; and Gapdh, sense
5'-CATTGACCTCAACTACATGG-3' and antisense
5'-TTGCCCACAGCCTTGGCAGC-3'. PCR products were labeled by adding
[-32P] dCTP, separated on a 5% polyacrylamide gel, and
detected by autoradiography.
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Results |
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We next investigated which intracellular signaling pathway mediates the
effect of Wnt7a on neurogenesis in cortical NPC cultures. Wnt7a activates
either the canonical ß-catenin-dependent pathway or non-canonical
pathways, depending on the cellular context
(Kengaku et al., 1998;
Lucas and Salinas, 1997
).
Expression of either of two stabilized mutants of ß-catenin (
N90
or S33Y) (Morin et al., 1997
)
markedly enhanced the effect of growth factor withdrawal on the increase of
TuJ1+ cell number (Fig.
2A). Most of the TuJ1- cells were Nestin+
and when the cells were cultured 3 days longer, many of them became
TuJ1+ (see Fig. S3 at
http://dev.biologists.org/supplemental/).
Expression of S33Y ß-catenin also increased the population of cells
positive for neurofilament (NF), which is largely restricted to postmitotic
neurons with elaborate neurites (Lee and
Cleveland, 1996
) (Fig.
2B,C). We also examined the role of the JNK pathway on
neurogenesis of NPCs, by use of a constitutively active form of JNK, the
fusion protein MKK7-JNK in which JNK1 is intramolecularly phosphorylated and
activated by MKK7. Expression of this construct did not result in an increase
in the percentage of neurons in NPC cultures
(Fig. 2A), although it did
cause an increase in the transcriptional activity of an AP1-dependent
luciferase reporter gene, which serves as a monitor of JNK pathway activity
(data not shown). These results thus suggest that activation of the canonical
Wnt pathway causes an increase in the neuronal population in cultured cortical
NPCs, whereas activation of JNK does not. Moreover, when the canonical Wnt
pathway was blocked in NPCs by ectopic expression of Axin, which inhibits Wnt
signaling by destabilizing ß-catenin
(Zeng et al., 1997
), the
proportion of TuJ1+ cells was substantially reduced compared with
NPCs infected with control retrovirus (see Fig. S2 at
http://dev.biologists.org/supplemental/),
suggesting that the endogenous activity of the canonical Wnt pathway plays an
important role in neuronal differentiation in neocortical NPCs in culture.
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|
After culture of NPCs for 6 days in the presence of Fgf2, we detected very
few (<1%) GFAP+ glial cells or cells positive for the
oligodendrocyte marker O4 among GFP+ NPCs, and the expression of
S33Y ß-catenin did not affect the proportion of these glial cells. Next,
we stained for dying cells by immunostaining with antibodies to cleaved
caspase 3 (Srinivasan et al.,
1998). We found that the percentage of cleaved caspase
3+ cells was less than 1% among GFP+ NPCs, regardless of
the presence or absence of Fgf2, and regardless of whether S33Y-ß-catenin
was ectopically expressed (3 days) or not. Therefore, neither selective cell
death nor suppression of glial differentiation appeared to account for the
increase in the neuronal population induced by activation of the canonical Wnt
pathway.
To determine whether Wnt signaling instructs the fate switch of NPCs, we carried out clonal analysis by using NPCs infected with control or S33Y ß-catenin-encoding retroviruses at a low titer, allowing us to trace the fate of each infected cell. Expression of S33Y ß-catenin significantly increased the proportion of neuron-only and neuron-containing clones and reduced that of non-neuronal clones (Fig. 4A), demonstrating that ß-catenin can induce neuronal fate instructively. By contrast, expression of S33Y ß-catenin did not substantially increase the size of the clones (Fig. 4B). Expression of S33Y ß-catenin also did not increase bromodeoxyuridine (BrdU) incorporation into cells measured after culture for 1 to 3 days in the presence of Fgf2, and in fact decreased it (data not shown), probably as a result of cell cycle arrest associated with neuronal differentiation.
|
Wnt signaling contributes to cortical neurogenesis in vivo
In addition to the in vitro culture experiments, we examined the role of
Wnt signaling in the developing mouse neocortex by gene transfer in utero.
Expression plasmids were injected into the lateral ventricles of mouse embryos
at E13.5, and were introduced into NPCs in the VZ by electroporation with the
electric pulses applied from outside the uterus. Two days after
electroporation of a plasmid harboring GFP, a large fraction (30-50%) of the
GFP+ cells remained in the VZ and most of these cells were negative
for TuJ1. By contrast, when a plasmid encoding S33Y ß-catenin and GFP was
introduced by electroporation, the population of GFP+ cells that
had migrated out of the VZ into the outer layers of the neocortex after 2 days
was markedly increased (Fig.
5A). Similar results were obtained by electroporation of plasmids
encoding Wnt7a and Fz5 (data not shown). In some cases, the VZ became thinner
at the location where these proteins were ectopically expressed, probably
because of premature neurogenesis of the NPCs
(Fig. 5A and data not
shown).
|
To test the hypothesis that the canonical Wnt pathway is necessary for neuronal differentiation in the developing neocortex in vivo, we blocked this pathway in cortical NPCs by in utero electroporation with a plasmid for either Axin or Dkk1 at E13.5. Ectopic expression of Axin increased the population of cells remaining in the VZ 2 days later (Fig. 5C) and reduced the levels of HuC/D expression (Fig. 5D). Expression of Dkk1 induced similar effects (Fig. 5E).
Direct regulation of the Ngn1 promoter by the ß-catenin/TCF complex
We next investigated the mechanism by which Wnt signaling regulates
neurogenesis. As our results implicated the ß-catenin/TCF complex in
neuronal differentiation, we searched for a proneural gene that might be under
the control of these transcription factors. One such candidate is the bHLH
transcription factor Ngn1, because this gene is expressed during early
neurogenesis in the neocortex, and its expression, together with that of the
Ngn2 gene, is essential for development of the neocortex
(Schuurmans and Guillemot,
2002). We found a consensus sequence for TCF binding
(van de Wetering et al., 1997
)
located at nucleotide (nt) positions -1167 to -1160 relative to the
transcription start site of the mouse Ngn1 gene. This region within
the promoter has been shown to be responsible for expression of the gene in
the dorsal neocortex during neurogenesis
(Murray et al., 2000
). To
determine whether this TCF binding element is functional, we compared the
activities of the Ngn1 gene promoter (nt -2670 to +74) containing
either an intact or mutated version of this DNA sequence
(Fig. 6A). Cultured NPCs were
transfected with a luciferase reporter construct under the control of the
wild-type or mutant Ngn1 gene promoter. We found that the
transcriptional activity of the mutant promoter was markedly reduced compared
with that of the wild-type (Fig.
6B).
|
Stage-specific effects of Wnt signaling
Our results clearly indicate that stabilized ß-catenin instructs
neuronal differentiation of cortical NPCs prepared from mouse E11.5 neocortex
and cultured for 3 days. However, Chenn and Walsh
(Chenn and Walsh, 2002) have
shown that ectopic expression of stabilized ß-catenin by the nestin
enhancer results in the expansion of NPC cell number and suppression of cell
cycle exit. This difference might be due to the timing at which stabilized
ß-catenin was expressed, as the nestin enhancer is known to become active
at around E8.5. To test this idea, we compared the effects of ß-catenin
on NPCs prepared from different stages of mouse neocortex development.
Expression of stabilized ß-catenin increased the population of
TuJ1+ cells in NPCs prepared from E13.5 neocortex, but reduced
somewhat the population of TuJ1+ cells among neuroepithelial cells
acutely prepared from E10.5 neocortex (Fig.
7). This suggests that the response of NPCs to the canonical Wnt
pathway depends on the stage of neural development.
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Discussion |
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In general, two models can explain how the fate of an uncommitted precursor
cell is influenced by extrinsic cues. In one model, extrinsic cues instruct
multipotent precursor cells to commit to a particular lineage. In the other
model, multipotent precursor cells choose their fate stochastically, and the
proliferation and/or survival of specific lineage-restricted cells is then
supported by extrinsic cues. For example, Pdgf treatment increases the size of
the neuronal population in cortical neuroepithelial cultures by acting as a
mitogen in the early phase of NPC differentiation to expand the pool of
neuronal progenitors (Erlandsson et al.,
2001). Our results support a model in which the canonical Wnt
pathway acts on cortical NPCs to instruct their neuronal differentiation,
rather than to expand neuronal progenitors selectively, based on the following
evidence. First, expression of a stabilized form of ß-catenin did not
induce overt cell proliferation in NPC cultures under the conditions examined,
as assessed by BrdU incorporation. Second, the frequency of cell death was
negligible in NPCs expressing S33Y ß-catenin. Activation of the Wnt
pathway thus does not appear to increase neuronal population by selective
proliferation or survival of cells committed to the neuronal fate. Third, the
frequency of glial differentiation in the presence of Fgf2 was low, and
expression of S33Y ß-catenin did not inhibit glial differentiation
induced by Fgf2 withdrawal. It is therefore unlikely that the Wnt pathway
increases the neuronal population indirectly by suppression of glial
differentiation. Fourth, expression of either Wnt7a or S33Y ß-catenin
reduced the population of uncommitted precursors, as shown in the neurosphere
assay, consistent with fate determination at the expense of NPCs. And, most
importantly, clonal analysis revealed that expression of S33Y ß-catenin
increased the ratio of neuronal clones to non-neuronal clones. In addition,
ectopic expression of S33Y ß-catenin overrode the inhibitory effect of
Fgf2 on neurogenesis. As Fgf2 has been shown to maintain NPCs, it is likely
that Wnt signaling directly regulates the fate of NPCs.
At present, it is not clear whether neuronal differentiation is actively `triggered' by extracellular cues, or is induced passively when inhibitory signals are downregulated. Our results showing that Wnt signaling overcomes the inhibitory effect of Fgf2 clearly demonstrate that active triggering of neuronal differentiation can indeed take place during neuronal differentiation.
How does the Wnt pathway induce neuronal differentiation? In this study, we
found that the canonical Wnt pathway regulates the Ngn1 promoter.
Ngn1 is a proneural bHLH transcription factor expressed in newly committed
neuronal progenitors and immature neurons, and plays an essential role in
neurogenesis and regional specification in the neocortex, together with Ngn2
(Schuurmans and Guillemot,
2002). We found that ectopic expression of stabilized
ß-catenin increased the level of Ngn1 mRNA. Importantly, a TCF binding
element located at nucleotide positions -1167 to -1160 was found to be
necessary for maximal transcriptional activity of the Ngn1 gene
promoter, and direct binding of ß-catenin to the promoter was detected in
NPCs. These results imply that a ß-catenin/TCF complex directly regulates
the Ngn1 promoter, and that the canonical Wnt pathway induces
neuronal differentiation through regulation of Ngn1. A recent report showing
reduced expression of Ngn1 in ß-catenin-deficient neural crest cells
further supports a model of ß-catenin-mediated regulation of Ngn1
(Hari et al., 2002
). It is
possible that other mechanisms than Ngn1 induction also contribute to Wnt
induction of neurogenesis; for example, Wnt signaling may turn off the
intracellular signaling events that inhibits neuronal differentiation. Notch
signaling is known to inhibit neurogenesis, and an antagonistic interaction
between the Notch and the Wnt pathways has been demonstrated. For example,
mouse Wnt3a appears to be necessary for oscillating Notch signaling activity
during somitogenesis (Aulehla et al.,
2003
). It would thus be of interest to determine whether Wnt
signaling inhibits the Notch pathway in NPCs. As expression of S33Y
ß-catenin blocked the inhibitory effect of Fgf2 on neuronal
differentiation, it is also conceivable that ß-catenin somehow inhibits
intracellular signaling downstream of the Fgf2 receptor. It is currently
difficult to examine this possibility, as the mechanism by which Fgf2 blocks
neurogenesis is unclear.
Recent experiments have suggested that Wnt signaling has the capacity to
promote self-renewal in various tissue stem cells including neural stem cells
and hematopoietic stem cells (Alonso and
Fuchs, 2003; Reya et al.,
2003
; Willert et al.,
2003
). In the central nervous system, cells located in the
midbrain or hippocampus are deleted in mice deficient in Wnt1 or Wnt3a,
respectively (Lee et al.,
2000
; McMahon and Bradley,
1990
; Thomas and Capecchi,
1990
). Mice lacking both Wnt1 and Wnt3a also manifest a reduction
in the size of the caudal midbrain, rostral hindbrain, cranial and spinal
ganglia, and dorsal neural tube (Ikeya et
al., 1997
; Megason and
McMahon, 2002
). Furthermore, ectopic expression of Wnt1 or
stabilized ß-catenin was shown to lead to a net increase in the size of
the precursor pool in the chick spinal cord, in part through transcriptional
regulation of cyclinD (Megason and
McMahon, 2002
), and infection of cortical explants with
Wnt7aHA-expressing retrovirus induced expansion of neuronal precursors which
accompanied expression of the Egf receptor
(Viti et al., 2003
).
Consistently, transgenic mice expressing stabilized ß-catenin in NPCs
under the control of the nestin enhancer or Brn4 promoter also exhibited
overgrowth of the brain and spinal cord, reflecting an expansion of the
precursor population without alteration of the primary patterning of cell
identities (Chenn and Walsh,
2002
; Zechner et al.,
2003
). By contrast, in the present study, activation of the
canonical Wnt pathway reduced the size of the precursor pool and promoted
neuronal differentiation in the developing neocortex. We speculate that this
difference might be attributable to differences in the developmental stage of
the NPCs. Indeed, activation of the canonical Wnt pathway promoted neuronal
differentiation of NPCs derived from E13.5 embryos, but not those acutely
dissected from E10.5 embryos. It is possible that the chromatin region
encompassing regulatory elements of genes crucial for neuronal differentiation
(such as Ngn1) undergoes a change during development from a closed to
an open state, as observed for the STAT-responsive element within the GFAP
promoter, which becomes accessible to STAT3 only at later stages of neural
development, owing to demethylation of the element
(Takizawa et al., 2001
).
Therefore, chromatin state of the Ngn1 promoter should be examined in
future studies. In any case, our results have revealed that Wnt signals
function in a stage-specific manner within the same region of the brain,
analogous to the stage-specific functions of Drosophila Wg in wing
disc development: Wg promotes cell proliferation and survival at early stages
but determines the specification of sensory bristles of wing margin at later
stages (Giraldez and Cohen,
2003
; Johnston and Sanders,
2003
; Phillips and Whittle,
1993
).
One of the most fundamental questions about stem cells is what determines the timing of the fate switch from self-renewal to differentiation? Tissue stem cells appear to know how many times they should divide before undergoing differentiation, as this determines the size of each tissue. One might think that the expression of a differentiation-triggering cue is induced at the timing of the fate switch. However, our results suggest that what changes at the timing of cell fate switch might not be the expression of the extracellular cues, but the responsiveness of the stem cells to the cues, at least in the case of cortical development. These findings may shed lights on the nature of the stem cells.
Hundreds of distinct neuronal cell types are generated during development
of the mammalian neocortex, establishing a diversity that is essential for the
formation of complex neuronal circuits. Several extracellular factors,
including PDGF (Erlandsson et al.,
2001; Johe et al.,
1996
; Williams et al.,
1997
), insulin-like growth factor 1
(Arsenijevic and Weiss, 1998
;
Arsenijevic et al., 2001
),
brain-derived neurotrophic factor (Ahmed et
al., 1995
), bone morphogenetic protein 2
(Li et al., 1998
) and
erythropoietin (Shingo et al.,
2001
) have been implicated in the regulation of cortical
neurogenesis, and interactions among these factors may be necessary to
generate neuronal diversity in the neocortex
(Song et al., 2000
). It will
be interesting to determine which types of neurons are generated by activation
of the Wnt pathway and to investigate the interactions of this pathway with
other extrinsic and intrinsic factors that participate in neurogenesis.
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ACKNOWLEDGMENTS |
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