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First published online November 7, 2008
doi: 10.1242/10.1242/dev.024570
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1 Institute of Molecular and Cellular Biosciences, University of Tokyo, 1-1-1
Yayoi, Bunkyo-ku, Tokyo 113-0032, Japan.
2 Department of Immunology, Tokai University School of Medicine, 143
Shimokasuya, Isehara, Kanagawa 259-1193, Japan.
* Author for correspondence (e-mail: ygotoh{at}iam.u-tokyo.ac.jp)
Accepted 3 October 2008
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SUMMARY |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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Key words: Dll1, Notch, Lateral inhibition, Cell-cell interaction, Neural precursor cell, Telencephalon
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INTRODUCTION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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;
Hirabayashi and Gotoh, 2005).
Neocortical NPCs successively produce different types of neurons depending on
the stage at which they differentiate, and this contributes to cortical layer
formation (Molyneaux et al.,
2007). A subset of NPCs is selected to differentiate into neurons
at a given time point during the neurogenic phase, whereas other NPCs maintain
their undifferentiated state. The ratio between undifferentiated and
differentiating NPCs should thus be a key factor in determining the type and
correct number of neurons produced at each stage. However, it remains largely
unclear by what mechanisms NPCs are selected to undergo neuronal
differentiation in the developing mammalian brain. The prevailing view is that
the ratio of asymmetric to symmetric cell division contributes to the
proportion of NPCs differentiating into neurons. This model assumes that
asymmetric (vertical/oblique) cell division produces one differentiating and
one undifferentiated daughter cell due to an asymmetric distribution of cell
fate determinants, whereas symmetric (planar) cell division at the apical
surface of the ventricular zone mainly produces two undifferentiated daughter
cells (Chenn and McConnell,
1995; Götz and Huttner,
2005). However, perturbation of the ratio of vertical/oblique to
planar division does not necessarily alter the rate of neuronal production
(Konno et al., 2008),
indicating that other mechanisms are also likely to contribute to the
selection of differentiating cells. It is not known whether cell-cell
interactions are important for the selection of differentiating cells in the
mammalian brain.
The Notch signaling pathway plays a central role in the maintenance of the
undifferentiated state of NPCs in the mammalian central nervous system (CNS)
(Gaiano and Fishell, 2002;
Yoon and Gaiano, 2005;
Louvi and Artavanis-Tsakonas,
2006), and has been implicated in the selection of differentiating
cells in a number of systems
(Artavanis-Tsakonas et al.,
1995; Beatus and Lendahl,
1998; Greenwald,
1998). Notch is a transmembrane receptor that is activated by the
binding of ligands (delta-like 1, 3, 4 and jagged 1, 2 in mammals) presented
by neighboring cells, and thus mediates signaling generated by cell-cell
interactions. This triggers the cleavage of the intracellular domain of Notch,
which then translocates to the nucleus and binds to Rbpj, converting it from a
transcriptional suppressor to an activator. During neurogenesis, Notch
activation of Rbpj induces the expression of basic helix-loop-helix (bHLH) Hes
proteins, which suppress proneural bHLH transcriptional regulators, such as
Neurogenins and Mash1, and, thereby, suppress neuronal differentiation
(Kageyama et al., 2005). Notch
signaling has been proposed to contribute to binary cell fate specification
from an equipotent/homogeneous population through a mechanism called lateral
inhibition (Heitzler and Simpson,
1991; Muskavitch,
1994; Wilkinson et al.,
1994; Artavanis-Tsakonas et
al., 1995; Chitnis,
1995; Heitzler et al.,
1996; Lewis, 1996;
Beatus and Lendahl, 1998;
Greenwald, 1998). This
mechanism is based on feedback whereby Notch activation suppresses the
expression of its ligand, Delta. If the expression levels of Delta are
slightly different among cells, this difference is amplified because
Delta-expressing cells receive fewer Notch signals and express more Delta,
while the surrounding cells receive more Notch signals and express less Delta.
This amplification ultimately segregates the equipotent/homogeneous cell
population into two distinct cell populations: Delta-positive
Notch-inactivated (differentiating) cells and Delta-negative Notch-activated
(undifferentiated) cells. This binary cell fate specification by the
Notch-Delta lateral inhibitory signaling pathway was first demonstrated in
Drosophila neuroectoderm
(Heitzler and Simpson, 1991;
Heitzler et al., 1996) and
C. elegans gonad (Wilkinson et
al., 1994), and then in other systems such as chick and
Xenopus retina (Dorsky et al.,
1997; Henrique et al.,
1997). It might also function in the mammalian CNS given that the
proneural bHLH genes (Mash1 and neurogenin 1/2) and anti-neural bHLH
Hes genes positively and negatively regulate, respectively, the expression of
delta-like 1 (Dll1) (Casarosa et
al., 1999; Castro et al.,
2006; Hatakeyama and Kageyama,
2006), and that Dll1 exhibits non-homogeneous (so-called
salt-and-pepper) expression patterns in the developing mouse CNS
(Lindsell et al., 1996).
However, the causal relationship between the expression levels of Dll1 and
cell fate in these systems has not been demonstrated. Thus, it is unclear
whether the Notch-Delta lateral inhibitory system operates in uncommitted NPCs
to determine which NPCs become neurons during the neurogenic phase in the
mammalian CNS. Although numerous studies have reported the requirement of
Notch ligands for the maintenance of undifferentiated NPCs, it is not clear
whether Notch ligands are required for neuronal differentiation (via cell-cell
interactions) in the CNS.
In this study, we found that expression of Dll1 and activation of Notch1 occur in different cells in a mutually exclusive manner in the ventricular zone of the embryonic mouse telencephalon. Importantly, by the overexpression and conditional deletion of Dll1, we found that different levels of Dll1 expression can determine the proportion of differentiating cells among NPCs through cell-cell interactions. In particular, Dll1 deletion in a small proportion of NPCs revealed a prerequisite role of Dll1 in neuronal differentiation. These results strongly support the notion that lateral inhibition of Notch signaling indeed operates in the developing mammalian brain and contributes to the selection of differentiating cells among uncommitted NPCs.
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MATERIALS AND METHODS |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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) were
crossed with mice expressing Cre under the control of the nestin promoter and
enhancer (nestin-Cre mice) (kind gift of Dr R. Kageyama)
(Isaka et al., 1999). Jcl:ICR
strain mice (ICR mice) were purchased from CLEA Japan. All mice were
maintained according to the protocol approved by the Animal Care and Use
Committee of the University of Tokyo.
Expression constructs and antibodies
The plasmids pMX-enhanced green fluorescent protein (EGFP) (pMX-GFP),
pMX-IRES-EGFP (pMX-IG) and pMX-SV40-puro were kindly provided by Dr T.
Kitamura (University of Tokyo, Tokyo, Japan). The Cre recombinase construct
was kindly provided by Dr S. Kato (University of Tokyo, Tokyo, Japan).
HA-tagged rat Dll1 was inserted into the
EcoRI/SnaBI sites of pMX-IG (pMX-Dll1-IG). The plasmids
pMX-SV40-GFP and pMX-Cre-SV40-GFP have been described previously
(Yoshimatsu et al., 2006).
HA-tagged rat Dll1 intracellular domain (DICD) (Cys560 to Val714) was
amplified by PCR from the HA-tagged rat Dll1 construct, verified by
DNA sequencing, and then inserted into the BamHI/EcoRI sites
of pMX-IG (pMX-DICD-IG). Recombinant retroviruses were produced using the pMX
vectors as described previously
(Hirabayashi et al.,
2004).
Antibodies used in immunocytochemistry and immunohistochemistry were: mouse monoclonal antibodies to β III-tubulin (TuJ1) (Babco) at 1:1000, nestin (BD Pharmingen) at 1:200, Gfap (Chemicon) at 1:500, phosphorylated histone H3 (pH3) (Cell Signaling Technology) at 1:1000, Pcna (Ab-1, Oncogene) at 1:500 and BrdU (BD Biosciences) at 1:50; and rabbit polyclonal antibodies to Dll1 (H-265, Santa Cruz Biotechnology) at 1:100, cleaved Notch1 (Val 1744, Cell Signaling Technology) at 1:100, Sox2 (Chemicon) at 1:1000, Pax6 (Chemicon) at 1:1000, Tbr2 (Chemicon) at 1:1000 and GFP (MBL) at 1:1000. Alexa Fluor-labeled secondary antibodies, TO-PRO-3 and Hoechst 33342 (for nuclear staining) were from Molecular Probes.
Primary NPC culture and immunostaining
Primary NPCs were prepared from the dorsal cerebral cortex of ICR mouse
embryos at E12.5 (E1 was defined as 12 hours after detection of the vaginal
plug) as described previously (Hirabayashi
et al., 2004). The cells were cultured in medium comprising a 1:1
(v/v) mixture of Dulbecco's modified Eagle's medium and F12 medium (Gibco)
supplemented with B27 (Invitrogen) and with or without human FGF2 (R&D
Systems). To obtain NPC-enriched populations, we plated the dissociated
neuroepithelium directly on non-coated 100-mm dishes in culture medium
containing FGF2 (20 ng/ml) or both FGF2 (20 ng/ml) and Egf (20 ng/ml,
Upstate), and cultured the cells for 3 days (neurosphere culture). The
resulting neurospheres were then dissociated and plated on
poly-D-lysine-coated dishes in culture medium containing FGF2 (20
ng/ml) to yield an NPC culture. For retroviral infection, cells were mixed
with recombinant viruses for 18 hours, washed with phosphate-buffered saline
(PBS) and then incubated in the absence or presence of a low dose of FGF2 (2
ng/ml). Clonal analysis was performed as described previously
(Hirabayashi et al., 2004).
For immunostaining, cells were fixed with 4% paraformaldehyde in PBS,
permeabilized with 0.1% Triton X-100 for 10 minutes, incubated with primary
antibodies overnight and then with secondary antibodies for 30 minutes, and
mounted in Mowiol (Calbiochem).
Immunohistochemistry
Immunohistochemistry was performed as described previously
(Hirabayashi et al., 2004;
Yoshimatsu et al., 2006). For
staining with anti-Dll1 and anti-cleaved Notch1, antigen retrieval was
performed by autoclave treatment of sections for 5-10 minutes at 105°C in
0.01 M sodium citrate buffer (pH 6.0) and Target Retrieval Solution (TRS)
(Dako), respectively. The TSA Kit (Molecular Probes) was used for signal
amplification of Dll1 or cleaved Notch1 staining. Staining with anti-Dll1 and
anti-cleaved Notch1 was performed using the ABC Kit (Vector Laboratories) and
TSA Kit. After antigen retrieval in TRS, the samples were incubated
sequentially with anti-cleaved Notch1, with biotinylated secondary antibody,
with ABC reagent (ABC Kit) and then with tyramide-biotin (TSA Kit). The
samples were then subjected to antigen retrieval by autoclaving for 10 minutes
at 105°C in 0.01 M sodium citrate buffer (pH 6.0) and incubated with
anti-Dll1, HRP-conjugated secondary antibody, then with streptavidin-Alexa
Fluor 488 (for detection of cleaved Notch1) and tyramide-Alexa Fluor 555 (TSA
Kit) (for detection of Dll1). The fluorescence images were obtained with a
confocal laser microscope (LSM510, Zeiss).
In situ hybridization
In situ hybridization on frozen brain sections was performed essentially as
described previously (Nomura and Osumi,
2004). The digoxigenin-labeled antisense riboprobe for detecting
mouse Dll1 corresponds to a 
1.6 kb region spanning exons 8 and
11.
Retrovirus infection in utero
The protocol for retrovirus infection in utero was a modification of a
method for in utero electroporation
(Tabata and Nakajima, 2001).
At E12.5, mice were anaesthetized and the uterine horns were exposed.
Recombinant retrovirus suspension (0.5-1.0 µl) with Fast Green (0.01%) was
injected into the cerebral ventricles of each littermate. The uterine horns
were returned to the abdominal cavity to allow the embryos to continue normal
development. Two or three days after the operation, the embryos were harvested
and the brains examined by immunohistochemical analysis.
BrdU labeling
For in vivo labeling of BrdU, a single injection of BrdU (50 mg/kg,
intraperitoneally) was performed 30 minutes prior to sacrifice. BrdU-positive
cells were detected by immunohistochemistry as described above.
Statistical analysis
Quantitative data are presented as the mean ± s.e.m. from
representative experiments. The experiments were repeated at least three times
with similar results. Values were compared using the unpaired Student's
t-test. P<0.05 was considered statistically
significant.
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RESULTS |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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), a recent
report has demonstrated the oscillatory expression of Dll1 mRNA at
low levels in proliferating NPCs (Shimojo
et al., 2008). In this study, we investigated the expression of
Dll1 protein in the developing mouse telencephalon by immunohistochemical
analyses. Dll1-immunopositive cells were detected in a subset of cells in the
ventricular zone (VZ), the intermediate zone (IZ) and the cortical plate (CP)
of neocortex and in the ganglionic eminences at E13.5
(Fig. 1A). At later stages,
such as at E16.5, Dll1-expressing cells were found in the VZ and IZ, but at
very low levels in the CP of the neocortex
(Fig. 1B). The expression
pattern of Dll1 protein was similar to that of Dll1 mRNA
(Fig. 1C,D). Importantly, most
of the Dll1-expressing cells in the VZ were co-immunostained with antibodies
to the proliferation marker Pcna and to the undifferentiated NPC markers
nestin and Sox2 (Fig. 1E-P).
Consistent with this, Dll1-expressing cells in the VZ were mostly devoid of
the neuronal marker β III-tubulin (TuJ1; Tubb3), whereas most of the
Dll1-expressing cells in the IZ (and those in the CP at E13.5) were
TuJ1-positive (Fig. 1Q-T).
These results suggest that Dll1 is expressed in a subset of undifferentiated
NPCs in the VZ, as well as in immature neurons localized at the IZ and CP.
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-secretase-catalyzed cleavage site of Notch1, and by the use
of a reporter gene that monitors the activity of Notch signaling
(Tokunaga et al., 2004;
Del Monte et al., 2007;
Mizutani et al., 2007). We
confirmed that only a subpopulation of NPCs in the VZ was immunostained for
active Notch1 (Fig. 2B,F).
However, a negative correlation between the Notch activity and Dll1 expression
in the developing mouse brain has not been demonstrated, although this would
provide key evidence to support the role of the Notch-Delta lateral inhibitory
system in this system of differentiation. We thus carried out
immunohistochemistry of active Notch1 and Dll1 together, and found that their
expression patterns in the VZ of the telencephalon were segregated into
distinct cells in a mutually exclusive manner
(Fig. 2A-H). In the VZ of the
neocortex at E13.5, 73.8% of Dll1-negative cells were positive for active
Notch1 within the nucleus, whereas only 24.7% of Dll1-positive cells were
positive for active Notch1 within the nucleus
(Fig. 2I). Dll1 expression and
Notch1 activation might have a rough correlation with the cell cycle, as
active Notch1- and Dll1-expressing cells tended to localize basally and
apically in the VZ, respectively (Fig.
2A-H). However, the percentage of active Notch1-positive cells was
significantly lower among Dll1-positive cells than among Dll1-negative cells
in both the basal and apical halves of the VZ (see Fig. S1 in the
supplementary material). These results suggest that the mutually exclusive
patterns of active Notch1 and Dll1 expression might be established at
different phases of the cell cycle. This negative correlation between active
Notch1 and Dll1 expression strongly supports the existence of the Notch-Delta
lateral inhibitory system in the developing mouse telencephalon.
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; Campos et al.,
2001). However, the expression of Dll1 in undifferentiated NPCs,
as observed in Fig. 1, suggests
an additional role of Dll1, i.e. in neuronal fate specification among NPCs
through the lateral inhibitory system. If this is the case, ectopic expression
of Dll1 in a subpopulation of NPCs should lead to neuronal differentiation of
these cells, as ectopic Dll1 activates Notch signaling and reduces Dll1
expression in the surrounding cells, resulting in a reduction of Notch
signaling in the Dll1-expressing cells
(Heitzler and Simpson, 1991;
Dorsky et al., 1997;
Henrique et al., 1997). We
examined this hypothesis by introducing Dll1 into an in vitro culture of NPCs.
NPC cultures (see Materials and methods) prepared from E12.5 mouse neocortex
were infected with retroviruses encoding either green fluorescent protein
(GFP) alone, or GFP together with Dll1, at a low titer so that only a
small number (less than 0.1%) of the cells were infected. Under this
condition, the fate of each infected clone could be traced as a cluster of
GFP-positive cells (Fig. 3A).
The expression of Dll1 significantly increased the proportion of clones
containing only TuJ1-positive neurons (pure TuJ1-positive clones) among
GFP-positive clones (Fig. 3B).
The expression of Dll1 also increased the percentage of TuJ1-positive cells
among GFP-positive cells (Fig.
3C). The expression of Dll1 did not appear to alter the size of
the clones (see Fig. S2 in the supplementary material). Since the stage of
this culture roughly corresponds to the neurogenic phase, the percentage of
cells expressing the astroglial marker Gfap among infected cells was small
(7%, regardless of Dll1 expression)
(Fig. 3D), indicating that the
Dll1-induced neuronal differentiation shown in
Fig. 3B was not due to a
suppression of an alternate (astroglial) fate of NPCs. The contribution of
cell death was also negligible in these cultures (less than 2% among
GFP-positive cells).
The fate switch of NPCs from neurogenic to astrogliogenic can be
recapitulated in an in vitro culture. When Dll1 was overexpressed in a
subpopulation of NPCs prepared from a 12 days in vitro (DIV) culture of E12.5
neocortical neuroepithelial cells, which correspond to the astrogliogenic
phase, the proportion of clones containing only Gfap-positive astrocytes was
reduced and the proportion of clones containing only TuJ1-positive neurons was
increased (Fig. 3E,F). This
suggests that high levels of Dll1 were sufficient to change the astrogliogenic
fate into the neurogenic fate when expressed at the astrogliogenic phase.
Interestingly, the levels of Dll1 mRNA were reduced at around the
onset of the astrogliogenic phase (Campos
et al., 2001; Irvin et al.,
2004). This reduction might contribute to the suppression of
neurogenesis during the astrogliogenic phase.
Dll1-induced neuronal differentiation requires cell-cell interactions among NPCs
If neuronal differentiation induced by Dll1 expression can be ascribed to
the lateral inhibition model, it should be dependent on cell-cell interaction
and the difference in Dll1 levels between the cells. To examine this, we
introduced Dll1 into a large proportion of NPCs by retroviral infection at a
high titer (more than 70% of NPCs were infected under this condition), so that
there would be no major differences in Dll1 levels among the cells. In this
case, the expression of Dll1 did not promote neuronal differentiation
(Fig. 4A). We further examined
whether cell-cell interactions are essential for Dll1-induced neuronal
differentiation by reducing the cell density. We introduced Dll1 into a small
proportion of NPCs by retroviral infection at a low titer, as in
Fig. 3, but changed the seeding
cell density. At a lower cell density (0.26x105
cells/cm2 at seeding), the expression of Dll1 no longer increased
the percentage of pure TuJ1-positive clones among GFP-positive clones
(Fig. 4B). These results
together support the idea that Dll1 induces neuronal differentiation of NPCs
via cell-cell interaction, consistent with the lateral inhibition model.
Reduction of the cell density did not significantly increase the percentage
of TuJ1-positive clones among GFP-positive clones in control cultures
(Fig. 4B), suggesting the
existence of a transacting signal that promotes neuronal differentiation in a
non-cell-autonomous manner. Such a mechanism might involve Wnt signaling, as
Wnt ligands, including Wnt7a, were expressed in this culture (data not shown),
and the prevention of Wnt signaling suppresses the neuronal differentiation of
NPCs cultured under similar conditions
(Hirabayashi et al.,
2004).
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;
Ikeuchi and Sisodia, 2003;
LaVoie and Selkoe, 2003;
Six et al., 2003;
Kolev et al., 2005). A recent
study has shown that expression of the Dll1 intracellular domain (DICD)
promotes neurogenesis cell-autonomously in P19 cells
(Hiratochi et al., 2007).
However, we found that overexpression of DICD, even when expressed in a small
number of NPCs, did not induce neuronal differentiation in primary
telencephalic NPC culture (Fig.
4C) or in vivo (see Fig. S3 in the supplementary material). These
results again suggest that neuronal differentiation induced by Dll1 expression
is not due to a cell-autonomous mechanism.
Different levels of Dll1 expression regulate neuronal fate specification in the developing mouse neocortex
In addition to the in vitro experiments, we wished to examine the effects
of Dll1 expression on NPC fate in vivo. We therefore introduced Dll1 into a
small number of NPCs in the telencephalon by injecting retroviruses encoding
GFP alone, or both GFP and Dll1, into the telencephalic ventricle at E12.5.
Two days after infection, the numbers of GFP-positive cells localized at each
area [VZ, subventricular zone (SVZ), IZ or CP] were determined. When control
retroviruses were introduced, 26.8±2.7% of GFP-positive cells were
found within the VZ 2 days after infection. When Dll1-expressing retroviruses
were introduced, the percentage of GFP-positive cells within the VZ was
markedly reduced (5.3±2.1%) (Fig.
5A-C). Dll1 expression also decreased the percentage of
GFP-positive cells located in the SVZ, and increased the percentages of
GFP-positive cells in the IZ and CP. All of the GFP-positive cells in the IZ
and CP were negative for the NPC marker Pax6, and more than 97% of these cells
were positive for TuJ1 in both control and Dll1-expression samples. In fact,
the expression of Dll1 significantly increased the proportion of TuJ1-positive
cells among GFP-positive cells in the whole neocortex
(Fig. 5D). These results
strongly suggest that Dll1 expression in a small population of cells at the VZ
promotes neuronal differentiation in the developing neocortex.
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) and increased early production of GABA-positive neurons at
ganglionic eminences (Yun et al.,
2002). However, it has remained unclear whether Dll1 is a major
Notch ligand in other parts of the telencephalon such as the neocortex,
especially given that other Notch ligands such as jagged 2 and Dll3
are also expressed in the developing neocortex
(Luo et al., 1997;
Campos et al., 2001;
Irvin et al., 2004). Thus, to
avoid the embryonic lethality at early developmental stages, we utilized a
conditional deletion of the Dll1 gene in the CNS. Conditional
deletion was achieved by crossing mice carrying both Dll1 alleles
flanked by loxP sequences (Dll1flox/flox)
(Hozumi et al., 2004) with
mice harboring a transgene of Cre recombinase under the control of
the nestin promoter and enhancer (Isaka et
al., 1999). In these mice, Dll1 immunoreactivity was already
decreased at E11.5 in telencephalon (Fig.
6A-D). By E13.5, the Dll1-deficient neocortex became thicker than
the control neocortex in the radial axis, but thinner in the tangential axis
(Fig. 6I,J,M,N, and data not
shown), with a slight increase in cell death (data not shown). The levels of
active (cleaved) Notch1 in NPCs were significantly reduced in the
Dll1 conditional KO mice at E11.5
(Fig. 6E,E',F,F').
These mice exhibited robust premature neurogenesis in the neocortex as well as
in the subcortical regions by E13.5, as detected by an increase in
TuJ1-positive cells and a reduction in Sox2-positive cells
(Fig. 6G-N). We also observed a
transient increase of Tbr2 (Eomes)-positive basal progenitors at E11.5 and a
decrease at E13.5 in the lateral region of Dll1-deficient neocortex
(Fig. 6W-Z, see also
Fig. 6O-V showing the aberrant
location of mitotic cells), probably owing to the premature neurogenesis and
subsequent exhaustion of NPCs. At E16.5, fewer NPCs were found in the VZ of
the Dll1 conditional KO mice as compared with the control mice (data
not shown), also suggesting the exhaustion of NPCs owing to the premature
neurogenesis. Since these phenotypes are consistent with the premature
neurogenesis observed in mice defective in the canonical Notch pathway (e.g.
Notch1- and Rbpj-deficient mice) (de la
Pompa et al., 1997), these results suggest that Dll1 is a major
ligand for the Notch receptor in the developing neocortex and that it
contributes to the maintenance of the undifferentiated state of NPCs. Given that Dll1 is a major Notch ligand in the developing telencephalon, we next examined whether different levels of endogenous Dll1 among undifferentiated NPCs could determine the fate of NPCs in vivo, and whether the Notch-Delta lateral inhibitory system indeed operates in vivo. We deleted the Dll1 gene in a small proportion of NPCs in vivo by injecting retroviruses harboring GFP alone, or GFP with Cre recombinase, into the telencephalic ventricle of Dll1flox/flox mice at E12.5. When examined 3 days after infection, expression of Cre recombinase increased the percentage of GFP-positive cells within the VZ (from 19.2% to 36.3%) and reduced that of GFP-positive cells within the IZ and CP (IZ, from 26.7% to 19.3%; CP, from 36.1% to 26.2%) (Fig. 7A-C). Since more than 96% of GFP-positive cells in the VZ were Pax6-positive and TuJ1-negative in both control and Cre-infected samples, this result indicates that the reduction of endogenous Dll1 in a small proportion of NPCs suppressed the neuronal differentiation of these cells. In fact, the deletion of Dll1 significantly increased the proportion of Pax6-positive cells among GFP-positive cells in the whole neocortex (Fig. 7D). Since the reduction of endogenous Dll1 in all NPCs dramatically promoted neuronal differentiation (Fig. 6), these results strongly suggest that different levels of endogenous Dll1 regulate the neuronal differentiation of NPCs via cell-cell interaction, most likely through the lateral inhibitory system.
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DISCUSSION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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;
Heitzler et al., 1996). By
contrast, it has been proposed that the Notch pathway in the developing
vertebrate CNS mainly contributes to the negative-feedback signal emanating
from nascent neurons to suppress excess neuronal differentiation of NPCs. This
view was supported by the observation that Dll1 mRNA is found
predominantly in nascent neurons/intermediate progenitors (neuron-committed
progenitors), but is scarce in NPCs in the developing retina, spinal cord and
telencephalon (Henrique et al.,
1997; Campos et al.,
2001; Minaki et al.,
2005; Kawaguchi et al.,
2008). A recent study of mind bomb 1, an activator of Dll1, also
supports this view because of its expression in nascent neurons/intermediate
progenitors (Yoon et al.,
2008). However, another study has recently revealed low-level
expression of Dll1 mRNA in NPCs
(Shimojo et al., 2008),
although its role in NPC differentiation remained unclear. In this study, we
present a series of results supporting the notion that the Notch-Delta pathway
contributes to the lateral signaling between NPCs that segregates equipotent
mouse neocortical NPCs into two alternative fates: NPCs or neurons. Firstly,
we found that Dll1 protein is expressed in a substantial population of NPCs
that are positive for the undifferentiated cell markers nestin and Sox2 and
for the proliferation marker Pcna in the developing mouse telencephalon.
Secondly, we have shown that active Notch1 and Dll1 in the VZ of the
developing telencephalon are expressed in different cells in a mutually
exclusive manner, extending previous findings showing non-homogenous
(salt-and-pepper) expression patterns of active Notch and Dll1 in the VZ of
various nervous systems. This is consistent with the prediction of the lateral
inhibition model: in the initially homogenous/equipotent cell population there
might be small differences in the levels of ligands and receptor activity
among cells, and these initial changes are amplified so that some cells
express high levels of ligands and low levels of receptor activity, whereas
other cells express low levels of ligands and high levels of receptor
activity. We do not yet know whether the levels of active Notch and Dll1 are
initially homogeneous in the NPCs in the earlier stages of development.
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) (this study). These opposing effects of Dll1 on neurogenesis
depending on the context can be explained by the lateral inhibitory signaling
that takes place non-cell-autonomously, as the feedback signaling from
surrounding cells ultimately reduces the Notch activity in the Dll1-expressing
cells (Heitzler and Simpson,
1991; Dorsky et al.,
1997; Henrique et al.,
1997). The requirement of cell-cell interaction for the induction
of neurogenesis by Dll1 is also supported by the observation that induction
was not found in cells cultured at a low cell density. Together, these results
strongly support the idea that the lateral inhibitory signals between NPCs
contribute to the determination of cell fate in addition to the
negative-feedback signals emanating from nascent neurons, and that different
levels of Dll1 among NPCs play a crucial determining role.
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).
Alternatively, some NPCs might be biased or predetermined to become neurons by
additional mechanisms leading to Dll1 induction. For instance,
Dll1 might be regulated by the Wnt-Lef1 pathway, as is the case in
the developing somites (Galceran et al.,
2004; Hofmann et al.,
2004). Since the Wnt pathway promotes neurogenesis in the
neocortex (Hirabayashi et al.,
2004), it would be interesting to examine the possible involvement
of Dll1 in this. We have previously reported that Stat3 induces Dll1
and maintains NPCs in an undifferentiated state in a non-cell-autonomous
manner (Yoshimatsu et al.,
2006). Since Stat3 appears to be expressed homogenously in most
NPCs, it might not provide a `biased' cue but rather confers `competence' to
NPCs to start lateral inhibition via expression of Dll1.
It is crucial to understand where Notch-Dll1 interaction takes place at a
subcellular level because the amount of Dll1 present at the location of Notch
interaction affects the process of lateral inhibition. We and others have
observed that Dll1 is localized at the apical junctions (data not shown)
(Mizuhara et al., 2005), but
further studies will be needed to reveal the site(s) at which it activates the
Notch receptor. If the Dll1-Notch interaction occurring between NPCs takes
place at some particular site within the VZ, migration after neuronal
commitment (or an increase in Dll1 levels) would remove Dll1-positive cells
from that site, which would restart the selection process among NPCs.
Therefore, the speed of migration of committed neurons might contribute to the
rate of neurogenesis.
To our knowledge, this is the first report describing the conditional
deletion of the Notch ligand Dll1 in the mammalian CNS. Although
several Notch ligands are expressed in the developing telencephalon,
Dll1 appears to play a major role among them, given that its
conditional deletion reduces the immunoreactivity of active (cleaved) Notch1
and results in premature neurogenesis, recapitulating the phenotype of mice
defective in Notch signaling (de la Pompa
et al., 1997).
Although this is not a major focus of this paper, one of the prominent
phenotypes of the CNS-specific Dll1 gene deletion is the severe
hemorrhage found throughout the entire brain as early as E11.5 (data not
shown). Since an endothelium-specific deletion of Notch1 using
Tie2-Cre causes lethality at E10.5, with vascular
defects and hemorrhage (Limbourg et al.,
2005), our result unexpectedly suggests that Dll1 expressed in
nestin-positive cells (most likely cells in a neural lineage) might serve as a
ligand for supporting this function of Notch in vascular development.
It has been proposed that in mammalian CNS development, the orientation of
the mitotic spindle of NPCs (radial glial cells) regulates the fate of
daughter cells. That is, horizontal or oblique cleavage is coupled to
asymmetric division that produces one neuron and one NPC, whereas vertical
cleavage is coupled to symmetric division that produces two NPCs
(Chenn and McConnell, 1995;
Götz and Huttner, 2005).
Indeed, depletion of Ags3 (Gpsm1), which is responsible for maintaining
correct spindle orientation, affects NPC fate
(Sanada and Tsai, 2005).
However, recent studies have shown that the vertical cleavage is predominant
at all stages of the neurogenic phase
(Stricker et al., 2006;
Konno et al., 2008;
Noctor et al., 2008), and that
spindle orientation might be important for the position of the daughter cells
but not for neuronal production rate, as revealed by manipulation of some of
the components responsible for spindle orientation [LGN (Gpsm2), Insc]
(Konno et al., 2008). This
implies that mechanisms other than spindle orientation/asymmetric division
also contribute to the determination of neuronal fate in NPCs. We propose that
owing to cell-cell interactions that mediate Notch-Dll1 lateral inhibition,
equipotent NPCs can differentiate into neurons at a certain rate even without
asymmetric division. This is based on the finding that differential Dll1
expression levels between NPCs are sufficient for determining the neuronal
fate. It is also conceivable that asymmetric division (caused by an asymmetric
inheritance of fate determinants, if any) can bias the outcome of lateral
inhibition between adjacent cells. However, because Dll1-deficient NPCs
surrounded by NPCs with an intact Dll1 gene are less likely to
undergo neurogenesis, the Notch-Dll1 lateral inhibitory mechanism might be
dominant over fate determination by asymmetric division and could be
prerequisite for neuronal differentiation.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/135/23/3849/DC1
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ACKNOWLEDGMENTS |
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REFERENCES |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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