1 Laboratory for Cell Culture Development, Brain Science Institute, RIKEN,
Saitama 351-0198, Japan
2 Department of Anatomy and Cell Biology, Graduate School of Medicine, Nagoya
University, Nagoya 466-8550, Japan
3 CREST, Japan Science and Technology Corporation (JST), Tokyo 103-0027,
Japan
Author for correspondence (e-mail:
tmiyata{at}med.nagoya-u.ac.jp)
Accepted 16 March 2004
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SUMMARY |
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Key words: Cerebral cortex, Cortical plate, Neuroepithelium, Subventricular zone, Asymmetric cell division, Cell migration, Cell cycle, Cell fate determination, Layer formation, Neurogenin2, Slice culture, Mouse
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Introduction |
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We have recently observed the formation of four-cell clones from single
DiI-labeled progenitor cells in retinal slices prepared from embryonic day (E)
17 mice (Saito et al., 2003).
Strikingly, the majority of the P/P divisions that formed the retinal
four-cell clones were found to be asymmetric, with paired mitotic daughters
differing in the trajectory of interkinetic nuclear movement, cell cycle
length, and the composition of their daughter cells (i.e. granddaughter
cells). Therefore, we sought to determine whether the cerebral wall might also
undergo similar asymmetric P/P divisions, focusing on the stage of development
just before and during the emergence of the cortical plate (CP)
(Fig. 1A). This stage is
characterized by a shift in the productivity in the dorsal telencephalon from
proliferation without differentiation (stage 1) to
differentiation balanced with proliferation (stage 2)
(Smart, 1973
), as well as a
transition from symmetric to asymmetric divisions
in the entire cycling population
(Takahashi et al., 1996b
). We
therefore asked whether the shift in the behavior of the entire population of
cells could be explained by possible asymmetric P/P divisions at the single
cell level.
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The origin of the NS-dividing progenitor population is also unclear.
Although NS-dividing cells seen in the pallium can arise from the ganglionic
eminence (He et al., 2001),
the VZ of the pallium is thought to be their primary origin
(Altman and Bayer, 1990
;
Takahashi et al., 1995b
). A
leading hypothesis predicts that the NS-dividing population arises from the VZ
by E13 in mice and by E15 in rats and stays mainly in the SVZ without
exhibiting interkinetic nuclear movement and without receiving a further
supply of cycling cells from the VZ (Altman
and Bayer, 1990
; Takahashi et
al., 1995b
; Nowakowski et al.,
2002
); this has yet to be rigorously demonstrated. In this study,
therefore, we asked whether P/P divisions at the ventricular surface provide
these NS-dividing cells. Studying the cellular and molecular mechanisms of
normal NS mitoses should also facilitate the analyses of abnormal NS-divisions
in mutant animals (Estivill-Torrus et al.,
2002
; Ferguson et al.,
2002
).
We used a slice culture system (Miyata
et al., 2001; Miyata et al.,
2002
; Saito et al.,
2003
) combined with immunohistochemistry and in vivo experiments
to monitor progenitor cells in the cerebral wall. We directly observed that
P/P divisions were the dominant form of division taking place at the
ventricular surface of the cerebral wall around the emergence of the CP. The
majority of the P/P divisions supplied one mitotic daughter cell from the
ventricular surface to a NS position within a single cell cycle, and the
S-to-NS supply of a mitotic daughter was achieved by the loss of a ventricular
process by the daughter cell by the end of G2 phase. Also, the NS mitoses were
mostly positive for Hu and generated a pair of Hu+ neuron-like
cells. Neurogenin2 (Ngn2), a basic helix-loop-helix-loop (bHLH) transcription
factor known to regulate neuronogenesis in the dorsal telencephalon
(Sommer et al., 1996
;
Fode et al., 2000
;
Nieto et al., 2001
) (reviewed
by Ross et al., 2003
), was
detected in a cell cycle-dependent manner, with the highest and broadest
expression during G1 phase and a weaker and more NS population-restricted
pattern towards M phase. Finally, cells infected with Ngn2 retrovirus showed a
significantly higher percentage for NS mitoses compared with those infected
with control virus. Accordingly, we propose that commitment to the neuronal
lineage, accompanied by the loss of the ventricular process, occurs in a
mitotic cell prior to the completion of G2 phase, and the asymmetric P/P
division that generates such a NS-dividing mitotic cell, as well as a
S-dividing mitotic cell, contributes to the efficient segregation of neurons
and cycling cells, a process required in the pre-CP stage.
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Materials and methods |
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Immunohistochemistry
Cultured slices were fixed in 4% paraformaldehyde for 10 minutes,
vibratome-sectioned, treated with antibodies, and subjected to confocal
microscopy, as described (Miyata et al.,
2001). For in vivo analyses, mice [E13-P0 (P0=day of birth)] were
transcardially perfused with PLP fixative
(McLean and Nakane, 1974
),
postfixed in the same fixative for 30-60 minutes on ice, immersed in 20%
sucrose, embedded in O.C.T. compound (Miles), and then frozen and coronally
sectioned (15 µm). Primary antibodies used: Ki67 (mouse IgG, Novocastra);
pH3 (rabbit, UpDate Technology); p-vimentin [mouse IgG (4A4), MBL]; GFP (rat
IgG, Nakarai; rabbit, MBL), Hu [mouse IgG (16A11), Molecular Probes]; p27
(mouse IgG, Transduction Laboratories); Ngn2 [rabbit, gift from Masato
Nakafuku (Cincinnati Childrens Hospital Research Foundation)]; BrdU
(mouse IgG, Sigma); Nestin [mouse IgG (Rat401), Hybridoma Bank];
anti-endothelial cell [mouse IgG (#16985), Chemicon]. Some sections were
treated with NeuroTrace 500/525 (Molecular Probes) for green fluorescent Nissl
staining.
Virus generation and infection
Control-GFP retrovirus, made by inserting IRES-EGFP from pIRES2-EGFP
(Clontech) into pLNCX2 (Clontech), and Ngn2/GFP retrovirus, made by inserting
ngn2 from pCS2-ngn2 (gift from Jacqueline E. Lee, University of Colorado at
Boulder, CO, USA) or pCLIG-ngn2 (gift from Ryoichiro Kageyama, Kyoto
University, Japan) vectors into control-GFP, were individually transfected
into 2MP34, an ecotropic packaging cell line
(Yoshimatsu et al., 1998
),
using LipofectAMINE (Invitrogen). Supernatant was collected and concentrated,
according to Nanmoku et al. (Nanmoku et
al., 2003
), and injected, through in utero surgery, into lateral
ventricles of E12 B6C3 mice. Prior to the retrovirus experiments, we used in
utero electroporation (Tabata and
Nakajima, 2003
) to examine the effect of expression of pCLIG-ngn2
and pCS2-ngn2 on mitosis position, and we found that the total number of NS
mitoses [both those expressing a plasmid marker (myc or GFP) as well as those
not expressing] was significantly increased (T. Miyata, unpublished). Because
myc+ or GFP+ cells were diffusely found in the VZ at 24
hours after electroporation and all were found to be neurons at 48-72 hours,
we considered that Ngn2, through its neuron-inducing activity, may have
changed the overall neuroepithelial structure in the VZ, leading to the
reporter-negative NS mitoses. To avoid such a technical problem, Ngn2/GFP
virus was used at a lower titer (3-10x108 cfu/ml) compared
with control-GFP (3-4x109 cfu/ml). GFP adenovirus, of which
GFP expression is driven by the CAG promoter (T. Muto, unpublished), was
prepared and injected, by in utero surgery, into the lateral ventricles of E13
B6C3 mice according to previously described methods
(Tamamaki et al., 2001
;
Hashimoto and Mikoshiba,
2004
).
Analysis
Region and stage
Immunohistochemical quantifications were performed using coronal sections
lying between the anterior margin of the foramen of Monro and the anterior tip
of the hippocampal formation proper. Because CP formation
(Fig. 1) is initiated
rostrolaterally at E13 and proceeds dorsomedially by the end of E14
(Smart, 1973;
Smart and McSherry, 1982
), we
mainly examined dorsolateral and dorsal areas in E13 sections and dorsal and
dorsomedial areas in E14 sections (dorsomedial was defined as an
area medial to the greatest curvature of the ventricular roof, presumably
corresponding to the location of future primary somatosensory representation;
dorsal and dorsolateral were defined by dividing
the remaining pallium into two parts dorsal may
correspond to future parietal areas). In vivo data obtained at E13 and those
from E14 mice were very similar, probably reflecting that despite the gradient
in the initiation of neuronogenesis, the proliferative processes associated
with cortical neuronogenesis proceed essentially identically across the
lateral to medial axis of the pseudostratified ventricular epithelium
(Miyama et al., 1999
;
Takahashi et al., 1999
), and
in some parts of the present study they are presented together. Quantification
in sections at E15 and older were performed in the dorsal region. For
time-lapse recording of DiI-labeled cells in slices, singly and moderately
labeled cells were randomly chosen in dorsolateral and dorsal areas in E13
slices and dorsal and dorsomedial areas in E14 slices.
Statistics
In a comparison of two groups (e.g. NS-dividing vs. S-dividing),
statistical significance was assessed by chi-square test or Mann-Whitney test.
The numbers of samples, as well as the number of cells scored in each sample,
are indicated in the figure legends and tables.
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Results |
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The type II pattern of division was more frequently (44/57, 77%) observed
than type I (13/57, 23%). Given the obtained frequencies, 100 P/P divisions at
the surface would result in 123 S divisions (23+23+77) and 77 NS divisions,
and expected percentage NS-division arising from the P/P surface divisions
should be 39% (77/200). However, this figure is greater than the percentage of
NS-divisions that we observed by immunostaining with phosphohistone3 (pH3) in
vivo (20±1% at E13, n=3; 15±1% at E14, n=3) as
well as values obtained by others (Smart,
1973; Takahashi et al.,
1993
), and we feel that the division patterns in slice culture are
biased from type I divisions towards type II divisions. Anti-pH3
immunostaining on sections made from E13-derived slices revealed
time-dependent increase in the percentage of NS divisions: 17±1% at 12
hours (mean±s.e.m., n=3); 20±4% at 24 hours
(n=6); and 31±1% at 36 hours (n=8). Nevertheless, the
mechanism that facilitates daughter cell choice of an NS or an S division must
exist in slices because the majority of daughter cells generated by the 57
cases of P/P division divided at the surface [also notably, all P/N divisions
(11 cases) were judged as PS-div+N]. There was a correlation
between the frequency of division type and the thickness of the cerebral wall
in which the original DiI-labeled progenitor divided
(Fig. 1D,E;
Table 2; see Fig. S1 at
http://dev.biologists.org/supplemental/).
The averaged thickness of slices where type II divisions were observed
(169±6 µm, mean±s.e.m.) was significantly
(P<0.05, Mann-Whitney test) greater than that for type I
(142±5 µm).
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In order to further examine these phenomena in vivo, we injected
GFP-adenoviruses into the lateral ventricles of E13 embryos and examined
fluorescence 18-19 hours later. As shown in
Fig. 3B,C, numerous
GFP+ NS mitoses, along with GFP+ S mitoses, were
observed in the dorsal and dorsolateral regions (in 5 embryos from 3
independent experiments). Because adenovirus infection occurs at the
ventricular surface within 4 hours after injection
(Hashimoto and Mikoshiba,
2004), the supply of NS-dividing cells from the surface to the SVZ
was confirmed. Cell cycle length in the dorsomedial region is reported to be
approximately 11.4 hours at E13 (Takahashi
et al., 1995a
), and it is expected to be longer at the thicker
(thus developmentally more advanced)
(Smart and McSherry, 1982
)
regions. Therefore, the detection of the GFP+ NS-dividing cells at
18-19 hours suggests that the S-to-NS supply of the mitotic daughters in vivo
may have occurred within a single cell cycle.
NS mitoses at E13 and E14 are committed to the neuronal lineage and may generate neuron pairs
In the slice culture system, successful immunohistochemical examination of
daughter cells generated by NS-dividing cells was possible in 14 cases; these
included three type-IIa four-cell clones, five singly labeled
BPPNS-div cells, two type-IIb four-cell clones, and four
singly labeled PNS-div cells. Twelve examples were subjected to
immunostaining with anti-Hu (16A11)
(Marusich et al., 1994); Hu,
an RNA-binding protein, has been used as a neuronal marker in the developing
cerebral wall (Okano and Darnell,
1997
; Miyata et al.,
2001
). In 11 samples (92%), Hu was detected in both of the
NS-derived daughter cells (Fig.
4). Upon division of the BPPNS-div cell, its
bp was inherited by one of the Hu+ daughter cells. Some (though not
all) of such process-inherited Hu+ cells were quickly inserted into
the CP (Fig. 4B). In one case
(a PNS-div cell in an E14 slice), both daughter cells were negative
for Hu (not shown). One pair of daughter cells from an E14 PNS-div
cell stained with p27, a cell cycle arrest marker (cyclin kinase inhibitor),
and both daughter cells were positive (data not shown). One type-IIb clone
(case shown in Fig. 2C) was
treated with BrdU for 6 hours before fixation and stained for BrdU; only one
granddaughter cell born at the surface was positive, whereas the remaining
three granddaughters (including those generated by the NS mitosis) were
negative (data not shown). These Hu+, p27+, or
BrdU daughter cells showed morphologies (translocating- or
locomoting-like multipolar) (Nadarajah et
al., 2001
; Tamamaki et al.,
2001
; Tabata and Nakajima,
2003
) reported for neurons. During the extended observation of
daughter cells generated from BPPNS-div or
PNS-div cells (over 30 hours, without immunostaining; n=6
at E13 and n=8 at E14), only one NS-derived daughter cell divided but
all others (13 cells) did not. These results strongly suggest that most of the
NS mitoses observed in E13-E14 slices may have generated paired neurons (N/N
division).
We next sought to determine whether N/N divisions at the NS position also
occur frequently in vivo, but the direct lineage tracing was technically very
difficult. We therefore took advantage of the observation that Hu is expressed
in the peripheral nervous system in some dividing cells that are committed to
the neuronal lineage (Marusich et al.,
1994). Frozen sections of E13 and E14 cerebral walls were doubly
stained with Hu and pH3 (Fig.
5A). Separate immunostaining revealed that NS-dividing cells at
E13 and E14 were mostly (85-95%) Nestin+ and were rarely stained
with an endothelial cell marker (5-6%). Remarkably, most of the
pH3+ NS mitoses (93% at E13; 81% at E14) were Hu+,
whereas the S mitoses were completely negative
(Fig. 5B). The Hu+
index for NS mitoses at E14 was significantly higher (P<0.0001,
chi-square test) than those obtained for NS mitoses in sections at E17 (38%)
and P0 (22%) when gliogenesis predominates over neuronogenesis
(LeVine and Goldman, 1988
).
These results indicate that nearly all of the NS-dividing cells that posses
Nestin+ neuroepithelial-like character at E13 and E14 were
committed to the neuronal lineage.
Cell cycle-dependent and lineage-restricted expression of Ngn2
In order to identify the molecular mechanisms that allow a mitotic daughter
cell to undergo NS division, we focused on Ngn2, a bHLH transcription factor
known to be important for commitment to the neuronal lineage
(Sommer et al., 1996;
Fode et al., 2000
;
Nieto et al., 2001
) (reviewed
by Ross et al., 2003
). We
first compared the expression of Ngn2 protein between S and NS mitoses by
using antibodies against Ngn2 (Mizuguchi
et al., 2001
) and p-vimentin
(Fig. 6A-D, Table 3). Ngn2 was sporadically
detected in the VZ and SVZ with varying intensity
(Fig. 6A), but Nestin, RC2 and
Pax6 were all diffusely positive throughout the same area (not shown). The
proportion of Ngn2-positive cells out of the total p-vimentin+
cells (mitoses) at the NS position (32%) was significantly
(P<0.0001, chi-square test) greater than that observed for the S
mitoses (4%).
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We used slice culture to further examine the relationship between Ngn2 expression and cell cycle progression. Ngn2 immunostaining of DiI-labeled daughter cells within 4-8 hours of their generation (n=7) revealed that some daughter cells were Ngn2+++ but others were Ngn2+ or negative (Fig. 6L,M). In addition, moderate Ngn2 immunoreactivity was detected in the marking pin-like cells (Fig. 6N,O, n=3/3) and pia-connected, bipolar-shaped cells (not shown, n=2/2) that were about to lose their ventricular attachment, a critical step preceding or during G2 phase in cells undergoing NS-division (Figs 2, 3, 4). In contrast, Ngn2 immunoreactivity was not detected in DiI-labeled cells that were captured during their G2-like movement towards the ventricular surface (n=21) (Fig. 6P,Q). These data obtained through combined approaches indicate that Ngn2 expression is limited to a certain cycling population from as early as G1 phase and further restricted in a lineage-dependent fashion consistent with a bias towards the NS-dividing population, and that Ngn2 expression in cycling cells is strongest in G1 phase and subsequently declines.
Conversion of S division to NS division by Ngn2 retrovirus
The above results prompted us to hypothesize that Ngn2 might be involved in
the morphological changes that cause a cycling daughter cell to lose its
ventricular attachment and migrate to the SVZ or IZ for NS division. To test
this possibility, we injected a retrovirus containing ngn2 and GFP genes,
along with injecting a control GFP virus, into lateral ventricles of mouse
embryos (Fig. 7A). Considering
the delayed onset of expression of introduced genes, injections were made at
E12 and examinations to identify the position of GFP and pH3 double positive
cells were mostly performed 48 hours later (E14)
(Fig. 7B-I). In the control
treated group (7 embryos from 3 independent experiments), the majority of
GFP+ dividing cells (45 cells per embryo on average) were found at
the ventricular surface (Fig.
7B-D), providing a percentage NS-division of 26±3%
(mean±s.e.m.) (Fig. 7I).
In contrast, embryos injected with Ngn2 virus (n=9 from 3 independent
experiments) showed a reversed S:NS ratio
(Fig. 7E-I). The proportion of
GFP+ dividing cells (10 cells per embryo on average) that were
found at the NS position (73±3%) was significantly higher
(P<0.001, Mann-Whitney test) than that seen in control treated
samples. We confirmed the expression of Ngn2 in GFP+ NS dividing
cells (2/2) (Fig. 7H), although
all cells were not stained to determine precise Ngn2 expression rates. A
similar predominance of NS divisions was detected at 24 hours (n=3,
75-90%) and 36 hours (n=4, 75-85%) after injection of Ngn2 virus at
E12. These results, together with the spatiotemporal pattern of Ngn2
expression (Fig. 6), strongly
suggest that Ngn2 is involved in the choice of mitosis position during
E13-E14.
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Discussion |
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In vivo, Hu immunoreactivity in almost all the Nestin+ mitoses
indicated the complete commitment of this secondary mitotic population to the
neuronal lineage during E13-E14 (Fig.
5). This means, however, that detection of Hu alone cannot be
considered as proof in identifying a cell as a neuron; one extreme
interpretation of our observation that NS mitoses produced paired
Hu+ cells in slices would be that many of the Hu+
NS-derived daughter cells were neuronal progenitor. Nevertheless, we still
consider that the most likely mitosis pattern taken by these NS-dividing cells
in vivo must be PNS-divN+N.
The number of NS mitoses is limited to 10-20% of the total mitoses during
this stage, this means that only a few, if any, of the NS-derived
Hu+ daughter cells as observed in slices can divide in vivo. A
previous study by Takahashi and colleague
(Takahashi et al., 1995b)
reported an increase in the relative size of the secondary
proliferative population, which corresponds to the NS-dividing
population, during E14-E15 (approximately 2.4 times expansion) and concluded
that only 14% of the NS-derived daughter cells could be neurons (thus the
division pattern should primarily be non-terminal). This study did not take
into account the possibility that mitotic cells are continuously supplied from
the ventricular surface to the NS position (to allow most of the preexisting
NS cells to undergo terminal N/N divisions while allowing the overall
NS-dividing population to expand); we demonstrated that this is, in fact, the
case at E14 (Figs 1,
2,
3,
Table 4). These lines of
evidence suggest that most Hu+ daughter cells that would be
generated from Hu+ NS mitoses in vivo may not subsequently divide.
In addition, the frequency of N/N division at the ventricular surface (i.e.
production of two daughter cells that did not divide beyond the time that many
other daughter cells had divided to form 3- or 4-cell clones) was low (3%,
Table 1) in the present study.
This rate would not sufficiently explain the existence of N/N divisions during
early corticogenesis in vivo that has been evidenced in mice
(Cai et al., 2002
) and
suggested in rats (Noctor et al.,
2001
). We must therefore conclude that the mode of division most
heavily favored by the in vivo NS mitoses during the pre-CP stage is N/N
(Fig. 8A).
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Because the E13-born CP neurons are the first to split the preexisiting
preplate, we were curious as to how they behave in reeler mice which
do not show such splitting (Caviness,
1982). Our pH3 immunostaining of frozen sections and use of slice
culture to monitor single cells revealed that NS-directed progenitors at E13
(BPPNS-div cells) are normal in both their mitosis
position and preceding morphological changes (nuclear movements and
bipolar-to-unipolar change as shown in Figs
2,
4) (T. Miyata,
unpublished).
Asymmetric P/P division for efficient neuron/progenitor segregation
The most effective means to expand the mitotic population is through paired
daughter cells generated from a progenitor cell individually giving rise to
paired mitotic daughters (P2P
4P). The type I P/P division that we
observed in the pre-CP stage (Fig.
1A,D, Table 2) may
account for such an expansion of the progenitor pool
(Fig. 8A). During the next
developmental phase, one can expect some degree of neuron release, for
example, production of 2 neurons and 2 mitotic daughters (2N+2P) after two
rounds of cell cycling, which could be obtained by asymmetric division at
either of the first or second rounds of division: two N/P divisions after
symmetric P/P division [P
P (
N/P) + P (
N/P), model 1] or
asymmetric P/P division followed by N/N and P/P division [P
P (
N/N)
+P (
P/P), model 2]. From our observation of the type II P/P divisions
(Fig. 1E,
Fig. 2A,C,
Table 2), it is suggested that
the cerebral wall at E13, just before the emergence of the CP, favors the
latter, often launching one mitotic daughter to the SVZ for subsequent
production of the earliest CP neurons, with much less frequent N/P divisions
(Table 1). Although direct
observation has not been possible so far, there may be lineage continuity from
type IIa to type IIb, making one mitotic daughter (PS) generated in
a type IIa division behave as a parent cell in type IIb
(Fig. 8A). Given the observed
mitotic positions, model 1 can be regarded as PS
PS
(
N/P)+PS (
N/P) and model 2 as
PS
PNS (
N/N)+PS (
P/P). The
most important difference between these two models is the rate of the
segregation of neurons and mitotic cells.
Model 1 requires neurons to evacuate from the ventricular surface to
prepare a vacant space for descending G2-phase cells, and it further requires
cycling cells to stay in a limited space until new-born neurons exit the VZ,
which takes up to 10 hours (Takahashi et
al., 1996a). Model 2 does not have these constraints. Considering
the high productivity of the VZ during the period of CP emergence
(Smart, 1973
;
Takahashi et al., 1996b
),
model 1 would not be the best way to avoid cellular congestion
(Smart, 1973
) in the VZ.
Therefore, the asymmetric P/P division that we observed can be considered to
be a means for the cerebral wall to combine most efficiently the supply of
early CP neurons and the expansion of the cycling cell population. The
contribution of NS-divisions to the production of late-born (upper-layer) CP
neurons has just been demonstrated most convincingly in the rat
(Noctor et al., 2004
). It
seems appropriate to consider the NS neuronogenic division as a histogenetic
tool that has already evolved in the rodent
(Smart, 1973
;
Haubensak et al., 2004
) and
may have eventually allowed primates to develop unique morphological features,
such as the outer subventricular zone
(Smart et al., 2002
), rather
than taking it as a primate-specific system
(Letinic et al., 2002
).
Morphological signs of fate commitment during cell cycle progression
Figures 8B,C summarize the
changes in the morphology of S- and NS-dividing progenitor cells observed in
E13-14 slices. The proportions of individual cell cycle phases indicated at
the top are principally based on the findings of Takahashi et al.
(Takahashi et al., 1995a). For
example, bipolar-to-unipolar changes that are specific to
BPPNS-div cells, observed approximately 2-5 hours before
M-phase (Fig. 2B,
Fig. 4A,B), are illustrated
below S- and G2-phases (Fig.
8C, upper panel) considering time for G2+M (2 hours) and S
(approximately 4 hours) in vivo. Similarly, the collapse of a ventricular
process in the marking pin-like PNS-div cells
(Fig. 2C,
Fig. 4C) is shown during S-G2
phases (Fig. 8C, lower panel).
This irreversible transformation prior to a NS-division may always be
accompanied by commitment to the neuronal lineage and also in most cases by a
decision for a N/N division. Previous transplantation experiments on ferrets
(McConnell and Kaznowski, 1991) indicated that commitment of a neuron to take
a deep-layer (layer VI) fate occurs in its parent cell late in S-phase or at
the S-to-G2 transition. Although we are currently not sure whether our
observation can directly explain these results, the detection of irreversible
cell fate determination at very similar time points with these two completely
different approaches is remarkable.
Another sign for intracellular events that might be accompanied by or
linked with fate determination was detected in earlier timing
(Fig. 8B,C, lower panels).
Whether a daughter cell that does not inherit its parents bp extends a
new process to the pial surface (PS-div) or not
(PNS-div) can be distinguished within 10 hours after the
cells birth (Fig. 1B,
Fig. 2C), and this choice was
suggested to occur in G1 phase (5.5 hours at E13 and 9.3 hours at E14 in vivo)
(Takahashi et al., 1995a).
G1 has also been noted as a phase when the strongest and widest Ngn2
expression was detected (Fig.
6); Ngn2 seems to be expressed in a subset (33%,
Table 3) of mitotic daughters
with varying intensity (Fig.
6G). Its expression is more restricted to fewer cycling cells from
S (approximately 23%) through G2 (approximately 20%), along with the
disappearance of strongly positive cells. Although the direct measurement of
Ngn2 labeling in G2-phase cells destined for NS division was difficult, the
probability of Ngn2 expression in cells during or just prior to S divisions,
which are roughly four- to eight-times more numerous than NS divisions, was
low (3-5%), and therefore the majority of the Ngn2+ G2 cells seem
to be determined to divide abventricularly. This spatiotemporal pattern
(Fig. 8D) is very comparable
with the above mentioned morphological signs that are closely associated with
NS divisions. Most likely, mitotic daughter cells infected with Ngn2 virus
(Fig. 7) may have taken such
morphological changes to take NS positions during their cell cycling. Because
P/N divisions that we observed in the present study (11 cases) were all judged
as PS-divPS-div+N and we found that Ngn2 was
expressed in some neurons (Table
3), Ngn2 might also be involved in differential daughter cell
movements in P/N divisions, which should be examined in the future. It also
remains to be determined regarding the nature of the Ngn2+
S-dividing cells.
At E15 in Ngn2/Mash1 double knock-out mice
(Nieto et al., 2001), the
position of S-phase cells is abnormally high (scattered throughout the
cerebral wall). This could be a paradox between their findings and our
demonstration of increased NS divisions by Ngn2 expression. One plausible
explanation would be that the extra VZ S-phase cells found in
Ngn2//Mash1/ mice may be of
the glial lineage (Nieto et al.,
2001
). Another possibility predicts that Ngn2 might have a
negative effect on the elongation of the ventricular process (ascent of the
nucleus) late in G1 phase, the absence of which should result in the extra VZ
S-phase cells regardless of a role in S-G2 phases for NS-division that we
propose. Because Ngn2 is expressed in the mouse retina where no NS divisions
are seen, it is likely that the NS-directing effect of Ngn2 may be exhibited
in the context of region-specific histogenesis.
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ACKNOWLEDGMENTS |
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Footnotes |
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* Present address: Laboratory for Asymmetry, Center for Developmental
Biology, RIKEN, Kobe 650-0047, Japan
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