Centre for Cellular and Molecular Dynamics, Department of Anatomy and Developmental Biology, University College London, Gower Street, London WC1E 6BT, UK
* Author for correspondence (e-mail: jonathan.clarke{at}ucl.ac.uk)
Accepted 25 April 2003
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SUMMARY |
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Key words: In vivo imaging, Vertebrate neurogenesis, Neural progenitor cell fate, Asymmetric division, Zebrafish
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INTRODUCTION |
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Progenitor behaviour is better understood in invertebrate nervous systems
and here asymmetric cell division appears to be a fundamental mechanism for
generating cell diversity. In these systems asymmetric division is often based
on the unequal partitioning of cytoplasmic determinants during division
(reviewed by Lu et al., 2000)
and this relies on mechanisms that co-ordinate the plane of progenitor
division with the sub-cellular localisation of fate determinants. In
Drosophila the neuroblast division is clearly asymmetric in that
cytoplasmic determinants are inherited asymmetrically by daughter cells and
daughter cells have different fates
(Hirata et al., 1995
).
However, the neuroblast division generates two progenitors, whereas in
vertebrate studies an asymmetric division is typically defined as one that
generates a progenitor and a neuron (e.g.
Mione et al., 1997
;
Noctor et al., 2001
;
Cai et al., 2002
). There are
also clear cases in Drosophila in which an asymmetric division
generates two daughter neurons that have different phenotypes
(Bossing et al., 1996
). In
vertebrates the analysis of sister cell phenotypes in vivo has been very
limited, but clonal analysis in the chick has provided evidence that
individual progenitors often give rise to clones of neurons of a single
subclass (Lumsden et al.,
1994
; Clarke et al.,
1998
), suggesting that in this system sister cells may become
symmetrically fated neurons in vivo. The role that cytoplasmic determinants
may play in asymmetric fate decisions in the vertebrate nervous system is not
certain, but at least some aspects of the molecular machinery are conserved
(e.g. Kubu et al., 2002
;
Petersen et al., 2002
;
Shen et al., 2002
;
Verdi et al., 1999
;
Wakamatsu et al., 1999
;
Wakamatsu et al., 2000
;
Zhong et al., 1997
;
Zhong et al., 2000
;
Zilian et al., 2001
) and
orientation of division has been correlated with asymmetric cell behaviours in
some vertebrate systems (Chenn and
McConnell, 1995
; Cayouette et
al., 2001
).
Many invertebrate neural progenitors undergo stereotyped, invariant
patterns of division (e.g. Skeath and Doe,
1996; Brewster and Bodmer,
1996
; Shankland,
1995
). Whether invariant lineages occur in the vertebrate nervous
system is not yet known. However, when progenitors of the cerebral cortex are
dissociated from the early embryonic brain and cultured at low density, they
can undergo a series of stereotyped cell divisions and fate decisions to
generate a family of differentiated cell types
(Qian et al., 1998
).
Furthermore, the normal sequence of neurogenesis followed by gliogenesis is
maintained by cortical progenitors in culture
(Qian et al., 2000
). These
results suggests that some progenitor cells could be programmed to generate
invariant lineage trees, but whether such stereotyped patterns of division
occur in the normal environment of the vertebrate brain is not certain.
Our understanding of the importance of asymmetric divisions and invariant lineage in the vertebrate brain is largely limited by its complexity, inaccessibility during the period of neurogenesis and its poor optical qualities. Here we use the relative simplicity, accessibility and superior transparency of the zebrafish CNS to overcome these difficulties. We have been able to follow the fate of neural progenitors through multiple rounds of cell division, covering a substantial period of zebrafish neurogenesis, reconstruct lineage trees and document the type of divisions that lead to neuronal birth.
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MATERIALS AND METHODS |
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BrdU labelling
BrdU (Sigma) was applied by pulse labelling in solution. Embryos were
placed in a solution of 2 mg/ml BrdU in embryo medium with 15% DMSO for 20-30
minutes on ice. Embryos were left to recover at 28.5°C for at least 15
minutes afterwards.
HuC-GFP is pan-neuronal and expressed in neurons soon after
birth
HuC is an RNA-binding protein thought to be expressed exclusively in
neurons (Park et al., 2000a),
and we have used the HuC-GFP transgenic line
(Park et al., 2000b
) in
several of our analyses. In order to be sure that HuC-GFP was expressed only
in post-mitotic cells, we labelled embryos with single pulses of BrdU at 24,
36, 60 and 72 hpf and checked for co-expression with HuC-GFP 30 minutes later.
HuC-GFP cells were never BrdU-positive. We also determined how soon neurons
expressed HuC-GFP after their birth. First we determined that 3 hours was the
shortest time interval between BrdU pulses at 24 or 36 hpf and co-expression
of the cell-cycle marker phosphohistone H-3 that marks the G2/M transition
(Juan et al., 1998
). We then
determined that the shortest interval between BrdU pulse and HuC-GFP
expression was 7 hours and were thus able to calculate the shortest interval
between G2/M transition and HuCGFP expression at these times. At both 24 and
36 hpf, HuC-GFP expression can be observed just 4 hours after G2/M transition.
Considering that mitosis lasts approximately 1 hour and transcription and
translation of HuC-GFP will take approximately 1-2 hours, we consider that
HuC-GFP expression is a very early marker of neuronal differentiation.
Cell counting
Embryos were stained by placing them in 5 µM Bodipy 505/515, (Molecular
Probes), or 5 µM Texas Red Bodipy Ceramide (Molecular Probes). Total cell
numbers were counted from confocal z-series of living embryos stained with
Bodipy 505/515, which outlines all cells. Counts were made from rhombomeres 4
and 5, which are largely lineage-restricted compartments
(Fraser et al., 1990). The
total number of non-neuronal cells was counted as HuC-GFP-negative cells in
transgenic specimens counter-stained with Texas Red Bodipy ceramide. Total
neuronal number was then calculated by subtracting these two numbers. The
interval in the z plane was always smaller than the smallest unit to be
counted, typically 3 µm. The unbiased disector method was used to count
cells (Sterio, 1984
) and was
performed using NIH image. Volume estimation was performed using Volocity
software (Improvision).
Cell-cycle length
In order to be confident of our lineage data, we wanted to estimate the
number of cell cycles we could expect the labelled progenitors to complete
within our observation period, so we have estimated the cell-cycle length at
36 hpf. First, using repetitive pulses of BrdU, we determined that BrdU needs
to be present for at least 6 hours in order for incorporation into all
progenitors. This suggests that the G2+M+G1 interval between S-phases is
approximately 6 hours. Second, we determined that a single BrdU pulse at 36
hpf is incorporated into 42.5% of the total progenitor population, thus
suggesting that S-phase occupies 42.5% of the cell cycle. Combining these data
gives a minimum length of the cell cycle of 14 hours at 36 hpf. Thus, if the
interval between observations is less than 14 hours, no cell should undergo
more than one division in this time.
Single cell injection
Single cells were injected with fluorescent dextran in the region of neural
plate or neural keel fated to generate rhombomeres 3-7 of the hindbrain. Cells
in wild-type and HuC-GFP-positive embryos were labelled with a mixture of 1%
Rhodamine Dextran (3000 MW, Molecular Probes) and 2% biotinylated dextran
(Molecular Probes) by iontophoresis (see
Clarke, 1999). To facilitate
passing the electrode through the skin, embryos older than 9 hpf were
incubated in a solution of 5 mg/ml pronase (Sigma), for one minute. Embryos
were returned to embryo medium and were stabilised for iontophoresis by being
placed on a drop of 2% methyl cellulose. Each specimen was examined after
10-20 minutes to ensure that only one healthy cell was labelled. Cells were
re-observed every 8-12 hours. At each observation point, the number of cells,
their morphology and their location were noted and cells were imaged from both
dorsal and lateral aspects if necessary.
Imaging
Live and fixed embryos were imaged on either a Leica confocal microscope or
a Zeiss Axioplan 2 fitted with a Hamamatsu Orca ER digital camera. Data was
collected using either Leica confocal or Openlab software (Improvision).
Deconvolution of z-stacks gathered on the Axioplan was performed using Openlab
software. 3D analysis of confocal stacks was performed using either NIH image
or Volocity (Improvision).
Immunochemistry
The following primary antibodies were used during this study. Mouse
monoclonal anti-zrf-1 (1/4, Oregon Monoclonal bank), rabbit polyclonal
anti-GFAP (1/100, kind gift of John Scholes), mouse monoclonal anti-acetylated
tubulin (1/1000, Sigma), mouse monoclonal anti-BrdU (1/200, Sigma), rabbit
polyclonal anti-GFP (1/1000, Torrey Pines Biolabs) and anti-phosphohistone H-3
(1/1000, Upstate Biotechnology). The following secondary antibodies were used:
Alexa 488 and 568 goat anti-mouse IgG and goat anti-rabbit IgG (Molecular
Probes) and peroxidase-conjugated goat anti-mouse IgG (Sigma). All secondary
antibodies were used at a concentration of 1/200. Texas Red Avidin (1/200,
Zymed) was used to visualise cells injected with biotinylated dextran.
-tubulin Gal4UAS-GFP DNA
-tubulin Gal4UAS-GFP DNA (Koster
and Fraser, 2001
) was injected into the cytoplasm at the one-cell
stage at a concentration of 20 ng/µl to generate embryos with a mosaically
labelled nervous system. The GFP translated using this construct was excluded
from the nucleus, which facilitated detailed imaging of cell morphology.
Acridine orange
The vital dye acridine orange [acridinium chloride hemi-(zinc chloride)
(Sigma)] was used to detect apoptotic corpses in live embryos. Embryos were
incubated in a solution at 5 µg/ml for 30 minutes and washed in embryo
medium several times before imaging.
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RESULTS |
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Individual progenitors can be followed through several rounds of
division to their terminal mitosis
In order to analyse the generation of neurons, we have labelled single
neural progenitors with fluorescent dextran and followed their fate over a
large period of embryogenesis. Each labelled progenitor was checked at the
time of injection to ensure that only one cell was labelled and then
re-observed every 8-12 hours until the embryo was 48-72-hours old (Figs
2,
3,
4). The 8-12-hour interval is
sufficient for only one round of division (see Materials and Methods). Most of
our lineage analyses (54/86) were performed in the HuC-GFP line, thus
eliminating the need to phenotype cells by morphology alone. We present our
lineage tree analyses from a starting point of approximately 15 hpf when the
hindbrain is at the neural rod stage of development. Prior to this the cells
of the neural plate have converged on the dorsal midline and most have
undergone a single midline division that deposits one progenitor on either
side of the neural midline (Kimmel et al.,
1994) (Fig. 2A). We
have monitored the development of 86 progenitors. The majority of these
progenitors (79/86) resulted in clones containing only neurons, and for each
of these clones we have successfully reconstructed their lineage tree. The
remaining 7/86 progenitors generated clones containing a mixture of neurons
and radial cells (putative progenitors) at the end of their analysis. We will
describe the analyses of these two classes of clones separately.
|
|
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The range of lineage trees that were characterised in their entirety is shown in Fig. 5A. The reconstructed trees for neuron-only clones fall into two clear categories. Most trees (46/68 or 68%) contain no divisions that generated a neuron and a further progenitor, whereas in the remainder (22/68 or 32%) one or two such asymmetric divisions are seen (Fig. 4, Fig. 5A). When present, these asymmetric divisions were usually the first division of the lineage (19/22) and usually one cell-cycle before the terminal divisions of that tree, thus generating a three neuron sub-clone motif (19/22) (see Fig. 4 and Fig. 5A). None of the fully reconstructed lineage trees contain a progenitor that follows a classic stem-cell mode of division; i.e. one that self-renews and generates a differentiated cell-type at each division. The clones were evenly distributed in the mantle layer of the hindbrain (Fig. 5B), suggesting that we have sampled progenitors from many regions of the VZ.
|
It is possible that the composition of our lineage trees could be
influenced by cell death. We have estimated cell death by analysing acridine
orange incorporation in rhombomeres 4 and 5. We find an average of 15 dead
cells at 15 hpf (n=3), 14 at 24 hpf (n=2), 12 at 36 hpf
(n=2) and only 3 at 48 hpf (n=2) in rhombomeres 4 and 5. If
clearance of corpses takes an average of 2 hours
(Cole and Ross, 2001), then the
predicted number of cells undergoing apoptosis between 24 and 36 hours is less
than 2% (84/4294) of the total and only 1.3% (72/5367) of the total cells for
the period between 36 and 48 hours. Furthermore, the number of cells in our
clones never decreased in the intervals between observations and we never
detected apoptosis by analysing time-lapse movies of HuC-GFP-positive embryos
or Bodipy 505/515-stained embryos between 15 and 36 hpf. These results suggest
that cell death plays a minor role in hindbrain development at these stages
and suggest that our reconstructed lineage trees are a true representation of
clonal development.
Most asymmetric divisions that generate a progenitor and a neuron
occur within the plane of the VZ
Previous studies have suggested that the plane of mitosis within the VZ is
correlated with the fate of the daughter cells, such that progenitors dividing
within the plane of the VZ generate two further progenitors, whereas mitoses
that occur perpendicular to the VZ produce an asymmetric division generating
one neuron and one progenitor (Chenn and
McConnell, 1995). In order to assess this possibility in the
zebrafish hindbrain we performed a large-scale analysis of mitotic
orientation. We have analysed time-lapse data of progenitors labelled with
fluorescent dextran (Fig. 6A-C)
and additional material stained with the vital dye Bodipy 505/515 that allows
us to visualize cells as they round up in the VZ and divide
(Fig. 6D-F). We concentrated on
the period between 18-30 hpf because our lineage analysis demonstrated that
28% (19/68) of divisions in this specific period are asymmetric in that they
generate both a neuron and a progenitor. However, during this time only 2%
(12/557) of progenitor divisions were perpendicular to the plane of the VZ;
i.e. divided apico-basally. We conclude that in the zebrafish hindbrain most
asymmetric divisions must derive from progenitors that divide in the plane of
the VZ.
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DISCUSSION |
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Probabilities and implications for mechanisms
We wanted to be confident that our lineage tree analysis could account
quantitatively for neurogenesis in the zebrafish hindbrain. The 86 original
progenitors in our lineage analysis produce a total of 236 neurons by 48 hpf.
This gives a factor of 2.74 neurons produced per progenitor. This data
predicts that the 1618 progenitor cells actually present in rhombomeres 4 and
5 at 15 hpf should generate 2.74x1618 (4440) neurons by 48 hpf. In fact,
our cell counts reveal that there are 4150 HuC-GFP-positive cells present at
48 hpf. This confirmed that the lineage data predicts actual neurogenesis with
an error of only 7%.
We have quantified progenitor divisions according to whether they generate
progenitors, neurons or a combination of the two.
Table 1 shows the relative
proportion of each of these division modes at the first and second rounds of
division in our lineage analysis, and Table
2 shows the percentage of neurons and non-neuronal cells generated
at each division. After the first division there seems to be an almost equal
probability that an individual cell will differentiate into a neuron (54%) or
re-enter the cell cycle (46%) (Table
2). However, there is also a strong bias towards divisions that
generate either two neurons (40%) or two progenitors (32%) with
progenitor/neuron divisions occurring at a frequency well below that predicted
by chance alone (Table 1). After the second division there is an overwhelming bias for daughter cells to
differentiate as neurons (90%) and a consequent strong bias for both daughter
cells to be neurons (86%). Our data shows that the vast majority of neurons
generated throughout hindbrain neurogenesis are born via a terminal neurogenic
division. Thus for the majority of hindbrain neurons the mechanism that
commits cells to a neuronal fate is not linked to a mechanism that determines
asymmetric division. The strategy of using symmetric divisions to first expand
the progenitor population and then to generate pairs of neurons through
terminal divisions is the quickest mechanism to generate many neurons in a
small number of rounds of division. Thus only two rounds of division are
needed for each progenitor to generate four neurons. A progenitor that divides
in an asymmetric stem cell mode would require four rounds of division to make
the same number of neurons. The majority of hindbrain progenitors thus appear
to behave like `transit amplifying cells' rather than stem cells. Transit
amplifying cells have previously been described in both neural and non-neural
progenitor pools where they characteristically undergo a limited number of
rounds of division before terminally differentiating
(Doetsch et al., 2002;
Potten and Loeffler,
1990
).
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Signalling via the Notch pathway has been shown to be an important
mechanism that restricts the premature differentiation of cells in the
zebrafish neural plate and neural tube
(Haddon et al., 1998;
Gray et al., 2001
;
Itoh et al., 2003
). Loss of
Notch-Delta signalling in the zebrafish mind bomb mutant leads to a
spinal cord apparently depleted of progenitors and composed entirely of
neurons by 24 hpf (Itoh et al.,
2003
). Our own observations suggest that although the mind
bomb hindbrain is also largely composed of prematurely differentiating
neurons, a small population of progenitors remain even at 60 hpf, perhaps
suggesting some progenitors are maintained by mechanisms other than the
Notch-Delta pathway (D.A.L. and J.D.W.C., unpublished).
Asymmetric divisions in other vertebrate systems
Our results demonstrate that progenitors exhibiting asymmetric stem
cell-like behaviour are rare during the major phase of embryonic hindbrain
neurogenesis. However, both birth-dating studies and clonal analyses (e.g.
Takahashi et al., 1996;
Mione et al., 1997
;
Cai et al., 2002
) suggest that
asymmetric stem cell divisions do occur in the mammalian cortex, but direct
evidence for divisions that generate one progenitor and one post-mitotic cell
in the intact brain is scarce. Evidence has recently been provided from
cortical slice preparations that radial glia, infected with retroviruses
expressing GFP and followed by time-lapse microscopy, can divide
(Noctor et al., 2001
). Clonal
analysis in this study revealed individual radial glial progenitors alongside
neurons at varying times after infection, suggesting that they divided
asymmetrically to generate the neurons
(Noctor et al., 2001
). In
vitro studies (Qian et al.,
1998
; Shen et al.,
2002
) have followed cells isolated from embryonic mouse cortex
between E10.5 and E13 and monitored their behaviour using video time-lapse
microscopy. In a minority of clones analysed a stereotyped asymmetric division
is described in which one daughter cell divides once more to generate two
neurons whereas the other daughter divided more than once more. In this case
the asymmetric divisions produces two progenitors with different fates
(Qian et al., 1998
) as is the
case for the division of the Drosophila neuroblast. In fact, the
majority of clones followed in this in vitro study expanded by divisions that
generated either two progenitors or two differentiated progeny
(Qian et al., 1998
). In a more
recent in vitro study from the same lab, some divisions generated both a
progenitor and a neuron under similar culture conditions
(Shen et al., 2002
). The
precise behaviour of these progenitors in vivo remains to be determined.
Correlation between cell fate and plane of division
Initial reports claimed cortical progenitors dividing within the plane of
the VZ generated daughter cells that adopted symmetrical fates, whereas
progenitors dividing perpendicular to the plane of the VZ generated daughters
with asymmetric fates (Chenn and McConnell,
1995). This study reported that the cell that left the VZ
expressed notch 1 asymmetrically with respect to its sister. However, the fate
of these cells was never followed for a sufficiently long period to determine
their phenotype unequivocally. Recently, others have readdressed this question
and conflicting reports have been published
(Cayouette et al., 2001
;
Silva et al., 2002
;
Das et al., 2003
). In one study
the authors demonstrate that in the rat retina the proportion of cells
dividing out of the plane of the VZ peaks at P0 at 21%. The daughter cells of
these divisions are shown to inherit Numb protein asymmetrically. However,
these observations are made long after the bulk of retinal neurons have been
generated and do not address the fate of the daughter cells
(Cayouette et al., 2001
). In a
chick retinal study the orientation of division was compared between regions
where neurons were being produced and regions where neurons were not being
produced. In this study no difference in the orientation of division between
these two regions is reported (Silva et
al., 2002
). In a more recent study in the zebrafish retina, a
change in the orientation of division between the central-peripheral to
circumferential axes was reported to coincide with the onset of neurogenesis
but no cells were seen to divide out of the plane of the VZ
(Das et al., 2003
). It is
suggested that this change in orientation is linked to the switch from
proliferative to neurogenic divisions. However, no evidence is provided that
asymmetric cell fate occurs in any of these studies, so a correlation between
fate, division mode and orientation cannot be made. Our study addresses this
issue more directly because our analysis of division orientation is made
during a period when we have demonstrated a relatively high proportion of
asymmetrically fated divisions. Our findings do not agree with the original
Chenn and McConnell model of apico-basal asymmetric divisions, and strongly
suggest that asymmetrically fated divisions usually occur in the plane of the
VZ.
Radial glia and late progenitors
Trevarrow et al. (Trevarrow et al.,
1990) described the stereotyped arrangement of many cell types
within a zebrafish rhombomere. One cell type formed a glial curtain of radial
processes on either side of the rhombomere boundaries. We have characterised
these cells further and found that their cell bodies are located in the VZ in
an area devoid of HuC-GFP expression and that they have a distinct radial
glial morphology. Importantly, we find that many of these cells express both
glial and mitotic markers. We conclude that at least a subset of these radial
glial cells are progenitor cells. Other studies have demonstrated similar
functions for radial glia in the mammalian forebrain
(Hartfuss et al., 2001
;
Malatesta et al., 2000
;
Miyata et al., 2001
;
Noctor et al., 2001
;
Noctor et al., 2002
;
Tamamaki et al., 2001
;
Heins et al., 2002
). It is
probable that radial glia are a heterogeneous population with subsets
expressing distinct molecular profiles and that this may reflect differences
in their behavioural phenotype (Hartfuss
et al., 2001
). We have direct evidence that some radial cells of
the glial curtain are neurogenic progenitors (A.T.G. and J.D.W.C.,
unpublished) and it is probable that other subsets will give rise to
oligodendrocyte precursors and mature astroglial cells.
We have focused our analysis on progenitor divisions that generate neurons
and for this purpose we defined an asymmetric division as one that generates
both a neuron and a progenitor. In the strict sense it is possible that
divisions that generate two progenitors or two neurons may also be asymmetric
if the daughter cells are intrinsically different to one another, but we have
not analysed this possibility here. The Drosophila neuroblast divides
asymmetrically to produce another neuroblast and a ganglion mother cell, which
is itself another progenitor but with a more limited potential (reviewed by
Lu et al., 2000). Also in the
fly, asymmetric divisions have been shown to generate two different types of
neuron (Bossing et al., 1996
),
thus helping to generate the diversity of differentiated cells. In the
vertebrate brain, neuronal diversity is thought to be largely generated by
extrinsic signals acting on neuronal progenitors
(Edlund and Jessell, 1999
)
rather than through the mechanisms of asymmetric division. In line with this,
single hindbrain progenitors in the chick embryo tend to generate clones of
neurons of a restricted subclass (Lumsden
et al., 1994
), and the identity of the neurons generated in these
clones is correlated with the position that the progenitors occupy in the
dorsoventral axis (Clarke et al.,
1998
). In the future we intend to address the issue of neuronal
phenotypes in our zebrafish clones to determine whether there are any
consistent asymmetries in the identity of sibling neurons, but for the moment
our analysis has centred on the decision of whether to make a neuron or not.
Our study has of course only described the fate of daughter cells rather than
their potential. We do not know that the daughter cells fated to become
neurons were determined at or before their terminal mitosis or that the
determining step occurred simultaneously in both daughters (but our
unpublished evidence shows that the time between neuronal birth and HuC-GFP
expression is very stereotyped in post-mitotic neurons). However, it is
possible that there could be an asymmetry in sibling potential that is not
apparent from simply monitoring their fate. This will be an important issue to
address in the future.
In conclusion, our work has provided the first in vivo analysis of neurogenesis at the level of both the individual cell and the population as a whole in a vertebrate. Our results suggest that in this vertebrate system the molecular determinants that control whether a cell will become a neuron are usually not linked to a mechanism that generates asymmetric divisions. We believe that this analysis has provided key insights into how the majority of embryonic neurons are generated in a vertebrate system and provides a good framework against which genetic manipulations can be targeted in the future.
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ACKNOWLEDGMENTS |
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