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First published online April 30, 2007
doi: 10.1242/10.1242/dev.002519
1 Divisions of Cell and Developmental Biology, Wellcome Trust Biocentre, College
of Life Sciences, University of Dundee, Dow Street, Dundee DD1 5EH, UK.
2 Divisions of Gene Regulation and Expression, Wellcome Trust Biocentre, College
of Life Sciences, University of Dundee, Dow Street, Dundee DD1 5EH, UK.
* Authors for correspondence (e-mail: jason{at}lifesci.dundee.ac.uk; k.g.storey{at}dundee.ac.uk)
Accepted 13 March 2007
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SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: FGF, Asymmetry, Live imaging, Mitotic spindle, Neurogenesis, Spinal cord, Chick, Stem cells
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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;
Price and Briscoe, 2004).
There is also evidence for crosstalk between patterning factors that regulate
cell type and genes that drive cell cycle exit and neuronal differentiation
(Scardigli et al., 2003;
Scardigli et al., 2001)
(reviewed by Bertrand et al.,
2002). Neuron production is itself regulated through the Notch
signalling pathway; differentiating neurons deliver lateral inhibitory
signals, such as Delta1, which ensure that neighbouring cells do not
differentiate simultaneously (reviewed by
Lewis, 1996). However, despite
this wealth of understanding at the level of gene regulation, the cell
behaviour underlying neuron production in the spinal cord remains poorly
characterized.
The spinal cord is so well-studied because it is a relatively simple
neuroepithelium. At early stages it comprises a bilaterally symmetrical tube
each side of which is just one cell wide, with each cell contacting both the
outer basal and inner apical (ventricular/luminal) surfaces. Within this
tissue nuclei migrate between these surfaces and their position reflects
progression through the cell cycle; mitosis occurs apically and nuclei move
towards the basal surface during G1. After the nuclei arrive at the basal
surface, cells then either re-enter S phase or exit the cell cycle and become
neurons. This process, known as interkinetic nuclear migration
(Frade, 2002), and subsequent
neurogenesis have not been monitored in the spinal cord in vivo.
Neurons can be produced by asymmetric, stem cell mode divisions that
produce a neuron and a progenitor or by symmetric terminal divisions that
produce two neurons. Retroviral labelling studies indicate that both
asymmetric and symmetric modes of neuron production underlie sensory neuron
generation in the latestage spinal cord
(Wildner et al., 2006).
However, the first neurons to be born in the chick spinal cord are reticular
spinal interneurons located in dorsal and intermediate regions, followed by
ventral motor neurons (Sechrist and
Bronner Fraser, 1991), and the modes of division that produce
these crucial components of the spinal cord have not been determined.
Work in the mammalian cortex suggests that whether a division generates
asymmetric or symmetric cell fates depends on the orientation of the mitotic
spindle and hence cleavage plane position with respect to the apical surface
of the neuroepithelium (Chenn and
McConnell, 1995). A parallel cleavage plane places one daughter
cell at the apical surface and the other more basally, whereas a perpendicular
cleavage plane generates daughter cells that inherit equal portions of apical
and basal membrane; mitotic spindle orientation may thus mediate cell-fate
choice, as a parallel cleavage plane could lead to asymmetric segregation of
determinants localized at apical or basal surfaces (reviewed by
Gotz and Huttner, 2005). Chenn
and McConnell propose that in the early cortex perpendicular cleavage plane
divisions give rise to two progenitors, whereas parallel divisions generate a
neuron and a progenitor (Chenn and
McConnell, 1995). This and other studies
(Kosodo et al., 2004) thus
suggest a link between division orientation and acquisition of a neurogenic
(neuron generating) ability.
Experimental evidence for the importance of mitotic spindle orientation for
cell-fate choice in the mammalian cortex and retina has come from recent work
in which its position has been manipulated by interfering with components of
an apical protein complex, including Inscuteable and AGS3
(Betschinger and Knoblich,
2004; Sanada and Tsai,
2005; Zigman et al.,
2005). Disruption of this apical complex leads to an increase in
perpendicular cleavage plane divisions and in symmetric cell fates. The
relationship between cleavage plane orientation and cell-fate choice has not
been addressed in the spinal cord, nor is it known whether this tissue, like
the cortex, displays a transition from asymmetric to symmetric modes of neuron
production as development proceeds. Neuron-generating divisions in the cortex
are further distinguished by longer cell cycle times, because of extension of
G1 phase (Calegari and Huttner,
2003; Takahashi et al.,
1995) (reviewed by Gotz and
Huttner, 2005). It has been suggested that a longer G1 allows time
for the asymmetric localization and accumulation of cell fate determinants.
Although overall cell cycle time gets longer as development proceeds in many
neuroepithelia, including the spinal cord
(Kauffman, 1968), it has yet
to be established whether this is a specific characteristic of all
neuron-generating divisions.
Here we have developed a novel long-term time-lapse imaging assay to monitor single cells in the early chick spinal cord as they transit mitosis and then either divide again or undergo neuronal differentiation. Our study defines key parameters of cell behaviour in this tissue and surprisingly reveals that neurons are generated concurrently by symmetric and asymmetric division. Furthermore, we find that mitotic spindle orientation does not correlate with acquisition of neurogenic ability, but serves within neurogenic cells to distinguish modes of neuron production.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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)
stage 9-10 were electroporated using standard techniques
(Itasaki et al., 1999), with a
concentration of 0.125 µg/µl enhanced yellow fluorescent protein
(eYFP)-
-tubulin (BD Sciences/Clontech) and/or Gap43-mRFP1
(Poggi et al., 2005). Embryos
were incubated at 38°C for 4 or 16 hours (giving slices for imaging at
HH10 and 12). Slices of 
150 µm were taken with a microknife, embedded
in rat tail collagen type I in coverslip-based petri dishes (WillCo-dish
glass-bottom dish, Intracel, Royston, UK; GWst-3522, coated with
poly-L-lysine, Sigma) and cultured in Neurobasal medium, without phenol red
(Gibco), supplemented with B-27 to a final 1x concentration with
L-glutamine and gentomycin [after Placzek and Dale
(Placzek and Dale, 1999)]
maintained at 37°C with 5% CO2/air for 4 hours before
imaging. For fibroblast growth factor (FGF) treatment, media containing 200
ng/ml Human FGF4 (R&D Systems) replaced normal medium at the start of the
experiment and was changed once (at 18 hours) approximately halfway through
the imaging period.
Slice imaging
Slices were imaged on a DeltaVision Spectris microscope workstation
(Applied Precision, LLC, Issaquah, WA) in a Solent environmental chamber kept
at 37°C. The chamber stage was buffered with 5% CO2/95% air mix
and maintained in a humid environment. Images were captured using a 40x
1.35 NA objective lens with the Hg-arc lamp. Thirty optical sections (exposure
time=50 milliseconds, 512x512 pixels, bin=2) spaced by 1 µm to 2.5
µm were imaged at either 7-minute or 1.5-minute intervals, for up to 38
hours. The point-visiting function in the software allowed up to eight slices
to be imaged during each experiment. Data was deconvolved and analyzed using
constrained iterative deconvolution
(McNally et al., 1999;
Swedlow, 1997). At the end of time-lapse experiments, slice culture dishes
were immediately washed in 37°C PBS and fixed in fresh 37°C 4%
formaldehyde (FA) for 40 minutes to preserve
microtubules/eYFP--tubulin, washed in PBS and 37°C PBS 0.5% Triton
X-100, and subsequently labelled with antibodies against GFP and neuronal
markers (see below).
Analysis of neuron production
Embryos at day 1.5 (HH10), day 2 (HH13), day 2.5 (HH16) and day 3 (HH18-19)
were fixed in fresh 4% FA and subjected to in situ hybridization for
NeuroM (Roztocil et al.,
1997), a bHLH transcription factor that marks newly born neurons
(Diez del Corral et al., 2002;
Roztocil et al., 1997) (see
below), using standard techniques. Sections through neural tube were then
processed for immunocytochemistry for HuC, an RNA binding protein
characteristic of differentiating neurons
(Marusich et al., 1994) (see
below), followed by staining with the chromatin marker DAPI.
Measurement of cell cycle and mitosis duration
Embryos at HH10 were exposed to 100 µl of 0.1 mM BrdU for 1 hour or 4
hours, fixed in 4% FA and processed and cryosectioned using standard
techniques (three embryos per treatment, four sections from each embryo).
Sections were treated using standard immunocytochemistry techniques,
post-fixed in 4% FA for 10 minutes and subjected to a standard BrdU detection
protocol (Gunhaga et al.,
2000). Total progenitor cell population was determined by
subtracting calculated proportion of differentiated neurons (3%, see Fig. S1
in the supplementary material) from total cell number, and simultaneous
equations were used to calculate cell cycle time
(Storey, 1989). Mitotic cells
were determined by distinct morphology of DAPI-stained nuclei undergoing
mitosis at the apical surface, and M-phase duration was calculated using cell
cycle time and mitotic index (see Fig. S2 in the supplementary material).
Cell death assessment
Embryos at 1.5 and 3 days were fixed in FA, processed and cryosectioned
using standard techniques. Sections through neural tube flanked by somites 1-3
were then processed for immunocytochemistry for cleaved Caspase-3 (see below)
and stained with DAPI. Apoptotic cells were scored in four sections in three
embryos for each timepoint, day 1.5 (1±0.6%) and day 3
(0.2±0.05%) (s.d. is the variance between embryos). Images of cells in
slice culture were analyzed for apoptosis (characteristic blebbing and
eruption of cells) at 16 hours in time-lapse movies (15 slices, four
experiments) (1.5±0.5%) (s.d. is the variance between experiments). The
number of cells undergoing apoptosis and the total number of labelled cells
visible at this time were recorded. This was used to calculate the proportion
of cells dying at a fixed timepoint, which could then be compared with that
observed in fixed tissue sections.
Measurement of cleavage plane orientation
Embryos at day 1.5 and 3 were fixed in fresh 4% FA and stained with DAPI.
The angle of the cleavage plane of cells in anaphase and telophase was
measured relative to the apical surface of the neural tube in sections through
neural tube flanked by somites 1-3 (in 10-12 sections from three to four
separate embryos). Images were captured on a Leica DM IRB, 20x 0.3 NA
lens with a Hamamatsu Snapper DIG16 camera. Data were analyzed onscreen using
OpenLab (Improvision) software. In slice cultures the apical surface was
identified by the position of the flattened end feet of cell processes, which
form the lumen wall. When the lumen is not absolutely vertical, stereoscopic
viewing of the images was used to determine the position of a dividing cell in
relation to the apical surface. Visualization of the lumen was also assisted
by brightfield images. To measure the angle of the cleavage plane relative to
the apical surface, data was rendered into a three-dimensional view (using the
volume viewing tool in Softworx software) and assigning the lumen (apical
surface) to the horizontal plane. The image was then rotated around 360°
in the y-axis to enable accurate measurement of the angle bisecting
the two daughter cells at cytokinesis [similar to the rendering and angle
measurements made in Haydar et al. (Haydar
et al., 2003)].
In situ hybridization and immunocytochemistry
Standard whole-mount in situ hybridization techniques were performed to
detect NeuroM. Immunocytochemistry was also performed using standard
procedures. Differentiated neurons were detected using mouse anti-HuC/HuD
(1:200) (Molecular Probes, A-21271) or mouse anti-neurofilament-associated
antigen 3A10 (1:50) (Developmental Studies Hybridoma Bank). Cleaved Caspase-3
(Asp175) rabbit monoclonal antibody (Cell Signalling, 9664) was used to detect
apoptotic cells, following the manufacturer's instructions. YFP signals in
slice cultures were enhanced with a rabbit anti-GFP (1:100) (MBL, 598) after
the imaging period. BrdU was detected with sheep anti-BrdU (1:100) (AbCam,
ab1893). Secondary antibodies used were anti-mouse Alexa Fluor-488 (1:500)
(Molecular Probes, A21121), anti-mouse Alexa Fluor-568 (1:200) (Molecular
Probes, A-11031), or anti-mouse Rhodamine Red (1:200) (Jackson ImmunoResearch,
715-295-150), anti-sheep FITC (1:200) (Jackson ImmunoResearch, 713-095-147),
anti-rabbit biotin (1:1000) (Jackson ImmunoResearch, 111-065-144) with
Cy3-conjugated streptavidin (1:4000) (Jackson ImmunoResearch, 016-160-084),
and anti-rabbit Alexa Fluor-488 (1:200) (Molecular Probes, A-21206).
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-tubulin and/or GAP43-mRFP1 into the neural tube of
day 1.5 chick embryos (stage HH10). Whole embryo slices were taken between the
level of somites 1-6 and each slice embedded in collagen on a coverslip-based
petri dish and maintained in serumfree medium
(Fig. 1A). Using a low
concentration of plasmid we labelled 5% of the cells located in dorsal
and intermediate regions of the neural tube, allowing easy visualization of
the microtubule cytoskeleton (eYFP--tubulin) and cell membrane
(GAP43-mRFP1) of individual cells. Time-lapse, three-dimensional fluorescence
images were recorded on a wide-field DeltaVision imaging system and subjected
to image deconvolution (McNally et al.,
1999; Swedlow, 1997). In all cases, we recorded three-dimensional
data stacks spanning at least 30 µm at 7-minute intervals for up to 38
hours, unless otherwise stated (see Materials and methods). This imaging
period corresponds to development from HH10/11 to HH16, during which
newly born neurons rise from 3 to 15% of cells in the neural tube (see Fig. S1
in the supplementary material).
To validate this approach we compared cell cycle time, length of mitosis
and incidence of cell death in this region of the neural tube at day 1.5 in
fixed embryos with that observed in neural tube in slices subjected to our
imaging protocol. In fixed tissue, the proportion of cells incorporating BrdU
at two separate timepoints indicated a cell cycle time of 16 hours (see
Materials and methods and Fig. S2 in the supplementary material). In living
slices, cell cycle times were directly determined from the length of time
between cell divisions. These show striking heterogeneity, ranging from 9
to 28 hours (87 cells in 20 slices; see Fig. S2 in the supplementary material)
but with an average (16 hours 10 minutes±4 hours) similar to that
calculated in our fixed tissue and in the zebrafish hindbrain
(Lyons et al., 2003). The
duration of mitosis in slices (28±9 minutes) was similar to that
calculated in the embryo (32±8 minutes), indicating that imaged cells
are not delayed at cell cycle checkpoints (see Materials and methods and Fig.
S2 in the supplementary material). Immunocytochemical detection of the cleaved
form of Caspase-3, characteristic of apoptotic cells, also indicated that
imaging does not increase the incidence of cell death: similar proportions of
apoptotic cells were observed in sections of fixed neural tube at day 1.5
(1±0.6%) and day 3 (0.2±0.05%) and in imaged slices at 16 hours
(1.5±0.5%) (see Materials and methods). These findings indicate that
cell behaviour in these tissue slices is comparable to that observed in the
embryo and that this tissue slice assay can be used for detailed analysis of
normal cell behaviour in the chick neural tube.
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-tubulin. Labelling the
cell membrane with GAP43-mRFP1 revealed that as cells round up on division at
the apical surface they retain a thin membranous process that is inherited by
one daughter cell in all divisions in which we were able to observe this
structure (n=12 cells) (Fig.
1B). Following initiation of the cleavage furrow revealed with
GAP43-mRFP1, a midbody identified with eYFP--tubulin forms between the
two halves of the cell and then becomes displaced towards the apical surface
(Fig. 1C,
Fig. 2A; see Movie 1 in the
supplementary material). This structure, a remnant of the mitotic central
spindle (Glotzer, 2005;
Nagele and Lee, 1979)
consisting of a dense concentration of microtubules and many
microtubule-associated proteins (Skop et
al., 2004), relocated in all 25 cases examined to form the new
apical process in each daughter cell (Fig.
1C, Fig. 2A; see
Movie 1 in the supplementary material). The resumption of interkinetic
movement following division is then accompanied by the movement of
eYFP--tubulin into the basal process
(Fig. 2B; see Movie 2 in the
supplementary material). Once nuclei of both sibling cells have migrated to
the basal side of the neuroepithelium, cells may then remain in this
configuration in contact with both apical and basal surfaces for up to 15
hours (n=5 cells) (Fig.
2A; see Movie 2 in the supplementary material). If a cell is going
to differentiate into a neuron, the endfoot of its apical process then
releases from the apical surface and the process appears to lose tension. The
apical process, as visualized with eYFP--tubulin, then gradually
withdraws across the surface of neighboring cells until it disappears into the
basally located cell body (Fig.
2A,B). A striking reorientation of the polarity of the cell is
then manifest, such that it is shifted by 90° and the cell now extends
processes along the dorsoventral, rather than mediolateral, axis of the neural
tube (Fig. 2A,B; see Movie 2 in
the supplementary material). Subsequent fixation and labelling demonstrated
that such cells express neuronal markers (neurofilament or HuC;
Fig. 2C) in 19/19 cases (six
slices, three separate experiments), confirming that these novel and dramatic
changes in cell morphology are indicative of neuron birth.
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-tubulin and
the cell membrane marker Gap43-mRFP1 to help visualize the cleavage furrow.
These movies allowed us to confirm that our measurement of final cleavage
plane position is accurate in vivo using data collected at 7-minute intervals
(see Fig. 5C; see Movie 1 in
the supplementary material). Having established that the range of cleavage plane orientations observed in vivo is representative of normal cell behaviour, we investigated the relationship between cleavage plane orientation and the subsequent fates of daughter cells. This was analyzed in 47 lineages and in all cases we were able to identify all cells at all time points and determine the position of basal and apical surfaces (Table 1). Of the 34 cells undergoing PP divisions, the majority (21/34, 62%) divided with a perpendicular cleavage plane (Fig. 3; see Movie 3 in the supplementary material; Table 1). By contrast, all six cells that divided to give rise to two neurons exhibited a perpendicular cleavage plane (see Table 1) (Fig. 4; see Movie 4 in the supplementary material). We conclude that there is a good correlation between generation of symmetric neuronal fates and a perpendicular cleavage plane and that symmetric progenitor-generating divisions are not exclusively perpendicular, although they show a tendency to divide with this orientation.
Strikingly, in contrast to the NN divisions the seven PN-generating
divisions had cleavage planes in any orientation. For example,
Fig. 2B (see Movie 3 in the
supplementary material) shows a lineage arising from a division that generated
a neuron and a progenitor with a cleavage plane orientated 34° relative to
the apical surface, followed by division of the daughter progenitor cell with
a cleavage plane at 14° to the apical surface. However, another PN
division had a cleavage plane at 75°
(Fig. 6; see Movie 5 in the
supplementary material). One explanation for this last division comes from
observations in the mammalian cortex where only small differences about the
perpendicular axis appear sufficient to asymmetrically apportion apical
membrane between daughter cells (Kosodo et
al., 2004). Indeed, we find that all but one of the NN divisions
are oriented greater than 80° and are thus closer to perpendicular than
the PN divisions (see dot plot of all cleavage plane orientations,
Fig. 6B). Furthermore,
statistical comparison of PN and NN division orientations indicates that these
are distinct groups (see Table
1).
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Cell cycle length predicts cell fate
Work in the developing mammalian cortex suggests that neuron-generating
divisions are characterized by a longer cell cycle than progenitor-generating
divisions and that this provides time for daughter cells to become different
(Calegari et al., 2005;
Calegari and Huttner, 2003;
Takahashi et al., 1995). Here
we use our direct visualization of mitosis and neuron formation to make these
measurements for the first time in the spinal cord. We find that in the chick
neural tube the cell cycle length of cells dividing to produce one neuron and
one progenitor is longer than that for cells that generate only further
progenitors (see Fig. 7A).
These data support a role for cell cycle extension in the acquisition of the
ability to generate neurons.
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). Here, FGF has been shown to inhibit expression of a bHLH
transcription factor, NeuroM
(Diez del Corral et al.,
2002), which acts within the neurogenesis cascade that leads to
neuronal differentiation (Roztocil et al.,
1997). We therefore next assessed whether this loss of
neuron-generating ability in response to FGF signalling is also accompanied by
a decrease in cell cycle length. All 30 lineages analyzed in FGF-treated
slices consisted of only progenitor-generating divisions. All of these lacked
the apical process release and withdrawal behaviour that is characteristic of
newly born neurons (Fig. 7B;
see Movie 6 in the supplementary material). Exposure to FGF did not alter the
range of cleavage plane orientations of divisions at the apical surface
(Fig. 7C). This finding
indicates that FGF does not attenuate neuron production by directing mitotic
spindle position. Importantly, the cell cycle length of cells in
neuroepithelium exposed to FGF was similar to that of normal
progenitor-generating PP divisions (Fig.
7A), but shorter than those of PN divisions
(Fig. 7A), consistent with all
cells behaving as progenitor generators. In a subset of FGF-treated lineages
we also observed additional novel cell behaviours, including incomplete
interkinetic nuclear migration movements and increased non-apical divisions
(Fig. 7D) characterized by even
shorter cell-cycle times that are statistically different from PP, PN and
apically dividing cells in FGF-treated tissue (see
Fig. 7A,B,B'; see Movie 6
in the supplementary material). These data indicate that loss of neurogenic
factors in the presence of FGF is accompanied by adoption of a more rapid cell
cycle characteristic of cells that only generate further progenitors. |
DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). A
similar membranous basal process has been observed in other neuroepithelia
(Das et al., 2003;
Miyata et al., 2001;
Saito et al., 2003) and the
relocation of eYFP-tubulin into this structure following mitosis is consistent
with a microtubule-based mechanism mediating interkinetic nuclear migration
movements (Messier and Auclair,
1974). The apical displacement and incorporation of the
microtubule-rich midbody into the new apical processes on completion of
cytokinesis is the first observation that the post-mitotic midbody gives rise
to a specific structure.
Importantly, we demonstrate that all cells in the early spinal cord,
whether prospective progenitors or neurons, possess an apical and basal
process and exhibit nuclear movement towards the basal surface following
mitosis. Prospective neurons with basally located nuclei retain this apical
process (see Minaki et al.,
2005) and may do so for many hours prior to the first
morphological manifestation of neuronal differentiation; the release of this
process from the apical surface. This step must thus involve loss of
junctional complexes, including adherens junctions (see
Afonso and Henrique, 2006),
between the differentiating cell and its neighbours. A similar behaviour has
also been reported prior to neuronal differentiation in the mammalian cortex,
although here prospective neurons do not retain apical contact but migrate
away and then form a new process that extends to the apical surface and is
retracted on differentiation (Noctor et
al., 2004). In the early spinal cord, this loss of apical contacts
is followed by reorganization of the microtubule cytoskeleton as the apical
process is withdrawn towards the cell body, which then becomes realigned along
the basal surface. Together, these observations indicate that an early step in
the birth of a neuron is a dramatic change in neuroepithelial cell polarity,
as indicated by loss of apical contacts, followed by a reorganization of the
microtubule cytoskeleton.
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;
Haubensak et al., 2004;
Miyata et al., 2004;
Noctor et al., 2004). The time
and position of neuron births from both symmetric and asymmetric divisions,
which we observe in dorsal and intermediate regions of the early rostral
spinal cord, suggest that these cells are reticular spinal interneurons
(Sechrist and Bronner Fraser,
1991). Symmetric division is also a strategy used to generate some
early neurons in the zebrafish hindbrain
(Lyons et al., 2003), but this
will quickly deplete the progenitor cell population and it is surprising that
this mode of neuron production should take place so early in neurogenesis.
This is the first study in which mitotic spindle orientation and subsequent
daughter cell fates have been monitored continuously in the vertebrate neural
tube. Overall, perpendicular cleavage plane divisions generate daughter cells
that adopt the same fates, but these can be either two neurons or two
progenitors. As this occurs in the same tissue at the same time, this suggests
that individual cells within this early neuroepithelium are not equivalent and
that only some cells have neuron-generating (neurogenic) potential. This
conclusion is supported by the peppered expression pattern of proneural bHLH
transcription factors such as Ngn2 within the early spinal cord
(Ma et al., 1999;
Diez del Corral et al., 2003)
(see Fig. S3 in the supplementary material). If this logic is applied to cell
fate outcomes following non-perpendicular cleavage planes, it suggests that
only those cells with neurogenic factors have the potential to generate a
neuron and a progenitor, whereas non-neurogenic cells will continue to give
rise to only progenitors, regardless of spindle orientation. Indeed, this is
consistent with the daughter cell fates adopted following divisions with a
parallel cleavage plane; most of these divisions give rise to two further
progenitors. Overall, these data suggest that the mechanism of mitotic spindle
orientation operates in the context of a heterogeneous cell population within
the early spinal cord and that here assignment of neurogenic fate is not
linked to division orientation.
Our data also reveal that NN division orientations are exclusively
perpendicular and statistically distinct from those of both PN and PP
divisions, whereas PN and PP division orientations are indistinguishable from
each other. This suggests that mitotic spindle orientation is only crucial in
neurogenic cells, where it distinguishes between stem cell and terminal modes
of neuron production. This conclusion is consistent with data in the mammalian
cortex showing that a slight deviation of the cleavage plane from 90° is
sufficient to generate asymmetric fates in neurogenic cells
(Kosodo et al., 2004), and
with the increase in neurons born of terminal divisions following imposition
of a perpendicular cleavage plane in this context
(Sanada and Tsai, 2005).
However, this contrasts with increased proliferation of some retinal cells
following imposition of perpendicular cleavage plane divisions
(Zigman et al., 2005), which
indicates that here mitotic spindle orientation can regulate the switch to
neuron production (see Harris,
2005).
Nevertheless, there is growing evidence that simply altering mitotic
spindle orientation is not sufficient to elicit neuron production, as observed
recently in the chick caudal neural tube prior to the normal onset of neuron
production (Roszko et al.,
2006) and, while our paper was under revision, in the
Drosophila optic lobe (Egger et
al., 2007). Furthermore, neurogenic determination prior to
terminal mitosis has recently been reported in the zebrafish retina where it
is mediated by ath5 expression
(Kay et al., 2005;
Poggi et al., 2005).
Determination of a neurogenic fate prior to mitoses that generate neurons in
the chick neural tube is also suggested by Ngn2-positive cells, which
rapidly incorporate BrdU (Murciano et al.,
2002), and by the expression pattern of Dyrk1a
(Hammerle et al., 2002).
Tis21, which distinguishes neurogenic divisions in mammalian
neural tissue (Iacopetti et al.,
1999; Kosodo et al.,
2004), can drive cell cycle exit
(Tirone, 2001) and it has been
suggested that it functions to extend the G1 phase of the cell cycle, a
characteristic of cells in the neuron-generating phase of cortical development
(Calegari and Huttner, 2003;
Takahashi et al., 1995)
(reviewed by Gotz and Huttner,
2005). It also regulates expression of the neurogenic gene
Math1, providing a link between neurogenic status and cell cycle
control (Canzoniere et al.,
2004). Based on studies in fixed tissue in the later hindbrain and
early neural plate, cell cycle times in the early chick spinal cord have been
estimated to be 8 hours (Hammerle et
al., 2002). Our calculations from BrdU labelling and direct
measurements reveal a longer average cell-cycle time and a surprising
heterogeneity in cell cycle times in the living neuroepithelium. Furthermore,
as in the cortex, we show that neurogenic divisions are indeed longer than
progenitor-generating divisions. The cell cycle preceding a symmetrical
terminal division is predicted to be even longer than asymmetric neurogenic
divisions (Calegari and Huttner,
2003). This may explain why to date we have not recorded the full
cell cycle in a symmetric terminal division. This extension of cell cycle
length in neurogenic divisions further helps to distinguish neurogenic from
non-neurogenic progenitors.
Consistent with the link between neurogenic factors and cell cycle
regulation (reviewed by Ohnuma and Harris,
2003), we further show that FGF-mediated inhibition of neuronal
differentiation, which involves repression of neurogenic genes, also entails
cell cycle acceleration, and a similar observation has been made in cortical
progenitor cells in vitro (Lukaszewicz et
al., 2002). FGF probably interferes with neurogenesis at multiple
levels (Olivera-Martinez and Storey,
2007), and we also see an increase in non-apical divisions and
breakdown of interkinetic nuclear migration in a subset of cells; a response
difference that may reflect cell cycle phase on first exposure to FGF
(Hitomi and Stacey, 1999).
Finally, the lack of effect of FGF on the range of division orientations at
the apical surface reinforces the observation that the mechanism of mitotic
spindle position and the assignment of neurogenic cell fate are separable
processes.
Development of a long-term live imaging assay in the spinal cord has allowed us to follow individual cells throughout neurogenesis. We have revealed great cell heterogeneity within this tissue, measured accurately mitotic spindle orientation, identified novel cell behaviour associated with neuronal differentiation and imaged continuously for long enough to ascertain cell fate outcomes following oriented divisions. Our observations confirm a link between cell cycle duration and neuron generation and support the important conclusion that although mitotic spindle orientation can regulate cell fate symmetry, it need not determine neurogenic cell fate. This new long-term, high-resolution imaging assay now opens the way to analysis of signalling dynamics on a cell-by-cell basis during neurogenesis in the spinal cord, and to detailed investigation of the links between the mechanics of mitosis and neuron production.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/134/10/1943/DC1
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ACKNOWLEDGMENTS |
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