MRC Laboratory for Molecular Cell Biology and Cell Biology Unit,
University College London, London WC1E 6BT, UK
* Present address: Stanford University School of Medicine, Fairchild Building,
299 Campus Drive, Stanford, CA 94305-5125, USA
Author for correspondence (e-mail:
m.cayouette{at}stanford.edu)
Accepted 20 February 2003
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SUMMARY |
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Key words: Numb, Retina, Rat, Cell division
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INTRODUCTION |
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It remains uncertain to what extent asymmetric segregation of cell-fate
determinants contributes to cell diversification in vertebrates. Evidence that
this mechanism may operate in mammalian CNS development came from a pioneering
study by Chenn and McConnell (Chenn and
McConnell, 1995) that used videomicroscopy to follow cell
divisions in the ventricular zone of explants of developing ferret cortex.
They showed that cells dividing with their mitotic spindle aligned
horizontally to the plane of the neuroepithelium (we call these `horizontal'
divisions) tend to generate two daughters that seem to remain neuroepithelial
cells (NECs). By contrast, cells that divide with their mitotic spindle
aligned vertically to the plane of the neuroepithelium (`vertical' divisions)
tend to generate a basal daughter cell that behaves like a postmitotic
neuroblast, whereas the apical daughter seems to remain an NEC. The vertical
divisions are asymmetric in that an antigen recognized by anti-Notch-1
antibodies segregates exclusively to the basal daughter cell, which raises the
possibility that asymmetric inheritance of cell-fate determinants might help
to diversify the daughter cells. However, a limitation of this study is that
the daughter cells were not followed for long enough to determine their final
fate.
Several vertebrate homologues of Numb have been identified
(Verdi et al., 1996;
Wakamatsu et al., 1999
;
Zhong et al., 1996
). One of
them, called mammalian Numb, is asymmetrically located at the apical pole of
dividing NECs in the developing mouse cortex
(Zhong et al., 1996
) and rat
retina (Cayouette et al., 2001
;
Zhong et al., 1996
), and has
been shown to rescue the Drosophila numb-mutant phenotype
(Zhong et al., 1996
). Because
some NECs in the developing mammalian cortex
(Chenn and McConnell, 1995
;
Zhong et al., 1996
;
Zhong et al., 1997
) and retina
(Cayouette et al., 2001
) divide
vertically, Numb would be expected to segregate asymmetrically to the apical
daughter cell in such divisions, whereas it would be expected to be
distributed symmetrically to both daughters in horizontal divisions. Numb can
decrease Notch signaling in mouse cells
(French et al., 2002
) and
Notch signaling is known to influence cell fate in both invertebrates and
vertebrates (Artavanis-Tsakonas et al.,
1999
; Justice and Jan,
2002
). Therefore, it is proposed that the two daughter cells of
such asymmetric mammalian divisions have different fates
(Zhong et al., 1996
;
Zhong et al., 1997
). In the
developing rodent retina, for example, expression of constitutively active
Notch promotes the development of Müller glial cells
(Furukawa et al., 2000
). This
raises the possibility that the basal daughter cell of a vertical division
(which does not inherit Numb and would, therefore, have unopposed Notch
signaling) might be predisposed to become a Müller cell
(Cayouette et al., 2001
).
Recent studies on mouse cortical progenitor cells in culture show that
asymmetric segregation of Numb influences the developmental fate of daughter
cells (Shen et al., 2002
),
which supports a role for Numb in binary cell-fate decisions. The importance
of Numb and its close relative Numblike in mammalian neurodevelopment is
demonstrated by the severe CNS phenotype observed when both genes are
inactivated in mice (Petersen et al.,
2002
).
In the present study, we have used long-term videomicroscopy to follow single NECs expressing GFP in newborn rat retinal explants. In this way, we were able to investigate directly whether the orientation of cell division correlates with the fate of the two daughter cells. We show unambiguously that some NECs in the living newborn retina divide vertically, confirming our previous observations on sections of fixed retina. Most importantly, we show that, at this age, horizontal divisions produce two daughter cells that usually differentiate into the same cell type, whereas vertical divisions produce two daughter cells that usually differentiate into different cell types. We also show that Numb overexpression in newborn rat retinal NECs (RNECs) decreases the number of interneurons and glia and increases the number of photoreceptor cells that develop, indicating that the concentration of Numb in RNECs can influence cell fate in the newborn retina. Last, and surprisingly, we show that at least some RNECs do not retract their basal process when they undergo mitosis. Taken together, these results indicate that the orientation of cell division in the neonatal rat retina affects the fate adopted by the daughter cells and that Numb can influence this fate.
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MATERIALS AND METHODS |
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For time-lapse videomicroscopy, retinal explants were infected with the LEGFP-N1 retrovirus (Clontech), which encodes GFP. About 25 µl of viral suspension was added directly on top of the explants. After an overnight incubation at 37°C, the medium was changed, and the culture dish sealed with Parafilm and transferred to the stage of an inverted fluorescence microscope enclosed in a custom-made 37°C incubator.
We used one of two retroviruses to label RNECs clones with alkaline
phosphatase. The CLE control retrovirus, which encodes alkaline phosphatase
alone, has been described previously
(Gaiano et al., 1999). The
CLE-Numb retrovirus was constructed by cloning the full length mouse
Numb cDNA into the CLE retroviral vector, so that an mRNA encoding
both Numb and alkaline phosphatase is expressed under the control of the
Xenopus EF1
promoter, and alkaline phosphatase is translated
using an internal ribosome entry site (IRES; see
Fig. 7C). Newborn retinal
explants were infected with either CLE or CLE-Numb retrovirus as described
above. The next day, the culture medium was changed, and the explants were
cultured for 10 days, changing the medium every 2-3 days. The explants were
then fixed for 2 hours in 4% paraformaldehyde, rinsed in PBS and the activity
of the alkaline phosphatase determined as previously described
(Gaiano et al., 1999
). After
rinsing in PBS, the explants were cryoprotected in 20% sucrose, embedded in
OCT, frozen and sectioned at 30 µm in a cryostat, as described previously
(Cayouette et al., 2001
).
Clones of retinal cells were analyzed by counting the number of each cell type
present in radial clusters, using morphology and position in the cell layers
to identify the different cell types.
|
Measurement of cell body size
The surface area of the body of GFP+ cells was measured using
NIH-Image Software. The software was calibrated for the camera, microscope and
objectives used to acquire the images so that measurements were consistent for
all cells analyzed.
Immunostaining
We used immunofluorescence to visualize Numb in retinal explants infected
with the retrovirus encoding either alkaline phosphatase alone or alkaline
phosphatase and Numb. Frozen sections of the explants were incubated for 45
minutes in blocking buffer (PBS + 0.1% Triton X-100 + 3% BSA) at room
temperature and then overnight at 4°C in rabbit polyclonal anti-Numb
antibodies (Upstate Biotech, J. McGlade, or Q. Zhong, diluted 1:200 in
blocking buffer) and rabbit anti-alkaline phosphatase antibodies (Accurate,
diluted 1:100 in diluting buffer). Bound antibodies were detected using
biotinylated goat anti-mouse Ig antibodies (Amersham, 1:100 in PBS), followed
by Streptavidin-FITC (Amersham, 1:100 in PBS) and chicken anti-rabbit Ig
antibodies conjugated to Alexa Fluor 594 (Molecular Probes, 1:100 in PBS).
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RESULTS |
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GFP+ RNECs were readily identified by their typical morphology (Fig. 2A) and their characteristic interkinetic nuclear migration (basal to apical rate = 1.2±0.6 µm minute1; mean±s.d., n=49). The plane of the interkinetic nuclear migration established the apico-basal axis of the neuroepithelium and allowed us to determine the plane of division with respect to this axis (Fig. 2B). The location of the cell division along this axis established the apical surface of the retina. We only studied cells that were strongly GFP+, in focus, and displayed interkinetic nuclear migration.
|
|
Of the 28 horizontal divisions that we followed, 14 produced daughter cells
that we could not follow long enough to assess their fates. In two of the
remaining 14 cases, one of the daughter cells died and the other divided
again; in neither case could we follow the daughter cells of the second
division to determine their fates. The remaining 12 horizontal divisions were
terminal in that both daughter cells did not divide again. More than 90% (11
out of 12) of these divisions produced two daughter cells that acquired
similar morphologies. Five examples, seen 60-90 hours after division, are
shown in Fig. 4A-E. In one
case, it was possible to stain the explant with propidium iodide (after the
end of video recording) and then find the two GFP+ daughter cells
in a confocal microscope 70 hours after the cell division that produced them.
The nuclei of the two cells were located in the photoreceptor layer and
resembled those of their GFP neighbors, which had the
distinctive heterochromatin pattern characteristic of photoreceptors
(Neophytou et al., 1997)
(Fig. 4E). In all cases, the
two GFP+ daughter cells resembled GFP+ photoreceptor
cells in confocal sections of a newborn retinal explant infected with a
GFP-encoding retrovirus and examined after 10 days in culture (compare
Fig. 4A-E with
Fig. 4F). In the single case
where the two daughter cells of a horizontal division acquired different
morphologies, one acquired a photoreceptor-like morphology, whereas the other
became an interneuron-like cell with a large body and small processes (not
shown). Thus, at this developmental age, it seems likely that most horizontal
divisions produce daughter cells that become photoreceptors.
|
Vertical divisions produce two daughter cells that usually become
different cell types
Of the 17 vertical divisions that we followed, six produced daughter cells
that we could not follow long enough to assess their fates. Of the remaining
11 vertical divisions, >80% (nine out of 11) generated two daughter cells
that acquired different morphologies. Five examples, seen 70-96 hours after
division, are shown in Fig.
5A-E. In one case, it was possible to stain the explant with
propidium iodide (after the end of video recording) and identify the two
GFP+ daughter cells in a confocal microscope 84 hours after the
division that produced them. One of the daughters was large and found among
cells that had a large nucleus, whereas the other was smaller and among cells
that had a small nucleus. This suggests that they were in the interneuron and
photoreceptor layers, respectively (Fig.
5E). In four cases, we were able to follow the fate of the apical
and basal daughters separately and, in each case, the basal daughter developed
as a Müller-like cell, similar to those seen in confocal sections of a
newborn retinal explant infected with a GFP-encoding retrovirus and examined
after 10 days in culture (compare Fig. 5D
with 5F). The apical daughter cell in these four divisions became
either neuron-like (Fig. 5A) or
photoreceptor-like (Fig. 5D).
Thus, it seems that at this developmental age most vertical divisions produce
two daughter cells that become different cell types.
|
Retinal neuroepithelial cells can maintain their basal process during
mitosis
Based on electron microscopic studies, it was thought that mitotic RNECs
round up and retract their basal process. However, recent studies of
fluorescently labeled radial glial cells in the developing cortex suggest that
these cells do not loose their basal process during division
(Miyata et al., 2001;
Noctor et al., 2001
).
Moreover, the daughter cell that inherits the basal process tends to become a
neuroblast (Miyata et al.,
2001
). Do RNECs also maintain their basal process during
division?
In most cases, we could not detect a basal process during mitosis. However, in nine out of 48 cells analyzed (19%) we detected a thin basal process that persisted throughout mitosis. One such cell is shown in Fig. 6A-D. Some of these nine cells divided horizontally, whereas others divided vertically. In one favorable case, we were able to confirm the presence of a basal process by confocal microscopy (Fig. 6E-G).
|
Overexpression of Numb increases the proportion of photoreceptor
cells and decreases the proportion of interneurons and Müller cells
One reason why vertical divisions tend to produce two daughter cells that
become different cell types may be because Numb is asymmetrically segregated
to the two daughters in such divisions. We
(Cayouette et al., 2001) and
others (Dooley et al., 2003
)
previously provided evidence that Numb is asymmetrically distributed on the
apical side of the rat retinal neuroepithelium. However, a recent paper raised
the possibility that the anti-Numb monoclonal antibody (Transduction Lab) used
in these two studies recognizes another protein, called Mortalin, which has
the same molecular weight as Numb (Rivolta and Holey, 2002). We therefore
repeated the staining for Numb in the newborn rat retina using rabbit
anti-Numb antibodies that were either obtained from J. McGlade or bought from
Upstate Biotech. In both cases, we found that Numb localized asymmetrically on
the apical side of the retinal neuroepithelium. The staining with the
antibodies from Upstate Biotech is shown in
Fig. 7A.
If asymmetric segregation of Numb influences cell-fate in the retina, one would expect to see a change in cell fate if Numb were overexpressed so that it was inherited by both daughter cells of vertical divisions. To test this expectation, we used retroviral vectors that encoded either alkaline phosphatase alone or alkaline phosphatase and Numb (Fig. 7C). We infected newborn retinal explants overnight and cultured them for 10 days. We then fixed and stained the explants to reveal clones of cells that had alkaline phosphatase activity. We analyzed the composition of 1704 Numb-infected clones and 1891 control clones using morphology and retinal layer position to identify the cell types (Fig. 7D-G). In explants infected with the retrovirus encoding Numb, Numb-immunostaining showed that many cells expressed Numb basally as well as apically (Fig. 7H); even in vertical divisions of these cells, both daughters would be expected to inherit Numb. Using the same antibody (from W. Zhong, Yale University), cells in explants infected with the control vector that encodes alkaline phosphatase alone did not express Numb basally (data not shown).
In accordance with previous studies by others, we detected clones of
various sizes and composition that contained photoreceptors, amacrine cells,
bipolar cells and Müller cells in various combinations (see
Fig. 7D-G). However,
Numb-infected explants contained fewer cells with a complex morphology than
did control explants expressing alkaline phosphatase alone
(Fig. 7I,J). As shown in
Fig. 8A, overexpression of Numb
had little effect on clone size, indicating that it did not significantly
influence either RNEC survival or proliferation at this stage of development.
Quantitative analysis of frozen sections indicated that clones in the control
explants were similar in size and composition to those reported previously
using similar retroviruses to infect newborn RNECs in vivo
(Turner and Cepko, 1987;
Turner et al., 1990
). This
indicates that our explants developed normally. By contrast, in frozen
sections of Numb-infected explants, there was an increase in both the
proportion of clones that contained photoreceptor cells only and the
proportion of photoreceptor cells among all the infected cells
(Fig. 8B,C). In these explants,
there was a corresponding decrease in the proportion of clones that contained
at least one nonphotoreceptor cell (amacrine, bipolar and Müller cell),
as well as in the proportion of nonphotoreceptor cell types among all the
infected cells (Fig. 8D,E). To
compare the Numb-overexpression findings with our time-lapse imaging results,
we analyzed separately the clones that contained only two cells. As shown in
Fig. 8F, there were
significantly more two-cell clones containing two photoreceptors and
significantly fewer two-cell clones containing a mixture of cell types in
Numb-infected explants than in control explants
(Fig. 8F). Thus, Numb
overexpression in newborn retinal explants apparently increased the
development of photoreceptors at the expense of the development of
interneurons and Müller cells.
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DISCUSSION |
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Orientation of cell division
There is increasing evidence that some NECs in vertebrate neuroepithelia
rotate their mitotic spindle and divide vertically during CNS development.
This evidence comes from studies of the developing cortex in mouse
(Estivill-Torrus et al., 2002;
Zhong et al., 1996
), ferret
(Chenn and McConnell, 1995
) and
chick (Wakamatsu et al.,
1999
), and the developing retina in rat
(Cayouette et al., 2001
) and
chick (Silva et al., 2002
).
Molecules that are asymmetrically distributed along the apico-basal axis
during NEC division would segregate asymmetrically to the two daughter cells
in vertical divisions.
Our present results confirm our previous findings that a proportion of
RNECs divide vertically in sections of fixed retina
(Cayouette et al., 2001). We
showed previously that the proportion of vertical divisions is <5% in the
embryonic rat retina,
20% in the newborn retina and
10% in the
postnatal day 4 (P4) retina. In the present study, the proportion of vertical
divisions we observed by time-lapse recording in newborn retina was 34%. There
are several possible explanations why we found a higher proportion of vertical
divisions in the present study. First, in our previous study, we included both
metaphase and anaphase cells in the analysis. The proportion of vertical
divisions was greater when we analyzed anaphase cells only, indicating that
some `horizontal' metaphase cells reorient their spindle and divide
vertically, as has been shown to occur in Drosophila embryos
(Kaltschmidt et al., 2000
;
Roegiers et al., 2001
). The
proportion of vertical divisions in the present study is based on analyses of
actual cell division, rather than spindle orientation, and is, therefore, a
more accurate measure. Second, although here we studied explants of newborn
retina, most of the cell divisions analyzed occurred at an age equivalent to
P2. It is possible that the proportion of vertical divisions peaks at P2 and
decreases by P4. Third, and perhaps most important, our time-lapse analysis is
almost certainly biased toward vertical divisions, because many more
horizontal than vertical divisions would be expected to occur out of the plane
of focus. This bias would result in an apparent increase in the proportion of
vertical divisions. Thus, we suspect that the true proportion of vertical
divisions in the first two postnatal days of rat retinal development is
20-30%.
Although we mainly followed cells that divided either vertically or
horizontally, four out of the 49 divisions in which we could follow the fate
of the two daughters occurred with an intermediate apical-basal orientation.
Such intermediate divisions could segregate cell components asymmetrically to
the two daughter cells. For example, because the apical domain of NECs
constitutes such a small portion of the total plasma membrane, even a slight
rotation of the mitotic spindle away from the horizontal could lead to unequal
distribution of apical components to the daughter cells during division
(Huttner and Brand, 1997).
Relationship between orientation of cell division and cell fate
Chenn and McConnell (1995)
were the first to use videomicroscopy to provide evidence that the plane of
NEC division can influence cell-fate choice. Studying the ventricular zone of
developing ferret cortex, they found that horizontal divisions generated two
daughter cells that seemed to remain NECs, whereas vertical divisions
generated a basal daughter cell that seemed to become a postmitotic neuroblast
and an apical daughter that seemed to remain an NEC. Because the proportion of
vertical divisions increased with developmental age, they proposed that early
in development horizontal divisions expand the NEC pool, whereas later in
development, vertical divisions produce one cell that differentiates and
another that remains an NEC and continues to divide. It was not clear whether
late cell divisions that produce two neuroblasts and deplete the NEC pool
occurred by horizontal, vertical or both types of cell division.
In our study of the newborn rat retina, we find that the great majority of both horizontal and vertical divisions are terminal divisions, with both daughter cells differentiating. In only two out of 49 divisions (both horizontal), one of the two daughter cells divided again. In both cases, the other daughter cell died and we were unable to follow the fate of the daughter cells produced by the second division.
The most important finding of the present study is that horizontal divisions of NECs in the newborn rat retina usually produce daughter cells that acquire the same photoreceptor-like morphology and size, whereas vertical divisions usually produce two daughter cells that acquire different morphologies and sizes. The probability that chance alone accounts for 11 out of 12 horizontal divisions producing daughter cells that acquired the same morphology and size, and nine out of 11 vertical divisions producing daughters that acquired distinct morphologies and sizes is less than 0.005, assessed by chi-square analysis. In several cases, we were able to show that the two differentiated daughters of horizontal divisions have a photoreceptor-like morphology and size, and that they end up in the photoreceptor layer. However, the two differentiated daughters of vertical divisions not only usually look different and have different sizes, but they also end up in different retinal layers.
For technical reasons, it was not possible to confirm our cell-type
assignments by immunostaining. However, daughter cells of horizontal divisions
tend to have similar cell-body size, which is also similar to the size of
GFP-infected photoreceptor cells in cryosections of retinal explants, which
strongly supports our photoreceptor assignments. Thus, it seems likely that,
at this stage of retinal development, horizontal divisions almost always
generate two photoreceptors. These findings are consistent with previous
results. Rod photoreceptors are the main cell type produced in the neonatal
rat retina and make up >70% of the cells in the adult rat retina. Two-rod
clones account for 79% of all the two-cell clones observed following
retroviral infection of newborn rat retina in vivo
(Turner and Cepko, 1987), and,
as discussed above, we estimate that about 70-80% of cell divisions in the
newborn rat retina are horizontal.
Analysis of cell-body size also supports our conclusion that vertical divisions in the newborn rat retina usually generate two daughters that become different. One daughter usually becomes a cell with the typical morphology and body size of a photoreceptor cell, whereas the other daughter usually becomes a cell with a morphology and body size similar to that of cells in the interneuron layer either interneurons or Müller cells. At this stage of development, it is likely that the interneurons produced are either amacrine cells or bipolar cells. It is important to devise ways to combine time-lapse analysis with molecular identification of the differentiated cells.
Although we have focused on the orientation of cell division along the
apico-basal axis of the retina, it is also possible that the orientation of
division in the plane of the retina could influence cell fate. In flies and
worms, for example, asymmetric divisions along various axes can influence
cell-fate choices (Hyman and White,
1987; Roegiers et al.,
2001
; Rose and Kemphues,
1998
). A recent study showed that the orientation of cell division
within the plane of the neuroepithelium changes along the radial axes of the
developing retina in both zebrafish and rat; in zebrafish, this switch
correlates with neurogenesis, which is consistent with the possibility that
cell-fate determinants may segregate asymmetrically along axes oriented in the
plane of the vertebrate retina (Das et al.,
2003
).
Role of Numb in cell-fate choice in newborn rat retina
How does the plane of cell division along the apico-basal axis influence
cell fate? One mechanism may involve the asymmetric segregation of Numb during
vertical division. Because Numb is localized apically in the rat RNECs
(Fig. 7A), only the apical
daughter of vertical divisions should inherit Numb
(Cayouette et al., 2001).
Because Numb can inhibit Notch signaling
(French et al., 2002
;
Frise et al., 1996
;
Guo et al., 1996
;
Spana and Doe, 1996
), and
Notch signaling can influence cell fate in the developing rat retina
(Bao and Cepko, 1997
;
Furukawa et al., 2000
), it is
possible that the two daughters of vertical RNEC divisions develop differently
because of differences in Notch signaling associated with differences in the
levels of Numb. Our finding that the overexpression of Numb in RNECs in
newborn retinal explants promotes photoreceptor development at the expense of
interneuron and Müller cell development is consistent with this. Although
we cannot exclude the possibility that overexpression of Numb decreases the
probability that a RNEC will divide vertically, experiments in flies makes
this unlikely. Our Numb overexpression results are consistent with our
observation that horizontal divisions, in which Numb is expected to be
distributed symmetrically to daughter cells, produce two photoreceptor-like
cells, whereas vertical divisions, in which Numb is expected to be
asymmetrically distributed produce two daughters that become different from
each other. These findings are also similar to the results seen following
overexpression of Numb in SOP cells in Drosophila
(Rhyu et al., 1994
). In
dividing SOP cells, Numb is normally asymmetrically segregated to the daughter
cell that becomes the IIb cell. The cell that does not inherit Numb becomes
the IIa cell, apparently because of unopposed Notch signaling
(Frise et al., 1996
;
Guo et al., 1996
). When Numb
is overexpressed in the SOP cell, both daughters become IIb cells
(Rhyu et al., 1994
). Thus, in
both SOP cells and RNECs, Numb overexpression reduces cell diversification.
Similarly, mouse cortical progenitor cells in culture can asymmetrically
distribute Numb when they divide, thus influencing the fate of the daughter
cells (Shen et al., 2002
).
Moreover, cortical progenitors isolated from Numb-knockout mice show
a 50% reduction in their ability to produce daughter cell pairs that adopt
different fate (Shen et al.,
2002
). In the same study, it was found that in terminal divisions
that produced two neurons in which Numb was asymmetrically distributed, the
neurons acquired different morphologies. This observation is similar to our
finding that vertical divisions, in which Numb would be asymmetrically
inherited, produce two daughters that adopt different morphologies.
Whereas Numb overexpression in newborn rat RNECs inhibits Müller-cell
development, it has been shown previously that excessive Notch signaling in
RNECs increases Müller-cell development
(Furukawa et al., 2000). On
the basis of the Müller-cell-promoting effect of excessive Notch
signaling and apical localization of Numb in RNECs, we hypothesized previously
that the basal daughter of a vertical RNEC division would tend to
differentiate into a Müller cell
(Cayouette et al., 2001
). Our
present results are consistent with this hypothesis but indicate that it is an
oversimplification. In the four vertical divisions in which we were able to
follow the fate of the apical and basal daughter cell separately, the basal
cell acquired a Müller-cell-like morphology, whereas the apical cell
acquired either a photoreceptor-like or a neuron-like morphology. However, not
all vertical divisions produce Müller-like cells (see, for example,
Fig. 5B). Thus, it seems likely
that the apical daughter cell of a vertical division of a newborn RNEC that
inherits Numb has an increased probability of becoming a photoreceptor cell,
whereas the basal daughter that does not inherit Numb has an increased
probability of becoming either an interneuron or a Müller cell. Numb
might act positively, to promote photoreceptor development, negatively, to
inhibit interneuron and Müller cell development, or in both ways.
In a recent study, Silva et al. (Silva
et al., 2002) found little correlation between cell-fate choice in
the chick retina and either the orientation of cell division or the
distribution of Numb. Although this seems at odds with our findings, there are
a number of reasons, apart from species difference, why the two studies come
to such different conclusions. One is that Silva et al.
(Silva et al., 2002
) studied
earlier stages of retinal development. Because there are very few vertical
divisions in the rat retina this early age, the mechanism we propose is
unlikely to operate. Another is that they use the term asymmetric division to
describe a division in which one daughter cell continues to divide and the
other differentiates, whereas we use it to describe a division in which one or
more molecules are asymmetrically segregated between the two daughter cells.
Silva et al. use the term symmetric division to describe a division in which
both daughter cells continue to divide but, without following the fate of the
two daughters, it is impossible to know whether they are the same or not; they
could have inherited different components from the mother cell, for example,
and have different fates. Similarly, cell divisions in which both daughter
cells differentiate can be asymmetric if the two daughters become different,
as shown in this study. Even at the early stages of retinal development
studied by Silva et al., the plane of division could, apparently, influence
cell-fate choice: in the rare divisions with a vertical spindle, the apical
daughter cell never expressed Numb and never differentiated into a retinal
ganglion cell.
Because Notch signaling has different functions in different tissues during
development, and at different developmental times in the same tissue, it seems
likely that Numb will also have different functions in different tissues and
at different developmental times (Cayouette
and Raff, 2002; Zhong,
2003
). Indeed, experiments in both flies and vertebrates indicate
that Numb proteins help make the two daughter cells of a division develop
along different pathways, rather than to specify a particular cell type. For
example, in Drosophila Numb functions in binary cell-fate decisions
but does not induce one particular fate; it can help a daughter cell to either
remain a progenitor cell or to develop into a neuron, glial cell or other cell
type (Gho et al., 1999
;
Rhyu et al., 1994
;
Roegiers et al., 2001
;
Spana et al., 1995
;
Van De Bor et al., 2000
).
Similarly, in rodent cortical progenitor cells dividing in culture, asymmetric
segregation of Numb is observed in divisions that produce two different cell
types, but it can be inherited by a cell that remains a progenitor cell or by
a cell that becomes a neuron (Shen et al.,
2002
).
Retention of the basal process during RNEC division
Although it was generally believed that NECs round up and retract their
basal process during division, it was shown recently that radial glial cells
maintain a basal process during mitosis
(Miyata et al., 2001;
Noctor et al., 2001
). This is
inherited asymmetrically by the daughter cell that becomes a postmitotic
neuroblast (Miyata et al.,
2001
). Moreover, in the fish retina, it has been shown recently
that mitotic cells always retain a thin basal process that can be inherited
asymmetrically (Das et al.,
2003
). In agreement with these results, we find that at least some
RNECs retain their basal process during mitosis. Although it is possible that
only a proportion of mitotic RNECs retain their basal process, it seems more
likely that they all do, but that some were too thin to be detected.
We saw RNECs that maintain their basal process during both horizontal and
vertical divisions. In favorable cases, the basal process was asymmetrically
inherited by one of the two daughter cells, in both kinds of divisions. We
could not determine whether the inheritance of the basal process influences
cell fate because the two daughter cells often stay so close to each other
that it is difficult to follow them separately for long periods. When we were
able to follow the two daughter cells separately during the first few hours
after division, the cell that inherited the process always migrated to lie
basal to its sibling. The differentiated cells in retinal clones are aligned
radially along the apico-basal axis
(Turner and Cepko, 1987;
Turner et al., 1990
) and it
may be that the retained basal process helps cells in a clone achieve this
radial organization.
In conclusion, it seems likely that the asymmetric segregation of cell-fate
determinants during division plays a much greater part in influencing
cell-fate decisions in vertebrate development than is generally appreciated.
There are increasing numbers of examples where a proportion of vertebrate
epithelial cells reorient their mitotic spindle to divide vertically to the
plane of the epithelium. These include ependymal cells
(Johansson et al., 1999), stem
cells of the oesophageal epithelium (Seery
and Watt, 2000
) and several NECs
(Cayouette et al., 2001
;
Chenn and McConnell, 1995
;
Chenn et al., 1998
;
Silva et al., 2002
;
Wakamatsu et al., 1999
;
Wakamatsu et al., 2000
;
Zhong et al., 1996
). The
challenge is to identify the putative cell-fate determinants and to determine
how they influence cell fate.
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ACKNOWLEDGMENTS |
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Footnotes |
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