2329
Development 130, 2329-2339
© 2003 The Company of Biologists Ltd
doi:10.1242/dev.00446
The orientation of cell division influences cell-fate choice in the developing
mammalian retina
Michel Cayouette*,† and Martin Raff
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@stanford.edu)
Accepted 20 February 2003
SUMMARY
Asymmetric segregation of cell-fate determinants during
cell division plays an important part in generating cell
diversity in invertebrates. We showed previously that cells
in the neonatal rat retina divide at various orientations and
that some dividing cells asymmetrically distribute the cellfate determinant Numb to the two daughter cells. Here,
we test the possibility that such asymmetric divisions
contribute to retinal cell diversification. We have used longterm videomicroscopy of green-fluorescent-protein (GFP)labeled retinal explants from neonatal rats to visualize the
plane of cell division and follow the differentiation of the
daughter cells. We found that cells that divided with a
horizontal mitotic spindle, where both daughter cells
should inherit Numb, tended to produce daughters that
became the same cell type, whereas cells that divided with
a vertical mitotic spindle, where only one daughter cell
should inherit Numb, tended to produce daughters that
became different. Moreover, overexpression of Numb in the
dividing cells promoted the development of photoreceptor
cells at the expense of interneurons and Müller glial cells.
These findings indicate that the plane of cell division
influences cell-fate choice in the neonatal rat retina and
support the hypothesis that the asymmetric segregation of
Numb normally influences some of these choices.
INTRODUCTION
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
A major challenge in developmental neurobiology is to
understand how the enormous diversity of cells in the
mammalian CNS is produced. In invertebrates such as
Drosophila and C. elegans, asymmetric segregation of cellfate-determining proteins and mRNAs to daughter cells makes
an important contribution to cell diversification (Knoblich,
2001; Lu et al., 2000; Rose and Kemphues, 1998). In the
Drosophila PNS, for example, asymmetric segregation of the
cell-fate determinant Numb to only one daughter of the sensory
organ precursor (SOP) cell is essential to confer distinct fates
to the daughter cells (Rhyu et al., 1994). Numb is a cell-cortexassociated protein that can inhibit Notch signaling, which is
one way it is thought to influence cell fate (Frise et al., 1996;
Guo et al., 1996; Spana and Doe, 1996).
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
Movie available online
Key words: Numb, Retina, Rat, Cell division
2330 M. Cayouette and M. Raff
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.
MATERIALS AND METHODS
Retinal explant culture
Newborn Sprague Dawley rats were killed and the eyes removed and
kept in cold HBSS until dissection. Retinal explants containing both
the neural retina and retinal pigment epithelium were prepared as
previously described (Cayouette et al., 2001). Each explant was
placed on a Millipore-CM organotypic filter in a T25 Falcon tissue
culture flask in Neurobasal medium containing 20% FCS and
penicillin/streptomycin. We made certain that the edge of the explant
was folded over so that we could visualize RNECs aligned along the
apico-basal axis of the folded retinal neuroepithelium (see Fig. 1A).
The explants were allowed to settle for a few hours in a CO2 incubator
at 37°C before they were infected with a retrovirus (see below).
Retroviral vectors and infection of retinal explants
All retroviruses were prepared in Phoenix packaging cells and were
concentrated by ultracentrifugation, as previously described (Kondo
and Raff, 2000).
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 EF1α 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.
Time-lapse recording
An inverted Zeiss Axioscope fluorescence microscope was enclosed
in a custom-made 37°C incubator. GFP+ cells were viewed with a 10X
objective. A field of interest was selected based on the presence of
several GFP+ cells with neuroepithelial morphology. The field was
imaged using a Hamamatsu CCD video camera connected to a
Macintosh computer equipped with OpenLab software (Improvision)
programmed to capture a frame every 7 minutes for the first 3648 hours. To minimize light damage, we then reprogrammed the
computer to capture a frame every 6-12 hours for the remainder of the
recording period. In some cases, after the first 36-48 hours of
recording, a mark was made on the filter and the culture flask returned
to a conventional CO2 incubator. Using the mark on the filter, we
found the recording area and took a picture of it every day to follow
the fate of the cells. By focusing through the explant after the
recording period, we excluded the possibility that processes of outof-focus cells were in the field of view. In some experiments, at the
end of the recording period, we fixed and stained the explant with
propidium iodide (5 µg ml–1 in PBS + 25 U RNase A). In a few cases,
we were able to use the mark on the filter to find the original recording
area in a MRC 1024 confocal microscope. This allowed us to identify
the two daughter cells we had followed to help determine which
retinal layer the differentiated cells ended up in.
Measurement of cell body size
The surface area of the body of GFP+ cells was measured using NIHImage 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
Asymmetric cell division in the developing rat retina 2331
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).
RESULTS
Some neuroepithelial cells divide
vertically in living retinal explants
To investigate directly the relationship
between the orientation of cell division
along the apico-basal axis and cell fate in
the newborn rat retina, we needed a system
in which we could both visualize the
orientation of division of individual RNECs
along this axis and follow the fate of the
daughter cells over days. First, we tried
cultures of slices of newborn retina, but
extensive cell death precluded their use.
Therefore, we used newborn retinal
explants. By folding over the edge of the
explant, cell divisions along the apico-basal
axis could be observed at the edge of the
fold (Fig. 1A). These explants seemed to
Fig. 1. Experimental setup for time-lapse video recording. (A) Retinal explants from
develop normally, with mitosis occurring at
newborn rats were cultured on organotypic filters, which allow the culture medium to
the apical surface (Fig. 1B) and distinct
diffuse through the explant by capillary action. The edge of the explant was folded over,
layers of photoreceptors and interneurons
which allowed RNECs infected with a GFP-encoding retrovirus to be observed along their
developing after 6 days in culture (Fig. 1C).
apico-basal axis. Areas of the folded edge are shown after (B) 3 hours and (C) 6 days.
We infected the explants overnight with a
Note that all mitotic cells are at the apical surface of the neuroepithelium in B and that
retrovirus encoding GFP and then placed
two, distinct cell layers have developed in C, just as observed in vivo. However, the retinal
ganglion cells die in the explant because they lack neurotrophic support from their normal
them on the stage of an inverted
target cells. Scale bar: 20 µm.
fluorescence microscope that was enclosed
within a 37°C incubator. We followed the
behavior of GFP+ RNECs (typically 1-10
the retina. We only studied cells that were strongly GFP+, in
cells per field) by time-lapse videomicroscopy.
+
focus,
and displayed interkinetic nuclear migration.
GFP RNECs were readily identified by their typical
We recorded >150 cell divisions in 30 explants and found
morphology (Fig. 2A) and their characteristic interkinetic
that RNECs divided at various orientations to the apiconuclear migration (basal to apical rate = 1.2±0.6 µm minute–1;
basal axis (Fig. 3; Movie 1 at http://dev.biologists.org/
mean±s.d., n=49). The plane of the interkinetic nuclear
supplemental/). Most of the 150 cell divisions occurred partly
migration established the apico-basal axis of the
out of the plane of focus, making it impossible to determine
neuroepithelium and allowed us to determine the plane of
their orientation. We could, however, unambiguously
division with respect to this axis (Fig. 2B). The location of the
determine the orientation of division in 49 cases. Of these,
cell division along this axis established the apical surface of
Fig. 2. GFP+ RNECs undergoing interkinetic nuclear migration. (A) A single GFP+ RNEC is seen at the edge of the folded explant, 24 hours
after infection with a GFP-encoding retrovirus. (B) Snapshots from a time-lapse video recording of a GFP+ RNEC undergoing interkinetic
nuclear migration along its apico-basal axis. The cell divided horizontally at the apical surface of the neuroepithelium at 16.5 hours.
Arrowheads indicate the plane of the cell division, perpendicular to the orientation of the mitotic spindle. Scale bars: 20 µm in A; 40 µm in B.
2332 M. Cayouette and M. Raff
Fig. 3. Horizontal and vertical divisions of GFP+ RNECs.
(A) Snapshots from a time-lapse video recording showing two cells
in the same field, one of which divided horizontally (cell 1) and one
of which divided vertically (cell 2). The dashed line indicates the
apical surface of the neuroepithelium and arrowheads the plane of
cell division (which is perpendicular to the orientation of the mitotic
spindle). See also Movie 1 at http://dev.biologists.org/supplemental/.
Scale bar: 40µm. (B) Higher magnification view of cell 1 dividing horizontally in time frame 12h08. (C) Higher magnification view of cell 2
dividing vertically in time frame 13h18. (D) Quantitative analysis of the distribution of mitotic spindle orientations relative to the plane of the
neuroepithelium in live retinal explants, as determined by time-lapse video microscopy of GFP+ RNECs.
58% were horizontal, 34% vertical and 8% intermediate in
orientation (Fig. 3B). Thus, a substantial proportion of RNECs
divided vertically in the living, newborn retina, confirming our
previous findings on sections of fixed retinal preparations
(Cayouette et al., 2001).
Horizontal divisions produce two daughter cells that
usually become photoreceptor-like cells
To determine if the orientation of cell division correlated with
the fate of the daughter cells, we followed the two daughter
cells of each GFP+ cell that divided either horizontally or
vertically for up to 5 days after the division. Most daughter
cells did not divide again: they did not undergo interkinetic
nuclear migration and they adopted a morphology that
indicated that they had started to differentiate.
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
Fig. 4. Horizontal divisions producing two daughter cells that
acquire a similar morphology and cell body size. (A-E) Five
examples of daughter cells produced by horizontal divisions
of newborn RNECs, seen 60-90 hours after the division. Note
the similarity in size and morphology of the two daughter
cells in each case. In the example shown in E, it was possible
to fix the explant after the recording, stain the nuclei with
propidium iodide and find the two GFP+ daughter cells again
in a confocal microscope. Both daughter cells have a similar
body size and morphology and are located in the
photoreceptor layer. (F) A two-photoreceptor clone in a
frozen section of a newborn retinal explant, 10 days after
infection with a retrovirus encoding GFP. (G) Quantification
of cell body size of GFP-infected photoreceptor cells (PR)
and interneuron layer cells (INL), measured in cryosections
of retinal explants 10 days after infection with a GFPencoding retrovirus. Results are mean±s.d. of 35 PR and 28
INL cells. (H) Cell-body sizes of pairs of daughter cells
produced by horizontal divisions of GFP-infected RNECs,
followed by video recording. The daughters of all 12
horizontal divisions followed are shown. Note that in 11 out
of 12 cases the daughter cells are similar in size to
photoreceptors. Scale bars: 20 µm in A-E; 10 µm in F.
Asymmetric cell division in the developing rat retina 2333
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.
We tried repeatedly to stain the explants as wholemounts
with antibodies after time-lapse recording to confirm the celltypes of the daughters, but it proved to be impossible to find
the daughter cells and assess their staining. In an alternative
way to help identify the differentiated cell types, we measured
the size of the cell body. First, we measured the size of the cell
body of GFP-infected photoreceptors and cells of the
interneuron layer in cryosections of newborn retinal explants.
As shown in Fig. 4G, photoreceptor cells had a cell-body size
of 58±8 arbitrary units (mean±s.d., n=35), whereas cells
located in the interneuron layer had a cell-body size of 113±25
arbitrary units (n=28). This difference in mean cell-body size
between interneuron layer cells and photoreceptors was highly
significant when analyzed by Student’s t-test (P<0.0001). In
the 11 out of 12 horizontal divisions that produced daughter
cells with similar morphology, the cell-body size of the two
daughters was similar (Fig. 4H). Daughter cells of horizontal
divisions had a cell-body size of 54±16 arbitrary units (n=24),
which was similar to that of photoreceptors (58±8 arbitrary
units, Fig. 4G,H). In the single case where the two daughter
cells of a horizontal division acquired different morphologies,
the daughters had different cell-body sizes, one similar to that
of photoreceptors and one similar to that of cells in the
interneuron layer (Fig. 4G,H). These results strongly suggest
that the majority of daughter cells of horizontal divisions in the
newborn rat retina become photoreceptor cells.
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.
Fig. 5. Vertical divisions producing two daughter cells that acquire
different morphologies. (A-E) Five examples of daughter cells
produced by vertical divisions in newborn RNECs, seen 70-96 hours
after the division. Note that the two daughters differ in size and
morphology. In the example shown in E, it was possible to fix and
stain the explant and find the two GFP+ daughter cells in a confocal
microscope as described for Fig. 4E. The larger daughter cell (arrow)
is located in a cell layer that contains big nuclei (presumably the
interneuron layer), whereas the smaller daughter cell (arrowhead) is
located in a cell layer that contains small nuclei (presumably the
photoreceptor layer). (F) A z-series confocal projection of a clone
containing a Müller cell (arrow) and a photoreceptor (arrowhead) in a
frozen section of a newborn retinal explant, observed 10 days after
infection with a retrovirus encoding GFP. The section was imaged in a
confocal microscope and a stacked z-series is shown.
(G) Quantification of cell body size of GFP-infected photoreceptors
and interneuron layer cells as measured in cryosections of retinal
explants. This is the same graph as shown in Fig. 4G. (H) Cell-body
sizes of pairs of daughter cells produced by vertical divisions of GFPinfected RNECs followed by video recording. The daughters of all 11
vertical divisions that were followed are shown. Note that in 9 out of
11 cases the daughter cells are different in size. The larger daughter is
much larger than a photoreceptor cell and is similar in size to an
interneuron layer cell. The smaller daughter is usually similar in size
to a photoreceptor cell. Scale bars: 10 µm in A-D; 2 µm in E,F.
2334 M. Cayouette and M. Raff
This conclusion was supported by measurements of cell-body
size. In the 9 out of 11 vertical divisions that produced daughter
cells with different morphologies, the cell-body size was
different (Fig. 5H). The smaller daughters had a cell-body size
of 52±13 arbitrary units (n=11), which was similar to that of
photoreceptors (58±8 arbitrary units), whereas the bigger
daughters had a cell-body size of 119±66 arbitrary units (n=11),
which was similar to that of interneuron layer cells (113±25
arbitrary units, Fig. 5G,H). In the two cases in which the
two daughter cells of a vertical division acquired similar
morphologies, the body size of the daughters was similar, and
also similar to that of photoreceptors (Fig. 5G,H). These results
indicate that the majority of vertical divisions in the newborn rat
retina produce one daughter that becomes either an interneuron
or a Müller cell and one daughter that becomes a photoreceptor.
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
Fig. 6. Maintenance of a basal process during RNEC division.
(A-D) Snapshots of a time-lapse video recording showing a GFP+
RNEC that maintained its basal process (arrows) during division.
This cell divided with a horizontal spindle. The cleavage plane is
indicated with arrowheads in D. The basal process seems to be
asymmetrically inherited by the daughter cell on the left.
(E-G) Confocal micrographs of a different GFP+ RNEC in
metaphase, labeled with propidium iodide (PI). The PI label is shown
in E, GFP in F and a merged image in G. The arrow in F points to the
metaphase plate and the arrowhead in G points to the basal process.
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).
In each case in which a basal process was detected in a
mitotic cell, it seemed to be inherited by only one of the two
daughter cells (Fig. 6D). The daughter cell that inherited the
process in either a horizontal or a vertical division always
moved to take up a position basal to the other daughter cell
(not shown).
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, Numbinfected 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
Asymmetric cell division in the developing rat retina 2335
Fig. 7. The effect of Numb overexpression on clonal
development in newborn rat retinal explants.
(A) Immunostaining of Numb in the newborn rat
retina using polyclonal antibodies (Upstate Biotech).
Punctate Numb staining is concentrated at the apical
side of both interphase and mitotic RNECs (arrow in
inset). (B) Control retinal section, stained as in A but
without the primary anti-Numb antibodies. (C) The
retroviral vector encoding Numb and alkaline
phosphatase. The full-length open-reading frame for
Numb was cloned in front of an internal ribosome
entry site (IRES), which is upstream of the coding
sequence for placental alkaline phosphatase (PLAP).
The bicistronic mRNA encoding Numb and PLAP is
transcribed from the Xenopus EF1α promoter.
(D-G) Examples of clones in frozen sections of a
newborn retinal explant infected with a control
retrovirus encoding alkaline phosphatase (PLAP)
alone and cultured for 10 days. Four clones
containing a photoreceptor (D), an amacrine cell (E),
a bipolar cell (F) and a Müller cell (G) are shown.
(H) Composite of a z-series of confocal images
showing Numb immunostaining in a frozen section
of a newborn retinal explant infected with the Numbexpressing retrovirus. There is strong Numb staining
throughout the two interphase RNECs shown. This
contrasts with the apical concentration of
endogenous Numb in nontransfected RNECs, shown
in A. (I,J) Wholemounts of newborn retinal explants
10 days after infection with either the control
retrovirus (I) or the Numb-expressing retrovirus (J).
Note that the control explant contains a larger
diversity of cell morphologies. Scale bars: 20 µm in
A,B; 10 µm in D-G; 5 µm in H; 100 µm in I,J.
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 Numboverexpression 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 twocell 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.
DISCUSSION
We have used long-term videomicroscopy to visualize the
division of GFP-labeled RNECs in explants of newborn rat
retina and to follow the subsequent differentiation of the
daughter cells that result. We found that RNECs that divide
horizontally tend to give rise to two daughter cells that both
become photoreceptor-like cells, whereas RNECs that divide
vertically tend to give rise to two daughter cells that become
different from each other. We also found that overexpression
of Numb in RNECs in newborn retina increases the proportion
of photoreceptors and decreases the proportion of
nonphotoreceptor cells that develop from the RNECs. Together
with our previous findings that Numb is normally segregated
to the apical daughter cell in vertical RNEC divisions
(Cayouette et al., 2001), these results are consistent with the
hypothesis that asymmetric segregation of Numb during
vertical divisions influences cell-fate choice in the newborn rat
retina.
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
2336 M. Cayouette and M. Raff
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
A
Average cells/clone
1.5
1
Control
Numb
0.5
0
C
100
95
90
85
80
75
70
65
60
55
50
% total infected cells
% Clones
B
Photoreceptor only
E
D
16
14
12
10
8
6
4
2
0
14
% total infected cells
% Clones
100
95
90
85
80
75
70
65
60
55
50
12
10
8
6
4
2
0
≥1 Neuron
≥1 Müller cell
>1 cell type
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
Photoreceptors
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
INL neuron
Müller cell
during division (Huttner and Brand,
1997).
% Clones
F
100
90
80
70
60
50
40
30
20
10
0
2 PR
1 PR + 1 other
Fig. 8. Quantitative clonal analysis of Numb-infected and control retinal explants.
Newborn explants were infected and analyzed as in Fig. 7. A total of 1891 control
clones and 1704 Numb clones in nine different retinal explants were analyzed. The
results are shown as mean±s.d. (A) Clone size. (B) Proportion of photoreceptor-only
clones. (C) Proportion of photoreceptors in the total population of infected cells
analyzed. (D) Proportion of clones containing at least one neuron, one Müller cell or
more than one cell type. (E) Proportion of interneurons or Müller cells in the total
population of infected cells analyzed. (F) Proportion of two-cell clones containing either
two photoreceptors (PR) or a photoreceptor and another cell type. In (B-F), all of the
differences between the control and Numb clones are significantly different when
analyzed by a non-parametric statistical test (P<0.01).
Asymmetric cell division in the developing rat retina 2337
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 celltype 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 cellfate 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
2338 M. Cayouette and M. Raff
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.
Asymmetric cell division in the developing rat retina 2339
We are grateful to Weimin Zhong for the Numb cDNA and antiNumb antibodies, Gord Fishell for the CLE retroviral vector, Jane
McGlade for anti-Numb antibodies, Mark Shipman for help with
confocal microscopy and time-lapse videomicroscopy, Bill Harris and
Ben Barres for comments on the manuscript, and members of the Raff
lab for stimulating discussion and support. M.C. was funded by a
Long-Term Fellowship from the Human Frontier Science Program
Organization and M.R. was funded by the Medical Research Council.
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