Article

Importance of Intrinsic Mechanisms in Cell Fate Decisions in the Developing Rat Retina

Stanford University, Stanford, California, United States
Neuron (Impact Factor: 15.05). 01/2004; 40(5):897-904. DOI: 10.1016/S0896-6273(03)00756-6
Source: PubMed
ABSTRACT
Cell diversification in the developing nervous system is thought to involve both cell-intrinsic mechanisms and extracellular signals, but their relative importance in particular cell fate decisions remains uncertain. In the mammalian retina, different cell types develop on a predictable schedule from multipotent retinal neuroepithelial cells (RNECs). A current view is that RNECs pass through a series of competence states, progressively changing their responsiveness to instructive extracellular cues, which also change over time. We show here, however, that embryonic day 16-17 (E16-17) rat RNECs develop similarly in serum-free clonal-density cultures and in serum-containing retinal explants--in the number of times they divide, the cell types they generate, and the order in which they generate these cell types. These surprising results suggest that extracellular signals may be less important than currently believed in determining when RNECs stop dividing and what cell types they generate when they withdraw from the cell cycle, at least from E16-17 onward.

Full-text

Available from: Michel Cayouette
Neuron, Vol. 40, 897–904, December 4, 2003, Copyright 2003 by Cell Press
ReportImportance of Intrinsic Mechanisms in
Cell Fate Decisions in the Developing
Rat Retina
vertebrate retina. The selective destruction of monoami-
nergic amacrine cells in the goldfish and frog retina, for
example, results in the overproduction of these cells by
RNECs, suggesting that negative feedback signals can
influence cell fate decisions (Negishi et al., 1982; Reh
Michel Cayouette,
1,3,
* Ben A. Barres,
2
and Martin Raff
1
1
MRC Laboratory for Molecular Cell Biology and
Cell Biology Unit
University College London
London, WC1E 6BT and Tully, 1986). Similarly, ganglion cells in the chick
retina apparently secrete a factor that prevents cellsUnited Kingdom
2
Department of Neurobiology from choosing the ganglion cell fate (Waid and McLoon,
1998). Conversely, newborn rat retinal cells promote rodStanford University
Fairchild Building photoreceptor development when cultured with embry-
onic retinal cells, suggesting that positive signals may299 Campus Drive
Stanford, California 94305 also contribute to cell fate choice (Watanabe and Raff,
1990, 1992). In these mixed-age experiments, however,
signals from older rat retinal cells cannot induce the
younger RNECs to make inappropriate cell fate choices
Summary
and produce late-born cell types prematurely, sug-
gesting that young and old RNECs are intrinsically differ-
Cell diversification in the developing nervous system
ent in the types of cells they can produce (Altshuler and
is thought to involve both cell-intrinsic mechanisms
Cepko, 1992; Belliveau and Cepko, 1999; Lillien and
and extracellular signals, but their relative importance
Cepko, 1992; Watanabe and Raff, 1990). These findings
in particular cell fate decisions remains uncertain. In
indicate that extracellular signals are not the only deter-
the mammalian retina, different cell types develop on
minant of cell fate choice in the retina and that the
a predictable schedule from multipotent retinal neuro-
developmental potential of RNECs changes over time.
epithelial cells (RNECs). A current view is that RNECs
A current view of retinal development that tries to
pass through a series of competence states, progres-
reconcile all of these findings suggests that, as RNECs
sively changing their responsiveness to instructive ex-
proliferate and mature, they pass through a series of
tracellular cues, which also change over time. We
competence states, progressively changing their re-
show here, however, that embryonic day 16–17 (E16–
sponsiveness to instructive extracellular signals that in-
17) rat RNECs develop similarly in serum-free clonal-
fluence what type of cell they become (Livesey and
density cultures and in serum-containing retinal ex-
Cepko, 2001; Watanabe and Raff, 1990). Although it is
plants—in the number of times they divide, the cell
now generally accepted that both cell-intrinsic mecha-
types they generate, and the order in which they gener-
nisms and extrinsic signals influence cell fate choice
ate these cell types. These surprising results suggest
in the developing retina, their relative contributions to
that extracellular signals may be less important than
particular cell fate decisions remain unclear.
currently believed in determining when RNECs stop
To test the relative importance of cell-intrinsic mecha-
dividing and what cell types they generate when they
nisms and extracellular signals in cell fate choice, we
withdraw from the cell cycle, at least from E16–17
have developed a serum-free and extract-free clonal-
onward.
density culture system, in which E16–17 rat RNECs
proliferate and differentiate. We have compared the de-
Introduction
velopment of clones in these cultures, in which the envi-
ronment is relatively homogeneous and constant, to the
It is a major challenge to understand how the enormous
development of clones in serum-containing explants of
diversity of cell types develop in the mammalian central
the same age retina, in which the environment is com-
nervous system (CNS). The retina is an attractive part
plex and changes over time. Our findings suggest that
of the CNS to take on this challenge, as it contains a
cell-intrinsic mechanisms play a major part in determin-
manageable number of cell types, which develop on a
ing when E16–17 rat RNECs stop dividing and what
predictable schedule. Pioneering cell lineage studies in
cell types they generate when they withdraw from the
the retina of vertebrates showed that photoreceptors,
cell cycle.
neurons, and Mu
¨
ller glial cells can all develop from a
single RNEC (Turner and Cepko, 1987; Holt et al., 1988;
Results
Wetts and Fraser, 1988), raising the question of how
individual multipotent RNECs choose between differ-
Embryonic Rat RNECs Proliferate
ent fates.
and Differentiate in Serum-Free
A number of studies suggest a role for extracellular
and Extract-Free Clonal-Density Cultures
signals in influencing cell fate choice in the developing
One way to study what influences the fate of RNECs
is to remove them from their normal environment and
*Correspondence: m.cayouette@stanford.edu
analyze their behavior in clonal-density culture, where
3
Present address: Stanford University, Department of Neurobiology,
one can maintain a relatively homogeneous and con-
Fairchild Building, 299 Campus Drive, D231, Stanford, California
94305-5125.
stant environment. We used E16–17 rat neural retinal
Page 1
Neuron
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cells and plated them at clonal density in a defined
culture medium in a culture flask coated with poly-
L-lysine and laminin. At this stage, RNECs still have
the potential to generate rod photoreceptors, amacrine
cells, bipolar cells, and Mu
¨
ller glia, but most cones,
ganglion cells, and horizontal cells have already been
generated. Two hours after plating, 65%–70% of the
cells were RNECs, as judged by their characteristic flat
and dark morphology, extensive lamellapodia, and ex-
pression of nestin (Figures 1A and 1B). Many of the
RNECs proliferated, and, after 7–10 days, had produced
clones that varied in size and contained only differenti-
ated cells, as judged by their morphology (Figure 1C),
lack of nestin staining (data not shown), and inability to
incorporate BrdU (data not shown).
The Expected Differentiated Cell Types Develop
in Clonal-Density Cultures
In vivo, photoreceptor cells display a characteristic pat-
tern of heterochromatin in their nucleus, which is not
seen in the other cell types in the retina (Figure 1D), and
previous studies showed that postmitotic rods acquire
this chromatin pattern before they express rhodopsin
(Neophytou et al., 1997). A large proportion of cells in
our clonal cultures displayed this chromatin pattern,
along with a typical rod cell size and morphology (Fig-
ures 1E and 2); as expected in such cultures (Harris and
Messersmith, 1992; Neophytou et al., 1997), these cells
did not express rhodopsin. When we stained medium-
density cultures with antibodies against the photorecep-
tor protein recoverin, however, we found that 97%
0.1% of the cells with this characteristic chromatin pat-
tern expressed recoverin after 10 days in culture (Figures
1E and 1F) and did not stain with any of the other cell
type-specific antibodies used in this study, as was the
Figure 1. The Various Markers Used to Distinguish the Differenti-
case for all the rod-like cells in our clonal-density cul-
ated Cell Types Produced in Our Dissociated-Cell Cultures
tures (Figure 2).
(A and B) Two E16–17 RNECs after 24 hr in clonal-density culture
Neurons in the clones could be readily distinguished
photographed in phase-contrast (A) or stained for the neuroepithelial
from rods by their larger size, longer processes, and
cell marker nestin ([B], red) and Hoechst to visualize the nucleus
(blue). (C) Phase-contrast micrograph of a clone containing a mixture
more homogeneous chromatin (see Figures 1E, 2I, and
of differentiated cell types after 7 days in culture. (D) A cryosection
2M). To help identify the two neuronal cell types ex-
of a P14 rat retina stained with Hoechst. Note the characteristic
pected to develop from E16–17 RNECs, amacrine, and
pattern of heterochromatin in photoreceptor (PR) cell nuclei (arrows)
bipolar cells, we used antibodies against syntaxin and
compared to the more homogeneous pattern in cells of the interneu-
islet-1. As expected (Barnstable et al., 1985; Galli-Resta
ron layer (INL). (E and F) Three rod cells (arrows) stained with
et al., 1997), staining cryosections of postnatal day 10
Hoechst (E) and anti-recoverin antibodies (F) after 10 days in vitro.
Note that the recoverin
photoreceptors display the same pattern
(P10) retina revealed that syntaxin antibodies stained
of heterochromatin as photoreceptors in vivo. (G) Fluorescence mi-
amacrine cells, while islet-1 antibodies stained bipolars,
crograph of a cryosection of P10 rat retina. Syntaxin is expressed
a subset of amacrines, and a subset of ganglion cells
in amacrine cells, as seen by the staining in the innermost layer of
(Figure 1G). Consistent with these results, we found two
the interneuron layer (INL), which contains amacrine cell bodies,
populations of syntaxin
amacrine cells in our cultures,
and of the inner plexiform layer (IPL), which contains amacrine ax-
one that was islet-1
(Figure 1H) and one that was islet-
ons. Islet-1 is expressed in bipolar cells, as seen by the nuclear
staining of the outermost layer of the INL (asterisk), which contains
1
(Figures 2E–2H and 2M–2P); both had a typical ama-
bipolar cell bodies. It is also expressed by a subset of amacrine
crine cell morphology (Figures 2E and 2M). Bipolar cells
(arrows) and ganglion cells (GCL). Nuclei are stained with Hoechst
in our cultures had a simpler morphology (Figures 2I
(blue). (H) Fluorescence micrographs of a syntaxin
/islet-1
ama-
and 2M) and, as expected from staining cryosections
crine cell and an islet-1
/syntaxin
bipolar cell (arrow) in a clonal
(Figure 1G), were islet-1
and syntaxin
(Figures 1H,
culture after 10 days. (I) Fluorescence micrograph of a cryosection
2I–2L, and 2M–2P); they did not, however, express late
of P10 rat retina. The nucleus of the Mu
¨
ller cells in the INL express
cyclin D3 (green). (J and K) Phase-contrast (J) and fluorescence (K)
differentiation markers, such as protein kinase C (Grefer-
micrographs showing a cyclin-D3
Mu
¨
ller cell (arrow), surrounded
ath et al., 1990) or 115A10 (Onoda and Fujita, 1987). The
by a number of cyclin-D3
cells after 10 days in clonal culture. Scale
islet-1
cells in our cultures were unlikely to be ganglion
bars, 10 m (D); 15 m (A, B, E, F, H, J, and K); 50 m (C); 60 m
cells, as they did not stain with Brn3b, a specific marker
(G and I).
for retinal ganglion cells (data not shown).
We identified Mu
¨
ller glial cells by their characteristic
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Intrinsic Mechanisms in Cell Fate Choice
899
Figure 2. Phase-Contrast and Fluorescence
Micrographs of Clones Produced by E16-17
RNECs in Clonal-Density Cultures after
7–10 Days
The cells were stained as indicated. (A–D) A
clone containing three rods, all of which show
a characteristic heterochromatin pattern and
are negative for islet-1 and syntaxin. (E–H) A
clone containing three rods (arrows) and a
syntaxin
/islet-1
amacrine cell. (I and J) A
clone containing three rods (arrows) and an
islet-1
/syntaxin
bipolar cell. (M–O) A clone
containing a rod (arrow), a syntaxin
/islet-1
amacrine cell, and an islet-1
/syntaxin
bipo-
lar cell. (Q–S) A clone containing two rods
(arrows) and a cyclin D3
Mu
¨
ller cell. Scale
bars, 8 m (A–D and I–L); 10 m (E–H); 15 m
(M–P); 20 m (Q–S).
glial morphology and by the expression of cyclin D3 in and Mu
¨
ller cells (Table 1). Although clonal development
in our cultures was remarkably similar to that expectedtheir nuclei. In cryosections of P10 retina, the expression
of cyclin D3 was consistent with it being specific for from previous studies in vivo, some of the cell types
(such as rods and bipolars) did not express late differen-Mu
¨
ller cells (Figure 1I), as previously reported (Dyer and
Cepko, 2000). As shown in Figures 1J, 1K, and 2Q–2S, tiation markers, suggesting that environmental cues
may be required for their phenotype maturation.small numbers of cyclin-D3
Mu
¨
ller cells developed in
our clonal-density cultures.
The clones that developed in our cultures were very Clone Development in Dissociated-Cell Culture
Closely Resembles that in Explant Culturesheterogeneous in both size and cell type composition,
much as observed in vivo (Turner and Cepko, 1987; To assess more accurately how closely RNEC develop-
ment in our E16–17 clonal-density cultures resembledTurner et al., 1990). Rods were the major cell type that
developed, and rod-only clones were the most abundant that in normal developing retina, we compared it to
clonal development in explant cultures of E16–17 rattype of clone (Figures 2A–2D and Table 1), as expected
from in vivo clonal analyses (Turner and Cepko, 1987; retinas containing 10% FCS. In these conditions, retinal
explants develop remarkably similarly to retinas in vivoTurner et al., 1990). We also found clones containing
mixtures of rods and amacrines (Figures 2E–2H), rods (Sheedlo and Turner, 1996). We infected the explants
with retroviral vectors that encoded either enhancedand bipolars (Figures 2I–2L), rods, amacrines, and bipo-
lars (Figures 2M–2P), rods and Mu
¨
ller cells (Figures 2Q– green fluorescent protein (GFP), which was used to ana-
lyze clone size (Figure 3A), or placental alkaline phos-2S), Mu
¨
ller cells and neurons, with or without rods, and
all four cell types (Table 1). As expected from previous phatase (PLAP), which was used to analyze the cell type
composition of the clones (Figure 3B).in vivo studies (and from our own clonal analysis in
explants—see below), we did not find any clones con- As shown in Figure 3C, clone sizes in explant and
clonal-density cultures were remarkably similar. Clonestaining just bipolars and Mu
¨
ller cells or just amacrine
Page 3
Neuron
900
Table 1. Clonal Analysis in Clonal-Density Cultures and Retinal Explants
Clonal-Density Cultures Explants
Total Number of Number of Clones with Total Number of
Cell Combination Clones (%) Dead Cells (%) Clones (%)
Photoreceptor only 545 (60.2) 38 (4.2) 415 (64.5)
Photoreceptor Amacrine 113 (12.5) 8 (0.9) 47 (7.3)
Photoreceptor Bipolar 132 (14.6) 10 (1.1) 86 (13.4)
Photoreceptor Mu
¨
ller 20 (2.2) 4 (0.4) 30 (4.7)
Photoreceptor Amacrine Bipolar 35 (3.9) 8 (0.9) 38 (5.9)
Photoreceptor Amacrine Bipolar Mu
¨
ller 13 (1.4) 4 (0.4) 6 (0.9)
Photoreceptor Bipolar Mu
¨
ller 9 (1.0) 1 (0.1) 8 (1.2)
Photoreceptor Amacrine Mu
¨
ller 11 (1.2) 0 (0) 6 (0.9)
Bipolar only 1 (0.1) 1 (0.1) 0 (0)
Amacrine only 14 (1.5) 0 (0) 4 (0.6)
Mu
¨
ller only 1 (0.1) 0 (0) 0 (0)
Bipolar Amacrine 8 (0.9) 0 (0) 3 (0.5)
Bipolar Amacrine Mu
¨
ller 3 (0.3) 0 (0) 0 (0)
Bipolar Mu
¨
ller 0 (0) 0 (0) 0 (0)
Amacrine Mu
¨
ller 0 (0) 0 (0) 0 (0)
Total 905 (100) 74 (8.2) 643 (100)
A total of ten dissociated-cell cultures of E16–17 neural retina from three separate experiments were analyzed after 7–10 days in culture. A
total of four separate retinal explants were analyzed after 10 days in culture. Numbers in parentheses represent the proportions.
in clonal-density cultures contained an average of 2.9 tion of clone sizes was very similar in the two types of
cultures (Figure 3C). By contrast, the distribution ofcells per clone, whereas clones in explants contained
an average of 3.2 cells per clone. Moreover, the distribu- clone sizes that developed from E20–21 RNECs was
Figure 3. Comparison of Clones that De-
velop in Explant versus Clonal-Density Reti-
nal Cultures and the Sequence of Develop-
ment of Different Cell Types in Clonal-
Density Cultures
(A) Example of a clone containing four rods,
10 days after an E17 retinal explant was in-
fected with a GFP-encoding retroviral vector.
(B) Example of a clone containing three rods
and one bipolar, 10 days after an E17 retinal
explant was infected with a PLAP-encoding
retroviral vector. (C) The distribution of clone
sizes in explants and clonal-density cultures
from E16–17 or E20–21 rat retina, after 7–10
days in culture. (D and E) Cellular composition
of clones in explants and clonal-density cul-
tures, as a percent of either the total number
of cells in clones analyzed (D) or the total
clones analyzed (E). Results are shown as
mean SD of four separate cultures. (F) Se-
quence of development of different cell types
in cultures of E16–17 retinal cells. Results are
shown as mean SD of three separate cul-
tures per time point. (G) BrdU was added to
the cultures at 2 hr, 18 hr, and 48 hr post-
plating, and the clones were then allowed to
develop for 7 days. Results are shown as
mean SD of three cultures per time point
(*significantly different from t 2 hr,
ANOVA test).
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Intrinsic Mechanisms in Cell Fate Choice
901
much more restricted than observed with E16–17 cells Does Instructive Intraclonal Signaling Play
a Part in Cell Fate Choice?(Figure 3C), consistent with previous evidence that the
proliferative potential of RNECs progressively decreases Detailed analysis of the clones that developed in clonal-
density cultures is helpful to address whether instructiveas the RNECs mature (Lillien and Cepko, 1992; Wata-
nabe and Raff, 1990). signals from previously differentiated cells might be re-
quired for other cell types to develop within the sameThe cell type compositions of the clones generated
from E16–17 RNECs in clonal-density cultures and in clone. As shown in Table 1, we found almost all possible
combinations of the four expected cell types withinexplants were also remarkably similar (Figures 3D and
3E). When analyzed as a proportion of the total number clones. Clones containing only rods, for example, were
the most common, suggesting that signals from otherof cells assessed in clones, the proportions of rods,
bipolars, amacrines, and Mu
¨
ller cells were strikingly sim- cell types were not required for E16–17 RNECs to de-
velop into rods in these cultures. Similarly, we foundilar in the two cases (Figure 3D). The proportions of
clones containing only rods, or at least one bipolar, clones containing only amacrine cells and rods, only
bipolar cells and rods, and only Mu
¨
ller cells and rods,one amacrine, or one Mu
¨
ller cell, were also remarkably
similar in the two cases (Figure 3E). suggesting that the development of RNECs into ama-
crine, bipolar, or Mu
¨
ller cells did not require signals fromAs RNECs change their developmental potential over
time (Altshuler and Cepko, 1992; Lillien and Cepko, 1992; other cell types, with the possible exception of signals
from rods. We also found, however, a small number ofWatanabe and Raff, 1990), one would expect that
E20–21 RNECs would produce fewer early-born cell clones containing amacrine, bipolar, and/or Mu
¨
ller cells
that did not contain photoreceptors, suggesting thattypes, such as amacrines, and more single-cell-type
clones than would E16–17 RNECs. This was the case. signals from rods may not be required for E16–17 RNECs
to develop into these cell types. Although cell deathOnly 7% 3% of clones produced by E20–21 RNECs
contained at least one amacrine cell, compared to could have confounded this analysis (Voyvodic et al.,
1995), we found that little cell death occurred in our17% 7% of clones produced by E16–17 RNECs (p
0.05, Student’s t test), and 75% 3% of clones pro- clones (Table 1). The 905 clones analyzed contained a
total of 2625 cells, and only 106 dead cells (4%) wereduced by E20–21 RNECs contained only rods, compared
to 64% 7% of clones produced by E16–17 RNECs observed among them, and only 74 clones contained
one or more dead cells (Table 1). Most importantly, no(p 0.05, Student’s t test). Even if we only compared
clones containing four cells in E16–17 and E20–21 cul- dead cells were seen in 507 out of 545 clones that con-
tained only rods, in 26 out of 27 clones that did nottures, 21% of the E20–21 clones contained amacrine
cells, whereas 33% of E16–17 clones contained ama- contain rods, or in 15 clones that contained only ama-
crines or Mu
¨
ller cells. Thus, cell death is unlikely to havecrine cells, suggesting that the reduced ability of E20–21
RNECs to give rise to early-born cell types was not significantly confounded our analyses.
caused only by their reduced proliferative capacity.
RNECs in Dissociated-Cell Culture Can Reorient
Their Mitotic Spindle before Division
Different Cell Types Are Born and Differentiate
We recently reported evidence that the plane of RNEC
in the Predicted Sequence
division in the newborn rat retina can influence the fate
in Clonal-Density Cultures
of the daughter cells (Cayouette and Raff, 2003). How
To help determine if the different cell types develop in
can we reconcile the apparent importance of the orienta-
the normal sequence in our clonal-density cultures, we
tion of cell division for cell fate choice with our present
either stained the cultures with cell type-specific mark-
findings that RNECs seem to diversify normally in clonal-
ers at different times or added bromodeoxyuridine
density cultures? To study the orientation of division of
(BrdU) to the culture at different times and analyzed the
RNECs in such cultures, we used time-lapse video mi-
cultures after 7 days. As shown in Figure 3F, we found
croscopy.
that amacrine cells and rods were the first differentiated
RNECs in our dissociated-cell cultures usually divided
cells detectable, followed by bipolar and Mu
¨
ller cells.
with their mitotic spindle oriented perpendicular to their
As expected, cells expressing nestin decreased over
long (putative apical-basal) axis (Figure 4A). In some
time and were mostly gone by 8 days. The proportion
cases, however, they rotated their spindle at metaphase
of BrdU-positive amacrine cells was greater when BrdU
to divide with their spindle oriented parallel to this axis
was added at 2 hr than when it was added at 48 hr
(Figure 4B). Very few cells divided with their spindle in
(Figure 3G), indicating that most amacrine cells were
an intermediate orientation. Thus, it seems that, even
born early. In contrast, the proportion of BrdU-positive
in dissociated-cell cultures, RNECs can position their
Mu
¨
ller cells was greater when BrdU was added at 48 hr
spindle so as to divide in either of two orientations,
suggesting that they could segregate asymmetricallythan when it was added at 2 hr (Figure 3G), indicating
localized proteins to only one of the two daughter cells
that most of these cells were born later than amacrine
during cell division.
cells. Large numbers of BrdU-positive rods were seen
at all time points. All of these results are consistent with
the birthdating experiments previously reported in vivo
Discussion
(Rapaport et al., 1996; Young, 1985), suggesting that the
various cell types are born and differentiate in the same
We are interested in how RNECs choose between alter-
general order as in vivo, although it will be important to
native fates to generate the different cell types in the
developing mammalian retina. To help address thisconfirm this by following individual clones over time.
Page 5
Neuron
902
Figure 4. RNECs in Dissociated-Cell Culture
Divide Either Parallel or Perpendicular to the
Long Axis of the Cell
(A) Snapshots of a time-lapse video recording
showing an RNEC dividing with its mitotic
spindle oriented perpendicular to the long
axis of the cell. Note that the nucleus mi-
grated to one pole of the cell to divide. (B)
Snapshots of a time-lapse video recording
showing an RNEC dividing with its mitotic
spindle oriented parallel to the long axis of
the cell. Its nucleus also migrated to one pole
of the cell before the cell divided. Scale bar,
10 m.
problem, we have developed a serum-free and extract- gene has been inactivated, but all other major retinal
cell types develop, suggesting that signals from rodsfree clonal-density culture system in which E16–17 rat
RNECs proliferate and differentiate in the presence of may not be required for the other retinal cell types to
develop in vivo (Mears et al., 2001). None of these find-a cocktail of growth factors and neurotrophins. We find
that the RNECs form clones of various sizes and cellular ings exclude the possibility that negative feedback sig-
nals operate to inhibit differentiation. Indeed, there iscompositions, even though they are in the same flask.
Unexpectedly, the clones that develop are remarkably strong evidence for such signals, as mentioned in the
Introduction. Moreover, our findings do not exclude thesimilar in size and composition to the clones that devel-
op in retinal explants of the same age cultured in serum, possibility that positive instructive signals play a crucial
part before E16–17 in the rat retina, either to influenceand the various cell types are born and differentiate in
a similar general sequence to that observed in vivo. cell fate directly when RNECs withdraw from the cell
cycle or to program cycling RNECs to influence theirFinally, we find that some RNECs in dissociated cell
cultures reorient their mitotic spindle through 90 before later decisions.
Extracellular signals are almost certainly required forthey divide. We discuss the possible implications of
these surprising findings below. cell survival and cell proliferation in the developing ret-
ina, as well as for the phenotype maturation of some cell
types after they initially differentiate. We rarely detectIntrinsic versus Extrinsic Mechanisms
of Cell Diversification differentiated cells expressing late cell type-specific
markers in our clonal-density cultures, suggesting thatThe finding that E16–17 RNECs produce clones of simi-
lar size and cellular composition in our clonal-density cell-cell interactions are required for the expression of
these proteins. There is strong previous evidence thatand explant cultures is unexpected, as the environments
in which the RNECs develop in the two culture systems opsin expression in photoreceptors requires cell-cell
interactions (Harris and Messersmith, 1992; Morrow etare very different. It suggests that cell-intrinsic mecha-
nisms may be more important than previously sus- al., 1998; Neophytou et al., 1997), as does the expression
of some differentiation markers in other retinal cell typespected in determining both when RNECs stop dividing
and differentiate and what cell types they produce when (Akagawa and Barnstable, 1986; Akagawa et al., 1987;
Reh, 1992).exiting the cell cycle, at least from E16–17 onward in
culture. As clonal development in retinal explants closely
resembles that in vivo (Sheedlo and Turner, 1996), it Orientation of Cell Division
in Dissociated-Cell Cultureseems likely that cell-intrinsic mechanisms are also im-
portant in vivo, at least after E16–17 in rats, but this We find that most RNECs proliferating in dissociated-
cell culture divide with their mitotic spindle orientedremains to be shown directly.
We also find in our clonal-density cultures that the perpendicular to their long axis. Remarkably, however,
some rotate their spindle through 90 so that it alignspresence of a particular cell type is not required for
the development of any other cell type within a clone, along the long axis. We previously showed that an anti-
gen recognized by a monoclonal anti-m-Numb antibodysuggesting that positive instructive signals from differ-
entiated retinal cells are unlikely to determine the choice is asymmetrically localized at one pole in many RNECs
in dissociated-cell cultures and can segregate to onlythat a RNEC makes when it withdraws from the cell
cycle. There is previous genetic evidence that this may one of the daughter cells when the RNEC divides (Cay-
ouette et al., 2001). Thus, our current finding raises thealso be true in vivo. Inactivation of the pax6 gene in the
developing mouse retina, for example, results in the possibility that spindle rotation and asymmetric segre-
gation of cell fate determinants may contribute to RNECexclusive development of amacrine cells, suggesting
that amacrine cells can develop without signals from fate choice, even in dissociated-cell cultures.
There are other examples where neural precursor cellsother cells (Marquardt et al., 2001). Remarkably, various
subtypes of amacrine cells also develop in these mice, asymmetrically segregate cell fate determinants, even
though the cells are not within a polarized epithelium.indicating that amacrine cells do not need to make syn-
aptic contact with, or receive signals from, other cell Drosophila neuroblasts can asymmetrically localize the
proteins Inscuteable, Prospero, and Staufen at mito-types in the retina to diversify into subtypes. Similarly,
rods are not found in the retina of mice in which the nrl sis in dissociated-cell culture, just as they do in vivo
Page 6
Intrinsic Mechanisms in Cell Fate Choice
903
(Broadus and Doe, 1997). Similarly, mouse cortical pro- it will be important to compare the lineage trees of clones
generated in explants with those generated in clonal-
genitors segregate m-Numb asymmetrically in dissoci-
density cultures, using long-term time-lapse video-
ated-cell culture, and this segregation seems to influ-
microscopy. If particular combinations of cell types arise
ence cell fate choice (Shen et al., 2002).
by stereotyped patterns of cell division in both types of
cultures, it would strongly suggest that intrinsic pro-
Preprogramming versus Stochastic Models
grams are at work.
Our findings strongly suggest that intrinsic mechanisms
play an important part in determining when E16–17
Experimental Procedures
RNECs stop dividing and differentiate and what cell
types they produce. The alternative explanation for our
Clonal-Density Culture of RNECs
Detailed protocols are available upon request. Retinas from Sprague
findings—that it is a coincidence that E16–17 RNECs
Dawley rats were dissected and dissociated following a previously
behave so similarly when in the retinal neuroepithelium
published method (Jensen and Raff, 1997). The cells were resus-
and when isolated in clonal-density culture—seems
pended in serum-free medium consisting of a 1:1 mixture of DMEM-
highly unlikely.
F12 medium with N2 supplement and of Neurobasal medium with
There are at least two types of cell-intrinsic mecha-
B27 supplement. This medium also contained 8-(4-chlorophe-
nylthio) adenosine 35-cyclic monophosphate (cpt-cAMP, 0.1 mM),
nisms that could explain our results. One is that RNECs
forskolin (25 M), N-acetyl-L-cystein (6.3 mg/ml), insulin (20 g/ml),
make decisions stochastically, with the probabilities of
FGF-2 (10 ng/ml), EGF (50 ng/ml), BDNF (10 ng/ml), NT-3 (10 ng/ml),
cell fate choices weighted toward certain cell types and
and penicillin/streptomycin. The cell suspension was filtered twice
changing over time. Late RNECs, for example, would
through an 8 m nylon mesh to obtain a single-cell suspension.
be heavily biased to produce rods, whereas early RNECs
Between 3000 and 5000 cells were plated in T-25 Falcon flasks
would be biased to produce cones and ganglion cells.
coated with poly-L-lysine (10 m/ml) and laminin (10 g/ml). After
a few hours, clumps of more than one cell were ringed and excluded
Such a stochastic model has been proposed for hemato-
from the study. Such clumps represented less than 1% of the
poiesis (Till et al., 1964). An alternative possibility is
plated cells.
that individual RNECs become differently programmed
before E16–17 and then step through their specific de-
Retinal Explant Cultures and Retroviral Infection
velopmental program independently of instructive sig-
E16–17 rat retinal explants were prepared as previously described
(Cayouette et al., 2001). The explants were allowed to settle for a
nals from the environment.
few hours in a CO
2
incubator at 37C before they were infected
We prefer the second possibility for several reasons.
with a retroviral vector encoding either enhanced green fluorescent
(1) It could more easily explain how a clone containing
protein (GFP) or placental alkaline phosphatase (PLAP). Retroviral
exclusively 33 rods could develop in vivo from an E14
vectors were prepared and used to infect explants as described
mouse RNEC (Turner et al., 1990). As pointed out by
previously (Cayouette and Raff, 2003).
Williams and Goldowitz (Williams and Goldowitz, 1992),
Histology and Immunostaining
the chance that such a clone would develop if all RNECs
The retinal explants were fixed and cryosectioned after 10 days in
were similar and differentiated stochastically is 1:13,000,
culture as previously described (Cayouette et al., 2001). The follow-
although weighted probabilities were not considered
ing antibodies were used for immunofluorescence: monoclonal
in this calculation. (2) There are other examples where
mouse anti-syntaxin (1:1000; Sigma); monoclonal mouse anti-islet-1
(produced by T. Jessell and obtained from the Developmental Stud-
neural precursor cells seem to be specified early and
ies Hybridoma Bank); rabbit anti-recoverin (1:1000; Berkeley Ab Co.),
then step through a prescribed developmental program.
and monoclonal mouse anti-cyclin D3 (1:100, Santa Cruz Biotech.).
Neuroblasts in the Drosophila CNS are a particularly
Primary antibodies were detected using the appropriate Alexa Fluor
impressive example. They go through a series of asym-
488 or 594 goat antibodies (Molecular Probes). In all cases, we
metrical divisions to produce a variety of cell types, even
counterstained the nuclei by incubating the cells for 5 min in Hoechst
in dissociated-cell culture (Furst and Mahowald, 1985;
33342 (Molecular Probes). For BrdU-labeling experiments, BrdU was
added to the culture at a concentration of 10 M. After 7 days
Luer and Technau, 1992), and they sequentially express
in culture, cells were fixed and BrdU incorporation detected as
different sets of transcription factors with each cell divi-
described (Neophytou et al., 1997).
sion (Isshiki et al., 2001). Similarly, dissociated cortical
progenitor cells can undergo stereotyped patterns of
Acknowledgments
cell division and differentiation to produce neurons and
We are grateful to Gord Fishell for the CLE retroviral vector; Bill
glial cells in a normal sequence in clonal cultures (Qian
Harris for insightful comments; and members of the Raff and Barres
et al., 1998, 2000). (3) Some vertebrate RNECs have
labs for stimulating discussions and support. This work was funded
been shown to be biased to produce amacrine cells:
by a Long-Term Fellowship from the Human Frontier Science Pro-
this is the case for embryonic rat RNECs that express
gram Organization and a senior postdoctoral fellowship from the
the antigenic epitope VC1.1 (Alexiades and Cepko, 1997)
Canadian Institute of Health Research (M.C.), the NIH-NEI (B.A.B,
grant R01 EY11310), the March of Dimes Foundation (B.A.B, grant
and for some Xenopus RNECs (Moody et al., 2000) and
1FY01-352), and the Medical Research Council UK (M.R.).
even some Xenopus blastomeres (Huang and Moody,
1995, 1997). Taken together, these findings and ours
Received: October 1, 2002
seem most consistent with the possibility that individual
Revised: June 18, 2003
RNECs become preprogrammed in various ways to di-
Accepted: November 12, 2003
Published: December 3, 2003
vide for a particular period of time or number of divisions
and to give rise to one or more specific cell types. If so,
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  • Source
    • "Confidence in the present findings comes from our ability here to count and identify all the cells, including displaced ACs, in large sets of individual clones, combined with the consistency of the statistical effects in morphants and mutants. The conclusion of minimal homeostatic compensation is independently supported by the fact that rat RPCs in clonal cultures give rise to clonal distributions that are similar to those in vivo (Cayouette et al., 2003); i.e., there is not an overproduction of GCs or ACs in these clones even though they are grown in the absence of any feedback cues. The model outlined here has three phases. "
    [Show abstract] [Hide abstract] ABSTRACT: Early retinal progenitor cells (RPCs) in vertebrates produce lineages that vary greatly both in terms of cell number and fate composition, yet how this variability is achieved remains unknown. One possibility is that these RPCs are individually distinct and that each gives rise to a unique lineage. Another is that stochastic mechanisms play upon the determinative machinery of equipotent early RPCs to drive clonal variability. Here we show that a simple model, based on the independent firing of key fate-influencing transcription factors, can quantitatively account for the intrinsic clonal variance in the zebrafish retina and predict the distributions of neuronal cell types in clones where one or more of these fates are made unavailable.
    Full-text · Article · Sep 2015 · Developmental Cell
  • Source
    • "These cell types are organized into three nuclear layers and generate two plexiform layer neuropils where most of the synapses are confined. Retinal progenitor cell (RPC) proliferation and fate choice are regulated by intrinsic mechanisms234 and extracellular signaling from differentiated cells567. During retinogenesis, the cell-cycle length increases over time in progenitor cells [8]. "
    [Show abstract] [Hide abstract] ABSTRACT: Nr2e1 is a nuclear receptor crucial for neural stem cell proliferation and maintenance. In the retina, lack of Nr2e1 results in premature neurogenesis, aberrant blood vessel formation and dystrophy. However, the specific role of Nr2e1 in the development of different retinal cell types and its cell-autonomous and non-cell autonomous function(s) during eye development are poorly understood. Here, we studied the retinas of P7 and P21 Nr2e1 (frc/frc) mice and Nr2e1 (+/+) ↔ Nr2e1 (frc/frc) chimeras. We hypothesized that Nr2e1 differentially regulates the development of various retinal cell types, and thus the cellular composition of Nr2e1 (frc/frc) retinas does not simply reflect an overrepresentation of cells born early and underrepresentation of cells born later as a consequence of premature neurogenesis. In agreement with our hypothesis, lack of Nr2e1 resulted in increased numbers of glycinergic amacrine cells with no apparent increase in other amacrine sub-types, normal numbers of Müller glia, the last cell-type to be generated, and increased numbers of Nr2e1 (frc/frc) S-cones in chimeras. Furthermore, Nr2e1 (frc/frc) Müller glia were mispositioned in the retina and misexpressed the ganglion cell-specific transcription factor Brn3a. Nr2e1 (frc/frc) retinas also displayed lamination defects including an ectopic neuropil forming an additional inner plexiform layer. In chimeric mice, retinal thickness was rescued by 34 % of wild-type cells and Nr2e1 (frc/frc) dystrophy-related phenotypes were no longer evident. However, the formation of an ectopic neuropil, misexpression of Brn3a in Müller glia, and abnormal cell numbers in the inner and outer nuclear layers at P7 were not rescued by wild-type cells. Together, these results show that Nr2e1, in addition to having a role in preventing premature cell cycle exit, participates in several other developmental processes during retinogenesis including neurite organization in the inner retina and development of glycinergic amacrine cells, S-cones, and Müller glia. Nr2e1 also regulates various aspects of Müller glia differentiation cell-autonomously. However, Nr2e1 does not have a cell-autonomous role in preventing retinal dystrophy. Thus, Nr2e1 regulates processes involved in neurite development and terminal retinal cell differentiation.
    Full-text · Article · Jun 2015 · Molecular Brain
  • Source
    • "The vertebrate retina, a highly organized neural structure with three major cellular layers, is an excellent model for studying cell fate decision [1][2][3]. During retinogenesis, multipotent retinal progenitor cells (RPCs) give rise to six types of neurons and one type of glial cells in a precise and conserved order [4]. "
    [Show abstract] [Hide abstract] ABSTRACT: The homeobox transcription factor orthodenticle homolog 2 (otx2) is supposed as an organizer that orchestrates a transcription factor network during photoreceptor development. However, its regulation in the process remains unclear. In this study, we have identified a zebrafish limb bud and heart-like gene (lbh-like), which is expressed initially at 30 hours post fertilization (hpf) in the developing brain and eyes. Lbh-like knockdown by morpholinos specifically inhibits expression of multiple photoreceptor-specific genes, such as opsins, gnat1, gnat2 and irbp. Interestingly, otx2 expression in the morphants is not significantly reduced until 32 hpf when lbh-like begins to express, but its expression level in 72 hpf morphants is higher than that in wild type embryos. Co-injection of otx2 and its downstream target neuroD mRNAs can rescue the faults in eyes of Lbh-like morphants. Combined with the results of promoter-reporter assay, we suggest that lbh-like is a new regulator of photoreceptor differentiation directly through affecting otx2 expression in zebrafish. Furthermore, knockdown of lbh-like increases the activity of Notch pathway and perturbs the balance among proliferation, differentiation and survival of photoreceptor precursors.
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