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The Journal of Neuroscience, March 15, 2000, 20(6):2247-2254
Late Retinal Progenitor Cells Show Intrinsic Limitations in the
Production of Cell Types and the Kinetics of Opsin Synthesis
Michael J.
Belliveau,
Tracy L.
Young, and
Constance L.
Cepko
Department of Genetics, and Howard Hughes Medical Institute,
Harvard Medical School, Boston, Massachusetts 02115
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ABSTRACT |
The seven major cell classes of the vertebrate neural retina arise
from a pool of multipotent progenitor cells. Several studies suggest a
model of retinal development in which both the environment and the
progenitor cells themselves change over time (Cepko et al., 1996 ). To
test this model, we used a reaggregate culture system in which a
labeled population of progenitor cells from the postnatal rat retina
were cultured with an excess of embryonic retinal cells. The labeled
cells were then assayed for their cell fate choices and their kinetics
of rod differentiation, as measured by opsin synthesis. The kinetics of
opsin synthesis remained unchanged, but fewer postnatal cells adopted
the rod cell fate when cultured with embryonic cells. There was an
increase in the percentage of bipolar cells produced by postnatal
progenitor cells, indicating a possible respecification of fate. The
increase in bipolar cells could occur even after progenitor cells had
completed their terminal mitoses. These alterations in cell fates
appeared to be caused at least in part by a secreted factor released by
the embryonic cells that requires the LIFR /gp130 complex for
signaling. Finally, although surrounded by 20-fold more embryonic
cells, the postnatal cells did not choose to adopt any fates normally
produced only by embryonic cells.
Key words:
progenitor cell; opsin; retina; bipolar cell; rod
photoreceptor; embryonic retinal cell; rhodopsin kinetics
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INTRODUCTION |
The vertebrate CNS is composed of a
multitude of cell types. How these cells are generated during
development is a central problem in neurobiology. A model system for
the study of cell fate decisions has been the neural retina. The seven
major retinal cell types arise from a pool of multipotent progenitor
cells (Turner and Cepko, 1987; Holt et al., 1988; Wetts and Fraser,
1988). Retinal cell types are generated in a particular order; e.g., in
rodents certain cell types are produced in the embryonic period, and
others are produced in the early postnatal period (for review, see
Altshuler et al., 1993). Because a degree of multipotency has
been shown to persist throughout retinal development, it has been
proposed that cell fate choices are influenced by extrinsic factors
(Turner et al., 1990; Reh, 1991). However, it has been unclear how much of the information regarding cell fate decisions is intrinsic to
progenitor cells and how much influence the environment can exert in
terms of cell types produced by progenitor cells. Similarly, it has
been unclear whether progenitor cells from different ages are
equivalent or different regarding their ability to make the different
cell types.
We showed previously that embryonic progenitor cells, when cultured in
the presence of excess postnatal retinal cells, could be altered in
their cell fate decisions (Belliveau and Cepko, 1999). There was a
significant decrease in the percentage of amacrine cells born from
embryonic day 16 (E16) progenitor cells, as well as an increase in the
percentage of cone photoreceptors. Because rod photoreceptors are the
major cell type produced postnatally, it was surprising to find that
there was no increase in the percentage of cells adopting the rod cell
fate. Moreover, the E16 cells did not adopt cell fates restricted to
postnatal ages, namely the bipolar cell and Müller glial cell
fate. Thus it appears that although extrinsic cues can alter the fate
of the cell types produced by embryonic progenitor cells, they are
limited by the intrinsic repertoire of the progenitor pool, (Belliveau
and Cepko, 1999; Cepko, 1999).
In this study, we further analyzed the ability of progenitor cells to
respond to extrinsic cues by culturing reaggregates in which postnatal
day 0 (P0) cells were surrounded by an excess of E16 cells. The primary
postmitotic fate of P0 progenitor cells is the rod photoreceptor fate
(Alexiades and Cepko, 1997; Ezzeddine et al., 1997; M. M. LaVail,
personal communication). Previous work had established that
diffusible molecules could influence rod development in
vitro. For example, secreted factors have been shown to inhibit
(Lillien, 1995; Kirsch et al., 1996; Ezzeddine et al., 1997; Neophytou
et al., 1997; Kirsch et al., 1998) or stimulate (Altshuler et al.,
1993; Kelley et al., 1994; Levine et al., 1997) rod development when
added to rodent retinal cultures. Perhaps the best evidence that a
retinal factor or factors may be required for rod development is from
culture experiments in which P0 retinal cells were placed at various
cell densities. As the cells became more sparse, they no longer adopted
the rod cell fate, and at least some adopted an alternative fate, that of bipolar cells (Altshuler and Cepko, 1992). Interestingly,
application of CNTF or related cytokines produced a similar effect on
P0 retinae cultured as organ explants (Ezzeddine et al., 1997). These
data together suggest a possible "tug of war" between the signals
that direct two fates: those of rods and bipolar cells.
A second aspect of rod development examined in the current study was
the regulation of the kinetics of rhodopsin expression. We showed
previously that embryonic rod precursor cells display a variable lag
between their terminal mitosis and expression of rhodopsin (Morrow et
al., 1998). In contrast, cells undergoing their terminal mitosis on or
after E19 displayed a fixed lag of ~6 d to rhodopsin expression. When
cultured, embryonic progenitor cells born before E19 displayed the same
long and variable lags seen in vivo. Surprisingly, embryonic
progenitor cells, when cultured in the presence of excess postnatal
retinal cells, did not change either their kinetics for rhodopsin
expression or the percentage of cells adopting the rod fate (Morrow et
al., 1998). These data suggest that there are intrinsic limitations
within embryonic progenitor cells regarding rod development.
To examine which properties of P0 progenitor cells are intrinsic, we
performed mixing experiments in which P0 retinal progenitor cells were
labeled with a short pulse of
[3H]thymidine, reaggregated with 20-fold
more E16 retinal cells, and cultured for >2 weeks. The fate choices of
the immediate progeny of the labeled P0 progenitor cells were then
assayed with cell type-specific antibodies. As was observed when E16
cells were examined in the presence of excess P0 cells, the kinetics of
rhodopsin expression by the P0 cells was not altered, suggesting again
that the kinetics are intrinsically controlled. However, in contrast to
what we observed when E16 cells were surrounded by P0 cells, in the
current study, the production of rods by P0 cells was dramatically altered by surrounding them with an excess of E16 cells. Fivefold fewer
P0 cells adopted the rod cell fate. The P0 cells previously fated to
become rods instead expressed markers for the bipolar cell fate. There
was also a small increase in the production of amacrine cells. However,
there was no production of cells that normally are generated
embryonically but not postnatally, such as cone photoreceptors. The
ability of the P0-born cells to be altered in their cell fate choice
was maintained after the cells had undergone their terminal mitosis.
Finally, we found evidence that embryonic cells secrete a
rod-inhibiting factor or factors. The factor(s) requires signaling
through the LIFR/gp130 complex that serves as a receptor for CNTF,
leukemia inhibitory factor (LIF), and other members of their cytokine
family. These data, together with previous reaggregate culture
experiments (Belliveau and Cepko, 1999), support the hypothesis that
progenitor cells pass through phases of competence that dictate their
response to extrinsic cues such that they produce particular
repertoires of cell types (Cepko et al., 1996). Once the cells have
passed through a phase, they cannot be induced to return to it. This is
distinct in some aspects from the observations made for cerebral cortical progenitor cells, as will be discussed.
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MATERIALS AND METHODS |
Animals. Timed pregnant Sprague Dawley rats were
purchased from Taconic Laboratories (Germantown, NY).
[3H]thymidine labeling. For all culture
experiments, the mitotic P0 cells were pulse-labeled with
[3H]thymidine. Retinae were dissected
free of surrounding tissues and placed into DF10 [45% DME, 45%
Ham's F12 Nutrient Mixture (Life Technologies, Gaithersburg, MD), 10%
FCS and penicillin/streptomycin (100 U/ml)] containing 5 µCi
[3H]thymidine/ml for 1 hr at 37°C.
Retinae were subsequently rinsed by three media changes. Labeled cells
were cultured as described below. Retinae were then dissociated as
described previously (Altshuler and Cepko, 1992). The interval from the
end of the [3H]thymidine labeling to the
beginning of the culture was 30-40 min.
Reaggregate cultures. The reaggregate pellet culture
protocol was performed as described previously (Belliveau and Cepko, 1999). [3H]thymidine-labeled P0 retinae
were dissociated as described above for the dissociation of fresh
retinal tissue. Cells were counted and pelleted in a microcentrifuge
tube containing 20-fold excess, unlabeled E16 retinal cells by
centrifugation for 7 min at 1150 × g. The total per
pellet was 5 × 105 to 1 × 106 cells. Pellets were transferred to
nucleopore polycarbonate membranes, 0.2 µm pore size (Costar,
Cambridge, MA), and cultured for 3, 5, 7, 10, 12, 15, or 17 d as
described previously in DF10. At the end of the culture period, pellets
were dislodged from the membranes, dissociated, and processed
autoradiographically and immunocytochemically as described below.
Gel cultures. P0 retinae, previously labeled with
[3H]thymidine, and E16 retinae were
dissociated as described above for the dissociation of fresh retinal
tissue into single cells. Cells were cast into a collagen gel matrix,
and cocultures of two gels were set up as described previously
(Altshuler and Cepko, 1992). P0 cells in the center well were plated at
high density (8 × 105 P0 cells/40
µl), and for controls with a gel of P0 cells surrounding the central
gel, the surrounding P0 cells were cast at 4 × 106 P0 cells/200 µl. For experiments in
which the center gel of P0 cells was cocultivated with E16 cells, the
P0 cells in the central gel were 8 × 105 P0 cells/40 µl, and the surrounding
E16 cells were 4 × 106 E16 cells/200
µl. All gels were cultured in defined medium (Bottenstein and Sato,
1979). Gel cultures incubated with the LIFR antagonist hLIF-05
(Hudson et al., 1996) included 5 µg/ml of the antagonist. Gel
cultures were maintained for 10 d. Cells were then dissociated and
processed autoradiographically and immunocytochemically as described
below. Autoradiography was performed to ensure that the P0 cells from
the central well were the population scored for marker expression.
Dissociation of cultures. Pellets were dissociated by
sinking the filters into the well medium to detach the cells,
transferring the pellets to Ca2+- and
Mg2+-free HBSS, and then processing them
as described above for the dissociation of fresh retinal tissue into
single cells. Gels were dissociated as described previously
(Altshuler et al., 1993). Once dissociated, the cells were
plated on coated eight-well glass slides (Cel-Line Associates) coated
with 100 µl of 10 µg/ml poly-D-lysine for 20 min. The
cells were allowed to attach at 37°C for 1-2 hr, then fixed in 4%
formaldehyde for 10-20 min. The slides were then processed for immunocytochemistry.
Immunocytochemistry. Slides were blocked for 1 hr in 2%
donkey serum, 2% goat serum, and 0.1% Triton X-100 detergent in PBS. Primary antibody incubation for 1 hr used blocking solution containing primary antibody [VC1.1 (1:1000, Sigma, St. Louis, MO), anti-mGluR2/3 (1:300, Chemicon, Temecula, CA), recoverin (1:1000) (Dizhoor et al.,
1991), cone opsins (1:10,000) (Wang et al., 1992; Chiu and Nathans,
1994), 115A10 (neat) (Onodo and Fujita, 1987), anti-calbindin (1:300,
Sigma), and anti-CRALBP (1:10,000) (De Leeuw et al., 1990). Anti-rhodopsin staining with Rho4D2 (1:250) (Molday, 1989) was performed as described previously (Ezzeddine and Cepko, 1997). Anti-BrdU staining was performed per the direction of the supplier (Amersham, Arlington Heights, IL). Previous studies detected no mGluR3
RNA or protein in the rat retina (Hartveit et al., 1995; Koulen et al.,
1996). Hence, the antibody was most likely detecting mGluR2 protein.
Specificity of antibody labeling was verified by immunohistochemistry
(data not shown).
Incubation in primary antibody was followed by three PBS rinses and 20 min in blocking solution containing Texas Red-conjugated donkey
anti-mouse or donkey anti-rabbit secondary antibodies (1:200, Jackson
ImmunoResearch, West Grove, PA). Nuclear staining was performed by
adding 4',6-diamidine-2-phenylindole-dihydrochloride (DAPI) to the wash
solution at a final concentration of 0.0005%. Slides were then
processed for autoradiography.
Autoradiography. Cells becoming postmitotic the day of
[3H]thymidine pulse administration were
identified as described previously (Young, 1985). Slides with
[3H]thymidine-labeled cells were dipped
in NTB2 autoradiography emulsion (Eastman Kodak, Rochester, NY). Slides
were exposed for 5 d at 4°C in the dark. Slides were then
developed for 5 min in D19 developer (Kodak), rinsed in distilled
water, and then fixed for 20 min in fixer (Kodak). Slides were washed
with distilled water for 10 min, then mounted in gelvatol. Slides were
examined using a Zeiss Axiophot using a 63× Plan NEOFLUAR objective.
The relative quantity of [3H]thymidine
per cell was estimated based on the number of silver grains, visualized
by light microscopy, inside and around the cell nucleus. Cells
displaying at least n/2 grains (where
n = the number of silver grains in the most heavily
labeled cell) were estimated to be "heavily labeled" and to have
been born on P0 (Morrow et al., 1998). The identity later assumed by
these cells was assessed by scoring individual heavily labeled cells
for expression of various cell type markers.
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RESULTS |
To study the relative roles of extrinsic and intrinsic regulation
of cell fate choices, we used a reaggregate culture protocol that
allowed for the mixing of test cells with a host population, culturing
for >2 weeks to allow for differentiation, and subsequent identification of individual cells from the test population born on a
particular day to assess that cell's fate choice. Reaggregate cultures
from embryonic and postnatal rat retinae have been shown to generate
rod photoreceptors with kinetics similar to those in vivo
(Watanabe and Raff, 1990; Morrow et al., 1998). Furthermore, interneurons, cone photoreceptors, and Müller glia have also been
shown to be generated in this culture system (Watanabe et al., 1997;
Belliveau and Cepko, 1999).
Necessity of the postnatal environment for the rod cell fate choice
but not for regulation of rhodopsin kinetics
The importance of secreted factors in determining how many
postnatal cells differentiate as rods has been shown in
vitro by previous experiments in which P0 cells were placed into
low-density collagen gel cultures (Altshuler and Cepko, 1992). In these
experiments, fewer cells expressed rhodopsin after 7 d in culture,
relative to cells in high density collagen gels or retinal explants
in vitro. If, however, these cells were also provided with
medium conditioned by postnatal retinae, the percentage of cells that expressed rhodopsin increased (Altshuler and Cepko, 1992). When birth-dated E16 cells cultured in the presence of excess postnatal retinal cells were assayed for their expression of rhodopsin, it was
observed that the E16-born cells appeared uninfluenced by the
environmental signals created by an excess of P0 cells with respect to
both the onset of rhodopsin expression and the percentage of such cells
committing to the rod cell fate. These data supported the notion that
the embryonic progenitor cells were intrinsically different from their
postnatal counterparts and could not respond to any factors present in
the postnatal environment (Morrow et al., 1998). These studies did not
distinguish whether there was an inhibitor in the embryonic environment
that, like the postnatal environment, might regulate either the
kinetics or final expression of rhodopsin among E16-born cells. To
examine this possibility, we mixed
[3H]thymidine-labeled P0 retinal
progenitor cells with a 20-fold excess of E16 retinal cells and
cultured them for 3-17 d (Fig. 1). After
culturing, the reaggregates were dissociated and processed immunocytochemically and autoradiographically. Cells were assayed for
the presence of silver grains and anti-rhodopsin labeling. Cells
containing >50% of the maximum grain count were regarded as having
undergone their terminal mitosis immediately after labeling (Morrow et
al., 1998) and were termed "birth-dated" or "heavily labeled."
This subset was selected for analysis. The control reaggregates displayed properties of rhodopsin expression similar to those seen
in vivo; birth-dated E16 cells displayed a lag of >1 week, whereas the birth-dated P0 cells first began to express rhodopsin by
3 d in vitro (DIV). Moreover, the final percentage of
rhodopsin-expressing cells was approximately fivefold higher in P0
control reaggregates cultured in the absence of E16 cells than in E16
control reaggregates cultured in the absence of P0 cells (Fig. 1).
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Figure 1.
Requirement of the postnatal environment for the
rod cell fate but not for the regulation of rhodopsin kinetics.
Reaggregate cultures of E16 ( ), P0 ( ), or P0-E16
(P0:E16, 1:20;  ) were cultured for the indicated
number of days. They were then dissociated and processed for
immunocytochemistry and autoradiography. Birth-dated cells were scored
for their labeling with anti-rhodopsin. Data are expressed as either
the percentage of heavily labeled cells expressing rhodopsin
(A) or as a percentage of the cells expressing
rhodopsin after 17 DIV (B). Values represent the
mean ± SD of three to six trials. For each trial,  100 heavily
labeled cells were assayed.
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When [3H]thymidine-labeled P0 cells were
reaggregated with an excess of unlabeled E16 cells, the percentage of
birth-dated P0 cells expressing rhodopsin was greatly decreased
relative to P0 reaggregate controls at all time points examined. As
shown, 6.3 ± 2.1% of cells initiating their terminal S-phase in
P0 explants expressed rhodopsin after 5 DIV with excess E16 cells,
versus 25.0 ± 5.9% for such cells cultured with an excess of
other P0 cells. After rod differentiation was completed in culture at
17 DIV, 16.0 ± 2.0% of birth-dated P0 progenitor cells expressed rhodopsin when cultured with E16 cells versus 67.0 ± 3.7% when cultured alone (Fig. 1). Although the values were lower, the kinetics of rhodopsin by birth-dated P0 cells appeared unaffected by the embryonic neighbors. When cultured either with 20-fold more E16 cells
or when cultured alone, the first birth-dated, rhodopsin-positive P0
cells were detected after 3 DIV, and the plateau of rhodopsin expression was reached by 7 DIV. In contrast, the first birth-dated, rhodopsin-positive E16 cells were detected after 10 DIV, and the plateau was reached by 15 DIV.
Cells fated to become rod photoreceptors adopt the bipolar cell
fate when cultured in the embryonic environment
What was the fate of the P0 cells that would normally have become
rods? Two strong possibilities were that the cells were becoming either
the predominant fates of embryonically born cells, such as horizontal
cells and/or cone photoreceptors, or that they were adopting an
alternative postnatal cell fate, such as bipolar cells. Another
possible fate was that of the amacrine cell, which is produced both
embryonically and postnatally in the rat (Alexiades and Cepko, 1997;
M. M. LaVail, personal communication). We showed previously that
the postnatal environment contained an activity that inhibited the
genesis of amacrine cells from E16 progenitor cells (Belliveau and
Cepko, 1999). This activity, which appears to be produced by
postmitotic amacrines themselves, could also be serving to regulate the
production of amacrine cells from the P0 progenitor cells. Moreover,
the E16 host age is near the peak of amacrine cell production
(Alexiades and Cepko, 1997; M. M. LaVail, personal communication).
Thus, in the mixed culture, the P0 progenitor cells would be moved out
of an amacrine-inhibiting environment and placed in one that supports
the production of amacrine cells. Bipolar cells, like amacrine cells,
are normal progeny of P0 progenitor cells. When P0 progenitor cells are
placed in low-density collagen gels, or alternatively cultured as
retinal explants in the presence of CNTF, there is both a decrease in rods and an increase in bipolar cells (Altshuler and Cepko, 1992; Ezzeddine et al., 1997). To determine whether there was an increase in
the production of either amacrine or bipolar cells, labeled P0 retinal
progenitor cells were mixed with a 20-fold excess of E16 retinal cells
and cultured for 15 d. After culturing, the reaggregates were
dissociated, processed immunocytochemically and autoradiographically,
and scored for the bipolar and amacrine cell fates, as defined by
labeling with either 115A10 and mGluR6, for the bipolar cell fate, or
VC1.1 and mGluR2, for the amacrine cell fate.
When P0 cells were cultured with a 20-fold excess of E16 cells, there
was an increase in the production of bipolar cells. In the control
reaggregates, 7.6 ± 2.2% and 6.0 ± 1.8% of the heavily
labeled P0 cells were 115A10 positive and mGluR6 positive after 15 DIV
(Fig. 2). In contrast, 26.5 ± 5.2%
and 22.1 ± 6.0% of the birth-dated P0 cells were 115A10 positive
and mGluR6 positive, respectively, when these cells were cultured with
20-fold more E16 cells (Fig. 2). There was no significant
increase in the production of amacrine cells. In the
control reaggregates, 9.5 ± 2.7% and 8.6 ± 2.0% of the
heavily labeled P0 cells were VC1.1 positive and mGluR2 positive,
respectively, after 15 DIV (Fig. 2). In contrast, 11.1 ± 2.8%
and 12.4 ± 2.2% of the birth-dated P0 cells were VC1.1 positive
and mGluR2 positive, respectively, when these cells were cultured with
20-fold more E16 cells (Fig. 2). Similarly, there was no increase in
the production of the fourth major cell type generated postnatally: the
Müller glial cell (Table 1).
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Figure 2.
Alteration of the fate of postmitotic cells
produced by postnatal progenitor cells when cultured with excess
embryonic cells. Reaggregate cultures were maintained for 15 DIV, then
dissociated and processed immunocytochemically and
autoradiographically. Birth-dated cells were scored for labeling with
the indicated antibodies. When P0 cells were cultured alone
(black bars), nearly 70% of the birth-dated cells
became rods. This value was reduced fivefold when
[3H]thymidine-labeled P0 progenitor cells were
reaggregated with 20-fold more E16 retinal cells on day 0 (gray bars) or day 2 (white bars).
Values represent the mean ± SD of three to six trials. For each
trial, 100 heavily labeled cells were assayed. *p < 0.01.
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As mentioned above, another possibility was that when the postnatal
progenitor cells were exposed to an embryonic environment, they
would be induced to adopt the normal fates of embryonically born cells.
Two embryonically born cell types shown to be produced in embryonic
reaggregate cultures are horizontal cells and cone photoreceptors
(Belliveau and Cepko, 1999). When labeled P0 cells were cultured with
20-fold more unlabeled E16 cells for 15 DIV, there was no production of
either of these cell types (Table 1), although they were produced in
control E16 reaggregate cultures cultured similarly (Table 1).
Alteration of fate can occur after terminal mitosis
It was shown previously that cells fated to become rod
photoreceptors could be respecified to the bipolar cell fate with the addition of CNTF (Ezzeddine et al., 1997). This respecification could
occur even after the terminal mitosis of the progenitor cell. Thus,
postmitotic cells born in the postnatal period displayed a degree of
plasticity in their cell fate choice. In contrast, the inhibition of
the E16-born cells from the amacrine cell fate by the postnatal
environment had to occur before the terminal M phase of the E16 cell.
After this time, the E16 cells were refractory to the influence of the
postnatal environment (Belliveau and Cepko, 1999). To determine whether
the E16 environment was capable of respecifying postmitotic cells born
on P0, the following experiment was performed. P0 retinal explants were
labeled with [3H]thymidine as described
previously. These explants were cultured for 2 d, then dissociated
and reaggregated with 20-fold more E16 cells. The delay between
[3H]thymidine labeling and the
subsequent dissociation and reaggregation should be sufficient for all
cells born on P0 to complete their final M phase (Alexiades and Cepko,
1996). Reaggregates were then maintained for 13 DIV, so that the total
length of culture of P0 retinae was 15 d. At this time, the
reaggregates were dissociated and processed for immunocytochemistry and autoradiography.
The decrease in the percentage in rod photoreceptors and the increase
in the percentage in bipolar cells when P0 cells were transferred to
the reaggregate on day 2 were nearly identical to when they were
transferred on day 0 (Fig. 2). These data suggest that, similar to the
application of CNTF, transfer to the E16 environment led to the
respecification of at least a subset of postmitotic cells fated to
become rods.
Reduction in production of rod photoreceptors is caused in part by
inhibitory signals produced by embryonic retinal cells
It has been shown previously that the postnatal retina produces a
factor or factors that are required for the differentiation of rod
photoreceptors. The importance of such factors for the ability of
postnatal cells to differentiate into rods is exemplified by previous
experiments in which P0 cells were placed into low-density collagen gel
cultures (Altshuler and Cepko, 1992). In these experiments, fewer cells
expressed rhodopsin after 7 DIV. If, however, these cells were also
provided with medium conditioned with postnatal retinae, the percentage
of cells expressing rhodopsin increased (Altshuler and Cepko, 1992).
Thus, in the experiments described here, when the P0 cells were
cultured in the embryonic environment, it could be that the
rod-promoting activity was absent. Alternatively, factors that inhibit
rod differentiation in vitro have also been isolated
(Lillien, 1995; Kirsch et al., 1996; Ezzeddine et al., 1997; Neophytou
et al., 1997; Kirsch et al., 1998). These factors could be present in
the embryonic tissue and inhibit rod cell development. To determine
whether the embryonic cells were releasing a soluble inhibitor that
could inhibit a P0 culture that had an environment that produced a
sufficient amount of positively acting cues to support high-level rod
production, we made cocultures of collagen gels. A high density of P0
cells placed in a collagen gel matrix were cultured in either the
presence or absence of E16 cells. E16 cells were placed in an adjacent
collagen gel that ringed the central gel containing the P0 cells (Fig.
3). After 10 DIV, the cells originating
from the P0 retinae in the central gel were examined for their
expression of rhodopsin. The embryonic cells appeared to be secreting
an inhibitory activity (Fig. 3). Of the P0 retinal cells cultured in
the absence of neighboring E16 cells, 44.2 ± 11.2% were
rhodopsin positive. This value decreased approximately threefold to
15.4 ± 5.5% when P0 cells were cultured in the presence of
neighboring E16 cells. These data suggested that E16 cells secrete a
factor or factors that inhibit the adoption of the rod cell fate. A
less likely, but possible, interpretation is that the E16 cells somehow
caused a depletion in the rod-inducing activity made by P0 cells.
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Figure 3.
An inhibitor of rod production is produced by E16
cells, and it signals through a LIFR/gp130 complex. P0 retinae were
dissociated and cast at a density of 8 × 105
cells/40 ml collagen gel. These gels were placed in the center of a
culture well in a defined medium with or without the LIFR antagonist
hLIF-05 (5 µg/ml). The gels were cultured in the presence of a
surrounding collagen gel containing E16 or P0 cells at 4 × 106 cells/200 ml for 10 DIV. No contact between
cells in the two gels was detected. The cells were then removed from
the center gel of P0 cells and processed for immunocytochemistry, and
the percentage of rhodopsin-positive cells was determined. Values
represent the mean ± SD of three trials. For each trial, >300
cells were scored.
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To test whether the inhibitor produced by E16 cells was a member of the
CNTF family of cytokines, an inhibitor of signaling in this pathway was
included in the gel cocultures. The antagonist hLIF-05 binds to LIFR
but has lost affinity for gp130 (Hudson et al., 1996). Oligomerization
and subsequent signaling through these receptors is thus lost.
Antagonism of CNTF, LIF, oncostatin-M, and cardiotrophin-1 by hLIF-05
has been demonstrated, and thus this antagonist can relieve inhibition
produced by any known member of this family of cytokines (Vernallis et
al., 1997). Initially, we tested whether hLIF-05 could block signaling
in the rat because antagonism in this species had not been established.
We found that it could block the rod-inhibiting activity of
exogenous CNTF added to a rat explant culture (S. E. St. Pierre
and C. L. Cepko, unpublished data). The antagonist was thus added
to control P0 gel cultures and to cocultures of P0 and E16 cells. The
percentage of rhodopsin-positive cells among the P0 cells cultured in
the presence of E16 cells and hLIF-05 was 44.4% ± 1.7%, which is
indistinguishable from the control levels of 44.2 ± 11.2% when
P0 cells were cultured in the absence of E16 cells. These data indicate
that the E16 inhibitor requires signaling through the LIFR/gp130
receptor. These data also eliminate the possibility that the decrement
in rhodopsin-positive cells is caused by the depletion of
P0-produced stimulators by neighboring E16 cells.
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DISCUSSION |
In this study we have continued our analysis of the potential of
retinal progenitor cells. Two aspects of retinal development were
examined: extrinsic regulation of cell fate choice and rhodopsin expression kinetics. We showed previously that embryonic rod precursors display a variable lag between their terminal mitosis and expression of
rhodopsin (Morrow et al., 1998). Cells undergoing their terminal mitosis on or after E19 display a fixed lag of ~6 d to rhodopsin expression. Embryonic progenitor cells born before E19 display longer
and variable lags in vivo or when cultured in
vitro. When cultured in the presence of excess postnatal retinal
cells, these cells did not change either their kinetics for rhodopsin
expression or the final percentage of cells that become rods (Morrow et
al., 1998). One interpretation is that the embryonic cells are
intrinsically programmed regarding the kinetics of rhodopsin expression
and the percentage of progenitor cells that can make rods. Here, we performed the inverse experiment and assayed the fate choice and rhodopsin kinetics of P0 retinal progenitor cells when cultured with an
excess of embryonic cells. As in previous reaggregates, there was no
change in the kinetics of rhodopsin expression, again suggesting that
rhodopsin kinetics are controlled intrinsically. This observation
further supports the idea that P0 progenitor cells that give rise to
rods are intrinsically different from the E16 progenitor cells that
give rise to rods. In contrast to our previous study of reaggregates in
which E16 cells were the target of the P0 environment, in the current
study an effect of the E16 environment on rod production by P0 cells
was observed. Many fewer P0-born cells adopted the rod fate. The
reduction in rod photoreceptors was accompanied by an increase in
bipolar cells. There was no significant alteration in the percentage of
two other postnatally born cell types: amacrine cells and Müller
glia. The respecification from the rod fate to the bipolar fate could occur even after the terminal mitosis of the progenitor cell. Moreover,
experiments in which P0 cells and E16 cells were cocultured in a system
that prevented cell-cell contact between the two populations provided
evidence that the observed cell fate switch was caused in part by a
factor or factors released by the embryonic cells. This factor requires
signaling through the LIFR/gp130 receptor complex and thus is likely
a member of the CNTF family of cytokines.
Postmitotic cells fated to become rods can be respecified by
coculturing with embryonic cells
It has been shown previously that there are both positive and
negative factors that are able to regulate the rat rod cell fate
in vitro (Altshuler et al., 1993; Kelley et al., 1994;
Lillien, 1995; Kirsch et al., 1996; Ezzeddine et al., 1997; Levine et
al., 1997; Neophytou et al., 1997; Kirsch et al., 1998). To date,
however, the role of these factors in the development of the retina
remains to be elucidated. Nearly 70% of the cells born on P0 are fated to become rods. When these cells are cocultured with 20-fold more E16
cells, this percentage drops almost fivefold. After examination of the
percentage of each cell type in the rat retina, we found that only one,
the bipolar cell, was increased in the culture described above. From
these data, we conclude that cells fated to become rods were being
respecified to the bipolar cell fate. Interestingly, such a
respecification has been observed previously because of other
manipulations. When P0 retinal cells were cultured in a collagen gel
matrix at a low density, fewer rods and more bipolars were detected
(Altshuler and Cepko, 1992). Similarly, P0 retinal explants cultured in
the presence of CNTF had a decrease in the percentage of rods and an
increased percentage of bipolar cells (Ezzeddine et al., 1997). Taken
together, these data suggest that later in retinal development there
might be a bipotential precursor cell that can adopt either the rod or
the bipolar cell fate and that this choice is regulated by extrinsic cues.
When could the P0 cells be respecified? In cultures in which E16
retinal cells were cocultured with 20-fold more P0 cells, the embryonic
cells could be respecified from the amacrine cell fate to the cone
photoreceptor fate, but only if they were in the new environment before
their terminal M phase (Belliveau and Cepko, 1999). Similar findings
were reported for cortical progenitor cells making laminar fate
decisions (McConnell and Kaznowski, 1991). On transplantation into the
ventricular zone of older animals, early progenitor cells produced
cells respecified to later fates (McConnell, 1988). This
respecification required that the progenitor cells underwent mitosis
within the new environment (McConnell and Kaznowski, 1991). Here, if
the P0 cells were reaggregated with the excess E16 cells after their
terminal M phase, they could still be respecified from the rod cell to
the bipolar cell fate. Similarly, P0 cells could be respecified from
the rod cell to the bipolar cell fate with the addition of CNTF, even
after the P0-born cells had completed their terminal M phase (Ezzeddine et al., 1997). It is interesting that not all retinal cell fates are
being fixed at the same time in the cell cycle.
Reduction in production of rod photoreceptors is caused in part by
inhibitory signals produced by embryonic retinal cells
It has previously been shown that the postnatal retina produces a
factor or factors that are required for the differentiation of rod
photoreceptors. The importance of such factors for the ability of
postnatal cells to differentiate into rods is exemplified by previous
experiments in which P0 cells were placed into low-density collagen gel
cultures (Altshuler and Cepko, 1992). In these experiments, fewer cells
expressed rhodopsin after 7 DIV. If, however, these cells were also
provided with factors produced by postnatal retinae, the percentage of
cells expressing rhodopsin increased (Altshuler and Cepko, 1992). Thus,
in the experiments described here, when the P0 cells were cultured in
the embryonic environment, it could be that the rod-promoting activity
was absent. Alternatively, or additionally, factors that inhibit rod
differentiation could have been present; factors added to retinal
cultures have been shown to inhibit rod development (Lillien, 1995;
Kirsch et al., 1996; Ezzeddine et al., 1997; Neophytou et al., 1997;
Kirsch et al., 1998). However, rod inhibitory factors had not been
shown to be present in embryonic tissue. To determine whether the
embryonic cells were releasing a soluble inhibitor while also providing an environment that produced a sufficient amount of positively acting
cues to support high level rod production, we made cocultures of
collagen gels. A high density of P0 cells placed in a collagen gel
matrix were cultured in either the presence or absence of E16 cells.
The E16 cells were placed in an adjacent collagen gel that ringed the
gel containing the P0 cells (Fig. 3). As a control for overall cell
density, the control culture had P0 cells ringed by a collagen gel with
P0 cells. After 10 DIV, the cells originating from the central gel of
P0 cells were examined for their expression of rhodopsin. The embryonic
cells appeared to be secreting an inhibitory activity, in that
threefold fewer cells adopted the rod fate in the coculture of P0 and
E16 cells (Fig. 3).
To determine whether the inhibitory activity provided by E16 cells was
caused by a member of the CNTF cytokine family, an antagonist of
this family was included in the P0-E16 gel cocultures. The antagonist
hLIF-05 was developed by Hudson et al. (1996) and was shown by
Vernallis et al. (1997) to block signaling through LIFR and gp130.
All known ligands that signal through this receptor have been shown to
suffer from antagonism by hLIF-05. Inclusion of hLIH-05 in the
coculture of E16 and P0 cells completely relieved the inhibition of
opsin-positive cells by E16 cells (Fig. 3). These data indicate that
the drop in rod production in the coculture of P0 cells with E16 cells
is caused at least in part by the production of a factor of the CNTF
family of cytokines by E16 cells.
Failure of the embryonic environment to alter rhodopsin expression
kinetics of postnatal rods
The kinetics of rhodopsin expression appear to be intrinsically
programmed. Previously, we described the relationship between a cell's
terminal mitosis and when it expressed rhodopsin (Morrow et al., 1998).
For the vast majority of rods, those born on or after E19, there was a
fixed lag of ~6 d between exit from the cell cycle and onset of
rhodopsin expression. Cells born before E19, however, exhibited a
variable lag and approximately simultaneous expression of rhodopsin.
Thus the earliest born rods wait the longest time before expressing
rhodopsin. These data were consistent with a possible extrinsic
regulation of rhodopsin expression, attributable to the presence of an
inhibitor in the early retina, or the requirement of a factor in the
later retina. When E16 retinal cells were mixed with 20-fold more P0
cells, they maintained their long delay from their terminal mitosis to
the onset of rhodopsin expression, although the neighboring cells were
beginning to express rhodopsin. The long delay displayed by the
embryonic cells appeared, then, to be intrinsically programmed. It was
still possible, however, that the postnatal environment might contain
an activity required for the 6 d kinetics of rhodopsin expression
exhibited by P0 cells. To examine this, a careful time course of
rhodopsin expression was determined for experimental and control
reaggregate cultures. Although fewer P0-born cells expressed rhodopsin,
the time course of rhodopsin expression remained unaffected by the
surrounding embryonic cells. Thus the kinetics of rhodopsin expression
appears to be intrinsically programmed. The mechanisms that regulate
rhodopsin kinetics are currently unknown but appear to be able to keep
time. A similar intrinsic mechanism has been described for
oligodendrocyte precursors in the rat optic nerve (Temple and Raff,
1986). In this example, accumulation of the cyclin-dependent kinase
inhibitor p27Kip1 is thought to regulate exit from the cell cycle and
subsequent differentiation (Durand et al., 1997, 1998; Gao et al.,
1997). Although it was initially believed that the intrinsic mechanism was counting cell divisions (Temple and Raff, 1986), it now appears that it measures time. The intrinsic mechanism regulating rhodopsin expression also appears to somehow measure time. Indeed, the counting described here appears to take place in both mitotic and postmitotic cells (Fig. 4). The purpose of the time
delay between the terminal mitosis and the expression of rhodopsin is
unclear. For a small population, this delay in the rat can be >2
weeks, and even for the vast majority, the lag is ~6 d (Morrow et
al., 1998). The increase in rhodopsin levels in the rod population
coincides with the formation of rod outer segments. As photoreceptor
outer segments form in the same location where the mitotic cells
undergo M phase, perhaps rhodopsin expression and outer segment
formation are coordinated and delayed until after cell production is
nearly complete. This delay is seen in all species studied (for review,
see Cepko, 1996).
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Figure 4.
A model for regulation of the kinetics of
rhodopsin expression. Retinal progenitor cells (white)
divide to produce a combination of mitotic and postmitotic progeny.
Cells fated to become rod cells (rod precursors; light
gray) are postmitotic. After a delay, the cells begin to
express rhodopsin and form outer segments (dark gray).
For cells born before E19 (A, B), the onset of rhodopsin
appears synchronous. Thus, early-born rod precursors wait longer than
later-born rod precursors before expressing rhodopsin. For cells born
on or after E19 (C), there is a fixed lag of, on
average, ~6 d, although the first rhodopsin-positive cells appear
48-72 hr after the terminal mitosis. The regulation of rhodopsin
expression can be thought of as having two phases. The first phase
lasts up to E19 in the rat and appears to involve a mechanism of
counting that does not depend on cell division and is capable of
keeping a postmitotic cell (A) and a mitotic cell
(B) in synchrony. The second phase lasts ~6 d
and begins either on E19 (for the early cells) or after the cell has
undergone its terminal mitosis. Heterochronic experiments suggest that
the control of timing is cell intrinsic.
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Early retinal environment does not recruit late progenitor cells to
early cell fates
Different cell types are being generated at different times during
retinal development. Because there has been evidence that environmental
signals can influence cell fate choices, it was possible that the early
retinal environment would lead to the adoption of early fates by late
progenitor cells. In fact, we found no early cell types produced by
late progenitor cells placed in an early environment, suggesting that
the cells have lost the competence and/or potential to adopt the early
cell fates. This is in agreement with previous reaggregate experiments,
in which early progenitor cells did not adopt fates of late progenitor cells (Belliveau and Cepko, 1999), and further supports a model in
which retinal progenitor cells pass through phases of competence at
which time they have the capacity to adopt a particular fate or fates.
Through some unknown mechanism, the progenitor cell moves out of that
phase and into a new one. This progression appears to be
unidirectional. Thus, once a cell has passed through a phase, it cannot
return to it.
Another possible explanation of our observations is that there was
differential survival or proliferation between the experimental and
control culture conditions. We find this possibility highly unlikely
for the following reasons. If the decrease in the percentage of rods
when P0 cells were cocultured with 20-fold more E16 cells was the
result of selective rod cell death, then one would expect that the
proportion of all of the other cell types produced by P0 cells would
increase. This would be caused simply by a change in the total number
of P0-born cells because rods are such a significant fraction of
P0-born cells. We did not observe this. Rather, the percentage of two
other cell types, amacrine cells and Müller glia, remained
unchanged between the two conditions. Thus, amacrine cells and
Müller glia, but not bipolar cells, would also have to die in the
same proportions as rods to produce the observed result. We found no
evidence to support this possibility. Similarly, a proliferative event
that would lead to a decrease in rods and an increase in bipolar cells
and no change in the other two cell types would require increased
production of bipolar cells, and, to a lesser extent, of amacrine cells
and Müller glia. Finally, the combination of differential
proliferation and survival, although possible, seems to be quite
unlikely, because it would need to be perfectly balanced to give the
observed results.
Comparison with other regions of the nervous system
The above data suggest that retinal progenitor cells of different
ages have distinct intrinsic biases toward particular cell fates and
that these biases are temporally regulated. Intrinsic differences among
chick retinal progenitor cells of different ages placed in culture have
also been observed (Austin et al., 1995). Although nearly 70% of E4
progenitor cells maintained in low-density collagen gel culture adopted
the ganglion cell fate, only 5% of E7 progenitor cells did so when
cultured similarly. Moreover, when late chick progenitor cells were
transplanted into early developing retina, they did not adopt early
fates, but rather remained restricted to later ones (Fekete et al.,
1990). Similarly, later cortical progenitor cells transplanted into
early ferret ventricular zone did not adopt the early fates but adopted
laminar fates appropriate for their age (Frantz and McConnell, 1996). In contrast, however, late-migrating chick neural crest cells substituted for an early-migrating population behaved like early migrating crest (Baker et al., 1997), suggesting that neural
crest cells do not become progressively restricted. In contrast, Raible and Eisen (1996) showed that late-migrating zebrafish crest cells did
not form neurons, either during normal development or when transplanted
into an early environment. These cells could form neurons, however, if
the early-migrating cells were ablated, suggesting that the
early-migrating cells inhibit the late-migrating cells from adopting
the neuronal fate (Raible and Eisen, 1996).
It appears that during retinal development, changes in both the
progenitor cells and the retinal environment serve to regulate the
production of cell types in the proper ratio and order. Few of the
molecules involved in this coordination have been identified. In
particular, early progenitor cells must be expressing a different set
of genes from later progenitor cells. It appears that at least those
progenitor cells biased to produce amacrine and horizontal cells can be
identified by their precocious expression of two markers of mature
amacrine and horizontal cells (Alexiades and Cepko, 1997), whereas
early chick retinal progenitor cells precociously express a marker of
the earliest born cell type, ganglion cells (Austin et al., 1995).
However, the presumed upstream genes that regulate expression of such
markers in subsets of progenitor cells have not been identified. The
advent of techniques that allow a comprehensive description of the
genes expressed by individual cells make this area of study ripe for investigation.
| |
FOOTNOTES |
Received Sept. 30, 1999; revised Nov. 23, 1999; accepted Dec. 29, 1999.
We are grateful to Drs. J. Hurley, J. Nathans, R. Molday, N. Onada, and
J. Saari for gifts of primary antibodies and to Ann Vernallis and John
Heath for the antagonist hLIF-05. This work was supported by National
Institutes of Health Grant EY08064 (C.L.C.) and a National Science
Foundation predoctoral fellowship (T.Y.).
Correspondence should be addressed to Dr. Constance L. Cepko,
Department of Genetics and Howard Hughes Medical Institute, Harvard
Medical School, 200 Longwood Avenue, Boston, MA 02115. E-mail:
cepko{at}rascal.med.harvard.edu.
| |
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ABSTRACT
INTRODUCTION
