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First published online April 24, 2009
doi: 10.1242/10.1242/dev.027607
Division of Anatomy, Department of Surgery, School of Medicine, University of California, San Diego, La Jolla, CA 92093, USA.
e-mails:
lily-wong{at}ouhsc.edu;
drapaport{at}ucsd.edu
Accepted 5 March 2009
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SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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Key words: Xenopus, Eye, Retinogenesis, Cell fate determination, Cellular competence, 5-Bromodeoxyuridine
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INTRODUCTION |
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SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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Landmark studies of retinal cell lineage were published nearly
simultaneously for the frog and rodent
(Wetts and Fraser, 1988
;
Holt et al., 1988;
Turner and Cepko, 1987;
Turner et al., 1990). These
demonstrated that retinal progenitor cells (RPCs) produced clones that vary
greatly in size and could be composed of any combination of cell types. The
dominant hypothesis accounting for these data was that RPCs produced
uncommitted offspring that were acted upon by signals in the environment and
instructed in what to mature as. Gradual change in environmental signals
during development was invoked to account for the temporal periods of genesis
for each retinal cell type (Harman and
Beazley, 1989; La Vail et al.,
1991; Prada et al.,
1991; Rapaport et al.,
2004). Specifically, RGCs are always the first cell type to be
born, followed by a group including Ho, Am and CPr, and culminating with a
group including RPr, BP and MG.
However, evidence that retinal cell fate is not defined by the environment
began rapidly accumulating. Heterochronic cell mixing experiments (in vitro or
in vivo) showed that raising young RPCs in an older environment (or vice
versa) did not change cell fate acquisition or the schedule of expression of
cell-specific markers (Watanabe and Raff,
1990; Belliveau and Cepko,
1999; Belliveau et al.,
2000; Rapaport et al.,
2001). Clonal density cell culture demonstrated that RPCs from
embryonic day (E) 16-17 rat retinas grown in serum free medium behave
similarly to those in retinal explants
(Cayouette et al., 2003).
Eventually the `induction by environmental cues' model of retinal cell fate
determination evolved to include the property of competence, defining the
ability of a cell to respond to inductive cues, and an intrinsic cell
property.
Numerous molecules have been shown to be involved in retinal cell fate
signaling (Harris, 1997;
Cepko, 1999). However, the
nature of competence and the mechanism of its control is more difficult to
study and largely unknown. Competence appears to function as a permissive
property intrinsic to RPCs that allows them to respond to instructive signals
for cell type production (Cepko et al.,
1996). In such a way Notch-Delta has been shown to keep RPCs in an
undifferentiated, proliferative state
(Austin et al., 1995;
Dorsky et al., 1995;
Dorsky et al., 1997), whereas
agents that antagonize Notch, such as Numb
(Wakamatsu et al., 1999;
French et al., 2002), may be
part of the mechanism instilling competence.
The order of retinal cell production opens a window into the regulation and
limits of cellular competence. Studies in a number of vertebrates
(La Vail et al., 1991;
Rapaport et al., 2004) show
that the birth of cells of each phenotype during retinogenesis is staggered,
demonstrating a temporal, though overlapping sequence. Several models of
cellular competence are consistent with this `order with overlap'. For
instance, the control of competence may be imprecise, even stochastic,
allowing the genesis of any cell type at any time but exhibiting a
developmentally changing bias towards or away from specific types. For
example, at any point in time cell types A, B, C, D and E could be generated,
but early on the probability of A might be high and D low, and vice versa late
in development. We call this the `random model' of competence. In another
scheme, RPCs may pass through `competence periods' to make one or more cell
types. Again, for example, early in development an RPC may be competent to
make cell types A and B, then subsequently move on to make C, D and E. We call
this the `phasic model'. In this model, one would expect shifting of cell
genesis order within a phase (A-B or B-A, or C-D-E or E-C-D, etc.), but not
between phases (never A-C-B, C-B-E, etc.). Finally, the most extreme form of
competence control is where RPCs pass sequentially through periods to make
each cell type separately. In our example, a single RPC would produce cell
type A, then B, C, etc., although each RPC does not necessarily make all cell
types. We call this the `stepwise model'. A strict stepwise model would fit
data that show a sequence of genesis with the ordinal position of each
phenotype invariant. Both the random and phasic models require RPCs to possess
multiple competence states simultaneously, whereas the stepwise model suggests
that an RPC adopts a single competence state at a given time to allow the
production of a specific cell type.
Besides ordered cell genesis, these models make different predictions concerning the stage of cell production of individual RPCs at a given developmental stage. If the development of the population is synchronous, most RPCs would be expected to undergo symmetric mitotic divisions at early stages, then all transition around the same time to asymmetric mitoses as daughters leave the cell cycle, and eventually end with terminal divisions at a late embryonic stage. Both the random and phasic models predict synchrony between the developmental staging of RPCs, but the stepwise model is compatible with asynchrony.
To determine the synchrony of cell production, and whether it is
sequential, we studied cell genesis in the frog, Xenopus laevis,
whose retinogenesis completes in 48 hours. Prior attempts to establish ordered
retinal cell birth in this species (Holt
et al., 1988; Stiemke and
Hollyfield, 1995) were unsuccessful. Given these two facts,
Xenopus presents a rigorous test of ordered retinogenesis, a
challenge that we sought to address by combining lineage and cell birthday
markers allowing us to look at the level of resolution of single RPCs. We
demonstrate that the population of Xenopus RPCs is heterogeneous, and
they generate all cell types at almost all periods of retinogenesis. We also
show an ordered and regular sequence of cell production: RGC, Ho, CPr, RPr,
Am, BP, MG. Although not all cell types are present in each clone, in each one
the cells display these ordinal relations. These findings support the model in
which RPCs follow an intrinsic program with stepwise changes in competence to
produce one cell type at a time in a conserved order.
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MATERIALS AND METHODS |
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SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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).
The jelly coat and vitelline membrane of embryos were removed manually at
stages 17-19. All animals were held and treated experimentally according to
University of California and NIH guidelines.
Labeling clones
A 0.7 kb fragment of gfp3 was excised from pCS2-nls-gfp3
(provided by S. Evans) using Xho1 and Xba1. This fragment
was subcloned into vector, pCS2-ITR [provided by S. Evans
(Fu et al., 1998)] to create
pCS2-gfp3-ITR. This expression vector containing the ITR sequences of
AAV has been demonstrated to allow even segregation of plasmids among
daughters. GFP was detected throughout the cytoplasm, including all cell
processes. We transfected pCS2-gfp3-ITR DNA mixed, 1:3 by weight, DNA
with the lipofectant, DOTAP (Roche). Approximately 65 pl of this mix was
injected with a micropipette connected to a Picospritzer pneumatic system
(General Valve Corporation, Fairfield, NJ, USA). Stage 20-21 embryos were
injected (Fig. 4A), and two
distant sites per vesicle were targeted, with two injections performed per
site. The lipofection technique has been shown to produce lengthy, stable
expression (Holt et al.,
1990), well beyond the 55 hours of this study, so dilution of the
lineage tag is unlikely to be a problem. This was confirmed by the presence of
lineage-labeled profiles in large clones that were BrdU+ (62.5%)
from a late stage of BrdU injection. In such cases these would be expected to
have undergone the highest number of cell divisions and to have most likely
diluted the lineage tracer.
Labeling mitotic cells
At early, middle or late stages of retinal development
(Fig. 4A),
gfp-transfected embryos were injected with three 15 nl pulses of BrdU
(5 mg/ml). We targeted the abdomen, which early in development is primarily
composed of yolk. BrdU only slowly becomes cleared from here providing
cumulative labeling. This was verified by ensuring that the ciliary marginal
zone, which proliferates throughout the life of the tadpole/frog, was entirely
labeled with BrdU, indicating no dilution. At analysis (stage 41),
BrdU– and BrdU+ cells represent those that were
post-mitotic and mitotic, respectively, at the time of injection.
Histology and immunohistochemistry
At stage 41, tadpoles were anesthetized and then fixed in 4%
paraformaldehyde in 0.1 M phosphate buffer (pH 7.4) overnight at 4°C. They
were then cryoprotected in 30% sucrose for at least an hour before 10 µm
coronal sections were cut through the eyes. Sections were screened for GFP
fluorescence using a Nikon SMZ1000 fluorescence stereoscope. If at least one
section demonstrated GFP+ profile(s) all sections from that case
were immunolabeled. Sections from all other retinas were excluded from further
consideration.
To immunostain incorporated BrdU, we denatured DNA with heat. Slides were
immersed in 250 ml of 10 mM sodium citrate buffer (pH 6.0) and heated in a
microwave oven (700 W) for 3 minutes. They remained in the hot buffer for 5
minutes, and were microwaved again. Slides then cooled for 30 minutes. The
sections were immunostained with antibodies to GFP (rabbit polyclonal,
Molecular Probes, 1:1500), BrdU (mouse monoclonal IgG1, Becton Dickinson,
1:15) and rhodopsin [4D2, mouse monoclonal IgG2b, 1:100, gift of R. Molday
(Molday and Mackenzie, 1983)].
Secondary antibodies were, respectively, goat anti-rabbit Alexa-594 (Molecular
Probes, 1:500, red), goat anti-mouse FITC (IgG1, Southern Biotechnology,
1:150, green), and goat anti-mouse Alexa-350 (IgG2b, Molecular Probes, 1:150,
blue).
Data collection and analysis
Sections were imaged with a Nikon E800 epifluorescence microscope; images
were captured with an Apogee KX85 CCD camera and analyzed using ImagePro
software (Media Cybernetics). All data were collected from the mature, central
retina. Approximately 50% of transfected eyes contained GFP-labeled cells that
were clustered. A cluster was often confined to one section and seldom spanned
more than two to three. To be considered a clone, a GFP+ cell
cluster must be isolated by at least seven cell diameters from any other
GFP+ cell(s). Retinal phenotypes were identified by morphology
(Fig. 1). RPr were
distinguished by anti-rhodopsin (4D2) immunoreactivity. Each section was first
examined for GFP+ cells, to identify clones. Once a clone was
defined, the phenotype of each cell was determined. Finally, the BrdU-labeling
status of each constituent was determined. The occasional complex clone
containing overlapping cells was examined with a BioRad MRC 1024 confocal
microscope and the information used to aid in cell classification.
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SUMMARY
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MATERIALS AND METHODS
RESULTS
DISCUSSION
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;
Jeon et al., 1998), we were
able to reliably identify cell types based on the laminar and sub-laminar
location of their cell bodies and the disposition of processes. Examples of
the seven major retinal cell types are shown in
Fig. 1. The inner nuclear layer
(INL) contains four phenotypes: Am, Ho, BP and MG
(Fig. 1B-E). The somas of Am
(Fig. 1C) and Ho
(Fig. 1E) are on opposite sides
of the INL, with processes that extend laterally into the inner plexiform
(IPL) and outer plexiform layers (OPL), respectively. BP
(Fig. 1D) and MG
(Fig. 1B) both extend radial
processes, but can be reliably distinguished because the former terminate in
the plexiform layers whereas the latter extend beyond, to the outer and inner
limiting membranes. RGCs (Fig.
1A) are easily identified by their cell bodies in the ganglion
cell layer (GCL), their processes in the IPL and their single axons extending
into the nerve fiber layer. The one distinction that could not be based on
morphology is between RPr and CPr (Fig.
1F,G). For this, we used an antibody, 4D2 (kindly provided by R.
Molday), that labels RPr only. As the somas of photoreceptors are confined to
the outer nuclear layer (ONL), RPr and CPr are readily distinguished by 4D2
immunoreactivity (RPr) (Fig.
1F), or its absence (CPr) (Fig.
1G). Experiments on dissociated retinal cells from stage 41
tadpoles confirm no cross-reactivity of this antibody with a cone-specific
marker (Chang and Harris, 1998)
and give an expected 1:1 ratio of RPr to CPr (data not shown).
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), although there was seldom any doubt. The gfp-DOTAP injection dosage was titrated, seeking that which gave a high number of single clusters, and few with more than one cluster, per eye. At this dose 30% of injected embryos had no discernible GFP fluorescence in either eye. We eventually analyzed 111 tadpoles that demonstrated GFP fluorescence in at least one eye. Fig. 3A shows the frequency of cell clusters per eye. Of the 222 optic vesicles transfected, 134 (60%) eyes had 162 GFP-labeled cell clusters. Of the retinas with labeled cells, the majority contained only one cluster (Fig. 3A). Both the infrequent occurrence of eyes with labeled cells and the low number of clusters per eye indicated that the dose used was on the cusp between allowing or not allowing a transfection `hit' and, when hit, a single occurrence was the predominant outcome.
To further demonstrate that the labeled cell clusters were clones, we
compared the size distribution that we obtained with that using a more
traditional technique of lineage tracing, single cell injection
(Holt et al., 1988). The
distribution of clone sizes (Fig.
3B) was very similar across techniques. Each demonstrated a median
cluster size of three cells, and a positive skew
(Fig. 3B). Based on many lines
of evidence, we conclude that the GFP-labeled cell clusters in this study can
justifiably be regarded as clones.
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(middle) and the mitotic index [bottom, modified from Holt et al.
(Holt et al., 1988)]. The proportion of BrdU+/GFP+ cells showed a gradual decrease from early (62%) through middle (41%) to late periods of retinogenesis (31%) (Table 1; Fig. 4B) (`all cell types' histogram). However, while a large proportion [45% (22/49)] of clones contained exclusively BrdU+ constituents early in retinogenesis (Table 2), 31% (15/49) were composed exclusively of BrdU– profiles, and 24% (12/49) had mixed BrdU+/BrdU– labeling. These data indicate that at this early period about half of RPCs had yet to begin to produce post-mitotic daughters, whereas one-third had already reached the end of their proliferative life. Similarly, during the middle period, 19% of clones had yet to produce a post-mitotic daughter, whereas 49% had generated all their progeny. Even at the late period, we found that 7% of clones were composed of all BrdU+ profiles, even though 55% were mitotically arrested (BrdU–). These data indicate that RPCs at early, middle and late developmental stages coexist at all stages of retinogenesis.
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;
Rapaport et al., 2004). To
determine whether the significant overlap in the timing of retinal cell birth
of different cell types in Xenopus is genuine or reflects
heterogeneity of the developmental schedule of each RPC that may have masked a
sequence, we analyzed the birth order within individual RPCs.
Clonal analysis reveals that retinal cell genesis follows a common sequence
Fig. 5 shows a graphical
display of 61 RPC clones. Each clone is represented by a row, with the boxes
representing each cell type. If a box has a circle in it, it means that the
clone has at least one cell of that phenotype, and if the circle is white it
means that all of them are BrdU+. A black circle means all clone
members of that phenotype are BrdU–. Finally, a half-black
half-white circle indicates more than one cell of that phenotype in the clone,
some BrdU–, some BrdU+. The number of
BrdU– and BrdU+ cells, if greater than one, is
indicated to the left and right of the circle, respectively.
Five of the clones shown contain exclusively BrdU+ cells (top), and five BrdU– cells (bottom). These were randomly selected from the larger cohort with these labeling patterns. (For a complete list of clones, BrdU injection times and other details, see Table S1 in the supplementary material.) Between them are all 51 heterogeneous clones, those that contain BrdU+ and BrdU– cells. These clones provide information on the order of genesis of retinal cell types. Take, for example, clone #90 (asterisk), which is illustrated in Fig. 2G. In this five-cell clone an RGC and an RPr are BrdU–, while an Am and two BP are BrdU+, indicating that the RGC and RPr were post-mitotic, whereas the RPC, at the time of BrdU administration (stage 31), had yet to produce the Am and BP. Similarly, in clone #146 (asterisk; shown in Fig. 2E), the two RPr, both BrdU–, were born before the Am and the BP, both of which are BrdU+. Each clone provides a `snapshot' of the ordinal relationships between two or more cell types. As we consider more and more clones, the full picture of the cell birth relationships in the frog retina becomes clear, and tells an unambiguous story, that a sequence of genesis is apparent at the clonal level of resolution. If a clone has one or more BrdU+ RGCs, then all other clone members are BrdU+ (10 of 10 clones) (see Table S1 in the supplementary material). Similarly, if one or more MG is BrdU–, then all other clone members are as well (12 of 13 clones, see Table S1 in the supplementary material; exception, clone #148). These data indicate that RGCs are the first cells in the frog retina to be generated, and MG the last. The columns, which represent each cell type (Fig. 5) are arranged, and produce a pattern whereby white circles transition to black circles, sweeping from the superior right corner to the inferior left. If the cell birth relations between individuals within a clone were random, then no matter how we arranged the rows or columns we would not be able to achieve a smooth transition from BrdU+ to BrdU– cells. The pattern allows us to assign ordinal relationships between cell types. For example, 17 heterogeneous clones containing at least one BrdU+ Am had BrdU– RGC (6), Ho (5), CPr (2) and RPr (5), but all the BP (6) and MG (3) were BrdU+. This indicates that Am are generated between these two groups. Performed for all heterogeneous clones, this analysis allows us to recognize the ordinal birth relationships between each phenotype. Specifically RGC is always the first cell type to become post-mitotic, followed by Ho, then CPr, then RPr, then Am, then BP, and finally MG.
Among the 51 clones with heterogeneous BrdU labeling, there were only four with ordinal relations that did not fit this sequence (arrows, Fig. 5). In clone #94, at the time of BrdU administration, the Ho was not yet born (BrdU+), but the CPr had been generated (BrdU–). Clones #59 and #44 contained more than one occurrence of two ordinally adjacent cell types, some BrdU+ and some BrdU–. In these cases at least one Am was post-mitotic when at least one RPr was still mitotic. Finally, in clone #148, three out of five MG were generated (BrdU–) before the single BP (BrdU+). In all four of these clones, all other cell types displayed BrdU labeling consistent with the overall sequence of genesis. It is possible that these few outliers indicate some variability in the order of genesis. However, such variation would seem to be very limited as it is always between adjacent phenotypes in the cell birth sequence. Although they need to be explored further, the rarity of `out-of-order' events makes them difficult to study. Their occurrence should not be the cause for rejecting the robust finding that the great majority of observations indicate that cell genesis order in the frog retina is regular, rigid and unidirectional.
The rigidity of cell birth sequence is further demonstrated in a
quantitative analysis. We made pairwise comparisons between two cell types
under the condition: when one was 100% BrdU– (reference cell
type) and the other 100% BrdU+ (comparison cell type). The results
are presented in a matrix (Fig.
6). The diagonal black-filled rectangles give the number of clones
with all individuals of that (reference) phenotype BrdU–. The
other rectangles in the row give the quantity of the reference clones in which
all members of that type (comparison) are BrdU+. For example, there
are 49 clones that contain exclusively BrdU– RGCs. Of these,
five contain all BrdU+ Ho, five all BrdU+ CPr, and so
on. This shows that all six non-RGC cell types are born after RGCs, and this
is the only cell type for which this can be said. In another example, there
are 41 clones containing exclusively BrdU– RPr. In these
there are no RGC, Ho or CPr that are BrdU+, indicating that these
cell types were born before or at the same time as RPr. However, these clones
do contain Am, BP and MG that are BrdU+. These cell types must be
born after RPr. For some pairwise comparisons the numbers are small:
specifically, those between CPr and RPr, and CPr and Am, are limited to only
one example each. However, the fact that these comparisons fit so well in the
overall context based on many more pairwise comparisons gives us confidence in
them. The `Totals' in the right and left columns indicate for each reference
phenotype the number of comparisons that fit and do not fit, respectively, our
cell production sequence. Of 89 comparisons, we find only a single incongruent
case (clone #94). 
2 tests comparing these data to what would
be expected if there were no order to cell genesis were significant at
P<0.01 or less for all rows, except the BP-MG comparison, which
failed to reach this significance level, probably because of the small number
of these pairs. However, when the reversed matrix was considered (reference
cell type all BrdU+, and comparison cell type all
BrdU–; data not shown), the sequential relationship between
BP-MG was highly significant (P<0.001). 2 tests on
paired cell types with ordinal relations (i.e. RGC-Ho, Ho-CPr, etc.), that
further account for clone size, were all significantly different from random
ordering at the P<0.01 level. Both graphical and quantitative
analyses demonstrate that retinal cell genesis in the frog follows a regular
and consistent order within clones.
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DISCUSSION |
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SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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)
(Fig. 3B). Many factors
influence clone size, including the length of time that RPCs remain
proliferative, cell cycle timing and mode of cell division. Given that the
proliferative life of a Xenopus RPC is maximally 48 hours, and cell
cycle time is about 12 hours (Rapaport,
2006), 1-16 cells per clone would be expected.
As in previous studies (Wetts and
Fraser, 1988; Holt et al.,
1988; Turner and Cepko,
1987; Turner et al.,
1990), we had a number of one-cell clones, which raises the
question as to how they are produced. Do they reflect a technical limitation?
Their ubiquity suggests otherwise. The only way to produce a one-cell clone
involves apoptosis, and the simplest mechanism is either for an RPC to die
after it produces a single post-mitotic daughter, or for one of a pair of
post-mitotic sisters to die. Cell death is a feature of the developing retina,
including RPCs (Voyvodic et al.,
1995). Despite its occurrence, apoptosis is unlikely to affect the
conclusions of this study. For it to produce apparent cell birth order, or to
vary the sequence, it must be targeted to specific phenotypes at specific
times, and no evidence for such `directed' cell death has ever been seen in
the retinas.
We see, as others have, many two-cell clones. These are significant because
they could represent daughters of a symmetric, terminal mitosis that, if RPCs
pass sequentially through single competence states, might be expected to be
composed of two cells of the same phenotype. However, this was clearly not the
case (see Fig. S1 and Table S1 in the supplementary material). Rather, by far
the most common pairing in two-cell clones is one cell along on the sequence
of cell genesis. Cell death may contribute to formation of heterogeneous
two-cell clones. However, these data are also consistent with the finding that
not all retinal cells are determined and/or committed at the time they leave
the cell cycle (Belecky-Adams et al.,
1996). It follows that competence extends into post-mitotic stages
– so long as the post-mitotic cell is undetermined, the competence clock
continues to run. Thus a heterogeneous two-cell clone could arise by a
terminal division in which one daughter is determined at cell division
(perhaps by asymmetric segregation of cellular components) and the other
sometime later while in a subsequent competence state. The fact that two-cell
clones are very commonly composed of two phenotypes that are immediate
neighbors in the sequence of genesis (see Fig. S1 in the supplementary
material) supports this idea.
Confirming the findings of others
(Wetts and Fraser, 1988;
Holt et al., 1988;
Turner and Cepko, 1987;
Turner et al., 1990), we find
that the output of RPCs is highly variable. Clones containing a variety of
combinations of two or more phenotypes are observed
(Fig. 5; see Tables S1 and S2
in the supplementary material) supporting the idea that, as a population, RPCs
are not restricted or limited in their ability to produce any cell type(s) at
the onset of retinogenesis. However, cell type composition within clones is
not altogether random (Alexiades and Cepko,
1997). Similarly, the cell type associations within our clones
demonstrate some preferential pairings (see Table S2 in the supplementary
material). For example, clones that contain RPr will also tend to include Am,
and clones containing RGCs have a lower probability of including MG. We are
not certain of the cause of high- and low-probability associations, but we
suggest that the explanation may include factors such as the proximity of
cells in the sequence of genesis, the proportion in the retinal population,
and exposure to environmental cues that may allow or prohibit the production
of cell type(s).
A significant and surprising heterogeneity of RPCs is seen in their
developmental staging. RPCs at the same embryonic age can be on very different
developmental schedules, even at extremes of their proliferative capacity
(Table 2). Thus, a clone of all
BrdU+ constituents can neighbor one with all BrdU–
members. This heterogeneity probably accounts for the failure of earlier
studies to detect sequential cell genesis in the frog
(Holt et al., 1988;
Stiemke and Hollyfield, 1995).
These studies analyzed the population of retinal cells, which does not provide
the resolution to reveal a sequence against the `noise' of the variability in
developmental timing we herein observe. Further, heterogeneity of RPC timing
suggests that the environment in which they grow does not determine their
schedule. Rather, it supports the hypothesis that the temporal shifting of
cellular competence is governed by a program intrinsic to each RPC. Finally,
given the heterogeneity of RPCs on many parameters, it is perhaps not
surprising that they also exhibit significant heterogeneity in gene expression
(Trimarchi et al., 2008). The
challenge will be to relate gene expression differences to different
properties of RPCs and the clones they produce.
In a retinal clone certain phenotypes are always generated before others
Clones made up of a mixture of BrdU– and BrdU+
cells capture a `snapshot' of the genesis relationship(s) between these
phenotypes. These snapshots indicate that cells in a clone are not generated
in a stochastic order. Were this the case, a two-cell clone of heterogeneous
BrdU labeling would just as likely have one cell BrdU+ as the
other. However, we observed that the potential identity of BrdU+
profile(s) of such a pair was limited by the identity of the
BrdU– profile. For example, six clones that had at least one
BrdU– Am had BrdU–, but not BrdU+
RPr. However, 11 clones with at least one BrdU– Am (some the
same clones) had BrdU+, but not BrdU– BP. In these
cases, Am are generated after RPr, but before BP. No case exhibiting different
ordinal relationships between these three cell types was seen.
The sequence of retinal cell genesis can be derived from the ordinal relationships within clones
Whereas clones give a `snapshot' of the ordinal relationships of genesis
between cell types, the totality of these pictures provides a coherent `movie'
of the entire retinal population. Specifically, our data establish that
retinal cell genesis follows a defined sequence:
RGC
HoCPrRPrAmBPMG. This sequence is rigid
in that there is almost no variation in any of these ordinal relationships.
Further, although cell genesis is dynamic and temporally shifting, it shifts
only in one direction. Once an RPC generates a phenotype different from those
previously produced it never goes back to generate the earlier cell types
again. This finding validates an earlier suggestion that competence shifts
unidirectionally (Cepko et al.,
1996). These data do not accord with a model whereby RPCs possess
multiple competence states simultaneously to form some subset of cell types.
Were this model to operate one would expect consistent ordinal relationships
between some cell types, but not between others. For example, if the phases of
retinal cell birth reported in a number of vertebrates
(Harman and Beazley, 1989;
La Vail et al., 1991;
Rapaport et al., 2004)
represent competency periods for subsets of cell types, we would have expected
to see variable ordinal relations within, but invariant relations between,
phases. We always observe rigid ordinal relationships and this accords only
with the stepwise model for shifting competence states of RPCs, a cell type at
a time.
The order of cell genesis in Xenopus retina apparent at the clonal
level is very similar to that of other vertebrates [wallaby
(Harman and Beazley, 1989);
monkey (La Vail et al., 1991);
rat (Rapaport et al., 2004)].
The only difference is that RPr are generated in close step with CPr in
Xenopus, whereas in the wallaby, rat and non-foveal region of monkey
retina, they are generated late, after Am. We collected only two heterogeneous
clones (94 and 138) that directly compared RPr and CPr
(Fig. 5). This low number
probably reflects the fact that these cell types are generated in close step
so that there is a very narrow window for BrdU administration to reveal the
birth order between these two cell types. When we administered BrdU at stage
22, and identified unlabeled (post-mitotic) cells in the ONL at stage 41, we
found unlabeled CPr in a 4:1 ratio with unlabeled RPr (unpublished
observation). This is consistent with Chang and Harris's
(Chang and Harris, 1998) report
that, despite significant overlap in the genesis periods of RPr and CPr in
Xenopus, the first CPr were born before the first RPr. In the
cone-dominated chick retina (60% CPr, 40% RPr), CPr and RPr were generated
before cells found in the INL
(Belecky-Adams et al., 1996).
The addition of an amphibian to the database of retinal cell genesis sequences
adds weight to the observation that they are highly similar in all
vertebrates, possibly conserved during evolution. However, the relatively
minor interspecies differences in ordinal position, particularly for CPr and
RPr, may reflect adaptation of cell production to lifestyle demands of the
species. The more RPr to be made, the later in development the peak of RPr
genesis occurs. Even though we do not think the demand on cell production in
terms of numbers affects the rigidity of their production, demonstration of a
rigid cell genesis sequence in other vertebrates will substantiate the
hypothesis that mechanism(s) for regulating cellular competence in retinal
development is conserved.
|
;
Zhu et al., 2006). Four
transcription factors are expressed in rigid sequence
(HunchbackKrüppelPdmCastor) as embryonic neuroblasts
produce daughters that can either commit to a specific neuronal lineage or
continue to divide. Indeed, a single division can give both outcomes – a
commitment with the daughter maintaining expression of the transcription
factor, becoming determined and differentiating, or a progression with the
other daughter remaining in the cell cycle, shifting expression to the next
transcription factor and establishing a unique lineage of different cell
types. In development of post-embryonic Drosophila brain, competence
of neuroblasts appears to be governed by the concentration of another
transcription factor, Chinmo. High levels lead to the production of neuron
types that are born early, whereas low to medium levels lead to late-born
types. As these transcription factors can regulate cell fate determination
temporally without specifying particular fate(s) they are thought to be
`competency genes'. Despite their identification in Drosophila, the
regulation of their activity is still poorly understood. Nevertheless, the
rigid sequential formation of cell types that is produced by regulating
competency is very similar to what we see in the frog retina. The molecular
mechanism(s) for competence regulation in invertebrate neurogenesis may be
conserved in the vertebrate CNS. An ortholog of the gene hunchback, a
player in the competence cascade in Drosophila neurogenesis, called
ikaros (IKAROS family zinc finger 1 – Mouse Genome
Informatics), has been shown to be necessary and sufficient to produce
early-born cell types (RGC, Ho and Am) in the developing mouse retina
(Elliott et al., 2008).
We do not yet understand the mechanism for maintaining and changing retinal
cell competence. However, we know that it does not involve counting cell
divisions because embryos in which cell division was inhibited during
retinogenesis form at least some cell types in the retina
(Harris and Hartenstein,
1991). This observation supports the presence of a clock or
program for shifting competence in post-mitotic neurons before determination
and differentiation. We speculate that cellular activities such as protein
and/or RNA accumulation or degradation may be a mechanism for shifting
competence. This is seen in rat optic nerve oligodendrocytes, in which an
intrinsic clock counts time (not cell divisions) to differentiation
(Gao et al., 1997), and the
concentration of the cyclin-dependent kinase p27 (Kip1) is likely to be part
of the counting mechanism (Raff,
2007).
We propose a model in which retinal cell competence regulates cell fate
determination. Fig. 7 depicts a
pool of heterogeneous RPCs, each at a unique developmental stage that cannot
be predicted by its neighbors, but partly defines its competence state. The
shape of the RPC indicates their developmental stage: circular, yet to make
post-mitotic daughters; oval, producing post-mitotic daughters. The temporal
switching of cellular competence is intrinsic to RPCs and their undetermined
post-mitotic daughters, and exerts control over the fate of these cells. This
does not imply that external environmental cues are uninvolved. They may act
as permissive signals that allow or prohibit the production of a cell type and
may control the number of each type produced. For example, extrinsic signals
may affect the mode of cell division (Mu
et al., 2005; Poggi et al.,
2005). The presence or absence of differentiating factors or
inhibitors can regulate the timing of commitment and/or differentiation
(Cepko et al., 1996;
Zhang and Yang, 2001;
Kay et al., 2005). Finally,
the duration of a competence state can be modulated during retinal development
(Kim et al., 2005). In our
model (Fig. 7), intrinsic
cellular competence acts as an instructive signal, whereas external
environmental signals act as permissive signals for retinal cell type
specification and production. An RPC may be multipotent over its proliferative
life, but is competent to make only a single cell type at a given time.
|
Footnotes |
|---|
Supplementary material for this article is available at http://dev.biologists.org/cgi/content/full/136/10/1707/DC1
* Present address: Department of Ophthalmology, College of Medicine,
University of Oklahoma, Health Sciences Center, and Dean A. McGee Eye
Institute, Oklahoma City, OK 73134, USA 
We thank R. Dorsky and W. Harris for their involvement at the inception of this study and for advice during execution and analysis. We thank R. Molday for the rhodopsin antibody and Sylvia Evans for the pCS2-ITR vector and gfp3 cDNA. We are appreciative of members of the Rapaport lab for technical support, and personnel at the NEI Core Grant for Vision Science (P30 EY12598-02) for confocal training and use. D.H.R. was funded by NEI EY11875. Deposited in PMC for release after 12 months.
|
REFERENCES |
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|
TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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