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Note to the Reader Trends in Neuroscience, Vol. 15: 368-373.
Print Friendly The idea that microenvironmental cues acting alone late in development
determine a cell's phenotype has dominated recent discussion of retinal
development and has successfully displaced any role for cell lineage in the
process of cell determination. We argue that there is, in fact, evidence
favoring a degree of lineage restriction during the development of the
vertebrate retina. We propose that environmental factors modulate a process
of progressive lineage restriction. In this model, progenitor cells are
viewed as having unequal potential and their progeny are viewed as being
committed to one of the major retinal cell classes before the stage at which
they become postmitotic. Much progress has been made in the last ten years in developing ways to
mark and analyze descendants of single progenitor cells—cellular clones—in
the vertebrate CNS. A compelling reason to study these families of cells is
that a careful analysis of their size, placement, and cell composition gives
us insight into genetic and developmental mechanisms that generate fully
differentiated cell and tissue types from progenitors that are initially
undifferentiated (1-5). In this article we focus on the retina, an
accessible part of the CNS with a comparatively simple layout that makes it
a particularly favorable tissue in which to explore the relationship between
a cell's lineage, its environment, and its phenotype. In a set of recent studies, progenitor cells in retina have been marked
at different stages of development, using a variety of methods (6-11).
Without exception, these studies have shown that the resulting clones of
retinal cells are tightly interknit clusters of cells that are aligned
radially across the retinal layers (Fig. 1). Whether by design or
happenstance, cells in a clone work together in adult retina, processing
information from the same region of visual space. A menagerie of clone types—dependence on time of labeling When progenitor cells are marked early in development by combining
genetically distinguishable 4- to 8-cell mouse blastocysts into a single
embryo (12), the resulting clones contain a balanced representation of the
major retinal cell types (10). In chimeric mice these clones can be
visualized as beautifully discrete and complete retinal building blocks
(Figure 1a). With minor exceptions, each clone contains the same ratio of
cell types as the retina itself. This result has been confirmed and extended
in a second vertebrate—the African clawed toad, Xenopus laevis. Huang and
Moody (11) injected a fluorescent dye into individual cells at a stage when
the entire embryo is made up of just 32 cells. They found that 10 of these
32 progenitors contribute to the pool of retinal progenitors, and no matter
which of these 10 cells they marked, the resulting clones in retina
contained nearly the same ratio of cell types as the retina as a whole.
These complementary results in mouse and Xenopus demonstrate that early in
development retinal progenitors have equivalent capacity to produce all
major retinal cell types. In this key respect, the pool of progenitors is
uniform. In marked contrast, the structure of clones differs greatly when
progenitor cells are labelled at later stages of retinal development in
these same two species (6, 7, 9). Clones are now extremely variable in their
cellular composition. One dramatic example of the range of variation is
illustrated by clones generated by progenitor cells infected with a
retroviral marker on day 14 of gestation (E14) in the mouse (7). One
progenitor gave rise to a cluster of 33 rods exclusively, whereas another
progenitor in the periphery of the same embryo gave rise to a large clone of
198 rods, 1 cone, 26 bipolar cells, 7 amacrine cells, and 2 Muller glial
cells. A similar profusion of different types and sizes of clones (see Fig.
1B) has been found in Xenopus retinas following injections of heritable
tracers—either horseradish peroxidase or fluorescent dye—during the middle
stage of retinal development (6, 9). Competing hypotheses: Lineage restriction and environmental regulation
There are two different explanations for the transformation from uniform
clones generated by progenitors early in development to highly variable
clones generated by progenitors labelled later in retinal development
(Figure 2). The first explanation is that at some point in retinal
development, an initially uniform pool of progenitors splits up into a
variety of subtypes—each with differing proliferative potential and
differing capabilities to make different types of retinal cells (Fig. 2A).
From this perspective, the clone of 33 rods was produced by an E14
progenitor that was only capable of generating rods. In this cell a genetic
switch instrumental in deciding the fate of all its progeny was presumably
stuck in the rod-only position. This is a possible instance of simple and
complete lineage restriction. The lineage restriction hypothesis views the
variation among clones generated at later stages of retinal development as
being a direct reflection of underlying variation in gene expression among
retinal progenitors—some become totally restricted, others become only
partially restricted, and some may retain their original pluripotence; some
produce large clones via frequent symmetrical divisions, and others produce
small clones via asymmetrical or differential divisions (Fig. 2A). The second and more widely accepted explanation for the bewildering
diversity among retinal clones at later stages of retinal development is
that this diversity is a direct reflection of underlying microenvironmental
heterogeneity (Fig. 2B). This idea was initially based on an exquisitely
detailed analysis of electron microscopic images of cells in embryonic mouse
retina undertaken by Hinds and Hinds (13). They found that the pool of
progenitors appeared homogeneous at the ultrastructural level, even quite
late in development. From this beginning the idea has evolved that
homogeneous progenitors produce postmitotic "blank slate" progeny. These
postmitotic, still uncommitted cells are then assigned a phenotype by
interacting with neighbors that have already been committed and that have
already begun to differentiate (6, 14). In essence, a spatio-temporal
cascade of inductive and inhibitory interactions among cells, both local and
global, is thought to be the key arbitrator of a cell's destiny (14-16).
Environmental differences give rise to the great variety of mixture of cell
types seen in neighboring clones. A corollary is that the large, modular
clones generated by progenitors at early stages of development are composed
of sets of these smaller and highly variable subclones. Lineage and environmental hypotheses lead to different predictions
about clone structure The wealth of data on retinal clones in mouse and frog now makes it
possible to assess the strengths and weaknesses of these two models. If the
potential of progenitors to make different cell types is progressively
restricted, then clones marked at later stages of development should contain
fewer combinations of cell types than either those generated early in
development or those generated by a process that randomly assigns a
phenotype to each member of the clone. Thus, the frequency of clones
containing only a few cell types should be much higher than expected by
chance (Fig. 3). An extreme example of restriction is the clone of 33 rods—a
combination which a random process would generate in the mouse with a
frequency of only 1 in 13,000. The opposite prediction follows from the environmental
hypothesis—progenitor cells are thought to remain fully pluripotent.
Consequently, sets of cells that these progenitors generate (members of a
clone) should include many different cell phenotypes. However, these clones
should not be random sets of different cell phenotypes. The retina is after
all, a highly regular structure (17), and radial arrays contain balanced
ratios of different retinal cell types. The environmental hypothesis holds
that this regularity is achieved by stereotypic patterns of interactions
among neighboring cells, a great many of which are inevitably members of the
same clone (Fig. 1). For example, if one of the first cells in a clone
differentiates as a ganglion cell, this cell will generate environmental
signals that lower the probability that its neighbors—including other
members of its clone—also become ganglion cells. Instead, this young
ganglion cell should signal its neighbors to differentiate as bipolar,
amacrine, or photoreceptor cells. Feedback interactions of this type will
generate a greater diversity of cell types within clones than would a random
process. There are several concrete experimental examples of the ways in
which such mechanisms reestablish a more nearly balanced representation
among cell types in retina following the selective ablation of specific
phenotypes (15, 18). Perhaps the best current example of the numerical and
phenotypic regularity that can be achieved by environmental interactions is
the complex of photoreceptor types in the ommatidium of the fly (19). Here,
a spatio-temporal gradient of cell production coupled to a series of ligand-receptor
interactions between neighboring cells triggers an invariant mosaic of three
different receptor types in ommatidia across the entire eye. A critical assessment using a random model of cell determination
Given these two predictions, a way to test the relative importance of
lineage and environment is to determine whether combinations of cell types
in clones are less diverse than predicted by chance (favoring lineage
restriction) or more diverse than predicted by chance (favoring
environmental regulation). We have tested these predictions using as a starting point the set of
clones published in the landmark study by Turner, Synder, and Cepko (7). We
performed a Monte Carlo simulation in which many thousands of
computer-generated clones were compared to the real data set. To run this
simulation, retinal cells of different types were assigned a selection
probability based on their proportion in the adult mouse retina (Fig. 3).
Adjustments were made to eliminate from consideration cells produced before
the stage at which Turner and colleagues made their retroviral injections
(E13 and E14). Cells were then randomly and repeatedly 'pulled out of a hat'
thereby generating sets of simulated clones that contained precisely the
same numbers of cells as observed by Turner and colleagues. The variety of
cell types in these simulated clones was categorized and compared to the
variety in the set of real clones. The cellular composition of simulated clones is strikingly different from
that of the set of 219 retrovirus-labelled clones (Fig. 3). An average of
20% of the Monte Carlo clones (44 of 219) contain all six of the most
numerous retinal cell types produced after E14. In contrast, only a single
retrovirus-labelled clone contained representatives of each of these six
common cell types. Similarly, 60 Monte Carlo clones contain five different
cell types, whereas fewer than a third as many of the retrovirus-labelled
clones contained five cell types. Yet, one would expect that if the
microenvironment modulates clone structure, then these canonical clones
containing representatives in each cell layer, should be even more common
than predicted by a merely random process. Complementing this first finding,
the simulation also reveals that as the mixture of cell types within clones
is restricted (lower part of Fig. 3), observed numbers of retrovirus-labelled
clones rise substantially above numbers predicted by the simulation. For
example, only 45 of the Monte Carlo clones contain just two or three
different cell types, whereas 126 of these more restricted clones were
observed in the real data set. In addition, many exceedingly improbable
clone types with only one or two cell types, such as all-cone clones, were
found following retroviral injections. Summing this work up, real clones
with low cellular diversity are more common, whereas clones with the high
cellular diversity are much less common than predicted either by a random
process. A similar analysis of retinal clones has also been performed in Xenopus
by Holt and colleagues (6). In this amphibian, 24% of all retinal cells are
photoreceptors, 54% are inner nuclear layer interneurons (predominantly
amacrine and bipolar cells), and the remaining 22% are ganglion cells. They
injected progenitors with HRP at early stages (before the production of an
appreciable number of postmitotic cells), and compared the observed and
expected mixtures of cell types in clones containing two or three cells.
Their chi-square analysis reveals that the observed combinations often
resemble those expected by chance—a finding consistent with neither lineage
restriction nor environmental regulation. However, in scanning their Table
3, one cannot help noticing that the most restricted clone types—in
particular, all-rod clones—are found more frequently than predicted by
chance. Perhaps even more intriguing, only 1 of 23 three-cell clones
contained a member in each of the three cell layers. Yet if inductive and
inhibitory interactions among cells in these tightly intertwined clones
(Fig. 1B) are influential—as they are, for instance, in ommatidia of the
fly—one would expect a much larger percentage of clones with a balanced
representation of cell types across all cell layers. A synthesis From the set of studies in mouse and frog we conclude that lineage
restriction does occur during retinal development, just as it does to a
certain degree both in cortex (see Box 1) and in the optic nerve (4).
Several lines of evidence suggest that decisions are made and biases are
introduced among members of the progenitor pool throughout development. For
instance, in vitro studies by Reh and colleagues (20) have demonstrated that
dividing cells taken from fetal retinas produce an abundance of ganglion
cells, whereas those taken from neonatal retinas and put in an identical in
vitro environment produce an abundance of rods. This work provides
compelling evidence that the average internal state of progenitors shifts
over time, possibly under the influence of changes in retinal environment,
or possibly due to internal changes associated with cell division. Along
with these temporal shifts, Drager and colleagues (21) have recently shown
molecular heterogeneity among progenitors in the mouse at a very early
stage. As early as E9, long before the production of any postmitotic cells,
progenitors in dorsal retina, but not ventral retina, express high levels of
alcohol dehydrogenase activity. We have highlighted findings that suggest lineage restriction plays a
role in retinal development. We have done this to counterbalance a growing
perception that cell phenotype in the CNS is almost entirely under
environmental control acting late in development. In a recent New York Times
article (22), Cepko summarized her group's work as showing that "once the
neurons have settled into a particular neighborhood, they learn what they
are meant to do from signals that surround them, rather than from an innate
genetic program." In the same vein, the title of the paper by Turner, Synder,
and Cepko on retinal clones (7) reads "Lineage-independent determination of
cell type in the embryonic mouse retina." Yet as we have shown in Figure 3,
the paper by Turner and colleagues points to a surprising degree of lineage
restriction. How can such a stark difference of interpretation arise, and
how can it be resolved? Is there a middle ground in which both factors can
be shown to play a role? One problem may be what is meant by "lineage restriction." If restriction
means that each progenitor gives rise to only a single cell type, then, yes,
the data rule out such a process. But as Jacobson and Moody (23) have
suggested, restriction is not necessarily all-or-nothing. Allowance should
be made for the possibility that there are developmental shifts in
probabilities that progenitors will make certain types of cells—restriction
may be lax. What role do we leave the environment? There can be no doubt that
inductive interactions have the most profound influence on cell potential
and phenotype—starting with the earliest interactions between germ layers.
These interactions, examined in depth by Leo Buss in The Evolution of
Individuality (24), are the key to creating multicellular organisms. At this
point, the relevant questions include, Which cells are influenced by their
environment, how, and in what sequence? We think that the environment
targets progenitor cells directly and then, either in a stepwise or graded
manner, restricts their potential. The diversification of retinal
progenitors may begin just after the first contact between the eye vesicle
and overlying ectoderm (21) and may cease only at the last cell division
(25, 26). Based on a quantitative comparison between our clones and
polyclones in chimeric mice and clones and subclones labeled with retrovirus
by Turner et al., we think it is likely that restriction of these
progenitors begins between E11 and E12 (10), roughly concurrent with the
production of the first postmitotic retinal cells (27). The progressive
restriction of progenitors may be fine-tuned by environmental signals. Although one may disagree with our reading of the results, it is
certainly premature to rule out a role for lineage. To critically assess the
role of cell lineage and environment will require in vivo transplantation of
single progenitor cells into host retinas at different stages of development
(5, 28). In vitro experiments, in which progenitors are placed in
well-defined environments, will also continue to help in determining the
rigidity of commitment and in discovering ways to control the state and
output of CNS progenitor cells. Acknowledgments: We thank S. Moody and S. Huang for sharing a draft of
their paper on clones in Xenopus retina before publication. We thank R.
Wetts and M. Luskin and the reviewers for their thoughtful comments.
Supported by the NEI. Selected references Fig. 1. Clones of cells in vertebrate retina. (A) is a cross-section
through the retina of an adult chimeric mouse. These chimeric mice are made
by combining genetically distinct mouse embryos in vitro. The resulting
double-genotype embryos are implanted into psudeo-pregnant mothers and born
normally at term (Ref. 12). The three narrow columns of unlabelled cells are
of Mus caroli genotype, whereas the more extensive heavily labeled
regions are made up of cells that have been labelled with a biotinylated DNA
probe that hybridized with a Mus musculus satellite DNA sequence
(Ref. 12). Cells within the radially-oriented arrays are in many cases
derived from single retinal ancestors. These clonal columns are often
sectioned obliquely, giving rise to clones that appear to be limited to one
layer in single sections (right-most clone in A). In Ref. 10 we discuss the
characterization of clones and polyclones in chimeric tissue. Combinations
of cells in these relatively large and uniformly shaped clones reflect the
underlying structure of the retina itself. (B, C, and D) Clones of retinal
cells in larval Xenopus frog at stage 41 taken from Ref. 6. The three clones
in B, C, and D were marked by Holt and colleagues by injecting single
progenitor cells with HRP at stages 22 to 27. These small clones of cells
contain a wide variety of combinations of different cell types. For example,
(B), left-most clone, contains two amacrine cells and one ganglion cell; (C)
the middle clone contains one cell in each layer; and (D) contains 3 cells,
all of which are photoreceptors In all photographs the photoreceptor layer
or outer nuclear layer (ONL) is at the top, the inner nuclear layer (INL),
is in the middle, and the ganglion cell layer (GCL) is at the bottom.
Calibration bar equals 25 um. (Fig. 1B,C, and D are taken with permission
from Ref. 6.) Box 1. Environment and lineage in the cerebral cortex: Both play a
role. Recent work on the development of the mammalian cortex has, as in the
retina, focused on the role of cell environment and cell lineage in
determining neuronal features that range from phenotypes of single cells to
areal projection patterns (1). Finlay and Slattery (2) initially suggested
that a uniform embryonic cortex differentiates into numerous
cytoarchitectonic divisions via differential cell death that is itself
regulated by ingrowing afferents. By transplanting small pieces of embryonic
rat neocortex to ectopic cortical sites, O'Leary and colleagues (3) have
demonstrated that projection phenotype of cortical cells are influenced by
the local cortical environment. Frost, Sur and their coworkers (4,5) have
shown that cortex is functionally pluripotent—both auditory and
somatosensory cortex can process visual information following early
alterations in cortical environment. Collectively, these studies have led to
the idea that the entire cortex is initially a uniform sheet. The highly
differentiated functional and structural state of adult cortex is thought to
arise gradually under the control of developing neuronal connections. Evidence for intrinsic, lineage-related determination of other cortical
properties, such as laminar destination and cell type, have come from
transplantation and retroviral lineage studies. Barbe and Levitt (6) have
found that neurons from embryonic limbic cortex are committed to expressing
a limbic system antigen even when transplanted into non-limbic neonatal
cortex. McConnell and Kaznowski (7) have found that laminar destiny is
determined during or before the final round of cell division. Finally,
Luskin, Parnavelas and coworkers (8,9) have shown that cortical progenitors
give rise to clones of a single phenotype, i.e., containing pyramidal cells
or interneurons, oligodendrocytes or astroglial. Their work provides support
for the idea that cell lineages are restricted along these phenotypic axes
at least 2-3 cycles before mitotic exhaustion. Cortical neurons are undoubtedly influenced by numerous environmental
cues, but the fact that apparently undifferentiated cells can be altered in
certain respects does not necessarily mean that those cells are unspecified.
Cells and cytoarchitectonic regions have many phenotypic traits, some of
which may be under relatively tight genetic control, while others depend on
environmental cues. Interpretations of results in cortex depend upon the
level of analysis, and to some degree on the willingness of observers to
entertain the idea that cortical development is more complex than we would
like it to be. In the pursuit of trying to find out how a cell in the vertebrate retina
decides what type of cell it's going to be, we and others (1-3) found that
sister cells had fates that could not be predicted by any simple or strict
lineage model. Williams and Goldowitz (4) in their recent TINS perspective
article do not argue this point, but they suggest that in retinal
histogenesis, lineage restrictions might nevertheless play a role, and might
better predict the numercial data on clone constitution than a model based
on cellular or microenvironmental interactions. While they present no
numerical model of their own, and their Monte Carlo simulation-based
challenge of the existing lineage data may be flawed (see the following
letter by C. Cepko), they could
still be right. They start by reminding us that late clones in the mouse
naturally comprise primarily only a few types of cell (rod, bipolars and
Muller glial cells), i.e. the ones that are born later. It seems natural,
therefore, to suspect some kind of lineage restriction takes place. If by
lineage restriction we mean that a cell carries a predisposition (in the
form of nuclear changes, cytoplasmic determinants or cell surface receptors)
that enables it to choose a more limited number of fates than its mother,
this certainly could be consistent with the data. Clearly, however, even
this hypothesis implies that a newly born retinal cell has at least a
repertoire of possibilities (the various late-born types of cell) open to
it, and the choice it eventually makes among these is ungoverned by lineage.
There is, however, another possibility that is consistent with the
predominance of rods, bipolars and Muller cells in late clones of the mouse,
a possibility that Williams and Goldowitz seem not to consider as clearly as
they might. This is that the environment changes with time! The addition of
certain differentiated cells, the release of trophic factors and the
maturation of the extracellular matrices may all influence the possible
fates of a fully pluripotent retinoblast. A decision between two
possibilities: a lineage-related restriction in fate on the one hand, and a
changing microenvironment influencing pluripotential cells on the other,
must therefore be resolved experimentally. A number of recent papers have suggested that the phenotype of a newly
born retinal cell can be influenced by the environment. Embryonic cells from
the rat retina, if removed and co-cultured with an excess of older retinal
cells, give rise to many more rods than they do when cultured alone (5).
Postmitotic chick retinal cells that would have become photoreceptors if
cultured at one time, become mutlipolar neurons if they are allowed to stay
in the retina for another day (6). In Xenopus, cell-cell interactions
seem to be required for photoreceptor determination (7). In the mouse, a
transiently expressed soluble factor can make cells choose a rod fate rather
than what appears to be a bipolar fate (8,9). Single mouse retinal cells
will become rods if they are cultured next to rat rods, but will grow
multipolar neurites and turn on ganglion cell markers if they are plated
next to cortical cells (10). These many examples of demonstrable flexibility
of cell fate cannot go down too well with a model of lineage restrication as
define above. Hartenstein and I (Ref. 11) also showed that various classes
of cell types in all three retinal layers can arise in embryos in which cell
division had been blocked from before the time when retinal histogenesis
normally begins. In these embryos, retinoblasts chose particular fates
without spinning off any of their normal postmitotic progeny. That cells
'deprived of lineage' can choose these various fates is another difficulty
with the lineage restriction idea. I don't want to suggest the Williams-Goldowitz idea has no validity, it
is just that the data that exist now would seem to argue against it, at
least in the retina. Things are clearly somewhat different in the cortex,
where cells seem to be restricted at least to particular laminae in the
S-phase prior to their final mitosis (12). I think we all agree that
cellularly inherited determinants or induced states are clearly an important
part of embryology. The questions facing us now concern the cellular and
molecular bases of the inductions that restrict neuron fate. When these
occur in devleopment may vary from tissue to tissue. In the cortex, the
central glia (13) and the neural crest (14), some restrictions happen while
the cells are still dividing. In the vertebrate retina, it seems that many
of these cell-type decisions happen postmitotically. William A Harris Dept of Biology, University of California at San Diego, La Jolla, CA
92093, USA References Many hypotheses concerning retinal cell fate dtermination have been based
upon the data generated by lineage analyses. When evaluating these
hypotheses, it is important to bear in mind the limitations in the type of
conclusions that can be drawn from studies of lineage. Lineage analysis is a
descriptive technique in which the fate of cells left in situ, rather
than the full potency of cells, is observed. What studies of lineage have
not, and cannot, resolve are the mechanisms underlying the observed clonal
compositions. Only by manipulating the environment can one resolve the
extent to which the autonomous programs of cells and environmental
interactions contribute to development. Every lineage study conducted to date has indicated that the vast
majority of early retinal progenitors are multipotent (Footnote
1). The existence of clones comprising multiple cell types rules out a
model in which each cell type is generated from a mitotic progenitor
committed to making only one cell type. While the finding of multipotency
rules this model out, it is consistent with several other models. As we
hypothesized in our paper on mouse retinal lineage (1), multipotency could
reflect cells responding to environmental cues to become or produce the many
types of daughters that are observed in retrovirally marked clones.
Moreover, complex clones could result from progenitors that are totipotent,
and thus equivalent, throughout development of the retina. Alternatively,
progenitors could change in potency during development. Changes in potency
could be due to environmental influences, autonomous 'programming', or both.
More extensive discussions of these possibilities have already been presentd
(Ref. 1, p. 843, and Ref. 2). The scenarios presented above were offered as hypotheses consistent with
the data, rather than as conclusions, as one cannot distinguish among them
using data generated by lineage analysis. Nonetheless, Williams and
Goldowitz (3) attempted to distinguish among these models on the basis of
lineage data using a Monte Carlo simulation to predict the composistion of
clones marked by retroviral infections. They believe that their analysis
ruled out a model in which environmental interactions direct the choice of
cell fate. However, what their simulation really tested was whether clones
comprised random assortments of cells that could be statistically predicted
by the frequency of each cell type in the adult retina. The failure of
clonal composition to be predicted by their model is due to the fact that
the assumptions of their model are inconsistent with the biology of retinal
development, as discussed below. However, even had their assumptions been
correct, many interpretations of their findings regarding mechanisms would
have been possible, such as variations in environmental influences,
differences in progenitors, or both. An examination of the model used for the Monte Carlo analysis shows that
a critical underlying assumption is inconsistent with what we know about
retina cell generation. By using the frequencies of cell types in the adult
retina to predict probabilities, Williams and Goldowitz made the assumption
that there is no temporal variation in the probabilities that a particular
cell type would be generated. However, retinal cell types are born in a
temporal sequence. This means that the strongest predictor for the fate of a
given cell, regardless of the mechanism by which that fate is achieved, is
the birthday of the cell, not the frequency of cells in the adult retina. An
analysis based upon the frequencies of cell types in the adult retina would
quite predictably lead to clonal compositions that would be more complex
than predicted for a system in which there is birth order. Thus, the result
of the Monte Carlo simulation had to differ from the results of the
retrovirus study regardless of whether commitment was environmentally
influenced. The problem created by the phenomenon of birthdate order is illustrated
by the example of the composition of two-cell clones. According to the
assumptions of Williams and Goldowitz, 70% of the cells in two-cell clones
should be rods, as this is the frequency of rods in the adult retina.
However, the birthdate data would predict that two-cell clones would most
likely be composed of cell types born shortly after infection with a
retrovirus. As predicted by the birthdate data, two-cell clones marked by
retroviral infection at embryonic day 13 (E13) or E14 have no rods or Muller
cells (cell types generated late in development), but do have the predicted
early-generated cell types, all of which are rare in the adult retina. Since lineage data cannot be manipulated in order to distinquish among
modesl of mechanisms, several labroratories, including our own, have
undertaken experiments designed to explore the potency of retinal
progenitors and define mechanisms of determination. The approaches are
varied, including heterochronic transplantation, assays of commitment and
differentiation in vitro, sutdies of the effects of peptide growth
factors, and identification, cloning and functional studies of genes
proposed to act in development. Data generated by such studies will
ultimately reveal what lineage analysis cannot, that is, the ptency of
progenitors at differnt times in development and the mechanisms by which
fate is achieved. Until such data are obtained, arguments about models
regarding mechanisms are not really very productive, and obscure the
interersting story that is currently unfolding regarding teh very resolvable
problem of fate determinaation in the vertebrate retina. Footnote 1. There are clones that comprise rods only. Interpretation of
such clones is difficult as the absence of other cell types in a clone can
be explained by a failure to express the marker used to identify the cell
type or cell death. These problems are particularly vexing in this case as
rods have a very low rate of cell death, non-photoreceptors neurons have an
appreciably higher rate of cell death, and rods are both extremely common
and late born. Constance Cepko Dept of Genetics, Harvard Medical School, 200 Longwood Av, Boston, MA
02115, USA. References We agree with Dr. Harris—environment plays an important role in
developmental decisions made by retinal cells. But the work he cites, while
demonstrating a role for one process (environmental modulation), does not
rule out a role for another process (lineage restriction or bias). As we
concluded in our review (1), this is not an "either-or" situation. Drs.
Harris (2) and Cepko (3) have left us and other readers with the conclusion
that cell type determination is entirely under environmental control and
that the environment targets a homogeneous pool of postmitotic retinal
cells. While many, if not most, retinal progenitors generated a variety of
cell types (5), the fact that clone composition is so variable late in
development (3) suggests to us that the progenitor pool is heterogeneous. In
other words, the potential of progenitors is non-equivalent—these cells
appear to be limited in the range of cell types they generate. There are a few points in Constance Cepko's letter that we wish to
comment on. First she notes that the temporal sequence of cell generation
needs to be taken into account in modeling clone structure. We recognized
this at the outset and therefore excluded from the simulation those cells
known to be generated prior to the retroviral injections (see the legend to
Fig. 3 in Ref. 1). Even considering later born cells, our conclusions remain
valid— there are rather obvious signs of lineage bias in the original
retroviral data. How else can one explain a clone made up of 33 rods? The
progenitor of this clone must have undergone five or more cycles of cell
division and all of the final progenitors deivided to produced nothing but
rods. Can this result really be written off in a foonote as a retroviral
artifact or as the result of highly selective cell death? If so, there are
unpleasant implications for the remainder of the retroviral data set. We
emphasize this clone because it is such a blatant example. However, there
are other signs of lineage bias. For instance, of a total of 70
multicellular clones containing an average of 57.6 cells that were labeled
at embryonic day 13 (E13), only three contain even a single retinal ganglion
cell. Yet ganglion cell production peaks at this stage and continues until
birth (6). This also indicates some form of restriction. Second, we agree with Cepko that small clones, which are probably
generated within days of the injection, would best be modeled separately
using data on cell genesis just after the time of the injection. However,
she draws an overly sharp distinction between early- and late-generated cell
types. Rods are actually generated as early as E13, and by E14 two to three
times as many rods are being generated as cones (Figs 4 and 6 or Ref. 7).
Averaged over the E13-E14 interval, 30% of all postmitotic cells being
produced differentiate as rods, 25% as amacrine cells, 20% as cones, and 20%
as ganglion cells (7,8). Yet of 105 one- and two-cell clones labeled by the
retrovirus, 61 are made up exclusively of cone photoreceptors (3). This
observation strikes us as being consistent with the exhaustion of a discrete
progenitor pool that undergoes its final round of cell division during this
period. We are uncomfortable with Cepko's statement that 'arguments about models
regarding mechanisms are not productive and obscure the interesting story
that is currently unfolding...' The ain of our review was to preserve a bit
of intellectual space for arguments in favor of lineage. Discussion and
dialectics should continue to influence the design of experiments on
mechanisms of cell determination. It would be more productive if these
studies could be interpreted in an environment in which more than one
hypothesis is considered. Finally, Cepko writes in her letter that she and her colleagues presented
'hypotheses consistent with the data, rather than conclusions'. However, the
title of her paper with Turner and Snyder (3), 'Lineage-independent
determination of cell type in the embryonic mouse retina' strikes us as a
conclusion, one that could have heritable effects on the way in which future
cycles of research are carried out. References Since 11 August 98
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