Note to the Reader R.R. Mize and R.S. Erzurumlu (Eds.) Progress is Brain Research, Vol. 108 1996 Elsevier Science B.V. All rights reserved.



Clonal Architecture of the Mouse Retina

Dan Goldowitz, Dennis S. Rice and Robert W. Williams
Center for Neuroscience and Department of Anatoiny and Neurobiology, University of Tennessee, 855 Monroe Avenue, Memphis, TN 38163. USA

 

Introduction

Vision depends critically on having the right numbers of cells, the right ratio of cell types, and the right distribution of cells across the surface of the retina (Walls, 1942; Rodieck, 1973, 1988; Wassle and Riemann, 1978; Sterling, 1983). In this paper we report and review recent findings on how lineage plays a role in the establishment of these important quantitative features in the mouse retina. We ask questions such as: What is the relationship between individual progenitor cells and the regional variation in retinal structure? What is the productive and differentiative capacity of a single progenitor cell? How many progenitor cells give rise to the retina? The underlying purpose of our work is to elucidate the genes and developmental interactions that are responsible for the intricate cellular architecture of the vertebrate retina. One endpoint will be a better understanding of patterns of gene expression associated with different retinal microenvironments and the differentiation of retinal cell types.

In mouse, evagination of the optic vesicle occurs on the 8th day of gestation (embryonic day 8.5 or E8.5). As in other vertebrates, these cells are fated to contribute to an amazing diversity of tissue types: the retina, the pigment epithelium, the optic nerve, and parts of the iris and ciliary body. It is not known precisely how many cells are involved in the evagination, but we estimate that the number is between 500 and 1000 (estimated from the work of Froriep, 1906). This population expands 10-20 fold (equivalent to about four or five rounds of symmetric division) before the first postmitotic cells are generated on E11 (Sidman, 1961; Driiger, 1985). The list of cell phenotypes produced by these cells includes photoreceptors, pigment producing cells, Muller glia, and the 20 or more neuronal types and subtypes within the inner nuclear and ganglion cells layers. By maturity, those progenitors have given rise to 5-10 million cells (Goldowitz and Williams, 1992).

How does one study these progenitor cells and the clones of differentiated cells that they generate? Several approaches involve labeling progenitor cells with a heritable marker. The marker can be an exogenous dye or retrovirus that is injected into the retina, or the marker can be an endogenous nuclear or cytoplasmic tag that is restricted to a subpopulation of retinal progenitors. Studying retinal progenitors in vitro under more controlled but less natural conditions is also highly informative (e.g., Adler and Hatlee, 1989; Reh and KIjayin, 1989; Anchan et al., 1991). Each method has advantages and disadvantages, some of which we consider below.

We have chosen to use endogenous cell markers to study retinal development from its inception. Only a subset of the retinal progenitor cells contain the endogenous marker. This is accomplished by making chimeric mice that are a mixture of marked and unmarked cells. Two embryos at the 8-cell stage are pushed together in vitro and the combined embryo is then transplanted into the uterus of a pseudopregnant female. Cells of one of the embryos contain the endogenous marker; cells of the other are either unmarked or contain another distinct marker. A complete description of this method is provided in a recent chapter (Goldowitz et al., 1992). All tissues in chimeras can be a mosaic of the two genotypes of cells. But the ratios of the two genotypes in a series of chimeras are highly variable. For example, in some chimeras, the retina is composed of a nearly balanced mixture (50:50), but in others, one genotype may make up less than 5% of the cell population (see Fig. 1). This variation from balanced to highly imbalanced contributions has proved to be a very valuable feature of the chimera system.

One of the most crucial aspects in the analysis of chimeras is the identification of cell genotype. In studies of chimeras made between chickens and quails, LeDourain and colleagues (LeDourain and Teillet, 1974; Balaban et al., 1988) have used prominent differences in the staining of nucleoli between these avian species. Hunt and colleagues (OGorman et a]., 1987) have used a selective quinacrine stain in transplantation chimeras made between Xenopus borealis and X. laevis. Several methods have now been developed to label cells of specific genotypes in mice (Goldowitz et al., 1992). Criteria for a good marker are:

 (I) The marker should be inherited by all progeny of a given progenitor cell.

 (2) The marker should not diffuse between cells.

 (3) The marker should be detectable in cells of all phenotypes at all stages of development.

 (4) The marker should not interfere with development.

 (5) The genotype marker should be compatible with other staining methods used to define cell phenotype and structure, particularly immu nohistochemical methods.

 (6) Specifically for the retina, the marker should be demonstrable in wholemounts because these preparations greatly improve the speed and reliability of data analysis.

Over the past 6 years we have explored several different methods to label cells in chimeric mice. Two different transgenic lines of mice, a globin transgenic line (Lo et al., 1987) and a beta-galactosidase expressing line (Friedrich and Soriano, 1991) have been particularly effective. The clonal structure of retina appears identical with the two marker systems. Much of our current work uses the constitutively expressed beta-galactosidase transgenic line (designated R0SA26). This transgene is expressed in all retinal cells with the important exception of the photoreceptors. But complementary labeling methods exploiting in situ marking of cells with the globin transgenic mouse have allowed us to study photoreceptors to yield an accurate view of the overall clonal architecture of the mouse retina.


 

Comparison of chimeras with other systems for studying cell lineage


 

The analysis of chimeric tissue complements studies that employ retroviruses and dyes to label clones in vertebrate retina (Wetts and Fraser, 1987; Holt et al., 1988; Turner et al., 1990; Huang and Moody, 1993; Fekete et al., 1994). Each method has advantages and disadvantages. Retroviral methods are often more suitable for studying individual clones because the frequency of transfection can be controlled by adjusting the titre of viruses injected into the embryonic eye. The age at which progenitors are marked can also be varied systematically. A risk with retroviral methods is that different types of viral constructs can be associated with different labeling patterns (Turner and Cepko, 1987; Fekete et al., 1994). For example, the viral construct CHAP does not label photoreceptors (Fekete et al., 1994). It appears from these studies that retroviral expression can be shut down in some members of a clone. A further problem is that the virus is generally not integrated immediately after injection (Fekete et al., 1994). As a result of this delay and the asymmetry of integration of the retrovirus (only one progeny of the transfected parent cell inherits the beta-galactosidase construct), the mean clone size is usually smaller than would be anticipated based upon the age at which the retroviral injection is made.

Labeling cells with dyes overcomes some of these problems (Wetts and Fraser, 1987; Holt et al., 1988; Huang and Moody, 1993). This method has been particularly useful in determining the percentage of cells in Xenopus retina derived from individual blastomeres at early stages of development (Huang and Moody, 1993). The drawback is that dye injection is impractical in vertebrate classes in which the clone size and volume increase exponentially during development and dyes are quickly diluted. Furthermore the injection of single cells, even in Xenopus embryos in which the dilution of dye is not a key concern, is technically difficult. Consequently, the total number of clones that have been examined with this method is relatively small.


 

Advantages and disadvantages of chimeras for studying cell lineage


 

There are two main advantages of chimeras. In terms of examining global clonal structure of retina, the chimeric system provides a large scale view of clones and polyclones across the entire retinal surface. The density of clones and polyclones can be very high, particularly in the balanced chimeras. Therefore, in a single retina one can ask whether there are systematic differences in the size of cohorts between center and periphery, or between nasal and temporal regions, or between right and left sides. One also can detect apparent boundaries to clonal expansion. An example of such a clonal restriction boundary is seen in the region of the optic fissure in the majority of our chimeric retinal wholemounts (Fig. 1B,C). In chimeric retinas that are composed of very few cells from one genotype, termed highly unbalanced chimeras, one can also estimate the size of single clones of the minority genotype and thereby arrive at estimates of the total number of cells that are the progenitors of retina (Rossant, 1990). Another unique advantage of chimeras is that one can generate a retinal environment in which two genotypes with variant phenotypes are juxtaposed. This juxtaposition may be between mutant (e.g., a photore ceptor degeneration mutant mouse) and nonmutant genotypes (Mullen and LaVail, 1976) or between mice with normal variation in retinal structure such as the total numbers of retinal ganglion cells (Williams et al., 1993). Consequently, chimeras have a unique role in determining the relative importance of intrinsic and extrinsic factors in controlling the development of retina.

 Among the disadvantages of chimeras are (i) the lack of control over ratios of the two genotypes that make up a tissue, and (ii) the inability to label individual progenitors or clones in a discrete manner or at different stages of development. These drawbacks make it impractical to use chimeras to address the competence of individual progenitor cells in retina. However the use of limited numbers of progenitor cells in blastocyst injection chimeras or the use of special strains of transgenic mice in which the expression of the cell marker is under inducible control may overcame these limitations.


 

Inter- and intra-species chimeras


 

A critical issue raised by Jacobson (1991) is that the two genotypes of cells in chimeras, particularly, interspecies chimeras, may not mix in a normal pattern. Clustering of like-genotype cells in interspecies chimeras could result from a secondary aggregation of clonally unrelated cells; an idea that recalls the classic laboratory demonstration of homotypic reaggregation of a mixture of cells from two species of sponges. This process is unlikely to be important. Our first evidence is that there is extensive intermixing of neurons and glia in the brains of interspecies chimeras (Goldowitz, 1989). This reduces the likelihood that selective affinity distorts clone structure in retina. The second line of evidence is more direct: cohorts of cells in chimeras generated between embryos belonging to the same species are just as sharply defined as those of interspecies chimeras (Fig. 1A). There is no qualitative or quantitative difference in the mean clone or cohort size (Fig. 2 of this paper compared to Fig. 2 of Williams and Goldowitz, 1992a). Finally, Reese et al. (1995) have analyzed clones in X-inactivation mosaics in which marked and unmarked cells are genetically identical, and they find similar clonal architecture as in chimeras. While there may be subtle differences in clone size and distribution among different types of chimeras (see below), fundamental traits appear insensitive to genotypic differences.


 

Global architecture of the mammalian retina


 

The first analysis of chimerism in the eye relied on easily detectable differences in pigmentation between albino and pigmented cells (Mintz, 1971). The pattern of chimerism in the pigment epithelium of these animals was reported to consist of radially arranged wedges centered near the posterior pole of the eye. The inference was made that the neural retina was also constructed out of a set of wedges of either pigmented or albino cells centered on the optic nerve head (Mintz, 1971; Sanyal and Zeilmaker, 1977), a reasonable deduction given the fact that pigmented and neural retina are derived from the same neuroectoderm (Herrup and Silver, 1986). Work in mosaic Xenopus (Hunt et al., 1987a,b, 1988) has shown much more clearly that large cohorts, derived by transplantation of pigmented cells into albino retinas at early stages, generate wedges oriented from center to periphery, like those illustrated in Fig. 3A.

The advent of methods to label genotypes of individual neurons and glia has made it possible to examine the clonal architecture of entire mouse retina directly. Our first study (Rice et al., 1995b) relied on visualizing, by in situ hybridization, DNA sequences found in only one of the two genotypes of cells in chimeric retinas (Lo et al., 1987). This DNA hybridization method worked well on 5-m thick sections of retina, but could not be successfully modified to reproducibly stain l00-m thick retinal wholemounts. But by using Friedrich and Soriano's (1991) transgenic mice, the beta-galactosidase positive cells can be stained easily in thick slabs of tissue, providing a means to study the clonal architecture in wholemounts. The analysis of retinas from R0SA26 chimeras is now yielding a striking picture of the size and distribution of clones and polyclones derived from the two parental strains across the entire retina (see Fig. 1B,C,G,H). Because the processes of Muller glia are well stained, borders between the two genotypes can be traced through all layers of the retina, including the acellular plexiform layers.

It is evident that the clonal structure of mouse retina is a patchwork of cells of the same genotype (blue transgenic cells and unlabeled non-transgenic cells) that range in size from small clusters containing fewer than 400 cells to large blocks con taining hundreds of thousands of cells (Fig. 2A). In general, there are no striking differences in the texture of the patchwork in dorsal or ventral retina, nor in nasal or temporal retina. Differences, however, between central and peripheral retina are present. The highly variable size of patches maybe a function of the length of time over which clones of mitotically active cells remain packed together. If a progenitor cell at E11 gives rise to a single clump of eight mitotically active daughter cells, and if all the postmitotic cousins stay together, then the final patch in adult retina may consist of several thousand cells. If, on the other hand, these eight daughters move apart, then the pattern will consist of a cluster of eight smaller patches of labeled cells situated within the same retinal sector. It appears that the retina is constructed in a jig-saw manner, and not like the slices of a pie. However, there is a tendency for cohorts to have a long axis that is aligned along the central-to-peripheral axis. This feature is most prominent in the peripheral retina. Clearly the pie-model of retinal development (see Fig. 3A) proposed by Mintz (1971) and Sanyal and Zeilmaker (1977) fails to account for the clonal patterns noted in chimeric mice. Our data is more consistent with the patchwork model illustrated in Fig. 3B.

Figure 1 Fig. I. (A) Cross section of the retina of an intraspecies chimera. Two clusters of globin-transgenic cells (asterisks) are aligned radially across the retinal layers. Micrographs (B-H) are of wholemounted chimeric retinas generated between a pigmented, beta-galactosidase-positive transgenic strain (R0SA26) and an albino strain (ICR). (B,C) The cells from the transgenic strain (blue precipitate in cytoplasm) comprise the majority of cells in these retinas from two chimeras. In both cases there is a preponderance of unlabeled cells (albino genotype) in the ventral temporal crescent (vtc, arrowheads). The optic fissure (arrows) extends from the optic nerve head to the ventral periphery. (D) Higher magnification view of clones, polyclones, and isolated cells in chimeric retina. Note that several transgenic cells (arrowheads) have dispersed into areas populated by albino cells. The asterisk marks one particularly large cohort of transgenic cells. (E) Ipsilaterally-projecting ganglion cells in the ventral temporal crescent of this chimeric retina contain the brown HRP-reaction product. Three of the labeled ganglion cells are transgenic cells that also contain the blue galactosidase reaction product (arrowheads). Two of these three double-labeled cells are more than 45 m from the nearest blue cohort (asterisk). Approximately 12 ganglion cells (brown precipitate) from the albino strain are present in this field. Two of these are marked with curved arrows. (F) Horizontal cells stained with a calbindin antibody are also dispersed from cohorts of like-genotype cells. A blue horizontal cell (arrow) is positioned between two blue clusters (asterisks). Horizontal cells that have the albino genotype (arrowheads) are also located in this region. (G) In this complete wholemount of a chimeric retina, the transgenic genotype makes up merely 1.5% of the total surface area and cell population. Despite this minute contribution, blue cells are widely distributed. The optic nerve head is marked by a curved arrow. (H) In this retina the transgenic genotype comprises less than 1% of the total retinal cell population. Unlike the previous example, most of the transgenic cells are located in the temporal quadrant. In both C and H there are large portions of the retina that do not contain any transgenic cells. Dorsal is up in the B, C, G, and H. Temporal is to the left in B, C and H and to the right in C. Scale bar = 50 m in A, 1 mm in B, C, G, and H, 100 m in D, and 25 m in E and F.



 

Figure 1 Fig. 2. (A) The size of isolated cohorts in 10 different chi meric retinas. Isolated cohorts were measured directly on a video microscope interfaced to a Macintosh computer. Dis persed cells are not included in the measurement of cohort boundary. The majority of cohorts are less than 2000ym2. Most cohorts have a surface area between 500 and lOOO m2. (B) The black bars represent a subsample of cohorts from the central retina whereas hatched bars represent cohorts from the peripheral half of retina in four highly imbalanced chimeric retinas (see Fig. IG,H for examples).

A consistent feature that we have discovered is a clonal raphe or seam in the ventral half of the retina (Fig. 1B,C). It consists of one or two bands of cells of the same genotype that extend with only limited interruptions from the optic nerve head to the ventral periphery of the retina. This seam probably results from the proliferation and migration of progenitors along the margins of the optic fissure of the retina. When the margins fuse (ca. E11-13), there is presumably only limited movement and/or mixing of progenitor cells and their progeny across this junction. Thus it appears that the closing of the optic fissure serves to mark a point in time (somewhere between E11-E13) when the free movement of neuroepithelial cells is restricted.

Another boundary, apparently more impenetrable to the expansion of neuroepithelial cells, is at the boundary between neural retina, ciliary body, and pigment epithelium. Clones rarely extend from retinal periphery into the adjacent pars plana of the ciliary body. This feature suggests that neuroepithelial progenitors in this region are split into two restricted lineages, one of which produces cells of the ciliary body, the other only cells of neural retina. This recalls the clonal restriction that occurs between cells in hindbrain rhombomeres (Fraser et al., 1990).


 

Clonal attributes of central and peripheral retina


 

In most vertebrates, the central part of the retina matures from days to weeks in advance of the peripheral part of retina (Mann, 1964). The central- to-peripheral developmental gradient is most striking in species that have large retinas and large differences in the density of cells across the retinal surface. This group includes many primates, carnivores, and birds. Given the big variation in cell density in different parts of the retina, from high densities in the area centralis or fovea to very low densities in the extreme periphery, one might anticipate large differences in the size and shape of retinal clones in center and periphery. If progenitors make a fixed number of progeny, then clones in the periphery (low cell density) might be expected to spread out over a much larger area than those in and around the fovea. Fekete et al. (1994) tested this idea in chicken, and have clearly demonstrated the predicted central-to-peripheral increment in clone area.

Figure 1 Fig. 3. Two models of the pattern of clones and polyclonal cohorts in chimeric retinas. Both models assume a developmental focus at or near the optic nerve head (black circle in A and B). The ventral temporal crescent (vtc) is outlined in both cases. (A) Cohorts are arranged in radial wedges organized around the disk. (B) Cohorts are arranged in a mosaic pattern with a weak radial alignment

What would one expect to see in the mouse--a species with a comparatively modest two-fold difference in cell density from the center to the periphery (Rice et al., 1995a)? Assuming that progenitors produce equal numbers of progeny, whether they are situated in the center of the retina or near its edge, then clonal surface area should be inversely proportional to cell density; lower densities corresponding to larger areas per clone. We have looked for such a gradient in the retinas of the R0SA26 to BALB/cJ chimeras (Fig. 2B). We measured surface areas of isolated cohorts (putative clones) of transgenic cells in central and peripheral retinas from four chimeras. Highly imbalanced cases were used because isolated clusters are easier to identify (e.g., Fig. 1G,H). The average size of the clones is somewhat larger in central than peripheral retina (3216 313 m2, n = 87 central; 2683 207 m2, n = 92 peripheral). However, this average obscures an interesting trend seen in the histogram (Fig. 2B). There are almost twice as many peripheral clones with areas be tween 3000 and 6000 m2 in the periphery than in the center. This is consistent with the idea that peripheral clones contain roughly the same numbers of cells as those in the center and are stretched over a larger expanse of surface in the periphery.

Most of the very large cohorts (>5500 m2) are found close to the center (Fig. 2B). There are at least two explanations for this apparently contradictory finding. (1) There may be less intermixing among progenitor cells in the central retina early in development than in the periphery. This might result in the production of large coherent clones in central retina. Those in the periphery might break up into many smaller clones; what Fekete et al. (1994) call "shotgun" clones (Fig. 1G,H). (2) The ratio of the two genotypes may differ between center and periphery, with consistently higher percentages of transgenic cells in the central retina. There is a clear bias in this direction in all 15 of our albino to pigmented retinas, including the two shown in Fig. 1B,C. Such a difference might distort our quantitative analysis, and make the central cohorts appear larger than those in the periphery. The mechanism that creates this biased colonization may be similar to the developmental timing differences that have been seen between neurons of the two genotypes in the dentate gyrus and cerebellum in interspecies chimeras (Goldowitz, 1989). The development of the albino retina is known to lag behind that of the pigmented retina (Webster and Rowe, 1991), and this could lead to heterochronic distortions of cell allocation. This idea is easily tested by making pigmented to pigmented, transgenic chimeras.


 

Lateral dispersion of early-generated cell types


 

In retina, there is a consensus that clones form tight radially aligned groups of cells. However, in our first study we demonstrated that single ganglion cells, amacrine cells, and horizontal cells were occasionally found situated in adjacent territory dominated by the other genotype (Rice et al., 1992; Williams and Goldowitz, 1992a). Reese et al. (1995) found similar dispersion of these first generated retinal cells in murine X-inactivation mosaics. Fekete et al. (1994) have provided additional evidence for tangential dispersion of cells in chicken retina. There are two possible explanations for this phenomenon. Cells may simply migrate short distances away from nearby, parental clones after their final cell division. If cells do migrate, then it is of interest to know whether they are doing this to produce a more orderly retinal mosaic or whether they are just moving passively in response to extrinsic forces such as the ingrowth of blood vessels and changes in retinal shape. Alternatively, isolated cells may be members of small clones that are generated by progenitor cells that become isolated or displaced from cells of the same genotype. If the tangentially dispersed cells are clonally related then these cells share a clonal history that is unique from radially-arrayed clones and may well have unique patterns of gene ex pression in the progenitor cells that establish these clones, implying that there may be at least two progenitor populations in the mammalian retina.

 To address these alternative mechanisms, we examined isolated horizontal, amacrine, and ganglion cells in wholemounts of R0SA26 transgenic chimeras (Fig. 1D-F). The frequency and type of isolated cells were quantified at many sites in single retinas (Fig. 4). If the process responsible for these isolated cells is based upon movement of early generated cells away from a parental clone, then we may expect a monotonic decline in their number as a function of distance away from the nearest cohort of like-genotype cells. Alternatively, if the process responsible for these isolated cells is an independent clonal event, then we may see more complex patterns of distribution, e.g., small clones containing early generated cell types with no clear spatial contiguity with larger cohorts of like-genotype cells.

In each retina studied, we find cells that are out of register with the boundary of the nearest cluster of R0SA26 cells (Fig. 1D-F). These cells were identified as ganglion cells, amacrine cells, and horizontal cells based upon the position and sizes of their cells bodies. The identity of ganglion cells and horizontal cells was also confirmed using histochemical techniques (Fig. 1E,F). Dispersion distances away from the nearest like-genotype cluster were determined for each class of cell (Fig. 4). Ganglion cells were found to disperse farther than any other cell type. These cells were found up to 120 m away from their nearest genetically identical cohort. Horizontal cells were found up to 60 m away from the nearest cohort. Amacrine cells were found up to 40 m away from the nearest cohort (see Fig. 4). The number of these isolated cells decreases as the distance away from the nearest cohort increases. This monotonic decline is consistent with the view that early generated retinal neurons move tangentially from their parental clone during development.

Figure 1 Fig. 4. Distance between the edge of the nearest cohort of transgenic cells to isolated ganglion cells, amacrine cells, and horizontal cells. Data were obtained from four highly imbalanced chimeric retinas. Isolated ganglion cells (black bars) are found more than 100 m away from the nearest transgenic cohorts. Horizontal (stippled bars) cells are found up to 60 m away. Cells in the inner portion of the inner nuclear layer, presumably amacrine cells (hatched bars) disperse up to 40 m.

The question arises if this lateral movement of ganglion, horizontal, and amacrine cells is driven primarily by an active or passive process. Lateral movement of ganglion cells is already known to occur during the development of the human fovea (Yuodelis and Hendrickson, 1986). Ganglion cells are displaced or migrate a considerable distance to build the fovea. But this phenomenon is assumed to be restricted to species with a well developed fovea. Our work suggests that small lateral movements of ganglion cells may be a more common feature of retinal development even in a species such as the mouse that does not have an obvious central retinal specialization. Our findings are consistent with a passive process of cell displacement that is due to two temporal and mechanical features of retinal development. The first feature is that the early (E11-E13) retinal neuroepithelium is more permissive to cell movement than the older neuroepithelium. Early-generated celIs that leave the neuroepithelium in this fluid environment become more dispersed. The second feature is the expansion of the retina. Thus, the cell type that is most dispersed in adults--ganglion cells--moves away from the tightly packed neuroepithelium at the earliest age. Like cells in layer I of cortex, ganglion cells are more able or prone to move tangentially than are later generated retinal cells.


 

The relationship between cell lineage and cell phenotype


 

One of the most important findings in the past decade in the field of retinal development was the clear demonstration by Cepko and colleagues, Holt and colleagues (Holt et al., 1988), and Wetts and Fraser (1987) that single retinal progenitor cells labeled even fairly late in development can produce a wide variety of retinal cell types. For example, progenitor cells in early postnatal rats are able to produce both bipolar cells and Muller glia (Turner and Cepko, 1987). This finding led to the notion that retinal progenitors are not only able to make many retinal cell types but they retain their ability to make all retinal cell types even late in development, and that the environment alone has a controlling influence on final cell phenotype.

 We tested this uniform progenitor cell hypothesis in two ways: (i) by analyzing the large clones in chimeras (Williams and Goldowitz, 1992a), and (ii) by a systematic quantitative analysis of the retroviral data set, Turner and Cepko (1987) and Turner et al. (1990) using a Monte Carlo procedure (Williams and Goldowitz, 1992b). In essence, what we did was to compare the mixture of phenotypes of cells generated by a computer program with the real clones generated from E13 onward by Turner and colleagues. Data on over 50,000 computer-generated clones were analyzed, pooled, and then compared to the real data set. Several different models of the sequence of retinal cell generation and cell death were tested in making the computer clones. Despite the impressive qualitative impression that the data gives of a heterogeneity of cell types in retroviral clones, our quantitative analysis has shown that this heterogeneity is substantially less than predicted even by a simple random model. The bottom line is that the cell type composition of retroviral clones differs in surprising ways from that predicted by exclusive environmental control. There are two important technical caveats to this conclusion. First it is possible that the retroviral clones were themselves skewed in cell composition. This could happen if particular cell classes migrated away from the clone centers and were scored as independent small clones, or if retroviral expression was suppressed in some cell types. Second, it is possible that the kinetics of cell production and cell death are so different from those assumed by the model as to seriously compromise the model's validity. However, from our recent studies of ganglion cell projection phenotypes (see below) and the work on amacrine cell phenotypes in Xenopus (Huang and Moody, 1995), there is mounting evidence that retinal neuroepithelial cells are partially restricted in the pheno types they can normally produce.

A special case for lineage restriction in ganglion cells has been proposed by Jacobson and Hirose (1978). They found that each retina in Xenopus is derived from blastomeres on both sides of the embryo. They raised the possibility that the bilateral origins of the retina might be associated with the division of ganglion cells into two populations; one with crossed projections, the other with uncrossed projections. This hypothesis can now be tested in chimeric mice by the examination of the projection phenotype and the genotype of individual ganglion cells (Rice et al., 1995b). The projection phenotype is assessed by backfilling cells with horseradish peroxidase that yields a brown reaction product in the cell soma (see Fig. 1E,H). The genotype of cells is determined by reacting the retina using the beta-galactosidase X-gal reaction. Transgenic ganglion cells contain distinct blue punctae of cytoplasmic label (Fig. 1 E). The wholemounted retina is examined to determine if the cells of the ventral temporal crescent (VTC) have a distinct clonal history and if individual cohorts of like-genotype cells are biased in their production of ganglion cells with ipsilateral or contra lateral projections.

Our analysis demonstrates that the ventral temporal crescent, the region that contains almost all ipsilaterally-projecting ganglion cells, is not a clonal compartment with sharp boundaries separating it from the rest of the retina. Small cohorts of cells appear to extend without interruption or irregularity across the inner border of the VTC (compare to the model in Fig. 3B). Individual clones probably contribute to both retinal "compartments". While there may not be a sharp demarcation, several chimeras have a clonal composition in the VTC that differs markedly from that of the rest of the retina (Fig. lB,C). In the most compelling case, about one-half of the border of the ventral-temporal crescent corresponds nicely to a large difference in the proportions of the two genotypes (Fig. IB). This difference in cell allocation suggests that the VTC may have clonal origins that are somewhat different from the rest of the retina. The question remains whether individual clones in the VTC produce ganglion cells of a single projection phenotype.

The fine grained analysis of single labeled and unlabeled ganglion cells within the ventral temporal crescent of mice is providing evidence for lineage restriction. This region contains the vast majority of the 1500 or so ganglion cells that project ipsilaterally, but it also contains a population of about 9000 contralaterally projecting cells (Drager and Olsen, 1980; Drilger, 1985; Rice et al., 1995a). If the ganglion cell mixture within single clones reflects the ratio of the two types of cells in this part of the retina, then clones should not differ significantly from the predicted 1:6 ratio. In contrast, if some clones consist largely of ipsilateral cells and contain few, if any, contralateral cells, then one can infer that phenotypes are not assigned randomly and that the progenitors that gave rise to the ganglion cells may have become restricted. We have recently found evidence for such restriction in our analysis of single clones. For example, the small, isolated clone in Fig. 1E (asterisk and arrowheads) contains at least three ipsilaterally projecting ganglion cells and at most a single contralaterally projecting ganglion cells (a blue, not HRP-labeled ganglion cell). While this analysis is still preliminary, these findings suggest some level of lineage restriction may control this aspect of ganglion cell phenotype. Similar findings are emerging in Xenopus in which it has been shown that there is a partial lineage restriction in the pro duction of amacrine cells and amacrine cell subtypes (Huang and Moody, 1995).

Analysis of highly imbalanced chimeras and the origins of the retina


 

One of the powers inherent in the analysis of chimeric tissue is that an estimate can be made of the number of progenitor cells that give rise to any tissue. The retina is particularly suitable for this analysis because it develops from a discrete and isolated neuroepithelium. The simplest estimate of progenitor cell number is made from the most imbalanced chimeric retina. This lowest percentage chimera illustrates what might be a single progenitor cell's proliferative potential and progeny. In our analysis of interspecies chimeras we found that the lowest percentage chimeras were between 1 and 2%, suggesting that the retinal progenitor pool is comprised at least 50-100 cells (Williams and Goldowitz, 1992a). However, these estimates were derived from sectioned material that precluded an analysis of the whole retina. A more accurate means to assess the percentage contribution of cells and their distribution is with a wholemount preparation.

 We find a limited number of labeled cells that colonize the retina in several of our transgenic chimeras. For example, in Fig. 1 G,H, two cases of low percentage chimeras are shown with cohorts of labeled cells that are restricted to a limited region of retinal space. This finding suggests that the original cells that gave rise to these clones are limited in number, since we know that the retina (at least before E13) provides a fairly fluid environment for cell movement; the more progenitor cells we start with the greater the likelihood that cohorts will be strewn all about retina. The retina pictured in 1H had the fewest number of labeled cells of all other retinas examined. The number of labeled cells was about 1000th of the total number of retinal cells (about 100,000 cells were labeled in a retina estimated to contain about 5-10 million cells). Interestingly, other very low percentage chimeras (such as the one pictured in G) had reasonable multiples of this "quantal" number. Thus, from the analysis of wholemounted chimeric retinas we were able to adjust upwards the estimated number of progenitor cells that establish retina to a pool of at least 1000 cells. The localized nature of the like-genotype cohorts in the retina pictured in Fig. 1H suggests that these cohorts may be the progeny of a single progenitor cell. Furthermore, the number of progenitor cells we estimate is similar to the approximate number of cells that compose the early anlage of the retina (estimated from Froriep, 1906).

If the blue transgenic cells in the retina pictured in Fig. 1H truly arise from a single progenitor cell, then the clonal allocation of cells in this retina provides a picture of what a single progenitor cell can do. A single progenitor cell produces spatially restricted subelones that appear to be focused on the optic disk. The distribution of subelones appears to be organized along the central-to-peripheral axis. The minimum size of a subelone is about 175 m2. This minimum corresponds to single subclones that contain about 120-250 cells, equal to the progeny that would be produced by 7 or 8 rounds of symmetric cell division. Since the first postmitotic cells are generated as early as E11, many of these progeny are the products of asymmetric divisions stretched out over nearly two weeks (E11-E19 = PO-P6) and as many as 20-25 cycles of cell division. Continuing in a retrospective vein, it is possible to estimate the time when these 1000 cells are set aside as the progenitor pool for retina. We have calculated the number of cells in the E11.5 retinal neuroepithelium to be about 12,000 (Goldowitz and Williams, 1992a). This means that a single progenitor cell (1/1000 of the retinal neuroepithelium) would produce 12 cells at E11.5. Working back in time, assuming a cell cycle of about one-half day, these 12 cells would take about two days to be generated. We conclude, then, that the original progenitor cells of retina are established around E9-E9.5. This period corresponds with the optic vesicle stage of retinal development and the time that Pax6, a master control gene for retinal development (Halder et al., 1995), is expressed in the cells of the optic vesicle (Walther and Gruss, 1991).

We can antedate the origins of retina to an even earlier time in development. In the analysis of interspecies chimeras there is a clear correlation between the percentage chimerism in the right and left retinas of an animal (Williams and Goldowitz 1992a). In intraspecies chimeras, where we have a more accurate picture of percentage chimerism, there is also a strong correlation (r = 0.98) between the percentage chimerism in right and left retinas. The consistency of these findings supports a notion that early in development (around E7-E7.5) the future retinas originate from a common pool of progenitor cells. Neuronal precursors congregate at the future midline and are split into two populations of precursors that generally rellect the original precursor pool. These events would best describe the right/left symmetry of cell allocation that is seen in neural retina as in other parts of the CNS.


 

Summary and conclusions


 

The study of chimeric retinas has yielded insight on the early development of retina. The close match in chimerism ratios between right and left retinas is significant and supports the idea that both retinas originate from a common population of progenitors. We are able to estimate numbers of progenitor cells that contribute to the formation of the retina and the approximate time at which this small group is isolated from surrounding prosencephalic cell fields. These cells undergo at least five rounds of division before the first retinal neurons are generated. The mouse retina is not built from the center outward. There is simultaneous expansion and differentiation in all parts of the retina and as a result clones are not arranged in wedges. Instead the mouse retina is a patchwork of clones that do not differ greatly in size from center to periphery. The most consistent radial feature in mouse retina is a raphe left at the line of fusion of the margins of the ventral fissure.

 Processes that shape the clonal patchwork are both passive and active, intrinsic and extrinsic. Certain features of the clonal architecture of the retina, such as the size differences of clones are primarily passive responses to extrinsic forces on progenitor cells and their progeny. The fifteen-fold range in the size of cohorts is not due to intrinsic differences in the proliferative capacity of individual progenitor cells, but is due to the extent of cell movement and mixing at early stages of development. In contrast, active or intrinsic processes are illustrated by the partial (and still controversial) restriction of retinal progenitors, the possible clonal differences between ganglion cells with crossed and uncrossed projections, and the consistent differences in ratios of albino and pigmented genotypes in peripheral and central retina.


 

Acknowledgements


 

Supported by RO1 EY-8868 and EY-9586 to D.G. and R.W.


 

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