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University of Wisconsin Press Asanuma, H. and Sakata, H. (1967) J. Neurophysiol. 30, 35-53 Asanuma, H. and Ros~n, I. (1972) Exp. Brain Res. 14, 243-256 Asanuma, H. (1975) Physiol. Rev. 55(2), 143-155 Zeki, S. (1974) J. Physiol. 236, 549-573 Van Essen, D. C., Maunsell, J. H. R. and Bixby, J. L. (1981) J. Comp. Neurol. 199, 293-326 Komatsu, H. and Wurtz, R. H. (1988) J. NeurophysioL 60, 580-601 Andersen, R. A. (1989) Annu. Rev. Neurosci. 12, 377-403 Fuster, J. M. (1973) J. NeurophysioL 36, 61-78 Goldman-Rakic, P. S. (1987) Handbook of Physiology 5 (Pt 1) (Plum, F., ed.), pp. 373-417, American Physiology Association Gross, C. G., Rocha-Miranda, C. E. and Bender, D. B. (1972) J. Neurophysiol. 35, 96-111 Bruce, C. J., Desimone, R. and Gross, C. G. (1981) J. NeurophysioL 46, 369-384 Baylis, G. C., Rolls, E. T. and Leonard, C. M. (1987) J. Neurosci. 4, 2051-2062 Albright, T. D., Desimone, R. and Gross, C. G. (1984) J. Neurophysiol. 51, 16-31 Zeki, S. and Shipp, S. (1988) Nature 335, 311-317 Rockel, A. J., Hiorns, R. W. and Powell, T. P. S. (1980) Brain 103, 221-244 Rakic, P. (1988) Science 241, 170-176 Stryker, M. P. (1988)in Neurobiology of Neocortex (Rakic, P. and Singer, W., eds), pp. 115-136, J. Wiley Durbin, R. and Mitchison, G. (1990) Nature 343, 644-647 Ma, P. M. (1991)J. Comp. Neurol. 309, 161-199 Golgi, C. (1874) Riv. Sper. Freniatr. Meal. Leg. Alienazioni /vlent. 1,405-425 Graybiel, A. M. and Ragsdale, C. W. (1978) Proc. NatlAcad. 5ci. USA 75, 5723-5726 Langley, J. N. (1895) J. PhysioL 18, 280-284 Sperry, R. W. (1963) Proc. NatlAcad. 5ci. USA 50, 703-710 Purves, D. and Lichtman, J. (1985) Principles of Neural

Development, Sinauer Associates 82 Langley, J. N. and Anderson, H. K. (1904) J. Physiol. 31, 365-391 83 Sur, M., Garraghty, P. E. and Roe, A. W. (1988) Science242, 1437-1441 84 Wiesel, T. N. and Hubel, D. H. (1963) J. Neurophysiol. 26, 1003-1017 85 Hubel, D. H. and Wiesel, T. N. (1965) J. NeurophysioL 28, 1041-1059 86 Purves, D., Snider, W. D. and Voyvodic, J. T. (1988) Nature 336, 123-128 87 Purves, D. (1988) Body and Brain: A Trophic Theory of Neural Connections, Harvard University Press 88 Lichtman, J. W. (1980) J. Physiol. 302, 121-130 89 Johnson, D. A. and Purves, D. (1981) J. Physiol. 318, 143-159 90 Hume, R. I. and Purves, D. (1983) J. Physiol. 338, 259-275 91 Forehand, C. J. and Purves, D. (1984) J. Neurosci. 4, 1-12 92 Shatz, C. J. (1990) Neuron 5, 745-756 93 Gilbert, C. D. (1983) Annu. Rev. Neurosci. 6, 217-247 94 Katz, L. C. and Callaway, E. M. (1992) Annu. Rev. Neurosci. 15, 31-56 95 Miller, K. D., Keller, J. B. and Stryker, M. P. (1989) Science 245, 605-615 96 Zahs, K. R. and Stryker, M. P. (1988) J. Neurophysiol. 59, 1410-1429 97 Stout, R. P. and Graziadei, P. P. C. (1980) Neuroscience 5, 2175-2186 98 Schlaggar, B. L. and O'Leary, D. D. M. (1991) Science 252, 1556-1560 99 Constantine-Paton, M. and Law, M. I. (1978) Science 202, 639-641 100 Constantine-Paton, M. (1981) in The Organization of the Cerebral Cortex (Schmidt, F. O., Warden, F. G., Adelman, G. and Denkis, S. G., eds), pp. 50-51, MIT Press 101 Turing, A. M. (1952) Philos. Trans. R. Soc. London Ser. B 237, 37-72 102 Murray, J. D. (1988) Sci. Am. 258, 80-86 103 Cott, H. B. and Huxley, J. S. (1940) Adaptive Coloration in Animals, Methuen

Lineage versusenvironmentb embryonicretina: a revisionistperspective Robert W. Williams and Dan Goldowitz RobertW. Williams and Dan 6oldowitz are at the Deptof Anatomyand Neurobiology, Collegeof Medicine (Universityof Tennessee),875 MonroeAve, Memphis, TN38163, USA.

The idea that microenvironmental cuesact alone/ate m development part of the CNS with a comparatively simple layout to determine a cell's pheno~/pe has dominated recent discussion of that makes it a particularly favorable tissue in which retinal development, and has successfully displaced the notion of any to explore the relationships between a cell's lineage, role for cell lineage in the processof cell determination. We argue that its environment and its phenotype. there is, in fact, evidence favoring a degree of lineage restriction In a set of recent studies, progenitor cells in the dunng the development of the vertebrate retina. We propose that retina have been marked at different stages of deenvironmental factors modulate a process of progressive lineage velopment, using a variety of methods (Refs 6-I0; restriction. In this model, progenitor cells are viewed as having Huang, S. and Moody, S. A., unpublished obserunequal potential, and their progeny are viewed as being commi~ed vations). Without exception, these studies have to one of the major retinal cell classesbefore the stage at which they shown that the resulting clones of retinal cells are become postmitotic. tightly interknit clusters of cells that are aligned radiM u c h progress has been made in the past ten years in d e v e l o p i n g methods to mark and analyse descendants of single p r o g e n i t o r cells - cellular clones - in the vertebrate CNS. A c o m p e l l i n g reason to study these families of cells is t h a t a careful analysis of their size, placement and cell composition gives us insight into the genetic and d e v e l o p m e n t a l mechanisms t h a t generate f u l l y differentiated cell and tissue types from initially u n d i f f e r e n t i a t e d progenitors 1-s. In this article w e focus on the retina, an accessible

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ally across the retinal layers (Fig. I). 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 W h e n p r o g e n i t o r cells are marked early in dev e l o p m e n t by c o m b i n i n g genetically distinguishable four- to eight-cell mouse blastocysts into a single e m b r y o 11, the resulting clones contain a balanced

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representation of the major retinal cell types I°. In chimeric mice these clones can be visualized as beautifully discrete and complete retinal building blocks (Fig. IA). 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. A fluorescent dye was injected into individual cells at a stage when the entire embryo is made up of just 32 cells (Huang, S. and Moody, S. A., unpublished observations). Ten of these 32 progenitors contributed to the pool of retinal progenitors, and no matter which of these ten cells were marked, the resulting clones in the retina contained nearly the same ratio of cell types as the g~cl " . . . . . retina as a whole. These complementary results in mouse and Xenopus demonstrate that, early in development, retinal progenitors have the 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 labeled 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 the 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 MLiller glial cells. A similar profusion of different types and sizes of clones (see Fig. 1. Clones of cells in vertebrate retina. (A) Cross-section through the retina Fig. I B) has been found in Xenopus retinas follow- of an adult chimeric mouse. These chimeric mice are made by combining ing injections of heritable tracers - either horse- genetically distinct mouse embryos in vitro. The resulting double-genotype radish peroxidase (I-IRP) or fluorescent dye - during embryos are implanted into pseudo-pregnant mothers and born normally at term 11. The three narrow columns of unlabeled cells (clear regions) are of Mus the middle stage of retinal development 6'9. caroli genotype, whereas the more extensive heavily labeled regions (dark areas) are made up of cells that have been labeled with a biotinylated DNA Competing hypotheses: lineage restriction and probe that hybridized with a Mus musculus satellite DNA sequence 11. Cells o

environmental regulation

There are two different explanations for the transformation from uniform clones generated by progenitors labeled early in development to highly variable clones generated by progenitors labeled later in retinal development (Fig. 2). The first explanation is that at some point during 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 rod cells was derived from 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 pluriTINS, Vol. 15, No. 10, 1992

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) that appears restricted to the onl]. 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 of development (relatively mature state). The three clones in (B), (C) and (D) were marked by injecting single progenitor cells with horseradish peroxidase at stages 22-27 of development (early pen'od of brain and eye development) 6. These small clones of cells contain a wide variety of combinations of different cell types. For example, (B) contains two amacrine cells and one ganglion ceil; (C) contains one ceil in each layer; and the clone in (D) contains three 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. Scale bar is 25 l~m. [(B), (C) and (D) are taken with permission from Ref. 6.]

potence; some progenitors 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 369

A

B

and weaknesses of the lineage restriction and environmental regulation models. If the potential of progenitors to make different cell types is progressively reDivisiom produce nonequi~ ,t daughtercells stricted, then clones marked at O0 later stages of development should Heterogeneousmidstage progenitors Homogeneousmidstage progenitors contain fewer combinations of cell types than if cell phenotype is either generated early in development or generated by a process oducemore erc Jaughtercells that randomly assigns a phenoof uuuu,=¢erc~li~ • (~0 type to each member of the clone. Thus, the frequency of interactions ~ Uncommitted Committed 0 0 ( 1 postmitotic postmitotic clones containing only a few cell cells cells types should be much higher than Feedback expected by chance (Fig. 3). An regulationfrom older postmitotic extreme example of restriction is ceilsontoyoung postmitoticcells the clone of 33 rods - a combination of cells which a random process would generate in the mouse with a frequency of only Differentiating retinal cells Differentiatingretinal cells one in 13 000. Fig. 2. Two simple models of the determination of ceil types in vertebrate retina. (A) Lineage The opposite prediction follows ,'estriction. Progenitor cells undergo partial and progressive lineage restriction in the range of cell from the environmental hypothtypes they are able to produce. This restriction is associated with an accentuation of differences between progenitor cells. In this lineage model, young postmitotic cells, near the bottom of the esis - progenitor cells are thought figure, are viewed as being committed to specific fates. (B) Environmental regulation. Progenitor to remain fully pluripotent. cells retain equivalence and pluripotency throughout retinal development. Young postmitotic cells Consequently, the sets of cells are uncommitted. In this environmental regulation model, the ultimate phenotype of a retinal cell- generated by these progenthe decision to become a rod, cone, bipolar cell, amacrine cell, ganglion cell or even a MOller glial itors (members of a clone) cell - is controlled by the microenvironmental interactions of uncommitted postmitotic cells with should include many different cell other more mature cells and global spatio-temporal environmental gradients. phenotypes. However, these clones should not be random clones at later stages of retinal development is that sets of different cell phenotypes. The retina is, after this diversity is a direct reflection of underlying all, a highly regular structure 17, and radial arrays microenvironmental heterogeneity (Fig. 2B). This contain balanced ratios of different retinal cell types. idea was initially based on an exquisitely detailed The environmental hypothesis holds that this analysis of electron microscope images of cells in regularity is achieved by stereotypic patterns of embryonic mouse retina that was undertaken by interactions among neighboring cells, a great many Hinds and Hinds12. They found that the pool of of which are inevitably members of the same clone progenitor cells appeared homogeneous at the (Fig. 1). For example, if one of the first cells in a ultrastructural level, even quite late in development. clone differentiates as a ganglion cell, this cell will From these initial observations, the idea has evolved generate environmental signals that lower the that homogeneous progenitors produce postmitotic probability that its neighbors, including other 'blank slate' progeny. These postmitotic, still un- members of its clone, also become ganglion cells. committed cells are then assigned a phenotype by Instead, this young ganglion cell should signal its interacting with neighbors that have already been neighbors to differentiate as bipolar, amacrine or committed and that have already begun to photoreceptor cells. Feedback interactions of this differentiate 6'~3. In essence, a spatio-temporal cas- type will generate a greater diversity of cell types cade of inductive and inhibitory interactions among within clones than would a random process. There cells, both local and global, is thought to be the key are several concrete experimental examples of the arbitrator of a cell's destiny ~3-~5. Environmental ways in which such mechanisms re-establish a more differences give rise to the great variety of mixture nearly balanced representation among cell types in of cell types seen in neighboring clones. A corollary retina after specific phenotypes have been selectively is that the large modular clones generated by ablated 14'is. Perhaps the best current example of progenitors at early stages of development are the numerical and phenotypic regularity that can be composed of sets of these smaller and highly achieved by environmental interactions is the comvariable subclones. plex of photoreceptor types in the ommatidium of the fly 19. Here, a spatio-temporal gradient of cell Lineage and environmental hypotheses lead to production coupled to a series of ligand-receptor different predictions about clone structure interactions between neighboring cells triggers an The wealth of data on retinal clones in mouse and invariant mosaic of three different receptor types in frog now makes it possible to assess the strengths ommatidia across the entire eye. Homogeneous pludpotent retinal progenitor cells

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Homogeneouspluripotent retinal progenitor cells

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T y p e s o f cells irl c l o n e s : Rod (R),Amacrine(A),Bipolar(B),Coi l , Ganglioncetl(G), Mellerglialcell (M)

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Fig. 3. Comparison of real and randomly generated sets of clones. Each row lists the expected and observed numbers of clones that contain different numbers of retinal cell types after progenitor cells in the mouse are labeled on embryonic days E13 and E14. Clones containing the greatest variety of cell types are listed at the top (piuripotency); those with the least variety are listed at the bottom (restriction). Rare clone types (for example, ABC), in which expected and observed numbers are typically less than 1.0, are not listed. The observed data set of 219 clones, containing an average of 45 cells per clone, is taken from Ref. 7. The expected numbers of clones were generated by a process in which different types of cells were randomly assigned to simulated clones by a program run on a computer. More than 10000 of these Monte Carlo-simulated clones were categorized according to cell type to obtain values expected in subsets of 219 clones. (Single-cell 'clones' listed in Turner et al. 7 have been

excluded from the analysis.) We corrected for the production of some types of retinal cells before E15 (see text and Ref. 16). The specific percentages of cells (assignment probabilities) used in running this simulation were 75% (rods), 6.64% (amacrine cells), 11.1% (bipolar cells), 1.44% (cones), 1.91% (ganglion cells)and2.94% (Miller gila). Since rods are so common in the mouse retina, most clones contain this cell type. In contrast, horizontal cells are rare (1 in 300 cells), and many are also generated before E15 (Ref. 16). An assessment of the role of fineage restriction in retinal development requires as a starting point a scheme for classifying cell phenotypes. We and others have chosen the most obvious scheme, major cell categories. However, given the diversity of types and subtypes of retinal cells, we should not discount the possibility that other classification schemes (e.g. transmitter phenotype, on- and off-center response polarity) may be as pertinent in developing a taxonomy of retinal clones.

A critical assessment using a random model of cell determination

of different types were assigned a selection probability based on their proportions in the adult mouse retina (Fig. 3). Adjustments were made to eliminate from consideration cells produced before the stage at which Turner et al. made their retroviral injections (E13 and E14). Cells were then randomly and repeatedly sampled, thereby generating sets of simulated clones that contained precisely the same numbers of cells as were observed by Turner et aL 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-labeled clones (Fig. 3). An average of

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 by using as a starting point the set of clones published in the landmark study by Turner et al. 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 TINS, Vol. 15, No. 10, 1992

371

Box 1. Environment and lineage in 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 ranging from the phenotypes of single cells to areal projection patterns a. Finlay and Slattery b 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 c and colleagues have demonstrated that projection phenotypes of cortical cells are influenced by the local cortical environment. Frost, Sur, and their co-workers d'e 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 pre-developed 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 Levittf 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 g have found that laminar destiny is determined during or before the final round of cell division. Finally, Luskin, Parnavelas and co-workers h'' have shown that cortical progenitors give rise to clones of a single

20% of the M o n t e Carlo-generated clones (44 out of 219) contain all six of the most numerous retinal cell types produced after E14. In contrast, only a single retrovirus-labeled clone contained representatives of each of these six common cell types. Similarly, 60 M o n t e Carlo-generated clones contain five different cell types, whereas fewer than a third as many of the retrovirus-labeled 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), the observed numbers of retrovirus-labeled clones become substantially greater than the numbers predicted by the simulation. For example, only 45 of the M o n t e Carlo-generated 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 clones, containing only one or two cell types, such as allcone clones, were found following retroviral injections. Summing this work up, real clones with low cellular diversity are more common and clones with high cellular diversity are much less common in their occurrence than would be predicted by a random process. 372

the cerebral cortex: both play a role phenotype, i.e. containing pyramidal cells or interneurons, oligodendrocytes or astroglia. 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 the 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. References a Rakic, P. (1988) Science241,170-176 b Finlay, B. L. and Slattery, M. (1983) Science 219, 1349-1351 c O'Leary, D. D. M. (1989) TrendsNeurosci. 12,400-406 d Frost, D. O. and Metin, C. (1987) Nature 317, 162-164 e Sur, M., Pallas, S. L. and Roe, A. W. (1990) Trends Neurosci. 13, 227-233 f Barbe,M. F. and Levitt, P. (1991) J. Neurosci. 11,519-533 g McConnell, S. K. and Kaznowski, C. E. (1990) Science254, 282-285 h Barfield, J. A., Parnavelas, J. G. and Luskin, M. B. (1990) Neurosci. Abstr. 16, 1272 i Parnavelas,J. G., Barfield, J. A., Franke, E. and Luskin,M. B. (1991) Cereb. Cortex 1,463-468

A similar analysis of retinal clones has also been performed by Holt et a/. 6 in Xenopus. 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. Holt e t a / . injected progenitors with HRP at early stages in development (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 that is inconsistent with both lineage restriction and environmental regulation. However, on analysing their results, 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 one 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 TIN& Vol. 15, No. 10, 1992

retinal development, just as it does to a certain degree both in the cortex (see Box I) 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, studies in vitro by Reh and colleagues 2° 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 the retinal environment, or possibly due to internal changes associated with cell division. Along with these temporal shifts, Dr~ger et al? ~ have recently shown molecular heterogeneity among progenitors in the mouse at a very early stage of development. 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 that acts late in development. In a recent N e w 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 et al. on retinal clones 7 reads: 'Lineage-independent determination of cell type in the embryonic mouse retina'. Yet as we have shown in Fig. 3, the paper by Turner eta/. indicates 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 that there is confusion over what is meant by 'lineage restriction'. If restriction means that each progenitor gi~es rise to only a single cell type, then, yes, the data of Turner et al. 7 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 the 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 Buss in The Evolution o f 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 directly targets progenitor cells, and then, either in a stepwise or graded manner, it restricts TINS, Vol. 15, No. 10, 1992

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 the clones and subclones labeled with retrovirus by Turner et aL7, we think it is likely that restriction of these progenitors begins between E11 and E12 (Ref. 10), which is roughly concurrent with the production of the first postmitotic retinal cells 16. The progressive restriction of progenitors may be fine-tuned by environmental signals. Although one may disagree with our interpretation of the results, it is certainly premature to rule out a role for lineage restriction. To assess the role of cell lineage and environment critically will require in vivo transplantation of single progenitor cells into host retinas at different stages of development 5'27. Experiments in vitro, in which progenitors are placed in well-defined environments, will also continue to help to determine the rigidity of commitment and to discover ways of controlling the state and output of CNS progenitor cells.

Selected references 1 Jacobson,M. (1985) Trends Neurosci. 8, 151-155 2 Doe, C. Q. and Scott, M. P. (1988) Trends Neurosci. 3, 101-106 3 Sanes,J. R. (1989) Trends Neurosci.12, 21-28 4 Raft, M. C. (1989) Science 243, 1450-1455 5 McConnell, S. (1991) Annu. Rev. Neurosci. 14, 269-300 6 Holt, C. E., Bertsch, T. W., Ellis, H. M. and Harris, W. A. (1988) Neuron 1, 15-26 7 Turner, D. L., Synder,E.Y. and Cepko, C. L. (1990) Neuron4, 833-845 8 Price,J., Turner, D. and Cepko, C. (1987) Proc. Natl Acad. Sci. USA 84, 154-160 9 Wetts, R. and Fraser,S. E. (1988) Science 239, 1142-1145 10 Williams, R. W. and Goldowitz, D. (1992) Proc. Natl Acad. Sci. USA 89, 1184-1188 11 Goldowitz,D. (1989) Neuron 3,705-713 12 Hinds,J. W. and Hinds, P. L. (1979) J. Comp. Neurol. 187, 495-512 13 Jessell, T. M. and Schacher, S. (1991) in Principles of Neuroscience (3rd edn) (Kandel, E. R., Schwartz, J. H. and Jessell, T. M., eds), pp. 887-907, Elsevier 14 Reh,T. A. and Tully, T. (1986) Dev. Biol. 114, 463-469 15 Adler, R. and Hatlee, M. (1989) Science 243,391-393 16 Young, R. W. (1985) Anat. Rec. 212, 199-205 17 W~ssle,H. and Reimann,R. J. (1978) Proc. R. Soc. Lond. Ser. B 200, 441-461 18 Negishi, K., Teranishi, T. and Kato, S. (1982) Science 216, 747-749 19 Hafen, E. and Basler, K. (1991) Development (Suppl. 1), 123-130 20 Anchan, R. M., Reh,T. A., Angello, J., Balliet,A. and Walker, M. (1991) Neuron 6, 923-936 21 Dr~ger, U. C., McCaffery, P. and Tempst, P. (1991) Soc. Neurosci. Abstr. 17, 186 22 Angler, N. (1992) 'A brain cell surprise: genes don't set function' in New York Times, January28 23 Jacobson, M. and Moody, S. A. (1984) J. Neurosci. 4, 1361-1369 24 Buss,L. W. (1987) The Evolution of Individuality, Princeton University Press 25 Turner, D. L. and Cepko, C. (1987) Nature 328, 131-136 26 LaVail, M. M., Rapaport, D. H. and Rakic,P. (1991)J. Comp. Neurol. 309, 86-114 27 Stent, G. S. (1985) Philos. Trans. R. 5oc. Lond. Set. B 312, 3-19

Acknowl~l&ement$ We thankS.Moody andS.Huan&for sharingwith usa draft of theirpaperon clonesin Xenopus retina before publication. We thank R. Wefts,M. Luskin, KadHerrupand the reviewersfor their

thoughtful

comments.Supported by the NationalEye Institute.

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