Proc. Natl. Acad. Sci. USAVol. 93, pp. 589-595, January 1996Colloquium Paper

This paper was presented at a colloquium entitled "Vision: From Photon to Perception," organized by John Dowling,Lubert Stryer (chair), and Torsten Wiesel, held May 20-22, 1995, at the National Academy of Sciences, in Irvine, CA.

Cell fate deternination in the vertebrate retinaCONSTANCE L. CEPKO, CHRISTOPHER P. AUSTIN, XIANJIE YANG, MACRENE ALEXIADES, AND DIALA EZZEDDINEHoward Hughes Medical Institute and Department of Genetics, Harvard Medical School, Boston, MA 02115

ABSTRACT In the vertebrate central nervous system, theretina has been a useful model for studies of cell fate deter-mination. Recent results from studies conducted in vitro andin vivo suggest a model of retinal development in which boththe progenitor cells and the environment change over time.The model is based upon the notion that the mitotic cellswithin the retina change in their response properties, or"competence", during development. These changes presagethe ordered appearance of distinct cell types during develop-ment and appear to be necessary for the production of thedistinct cell types. As the response properties of the cellschange, so too do the environmental signals that the cellsencounter. Together, intrinsic properties and extrinsic cuesdirect the choice of cell fate.

The mechanisms that lead to specification of cell fates duringdevelopment are starting to come into focus. It is clear that acell responds to information from the environment and thatthis response depends upon the complement of genes ex-pressed within the cell. Since gene expression varies amongcells, some cells will interpret a particular environmental cueto give one fate, and another cell will interpret the same cueto give another. This is well illustrated by recent work on theresponse of neurepithelial cells to the secreted signaling mol-ecule sonic hedgehog (Shh). Neurepithelial cells in the ventralspinal cord respond to Shh by becoming floorplate or motorneurons (1, 2), while neurepithelial cells in the more rostralmesencephalon respond by becoming dopaminergic neurons(3). The ability of a cell to respond to a set of environmentalcues can be thought of as an aspect of its "competence". Thisis not to be confused with its potential, which is expressed overa greater time scale and/or through its progeny (4). Forexample, murine blastomeres clearly have the potential toproduce all of the cell types of a mouse (5). However, earlyblastomeres would be incompetent to respond to cues that, forexample, a retinal progenitor might respond to by making a rodphotoreceptor. While it is generally appreciated that theprocess of development leads to progressive restriction, orgradual loss of potency, development can also be viewed as aseries of changes in competence. In the central nervous system(CNS) of vertebrates, the retina is an accessible model systemwhich recently has served to highlight the changes in compe-tence that occur during production of a series of cell types andhas allowed a description of environmental factors that elicitresponses from competent retinal progenitors.

The Retina as a Model of CNS Development

The retina is a relatively simple, thin sheet of neural tissue thatlines the back of the eye. More has been learned about theanatomy, physiology, metabolism, and development of the

The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement" inaccordance with 18 U.S.C. §1734 solely to indicate this fact.

retina than any other CNS structure (6). This is due in part tothe relative simplicity and accessibility of the tissue. Moreover,it is a system where total control of the input signal can beachieved. Light is the stimulus for the photoreceptor cells, therods and the cones. Photoreceptors synapse with two types ofinterneurons, bipolar and horizontal cells. Further informationis extracted through synapses between bipolar cells and an-other class of interneurons, the amacrine cells. Finally, retinalganglion cells, the output neurons of the retina, transmit theresult of all of the information processing to various targetlocations within the brain.The retina must solve several problems during development

(7). The proper cell types in the proper ratios must beproduced, whereupon they must migrate to the proper layer,differentiate, and form synaptic connections. The earliest stepin this process, production of retinal cells, is by neurepithelialcells ("progenitors"), which line the former surface of theneural tube, forming a layer known as the retinal ventricularzone. Newly postmitotic cells leave the ventricular zone andmigrate relatively short and variable distances to one of threecellular layers. Newborn neurons, which make up six of theseven major classes of cells, then form synapses, almostexclusively with other retinal neurons. Effective informationprocessing and patterning is critically dependent upon theseearly processes. Control over the genesis of the different celltypes, as opposed to cell death, appears to contribute greatlyto achieving the correct ratios of cell types. For example,retinal ganglion cells make up 2.7% of cells in the adult mouseretina, whereas rod photoreceptors account for 70% of all cells(8, 9). Death occurs in -50% of ganglion, amacrine, andbipolar cells, whereas only 5% of photoreceptors die (10).Since the numbers of cells that die are relatively modestcompared to the differences in the final numbers of each celltype, much of the control of final cell numbers must be exertedthrough cell genesis rather than cell death. Many of the studiesof retinal development are focused on these early processes ofretinal development.Two aspects of the descriptive studies of retinal development

most salient for the following discussion of cell fate determi-nation are (i) the order of generation of retinal cell types and(ii) the lineal relationships among retinal cell types.

Retinal cells are generated in sequence, with the firstbecoming postmitotic as the optic cup forms. The order inwhich cell types are "born", as defined by the day in which theyundergo their last S phase, has been examined using [3H]thy-midine labeling and autoradiography (Fig. 1). The day onwhich a cell undergoes its last S phase allows a strong predic-tion to be made concerning the type of cell that it will become.It is not clear why there is an order and why the order is as itis for any given area or species, but it is a common feature ofdevelopment of the CNS. Some clues can be sought in thephylogenetic comparison of the order of birth of retinal cell

Abbreviations: CNS, central nervous system; En, embryonic day n; Pn,postnatal day n; NF, neurofilament; CNTF, ciliary neurotrophicfactor; LIF, leukemia inhibitory factor.

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FIG. 1. Order of birth of retinal cells in the mouse retina. A pulseof [3H]thymidine was administered to animals each day during devel-opment of the retina. Mature retinae were then processed for auto-radiography to reveal the labeled cells. Cells that were in S phaseduring the pulse would incorporate the label. Those that continued todivide would dilute the label, and those that underwent their last Sphase would retain the highest levels of label. By analyzing matureretinae for the presence of heavily labeled cells, the day of birth of eachcell type is revealed. The percentage of cells born on a given day thatare each type is shown on the ordinate. The data shown are for mouseretina. [Modified and reproduced with permission from ref. 11 (copy-right Wiley, New York).]

types in disparate species (12). It appears that there are someconserved aspects of the order of birth. For example, ganglioncells are the first born in many species. However, cones,horizontal cells, and amacrine cells can be born at about thesame time, although none before the first ganglion cells.Overlap in the birth of different cell types, and extremedifferences in the numbers of different cell types, precludesimple models in which there is a set order of recruitment of

different cells into the different cell fates, as in the develop-ment of the Drosophila retina (13, 14).

Lineal relationships among retinal cells have been defined.Several groups have performed lineage analysis of retinae ofvarious species using either intracellular injection of tracers orretroviruses (Fig. 2). These studies have yielded similar resultswith respect to clonal composition (15-19). In all species,retinal progenitors appear to be multipotent. Infection orinjection of mitotic retinal progenitors can produce clones withone to six cell types. Clones can also vary a great deal in termsof their size. In the rodent, clones composed of from 1 to 234cells have been observed from infection at embryonic day 14(E14).The multipotency of retinal progenitors appears to extend to

the last cell division. Clones of only two cells can consist of twodifferent cell types. For example, in the rodent retina, cells asdistinctive as rod photoreceptors and muller glia (the onlynonneuronal cell type generated by retinal progenitors) can bethe members of a two-cell clone (15). Even in the prenatalperiod of mouse development, two-cell clones can arise andconsist of two different cell types (16). The only apparentexception to this is that of rod photoreceptors in mice and rats.Rods account for 70% of the cells in the rodent retina, andthere are many multicellular clones (up to 33 cells in one clone)that are exclusively rods (15, 16). This makes possible thehypothesis that there is a committed, mitotic progenitor thatmakes only rods. Lineage analysis is a technique that cannotaddress this issue, and other studies, described below, wereundertaken to directly address it.The observations that distinctive retinal cell types can be

born at the same time and that retinal progenitor cells aremultipotent favor the role of extrinsic cues in directing cellfates. However, as mentioned above, intrinsic properties ofprogenitor cells must contribute to choice of cell fate as wellin that cells must be competent to respond to extrinsic cues toproduce the appropriate cell types. To begin to define thefactors making up the environment and the competence ofcells to respond to these factors, we and others have under-taken studies of cell fate determination using in vitro culturesystems (20-25). One of the major advantages of the retina forsuch studies is that it is fairly autonomous in its development.While many areas of the CNS are intimately intertwined withother areas of the CNS during development, the retina is not.It is separated from the rest of the CNS, connected only by the

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FIG. 2. Lineage analysis of rat retinal cells. P0 rat retinae (Left and Center) or an E14 mouse retina (Right) were infected in vivo withreplication-incompetent retroviruses encoding either 83-galactosidase (Left and Right) or human placental alkaline phosphatase (Center). Atmaturity, the retinae were processed histochemically to reveal the presence of infected cells, and cross-sections were made on a cryostat. Clonesof infected cells are arranged radially as a result of siblings migrating radially from the ventricular zone to their final location in the indicated layers.Cells were identified on the basis of their morphology and location within the retina. Retinae are shown with the photoreceptor outer segmentlayer at the top of the photograph. r, rod; b, bipolar cell; m, muller glial cell; g, ganglion cell; a, amacrine cell. [Left, reproduced with permissionfrom ref. 15 (copyright Macmillan Magazines); Right, reproduced with permission from ref. 16 (copyright Cell Press.]

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optic nerve, and is not dependent upon other areas of the CNSfor generation of any retinal cell type. The retina can beexplanted as an intact tissue and cultured such that the correctcell types are generated and differentiate to the point wherethe various layers of cell bodies and synapses form (23). Evendevelopment of the outer segments of photoreceptors, a veryelaborate and sensitive process, begins to occur in explantcultures. Alternatively, retinal cells can be dissociated andcultured as well-separated cells, either in tissue culture mono-layers (22, 25) or in three-dimensional collagen gels (21). Thelatter method has been used extensively by our laboratory toprobe the role of cell-cell interactions during development ofrod photoreceptors and ganglion cells.The studies described below were undertaken to answer

questions of retinal development at two levels. One levelconcerns the details of development of each cell type. Theother level concerns the overall scheme of retinal developmentand the intrinsic properties of retinal progenitors. In regard toan individual cell fate decision, for example, for rods, we askthe following questions. What, if any, environmental factorsare required for rod photoreceptor development? How do thefactors interact with each other and the cell to yield rods? Howmany steps are there to form a rod from a mitotic, multipotentprogenitor? Is there more than one type of progenitor capableof making rods, one of which is restricted to making only rods?Similar questions have been asked concerning generation ofganglion cells, amacrine cells, and bipolar cells. The overallgoal is to then integrate these findings to understand howintrinsic properties of progenitors contribute to the productionof each cell type. Do the original, totipotent retinal progenitorschange during development? If so, how? Do these changesindicate a gain or loss of competence to respond to theenvironmental cues defined by the studies of each cell type?Do they indicate loss of potency to make certain cell types? Apicture of the overall scheme of retinal development is begin-ning to emerge from these studies. Recent results will besummarized below, and a model based upon these findings willbe presented.

Rod Development

In rodents, rods are born primarily in the late embryonic andearly postnatal period (Fig. 1). The first known marker specificto differentiating rods is rhodopsin, the visual pigment of rodcells, composed of the apoprotein, opsin, and the chro-mophore, 11-cis-retinal. There can be a long delay betweenbirth of a cell fated to be a rod and opsin expression. Theearliest rods are born on E16 in the rat, but significant opsinexpression does not occur until about 8 days later, on postnatalday 2 (P2) (26). When the kinetics of opsin expression wasexamined for cells born on P1 in the rat, it was determined thatthe majority took more than 4 days to turn on opsin (ref. 27 andE. Morrow and C.L.C., unpublished results). These findingsmight suggest that the environment is limiting for opsinexpression in the embryonic period and that there is a set ofinteractions that takes longer than 4 days, even when theenvironment is permissive, as it is in the postnatal period.Watanabe and Raff (27) investigated the influence of the

environment on rod photoreceptor development in vitro. Theyfound that rat E15 cells mixed in vitro with a 50-fold excess ofP1 cells were unaltered in their timing of opsin expression.However, when opsin was expressed, it was expressed by40-fold more E15 cells than in the absence of P1 cells. Theseresults suggest that E15 cells did not respond to the cuesprovided by the postnatal environment until they reached theage at which they normally express opsin. The significance ofthis "age" may be that it is a state of competence that allowscells to respond to environmental cues. Cells apparentlycannot be hurried along the path to achieve this competence;the progression may be controlled solely by intrinsic factors, as

suggested by the authors. The other finding reported in thisstudy, that postnatal cells can raise the number of opsin-expressing cells among those that originate as E15 cells,suggests that environmental factors are limiting when E15 cellsmake it to this point on their own and that postnatal cellsproduce the limiting components.

In keeping with the findings of Watanabe and Raff (27), weobserved stimulation of the number of cells expressing opsinafter P2 by soluble factors produced by the retina. Thestimulators were low molecular weight and heat resistant (28).Candidates for the factors are taurine (28), a derivative ofcysteine, vasoactive intestinal peptide, a neuropeptide (J.LoTurco and C.L.C., unpublished results), and retinoic acid(29). Taurine and vasoactive intestinal peptide were found tobe additive in stimulation of the number of cells expressingopsin.

Differentiation of other retinal cells, such as ganglion cells(30) and amacrine cells (31), occurs immediately upon theirbirth (see below). Why is there such a lengthy delay betweenbecoming postmitotic and overt rod differentiation, and whendoes commitment to the rod fate occur? The definition ofcommitment is that the fate cannot be altered by differentenvironments. Exposure of cells to different environments andexamination of whether they still express the rod marker,opsin, is thus required to probe the issue of commitment. Wehave used growth factor treatment of cells in vitro for thispurpose. Although we were looking for stimulatory growthfactors, the factors we have defined to date are those thatinhibit rod development. We found that cells normally fated tobe rods according to their birthdates can be blocked fromdifferentiating as rods.The factors that have allowed this insight are ciliary neuro-

trophic factor (CNTF; ref. 32) and leukemia inhibitory factor(LIF; ref. 33). Treatment with either CNTF or LIF, whichshare a common receptor (34) and signal through the STATfamily of transcription factors (35), reveals that cells that arefated to be rods according to their birthday, but which are notexpressing detectable levels of opsin at the time of factoraddition, can be prevented from expressing opsin (D.E., X.Y.,J. LoTurco, and C.L.C., unpublished results). Treatment ofcells with CNTF or LIF leads to a 5-fold stimulation in thenumber of bipolar cells, and thus the cells that were fated tobe rods may become bipolar neurons. Cells that are expressingopsin at the time of CNTF or leukemia inhibitory factoraddition are resistant in that they continue to express opsin anddo not become bipolar cells. Similarly, treatment with epider-mal growth factor or transforming growth factor a, whichsignal through the epidermal growth factor receptor to stim-ulate the ras pathway (36), appears to block some opsin-negative cells from becoming opsin-positive. Finally, retinaland brain extracts contain an inhibitor of rod development thatsimilarly blocks development of opsin-positive cells (28) andstimulates bipolar development (D.E., X.Y., J. LoTurco, andC.L.C., unpublished results). This activity(ies) appears to bedistinct from CNTF or other ligands that stimulate STATphosphorylation (D. Feldheim and C.L.C., unpublished re-sults). In addition to these data concerning treatment withinhibitors, another culture condition, culture of dissociatedcells at low density, leads to a reduction in the number ofopsin-positive cells and an increase in the number of bipolarcells (21).

All of the above data are consistent with the followingmodel. Cells enter a state of competence where they cancommit to the rod fate. While in this state, they interact withboth inhibitory and stimulatory environmental factors to reacha decision concerning commitment to the rod fate. Commit-ment appears to occur in postmitotic cells, as cell division is notrequired for the changes that result from CNTF treatment.Immediately after commitment, cells synthesize detectablelevels of opsin protein. If they fail to become committed while

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in a state of competence for rod signals, due to either excessinhibition or insufficient stimulation, they may become bipolarcells. It is not clear whether this is the result of a binary decisionin the sense that cells are poised to become either a rod or abipolar. If so, then anything that favors one fate does so at theexpense of the other fate. Other alternatives, such as twodistinct states of competence, one for responding to factorssignaling commitment to the rod fate and one for respondingto signals for the bipolar fate, are also possible (as discussedbelow). In either case, the factors, if any, that would commitcells to the bipolar fate must not be limiting in the variousculture conditions.

Ganglion Cell Development

Issues concerning differentiation in response to environmentcues were investigated for ganglion cells, the first born retinalcell type (37). Cells were dissociated from E4 chickens, thetime when ganglion cell genesis is near its peak (38), and werecultured at various densities suspended in a collagen gel inserum-free medium for 24 hr. A significant overproduction ofcells expressing ganglion cell antigens was seen. While - 15%of the starting population expressed ganglion cell markers, upto 70% of the cultured cells expressed ganglion cell markersafter 24 hr. In vivo, the highest percentage of cells expressingthe markers was about 17%. The overproduction was inverselycorrelated with density, suggesting that inhibition was control-ling the production of cells expressing the markers. In addition,when cells were cultured as explants (intact retinae), there wasonly a small increase in the number of cells expressing themarkers, to about 20%. Coculture experiments and transfer ofconditioned medium did not lead to stimulation of markerexpression in explants or high-density gel cultures, leading tothe idea that contact-mediated inhibition was controlling theexpression of the markers.As the neurogenic gene Notch had been shown in Drosoph-

ila, Xenopus, and mouse to play a role in contact-mediatedinhibition of neurogenesis (39), we examined whether it playeda role in controlling ganglion cell genesis. Antisense and senseoligonucleotides directed against three different regions of theNotchl gene were individually injected into chicken eyes invivo. The antisense, but not the sense oligonucleotides, led toa 74% or greater overproduction of ganglion cells. In theperipheral retina, where development lags relative to centralretina, antisense oligonucleotides led to precocious develop-ment of ganglion cells. When Notchl RNA levels were exam-ined, Notchl RNA was decreased specifically. To examine ifthe opposite perturbation, an increase in Notch signaling,would reduce the number of cells differentiating as ganglioncells, an intracellular domain of Notch, shown to signal in aligand-independent manner in other systems, was transducedinto the retina with a retrovirus vector. Infection of earlyembryonic chicken eyes led to a 41-94% reduction in thenumber of ganglion cells relative to infection with a controlvirus.One additional line of evidence supports the notion that

Notch signaling is a controlling element in development ofganglion cells (37). One ligand for Drosophila Notch is the cellsurface molecule, Delta. As no chicken homologue of Deltawas available, we examined whether Drosophila Delta couldinhibit chicken ganglion cell genesis. Coculture of chickenretinal cells with a 50-fold excess of Drosophila cells expressingDelta led to only 9% of the chicken cells expressing ganglioncell antigens. This was not due to a nonspecific effect ofDrosophila cells or of generic cell-cell contact, as coculturewith Drosophila cells lacking Delta led to 76% of the chickencells expressing ganglion cell markers. Thus, at least in thiscase, Delta inhibited ganglion cell genesis, and presumably achicken ligand for Notch, such as the recently identified chickDeltal (40), plays a similar role in vivo.

We also addressed whether retinal cells from different agesvaried in their competence to produce ganglion cells whenplaced in a low-density collagen gel environment (37). Alow-density culture was made using cells taken from differentdays during retinal development, beginning with E2 andending with E7. As shown in Fig. 3, cells varied greatly in theirresponse to the low-density environment. Cells originatingfrom E2 and E7 were very poor in their response. The peakresponse was obtained from cells taken from E3-E6, whichcorresponds to the period of ganglion cell genesis in vivo. Asthe cultures were made with all of the cells from each age-thatis, progenitors and all postmitotic differentiating cells presentat a given age-the response to the low-density environmentcould have been due to the environmental signals carried intothe culture by the total population of cells. Alternatively, thediffering responses of the progenitor cells from the differentages could have been due to intrinsic differences in progenitorcells. Mixing experiments with cells from different agesshowed that the response to the low-density environment wasan intrinsic property of the progenitors in the low-densityenvironment. These data are consistent with the progenitorsgaining and then losing competence to produce ganglion cells.Are there molecular or biochemical markers that can be

used to indicate that a cell has the competence to become aganglion cell? An interesting observation concerning the ex-pression of NFs by mitotic progenitors of the chicken retinawas made several years ago by Sechrist (41) and might be a leadto such markers. Sechrist examined chicken retina for thepresence of NFs using silver staining and electron microscopy.He found that -10% of the cells in the mitotic region of theE3-E4 chicken retina expressed NFs. He further showed thatthese cells had recently incorporated [3H]thymidine. We madesimilar observations using antisera to the low molecular weightsubunit of NF to stain E5 chicken retinae pulsed for 1 hr with[3H]thymidine. We similarly found that 5-10% of cells in Sphase expressed this subunit of NFs (42). In the study shownin Fig. 3, the data regarding the expression of NFs by S phasecells taken from different ages is shown. Throughout theperiod of ganglion cell genesis in vivo (E3-E6), which is alsothe period when the cells appear to be competent to respondto the low-density environment to produce ganglion cells,

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FIG. 3. Progenitors are temporally regulated in their response tolow-density culture. Chicken retinae from E2 (stage 16), E3 (stage 20),E4 (stage 24), E5 (stage 27), E6 (stage 29), and E7 (stage 31) wereincubated as explants for 1 hr in [3H]thymidine at 5,tCi/ml (1 Ci =37 GBq) to label progenitor cells. They were then dissociated andcultured at low density (0.25 x 105/25,ul) in collagen gels for 24 hr,stained for the ganglion cell-specific marker, low molecular weightneurofilament (NF), and developed for autoradiography. 3H+ cellsthat were also NF+ were scored. "Before Culture", cells were fixed andstained immediately after dissociation; "After Culture", cells werecultured for 24 hr, then fixed and stained. The percentage of 3H-labeled cells that were NF+ is shown (mean ± SEM for threeexperiments). (Reproduced from ref. 37).

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there is a small percentage of mitotic cells that express NFs.This is clearly not a general feature of retinal progenitors, asvirtually no mitotic cells express NFs after E7. Similarly,Sechrist (41) reports that very few progenitor cells after E4expressed assembled NFs. Lineage analysis suggests that thereis no mitotic progenitor committed to making only ganglioncells, though the data on this issue are not robust since very fewganglion cells exist in the lineage data published to date.However, it is possible that the expression of the low molecularweight NF in a subset of retinal progenitors is a marker of thecompetence of those cells to produce ganglion cells. Themarker may be expressed only in a subpopulation of competentcells or may be expressed only in a portion of S phase in a largergroup of competent cells, given that 70% of E4 progenitors areable to respond to the low-density culture environment anddifferentiate as ganglion cells, but only 5-10% of S phase cellsexpress NF. The possibility that NF provides a marker ofcompetence for any cells is strenthened when consideration ofthe expression of amacrine and horizontal cell markers inembryonic rat cells is considered (discussed below).

Amacrine and Horizontal Cell Development

We recently embarked upon studies of the development ofamacrine cells within the rat retina. We began our studies usingtwo monoclonal antibodies, HPC (31) and VC1.1 (43), whichwere reported as markers of mature amacrine cells and alsopossibly of horizontal cells. HPC has been shown to react withsyntaxin (44), a synaptic vesicle docking protein (45), andVC1.1 has been shown to recognize an N-linked carbohydrate(46). One of the questions that we are seeking to answer in thestudy of each cell type is the time course of its differentiation.In staining embryonic retinae with these antibodies to answerthis question, we found that both antibodies stained cells in themitotic region of the retina. To determine whether the anti-bodies recognized mitotic cells, as opposed to newly postmi-totic cells leaving the ventricular zone, we labeled retinae ofdifferent ages with [3H]thymidine for 1 hr and performedimmunohistochemistry with HPC and VC1.1, followed byautoradiography (Fig. 4).A high percentage of S phase cells express HPC and VC1.1

from E14 to E18, the period of horizontal and amacrine cellgenesis. Are cells expressing these markers committed toproducing only amacrine cells, only horizontal cells, or a

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mixture of the two cell types? Lineage analysis suggests thatnone of these is the case (15, 16). Horizontal and amacrine cellsare found predominantly in clones with other cell types,including those that are born after E18.

If amacrine and horizontal cells are not the only progeny ofthe mitotic VC1.1 cells, is there any specificity in the types ofprogeny produced by this subpopulation of progenitors? Pre-liminary results suggest that the postmitotic daughters of E14,E16, and E18 VC1.1+ cells are almost always amacrine andhorizontal cells. However, classical birthdating experimentshave shown that cone photoreceptors and ganglion cells arealso being produced between E14 and E18 (8, 11). Thesefindings suggest that distinct progenitor types are biased intheir production of cell types, with embryonic VC1.1+ pro-genitors making amacrine and horizontal cells and VC1.1-progenitors making cone and ganglion cells. The bias may bea reflection of the competence of the subpopulations ofprogenitors to make the various types of progeny. Interest-ingly, mitotic daughters of VC1.1+ progenitors can be VC1.1+or VC1.1-, in keeping with the lineage analysis and the idea ofthe dynamic nature of competence.Markers expressed by other subpopulations of mitotic ret-

inal progenitor cells may allow further definition of compe-tence states. It appears that the receptor tyrosine kinase, flkl,is expressed on subsets of retinal progenitors (X.Y. and C.L.C.,unpublished results). f1k-1 is expressed on a small subset ofmature amacrines, and thus its expression in a relatively highpercentage of progenitors is presumably not of the samesignificance as expression ofVC1.1, syntaxin, or NFs. It is morelikely that flkl has a function in progenitors, perhaps in the cellfate or differentiation process. Other molecules whose expres-sion marks subsets of progenitors have been partially charac-terized (47-49), and still others will undoubtably be discov-ered, as expression patterns of many genes are being explored.Some of these will be transcription factors whose role may beto control or direct the response of cells to various signalingevents.

Model of Retinal Cell Fate Determination

Our current model is that retinal progenitors undergo a seriesof state changes in which a state is defined by the competenceto respond to environmental cues to produce one, or a few,particular cell types (Fig. 5). Each state of competence is

E14 E17 E20 P0 P3 P6Age

FIG. 4. VC1.1 is expressed on a subset of progenitor cells in a temporally regulated manner. Retinae explanted from rat embryos and neonatesat the indicated times were pulse labeled in vitro as explants for 1 hr with [3H]thymidine. They were then dissociated and stained with monoclonalantibody VC1.1 (43), which recognizes an N-linked carbohyrate, and processed for autoradiography. The percentage of 3H-labeled cells that wasreactive with VC1.1 antibody is indicated (mean ± SEM).

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endowed upon a cell by expression of a combination oftranscription factors. These factors may direct synthesis ofsurface receptors or elements in signal transduction cascadesso that a cell can respond to a particular set of cues. In addition,the transcription factors respond directly and/or direct theresponse to signal transduction cascades in order for differ-entiation to begin. A state of competence is transient. Itappears that when a cell moves from one state to the next, itcannot go back to a previous state, as discussed above con-cerning the competence of chicken retinal cells to makeganglion cells (Fig. 3 and ref. 37), as suggested by otherexperiments carried out in vitro (20, 50), and by transplantationin vivo (D. Fekete and C.L.C., unpublished results). Commit-ment is achieved when extrinsic factors allow stabilization ofthe network of transcription factors and/or lead to productionof a stable group of factors so that the cell is no longerdependent upon environmental cues to move forward in aprogram of differentiation. The transition from one state ofcompetence to the next may be due to extrinsic cues or anintrinsic program.The above hypothesis concerns specific signals between a

competent cell and its environment. Over the past few years,as many specific receptors and ligands have been identified, ithas been noted that signaling through these receptors triggersrelatively few signal transduction cascades. For example, theras cascade is triggered by most receptor tyrosine kinases (36,51, 52), and phosphorylation of the STAT family of transcrip-tion factors occurs as a result of signaling through the cytokinereceptors (35). In addition to the apparent convergence ofmany specific signals into these pathways, disparate cell typeshave another common signal transduction pathway that iscritical to differentiation. Signaling through the Notch/glp/linreceptor family has been shown to regulate differentiation inmany types of cells in both invertebrates and vertebrates (39).Finally, a recently described barrier to differentiation, repres-sion by the transcription factor, yan, has been hypothesized tocontrol differentiation in many types of Drosophila cells (53,54). As yan is downstream of ras and is a target of themitogen-activated protein kinase, a need to reduce yan activitycould explain the fact that differentiation of many cell types

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FIG. 5. A model for the behavior of progenitor cells in thedeveloping retina. Retinal progenitors are proposed to undergo a

series of changes in intrinsic properties. These properties are revealedby the competence of cells, or the ability to respond to environmentalcues to produce different retinal cell types. Each state is depicted bya distinct color in the figure. One state of competence might occur forproduction of each cell type, or perhaps cells can produce two or threecell types in a particular state of competence. The commitment of a

competent cell to become, or produce, a particular cell type iscontrolled by environmental signals. Movement of cells from one stateto the next appears to be in one direction only (see text).

involves stimulation of the ras/mitogen-activated protein ki-nase pathway (36, 51, 52). Although a vertebrate homologue ofyan has not been identified, yan is an ETS domain transcriptionfactor, and since a number ofETS domain transcription factorshave been found in vertebrate genomes (55), a yan homologuewill most likely be found.How does stimulation of a few common pathways lead to the

generation of so many types of cells? The developmentalhistory of each cell, which contributes to its state of compe-tence, has to be critical in the choice of cell fate. There mustbe a selection within the cell ofwhich genes will respond to thesignal transduction cascades. Such genes are just beginning tobe identified-for example, phyllopod in the Drosophila eye(56, 57). In addition, some of the genes that contribute tocompetence and/or control the response to extracellular cueshave been identified, such as the homeodomain gene, rough(58, 59), also in the Drosophila eye. Given the fairly limitednumber of signal transduction cascades identified to date, thecontribution of the developmental history and competence tothe generation of diversity cannot be overstated.

We thank David Cardozo, Eric Morrow, Michael Belliveau, Zheng-zheng Bao, David Feldheim, and Jeff Golden for helpful comments onthe manuscript and the past and present members of the CepkoLaboratory for stimulating discussions concerning the ideas and datadiscussed herein. The authors also gratefully acknowledge the help ofMichael Belliveau in the preparation of the figures.

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