Proc. Natl. Acad. Sci. USAVol. 93, pp. 596-601, January 1996Colloquium Paper

This paper was presented at a coUloquium 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.

Molecular biology of retinal ganglion cellsMENGQING XIANG*t, HAO ZHOU*, AND JEREMY NATHANS*t#§Departments of *Molecular Biology and Genetics, tNeuroscience, §Ophthalmology, tHoward Hughes Medical Institute, Johns Hopkins University School ofMedicine, Baltimore, MD 21205

ABSTRACT Retinal ganglion cells are the output neuronsthat encode and transmit information from the eye to thebrain. Their diverse physiologic and anatomic properties havebeen intensively studied and appear to account well for anumber of psychophysical phenomena such as lateral inhibi-tion and chromatic opponency. In this paper, we summarizeour current view of retinal ganglion cell properties and posea number of questions regarding underlying molecular mech-anisms. As an example of one approach to understandingmolecular mechanisms, we describe recent work on severalPOU domain transcription factors that are expressed insubsets of retinal ganglion cells and that appear to be involvedin ganglion cell development.

This paper reviews our current knowledge of retinal ganglioncell structure and function with an emphasis on those areas inwhich molecular biological approaches may be expected toprovide new insights. We begin with an overview of thephysiological, anatomical, and psychophysical experimentsthat have revealed the diversity of ganglion cell properties andthe significance of that diversity for visual perception. Al-though little is currently known about the molecular basis ofthis diversity, it is likely that many of the relevant moleculeswill be identified in the near future. As an illustration of onearea in which significant progress seems likely, we concludewith a description of recent work on transcription factors inretinal ganglion cells.

Physiological Properties of Retinal Ganglion Cells

Ganglion cells are the output units of the retina. Because theircell bodies and axons are relatively accessible, they were amongthe first vertebrate neurons for which single unit responseswere determined. In 1938 Hartline (1) recorded from individ-ual axons at the vitreal surface of the frog retina whilestimulating the retina with a spot of light. These seminalexperiments introduced the concept of a receptive field, definedby Hartline as the region of the retina that must be illuminatedin order to obtain a response in a given fiber. As shown in Fig. 1,these experiments also revealed a multiplicity of response prop-erties among retinal ganglion cells, including both activation andinhibition: "This diversity of response among fibers from closelyadjacent regions of the same retina is extreme and unmistakable;it does not depend upon local conditions of stimulation oradaptation, but appears to be an inherent property of theindividual ganglion cells themselves" (1).

In 1953 Barlow and Kuffler (2, 3) independently discoveredthat many ganglion cells have an antagonistic spatial organi-zation in which either an excitatory center is paired with aninhibitory surround or an inhibitory center is paired with anexcitatory surround. The center-surround organization filters

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.

the image by emphasizing spatial contrast. This type of spa-tially antagonistic filtering had been predicted in the 19thcentury by both Hering and Mach (4, 5) on psychophysicalgrounds, and it accounts for the illusory black dots seen in theHermann grid in Fig. 2. In the retinas of old world primates,many ganglion cells also relay chromatic information by re-porting either the difference between red and green coneinputs or the difference between blue cone input and a sum ofred and green (= yellow) cone inputs. For reasons that are notobvious, most ganglion cells of the red vs. green type have bothchromatically and spatially opponent receptive fields, whereasmost ganglion cells of the blue vs. red + green type have nearlycoextensive excitatory and inhibitory zones and therefore a muchsmaller degree of spatial opponency (6). The channeling ofchromatic information into two pathways with red vs. green andblue vs. yellow color opponent organization was deduced onpsychophysical grounds by Hering (4). It accounts for the chro-matic afterimages generated by selective desensitization of one oranother limb of the opponent processing system (Fig. 2).

Hartline's original recordings showed that in some ganglioncells a prolonged light stimulus evoked a steady response,whereas in others it evoked a transient (i.e., nonlinear) re-sponse (Fig. 1). The latter type of response filters the image byemphasizing temporal changes. Beginning in the mid-1960s,this distinction was systematically investigated in both cat andmonkey retinas (reviewed in refs. 6 and 7). In the cat two majorganglion cell types were identified and termed X and Y, theformer responding both spatially and temporally in a linearmanner and the latter responding nonlinearly (8). In primates,a similar dichotomy was found in temporal response proper-ties, with one class, now referred to as parvocellular or P-typeganglion cells, responding linearly, and a second class, nowreferred to as magnocellular or M-type ganglion cells, respond-ing nonlinearly (9). P- and M-type cells have been found todiffer in a number of properties. In general terms, P cells arecharacterized by relatively slow conduction velocities, insen-sitivity to small changes in luminance contrast, and high spatialresolution, especially near the fovea. Most P cells have achromatically opponent receptive field organization as de-scribed above. By contrast, M cells are characterized byrelatively fast conduction velocities, sensitivity to smallchanges in luminance contrast, and low spatial resolution. Mcells have achromatic center-surround receptive fields andtherefore detect luminance but not chromatic contrast. Thedistinction drawn between cat X and Y cells in spatial responseproperties does not appear to carry over to the primate P/Msystem as all P-type and most M-type ganglion cells show linearspatial summation (10). The distinct P and M systems appear

Abbreviations: IPL, inner plexiform layer; LGN, lateral geniculatenucleus.ITo whom reprint requests should be addressed at: 805 PreclinicalTeaching Building, 725 North Wolfe Street, Johns Hopkins Univer-sity School of Medicine, Baltimore, MD 21205.

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FIG. 1. Light responses obtained from isolated ganglion cell axons in the frog retina (reproduced from ref. 1). The interval between the regularmarks at the bottom of each trace correspond to 0.2 sec. The duration of illumination is indicated by the blackened portion of the strip near thebottom of each trace. The three cells reveal responses to the onset of illumination, the cessation of illumination, steady illumination, or variouscombinations of these. (Note: in trace A the apparent activity following cessation of illumination is from another cell.)

to represent a critical point at which the image is divided intoseparate and parallel streams.

Morphologic and Anatomic Properties of Retinal GanglionCells

From the earliest histologic studies of the vertebrate retina ithas been apparent that each major class of cells-photoreceptor, bipolar, horizontal, amacrine, and ganglion-contains within it morphologically distinct subtypes (11). Amajor theme during the past century of retina research hasbeen the identification of functional correlates for these mor-phologic differences (12). Among ganglion cells, one correla-tion that is now well established (and is perhaps not surprising)is between the area of the dendritic field and the area of thereceptive field, the former appearing to coincide with and todetermine the extent of the latter. Both dendritic field size andcell body size differ markedly between physiologically distinctganglion cell types. For example, in the cat, X and Y cellscorrespond, respectively, to the medium (,B) and large (a) celltypes, and in the monkey, P- and M-type cells correspond,respectively, to the small (midget) and large (parasol) cell types(reviewed in refs. 6 and 13). For P and M cells, both dendriticfield and soma size increase progressively with increasingretinal eccentricity, and this increase is matched by a corre-sponding increase in the size of the receptive field. Theeccentricity-dependent change in receptive field size accountsfor the absence of an illusory dark spot in the one intersectionof the Hermann grid upon which the observer fixates (Fig. 2).In the human retina, receptive field sizes have been measuredpsychophysically by determining the threshold for detection ofa small test flash in the presence of a superimposed circularbackground of varying diameter and constant brightness (14).When the superimposed background is confined to the exci-tatory center of a center-surround receptive field it producesa persistent activation, thereby decreasing the sensitivity of thecell to dim test flashes. When the superimposed background isenlarged so that it also includes the inhibitory surround, thelevel of persistent activation is reduced and the sensitivity ofthe cell approaches that seen with the test flash alone. Thispsychophysical measure closely matches the eccentricity-dependent size of primate M-type ganglion cell dendritic fields(15) and receptive fields (16).A second correlation between ganglion cell structure and

function relates the level at which the ganglion cell dendritesarborize in the inner plexiform layer and the inputs that the cellreceives. By examining the morphologies of individual gan-glion cells after recording their light responses, it was discov-ered that ganglion cells with OFF centers have dendritic arborsin the outer part of the inner plexiform layer (IPL), whereasganglion cells with ON centers have dendritic arbors in theinner part of the IPL (reviewed in ref. 12). Further subdivisionswithin the IPL are evident upon close examination of ganglion,bipolar, and amacrine cell dendritic morphologies (Fig. 3).

These are likely to be related, at least in part, to the segregationof chromatic inputs. In one well characterized example, theblue ON/yellow OFF color opponent type of ganglion cell hasbeen shown to be bistratified (17). One dendritic tree is locatedat that level in the inner part of the IPL where the processesof blue cone bipolar cells terminate, and the second dendritictree is located in the outer part of the IPL where it presumablyreceives inhibitory signals from bipolar cells driven by red andgreen cones.A third structure-function correlation can be seen in the

different projections made by retinal ganglion cells, with theresult that distinct aspects of the retinal image are delivered todifferent destinations in the brain (reviewed in refs. 6 and 7).The two principal projections from the retina are to themidbrain and to the dorsal lateral geniculate nucleus (LGN) ofthe thalamus, the latter projecting to the primary visual cortex.In amphibia and other lower vertebrates the midbrain projec-tion (the retinotectal pathway) constitutes the major outputpathway from the retina and mediates simple visually guidedbehaviors. In primates, the analogous pathway is devotedprincipally to the control of eye and head movements. Manyganglion cells that project to the midbrain exhibit receptivefields with a high degree of selectivity-for example, tomovement in a particular direction.

Ganglion cell axons navigate with extraordinary precision tocontact their appropriate targets within the brain. At the opticchiasm, most axons from the nasal but not the temporal halfof each retina cross the midline to follow the contralateraloptic tract. Central to the chiasm, ganglion cell axons in theprimate retinothalamic tract undergo further segregation.Axons from M-type ganglion cells project to the ventral twolayers of the LGN while axons from the P-type ganglion cellsproject to the dorsal four layers; axons derived from thecontralateral eye innervate the first, fourth, and sixth layers ofthe LGN, while those derived from the ipsilateral eye inner-vate the second, third, and fifth layers; and within each layerof the LGN the pattern of innervation generates a preciseretinotopic map that is aligned with each of the retinotopicmaps above and/or below it.

Molecular Biological Questions

The diversity of ganglion cell properties and the precision withwhich these properties are programmed invite numerous ques-tions regarding underlying molecular mechanisms. We listsome of these questions below.

(i) What determines the synaptic specificity of each ganglioncell for the various classes of bipolar and amacrine cells? Whatattractive or repulsive molecules determine the levels in theIPL where ganglion cells and the various classes of bipolar andamacrine cells synapse? What molecules determine the den-dritic field size for each type of ganglion cell?

(ii) How do different ganglion cell classes differ in the typesof neurotransmitters they use and in the properties and

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FIG. 2. Psychophysical demonstrations of chromatic and spatialsignal processing in the retina. (Upper) Spatial opponent processingdemonstrated by the Hermann grid. Viewing the figure at one-halfarm's length produces the illusion of gray dots at the intersectionsformed by four black corners. The effect can be understood withreference to excitatory center-inhibitory surround receptive fields.More light falls on the inhibitory annulus of a ganglion cell that has itsreceptive field centered over the image of an intersection compared toa ganglion cell that has its receptive field centered in the white spacebetween two adjacent black squares. Therefore, the former cell will beinhibited to a greater extent than the latter, with the result that thewhite area at the intersection will appear relatively dimmer. When thefigure is viewed at one-half arm's length, illusory gray dots are seen atall intersections except for the one upon which the observer fixates, aneffect that arises from the smaller receptive field sizes in the centralretina. (Lower) Color opponent processing demonstrated by theinduction of chromatic afterimages. To achieve the full effect, theviewer should fixate on the central black dot for ten seconds whilethe figure is illuminated by intense white light (e.g., sunlight). If theobserver then views a white piece of paper, an afterimage is seen inwhich each square appears as its opponent color. The effect occursbecause within the retinal region illuminated by each colored squarethose cones and/or cone pathways that were most strongly stimulatedwere selectively desensitized. The desensitization must occur withinthe retina because the afterimage appears to move in space as the eyemoves. Consistent with a retinal origin, if the figure is viewed with onlyone eye the afterimage will be confined to that eye. The observedafterimage colors reveal two systems for chromatic analysis: red vs.green and blue vs. red + green (= yellow).

regulation of their postsynaptic receptors? Do ganglion cellsexhibit physiological alterations in synaptic efficacy and, if so,by which mechanisms?

(iii) What are the identities of the guidance molecules thatlead ganglion cell axons across the retinal surface to the opticnerve, determine which axons cross the midline at the opticchiasm, direct different axons to the midbrain or thalamus (aswell as to other destinations), and produce the precise ar-rangement of synaptic contacts within the midbrain and LGN?

(iv) What genetic regulatory circuits distinguish retinalganglion cell types and how are these set up during develop-ment? How are the numbers of different ganglion cell typesdetermined, and what are the mechanisms by which thesediffer between species? How are the numbers and morphol-ogies of each type of ganglion cell programmed to vary as afunction of retinal eccentricity?

Transcription Factors in Retinal Ganglion Cells

Many of the questions posed above are under active investi-gation. As an illustration of one area in which some progresshas been made, we discuss below current work on the identi-fication and characterization of transcription factors that arelikely to be involved in controlling ganglion cell development.The specification of a final differentiated cellular phenotype

consists, in large part, of the selective transcriptional activationof particular genes. Work on myoblast differentiation in themouse (18) and on early embryonic development in Drosophila(19) suggests that this is accomplished by a combinatorialnetwork of interacting transcription factors. These act both tostably set the cell along a particular pathway of differentiationand to activate a battery of downstream genes, the products ofwhich are the structural proteins, enzymes, etc., that function-ally distinguish one cell type from another.A number of transcription factors have been localized to the

retina; most are also present in a variety of neural, and in somecases nonneural, tissues. Pax6 is the best characterized of thesefactors. It contains both a PAX domain and a homeodomainand is expressed in all or nearly all ocular tissues including thelens, iris, and retina (20). In mice, homozygous Pax6 mutantslack eyes and nasal primordia (21). In the heterozygouscondition, mutations in the murine Pax6 gene cause a small eyephenotype, and mutations in the human PAX6 gene causeaniridia (22, 23). SOHo-1, a homeodomain gene identified inchickens, is expressed in all layers of the developing retina aswell as in other sensory organs including the otocyst and dorsalroot and facial ganglia (24). Several homeodomain genes thatare highly homologous to the Drosophila NK-2 gene-Nkx2.2,TTF1, and DLb-are expressed in the developing retina and ina complex pattern in other regions of the developing centralnervous system (CNS) (25, 26). Isll, which contains both a LIMdomain and a homeodomain, is expressed in endocrine organs,in the brain and spinal cord, and in the retina in subsets of cellsin the inner nuclear and ganglion cell layers (27). ChxlO, ahomeodomain gene, is expressed in retinal neuroblasts but notin the developing ganglion cell layer; in the adult retina it isconfined to the inner nuclear layer (28). Two transcriptionfactors that do not contain homeodomains have been charac-terized in the retina. NRL, a member of the basic regionleucine zipper family, is expressed only in the retina, where itis present in most or all neurons (29). Mash-1, a member of thebasic region helix-loop-helix family, is expressed in manyregions of the developing CNS; in the developing retina, it ispresent in neuroblasts and is absent from the ganglion celllayer (30). A number of more ubiquitous transcription factorshave also been found in the retina but are unlikely to play a rolein distinguishing cell types.With respect to the generation and differentiation of retinal

ganglion cells, four POU domain transcription factors arelikely to be important, based on their expression in subsets of

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FIG. 3. Ganglion and amacrine cells in the dog retina (from ref. 11). A, B, and C, amacrine cells; a-i, ganglion cells.

ganglion cells in a variety of vertebrate retinas. The POUdomain family of transcription factors is defined by the pres-ence of a bipartite DNA binding domain consisting of aPOU-specific domain of -70 amino acids and a POU-specifichomeodomain of '60 amino acids, separated by a 10- to30-amino acid linker. More than 10 distinct POU domainfamily members have been identified thus far in vertebrates,including both ubiquitously expressed factors such as Oct-1and tissue-specific factors such as the pituitary-specific factorPit-1 (reviewed in ref. 31). Three of the four POU domainfactors implicated in ganglion cell development-Brn3a,Brn3b, and Brn3c-are highly homologous members of theclass IV POU domain subfamily. The fourth, RPF-1, is a newlydiscovered member of the class VI POU domain subfamily.The first member of the Brn3 subfamily was identified in

developing rat brain cDNA (32). Subsequent experiments ledto the identification and characterization of the three Brn3genes in mice (33-35) and in humans (36, 37). The Brn3proteins are closely related to Unc86, a protein involved in thedevelopment of sensory neurons in Caenorhabditis elegans (38,39). In the adult mouse, each Brn3 gene is expressed in a smallnumber of midbrain nuclei, in the dorsal root and trigeminalganglia, and in the retina. Within the retina, expression isconfined to subsets of cells within the ganglion cell layer (Fig.4). In cat and rabbit retinas, all of the Brn3-expressing cellsappear to be ganglion cells rather than displaced amacrine cells(which constitute an appreciable fraction of the cells in theganglion cell layer) as determined by double immunostainingwith AB5, an antibody previously shown to label only ganglioncells (40). In all retinas examined thus far, a characteristic andreproducible heterogeneity is observed in the intensity ofganglion cell immunolabeling. In the developing mouse retina,the Brn3 proteins are found in the ganglion cell layer beginningbetween embryonic days 12 and 15, the time at which this layerfirst separates from the underlying layer of dividing neuro-blasts (M.X. and J.N., unpublished).

In cat and macaque retinas, it has been possible to correlatethe pattern of expression of the Brn3 genes with the knownmorphologic and anatomic classes of ganglion cells (37). In thecat, Brn3a is found at high levels in small (y) ganglion cells andat lower levels in medium (,3) and large (a) cells; Brn3b isfound at high levels in all ganglion cells; and Brn3c is foundonly in small ganglion cells. A similar pattern is seen in themouse retina where Brn3a and Brn3b are present in '40% ofcells in the ganglion cell layer and largely colocalize; anti-Brn3cimmunoreactivity is present in '15% of cells in the ganglioncell layer and these constitute a subset of the cells that containBrn3a and Brn3b.

In the macaque retina, immunostaining also reveals colo-calization of Brn3a and Brn3b. The density of immunostainedcells in the ganglion cell layer falls steeply in going from thecenter to the periphery of the retina, a distribution that roughlymatches the overall distribution of retinal ganglion cells in theprimate retina (Fig. 5). Immunolabeling of macaque retinaefollowing retrograde tracing from the lateral geniculate nu-cleus shows high levels of Brn3a in a minority of P-typeganglion cells and low levels in all of the remaining P- andM-type ganglion cells. In the same retinae, high levels of Brn3bwere seen in nearly all P-type ganglion cells and low levels wereseen in nearly all M-type ganglion cells (Fig. 6). Brn3c-containing cells have not yet been mapped in the macaque

retina because of their low level of immunoreactivity withcurrently available antibodies.The Brn3 proteins are also expressed in the developing

dorsal root and trigeminal ganglia (33, 35), reminiscent of theexpression pattern of the chicken homeobox gene SOHo-1(24). In adult mice, each of the anti-Brn3 antibodies stains asubset of cells within these ganglia (37). Anti-Brn3a antibodieslabel most of the neurons; anti-Brn3b antibodies label <50%of the neurons; and anti-Brn3c antibodies label only occasionalneurons. The expression of these transcription factors in boththe somatosensory and visual systems is intriguing, given that

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FIG. 4. Anti-Brn3a, anti-Brn3b, and anti-Brn3c immunoreactivityin the mouse retina [reproduced with permission from ref. 37 (copy-right 1995, Society for Neuroscience)]. Immunoperoxidase stainingwith affinity-purified polyclonal antibodies specific for each of theBrn3 transcription factors (Left) and 4',6-diamidino-2-phenylindole(DAPI) staining of the same sections (Right). (A and B) Anti-Brn3a.(C and D) Anti-Brn3b. (E and F) Anti-Brn3c. Immunoreactive nucleiare found almost exclusively within the ganglion cell layer; the rareimmunostained cells found in the inner nuclear layer are presumed torepresent displaced ganglion cells. Note that the purple horseradishperoxidase reaction product partially quenches DAPI fluorescencewhen the two are present in the same nucleus. OS, outer segment; IS,inner segment; ONL, outer nuclear layer; OPL, outer plexiform layer;INL, inner nuclear layer; GCL, ganglion cell layer.

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FIG. 5. Distribution of anti-Brn3a immunoreactivity among gan-

glion cells in the central (A) and peripheral (B) macaque retina

[reproduced with permission from ref. 37 (copyright 1995, Society for

Neuroscience)].

both systems divide a complex sensory input into parallelstreams (41). These data suggest a homology in the develop-ment of these two sensory systems, based on a partial overlap

of transcriptional regulators.

RPF-1, the fourth POU domain sequence implicated in

ganglion cell development, was identified in human and mouse

genomic DNA and subsequently found in the human retina

where it is expressed in subsets of ganglion and amacrine cells

(H.Z. and J.N., unpublished data). As described above for the

pattern of immunostaining for the Brn3 factors, immunostain-

ing for RPF-1 shows a characteristic heterogeneity of nuclear

staining intensity. In the cat retina, the highest levels of RPF-1

are found in medium (13) and small (-y) ganglion cells; the large

(a) ganglion cells contain little or no RPF-1. In the macaque,

many ganglion cells contain low levels of RPF-1 and a minority

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FIG. 6. Anti-Brn3b immunolabeling of backfilled macaque retinalganglion cells [reproduced with permission from ref. 37 (copyright1995, Society for Neuroscience)]. A macaque retina was immuno-stained with anti-Brn3b after retrograde transport of Texas Red-conjugated dextran from an injection that included both the parvo-cellular and magnocellular layers of the LGN. (A) Anti-Brn3b immu-noreactivity visualized with horseradish peroxidase. (B) Texas Redfluorescence. Large arrow points to a large backfilled cell; small arrowspoint to two smaller backfilled cells.

of cells in the ganglion cell layer contain high levels of RPF-1.In contrast to the eccentricity-dependent decrease in overallcell density in the ganglion cell layer, the density of cells thatcontain high levels of RPF-1 changes little with retinal eccen-tricity.The most direct evidence that any of the POU domain

transcription factors play a role in ganglion cell developmentcomes from recent experiments in which the Brn3b gene hasbeen inactivated by homologous recombination in embryonicstem cells (M.X., J.N., L. Gan, and W. Klein, unpublisheddata). Mice that are homozygous for the mutant allele areviable but show specific defects in retinal structure. WhileBrn3b knockout retinae resemble those of the wild type inoverall structure, they have 70% fewer ganglion cells. Otherneurons within the retina and brain appear to be minimally ornot at all affected.An intriguing aspect of the Brn3 and RPF-1 immunolabeling

patterns is the characteristic heterogeneity in nuclear labelingintensity. This heterogeneity in levels of transcription factorssuggests that stable differentiated states may be determinednot only by the presence or absence of different transcriptionfactors but by the maintenance of these factors at particularintermediate levels. A graded mechanism of this general typehas been shown to mediate anterior-posterior fate determi-nation in the Drosophila embryo, in which case concentrationgradients of a small set of maternally derived regulatoryproteins determine the level of expression of a larger set oftarget genes at different positions in the embryo (19).The authors thank Dr. Stewart Hendry for helpful comments on the

manuscript. This work was supported by the National Eye Institute(National Institutes of Health) and the Howard Hughes MedicalInstitute.

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