Rod–Cone Interactions

PII: S1350-9462(01)00006-4

Rod–Cone Interactions:Developmental and Clinical Significance

Saddek Mohand-Said, David Hicks, Thierry Leveillard, Serge Picaud, Fernanda Portoand Jose A. Sahel*

Laboratoire de Physiopathologie Cellulaire et Moleculaire de la Retine, EMI 99-8 INSERM UniversiteLouis Pasteur, Clinique Medicale A, Hopitaux Universitaires de Strasbourg, 1 Place de l’Hopital 67091

Strasbourg Cedex, France

Abstract}During the last decade, numerous research reports have considerably improved our knowledge about thephysiopathology of retinal degenerations. Three non-mutually exclusive general areas dealing with therapeutic approacheshave been proposed; gene therapy, pharmacology and retinal transplantations. The first approach involving correction ofthe initial mutation, will need a great deal of time and further development before becoming a therapeutic tool in humanclinical practice. The observation that cone photoreceptors, even those seemingly unaffected by any described anomaly, diesecondarily to rod disappearance related to mutations expressed specifically in the latter, led us to study the interactionsbetween these two photoreceptor populations to search for possible causal links between rod degeneration and cone death.These in vivo and in vitro studies suggest that paracrine interactions between both cell types exist and that rods are necessaryfor continued cone survival. Since the role of cones in visual perception is essential, pending the identification of the factorsmediating these interactions underway, rod replacement by transplantation and/or neuroprotection by trophic factors oralternative pharmacological means appear as promising approaches for limiting secondary cone loss in currently untreatableblinding conditions. # 2001 Elsevier Science Ltd. All rights reserved

1. INTRODUCTION

Retinitis pigmentosa (RP), the leading cause ofinherited blindness in the developed world, is alsotermed rod–cone dystrophy. This terminology,

proposed decades ago, is now supported in mostsubtypes by an accumulating body of evidencefrom molecular genetics studies, establishing thatnot only are most mutations expressed exclusivelyin rods but also that secondary degeneration of

CONTENTS

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 451

2. Cone–rod interactions during development and differentiation. . . . . . . . . . . . . . . . . . 452

3. Sequential degeneration of rods and cones in animal and human conditions . . . . . . . . . . 453

4. Lessons from retinal transplantation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4574.1. RPE transplantation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4574.2. Neuronal transplantation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4584.3. Paracrine effect of retinal neuronal transplantation . . . . . . . . . . . . . . . . . . . . 4584.4. Indirect pharmacological neuroprotection of cones . . . . . . . . . . . . . . . . . . . . 461

5. Perspectives and conclusions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 463

Acknowledgements. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 464

References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 464

*Corresponding author. Tel.: +33-3-90-24-34-28; fax: +33-3-90-24-34-17; e-mail: [email protected].

Progress in Retinal and Eye Research Vol. 20, No. 4, pp. 451 to 467, 2001# 2001 Elsevier Science Ltd. All rights reservedPrinted in Great Britain1350-9462/01/$ - see front matter

genetically normal cones occurs almost invariablyafter rod depletion. Loss of cone-mediated lightadapted vision is actually the key event leading toblindness in these patients and therefore, preven-tion of cone cell death and by inference, retentionof cone function, might represent a very promisingtherapeutic approach for humans affected withthis currently untreatable group of diseases. Thissurvey reviews current knowledge and concepts ontrophic rod–cone interactions during developmentand disease and their clinical significance.

2. CONE–ROD INTERACTIONS DURING

DEVELOPMENT AND DIFFERENTIATION

Cell–cell interactions operate during cell speci-fication, patterning and differentiation of theretina in both invertebrates and vertebrates. Therole of cell–cell interactions and the signallingpathways involved in photoreceptor patterninghave been most extensively studied in Drosophila,in which the analytical power of genetics coupledwith the crystalline arrangement of the 750ommatidia within the compound eye have beenthe driving forces. A full treatment of the subject isbeyond the scope of the present article (reviewed inFreeman, 1997) and we will only mention themajor breakthroughs that have contributed to ourcurrent knowledge of insect photoreceptor devel-opment and differentiation. Fly eyes develop froma proliferating epithelial monolayer (the imaginaldisk), in which a groove (the morphogeneticfurrow) then starts to sweep anteriorly across thedisk, leaving rows of developing ommatidia in itswake. Ommatidial development is sequential witha gradient of increasing maturity. One of the majorbreakthroughs has been the demonstration thatthe hedgehog (hh) family of signalling molecules isintimately involved in controlling both the timingand fate of photoreceptor differentiation along thedeveloping morphogenetic furrow (Ma et al., 1993;Heberlein et al., 1993, 1995; Heberlein and Moses,1995). Several members of this family have beencloned, including sonic hh (Shh), desert hh (Dhh)and indian hh (Ihh). In addition, a transmembraneprotein present in hh responsive cells, patched(ptc), has also been described (Capdevila et al.,1994). Given the importance of hh in Drosophila

eye development, it was only natural that its roleshould be investigated in vertebrates. Studies inzebrafish showed that ectopic expression of Shhdisrupted optic stalk and retinal formation (Ekkeret al., 1995), and perturbation of the intracellularsignalling cascade through blocking protein kinaseA function prevented the formation of the opticstalk (Concordet et al., 1996). Deletion of Shhthrough homologous recombination led to similarphenotypes (Chiang et al., 1996). Jensen andWallace (1997) showed that Shh was mitogenicfor retinal precursors in reaggregate but notmonolayer mouse embryonic retinal cultures,leading to increased numbers of many cell types.Corresponding temporal and spatial expression ofptc was also observed, and their results hencesuggest a role for this pathway in regulating retinalcell numbers. Somewhat different results wereobtained by Levine et al. (1997) using a mixedmonolayer rat retinal culture system, in whichselective stimulation of rod photoreceptor differ-entiation was observed. Furthermore, this latterstudy demonstrated the presence of Ihh in theadjoining RPE, suggesting this may function insignalling between the RPE and photoreceptors.

There are many further examples of the role ofcell–cell interactions in determining cell fate withinthe retina. The epidermal growth factor receptor(EGFR) signalling pathway has been shown to bevery important in retinal development both ininvertebrates and vertebrates. Once again mostdetailed genetic analyses have been possible inDrosophila, where the crucial role of EGFR inretinal cell type determination and differentiationhas been intensively investigated (Baker andRubin, 1989; Freeman, 1996; Kumar et al.,1998). Within the rat retina, EGF can switch cellfates away from rods and towards Muller glia(Lillien, 1995). It has also been shown that EGFcan delay rod differentiation through stimulatingactivation of a transcription factor, Mash-1(Ahmad et al., 1998), and that EGF actuallyinduces rod cell death at later developmentalstages (Fontaine et al., 1998).

Considering in a stricter sense those interactionswhich may occur between cones and rods,substantial data are also available. In most species,cone cells are among the first retinal cell types toleave the cell cycle, whereas rods are generally

S. Mohand-Said et al.452

among the last to do so (Cepko et al., 1996). Theconcept that cones may constitute a defaultphotoreceptor phenotype came from studies byAdler (Adler and Hatlee, 1989; Repka and Adler,1992), in which examination of monodispersedcultures established from early embryonic chickretinas revealed high numbers of cones, whereasthis proportion decreased as a function of increas-ing embryonic age of the donor. Such datasuggested that the cone phenotype would form inthe absence of environmental cues, constituting adefault pathway. Somewhat similar conclusionswere reached by Harris and Messersmith (1992),using cultures of embryonic Xenopus retina inwhich two successive inductions were necessary tospecify rod fate. However, evidence for a ratherdifferent scheme of events is also emerging that roddifferentiation is a necessary prerequisite for conedifferentiation. In Drosophila, it has recently beenshown that specification of opsin expression in R7may occur autonomously, whereas this partner isresponsible for the decision of R8 (the founderphotoreceptor of the ommatidial unit and con-sidered analogous to cones) to express eitherinduced or default cell fates (Chou et al., 1999).Evidence from Stenkamp, Raymond and collea-gues suggests that highly ordered inductive inter-actions occur between cones and rods to organisethe differentiating photoreceptor mosaic in thegoldfish retina. They showed that the first photo-receptors to differentiate are in fact a precociouspopulation of rods, present in a small ventrona-sally located patch. Additional rods were recruitedin proximity to this region, extending graduallyacross the retina. Cone opsin mRNA expressioncommenced after that of rod opsin, and followed apredictable order of spectral cell types: redfollowed by green, blue and ultraviolet (Raymondet al., 1995; Stenkamp et al., 1996). So, despite thefact that cones leave the cell cycle earlier than rodsin fish as in many other species (Raymond andFernald, 1981), it appears to be the latter thattrigger and coordinate photoreceptor differentia-tion. The authors additionally suggested that theprecocious rod population may be distinct fromrods generated later (Stenkamp et al., 1996).Similar conclusions for two distinct rod photo-receptor populations were drawn for differentiat-ing rat retina (Morrow et al., 1998).

The sequence of cone differentiation is thoughtto be due to either lateral cell–cell interactions orliberation of diffusible molecules. An analogousbut controversial situation exists in mammals,since conflicting data in primate retinas haveindicated the initial expression of either red–greencone (Wikler and Rakic, 1991) or rod and bluecone opsin (Bumsted et al., 1997). Very recentstudies in mouse retina support the former,demonstrating that green cones represent a defaultpathway among the general cone population (Nget al., 2001).

Additional data in bovine retina also indicatethat rods may trigger the onset of cone function-ality. Although cones exit the cell cycle early in thedevelopment of the bovine retina, the transcrip-tional levels of two cone-specific messenger RNAs(red cone opsin, blue cone g sub-unit of cGMPphosphodiesterase) remain uniformly low formany weeks. It is not until the rod photoreceptorprecursors stop dividing much later in embryogen-esis and commence their own differentiation(increased transcription of rod opsin, rod b sub-unit of cGMP phosphodiesterase mRNAs) thatcones follow suit as though having waited for someinductive or permissive signal (DesJardin et al.,1993; van Ginkel and Hauswirth, 1994; van Ginkelet al., 1995). Finally, unidentified diffusible poly-peptides secreted by quail embryonic neural retinalcultures specifically stimulate differentiation of thecone homologue within pineal glands (Araki,1997). In summary to this section, one couldspeculate that crosstalk between rods and conesseems to be common place and occurs throughoutthe different steps of development, differentiationand maintenance.

3. SEQUENTIAL DEGENERATION OF RODS

AND CONES IN ANIMAL AND HUMAN

CONDITIONS

Mutations in virtually every identified protein ofphotoreceptor outer segments (OS) have beenfound associated with RP phenotypes. Theseinclude mutations in the visual pigment rhodopsin(Berson et al., 1991; Rosenfeld et al., 1992; Liet al., 1994a,b), enzymes of the phototransductioncascade [transducin a-subunit (Dryja et al., 1996),

Rod–Cone Interactions 453

guanylate cyclase (Perrault et al., 1996), cGMP-dependent phosphodiesterase (McLaughlin et al.,1993), arrestin (Fuchs et al., 1995)] and structuralor trafficking proteins [peripherin/rds, ROM-1(Kajiwara et al., 1994, Dryja et al., 1997),ABCR-RIM (Allikmets et al., 1997)].

Yet clues to understanding and eventuallycounteracting the events leading to photoreceptorcell death are still awaited. Most mutationsselectively affect rods. These cells, which areresponsible for peripheral and scotopic vision,die for unknown reasons by apoptosis (Changet al., 1993), a mechanism of programmed celldeath often related to growth factor deprivationand/or disorganisation of the cell cycle. Cones,which are involved in colour, photopic and highcontrast vision, are rarely affected directly by theidentified mutations. And yet, in many cases, thesecells degenerate secondarily to rods, accountingfor loss of central vision and blindness. Cideciyanet al. (1998) found that degeneration of cones wasobservable in patients harbouring rhodopsinmutations once rod degeneration (as determinedby electrophysiological criteria) exceeded 75%. Inthis study, examination of rod and cone dysfunc-tion in 18 different human rhodopsin mutationsdemonstrated that cone loss was spatially andtemporally correlated to that of rods. The samesequence has been described in many animalmodels with spontaneous or targeted mutationsleading to rod dysfunction and death.

Animal models of inherited retinal degenerationshow very similar aetiologies: the retinal degenera-tion (rd) mouse is a naturally occurring mutantstrain exhibiting mutations in the gene coding forthe b sub-unit of rod cGMP phosphodiesterase(Farber, 1995), as seen in some forms of humanRP (McLaughlin et al., 1993). In this animal, roddegeneration is rapid and practically completewithin one month of postnatal age, and is followedby more gradual progressive cone disappearance(Carter-Dawson et al., 1978). The retinal degen-eration slow (rds) mouse strain exhibits defects inthe gene coding for peripherin/rds, as observed inhuman RP (Jacobson et al., 1996; Cheng et al.,1997). Although photoreceptor OS formation ishighly perturbed, rod cell loss is much slower thanin the former mutant, occurring over manymonths rather than days. Nevertheless, rods will

eventually die by apoptosis (Chang et al., 1993).Many transgenic strains have been prepared inwhich abnormal rhodopsin genes have beeninserted, resembling the different forms of humanRP. These include mice (Kedzierski et al., 1997),rats (Liu et al., 1999) and pigs (Petters et al., 1997),and invariably rod cells degenerate and die as aresult of the mutation. In all these above examplesfor which data is available, subsequent to theinitial phase of rod cell loss there is a second waveof cone cell death. The rd mouse exhibits delayedcone degeneration following rod death (Carter-Dawson et al., 1978; Farber, 1995). Transgenicmice in which rod photoreceptors are ablated withtoxic transgenes show secondary cone defects(McCall et al., 1996). Transgenic pigs containingmutant rhodopsin genes reveal cone destructionparalleling that observed in rods (Petters et al.,1997). Huang et al. generated chimeric mice byaggregation of embryos of normal and transgenicmice expressing a mutant rhodopsin (Huang et al.,1993). In the chimeras, mosaicism was observed indifferent parts of the mice, including the retinas,where patches of photoreceptors containing thenormal gene were adjacent to patches expressingthe mutated gene. The degeneration of theseretinas was slower than in the matched transgeniccontrols, but was uniform, affecting the normal aswell as the mutant cells. (Huang et al., 1993).These findings point towards a potential mechan-ism of cell–cell interaction which will be furtherdiscussed below.

Irrespective of the mutation, the final commonpathway of rod degeneration is apoptosis; themechanism underlying cone death remains un-known. It is easy to imagine one of at least twonon-mutually exclusive scenarios to account forthis delayed cone loss. In the first, rod breakdownwould adversely affect neighbouring conesthrough non-specific environmental influences.The progressive disintegration of the surroundingfar more numerous rods might leave the coneouter segments more vulnerable to toxic insults.Rod cell death in RP retinas is usually accom-panied by changes in the neighbouring cones,including outer segment shortening, cytoplasmicdensification, axonal elongation, and ultimately,cone cell death (Li et al., 1996). Although rods dieby apoptosis which normally avoids release of


potentially toxic cellular metabolites which wouldcreate problems of poisoning or inflammation, andhence prevents extension of cellular breakdown tosurrounding regions of tissues, it seems never-theless possible that degeneration of the abundantrods (about 20-fold more numerous than cones inmany mammalian species including man) couldexert adverse effects upon the adjacent cones. Itwas suggested that cone cell death may betriggered by toxic products of rod cell degenera-tion (Bird, 1992). Certainly, if rods were releasinggeneral toxic by-products one would expect othernearby cell types, such as the immediately post-synaptic partners of photoreceptors, the bipolarcells, to die as well. Although there are somemodifications of inner retinal neurones followingphotoreceptor loss they do not undergo wide-spread death.

Among such secreted proteins, interphotorecep-tor retinoid binding protein (IRBP) is primarily aproduct of the rods, with less of a contributionfrom the cones (Hollyfield et al., 1985; Rodrigueset al., 1986; Porello et al., 1991). IRBP is a majorcomponent of the IPM (Bunt-Milam and Saari,1983) and is thought to transport retinoidsbetween the RPE and photoreceptors in the visualcycle (Saari, 1990). There is evidence that IRBPimmunoreactivity is decreased or absent in RPretinal regions where rod cells have been lost(Bridges et al., 1985; Rodrigues et al., 1985; Liet al., 1994b; Milam et al., 1996). A decrease inIRBP due to the death of rods as well as a loss ofthe RPE-recycled products of rod outer segmentshedding and degradation may contribute to theobserved cone outer segment shortening andultimately, the cone cell death found in RP retinasbecause of a supposed associated local deficiencyin vitamin A, as it is known that vitamin Adeficiency causes photoreceptor outer segmentshortening and cell death (Dowling and Gibbons,1961; Carter-Dawson et al., 1979).

Excitotoxicity could be also implicated in thedegenerative process, at least in the early stages ofthe disease. Biochemical analysis of the rd mouseretina showed high levels of glutamate in Mullerglial cells prior to the onset of photoreceptordegeneration (Fletcher and Kalloniatis, 1997).Preliminary data, obtained by our team, demon-strated a delay of photoreceptor degeneration in

the rd mouse model after AMPA glutamate-receptor inhibitor injections. These results explain,at least partially, the results obtained in the sameanimal model following Diltiazem (a calciumchannel blocker) treatment (Frasson et al., 1999).

Still, this mechanism of cone cell death shouldnot continue once all rods have disappeared.Furthermore, each cone is individually surroundedby an insoluble glycocalyx that forms a privilegedstructural microdomain linking each cone to anRPE cell. Death by toxicity may therefore act asan early limited event.

Changes in the structural and biochemicalmicroenvironment might also induce cone celldegeneration, e.g. loss of structural support byneighbouring rods, alterations of the interphotor-eceptor matrix which contains factors promotingcone survival (Hewitt et al., 1990). Recent electro-physiological analyses of transgenic pigs contain-ing mutant rhodopsin genes indicate failure ofcone circuitry maturation, localised to hyperpolar-ising cells post-synaptic to the middle wavelength-sensitive cones. These abnormalities occur prior toany alteration of cone photoreceptor physiology(Banin et al., 1999). These findings point towards apotential mechanism of cell–cell interaction thatcould be linked to the demonstration of intenseneurite sprouting by rods, horizontal and bipolarcells in post-mortem retinas from patients with RP(Milam et al., 1998; Fariss et al., 2000). Electronmicroscopy reveals that the rod neurites containmultivesicular bodies, along with numerous smallvesicles as found in normal rod synapses, but theydo not appear to form true synapses with innerretinal neurons (Li et al., 1995). Failure toestablish appropriate synaptic connections is awell known mechanism of neuronal degeneration(Oppenheim, 1991; Jacobson et al., 1997). Second-ary changes at the level of the RPE and of glialcells including microglia (migration, proliferation,etc.) have been constantly described and couldimpede proper PR development, differentiationand survival.

Another explanation, which if true could havefar-reaching implications, is that rods producesome kind of signal that is essential for maintain-ing cone viability, so that the disappearance ofrods for whatever reason would deprive the conesof this signal and trigger their degeneration.


The repercussion upon cones of pathogenicmechanisms initially affecting rods is not restrictedto rod–cone dystrophies. Although the pathogenicevents might be different in RP and maculardegenerations, the importance of rod survival andfunction for RPE and cone physiology and survivalis receiving increasing appraisal. This may even berelevant in the progression of age-related maculardegeneration (ARMD). Age-related macular degen-eration, the leading cause of blindness after 50 years,affects more than 8 million people in countries whereepidemiological data are available. In a minority ofpatients with neovascular complications, ablativetreatments (laser, photodynamic therapy, surgery orradiation therapy) can postpone the loss of centralvision for an average interval of 18 months. Notreatment is available for the more frequent atrophicform. The RPE, Bruch’s membrane, and the choroidare vitally important for the well-being of photo-receptors. It is the dysfunction and death ofphotoreceptors, through an atrophic process or aneovascular event, that account for the vision lossassociated with ARMD. Although most histopatho-logic and clinical studies incriminate RPE cells as theprimary site of lesions in ARMD, some data pointtoward initial photoreceptor damage at the level of adense rim of rods surrounding the fovea (58).

These include:

(a) Evidence by Curcio et al. (1993), (Curcioet al., 1996a, b), from several carefullyperformed quantitative post-mortem studies,showing early rod cell death in aging andARMD. In maculas of aged donors lackinggrossly visible drusen and pigmentary change(i.e., without ARMD), the number of cones inthe cone-dominated part of the macula wasstable, while the number of rods decreased by30% (Curcio et al., 1993). The greatest lossoccurs in the parafovea (1–3mm from thefovea or 3.5–108 from the point of fixation).The foveal cone mosaic of eyes with largedrusen and thick basal deposits appearedsurprisingly similar to that of age-matchedcontrols, and the total number of foveal coneswas normal (Curcio et al., 1996a,b). Incontrast, in the parafovea, cones appearedlarge and misshapen, and few rods remained.Furthermore, in eyes with late ARMD,

virtually all surviving photoreceptors in themacula were cones, a reversal of the normalpredominance of rods.

(b) The predictive value of scotopic functionaltesting in early ARMD (Sunness et al., 1989)and most recently by Owsley et al. (2000), inwhich regional differences in the retinal dark-adapted sensitivity loss of ARMD patientswere observed, incriminated rod vulnerability.Older patients with a normal fundus appear-ance have scotopic impairment greater thanphotopic impairment in 80% of the cases, andfurthermore, scotopic sensitivity throughoutadulthood declines faster than does photopicsensitivity (Jackson and Owsley, 2000). Withrespect to ARMD patients, 87% of themshowed the magnitude of mean scotopicsensitivity loss that exceeded the magnitudeof mean photopic sensitivity loss. The deficitin scotopic sensitivity in early ARMD wassignificantly most severe within 188 of fixationas compared to the age-matched controlswithout ARMD, suggesting that the emer-gence of regional sensitivity impairmentswithin the parafovea may be an early sign ofARMD.

(c) Abnormal kinetics of rod-function with agingand ARMD. In older adults with goodmacular health, the rod-mediated portion ofdark adaptation is significantly slower thanyounger adults (Jackson, 1999a). In earlyARMD patients, rod-mediated dark adapta-tion is much slower (13min on an average)than in normal age-matched controls (Jack-son, 1999b).

(d) Difficulty with activities performed at nightand under low illumination (e.g., driving, andreading) reported by older patients includingthose with ARMD (Kuyk and Elliott, 1999).

(e) Identification of mutations in rod photorecep-tors in retinal dystrophies displaying RPEalterations (Allikmets et al., 1997; Zhang etal., 1999). Heterozygous mutations in theABCR gene have been identified in 26 out of167 unrelated ARMD patients with age-related macular degeneration (Allikmets etal., 1997). Recessive mutations in the samegene lead to Stargardt disease, a form ofmacular dystrophy with early onset.


The sequence of events encountered in RP (i.e.cones die after rods) might, therefore, also berelevant to ARMD. In both conditions, preventionof cone cell death represents a very worthy andpromising approach. Recent circumstantial evi-dence and experimental findings suggest that notonly do such rod–cone interactions exist but thatwe can moreover intervene to limit or preventsecondary cone death.

4. LESSONS FROM RETINAL

TRANSPLANTATION

During the last decade, numerous animal studiesconcerning retinal transplantation have beenpublished, and preliminary results led to humantrials beginning in 1994. However, many majorquestions remain unresolved and a lot of experi-mental work is needed to conceive coherentstrategies founded on scientific data in order tojustify this new therapeutic approach and tovalidate it clinically. Within the retinal transplan-tation field, two different strategies can bedistinguished, the first represented by RPE graft-ing which is proposed to exert beneficial effectsupon the apposing photoreceptors, and the secondby retinal neuronal transplantation hoping todirectly replace defective tissue.

4.1. RPE transplantation

Publications concerning photoreceptor survivalafter RPE transplantation in animal models arenumerous. Such surgery has already been per-formed in humans, although no objective func-tional or anatomical benefits have yet beenreported. Some authors recommend this strategyas an adjuvant treatment in the surgery ofneovascular membranes in complicated forms ofthe ARMD.

The first promising results were observed in theRCS rat, which carries a genetic disorder localisedwithin the RPE manifested as phagocytic failureand rod degeneration (the mutation has recentlybeen identified as a receptor tyrosine kinase, Mertk(D’Cruz et al., 2000)). Using this model of retinaldegeneration, Li and Turner (1988), Sheedlo et al.(1989) and Gouras and Lopez (1989) noted

considerable delay in host retinal photoreceptorloss following transplantation of healthy RPE, andobserved a correlation between the density ofgrafted cells and the number of surviving photo-receptors. 48 hours after surgery, phagosomalmaterial was seen and normal metabolic para-meters could be measured (Sheedlo et al., 1989,1991). Similar results were reported when humanRPE was transplanted into the RCS retina. Someevidence of improvement in functional parameterswas also recorded by the group of Coffey andLund (Whiteley et al., 1996), and by Jiang andHammasaki (1994). The effects provided by thesetransplantations in the RCS rat retina couldpossibly be due to the release of trophic factors,since the extent of photoreceptor survival exceededthe borders of transplanted RPE. Some authorshave reported defects in FGF receptor numbers inthe RCS rat (Malecaze et al., 1993), and othershave reported that the FGF can stimulate OSphagocytosis (McLaren and Inana, 1997). It hasbeen known for many years that protective effectsare exerted by FGF-2 in the RCS rat (Faktorovichet al., 1990), and other neurotrophic factors suchas CNTF, BDNF and interleukin-1b are alsoknown to improve photoreceptor survival inanimal models of phototoxicity and murinemutants (LaVail et al., 1992, 1998). The recentidentification of the gene defect in RCS rats andsome human RP patients as the receptor tyrosinekinase, Mertk, lends further weight to the possi-bility that RPE exerts trophic effects upon photo-receptors (D’Cruz et al., 2000; Gal et al., 2000).

Nevertheless, the RPE transplantation presentssome drawbacks, and application to humanpathology should await progress in our under-standing of the basic science prior to therapeuticaluse. If beneficial effects are mediated by trophicfactors, such effects sometimes vary from onemodel to another. For example, FGF-2 promotesrat rod differentiation (Hicks and Courtois, 1992)and survival (Fontaine et al., 1998) in vitro, while itinduces rod apoptosis in chicken photoreceptors(Yokoyama et al., 1997). Furthermore, the resultsobserved in RCS rat cannot easily be transposed tohuman retinal disease because there is for themoment no strict equivalent. Another importantprecaution in the RPE transplantation is the needfor permanent immunosupressive therapy to


override rejection phenomena of these immune-competent cells and to permit graft survival.

4.2. Neuronal transplantation

Neuronal retinal transplantation has beengeared essentially towards combatting inheritedretinal dystrophies such as RP, and transplanta-tion of embryonic or adult retinal cells is used withfunctional recovery as its main aim. Most experi-mental data have been provided by the labora-tories of del Cerro, Aramant and Gouras. Theynoted that injections of embryonic dissociated cellsor embryonic retinal sheets into the subretinalspace survived for long periods and exhibited acertain degree of differentiation (del Cerro et al.,1985; Aramant et al., 1990; Gouras and Lopez,1989).

Recently, Seiler and Aramant recorded re-sponses of the grafted tissue after light stimulationSeiler et al., 1999), and the team of Lund reportedintegration of grafts with the host retina afterinjecting retinal cell suspensions from young donormice in the sub-retinal space of aged rd mice(Kwan et al., 1999). They noted a new synapticlayer at the graft–host interface, containing sub-stantial numbers of photoreceptor synapses thatmediated a simple light–dark preference (Kwan etal., 1999). Unfortunately, transplanted embryonictissue develops many rosettes similar to those seenin retinoblastomas and retinal dysplasias. Toavoid this, Ehinger used an approach consistingof implantation of entire retinas. In this case,tissue organisation was respected, but connectionswith the host retina were prevented through thepresence of all retinal layers. Most recently, hereported a long-time survival (10 months) of full-thickness embryonic transplants with normallaminated morphology development in the hostrabbit retina (Ghosh et al., 1999). In this mostrecent study, a few direct synaptic contactsbetween graft and host neuronal types wereobserved.

The majority of these studies were done usingsmall animal models, essentially rats or mice. Theinconvenience of these models is their small size,making grafting surgery sometimes uncertain withfrequent traumatic complications inducing greatvariability in the data. In addition, ocular func-

tional testing is complex and provides non-inter-pretable data. Fortunately, other larger animalmodels are actually available, namely a strain oftransgenic pig exhibiting dominant mutations ofthe rhodopsin gene (Petters et al., 1997). Thisanimal not only carries a similar retinal disease tohuman RP sufferers, but its size allows comfor-table, reproducible surgery and easy and reliablefunctional testing.

4.3. Paracrine effect of retinal neuronal transplantation

Although the concept of photoreceptor and/orRPE surgical replacement in retinal diseases isattractive, many functional aspects of the trans-planted tissue and its integration into the complexvisual chain have to be resolved before its routineapplicability becomes feasible. An alternativescheme which does not require reconstruction offunctional pathways hypothesises that trans-planted rods act as reservoirs of trophic factorsnecessary for cone survival. The end goal of suchan hypothesis is naturally the purification andidentification of such molecules, but in the short tomid-term suggest that transplantation could beuseful in limiting cone degeneration.

We studied the role of cellular interactions in themechanism of secondary cone degeneration ininherited retinal degenerations in which themutation specifically affects rods. Rod degenera-tion caused by a mutation in the b sub-unit ofrod cGMP phosphodiesterase has been describedin both the retinal degeneration (rd) mouse andin human RP. In the rd mouse, a differential effectof the mutation on rods and cones was demon-strated by Carter-Dawson et al. i.e. cones die afterrods (Carter-Dawson et al., 1978). This modelhence appeared appropriate to test the hypothesisof the dependence of cones on the viability ofrods.

Using this animal model, we performed in vivo(transplantations) and in vitro (co-culture) studies.The first results we obtained confirmed the link ofthe secondary cone loss to a lack of survivalfactor(s) provided or requiring the presence ofrods. Transplantation of rod-rich photoreceptorsheets, isolated by using the vibratome sectioningtechnique described by Silverman and Hughes(1989, Fig. 1), to the sub-retinal space of 5 week


old rd mice (age when >98% rods havedisappeared) induced a significant increase in hostcone survival 2 weeks after surgery (Fig. 2)(Mohand-Said et al., 1997). We observed a 30–40% increase of cone density in the central retinawhen compared to sham-operated eyes. In thisstudy, pre-labelling of the grafts in a sub-group ofthe animals allowed us to verify the reproducibilityof the surgical technique. The grafts were identi-fied in more than 90% of the cases. In addition,pre-labelling excluded the possibility that improve-ment in host cone survival could be due to aneventual colonisation of the host retina by the fewcones contained in the injected transplants. Thecomparison to sham-operated controls ruled outthe hypothesis of a non-specific effect related to arelease of inflammatory mediators induced by thesurgical trauma itself.

Fig. 1. Isolation and sub-retinal transplantation of a sheet of rod-rich photoreceptors. (a) Donor retina is flat mounted andstuck on a block of gelatin, photoreceptors facing down. (b) Inner retinal layers are progressively removed by the vibratome.(c) The remaining pure photoreceptor layer is removed, attached to a slice of gelatin. (d) Small grafts are prepared andloaded into the canula of the sub-retinal injector. (e) The canula is pushed into the sub-retinal space through a cornealincision and the irido-corneal sinus, where the transplant is injected (f). Reproduced by permission of the American Medical

Association. Reprinted from Arch. Opthalmol. 118, 807–811 (2000) # 2000 American Medical Association.

Fig. 2. Estimates of host rd mouse cone photoreceptorswithin the central field of paired transplanted and sham-operated animals two weeks after surgery, *: p50:05. Forthe following experiments, we have improved the methodof quantification in order to reduce the variability of the

results obtained in this study.


The fact that the effect was recorded in thecentral part of the host retina, far from the graftlocation, suggested the diffusible nature of themediator(s) of the survival effect. We developed anorgan co-culture model of whole retinas from 5week old rd mice separated from test tissues by asemi-permeable membrane (Fig. 3). Since previousstudies have shown that unequivocal demonstra-tion of neuroprotective effects on cones is renderedvery difficult by the extreme variability of cone cell

distribution across the mouse retina (LaVail et al.,1997), we developed a more reliable quantitativetechnique. Stereological counting is accepted asthe most unbiased and reproducible method forenumerating neurones in the central nervoussystem (Gundersen et al., 1988; Coggeshall andLekan, 1996). Its adaptation to the retina, basedon a pseudo-random sampling of the total retinalsurface (Fig. 4), allowed an accurate and repro-ducible estimation of the total number of immu-nochemically identified cones. This method greatlyreduced the large variation observed in otherstudies relying on the sampling of relatively fewtissue sections (Farber, 1995; LaVail et al., 1997).The precision of this counting method allows anaccurate estimation of cones and the detection ofsmall changes in their numbers. Using thisunbiased stereological approach, examination oforgan cultured retinas after one week in vitrorevealed significantly greater numbers of survivingcones, indicating prevention of around 50% of

Fig. 3. Co-culture system: dissociated retinal cells orretinal explants are cultured in the same medium as 5 weekrd mice retinal explants, in communication through a

semi-permeable insert.

Fig. 4. Stereological counting. (a) Systematic random sampling of the retinal surface for stereological counting. (b)Counting was performed using a stereological dissector. (c) Example of a counted retina: distribution of counted samples.


cone loss if cultured in the presence of retinascontaining rods (retinal cell cultures from youngnormal or rd mice, and retinal explants from adultnormal mice) as compared to controls or co-cultures with rod-deprived retinas (retinal explantsfrom aged rd mice) (Fig. 5) (Mohand-Saidet al., 1998). In addition, the fact that evennon-functional rods improved the survival ofcones (5 weeks rd retinas co-cultured with retinalcell cultures from 8 day old rd mice), represents aresult of capital importance. This justifies andopens other approaches aiming at delaying thesecondary cone loss by maintaining the non-functional rods alive. These approaches will bediscussed in the next chapter.

Recently, we have extended our studies oftransplantation effects on cone survival (Mo-hand-Said et al., 2000). In order to see whetherthe survival of host cones is due specifically to rodsor should be provided by other retinal cells, fivegroups of 5 week old rd mice were used; non-operated controls, those grafted with pure sheetsof rods isolated from young normal-sighted mice,those grafted with inner retinal cells from youngnormal donors, those receiving transplants ofentire retina from aged rd mice (rodless retina),and finally sham-operated controls receiving slabsof gelatin.

In parallel, different aspects of the procedureswere improved, especially the use of a calibratedtrephine for transplant preparation and the devel-

opment of a new sub-retinal injector based on amechanic, non-hydraulic, pressure which allowsgreater control of transplant insertion. The micewere allowed to survive two weeks post-surgery,sacrificed and the total cone numbers in theirretinas estimated by using the stereological meth-od. Only mice receiving rod-rich transplantsdemonstrated statistically significant greater conenumbers, with rescue of 40% host cones normallydestined to die during this period (Fig. 6). As in thefirst study, pre-labelling of the photoreceptortransplants with lipophilic fluorescent dyes con-firmed the success of the surgical procedure.Photoreceptor grafts were detected in 100% ofthe group receiving such transplants, and were ofvariable sizes and shapes, routinely appearing as asheet or scattered islands covering 510% of thetotal retinal surface. The grafts were located in themid- to far-periphery of the host retina. Label wasalways confined to the grafts, indicating that cellsdid not spread out to colonise host tissue (Fig. 7).The results of this study demonstrate that conesurvival depends specifically on rods and showedthat transplantation of normal rods allows neuro-protection against secondary cone death.

4.4. Indirect pharmacological neuroprotection of cones

The paracrine effect of rods on cone survivalsuggested that protecting non-functional rods mayalso allow cone survival. This hypothesis was

Fig. 5. (A) Histogram showing residual cone numbers in individual retinas of 7 week rd mice following the differentexperimental treatments. (B) The presence of a photoreceptor containing explants or cell cultures led to the significantreduction (�50%) of the cone population loss. (&) 5 week rd retinas, (&) control, ( ) 8 day C57 retinas, ( ) adult C57

retinas, ( ) 8 day rd, ( ) adult rd retinas.


supported by the observed cone neuroprotectionin the organ co-culture model described above.When 5 week old rd mouse retina was co-culturedin the presence of one week old rd mouse retinathat still contained many mutant rods, theirnumber of cones was increased by 40–50% withrespect to control experiments (Mohand-Saidet al., 1998). These observations suggest that rods,even those carrying function-compromising muta-tions but still viable, allow increased cone survival.Such a mechanism may have occurred in theprevious reports describing rod photoreceptorrescue with trophic factors (LaVail et al., 1998).In our search for rod neuroprotective molecules,we showed that the administration of GDNF canproduce a significant rod survival and delay theloss in cone function in the rd mouse (Fig. 8)(Frasson et al., 1999a). Another study from ourgroup has established, in the same animal model,

Fig. 6. (A) Histogram showing residual cone numbers in individual retinas of 7 week rd mice following the differentexperimental treatments. (B) Photoreceptor transplantation led to the significant protection of �40% of the cone populationnormally lost during this interval. (&) 5 week unoperated rd retina, (&) control (7 week unoperated rd retinas), ( ) retinas

transplanted with 8 day C57 photoreceptors or ( ) 8 day C57 Inner retinas, ( ) adult C57 retinas, ( ) gelatin.

3

Fig. 7. Immunolabelling of flat-mounted transplanted rdretinas, viewed from above. (A) This large graft was pre-labelled with PKH26-GL fluorescing orange/red (G),clearly visible against the host retina in which greenspecks (arrow) are PNA-labelled cones. (B) This graft waspresent as scattered islands of cells, labelled with anti-rodopsin antibody (red, G) visible against the host retinalbackground exhibiting green PNA-labelled cones (arrow).

Scale bar=20mm.


that a calcium-channel blocker can transientlyrescue rod and cone photoreceptors with apreservation of cone function (Fig. 9) (Frassonet al., 1999b). Photoreceptor survival has also beendemonstrated by others applying an anti-capsase-3inhibitor in transgenic rats with the rhodopsinmutation S334ter (Liu et al., 1999). These studiesindicate that targeting common molecular eventsin rod degenerating pathways may provide phar-macological treatments applicable to differentforms of diseases.

5. PERSPECTIVES AND CONCLUSIONS

Gene therapy, which theoretically represents themost logical and coherent approach to treatinginherited retinal degenerations, will take a longtime to become feasible and take its place amongthe usual therapeutic tools in medical practice. Inthe meantime, retinal transplantation could repre-sent a rapidly accessible alternative. The demon-stration by our studies of the paracrine effects oftransplanted rods on host cones in the rd mouse,

Fig. 8. (A) Rod numbers determined from transverse sections of GDNF, PBS, and non-injected rd retinas.*: comparisonbetween non-injected retinas and GDNF-or PBS-treated retinas, **: comparison between GDNF- and PBS-treated retinas.

(B) ERGs recorded from GDNF-injected and (Control) non-injected eyes.

Fig. 9. (A) Quantification of diltiazem-induced rod cell rescue in flat-mounted retinas of rd mice at post natal day 36. (B)Effect of Diltiazem on ERG b-wave. In all control untreated rd mice, the b-wave disappeared by post-natal day 24, whereas

an ERG signal could still be mesured in all diltiazem-treated rd mice on PND 24, and in some on PND 36.


the lack of immune response to photoreceptorsand the progress in retinal microsurgery (Kaplanet al., 1997) open the way to human clinical trials.

Rescue of cones might not be affected by thetype of mutation leading to rod cell death. This, inview of the number of mutations already describedin RP is no small advantage since in most patientsmutations do not affect cones. Many issues, suchas the long term survival of transplants, theduration of the effect and the functionality ofsurviving cones, remain currently unresolved.Larger animal models might provide decisiveclues. They will also offer the opportunity toextend these findings to different mutations anddifferent species (e.g. rat, pig).

The demonstration of the role of rods, eventhose that are non-functional, in cone survivalshould lead to designing strategies aimed atpostponing or blocking the death of rod cells,thereby improving the survival of cones. Bothtrophic factors (LaVail et al., 1998; Frasson et al.,1999a) and pharmacological agents (Liu et al.,1999; Frasson et al., 1999b) were already found tohave protective effects on rods. Cone survivalcould therefore be obtained through pharmacolo-gical approaches, possibly using already existingdrugs or gene delivery of trophic factors.

Acknowledgements}We would like to thank M. Simonutti fortechnical assistance, and the following organisations for theirgenerous financial support of this work: INSERM, LeMinistere de l’Education Nationale, de la Recherche et de laTechnologie, la Federation Nationale des Aveugles et Handi-capes Visuels de France, la Fondation de l’Avenir, l’AssociationFranc,aise contre les Myopathies, l’Etablissement Francais desGreffes, Retina France, Adret Alsace, Novartis, FoundationFighting Blindness.

REFERENCES

Adler, R. and Hatlee, M. (1989) Plasticity and differentiation ofembryonic retinal cells after terminal mitosis. Science243, 391–393.

Ahmad, I., Dooley, C. M. and Afiat, S. (1998) Involvement ofMash-1 in EGF-mediated regulation of differentiation inthe vertebrate retina. Dev. Biol. 194, 86–98.

Allikmets, R., Shroyer, N. F., Singh, N., Seddon, J. M., Lewis,R. A., Berstein, P. S., Peiffer, A., Zabriskie, N. A., Li, Y.,Hutchinson, A., Dean, M., Lupski, R. and Leppert, M.(1997) Mutation of the Stargardt gene (ABCR) in age-related macular degeneration. Science 277, 1805–1807.

Araki, M. (1997) Diffusible factors produced by cultured neuralretinal cells enhance in vitro differentiation of pineal cone

photoreceptors of developing quail retinas. Dev. BrainRes. 104, 71–78.

Aramant, R., Seiler, M., Ehinger, B., Bergstrom, A., Gustavii,B., Brundin, P. and Adolph, A. R. (1990) Transplanta-tion of human embryonic retina to adult rat retina.Restorative Neurol. Neurosci. 2, 9–22.

Baker, N. E. and Rubin, G. M. (1989) Effect on eyedevelopment of dominant mutations in Drosophilahomologue of the EGF receptor. Nature 340, 150–153.

Banin, E., Cideciyan, A. V., Aleman, T. S., Petters, R. M.,Wong, F., Milam, A. H. and Jacobson, S. G. (1999)Retinal Rod Photoreceptor-Specific Gene MutationPerturbs Cone Pathway. Development. Neuron. 223,549–557.

Berson, E. L., Rosner, B., Sandberg, M. A. and Dryja, T. P.(1991) Ocular findings in patients with autosomaldominant retinitis pigmentosa and rhodopsin, proline-347-leucine. Am. J. Ophthalmol. 111, 614–623.

Bird, A. C. (1992) Investigation of disease mechanisms inretinitis pigmentosa. Ophthalmol. Paed. Genet. 13, 57–66.

Bridges, C. D., O’Gorman, S., Fong, S. L., Alvares, R. A. andBerson, E. (1985) Vitamin A and interstitial retinol-binding protein in an eye with recessive retinitispigmentosa. Invest. Ophthalmol. Vis. Sci. 26, 684–691.

Bumsted, K., Jasoni, C., Szel, A. and Hendrickson, A. (1997)Spatial and temporal expression of cone opsins duringmonkey retinal development. J. Comp. Neurol. 78, 117–134, Erratum: J. Comp. Neurol. 380, 291.

Bunt-Milam, A. H. and Saari, J. C. (1983) Immunocytochem-ical localization of two retinoid-binding proteins invertebrate retinas. J. Cell Biol. 97, 703–712.

Capdevila, J., Estrada, M. P., Sanchez-Herrero, E. andGuerrero, I. (1994) The Drosophila segment polaritygene patched interacts with decapentaplegic in wingdevelopment. EMBO J. 13, 71–82.

Carter-Dawson, L. D., Kuwabara, T., O’Brien, P. J. and Bieri,J. G. (1979) Structural and biochemical changes invitamin A-deficient rat retinas. Invest. Ophthalmol. Vis.Sci. 18, 437–446.

Carter-Dawson, L. D., LaVail, M. M. and Sidman, R. L. (1978)Differential effect of the rd mutation on rods and cones inthe mouse retina. Invest. Ophthalmol. Vis. Sci. 17, 489–498.

Cepko, C. L., Austin, C. P., Yang, X., Alexiades, M. andEzzedine, D. (1996) Cell fate determination in thevertebrate retina. Proc. Natl. Acad. Sci. USA 93, 589–595.

Chang, G. -Q., Hay, Y. and Wong, F. (1993) Apoptosis : finalcommon pathway of photoreceptor death in rd, rds andrhodopsin mutant mice. Neuron 11, 595–605.

Cheng, T., Peachey, N. S., Li, S., Goto, Y., Cao, Y. and Naash,M. I. (1997) The effect of peripherin/rds haploinsuffi-ciency on rod and cone photoreceptors. J. Neurosci. 17,8118–8128.

Chiang, C., Litingtung, Y., Lee, E., Young, K. E., Corden,J. L., Westphal, H. and Beachy, P. A. (1996) Cyclopiaand defective axial patterning in mice lacking sonichedgehog gene function. Nature 383, 407–413.

Chou, W-H., Huber, A., Bentrop, J., Schulz, S., Schwab, K.,Chadwell, L. V., Paulsen, R. and Britt, S. G. (1999)Patterning of the R7 and R8 photoreceptor cells ofDrosophila: evidence for induced and default cell-fatespecification. Development 126, 607–616.

Cideciyan, A. V., Hood, D. C., Huang, Y., Banin, E., Li, Z. Y.,Stone, E. M., Milam, A. H. and Jacobson, S. G. (1998)Disease sequence from mutant rhodopsin allele to rod


and cone photoreceptor degeneration in man. Proc. Natl.Acad. Sci. USA 95, 7103–7108.

Coggeshall, R. E. and Lekan, H. E. (1996) Methods fordetermining numbers of cells and synapses: A case formore uniform standards of review. J. Comp. Neurol. 364,6–15.

Concordet, J. P., Lewis, K. E., Moore, J. W., Goodrich, L. V.,Johnson, R. L., Scott, M. P. and Ingham, P. W. (1996)Spatial regulation of a zebrafish patched homologuereflects the roles of sonic hedgehog and protein kinase Ain neural tube and somite patterning. Development 122,2835–2846.

Curcio, C. A., Medeiros, N. E. and Millican, C. J. (1996a)Photoreceptor loss in age-related macular degeneration.Invest. Ophthalmol. Vis. Sci. 37, 1236–1249.

Curcio, A., Millican, C. L., Allen, K. A. and Kalina, R. E.(1996b) Ageing of the human photoreceptor mosaic:evidence for selective vulnerability of rods in centralretina. Invest. Ophthalmol. Vis. Sci. 34, 3278–3296.

D’Cruz, P. M., Yasumura, D., Weir, J., Matthes, M. T.,Abderrahim, H., LaVail, M. M. and Vollrath, D. (2000)Mutation of the receptor tyrosine kinase gene Mertk in theretinal dystrophic RCS rat. Hum. Mol. Genet. 9, 645–651.

del Cerro, M., Gash, D. M., Rao, G. N., Notter, M. F.,Wiegand, S. J. and Gupta, M. (1985) Intraocular retinaltransplants. Invest. Ophthalmol. Vis. Sci. 26, 1182–1185.

DesJardin, L. E., Timmers, A. M. and Hauswirth, W. W. (1993)Transcription of photoreceptor genes during foetalretinal development. Evidence for positive and negativeregulation. J. Biol. Chem. 268, 6953–6960.

Dowling, J. E. and Gibbons, I. R. (1961) The effect of vitaminA deficiency on the fine structure of the retina. In Thestrucutre of the eye (ed. G. Smelser), pp. 85–99. AcademicPress, New York.

Dryja, T. P., Hahn, L. B., Kajiwara, K. and Berson, E. L.(1997) Dominant and digenic mutations in the peripher-in/rds and ROM1 genes in retinitis pigmentosa. Invest.Ophthalmol. Vis. Sci. 38, 1972–1982.

Dryja, T. P., Hahn, L. B., Reboul, T. and Arnaud, B. (1996)Missense mutation in the gene encoding the alphasubunit of rod transducin in the Nougaret form ofcongenital stationary night blindness. Nat. Genet. 13,358–360.

Ekker, S. C., Ungar, A. R., Greenstein, P., von Kessler, D. P.,Porter, J. A., Moon, R. T. and Beachy, P. A. (1995)Patterning activities of vertebrate hedgehog proteins inthe developing eye and brain. Curr. Biol. 5, 944–955.

Faktorovich, E. G., Steinberg, R. H., Yasumura, D., Matthes,M. T. and LaVail, M. M. (1990) Photoreceptordegeneration in inherited retinal distrophy delayed byfibroblast growth factor. Nature 347, 83–86.

Farber, D. B. (1995) From mice to men: The cyclic GMPphosphodiesterase gene in vision and disease. Invest.Ophthalmol. Vis. Sci. 36, 261–275.

Fariss, R. N., Zong-Yi, Li. and Milam, A. H. (2000)Abnormalities in rod photoreceptors, amacrine cells,and horizontal cells in human retinas with retinitispigmentosa. Am. J. Ophthalmol. 129, 215–223.

Fletcher, E. L. and Kalloniatis, M. (1997) Neurochemicaldevelopment of the degenerating rat retina. J. Comp.Neurol. 388, 1–22.

Fontaine, V., Kinkl, N., Sahel, J., Dreyfus, H. and Hicks, D.(1998) Survival of purified rat photoreceptors in vitrois stimulated directly by fibroblast growth factor-2.J. Neurosci. 18, 9662–9672.

Frasson, M., Picaud, S., Leveillard, T., Mohand-Said, S.,Dreyfus, H., Hicks, D. and Sahel, J. A. (1999a) Glial cellline-derived neurotrophic factor promotes functionalrescue of photoreceptors in a mouse model of inheritedretinal degeneration. Invest. Ophthalmol. Vis. Sci. 11,2724–2734.

Frasson, M., Sahel, J. A., Fabre, M., Simonutti, M., Dreyfus,H. and Picaud, S. (1999b) Retinitis pigmentosa: rodphotoreceptor rescue by a calcium-channel blocker in therd mouse. Nat. Med. 10, 1183–1187.

Freeman, M. (1996) Reiterative use of the EGF receptortriggers differentiation of all cell types in the Drosophilaeye. Cell 87, 651–660.

Freeman, M. (1997) Cell determination strategies in theDrosophila eye. Development 2, 261–270.

Fuchs, S., Nakazawa, M., Maw, M., Tamai, M., Oguchi, Y.and Gal, A. (1995) A homozygous 1-base pair deletion inthe arrestin gene is a frequent cause of Oguchi disease inJapanese. Nat. Genet. 10, 360–362.

Gal, A., Li, Y., Thompson, D. A., Weir, J., Orth, U., Jacobson,S. G., Apfelstedt-Sylla, E. and Vollrath, D. (2000)Mutations in MERTK, the human orthologue of theRCS rat retinal dystrophy gene, cause retinitis pigmento-sa. Nat. Genet. 26, 270–271.

Ghosh, F., Bruun, A. and Ehinger, B. (1999) Graft–hostconnection in long-term full-thickness embryonic rabbitretinal transplant. Invest. Ophthalmol. Vis. Sci. 40, 126–132.

Gouras, P. and Lopez, R. (1989) Transplantation of retinalepithelial cells. Invest. Ophthalmol. Vis. Sci. 30, 1681–1683.

Gundersen, H. J. G., Bendtsen, T. F., Korbo, L., Marcussen,N., Moller, A., Nielsen, K., Nyengaard, J. R., Pakken-berg, B., Sorensen, F. B., Vesterby, A. and West, M. J.(1988) Some new simple and efficient stereologicalmethods and their use in pathological research anddiagnosis. Acta Pathol. Microbiol. Immunol. Scand. 96,379–384.

Harris, W. A. and Messersmith, S. L. (1992) Two cellularinductions involved in photoreceptor determination inthe Xenopus retina. J. Neurosci. 9, 357–372.

Heberlein, U. and Moses, K. (1995) Mechanisms of Drosophilaretinal morphogenesis: the virtues of being progressive.Cell 81, 987–990.

Heberlein, U., Singh, C. M., Luk, A. Y. and Donohoe, T. J.(1995) Growth and differentiation in the Drosophila eyecoordinated by hedgehog. Nature 373, 709–711.

Heberlein, U., Wolff, T. and Rubin, G. M. (1993) The TGFbeta homologue dpp and the segment polarity genehedgehog are required for propagation of a morphoge-netic wave in the Drosophila retina. Cell 75, 913–926.

Hewitt, A. T., Lindsey, J. D., Carbott, D. and Adler, R. (1990)Photoreceptor survival-promoting activity in interpho-toreceptor matrix preparations: characterization andpartial purification. Exp. Eye Res. 50, 79–88.

Hicks, D. and Courtois, Y. (1992) Fibroblast growth factorstimulates photoreceptor differentiation in vitro. J.Neurosci. 12, 2022–2033.

Hollyfield, J. G., Fliesler, S. J., Rayborn, M. E., Fong, S. L.,Landers, R. A. and Bridges, C. D. (1985) Synthesis andsecretion of interstitial retinol-binding protein by thehuman retina. Invest. Ophthalmol. Vis. Sci. 26, 58–67.

Huang, P. C., Gaitan, A. E., Hao, Y., Peters, R. M. and Wong,F. (1993) Cellular interactions implicated in the mechan-ism of photoreceptor degeneration in transgenic miceexpressing a mutant rhodopsin gene. Proc. Natl. Acad.Sci. USA 90, 8484–8488.


Jackson, G. R., Edwards, D. J., McGwin, G. and Owsley, C.(1999b) Changes in dark adaptation in early AMD.Invest. Ophthalmol. Vis. Sci. 40, S739. Abstract nr 3911.

Jackson, G. R. and Owsley, C. (2000) Scotopic sensitivityduring adulthood. Vision Res. 40, 2467–2473.

Jackson, G. R., Owsley, C. and McGwin, G. (1999a) Aging anddark adaptation. Vision Res. 39, 3975–3982.

Jacobson, M. D., Weil, M. and Raff, M. C. (1997) Programmedcell death in animal development. Cell 88, 347–354.

Jacobson, S. G., Cideciyan, A. V., Maguire, A. M., Bennett, J.,Sheffield, V. C. and Stone, E. M. (1996) Preferential rodand cone photoreceptor abnormalities in heterozygoteswith point mutations in the RDS gene. Exp. Eye Res. 63,603–608.

Jensen, A. M. and Wallace, V. A. (1997) Expression of sonichedgehog and its putative role as a precursor cell mitogenin the developing mouse retina. Development 124,363–371.

Jiang, L. Q. and Hammasaki, D. (1994) Corneal electroretino-graphic function rescued by normal pigment epithelialgrafts in retinal degenerative Royal College of Surgeonsrats. Invest. Ophthalmol. Vis. Sci. 35, 4300–4309.

Kajiwara, K., Berson, E. L. and Dryja, T. P. (1994) Digenicretinitis pigmentosa due to mutations at the unlinkedperipherin/RD and ROM I loci. Science 264, 1604–1608.

Kaplan, H. J., Tezel, T. H., Berger, A. S., Wolf, M. L. and DelPriore, L. V. (1997) Human photoreceptor transplanta-tion in retinitis pigmentosa. A safety study. Arch.Ophthalmol. 115, 1168–1172.

Kedzierski, W., Lloyd, M., Birch, D. G., Bok, D. and Travis,G. H. (1997) Generation and analysis of transgenic miceexpressing P216L-substituted rds/peripherin in rodphotoreceptors. Invest. Ophthalmol. Vis. Sci. 38, 498–509.

Kumar, J. L., Tio, M., Hsiung, F., Akopyan, S., Gabay, L.,Seger, R., Shilo, B. -Z. and Moses, K. (1998) Dissectingthe roles of the Drosophila EGF receptor in eyedevelopment and MAP kinase activation. Development125, 3875–3885.

Kuyk, T. and Elliott, J. L. (1999) Visual factors and mobility inpersons with age-related macular degeneration. J. Re-habil. Res. Dev. 36, 132–303.

Kwan, A. S. L., Wang, S. and Lund, R. D. (1999)Photoreceptor layer reconstruction in a rodent model ofretinal degeneration. Exp. Neurol. 159, 21–33.

LaVail, M. M., Matthes, M. T., Ysumura, D. and Steinberg, R.H. (1997) Variability in rate of cone degeneration in theretinal degeneration (rd/rd) mouse. Exp. Eye Res. 65, 45–50.

LaVail, M. M., Yasumura, D., Matthes, M. T., Lau-Villacorta,C., Unoki, K., Sung, C. H. and Steinberg, R. H. (1998)Protection of mouse photoreceptors by survival factors inretinal degenerations. Invest. Ophthalmol. Vis. Sci. 39,592–602.

LaVail, M. M., Unoki, K., Yasumura, D., Matthes, M. T.,Yancopoulos, G. D. and Steinberg, R. H. (1992) Multiplegrowth factors, cytokines and neurotrophins rescuephotoreceptors from the damaging effects of constantlight. Proc. Natl. Acad. Sci. USA 89, 11249–11253.

Levine, E. M., Roelink, H., Turner, J. and Reh, T. A. (1997)Sonic hedgehog promotes rod photoreceptor differentia-tion in mammalian retinal cells in vitro. J. Neurosci. 17,6277–6288.

Li, L. and Turner, J. E. (1988) Transplantation of retinalpigment epithelial cells to immature adult rat hosts:short- and long-term survival characteristics. Exp. EyeRes. 47, 771–785.

Li, T., Adaman, M., Roof, D. J., Berson, E. L., Dryia, T. P.,Roessler, B. J. and Davidson, B. L. (1994a) In vivotransfer of a reporter gene to the retina mediated by anadenoviral vector. Invest. Ophthalmol. Vis. Sci. 35,2543–2549.

Li, T., Snyder, W. R., Olsson, J. E. and Dryja, T. P. (1996)Transgenic mice carrying the dominat rhodopsin muta-tion P347S: evidence for defective vectorial transport ofrhodopsin to the outer segments. Proc. Natl. Acad. Sci.USA 93, 14176–14181.

Li, Z. Y., Jacobson, S. G. and Milam, A. H. (1994b) Autosomaldominant retinitis pigmentosa caused by the threonine-17-methionine rhodopsin mutation: retinal histopathol-ogy and immunocytochemistry. Exp. Eye Res. 58,397–408.

Li, Z. Y., Kljavin, I. J. and Milam, A. H. (1995) Rodphotoreceptor neurite sprouting in retinitis pigmentosa.J. Neurosci. 15, 5429–5438.

Lillien, L. (1995) Changes in retinal cell fate induced byoverexpression of EGF receptor. Nature 377, 158–162.

Liu, C., Li, Y., Peng, M., Laties, A. M. and Wen, R. (1999)Activation of caspase-3 in the retina of transgenic ratswith the rhodopsin mutation s334ter during photorecep-tor degeneration. J. Neurosci. 19, 4778–4785.

Ma, C., Zhou, Y., Beachy, P. A. and Moses, K. (1993) Thesegment polarity gene hedgehog is required for progres-sion of the morphogenetic furrow in the developingDrosophila eye. Cell 75, 927–938.

Malecaze, F., Mascarelli, F., Bugra, K., Fuhrmann, G.,Courtois, Y. and Hicks, D. (1993) Fibroblast growthfactor receptor deficiency in dystrophic retinal pigmentedepithelium. J. Cell Physiol. 154, 631–642.

McCall, M. A., Gregg, R. G., Merriman, K., Goto, Y.,Peachey, N. S. and Stanford, L. R. (1996) Morphologicaland physiological consequences of the selective elimina-tion of rod photoreceptors in transgenic mice. Exp. EyeRes. 63, 35–50.

McLaren, M. J. and Inana, G. (1997) Inherited retinaldegeneration: basic FGF induces phagocytic competencein cultured RPE cells from RCS rats. FEBS Lett. 412,21–29.

McLaughlin, M. E., Sandberg, M. A., Berson, E. L. and Dryja,T. P. (1993) Recessive mutations in the gene encoding thebeta-subunit of rod phosphodiesterase in patients withretinitis pigmentosa. Nat. Gen. 4, 130–134.

Milam, A. H., Li, Z. -H. and Fariss, R. N. (1998) Histopathol-ogy of the human retina in Retinitis Pigmentosa. Prog.Retinal Eye Res. 17, 175–205.

Milam, A. H., Li, Z. Y., Cideciyan, A. V. and Jacobson, S. G.(1996) Clinicopathologic effects of the Q64ter rhodopsinmutation in retinitis pigmentosa. Invest. Ophthalmol. Vis.Sci. 37, 753–765.

Mohand-Said, S., Deudon-Combe, A., Hicks, D., Fintz, A. -C.,Simonutti, M., Leveillard, T., Dreyfus, H. and Sahel, J.A. (1998) Normal rod photoreceptors increase conesurvival in the retinal degeneration (rd) mouse. Proc.Natl. Acad. Sci. USA 95, 8357–8362.

Mohand-Said, S., Hicks, D., Leveillard, T., Dreyfus, H. andSahel, J. (2000) Selective transplantation of rods delayscone loss in a retinitis pigmentosa model. Arch. Ophthal-mol. 118, 807–811.

Mohand-Said, S., Hicks, D., Simonutti, M., Tran-Minh, D.,Deudon-Combe, A., Dreyfus, H., Silverman, M., Mo-singer, L., Ogilvie, J., Tenkova, T. and Sahel, J. (1997)Photoreceptor transplants increase host cone survival in


the retinal degeneration (rd) mouse. Ophthalmic Res. 29,290–297.

Morrow, E. M., Belliveau, M. J. and Cepko, C. L. (1998) Twophases of rod photoreceptor differentiation during ratretinal development. J. Neurosci. 18, 3738–3748.

Ng, L., Hurley, J. B., Dierks, B., Srinivas, M., Salto, C.,Vennstrom, B., Reh, T. A. and Forrest, D. (2001) Athyroid hormone receptor that is required for thedevelopment of green cone photoreceptors. Nat. Genet.27, 94–98.

Oppenheim, R. W. (1991) Cell death during development of thenervous system. Annu. Rev. Neurosci. 14, 453–501.

Owsley, C., Jackson, G. R., Cideciyan, A. V., Huang, Y., Fine,S. L., Ho, A. C., Maguire, M. G., Lolley, V. andJacobson, S. G. (2000) Psychophysical evidence for rodvulnerability in age-related macular degeneration. Invest.Ophthalmol. Vis. Sci. 41, 267–273.

Perrault, I., Rozet, J. M., Calvas, P., Gerber, S., Camuzat, A.,Dollfus, H., Chatelin, S., Souied, E., Ghazi, I., Leowski,C., Bonnemaison, M., Le Paslier, D., Frezal, J., Dufier,J. -L., Pittler, S., Munnich, A. and Kaplan, J. (1996)Retinal-specific guanylate cyclase gene mutations inLeber’s congenital amaurosis. Nat. Gen. 14, 461–464.

Petters, R. M., Alexander, C. A., Wells, K. D., Collins, B.,Sommer, J. R., Blanton, M. R., Rojas, G., Hao, Y.,Flowers, W. L., Banin, E., Cideciyan, A. V., Jacobson,S. G. and Wong, F. (1997) Genetically engineered largeanimal model for studying cone photoreceptor survivaland degeneration in retinitis pigmentosa. Nat. Biotech.15, 965–970.

Porello, K., Bhat, J. P. and Bok, D. (1991) Detection ofinterphotoreceptor retinoid binding protein (IRBP)mRNA in human and cone-dominant squirrel retinas byinsitu hybridization. J. Histochem. Cytochem. 39, 171–176.

Raymond, P., Barthel, L. K. and Curran, G. A. (1995)Developmental patterning of rod and cone photorecep-tors in embryonic zebrafish. J. Comp. Neurol. 359,537–550.

Raymond, P. and Fernald, R. (1981) Genesis of rods in teleostretina. Nature 271, 360–362.

Repka, A. and Adler, R. (1992) Differentiation of retinalprecursor cells born in vitro. Dev. Biol. 153, 242–249.

Rodrigues, M. M., Hackett, J., Gaskins, R., Wiggert, B., Lee,L., Redmond, M. and Chader, G. J. (1986) Interphotor-eceptor retinoid-binding protein in retinal rod cellsand pineal gland. Invest. Ophthalmol. Vis. Sci. 27,844–850.

Rodrigues, M. M., Wiggert, B., Hackett, J., Lee, L., Fletcher,R. T. and Chader, G. J. (1985) Dominantly inheritedretinitis pigmentosa. Ultrastructure and biochemicalanalysis. Ophthalmology 92, 1165–1172.

Rosenfeld, P. J., Cowley, G. S., McGee, T. L., Sandberg, M. A.,Berson, E. L. and Dryja, T. P. (1992) A null mutation inthe rhodopsin gene causes rod photoreceptor dysfunction

and autosomal recessive retinitis pigmentosa. Nat. Genet.3, 209–213.

Saari, J. C. (1990) Enzymes and proteins of the mammalianvisual cycle. In Progress in Regional Research, Vol. 9 (eds.N. N. Osborne and G. J. Chader) pp. 363–381. PergamonPress, Oxford.

Seiler, M. J., Aramant, R. B. and Ball, S. L. (1999)Photoreceptor function of retinal transplants implicatedby light-dark shift of S-antigen and rod transducin.Vision Res. 39, 2589–2596.

Sheedlo, H. J., Gaur, V., Li, L., Seaton, A. D. and Turner, J. E.(1991) Transplantation to the disease and damagedretina. Trends Neurosci. 14, 347–350.

Sheedlo, H. J., Li, L. and Turner, J. E. (1989) Functional andstructural characteristics of photoreceptor cell rescued inRPE-cell grafted retinas of RCS dystrophic rats. Exp.Eye Res. 47, 911–916.

Silverman, M. S. and Hughes, S. E. (1989) Transplantation ofphotoreceptors to light-damaged retina. Invest. Ophthal-mol. Vis. Sci. 30, 1684–1690.

Stenkamp, D. L., Hisatomi, O., Barthel, L. K., Tokunaga, F.and Raymond, P. (1996) Temporal expression of rod andcone opsins in embryonic goldfish retina predicts thespatial organization of the cone mosaic. Invest. Ophthal-mol. Vis. Sci. 37, 363–376.

Sunness, J. S., Massof, R. W., Johnson, M. A., Bressler, N. M.,Bressler, S. B. and Fine, S. L. (1989) Diminished fovealsensitivity may predict the development of advanced age-related macular degeneration. Ophthalmology 96,375–381.

Van Ginkel, P. R. and Hauswirth, W. W. (1994) Parallelregulation of foetal gene expression in different photo-receptor cell types. J. Biol. Chem. 269, 4986–4992.

Van Ginkel, P. R., Timmers, A. M., Szel, A. and Hauswirth,W. W. (1995) Topographical regulation of cone androd opsin genes: parallel position dependent levelsof transcription. Dev. Brain Res. 89, 146–149.

Whiteley, S. J., Litchfield, T. M., Coffey, P. J. and Lund, R. D.(1996) Improvement of the pupillary light reflex of RoyalCollege of Surgeons rats following RPE cell grafts. Exp.Neurol. 140, 100–104.

Wikler, K. C. and Rakic, P. (1991) Relation of an array ofearly-differentiating cones to the photoreceptor mosaic inthe primate retina. Nature 351, 397–400.

Yokoyama, Y., Ozawa, S., Seyama, S., Namiki, H., Hayashi,Y., Kaji, K., Shirama, K., Shioda, M. and Kano, K.(1997) Enhancement of apoptosis in developing chickneural retina cells by basic fibroblast growth factor. J.Neurochem. 68, 2212–2215.

Zhang, K., Garibaldi, D. C., Kniezeva, M., Albini, T., Chiang,M. F., Kerrigan, M., Sunnes, J. S., Han, M. andAllikmets, R. (1999) A novel mutation in the ABCRgene in four patients with autosola recessive Stargardtdisease. Am. J. Ophthalmol. 128, 720–724.


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Rod–Cone Interactions