Dual immunodetection and hybridisation in situ of opsin mRNA and rhodopsin protein in retinal sections

We describe a simple method for combining in situ hybridisation and immunohistochemistry on the same retinal section. The technique was developed using a radiolabelled cDNA probe for opsin and an antibody (ROS1F4) against rhodopsin. This method retains the antigenic sites if immunocytochemistry is performed prior to in situ hybridisation. Opsin mRNA was found in the photoreceptor inner segment with rhodopsin immunolocalised to the photoreceptor outer segments. The technique should be applicable to numerous situations including analysis of the sequence of events in the expression and synthesis of the various opsins during retinal development and degeneration.     

Iron-induced fluorescence in the retina: dependence on vitamin A

PURPOSE: Intravitreal iron injection induces fluorophore formation in the photoreceptor outer segments, followed by an accumulation of inclusions with lipofuscin-like fluorescence in the retinal pigment epithelium (RPE). The accumulation of RPE lipofuscin during aging is dependent on vitamin A availability. Experiments were conducted to determine whether iron-induced fluorophore formation in the outer segments and in RPE is also dependent on vitamin A, and thus whether oxidation promotes the participation of vitamin A in lipofuscin formation. METHODS: For 23 weeks, beginning at weaning, albino Fischer rats were fed diets containing vitamin A either in the form of retinyl palmitate (+A), which can be metabolically converted into the retinoids involved in vision, or retinoic acid (-A), which does not support visual function. After 23 weeks, when rhodopsin levels had decreased more than 90% in the -A rats, some animals in this group were given an intramuscular injection of all-trans retinol and were allowed to recover from retinoid deficiency for 7 days (-A+A). Animals in all three treatment groups were then given an intravitreal injection of ferrous sulfate. Both 1 day and 7 days after the iron injections, the retinas and RPEs were examined for fluorophores with excitation and emission properties similar to those of RPE lipofuscin fluorophores. RESULTS: In retina sections examined with fluorescence microscopy 24 hours after the ferrous sulfate treatment, the photoreceptor outer segments of rats in all of the treatment groups displayed a fluorescence with a blue emission maximum. This outer-segment fluorescence was not present in untreated eyes. The in situ outer-segment fluorescence was correlated with the appearance of blue-emitting fluorophores in organic solvent extracts of the retinas. One week after the iron injections, the RPE cells of the +A animals became filled with inclusions that displayed a golden-yellow fluorescence emission when excited by blue light. Very little of this lipofuscin-like fluorescence was observed in the RPE of the -A rats 1 week after iron treatment. However, in the -A rats that had been repleted with vitamin A, the ability of iron to induce the RPE fluorescence was restored. Several orange-emitting fluorophores were present in organic solvent extracts of the RPE-choroids of the +A rats. The amounts of these fluorophores were not appreciably affected by the iron treatment. These orange-emitting compounds were not observed in extracts of any eyes in the -A or -A+A groups. CONCLUSIONS: The results of this study suggest that oxidation of the photoreceptor outer-segment lipids generates blue-emitting fluorophores that are not directly involved in RPE lipofuscin fluorophore formation. The findings also indicate that retinoids are direct precursors of RPE lipofuscin fluorophores, and that oxidative stress to the retina promotes participation of vitamin A in the formation of some of the compounds responsible for RPE lipofuscin fluorescence.     

Evidence from normal and degenerating photoreceptors that two outer segment integral membrane proteins have separate transport pathways

Detachment of the neural retina from the retinal pigment epithelium induces photoreceptor degeneration. We studied the effects of this degeneration on the localization of two photoreceptor outer segment-specific integral membrane proteins, opsin and peripherin/rds, in rod photoreceptors. Results from laser scanning confocal microscopic and electron microscopic immunolocalization demonstrate that these two proteins, normally targeted to the newly-forming discs of the outer segments, accumulate in different sub-cellular compartments during photoreceptor degeneration: opsin immunolabeling increases throughout the photoreceptor cell's plasma membrane, while peripherin/rds immunolabeling occurs within cytoplasmic vesicles. The simplest hypothesis to explain our results is that these proteins are transported in different post-Golgi transport vesicles and separately inserted into the plasma membrane. More complex mechanisms involve having the two co-transported and then opsin finds its way into the plasma membrane but peripherin/rds does not, remaining behind in vesicles. Alternatively, both insert into the plasma membrane but peripherin/rds is recycled into cytoplasmic vesicles. We believe the data most strongly supports the first possibility. Although the transport pathways for these proteins have not been fully characterized, the presence of peripherin/rds-positive vesicles adjacent to the striated rootlet suggests a transport role for this cytoskeletal element. The accumulation of these proteins in photoreceptors with degenerated outer segments may also indicate that their rate of synthesis has exceeded the combined rates of their incorporation into newly forming outer segment disc membranes and their degradation. The accumulation may also provide a mechanism for rapid recovery of the outer segment following retinal reattachment and return of the photoreceptor cell to an environment favorable to outer segment regeneration.     

Disease sequence from mutant rhodopsin allele to rod and cone photoreceptor degeneration in man

Mutations in the gene encoding rhodopsin, the visual pigment in rod photoreceptors, lead to retinal degeneration in species from Drosophila to man. The pathogenic sequence from rod cell-specific mutation to degeneration of rods and cones remains unclear. To understand the disease process in man, we studied heterozygotes with 18 different rhodopsin gene mutations by using noninvasive tests of rod and cone function and retinal histopathology. Two classes of disease expression were found, and there was allele-specificity. Class A mutants lead to severely abnormal rod function across the retina early in life; topography of residual cone function parallels cone cell density. Class B mutants are compatible with normal rods in adult life in some retinal regions or throughout the retina, and there is a slow stereotypical disease sequence. Disease manifests as a loss of rod photoreceptor outer segments, not singly but in microscopic patches that coalesce into larger irregular areas of degeneration. Cone outer segment function remains normal until >75% of rod outer segments are lost. The topography of cone loss coincides with that of rod loss. Most class B mutants show an inferior-nasal to superior-temporal retinal gradient of disease vulnerability associated with visual cycle abnormalities. Class A mutant alleles behave as if cytotoxic; class B mutants can be relatively innocuous and epigenetic factors may play a major role in the retinal degeneration.     

Reduction of all-trans-retinal limits regeneration of visual pigment in mice

Absorption of photons by pigments in photoreceptor cells results in photoisomerization of the chromophore, 11-cis-retinal, to all-trans-retinal and activation of opsin. Photolysed chromophore is converted back to the 11-cis-configuration via several enzymatic steps in photoreceptor and retinal pigment epithelial cells. We investigated the levels of retinoids in mouse retina during constant illumination and regeneration in the dark as a means of obtaining more information about the rate-limiting step of the visual cycle and about cycle intermediates that could be responsible for desensitization of the visual system. All-trans-retinal accumulated in the retinas during constant illumination and following flash illumination. Decay of all-trans-retinal in the dark following constant illumination occurred without substantial accumulation of all-trans-retinal, generated by constant approximately equal to visual pigment regeneration (t1/2 approximately 5 and t1/2 approximately 7 min, respectively). All-trans-retinal, generated by constant illumination, decayed approximately 3 times more rapidly than that generated by a flash and, as shown previously, the rate of rhodopsin regeneration following a flash was approximately 4 times slower than after constant illumination. The retinyl ester pool (> 95% all-trans-retinyl ester) did not show a statistically significant change in size or composition during illumination. In addition, constant illumination increased the amount of photoreceptor membrane-associated arrestin. The results suggest that the rate-limiting step of the visual cycle is the reduction of all-trans-retinal to all-trans-retinol by all-trans-retinol dehydrogenase. The accumulation of all-trans-retinal during illumination may be responsible, in part, for the reduction in sensitivity of the visual system that accompanies photobleaching and may contribute to the development of retinal pathology associated with light damage and aging.     

Cell-type- and developmental-stage-specific metabolism and storage of retinoids by embryonic chick retinal cells in culture

Biological functions of retinoids in the vertebrate retina include the role of 11-cis retinaldehyde as visual pigment chromophore, and possible effects of retinoic acid in histogenesis and cell survival. Qualitative and quantitative regulation of retinoid availability for these complex processes could involve several cell types, including retinal pigment epithelium, Müller glia and retinal photoreceptors and non-photoreceptor neurons; their relative contributions, however, have not been fully elucidated. Using purified cultures, we have carried out a study of cell-type-specific metabolism and storage of retinoids in chick embryo retinal photoreceptors and other neuronal cells, as compared to those of retinal glia. Retinal glia were found to synthesize both retinoic acid and retinyl esters, and to hydrolyse the latter; they also displayed retinol dehydrogenase activities. Cultured neurons and photoreceptors also synthesized and hydrolysed retinyl esters; their capacity for retinaldehyde synthesis from a retinol or retinyl ester substrate suggested the presence of retinol dehydrogenase activity. Retinoic acid was not synthesized in differentiated neuronal cultures, although some synthesis was detectable at early culture stages when the cells were still morphologically undifferentiated. These findings indicate that cell-type-specific metabolic activities are expressed during retinal cell differentiation in vitro, and that embryonic retinal photoreceptors and nonphotoreceptor neurons are active participants in the metabolism and storage of retinoids.     

A comparison of the efficiency of G protein activation by ligand-free and light-activated forms of rhodopsin

Activation of the photoreceptor G protein transducin (Gt) by opsin, the ligand-free form of rhodopsin, was measured using rod outer segment membranes with densities of opsin and Gt similar to those found in rod cells. When GTPgammaS was used as the activating nucleotide, opsin catalyzed transducin activation with an exponential time course with a rate constant k(act) on the order of 2 x 10(-3)s(-1). Comparison under these conditions to activation by flash-generated metarhodopsin II (MII) revealed that opsin- and R*-catalyzed activation showed similar kinetics when MII was present at a surface density approximately 10(-6) lower than that of opsin. Thus, in contrast to some previous reports, we find that the catalytic potency of opsin is only approximately 10(-6) that of MII. In the presence of residual retinaldehyde-derived species present in membranes treated with hydroxylamine after bleaching, the apparent k(act) observed was much higher than that for opsin, suggesting a possible explanation for previous reports of more efficient activation by opsin. These results are important for considering the possible role of opsin in the diverse phenomena in which it has been suggested to play a key role, such as bleaching desensitization and retinal degeneration induced by continuous light or vitamin A deprivation.     

Dark adaptaion processes in the amphibian rod

Rod dark adaptation in the amphibian retina appears to be due to three processes: 1. background adaptation, occurring immediately after the extinction of an adapting or bleaching light, 2. intermediate adaptation, that frequently lasts 30 min or more and 3. opsin adaptation, which in the isolated retina where regeneration of rhodopsin is insignificant, is observed a a permanent loss of sensitivity after the completion of intermediate adaptation. Intermediate adaptation is characterized by a linear relation between log threshold and the amount of "retinal" present, a similar relation is obtained between log threshold and the amount of rhodopsin bleached in opsin adaptation. These adaptation processes are discussed in terms of a model of the rod outer segment.     

Chlamyrhodopsin represents a new type of sensory photoreceptor

In order to find optimal light conditions for photosynthetic growth, the green alga Chlamydomonas uses a visual system. An optical device, a rhodopsin photoreceptor and an electrical signal transduction chain that mediates between photoreceptor and flagella comprise this system. Here we present an improved strategy for the preparation of eyespot membranes. These membranes contain a retinal binding protein, which has been proposed to be the apoprotein of the phototaxis receptor. The retinal binding protein, which we named chlamyopsin, was purified and opsin-specific antibodies were raised. Using these antibodies, the opsin was localized in the eyespot region of whole cells during growth and cell division. The opsin cDNA was purified and sequenced. The sequence reveals that chlamyopsin is not a typical seven helix receptor. It shows some homology to invertebrate opsins but not to opsins from halobacteria. It contains many polar and charged residues and might function as a light-gated ion channel complex. It is likely that this lower plant rhodopsin diverged from animal opsins early in opsin evolution.     

Integrated Child Development Services Programme--need for reappraisal

BACKGROUND: Lipofuscin granules in the retinal pigment epithelium are lipid protein aggregates which are thought to represent the lifelong accumulation of the non-degradable end products from the phagocytosis of photoreceptor outer segments. Given the increasing evidence for a key role for vitamin A in the formation of ocular lipofuscin, the fluorophores generated by reacting vitamin A with lipid were assessed. METHODS: Reaction mixtures consisting of vitamin A (retinol) or its aldehyde (retinal) and (a) isolated rod outer segments, (b) the lipid extract of rod outer segments, (c) protein, or (d) liposomes were incubated at either pH 4.5 or 7.0 for up to 42 days. The fluorescence characteristics and mobility of the chloroform soluble fluorophores generated were compared with those extracted from purified human lipofuscin. Finally, the effect of lysosomal degradation on fluorophores generated in the above mixtures was assessed. RESULTS: Major spectral changes were observed when ROS or liposomes were incubated with retinal. These changes were pH dependent and did not occur if retinal was replaced with retinol. A number of the fluorophores generated exhibited similar fluorescence characteristics and chromatographic mobility to those of lipofuscin. Neither the presence of protein nor exposure to lysosomal enzymes had any effect on the spectral profile or fluorophore mobility of the fluorophores generated. CONCLUSIONS: These results suggest that some of the chloroform soluble fluorophores of lipofuscin are formed as a direct reaction product of retinal and lipid.     

Non-cell-autonomous photoreceptor degeneration in rds mutant mice mosaic for expression of a rescue transgene

The inherited retinal dystrophies represent a large and heterogenous group of hereditary neurodegenerations, for many of which, the molecular defect has been defined. However, the mechanism of cell death has not been determined for any form of retinal degeneration. The retinal degeneration slow (rds-/-) mutation of mice is associated with nondevelopment of photoreceptor outer segments and gradual death of photoreceptor cell bodies, attributed to the absence of the outer segment protein rds/peripherin. Here, we examined the effects of a transgene encoding normal rds/peripherin that had integrated into the X-chromosome in male and female rds-/- mutant retinas. In 2-month-old transgenic males and homozygous-transgenic females on rds-/-, we observed virtually complete rescue of both the outer segment nondevelopment and photoreceptor degeneration. In contrast, hemizygous-transgenic rds-/- female littermates showed patchy distributions of the transgene mRNA, by in situ hybridization analysis, and of photoreceptor cells that contain outer segments. This pattern is consistent with random inactivation of the X-chromosome and mosaic expression of the transgene. Surprisingly, we observed significant photoreceptor cell loss in both transgene-expressing and nonexpressing patches in hemizygous female retinas. These observations were supported by nuclease protection analysis, which showed notably lower than predicted levels of transgene mRNA in retinas from hemizygous females compared with male and homozygous female littermates. This phenotype suggests an important component of non-cell-autonomous photoreceptor death in rds-/- mutant mice. These results have significance to both the etiology and potential treatment of human inherited retinal degenerations.     

Glucose metabolism in photoreceptor outer segments. Its role in phototransduction and in NADPH-requiring reactions

Glucose metabolism in the photoreceptor rod outer segment produces both ATP (GTP) and NADPH to support phototransduction and NADPH-requiring processes in this organelle. Glycolysis in isolated bovine rod outer segments produces 44.0 +/- 6.4 nmol of ATP/min/mg of protein or 5.7 mM ATP/min. This rate of ATP production is more than sufficient to maintain the basal rate of cGMP synthesis (0.86 mM cGMP/min) in the dark requiring 1.7 mM ATP/min. Following photoexcitation, the 4.5-fold increase in the turnover of cGMP requires an ATP synthesis rate of up to 7.7 mM ATP/min (Ames, A., Walseth, T. F., Heyman, R. A., Barad, M., Graeff, R. M., and Goldberg, N. D. (1986) J. Biol. Chem. 261, 13034-13042). Under these conditions the rate of ATP production by glycolysis as measured in isolated rod outer segments is not sufficient for the regeneration of cGMP. Additional energy is most likely provided by the phosphocreatine shuttle which transports high energy phosphate groups in the form of creatine phosphate from the rod inner segment to the rod outer segment for conversion to ATP. The hexose monophosphate pathway in bovine rod outer segments can produce up to 39.8 +/- 2.2 nmol of NADPH/min/mg of protein. This rate of NADPH production is sufficient to support both the reduction of retinal to retinol (1.2 +/- 0.2 nmol of NADPH/min/mg of protein) following the photobleaching of rhodopsin and glutathione reduction (1.1 +/- 0.1 nmol of NADPH/min/mg of protein) for the protection of rod outer segments from oxidative damage. These studies provide insight into the contribution of anaerobic glycolysis and the hexose monophosphate pathway in providing energy and nucleotides for phototransduction and other outer segment processes.     

Photoreceptor thresholds and visual pigment levels in normal and vitamin A-deprived Xenopus tadpoles

1. Measurements of the aspartate-isolated PIII component of the electroretinogram (ERG) were used to estimate photoreceptor threshold in dark-adapted tadpoles of the clawed toad, Xenopus laevis raised on a normal diet. Spectral sensitivity functions established that the rod mechanism governed absolute dark thresholds from stage 40 to the end of premetamorphic development. 2. Parallel measures of rod outer-segment dimensions and visual pigment levels demonstrated a) that visual pigment concentration remained constant at all tadpole stages, and b) that the fall in threshold over the course of premetamorphic development depended exclusively on the increased probability of quantal absorption that accompanied the growth of the rod outer segments. 3. When tadpoles were obtained from vitamin A-deficient females and raised subsequently on a vitamin A-free diet, the first appearance of the ERG was delayed and its absolute threshold raised, with respect to controls. 4. Histological examination of vitamin A-deprived retinas indicated that rod outer segments retained their structural integrity in spite of up to a 75% loss of visual pigment. 5. The threshold rise which accompanied a fall in visual pigment levels, whether effected by dietary deficiency or by bleaching, was greater than could be attributed solely to a reduction in the probability of quantal absorption.     

Effect of 2,3,7,8-tetrachlorodibenzo-p-dioxin on the lymphatic absorption of a single oral dose of [3H]retinol and on the intestinal retinol esterification in the rat

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) alters the turnover of vitamin A in the body and inhibits the normal hepatic accumulation of dietary vitamin A. Vitamin A is absorbed in the small intestine, where it is incorporated into chylomicrons as retinyl esters for release into the lymph and further distributed via blood to the liver for storage. The aim of the present study was to investigate if the decreased hepatic vitamin A levels in TCDD-exposed rats could be due to impaired intestinal absorption of vitamin A via lymph. Male Sprague-Dawley rats were given a single oral dose of TCDD (10 microg/kg). Five days after administration, the main intestinal lymph duct of the rats was cannulated. After a 24-h recovery from surgery, the rats were each given a single dose of [3H]retinol in corn oil via gavage and the lymph was collected for 24 h. The cumulative radiolabel recovered in the intestinal lymph was significantly lower in TCDD-treated than in control rats during the first 6 h of absorption. However, no significant differences in radiolabel recovered in lymph were seen when looking at the entire 24-h collection period. In the intestinal mucosa, retinol esterification catalyzed by the enzyme lecithin:retinol acyl transferase (LRAT) or acyl coenzyme A (CoA):retinol transferase (ARAT) was not statistically different between the groups. However, mucosal retinyl palmitate levels were significantly increased in TCDD-treated rats. In conclusion, a small and transient reduction was found of the uptake of vitamin A into the lymph of TCDD-treated rats. It is obvious that this finding cannot explain the TCDD-induced decrease in hepatic vitamin A levels in the rat. Rather, a combination of inhibited retinol esterification in hepatic stellate cells, increased release of endogenous vitamin A, and increased hepatic catabolism of retinoids could explain the effect of TCDD on liver retinoid levels.     

Molecular characterization of a novel short-chain dehydrogenase/reductase that reduces all-trans-retinal

The reduction of all-trans-retinal in photoreceptor outer segments is the first step in the regeneration of bleached visual pigments. We report here the cloning of a dehydrogenase, retSDR1, that belongs to the short-chain dehydrogenase/reductase superfamily and localizes predominantly in cone photoreceptors. retSDR1 expressed in insect cells displayed substrate specificities of the photoreceptor all-trans-retinol dehydrogenase. Homology modeling of retSDR1 using the carbonyl reductase structure as a scaffold predicted a classical Rossmann fold for the nucleotide binding, and an N-terminal extension that could facilitate binding of the enzyme to the cell membranes. The presence of retSDR1 in a subset of inner retinal neurons and in other tissues suggests that the enzyme may also be involved in retinol metabolism outside of photoreceptors.     

Effects of the intermediate retinoid metabolite retinal on the pattern of the dental lamina in vitro

Retinoids have important roles in pattern formation during embryonic development and might act as endogenous morphogens. They are necessary for normal odontogenesis and excess retinol alters the pattern of odontogenesis producing supernumerary buds of the dental lamina in the diastema region of the mouse mandible. Because the metabolism of retinoids in the developing mandible is unknown, the effects of retinal (an intermediate metabolite in the local conversion of retinol to retinoic acid) on the patterning of odontogenesis were examined. Retinal produces supernumerary buds and enhanced epithelial proliferation in day-9 mandibles in vitro. The endogenous levels of retinal in the mandible at the time of initiation of odontogenesis were also measured by high-performance liquid chromatography. Retinal was detected only at day 10 and not at later stages of development. Local metabolism of this intermediate retinoid may be a rate-determining step in the production of active retinoid metabolites that may control the pattern of the dentition, which is established at the time of the appearance of the dental lamina at embryonic day 12.     

Rod outer segment renewal in the retinae of deep-sea fish

Outer segment renewal involves the synthesis of disc material in the photoreceptor inner segments, the shedding of the tips of the photoreceptor outer segments, and their phagocytosis by the retinal pigment epithelial cells. It has been suggested that in the retinae of deep-sea fish no renewal of outer segments may take place. In order to assess outer segment renewal in deep-sea fish retinae we counted (i) periciliary vesicles in rod inner segments as a parameter for disc-synthesis activity and (ii) phagosomes in retinal pigment epithelial cells as a parameter of shedding and phagocytosis in 12 species of deep-sea fish with multibank or single bank retinae. We also measured the lengths of rod outer segments in order to evaluate the balance between synthesis and phagocytotic activity. In four of these species (Synaphobranchus kaupi, Nematonurus armatus, Coryphaenoides guentheri and Halosauropsis macrochir) we further recorded size-related changes of these parameters and their relation to the position of a given rod within the banks in the retina. The number of periciliary vesicles was highest in inner segments of the most vitread bank and in the periphery of the retina. Phagosomes were most abundant in retinal pigment epithelial cells of the central retina. Long rod outer segments were most frequently recorded in the peripheral retina indicating that in this region new synthesis may outbalance shedding. Vitread rod outer segments were only slightly longer than sclerad ones. Larger animals had shorter rod outer segments than small ones. We present evidence that rod outer segment renewal takes place in the retina of all deep-sea fish. Vitread rods may be more active in this respect than sclerad ones.     

Switch in rod opsin gene expression in the European eel, Anguilla anguilla (L.)

The rod photoreceptors of the European eel, Anguilla anguilla (L.), alter their wavelength of maximum sensitivity (lambda max) from c.a. 523 nm to c.a. 482 nm at maturation, a switch involving the synthesis of a new visual pigment protein (opsin) that is inserted into the outer segments of existing rods. We artificially induced the switch in rod opsin production by the administration of hormones, and monitored the switch at the level of mRNA accumulation using radiolabelled oligonuleotides that hybridized differently to the two forms of eel rod opsin. The production of the deep-sea form of rod opsin was detected 6 h after the first hormone injection, and the switch in rod opsin expression was complete within four weeks, at which time only the mRNA for the deep-sea opsin was detectable in the retinal cells. It is suggested that this system could be used as a tractable model for studying the regulatory control of opsin gene expression.     

Spatial and temporal expression of cone opsins during monkey retinal development

The primate retina requires a coordinated series of developmental events to form its specialized photoreceptor topography. In this study, the temporal expression of cone photoreceptor opsin was determined in Macaca monkey retina. Markers for mRNA and protein that recognize short wavelength (S) and long/medium wavelength (L/M) opsin were used to determine (1) the temporal and spatial patterns of opsin expression, (2) the spatial relationship between S and L/M cones at the time of initial opsin expression, and (3) the relative time of cone and rod opsin expression (Dorn et al. [1995] Invest. Ophthalmol. Vis. Sci. 36:2634-2651). Adult cone outer segments were recognized by either L/M or S opsin antiserum. Of all adult cone inner segments, 88-90% contained L/M opsin mRNA, whereas 10-12% contained S opsin mRNA. Fetal cones initially showed cell membrane as well as outer segment labeling for opsin protein, but cell membrane labeling disappeared by birth. No cones at any age contained markers for both S and L/M opsin mRNA or protein. S and L/M opsin protein appeared in the fovea at fetal day 75. Once opsin expression progressed beyond the fovea, both mRNA and protein for S opsin were consistently detected more peripherally than L/M opsin. Cones at the peripheral edge of S opsin expression had basal telodendria that appeared to reach toward neighboring cones. Because interactions between cone populations could organize the cone mosaic, the spatial relationship between S cones and the first cones to express L/M protein was analyzed quantitatively by using double-label immunocytochemistry. No consistent relationship was found between these two cone populations. Cones are generated at least 1 week before rods across monkey retina. However, rod opsin protein appears in and around the fovea at fetal day 66, 1 week before cone opsin protein. This suggests that independent local factors control differentiation in these two photoreceptor populations.