Identification and localization of an immunoreactiveAMPA-type glutamate receptor subunit (GluR4) with respect to identified photoreceptor synapses in the outer plexiform layer of goldfish retina

L-glutamate, the main excitatory synaptic transmitter in the retina, is released from photoreceptors and evokes responses in second-order retinal neurons (horizontal, bipolar cells) which utilize both ionotropic and metabotropic types of glutamate receptors. In the present study, to elucidate the functional roles of glutamate receptors in synaptic transmission, we have identified a specific ionotropic receptor subunit (GluR4) and determined its localization with respect to photoreceptor cells in the outer plexiform layer of the goldfish retina by light and pre-embedding electron-microscopical immunocytochemistry. We screened antisera to mammalian AMPA (alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate)-preferring ionotropic glutamate receptors (GluR 1–4) of goldfish retina by light- and electron-microscopical immunocytochemistry. Only immunoreactive (IR) GluR4 was found in discrete clusters in the outer plexiform layer. The cones contacted in this manner were identified as long-wavelength (“red”) and intermediate-wavelength (“green”) cones, which were strongly immunoreactive to monoclonal antibody FRet 43 and antisera to goldfish red and green-cone opsins; and short-wavelength (“blue”) cones, which were weakly immunoreactive to FRet 43 but strongly immunoreactive with antiserum to blue-cone opsin. Immunoblots of goldfish retinal homogenate with anti-GluR4 revealed a single protein at Mr=110 kDa. Preadsorption of GluR4 antiserum with either the immunizing rat peptide, or its goldfish homolog, reduced or abolished staining in retinal sections and blots. Therefore, we have detected and localized genuine goldfish GluR4 in the outer plexiform layer of the goldfish retina. We characterized contacts between photoreceptor cells and GluR4-IR second-order neurons in the electron microscope. IR-GluR4 was localized to invaginating central dendrites of triads in ribbon synapses of red cones, semi-invaginating dendrites in other cones and rods, and dendrites making wide-cleft basal junctions in rods and cones; the GluR4-IR structures are best identified as dendrites of OFF-bipolar cells. The results of our studies indicate that in goldfish retina GluR4-expressing neurons are postsynaptic to all types of photoreceptors and that transmission from photoreceptors to OFF-bipolars is mediated at least in part by AMPA-sensitive receptors containing GluR4 subunits.     

Activation of the blue opsin gene in cone photoreceptor development by retinoid-related orphan receptor beta

Color vision requires the expression of opsin photopigments with different wavelength sensitivities in retinal cone photoreceptors. The basic color visual system of mammals is dichromatic, involving differential expression in the cone population of two opsins with sensitivity to short (S, blue) or medium (M, green) wavelengths. However, little is known of the factors that directly activate these opsin genes and thereby contribute to the S or M opsin identity of the cone. We report that the orphan nuclear receptor RORbeta (retinoid-related orphan receptor beta) activates the S opsin gene (Opn1sw) through binding sites upstream of the gene. RORbeta lacks a known physiological ligand and activates the Opn1sw promoter modestly alone but strongly in synergy with the retinal cone-rod homeobox factor (CRX), suggesting a cooperative means of enhancing RORbeta activity. Comparison of wild-type and mutant lacZ reporter transgenes showed that the RORbeta-binding sites in Opn1sw are required for expression in mouse retina. RORbeta-deficient mice fail to induce S opsin appropriately during postnatal cone development. Photoreceptors in these mice also lack outer segments, indicating additional functions for RORbeta in photoreceptor morphological maturation. The results identify Opn1sw as a target gene for RORbeta and suggest a key role for RORbeta in regulating opsin expression in the color visual system.     

Photoreceptor cell death, proliferation and formation of hybrid rod/S-cone photoreceptors in the degenerating STK38L mutant retina

A homozygous mutation in STK38L in dogs impairs the late phase of photoreceptor development, and is followed by photoreceptor cell death (TUNEL) and proliferation (PCNA, PHH3) events that occur independently in different cells between 7-14 weeks of age. During this period, the outer nuclear layer (ONL) cell number is unchanged. The dividing cells are of photoreceptor origin, have rod opsin labeling, and do not label with markers specific for macrophages/microglia (CD18) or Müller cells (glutamine synthetase, PAX6). Nestin labeling is absent from the ONL although it labels the peripheral retina and ciliary marginal zone equally in normals and mutants. Cell proliferation is associated with increased cyclin A1 and LATS1 mRNA expression, but CRX protein expression is unchanged. Coincident with photoreceptor proliferation is a change in the photoreceptor population. Prior to cell death the photoreceptor mosaic is composed of L/M- and S-cones, and rods. After proliferation, both cone types remain, but the majority of rods are now hybrid photoreceptors that express rod opsin and, to a lesser extent, cone S-opsin, and lack NR2E3 expression. The hybrid photoreceptors renew their outer segments diffusely, a characteristic of cones. The results indicate the capacity for terminally differentiated, albeit mutant, photoreceptors to divide with mutations in this novel retinal degeneration gene.     

Human Blue Cone Opsin Regeneration Involves Secondary Retinal Binding with Analog Specificity

Human color vision is mediated by the red, green, and blue cone visual pigments. Cone opsins are G-protein-coupled receptors consisting of an opsin apoprotein covalently linked to the 11-cis-retinal chromophore. All visual pigments share a common evolutionary origin, and red and green cone opsins exhibit a higher homology, whereas blue cone opsin shows more resemblance to the dim light receptor rhodopsin. Here we show that chromophore regeneration in photoactivated blue cone opsin exhibits intermediate transient conformations and a secondary retinoid binding event with slower binding kinetics. We also detected a fine-tuning of the conformational change in the photoactivated blue cone opsin binding site that alters the retinal isomer binding specificity. Furthermore, the molecular models of active and inactive blue cone opsins show specific molecular interactions in the retinal binding site that are not present in other opsins. These findings highlight the differential conformational versatility of human cone opsin pigments in the chromophore regeneration process, particularly compared to rhodopsin, and point to relevant functional, unexpected roles other than spectral tuning for the cone visual pigments.     

Circadian photoreception in humans and mice

Circadian rhythms are the endogenous oscillations, occurring with a periodicity of approximately twenty-four hours, in the biochemical and behavioral functions of organisms. In mammals, the phase and period of the rhythm are synchronized to the daily light-dark cycle by light input through the eye. Certain retinal degenerative diseases affecting the photoreceptor cells, both rods and cones, in the outer retina reveal that classical opsins (i.e., rhodopsin and color opsins located in these cells) are essential for vision, but are not required for circadian photoreception. The mammalian cryptochromes and melanopsin (and possibly other opsin family pigments) have been proposed as circadian photoreceptor pigments that exist in the inner retina. Genetic analysis indicates that the cryptochromes, which contain flavin and folate as the light-absorbing cofactors, are the primary circadian photoreceptors. The classical photoreceptors in the outer retina, and melanopsin or other minor opsins in the inner retina, may perform redundant functions in circadian rhythmicity.     

Alpha transducin is present in blue-, green-, and red-sensitive cone photoreceptors in the human retina

Phototransduction in vertebrate rod and cone photoreceptor cells involves G protein-mediated light stimulation of cGMP hydrolysis. Enzymes of the cGMP hydrolysis cascades of rods and cones are products of different genes. Three different classes of cones in the human retina are maximally sensitive to either blue, green, or red light. Distinct opsin genes are expressed in each type of cone. The distribution of cone types in human retina was determined using anti-peptide antibodies that recognize specific amino acid sequences in green/red opsin and blue opsin. These antibodies together with an anti-peptide antibody against Tc alpha were used in double labeling experiments to demonstrate the presence of the Tc alpha peptide in all types of cones. cDNA clones corresponding to human rod and cone transducin alpha subunit (Tr alpha and Tc alpha) genes were isolated. Southern blot analyses of human genomic DNA suggest that there is only one rod T alpha gene but more than one cone T alpha gene. The multiple Tc alpha genes could be closely related genes or different Tc alpha alleles, or one could be a pseudogene.     

Toward the generation of rod and cone photoreceptors from mouse, monkey and human embryonic stem cells

We previously reported the differentiation of mouse embryonic stem (ES) cells into retinal progenitors. However, these progenitors rarely differentiate into photoreceptors unless they are cultured with embryonic retinal tissues. Here we show the in vitro generation of putative rod and cone photoreceptors from mouse, monkey and human ES cells by stepwise treatments under defined culture conditions, in the absence of retinal tissues. With mouse ES cells, Crx+ photoreceptor precursors were induced from Rx+ retinal progenitors by treatment with a Notch signal inhibitor. Further application of fibroblast growth factors, Shh, taurine and retinoic acid yielded a greater number of rhodopsin+ rod photoreceptors, in addition to default cone production. With monkey and human ES cells, feeder- and serum-free suspension culture combined with Wnt and Nodal inhibitors induced differentiation of Rx+ or Mitf+ retinal progenitors, which produced retinal pigment epithelial cells. Subsequent treatment with retinoic acid and taurine induced photoreceptor differentiation. These findings may facilitate the development of human ES cell-based transplantation therapies for retinal diseases.     

Differential CRX and OTX2 expression in human retina and retinoblastoma

The histogenesis of retinoblastoma tumors remains controversial, with the cell-of-origin variably proposed to be an uncommitted retinal progenitor cell, a bipotent committed cell, or a cell committed to a specific lineage. Here, we examine the expression of two members of the orthodenticle family implicated in photoreceptor and bipolar cell differentiation, cone-rod homeobox, CRX, and orthodenticle homeobox 2, OTX2, in normal human retina, retinoblastoma cell lines and retinoblastoma tumors. We show that CRX and OTX2 have distinct expression profiles in the developing human retina, with CRX first expressed in proliferating cells and cells committed to the bipolar lineage, and OTX2 first appearing in the photoreceptor lineage. In the mature retina, CRX levels are highest in photoreceptor cells whereas OTX2 is preferentially found in bipolar cells and in the retinal pigmented epithelium. Both CRX and OTX2 are widely expressed in retinoblastoma cell lines and in retinoblastoma tumors, although CRX is more abundant than OTX2 in the differentiated elements of retinoblastoma tumors such as large rosettes, Flexner-Wintersteiner rosettes and fleurettes. Widespread expression of CRX and OTX2 in retinoblastoma tumors and cell lines suggests a close link between the cell-of-origin of retinoblastoma tumors and cells expressing CRX and OTX2.     

A rod-dominated visual system in leptocephalus larvae of elopomorph fishes (Elopomorpha: Teleostei)

The nature and distributions of photoreceptor cell types were investigated in the retinas of 12 species (5 families) of elopomorph anguilliform leptocephalus larvae. Anti-opsin immunofluorescence, light microscopy and transmission electron microscopy (TEM) were used to assess opsin distribution across the retinas and to associate photoreceptor morphology and opsin content. Retinas of all species were immunoreactive with anti-rhodopsin throughout, while anti-cone opsin immunoreactivity was restricted only to the ventral region of the retina in all specimens. Rod and cone photoreceptors were morphologically indistinguishable at low magnifications; TEM revealed that nearly all photoreceptors had rod-like ultrastructure, with only rare examples of cone-like cells identified in the ventral retina. These results indicate a rhodopsin/rod-dominated retina in leptocephalus larvae of anguilliform eels in the teleost subdivision Elopomorpha, contrasting with the cone-dominated retinas of nearly all other species of teleost larvae. This distinctive developmental pattern shared among elopomorph larvae has important evolutionary and ecological implications, indicating a shared ancestor and/or ecological characteristics that are very different from most other teleost larvae.     

Opsin expression patterns coincide with photoreceptor development during pupal development in the honey bee,Apis mellifera

The compound eyes of insects allow them to catch photons and convert the energy into electric signals. All compound eyes consist of numerous ommatidia, each comprising a fixed number of photoreceptors. Different ommatidial types are characterized by a specific set of photoreceptors differing in spectral sensitivity. In honey bees, males and females possess different ommatidial types forming distinct retinal mosaics. However, data are lacking on retinal ontogeny and the mechanisms by which the eyes are patterned. In this study, we investigated the intrinsic temporal and circadian expression patterns of the opsins that give rise to the ultraviolet, blue and green sensitive photoreceptors, as well as the morphological maturation of the retina during pupal development of honey bees. qPCR and histological labeling revealed that temporal opsin mRNA expression differs between sexes and correlates with rhabdom elongation during photoreceptor development. In the first half of the pupal stage, when the rhabdoms of the photoreceptors are still short, worker and (dorsal) drone retinae exhibit similar expression patterns with relatively high levels of UV (UVop) and only marginal levels of blue (BLop) and green (Lop1) opsin mRNA. In the second half of pupation, when photoreceptors and rhabdoms elongate, opsin expression in workers becomes dominated by Lop1 mRNA. In contrast, the dorsal drone eye shows high expression levels of UVop and BLop mRNA, whereas Lop1 mRNA level decreases. Interestingly, opsin expression levels increase up to 22-fold during early adult life. We also found evidence that opsin expression in adult bees is under the control of the endogenous clock. Our data indicate that the formation of the sex-specific retinal composition of photoreceptors takes place during the second half of the pupal development, and that opsin mRNA expression levels continue to increase in young bees, which stands in contrast to Drosophila, where the highest expression levels are found during the late pupal stage and remain constant in adults. From an evolutionary perspective, we hypothesize that the delayed retinal maturation during the early adult phase is linked to the delayed transition from indoor to outdoor activities in bees, when vision becomes important.     

Embryonic appearance of rod opsin in the urodele amphibian eye

Notophthalmus (Triturus) viridescens, a urodele amphibian (newt) common to the Eastern United States, is a promising subject for developmental and regeneration studies. We have available a monoclonal antibody shown to be specific in many vertebrates for rod opsin, the membrane apoprotein of the visual pigment rhodopsin. This antibody to an N-terminal epitope, by rigorous biochemical and immunological criteria, recognizes only rod photoreceptor cells of the retina in light-and electron-microscopic immunocytochemistry. To determine the ontogeny and localization of rhodopsin in developing rods as an indicator of function in the embryonic urodele retina, we have utilized this antibody in the immunofluorescence technique on sections of developing N. viridescens. It was applied to serial sections of the eye region of Harrison stage 28 (optic vesicle) through stage 43 (most adult retina histology complete) embryos, and subsequently visualized with biotinylated species antibody followed by extravidin fluorescein isothiocyanate. The first positive reaction to rhodopsin could be detected in two to four cells (total) of the stage 37 embryonic eye, in the region of the central retinal primordium where the photoreceptors will be found. Some indications of retinal outer nuclear and inner plexiform layers could be seen at this time. Later embryonic stages demonstrated increasing numbers of positive cells in the future photoreceptor outer nuclear layer and outer and inner segments, spreading even to the peripheral retina. Nevertheless, by stale 43, no positive cells could be found at the dorsal or ventral retinal margins. Thus, biochemical differentiation of a photoreceptor population in the urodele retina occurs at a stage before retinal histogenesis is complete. The total maturation of retinal rods occurs topographically over a long period until the adult distribution is achieved. Correspondence to: D.S. McDevitt     

Immunoassay of rod visual pigment (opsin) in the eyes of rds mutant mice lacking receptor outer segments

In 020/A mice, homozygous for the retinal degeneration slow (rds) gene, the photoreceptor cells fail to develop outer segments, and in the absorption spectra of retinal extracts the rhodopsin peak is lacking. Application of an enzyme-linked immunoassay using antisera against bovine opsin shows, however, that opsin is present in the homozygous mutant retina (0.010 nmol/eye) at 3% of the level of the normal retina (0.38 nmol/eye) of Balb/c mice. In the retina of heterozygous mice the opsin level (0.19 nmol/eye) is about half of the normal. Detection of opsin in the rds mutant retina demonstrates the functional basis for the reported electroretinographic response and light-mediated reduction in cyclic nucleotide levels in this mutant.