Mechanistic insights into the evolution of the differential expression of tandemly arrayed cone opsin genes in zebrafish.

The genome of many organisms contains several loci consisting of duplicated genes that are arrayed in tandem. The daughter genes produced by duplication typically exhibit differential expression patterns with each other or otherwise experience pseudogenization. Remarkably, opsin genes in fish are preserved after many duplications in different lineages. This fact indicates that fish opsin genes are characterized by a regulatory mechanism that could intrinsically facilitate the differentiation of the expression patterns. However, little is known about the mechanisms that underlie the differential expression patterns or how they were established during evolution. The loci of green (RH2)- and red (LWS)-sensitive cone opsin genes in zebrafish have been used as model systems to study the differential regulation of tandemly arrayed opsin genes. Over a decade of studies have uncovered several mechanistic features that might have assisted the differentiation and preservation of duplicated genes. Furthermore, recent progress in the understanding of the transcriptional process in general has added essential insights. In this article, the current understanding of the transcriptional regulation of differentially expressed tandemly arrayed cone opsin genes in zebrafish is summarized and a possible evolutionary scenario that could achieve this differentiation is discussed.

Interactions of cone abundancies, opsin expression, and environmental lighting with emmetropization in chickens.

We had previously found that M to L cone abundancy ratios in the chicken retina are correlated with vitreous chamber depth and refractive state in chickens eyes, when they have normal visual exposure but not when they develop deprivation myopia. The finding suggests an interaction between cone abundancies and emmetropization. In the current study, we analyzed how stable this correlation was against changes in environmental variables and strain differences. We found that the correlation was preserved in two chicken strains, as long as they were raised in the laboratory facilities and not in the animal facilities of the institute. To determine the reasons for this difference, spectral and temporal lighting parameters were better adjusted in both places, whereas temperature, humidity, food, diurnal lighting cycles and illuminance were already matched. It was also verified that both strains of chickens had the same cone opsin amino acid sequences. The correlation between M to L cone abundancy and ocular biometry is highly susceptible to changes in environmental variables. Yet undetermined differences in lighting parameters were the most likely reasons. Other striking findings were that green cone opsin mRNA expression was downregulated when deprivation myopia developed. Similarly, red opsin mRNA was downregulated when chicks wore red spectacles, which made them more hyperopic. In summary, our experiments show that photoreceptor abundancies, opsin expression, and the responses to deprivation, and therefore emmetropization, are surprisingly dependent on subtle differences in lighting parameters.

Transcriptional co-regulation of evolutionarily conserved microRNA/cone opsin gene pairs: implications for photoreceptor subtype specification.

The vertebrate retina contains two types of photoreceptor cells, rods and cones, which use distinct types of opsins and phototransduction proteins. Cones can be further divided into several subtypes with differing wavelength sensitivity and morphology. Although photoreceptor development has been extensively studied in a variety of vertebrate species, the mechanism by which photoreceptor subtypes are established is still largely unknown. Here we report two microRNAs (miRNAs), miR-726 and miR-729, which are potentially involved in photoreceptor subtype specification. In the medaka Oryzias latipes, the genes encoding miR-726 and miR-729 are located upstream of the red-sensitive opsin gene LWS-A and the UV-sensitive opsin gene SWS1, respectively, and are transcribed in the opposite direction from the respective opsin genes. The miR-726/LWS pair is conserved between teleosts and tetrapods, and the miR-729/SWS1 pair is conserved among teleosts. in situ hybridization analyses and fluorescence reporter assays suggest that these miRNAs are co-expressed with the respective opsins in specific cone subtypes. Potential targets of miR-726 and miR-729 predicted in silico include several transcription factors that regulate photoreceptor development. Functional analyses of cis-regulatory sequences in vivo suggest that transcription of the paired microRNA and opsin genes is co-regulated by common cis-regulatory modules. We propose an evolutionarily conserved mechanism that controls photoreceptor subtype identity through coupling between transcriptional and post-transcriptional regulations.

The mammalian cone visual cycle promotes rapid M/L-cone pigment regeneration independently of the interphotoreceptor retinoid-binding protein.

Rapid regeneration of the visual pigment following its photoactivation is critical for the function of cone photoreceptors throughout the day. Though the reactions of the visual cycle in the retinal pigment epithelium (RPE) that recycle chromophore for rod pigment regeneration are well characterized, the corresponding mechanisms that enable rapid regeneration of cone pigment are poorly understood. A key remaining question is the relative contribution of the recently discovered cone-specific retina visual cycle and the classic RPE-dependent visual cycle to mammalian cone pigment regeneration. In addition, it is not clear what role, if any, the abundant interphotoreceptor retinoid-binding protein (IRBP) presumed to facilitate the traffic of chromophore, plays in accelerating mammalian cone pigment regeneration. To address these issues, we used transretinal recordings to evaluate M/L-cone pigment regeneration in isolated retinas and eyecups from control and IRBP-deficient mice. Remarkably, the mouse retina promoted M/L-cone dark adaptation eightfold faster than the RPE. However, complete cone recovery required both visual cycles. We conclude that the retina visual cycle is critical for the initial rapid regeneration of mouse M/L-cone pigment during dark adaptation, whereas the slower RPE visual cycle is required to complete the process. While the deletion of IRBP reduced the amplitude and slowed the kinetics of mouse M/L-cone photoresponses, cone adaptation in bright, steady light and the kinetics of cone dark adaptation were not affected in isolated retina or in intact eyecup. Thus, IRBP does not accelerate cone pigment regeneration and is not critical for the function of mouse M/L-cones in bright light.

Thyroid hormone controls cone opsin expression in the retina of adult rodents.

Mammalian retinas display an astonishing diversity in the spatial arrangement of their spectral cone photoreceptors, probably in adaptation to different visual environments. Opsin expression patterns like the dorsoventral gradients of short-wave-sensitive (S) and middle- to long-wave-sensitive (M) cone opsin found in many species are established early in development and thought to be stable thereafter throughout life. In mouse early development, thyroid hormone (TH), through its receptor TRß2, is an important regulator of cone spectral identity. However, the role of TH in the maintenance of the mature cone photoreceptor pattern is unclear. We here show that TH also controls adult cone opsin expression. Methimazole-induced suppression of serum TH in adult mice and rats yielded no changes in cone numbers but reversibly altered cone patterns by activating the expression of S-cone opsin and repressing the expression of M-cone opsin. Furthermore, treatment of athyroid Pax8(-/-) mice with TH restored a wild-type pattern of cone opsin expression that reverted back to the mutant S-opsin-dominated pattern after termination of treatment. No evidence for cone death or the generation of new cones from retinal progenitors was found in retinas that shifted opsin expression patterns. Together, this suggests that opsin expression in terminally differentiated mammalian cones remains subject to control by TH, a finding that is in contradiction to previous work and challenges the current view that opsin identity in mature mammalian cones is fixed by permanent gene silencing.

Visual opsin expression and morphological characterization of retinal photoreceptors in the pouched lamprey (Geotria australis, Gray).

Lampreys are extant members of the agnathan (jawless) vertebrates that diverged ~500 million years ago, during a critical stage of vertebrate evolution when image-forming eyes first emerged. Among lamprey species assessed thus far, the retina of the southern hemisphere pouched lamprey, Geotria australis, is unique, in that it possesses morphologically distinct photoreceptors and expresses five visual photopigments. This study focused on determining the number of different photoreceptors present in the retina of G. australis and whether each cell type expresses a single opsin class. Five photoreceptor subtypes were identified based on ultrastructure and differential expression of one of each of the five different visual opsin classes (lws, sws1, sws2, rh1, and rh2) known to be expressed in the retina. This suggests, therefore, that the retina of G. australis possesses five spectrally and morphologically distinct photoreceptors, with the potential for complex color vision. Each photoreceptor subtype was shown to have a specific spatial distribution in the retina, which is potentially associated with changes in spectral radiance across different lines of sight. These results suggest that there have been strong selection pressures for G. australis to maintain broad spectral sensitivity for the brightly lit surface waters that this species inhabits during its marine phase. These findings provide important insights into the functional anatomy of the early vertebrate retina and the selection pressures that may have led to the evolution of complex color vision.

The topography of cone photoreceptors in the retina of a diurnal rodent, the agouti (Dasyprocta aguti).

The presence, density distribution, and mosaic regularity of cone types were studied in the retina of the diurnal agouti, Dasyprocta aguti. Longwave-sensitive (L-) and shortwave-sensitive (S-) cones were detected by antibodies against the respective cone opsins. L- and S-cones were found to represent around 90 and 10% of the cone population, respectively. There was no evidence for L- and S-opsin coexpression in agouti cones. L-cone densities were highest, up to 14,000/mm2, along a horizontal visual streak located about 2-3 mm dorsal to the optic nerve, and the L-cone distribution showed a dorsoventral asymmetry with higher densities in ventral (about 10,000/mm2) than in dorsal (about 4000/mm2) retinal regions. This L-cone topography parallels the agouti's ganglion cell topography. S-cones had a peak density of 1500-2000/mm2 in the central retinal region but did not form a visual streak. Their distribution also showed a dorsoventral asymmetry with densities around 600/mm2 in dorsal and around 1000/mm2 in ventral retinal regions. The patterning of cone arrays was assessed by the density recovery profile analysis. At all eccentricities evaluated, the S-cone mosaic less efficiently packed than the L-cone mosaic. Rod densities ranged from 47,000/mm2 in peripheral to 64,000/mm2 in central retina, and rod:cone ratios were 4:1-9:1. The comparatively low rod density and high cone proportion appear well adapted to the diurnal lifestyle of the agouti.

Expressions of visual pigments and synaptic proteins in neonatal chick retina exposed to light of variable photoperiods.

Light causes damage to the retina, which is one of the supposed factors for age-related macular degeneration in human. Some animal species show drastic retinal changes when exposed to intense light (e.g. albino rats). Although birds have a pigmented retina, few reports indicated its susceptibility to light damage. To know how light influences a cone-dominated retina (as is the case with human), we examined the effects of moderate light intensity on the retina of white Leghorn chicks (Gallus g. domesticus). The newly hatched chicks were initially acclimatized at 500 lux for 7 days in 12 h light: 12 h dark cycles (12L:12D). From posthatch day (PH) 8 until PH 30, they were exposed to 2000 lux at 12L:12D, 18L:6D (prolonged light) and 24L:0D (constant light) conditions. The retinas were processed for transmission electron microscopy and the level of expressions of rhodopsin, S- and L/M cone opsins, and synaptic proteins (Synaptophysin and PSD-95) were determined by immunohistochemistry and Western blotting. Rearing in 24L:0D condition caused disorganization of photoreceptor outer segments. Consequently, there were significantly decreased expressions of opsins and synaptic proteins, compared to those seen in 12L:12D and 18L:6D conditions. Also, there were ultrastructural changes in outer and inner plexiform layer (OPL, IPL) of the retinas exposed to 24L:0D condition. Our data indicate that the cone-dominated chick retina is affected in constant light condition, with changes (decreased) in opsin levels. Also, photoreceptor alterations lead to an overall decrease in synaptic protein expressions in OPL and IPL and death of degenerated axonal processes in IPL.

Rax Homeoprotein Regulates Photoreceptor Cell Maturation and Survival in Association with Crx in the Postnatal Mouse Retina.

The Rax homeobox gene plays essential roles in multiple processes of vertebrate retina development. Many vertebrate species possess Rax and Rax2 genes, and different functions have been suggested. In contrast, mice contain a single Rax gene, and its functional roles in late retinal development are still unclear. To clarify mouse Rax function in postnatal photoreceptor development and maintenance, we generated conditional knockout mice in which Rax in maturing or mature photoreceptor cells was inactivated by tamoxifen treatment (Rax iCKO mice). When Rax was inactivated in postnatal Rax iCKO mice, developing photoreceptor cells showed a significant decrease in the level of the expression of rod and cone photoreceptor genes and mature adult photoreceptors exhibited a specific decrease in cone cell numbers. In luciferase assays, we found that Rax and Crx cooperatively transactivate Rhodopsin and cone opsin promoters and that an optimum Rax expression level to transactivate photoreceptor gene expression exists. Furthermore, Rax and Crx colocalized in maturing photoreceptor cells, and their coimmunoprecipitation was observed in cultured cells. Taken together, these results suggest that Rax plays essential roles in the maturation of both cones and rods and in the survival of cones by regulating photoreceptor gene expression with Crx in the postnatal mouse retina.