Spectral tuning by opsin coexpression in retinal regions that view different parts of the visual field

Vision frequently mediates critical behaviours, and photoreceptors must respond to the light available to accomplish these tasks. Most photoreceptors are thought to contain a single visual pigment, an opsin protein bound to a chromophore, which together determine spectral sensitivity. Mechanisms of spectral tuning include altering the opsin, changing the chromophore and incorporating pre-receptor filtering. A few exceptions to the use of a single visual pigment have been documented in which a single mature photoreceptor coexpresses opsins that form spectrally distinct visual pigments, and in these exceptions the functional significance of coexpression is unclear. Here we document for the first time photoreceptors coexpressing spectrally distinct opsin genes in a manner that tunes sensitivity to the light environment. Photoreceptors of the cichlid fish, Metriaclima zebra, mix different pairs of opsins in retinal regions that view distinct backgrounds. The mixing of visual pigments increases absorbance of the corresponding background, potentially aiding the detection of dark objects. Thus, opsin coexpression may be a novel mechanism of spectral tuning that could be useful for detecting prey, predators and mates. However, our calculations show that coexpression of some opsins can hinder colour discrimination, creating a trade-off between visual functions.     

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.     

The Python pit organ: imaging and immunocytochemical analysis of an extremely sensitive natural infrared detector

The Python infrared-sensitive pit organ is a natural infrared imager that combines high sensitivity, ambient temperature function, microscopic dimensions, and self-repair. We are investigating the spectral sensitivity and signal transduction process in snake infrared-sensitive neurons, neither of which is understood. For example, it is unknown whether infrared receptor neurons function on a thermal or a photic mechanism. We imaged pit organs in living Python molurus and Python regius using infrared-sensitive digital video cameras. Pit organs were significantly more absorptive and/or emissive than surrounding tissues in both 3-5 microns and 8-12 microns wavelength ranges. Pit organs exhibited greater absorption/emissivity in the 8-12 microns range than in the 3-5 microns range. To directly test the relationship between photoreceptors and pit organ infrared-sensitive neurons, we performed immunocytochemistry using antisera directed against retinal photoreceptor opsins. Retinal photoreceptors were labeled with antisera specific for retinal opsins, but these antisera failed to label terminals of infrared-sensitive neurons in the pit organ. Infrared-receptive neurons were also distinguished from retinal photoreceptors on the basis of their calcium-binding protein content. These results indicate that the pit organ absorbs infrared radiation in two major atmospheric transmission windows, one of which (8-12 microns) matches emission of targeted prey, and that infrared receptors are biochemically distinct from retinal photoreceptors. These results also provide the first identification of prospective biochemical components of infrared signal transduction in pit organ receptor neurons.     

Zebrafish cone-rod (crx) homeobox gene promotes retinogenesis

The mammalian Cone-rod homeobox (Crx) gene is a divergent member of the Otx gene family known to be involved in differentiation and survival of retinal photoreceptors and photoentrainment of circadian rhythms. Zebrafish have two genes in the Otx5/crx orthology class, and we previously showed that crx can transactivate rhodopsin expression in vitro, and that otx5 (orthodenticle-related gene), but not crx, regulates expression of circadian genes in the pineal. Here, we show that zebrafish crx does not regulate expression of opsins and other photoreceptor-specific genes in the pineal. We further show that crx is expressed in proliferating retinal progenitors and may be involved in patterning the early optic primordium and in promoting the differentiation of retinal progenitors, including photoreceptors. These results suggest novel functions for zebrafish crx during retinal specification and differentiation.     

Evolution of vertebrate retinal photoreception

Recent findings shed light on the steps underlying the evolution of vertebrate photoreceptors and retina. Vertebrate ciliary photoreceptors are not as wholly distinct from invertebrate rhabdomeric photoreceptors as is sometimes thought. Recent information on the phylogenies of ciliary and rhabdomeric opsins has helped in constructing the likely routes followed during evolution. Clues to the factors that led the early vertebrate retina to become invaginated can be obtained by combining recent knowledge about the origin of the pathway for dark re-isomerization of retinoids with knowledge of the inability of ciliary opsins to undergo photoreversal, along with consideration of the constraints imposed under the very low light levels in the deep ocean. Investigation of the origin of cell classes in the vertebrate retina provides support for the notion that cones, rods and bipolar cells all originated from a primordial ciliary photoreceptor, whereas ganglion cells, amacrine cells and horizontal cells all originated from rhabdomeric photoreceptors. Knowledge of the molecular differences between cones and rods, together with knowledge of the scotopic signalling pathway, provides an understanding of the evolution of rods and of the rods'' retinal circuitry. Accordingly, it has been possible to propose a plausible scenario for the sequence of evolutionary steps that led to the emergence of vertebrate photoreceptors and retina.     

Evolution of color vision.

Color vision is achieved by comparing the inputs from retinal photoreceptor neurons that differ in their wavelength sensitivity. Recent studies have elucidated the distribution and phylogeny of opsins, the family of light-sensitive molecules involved in this process. Interesting new findings suggest that animals have evolved a strategy to achieve specific sensitivity through the mutually exclusive expression of different opsin genes in photoreceptors.     

Knockout of DLIC1 leads to retinal cone degeneration via disturbing Rab8 transport in zebrafish

Retinal photoreceptors execute phototransduction functions and require an efficient system for the transport of materials (e.g. proteins and lipids) from inner segments to outer segments. Cytoplasmic dynein 1 is a minus-end-directed microtubule motor and participates in cargo transport in the cytoplasm. However, the roles of dynein 1 motor in photoreceptor cargo transport and retinal development are still ambiguous. In our present study, the light intermediate chain protein DLIC1 (encoded by dync1li1), links activating adaptors to bind diverse cargos in the dynein 1 motor, was depleted using CRISPR-Cas9 technology in zebrafish. The dync1li1^?/? zebrafish displayed progressive degeneration of retinal cone photoreceptors, especially blue cones. The retinal rods were not affected in dync1li1^?/? zebrafish. Knockout of DLIC1 resulted in abnormal expression and localization of cone opsins in dync1li1^?/? retinas. TUNEL staining suggested that apoptosis was induced after aberrant accumulation of cone opsins in photoreceptors of dync1li1^?/? zebrafish. Instead of Rab11 transport, Rab8 transport was disturbed in dync1li1^?/? retinas. Our data demonstrate that DLIC1 is required for function maintenance and survival of cone photoreceptors, and hint at an essential role of the cytoplasmic dynein 1 motor in photoreceptor cargo transport.     

Interaction of 11-cis-retinol dehydrogenase with the chromophore of retinal g protein-coupled receptor opsin

Vertebrate opsins in both photoreceptors and the retinal pigment epithelium (RPE) have fundamental roles in the visual process. The visual pigments in photoreceptors are bound to 11-cis-retinal and are responsible for the initiation of visual excitation. Retinochrome-like opsins in the RPE are bound to all-trans-retinal and play an important role in chromophore metabolism. The retinal G protein-coupled receptor (RGR) of the RPE and Müller cells is an abundant opsin that generates 11-cis-retinal by stereospecific photoisomerization of its bound all-trans-retinal chromophore. We have analyzed a 32-kDa protein (p32) that co-purifies with bovine RGR from RPE microsomes. The co-purified p32 was identified by mass spectrometric analysis as 11-cis-retinol dehydrogenase (cRDH), and enzymatic assays have confirmed the isolation of an active cRDH. The co-purified cRDH showed marked substrate preference to 11-cis-retinal and preferred NADH rather than NADPH as the cofactor in reduction reactions. cRDH did not react with endogenous all-trans-retinal bound to RGR but reacted specifically with 11-cis-retinal that was generated by photoisomerization after irradiation of RGR. The reduction of 11-cis-retinal to 11-cis-retinol by cRDH enhanced the net photoisomerization of all-trans-retinal bound to RGR. These results indicate that cRDH is involved in the processing of 11-cis-retinal after irradiation of RGR opsin and suggest that cRDH has a novel role in the visual cycle.     

Inner retinal photoreceptors (IRPs) in mammals and teleost fish.

Research over the past decade has provided overwhelming evidence that photoreception in the vertebrate eye is not confined to the rods and cones. The discovery of non-rod, non-cone ocular photoreceptors in mammals and fish arose from quite different lines of investigation. In transgenic mice entirely lacking functional rod and cone photoreceptors a range of responses to light, including the regulation of the circadian system and a pupillary light reflex, are preserved. Electrophysiological and imaging approaches were then able to characterise a coupled plexus of directly light sensitive ganglion cells. Most recently action spectroscopy has shown that a novel 'blue-light' sensitive photopigment based upon opsin/vitamin A (OP480) mediates these responses to light. Several candidate genes have emerged for OP480, with melanopsin being by far the strongest. A definitive link, however, between this gene and OP480 has still to be established. In contrast to the mammals, the discovery of inner retinal photoreceptors (IRPs) in fish started with the discovery of a new gene family (VA opsin). The teleost VA opsins form functional photopigments and are expressed in several different types of inner retinal neuron, including retinal horizontal cells. Recent studies have investigated the electrical properties of these photosensitive neurones, but their light-sensing role remains a matter of speculation. Thus the study of IRP is developing along quite separate lines. In the mammals the research is directed towards a molecular identification of the photopigment (OP480) and its cascade, whilst in fish the major effort is directed towards identifying a role for these novel photoreceptors using physiological approaches. The discovery of IRPs in the vertebrates tells us that despite 150 years of research, we still have much to learn about how the eye processes light.     

Unexpected diversity and photoperiod dependence of the zebrafish melanopsin system

Animals have evolved specialized photoreceptors in the retina and in extraocular tissues that allow them to measure light changes in their environment. In mammals, the retina is the only structure that detects light and relays this information to the brain. The classical photoreceptors, rods and cones, are responsible for vision through activation of rhodopsin and cone opsins. Melanopsin, another photopigment first discovered in Xenopus melanophores (Opn4x), is expressed in a small subset of retinal ganglion cells (RGCs) in the mammalian retina, where it mediates non-image forming functions such as circadian photoentrainment and sleep. While mammals have a single melanopsin gene (opn4), zebrafish show remarkable diversity with two opn4x-related and three opn4-related genes expressed in distinct patterns in multiple neuronal cell types of the developing retina, including bipolar interneurons. The intronless opn4.1 gene is transcribed in photoreceptors as well as in horizontal cells and produces functional photopigment. Four genes are also expressed in the zebrafish embryonic brain, but not in the photoreceptive pineal gland. We discovered that photoperiod length influences expression of two of the opn4-related genes in retinal layers involved in signaling light information to RGCs. Moreover, both genes are expressed in a robust diurnal rhythm but with different phases in relation to the light-dark cycle. The results suggest that melanopsin has an expanded role in modulating the retinal circuitry of fish.     

The putative brain photoperiodic photoreceptors in the vetch aphid,Megoura viciae

In an attempt to identify the brain photoreceptors that mediate the photoperiodic response of the vetch aphid, Megoura viciae, we utilised immunocytochemical techniques and employed 20 antibodies directed against invertebrate and vertebrate opsins and phototransduction proteins. A sub-set of these antibodies (to Drosophila rhodopsin 1: RH1-1; vertebrate cone opsins: COS-1; CERN-874; CERN-933; vertebrate rod opsin: CERN-901; vertebrate arrestin: AB-Arr; vertebrate transducin+arrestin+rhodopsin kinase+cGMP phosphodiesterase: CERN-911; and vertebrate cellular retinoid binding protein: CRALBP) consistently labelled an anterior ventral neuropile region of the protocerebrum. These anatomical findings, coupled with previous localised illumination and micro-lesion studies, provide strong evidence that this region of the aphid brain houses the photoperiodic photoreceptors. The present study also confirms that the medial (Group I) neurosecretory cells are not the photoperiodic photoreceptors.     

Rhabdom evolution in butterflies: insights from the uniquely tiered and heterogeneous ommatidia of the Glacial Apollo butterfly, Parnassius glacialis

The eye of the Glacial Apollo butterfly, Parnassius glacialis, a 'living fossil' species of the family Papilionidae, contains three types of spectrally heterogeneous ommatidia. Electron microscopy reveals that the Apollo rhabdom is tiered. The distal tier is composed exclusively of photoreceptors expressing opsins of ultraviolet or blue-absorbing visual pigments, and the proximal tier consists of photoreceptors expressing opsins of green or red-absorbing visual pigments. This organization is unique because the distal tier of other known butterflies contains two green-sensitive photoreceptors, which probably function in improving spatial and/or motion vision. Interspecific comparison suggests that the Apollo rhabdom retains an ancestral tiered pattern with some modification to enhance its colour vision towards the long-wavelength region of the spectrum.     

Generation of three-dimensional retinal organoids expressing rhodopsin and S- and M-cone opsins from mouse stem cells

Purpose

Three-dimensional retinal organoids can be differentiated from embryonic stem cells/induced pluripotent stem cells (ES/iPS cells) under defined medium conditions. We modified the serum-free floating culture of embryoid body-like aggregates with quick reaggregation (SFEBq) culture procedure to obtain retinal organoids expressing more rod photoreceptors and S- and M-cone opsins.

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.     

Visual photoreceptor subtypes in the chicken retina: melatonin-synthesizing activity and in vitro differentiation

The chicken retina contains five visual photoreceptor subtypes, based on the specific opsin gene they express. In addition to the central role they play in vision, some or all of these photoreceptors translate photoperiodic information into a day-night rhythm of melatonin production. This indolic hormone plays an important role in the photoperiodic regulation of retinal physiology. Previous studies have stopped short of establishing whether melatonin synthesis takes place in all the photoreceptor spectral subtypes. Another issue that has been left unsettled by previous studies is when during development are retinal precursor cells committed to a specific photoreceptor subtype and to a melatoninergic phenotype? To address the first question, in situ hybridization of the five opsins was combined with immunofluorescent detection of the melatonin-synthesizing enzyme hydroxyindole O-methyltransferase (HIOMT, EC.2.1.1.4). Confocal microscopy clearly indicated that all photoreceptor spectral subtypes are involved in melatonin synthesis. To tackle the second question, retinal precursor cells were dissociated between embryonic day 6 (E6) and E13 and cultured in serum-free medium for 4 days to examine their ability to autonomously activate the expression of opsins and HIOMT. Real-time PCR on cultured precursors indicated that red-, green- and violet-sensitive cones are committed at E6, rods at E10 and blue-sensitive cones at E12. HIOMT gene expression was programmed at E6, probably reflecting the differentiation of early cones. The present study provides a better characterization of photoreceptor subtypes in the chicken retina and describes a combination of serum-free culture and real-time PCR that should facilitate further developmental studies.     

Meckelin 3 Is Necessary for Photoreceptor Outer Segment Development in Rat Meckel Syndrome

Ciliopathies lead to multiorgan pathologies that include renal cysts, deafness, obesity and retinal degeneration. Retinal photoreceptors have connecting cilia joining the inner and outer segment that are responsible for transport of molecules to develop and maintain the outer segment process. The present study evaluated meckelin (MKS3) expression during outer segment genesis and determined the consequences of mutant meckelin on photoreceptor development and survival in Wistar polycystic kidney disease Wpk/Wpk rat using immunohistochemistry, analysis of cell death and electron microscopy. MKS3 was ubiquitously expressed throughout the retina at postnatal day 10 (P10) and P21. However, in the mature retina, MKS3 expression was restricted to photoreceptors and the retinal ganglion cell layer. At P10, both the wild type and homozygous Wpk mutant retina had all retinal cell types. In contrast, by P21, cells expressing rod- and cone-specific markers were fewer in number and expression of opsins appeared to be abnormally localized to the cell body. Cell death analyses were consistent with the disappearance of photoreceptor-specific markers and showed that the cells were undergoing caspase-dependent cell death. By electron microscopy, P10 photoreceptors showed rudimentary outer segments with an axoneme, but did not develop outer segment discs that were clearly present in the wild type counterpart. At p21 the mutant outer segments appeared much the same as the P10 mutant outer segments with only a short axoneme, while the wild-type controls had developed outer segments with many well-organized discs. We conclude that MKS3 is not important for formation of connecting cilium and rudimentary outer segments, but is critical for the maturation of outer segment processes.     

Simultaneous Expression of UV and Violet SWS1 Opsins Expands the Visual Palette in a Group of Freshwater Snakes

Snakes are known to express a rod visual opsin and two cone opsins, only (SWS1, LWS), a reduced palette resulting from their supposedly fossorial origins. Dipsadid snakes in the genus Helicops are highly visual predators that successfully invaded freshwater habitats from ancestral terrestrial-only habitats. Here, we report the first case of multiple SWS1 visual pigments in a vertebrate, simultaneously expressed in different photoreceptors and conferring both UV and violet sensitivity to Helicops snakes. Molecular analysis and in vitro expression confirmed the presence of two functional SWS1 opsins, likely the result of recent gene duplication. Evolutionary analyses indicate that each sws1 variant has undergone different evolutionary paths with strong purifying selection acting on the UV-sensitive copy and d_N/d_S ∼1 on the violet-sensitive copy. Site-directed mutagenesis points to the functional role of a single amino acid substitution, Phe86Val, in the large spectral shift between UV and violet opsins. In addition, higher densities of photoreceptors and SWS1 cones in the ventral retina suggest improved acuity in the upper visual field possibly correlated with visually guided behaviors. The expanded visual opsin repertoire and specialized retinal architecture are likely to improve photon uptake in underwater and terrestrial environments, and provide the neural substrate for a gain in chromatic discrimination, potentially conferring unique color vision in the UV–violet range. Our findings highlight the innovative solutions undertaken by a highly specialized lineage to tackle the challenges imposed by the invasion of novel photic environments and the extraordinary diversity of evolutionary trajectories taken by visual opsin-based perception in vertebrates.     

Disease mechanisms and neuroprotection by tauroursodeoxycholic acid in Rpgr knockout mice

Mutations in the retinitis pigmentosa GTPase regulator (RPGR) gene are the predominant cause of retinitis pigmentosa. RPGR plays a critical role as a scaffold protein in the regulation of protein trafficking from the basal body to the axoneme, where the cargoes are transported to the outer segments (OSs) of photoreceptors. This trafficking process is controlled directly by intraflagellar transport complexes and regulated by the RPGR protein complex, although the precise mechanisms have yet to be defined. We used an Rpgr conditional knockout (cko) mouse model to investigate the disease mechanisms during retinal degeneration and to evaluate the protective effects of tauroursodeoxycholic acid (TUDCA). Rhodopsin, cone opsins and transducin were mislocalized in Rpgr cko photoreceptors, while localization of NPHP4 to connecting cilia was absent, suggesting that RPGR is required for ciliary protein trafficking. Microglia were activated in advance of retinal degeneration in Rpgr cko mouse retinas. TUDCA treatment suppressed microglial activation and inflammation and prevented photoreceptor degeneration in Rpgr cko mice. Our data demonstrated that TUDCA has therapeutic potential for RPGR-associated RP patients.