Discovery and design of photocyclic animal opsins: potential application to gene therapy from non-visual opsin research

Photoreception—the detection of light for image formation (vision) as well as for nonimage-forming purposes (circadian regulation and DNA repair)—is critical to the survival of most animals. In vertebrates, photoreception is mediated by opsin proteins, which are classified, according to their function, into visual and nonvisual opsins. Here, we provide the most comprehensive study to date on the evolution of the opsin gene family in the largest class of vertebrates, actinopterygians, with a particular focus on the understudied nonvisual opsins. Based on an in-depth analysis of 535 high-quality genomes, we document great variation in gene numbers in the different opsin gene subfamilies across ray-finned fishes and show that visual opsins are more prone to duplications and losses than nonvisual opsins. We provide evidence that visual and nonvisual opsins coevolve in ray-finned fishes, both in terms of copy numbers and selective pressures acting on their coding sequences, probably in response to the different photic environments they inhabit. Species that live in dim light or in the dark (such as in caves or the deep sea) had reduced visual and nonvisual opsin gene repertoires, while polar species feature accelerated evolution in both. Fishes that rely on electroreception show a slight reduction in the number of visual and nonvisual opsin genes and accelerated evolution of the remaining genes. We further found that genes of the phototransduction cascade coevolve with opsins. Finally, the finding that nonvisual opsins are mainly expressed in the testes and ovaries (after the eyes) supports a function in gamete biology.

Knowledge of crustacean vision is lacking compared to the more well-studied vertebrates and insects. While crustacean visual systems are typically conserved morphologically, the molecular components (i.e. opsins) remain understudied. This review aims to characterize opsin diversity across crustacean lineages for an integrated view of visual system evolution. Using publicly available data from 95 species, we identified opsin sequences and classified them by clade. Our analysis produced 485 putative visual opsins and 141 non-visual opsins. The visual opsins were separated into six clades: long wavelength sensitive (LWS), middle wavelength sensitive (MWS) 1 and 2, short wavelength or ultraviolet sensitive (SWS/UVS) and a clade of thecostracan opsins, with multiple LWS and MWS opsin copies observed. The SWS/UVS opsins were relatively conserved in most species. The crustacean classes Cephalocarida, Remipedia and Hexanauplia exhibited reduced visual opsin diversity compared to others, with the malacostracan decapods having the highest opsin diversity. Non-visual opsins were identified from all investigated classes except Cephalocarida. Additionally, a novel clade of non-visual crustacean-specific, R-type opsins (Rc) was discovered. This review aims to provide a framework for future research on crustacean vision, with an emphasis on the need for more work in spectral characterization and molecular analysis. This article is part of the theme issue 'Understanding colour vision: molecular, physiological, neuronal and behavioural studies in arthropods'.

Opsins are universal photoreceptive proteins in animals and can be classified into three types based on their photoreaction properties. Upon light irradiation, vertebrate rhodopsin forms a metastable active state, which cannot revert back to the original dark state via either photoreaction or thermal reaction. By contrast, after photoreception, most opsins form a stable active state which can photoconvert back to the dark state. Moreover, we recently found a novel type of opsins whose activity is regulated by photocycling. However, the molecular mechanism underlying this diversification of opsins remains unknown. In this study, we showed that vertebrate rhodopsin acquired the photocyclic and photoreversible properties upon introduction of a single mutation at position 188. This revealed that the residue at position 188 contributes to the diversification of photoreaction properties of opsins by its regulation of the recovery from the active state to the original dark state.

Opsins are universal photoreceptive proteins in animals and can be classified into three types based on their photoreaction properties. Upon light irradiation, vertebrate rhodopsin forms a metastable active state, which cannot revert back to the original dark state via either photoreaction or thermal reaction. By contrast, after photoreception, most opsins form a stable active state which can photoconvert back to the dark state. Moreover, we recently found a novel type of opsins whose activity is regulated by photocycling. However, the molecular mechanism underlying this diversification of opsins remains unknown. In this study, we showed that vertebrate rhodopsin acquired the photocyclic and photoreversible properties upon introduction of a single mutation at position 188. This revealed that the residue at position 188 contributes to the diversification of photoreaction properties of opsins by its regulation of the recovery from the active state to the original dark state.

Cone visual pigments

Opsins underlie visual and non-visual photoreceptions in animals. Vertebrate and arthropod visual opsins belong to different opsin groups and convergently show spectral diversity ranging from the UV to the red region for color vision. Recently, uncharacterized opsins called arthropsin have been identified from various protostome genomes. Arthropsin is clustered with arthropod and mollusk visual opsins and vertebrate blue-sensitive non-visual opsin Opn4. Here, we show that arthropsins have unexpected spectral diversity ranging from the UV to the red region. In particular, water flea (Daphnia magna) expresses red-sensitive arthropsins in the optic lobe and brain. Among the non-visual opsins characterized so far, these arthropsins exhibit the most red-shifted spectral sensitivity. Moreover, the molecular mechanism responsible for the red shift of arthropsins is different from that of red-sensitive vertebrate visual opsins. We characterize arthropsin as a different type of non-visual opsin which acquired the spectral diversity independently of vertebrate and arthropod visual opsins.

Opsins are universal photoreceptive proteins in animals. Vertebrate rhodopsin in ciliary photoreceptor cells photo-converts to a metastable active state to regulate cyclic nucleotide signaling. This active state cannot photo-convert back to the dark state, and thus vertebrate rhodopsin is categorized as a mono-stable opsin. By contrast, mollusk and arthropod rhodopsins in rhabdomeric photoreceptor cells photo-convert to a stable active state to stimulate IP3/calcium signaling. This active state can photo-convert back to the dark state, and thus these rhodopsins are categorized as bistable opsins. Moreover, the negatively charged counterion position crucial for the visible light sensitivity is different between vertebrate rhodopsin (Glu113) and mollusk and arthropod rhodopsins (Glu181). This can be explained by an evolutionary scenario where vertebrate rhodopsin newly acquired Glu113 as a counterion, which is thought to have led to higher signaling efficiency of vertebrate rhodopsin. However, the detailed evolutionary steps which led to the higher efficiency in vertebrate rhodopsin still remain unknown. Here, we analyzed the xenopsin group, which is phylogenetically distinct from vertebrate rhodopsin and functions in protostome ciliary cells. Xenopsins are blue-sensitive bistable opsins that regulate cAMP signaling. We found that a bistable xenopsin of Leptochiton asellus had Glu113 as a counterion but did not exhibit elevated signaling efficiency. Therefore, our results show that vertebrate rhodopsin and L. asellus xenopsin regulate cyclic nucleotide signaling in ciliary cells and displaced the counterion position from Glu181 to Glu113 via convergent evolution, whereas subsequently only vertebrate rhodopsin elevated its signaling efficiency by acquiring the mono-stable property.

Ex Vivo Adenoviral Vector Gene Delivery Results in Decreased Vector-associated Inflammation Pre- and Post–lung Transplantation in the Pig

Photoreception is common in animals without a visual system. In animals with visual systems, it is sometimes presumed that the same photoreceptors and pathways will accommodate both visual and non-visual light detection. However, mounting evidence reveals that most animals exhibit broad extra-visual photoreceptive functions that are wholly independent of the visual system. One of these functions is the synchronization of the circadian clock to light–dark signals, or photoentrainment. In mammals, behavioral photoentrainment is achieved exclusively through visual and non-visual opsin proteins within the retina, and molecular photoentrainment of individual cells occurs using non-visual opsins in some peripheral tissues. This is in contrast to insects and fish where nearly all peripheral organs are directly photoentrainable. This review will summarize the family of opsins in mammals and focus on the role of non-visual opsins in circadian photoreception. Particular emphasis will be given to photoentrainment in other vertebrates in order to compare and contrast the use of the wide range of non-visual opsins in circadian photoentrainment throughout the animal kingdom.

Opsins play a key role in the ability to sense light both in image-forming vision and in non-visual photoreception (NVP). These modalities, in most animal phyla, share the photoreceptor protein: an opsin-based protein binding a light-sensitive chromophore by a lysine (Lys) residue. So far, visual and non-visual opsins have been discovered throughout the Metazoa phyla, including the photoresponsive Hydra, an eyeless cnidarian considered the evolutionary sister species to bilaterians. To verify whether light influences and modulates opsin gene expression in Hydra, we utilized four expression sequence tags, similar to two classic opsins (SW rhodopsin and SW blue-sensitive opsin) and two non-visual opsins (melanopsin and peropsin), in investigating the expression patterns during both diurnal and circadian time, by means of a quantitative RT-PCR. The expression levels of all four genes fluctuated along the light hours of diurnal cycle with respect to the darkness one and, in constant dark condition of the circadian cycle, they increased. The monophasic behavior in the L12:D12 cycle turned into a triphasic expression profile during the continuous darkness condition. Consequently, while the diurnal opsin-like expression revealed a close dependence on light hours, the highest transcript levels were found in darkness, leading us to novel hypothesis that in Hydra, an "internal" biological rhythm autonomously supplies the opsins expression during the circadian time. In conclusion, in Hydra, both diurnal and circadian rhythms apparently regulate the expression of the so-called visual and non-visual opsins, as already demonstrated in higher invertebrate and vertebrate species. Our data confirm that Hydra is a suitable model for studying ancestral precursor of both visual and NVP, providing useful hints on the evolution of visual and photosensory systems.

This study was carried out to identify and estimate physiological function of a new type of opsin subfamily present in the retina and whole brain tissues of Japanese eel using RNA–Seq transcriptome method. A total of 18 opsin subfamilies were identified through RNA–seq. The visual opsin family included Rh2, SWS2, FWO, DSO, and Exo-Rhod. The non-visual opsin family included four types of melanopsin subfamily (Opn4x1, Opn4x2, Opn4m1, and Opn4m2), peropsin, two types of neuropsin subfamily (Opn5-like, Opn5), Opn3, three types of TMT opsin subfamily (TMT1, 2, 3), VA-opsin, and parapinopsin. In terms of changes in photoreceptor gene expression in the retina of sexually mature and immature male eels, DSO mRNA increased in the maturation group. Analysis of expression of opsin family gene in male eel brain before and after maturation revealed that DSO and SWS2 expression in terms of visual opsin mRNA increased in the sexually mature group. In terms of non-visual opsin mRNA, parapinopsin mRNA increased whereas that of TMT2 decreased in the fore-brain of the sexually mature group. The mRNA for parapinopsin increased in the mid-brain of the sexually mature group, whereas those of TMT1 and TMT3 increased in the hind-brain of the sexually mature group. DSO mRNA also increased in the retina after sexual maturation, and DSO and SWS2 mRNA increased in whole brain part, suggesting that DSO and SWS2 are closely related to sexual maturation.

IntroductionLight plays a key role in regulating circadian rhythms and downstream physiological and behavioural functions. However, excessive exposure to artificial blue light (450–500 nm) can disrupt sleep, metabolism and neural integrity. Visual opsins mediate light-dependent signalling, but organisms also express non-visual opsins whose roles in blue-light-induced neural stress are not well understood.MethodsWe used Drosophila melanogaster knockout lines lacking either visual rhodopsin 1 (Rh11) or non-visual rhodopsin 7 (Rh71), alongside wild-type (w1118) controls. Flies were continuously exposed to 488 nm blue light (1,320 lux; 1,120 μW·cm−2) from egg deposition until they were 20 days old. DNA damage (γ-H2Av immunostaining) and vacuole formation were quantified in brain regions associated with sensory processing and neurotransmission.ResultsRh11 flies exhibited the highest levels of DNA damage and vacuolisation compared to the w1118 and Rh71 lines. These effects were most pronounced in neuropils linked to sensory integration and synaptic activity.DiscussionOur findings demonstrate that the visual opsin Rh1 plays a predominant role in blue-light-induced DNA damage and neurodegeneration in the Drosophila central nervous system. This suggests that it is visual, rather than non-visual, opsins that mediate the neurotoxic effects of exposure to artificial light.

Non-visual opsins were discovered in the early 1990s. These genes play roles in circadian rhythm in mammals, seasonal reproduction in birds, light avoidance in amphibian larvae, and neural development in fish. However, the interpretation of such studies and the success of future work are compromised by the fact that non-visual opsin repertoires have not been properly characterized in any of these lineages. Here, we show that non-visual opsins from tetrapods and ray-finned fish are distributed among 18 monophyletic subfamilies. An amphibian sequence occurs in every subfamily, whereas mammalian orthologs occur in only seven. Species in the major ray-finned fish lineages, Holostei, Osteoglossomorpha, Otomorpha, Protacanthopterygii, and Neoteleostei, have large numbers of non-visual opsins (22-32 genes) as a result of gene duplication events including, but not limited to, the teleost genome duplication (TGD). In contrast to visual opsins, where lineage-specific duplication is common, the ray-finned fish non-visual opsin repertoire appears to have stabilized shortly after the TGD event and consequently even distantly related species have repertoires of similar size and composition. Most non-visual opsins have been named without the benefit of a phylogenetic perspective and, accordingly, major revisions are proposed.

Many lizards (Squamata), as well as the tuatara (Rhynchocephalia), are distinguished among vertebrate groups for the presence of the parietal eye, or "third eye", a structure derived from the pineal complex containing a simplified retina with photoreceptor cells. The parietal eye expresses nonvisual opsins that differ from the visual opsin repertoire of the lateral eyes. These are pinopsin (OPNP), parapinopsin (OPNPP), and parietopsin (OPNPT), all being evolutionary close to visual opsins. Here, we searched over 60 lepidosaurian genomes for pineal nonvisual opsins to check for the evolutionary trajectory of these genes in reptiles. Unexpectedly, we identified a novel opsin gene, which we termed "lepidopsin" (OPNLEP), that is present solely in the genomes of the tuatara and most lizard groups but absent from other vertebrates. Remnants of the gene are found in the coelacanth and some ray-finned fishes, implying that OPNLEP is an ancient opsin that has been repeatedly lost during vertebrate evolution. We found that the tuatara and most lizards of the Iguania, Anguimorpha, Scincoidea, and Lacertidae clades, which possess a parietal eye, harbor all pineal opsin genes. Lizards missing the parietal eye, like geckos, teiids, and a fossorial amphisbaenian, lack most or all pineal nonvisual opsins. In summary, our survey of pineal nonvisual opsins reveals (i) the persistence of a previously unknown ancient opsin gene-OPNLEP-in lepidosaurians; (ii) losses of nonvisual opsins in specific lizard clades; and (iii) a correlation between the presence of a parietal eye and the genomic repertoire of pineal nonvisual opsins.

Gene transfer approaches to the healing of bone and cartilage.