Daisuke Kojima

Exo-rhodopsin: a novel rhodopsin expressed in the zebrafish pineal gland.

The zebrafish, a useful animal model for genetic studies, has a photosensitive pineal gland, which has an endogenous circadian pacemaker entrained to environmental light-dark cycles [G.M. Cahill, Brain Res. 708 (1996) 177-181]. Although pinopsin has been found in the pineal glands of birds and reptiles, the molecular identity responsible for fish pineal photosensitivity remains unclear. This study reports identification of a novel opsin gene expressed in the zebrafish pineal gland. The deduced amino acid sequence is similar to, but not identical (74% identity) with that of canonical rhodopsin in the zebrafish retina. This novel rhodopsin is expressed in the majority of pineal cells but not in retinal cells, and hence named exo-rhodopsin after extra-ocular rhodopsin. This study first shows that two different rhodopsin genes are expressed in an individual animal each within a unique location. A phylogenetic analysis indicated that the exo-rhodopsin gene was produced by a duplication of the rhodopsin gene at an early stage in the ray-finned fish lineage. As expected, the exo-rhodopsin gene was found in the medakafish and European eel genomes, suggesting strongly that exo-rhodopsin is a pineal opsin common to teleosts. Identification of exo-rhodopsin in the zebrafish provides an opportunity for studying the role of pineal photoreceptive molecules by using genetic approaches.

A Novel Go-mediated Phototransduction Cascade in Scallop Visual Cells

Scallop retinas contain ciliary photoreceptor cells that respond to light by hyperpolarization like vertebrate rods and cones, but the response is generated by a different phototransduction cascade from those of rods and cones. To elucidate the cascade, we investigated a visual pigment and a G-protein functioning in the hyperpolarizing cell. Sequencing of cDNAs andin situ hybridization experiments showed that the hyperpolarizing cells express a novel subtype of visual pigment, which showed significant differences in amino acid sequence from other visual pigments. Cloning cDNA genes of G-protein and immunohistochemical analysis revealed the presence of an alpha subunit of a Gotype G-protein, 83% identical in amino acid sequence to mammalian Go(α) in the nervous system, in the photoreceptive region of the cells. The results demonstrate that a novel, Go-mediated, phototransduction cascade is present in the hyperpolarizing cells. The phototransduction cascade in the scallop hyperpolarizing cell provides an alternative system to investigate Go-mediated transduction pathways in the nervous system. Molecular phylogenetic analysis strongly suggests that the Go-mediated phototransduction system emerged before the divergence of animals into vertebrate and invertebrate in the course of evolution.

UV-Sensitive Photoreceptor Protein OPN5 in Humans and Mice

A variety of animal species utilize the ultraviolet (UV) component of sunlight as their environmental cues, whereas physiological roles of UV photoreception in mammals, especially in human beings, remain open questions. Here we report that mouse neuropsin (OPN5) encoded by the Opn5 gene exhibited an absorption maximum (λmax) at 380 nm when reconstituted with 11-cis-retinal. Upon UV-light illumination, OPN5 was converted to a blue-absorbing photoproduct (λmax 470 nm), which was stable in the dark and reverted to the UV-absorbing state by the subsequent orange light illumination, indicating its bistable nature. Human OPN5 also had an absorption maximum at 380 nm with spectral properties similar to mouse OPN5, revealing that OPN5 is the first and hitherto unknown human opsin with peak sensitivity in the UV region. OPN5 was capable of activating heterotrimeric G protein Gi in a UV-dependent manner. Immuno-blotting analyses of mouse tissue extracts identified the retina, the brain and, unexpectedly, the outer ears as the major sites of OPN5 expression. In the tissue sections of mice, OPN5 immuno-reactivities were detected in a subset of non-rod/non-cone retinal neurons as well as in the epidermal and muscle cells of the outer ears. Most of these OPN5-immuno-reactivities in mice were co-localized with positive signals for the alpha-subunit of Gi. These results demonstrate the first example of UV photoreceptor in human beings and strongly suggest that OPN5 triggers a UV-sensitive Gi-mediated signaling pathway in the mammalian tissues.

Two isoforms of chicken melanopsins show blue light sensitivity

Melanopsin is a vertebrate non‐visual opsin and functions as a circadian photoreceptor in mammalian retinas. Here we found the expression of two kinds of melanopsin genes in the chicken pineal gland and identified the presence of five isoforms derived from these two genes. Reconstitution of the recombinant proteins with 11‐cis‐retinal revealed that at least two of these melanopsin protein isoforms can function as blue‐sensitive photopigments with absorption maxima at 476–484 nm. These values are consistent with maximal sensitivities of action spectra determined from the physiological and behavioral studies on mammalian melanopsins. The melanopsin isoforms found in this study may function as pineal circadian photoreceptors.

The primary structure of iodopsin, a chicken red-sensitive cone pigment

A purified iodopsin was digested by CNBr or several proteolytic enzymes into fragments, the amino acid sequences of which were determined. A partial sequence of the C‐terminal fragment was utilized for synthesizing an oligonucleotide probe which identified the iodopsin cDNA (1339 bases). The deduced amino acid sequence (362 residues) had 80%, 42% or 43% homology to that of human red‐sensitive cone pigment, cattle or chicken rhodospin, respectively. Although the hydropathy profile implies that iodopsin, like rhodopsin, has 7 transmembrane α‐helical segments, iodopsin may have a hydrophilic pocket near the seventh segment on the basis of the unexpected cleavages in the middle of the segment VII by chymotrypsin under nondenaturing conditions.

Identification of Nonvisual Photomotor Response Cells in the Vertebrate Hindbrain

Nonvisual photosensation enables animals to sense light without sight. However, the cellular and molecular mechanisms of nonvisual photobehaviors are poorly understood, especially in vertebrate animals. Here, we describe the photomotor response (PMR), a robust and reproducible series of motor behaviors in zebrafish that is elicited by visual wavelengths of light but does not require the eyes, pineal gland, or other canonical deep-brain photoreceptive organs. Unlike the relatively slow effects of canonical nonvisual pathways, motor circuits are strongly and quickly (seconds) recruited during the PMR behavior. We find that the hindbrain is both necessary and sufficient to drive these behaviors. Using in vivo calcium imaging, we identify a discrete set of neurons within the hindbrain whose responses to light mirror the PMR behavior. Pharmacological inhibition of the visual cycle blocks PMR behaviors, suggesting that opsin-based photoreceptors control this behavior. These data represent the first known light-sensing circuit in the vertebrate hindbrain.

Differential expression of duplicated VAL‐opsin genes in the developing zebrafish

Non‐visual opsins mediate various light‐dependent physiological events. Our previous search for non‐visual opsin genes in zebrafish led to the discovery of VAL‐opsin (VAL‐opsinA) in deep brain cells and retinal horizontal cells of the adult fish. In this study, we report the identification and characterization of its duplicated gene, VAL‐opsinB, in zebrafish. A molecular phylogenetic analysis indicates that VAL‐opsinB is orthologous to a previously reported salmon gene and that the duplication of the VAL‐opsin gene occurred in the teleost lineage. The recombinant protein of zebrafish VAL‐opsinB forms a green‐sensitive photopigment when reconstituted with 11‐cis‐retinal. VAL‐opsinB expression was detected in a limited number of cells of the brain and the eye, and the expression pattern is distinct from that of the VAL‐opsinA gene. Such a differential expression pattern suggests that VAL‐opsinA and VAL‐opsinB are involved in different physiological events in zebrafish.

Pineal expression-promoting element (PIPE), a cis-acting element, directs pineal-specific gene expression in zebrafish

The pineal gland, sharing morphological and biochemical similarities with the retina, plays a unique and central role in the photoneuroendocrine system. The unique development of the pineal gland is directed by a specific combination of the expressed genes, but little is known about the regulatory mechanism underlying the pineal-specific gene expression. We isolated a 1.1-kbp fragment upstream of the zebrafish exo-rhodopsin (exorh) gene, which is expressed specifically in the pineal gland. Transgenic analysis using an enhanced green fluorescent protein reporter gene demonstrated that the proximal 147-bp region of the exorh promoter is sufficient to direct pineal-specific expression. This region contains three copies of a putative cone rod homeobox (Crx)/Otx-binding site, which is known to be required for expression of both retina- and pineal-specific genes. Deletion and mutational analyses of the exorh promoter revealed that a previously uncharacterized sequence TGACCCCAATCT termed pineal expression-promoting element (PIPE) is required for pineal-specific promoter activity in addition to the Crx/Otx-binding sites. By using the zebrafish rhodopsin (rh) promoter that drives retina-specific expression, we created a reporter construct having ectopic PIPE in the rh promoter at a position equivalent to that in the exorh promoter by introducing five nucleotide changes. Such a slight modification in the rh promoter induced ectopic enhanced green fluorescent protein expression in the pineal gland without affecting its retinal expression. These results identify PIPE as a critical cis-element contributing to the pineal-specific gene expression, in combination with the Crx/Otx-binding site(s).

Homeobox transcription factor Six7 governs expression of green opsin genes in zebrafish

Colour discrimination in vertebrates requires cone photoreceptor cells in the retina, and high-acuity colour vision is endowed by a set of four cone subtypes expressing UV-, blue-, green- and red-sensitive opsins. Previous studies identified transcription factors governing cone photoreceptor development in mice, although loss of blue and green opsin genes in the evolution of mammals make it difficult to understand how high-acuity colour vision was organized during evolution and development. Zebrafish (Danio rerio) represents a valuable vertebrate model for studying colour vision as it retains all the four ancestral vertebrate cone subtypes. Here, by RT-qPCR and in situ hybridization analysis, we found that sine oculis homeobox homolog 7 (six7), a transcription factor widely conserved in ray-finned fish, is expressed predominantly in the cone photoreceptors in zebrafish at both the larval and the adult stages. TAL effector nuclease-based six7 knock-out revealed its roles in expression of green, red and blue cone opsin genes. Most prominently, the six7 deficiency caused a loss of expression of all the green opsins at both the larval and adult stages. six7 is indispensable for the development and/or maintenance of the green cones.

Probing pineal-specific gene expression with transgenic zebrafish.

The pineal gland of zebrafish (Danio rerio) contains light‐sensitive photoreceptor cells and plays an important role in the neuroendocrine system. The zebrafish exorhodopsin gene encodes a pineal‐specific photoreceptive protein, whose promoter region harbors a cis‐acting element, pineal expression‐promoting element (PIPE), directing pineal‐specific gene expression. For in vivo genetic studies on PIPE‐binding proteins and their regulatory mechanisms, we generated a transgenic zebrafish line, Tg(P20‐rh/P:gfp), that expresses green fluorescent protein (GFP) under the control of the zebrafish rhodopsin promoter fused with 20 PIPE repeats. In Tg(P20‐rh/P:gfp) fish, PIPE‐dependent gene expression is visualized by GFP fluorescence in the pineal gland along with PIPE‐independent GFP signals in the retinal rod photoreceptors. The transgenic fish exhibit detectable and reproducible GFP fluorescence in the larval pineal gland by 5 days postfertilization. Antisense morpholino‐mediated knock‐down of a pineal transcription factor gene, otx5, suppresses pineal GFP expression in the transgenic line. In a pilot screen of N‐ethyl‐N‐nitrosourea‐treated fish of the GFP transgenic line, we isolated potential dominant mutations that cause attenuation of pineal GFP fluorescence with a marginal effect on the retinal GFP signal. The results suggest that the Tg(P20‐rh/P:gfp) line will be useful for detecting deficits in PIPE‐dependent gene expression in the pineal gland.