Sayuri Tomonari

Opn5 is a UV-sensitive bistable pigment that couples with Gi subtype of G protein

Opn5 (neuropsin) belongs to an independent group separated from the other six groups in the phylogenetic tree of opsins, for which little information of absorption characteristics and molecular properties of the members is available. Here we show that the chicken Opn5 (cOpn5m) is a UV-sensitive bistable pigment that couples with Gi subtype of G protein. The recombinant expression of cOpn5m in HEK 293s cells followed by the addition of 11-cis- and all-trans-retinal produced UV light-absorbing and visible light-absorbing forms, respectively. These forms were interconvertible by UV and visible light irradiations, respectively, indicating that cOpn5m is a bistable pigment. The absorption maxima of these forms were estimated to be 360 and 474 nm, respectively. The GTPγS binding assay clearly showed that the visible light-absorbing form having all-trans-retinal activates Gi type of G protein, whereas no Gt or Gq activation ability was observed. Immunohistochemical studies using an antibody against cOpn5m clearly showed that this pigment is localized within some types of amacrine cells and some cells in the ganglion cell layer of the retinas, the vast majority of cells in the pineal gland and serotonin-positive cells in the paraventricular organ. Because cOpn5m is the only UV-sensitive opsin among the opsins found so far in chicken, this study provides the molecular basis for UV reception in chicken.

Identification of chick rax/rx genes with overlapping patterns of expression during early eye and brain development.

We have isolated chick rax/rx cDNAs, cRaxL (chick Rax/Rx-like) and cRax, (chick Rax) and examined their expression patterns during early eye and brain development. The cRaxL cDNA encodes a 228 amino acid protein that is most closely related to the zebrafish Rx1 and Rx2. The cRax cDNA encodes a 317 amino acid protein, which shares higher homology with the Xenopus Rx. In addition to the homeodomain, the octapeptide and paired tail domains are conserved between the cRax and other vertebrate Rax/Rx, while cRaxL lacks the octapeptide containing N-terminal region which is conserved among all other members of the rax/rx gene family identified so far. The chick rax/rx genes are expressed in overlapping domains in the anterior neural ectoderm which corresponds to the forebrain and retina field, and later in the optic vesicle. cRax mRNA can be detected earlier than cRaxL prior to the formation of the notochord and its expression domain appears broader than that of cRaxL.

A Non-Canonical Photopigment, Melanopsin, Is Expressed in the Differentiating Ganglion, Horizontal, and Bipolar Cells of the Chicken Retina

Vertebrate melanopsin is a photopigment in the eye, required for photoentrainment. Melanopsin is more closely related to opsin proteins found in invertebrates, than to the other photo‐pigments. Although the invertebrate melanopsin‐like protein is localized in rhabdomeric photoreceptors in the invertebrate eye, it has been shown to be expressed in a subset of retinal ganglion cells in the mouse and in horizontal cells in the frog, indicating its diversified expression pattern in vertebrates. Here we show that two types of melanopsin transcripts are expressed in the developing chicken retina. Melanopsin is firstly expressed by a small subset of ganglion cells, and then prominently expressed by horizontal cells and later by bipolar cells in the developing chicken retina. This suggests that a subset of ganglion, horizontal, and bipolar cells in the chicken retina may have rhabdomeric properties in their origins. Developmental Dynamics 234:783–790, 2005.

Identification of cis-element regulating expression of the mouse Fgf10 gene during inner ear development

Fibroblast growth factor (FGF) signaling is crucial for the induction and growth of the ear, a sensory organ that involves intimate tissue interactions. Here, we report the abnormality of Fgf10 null ear and the identification of a cis‐regulatory element directing otic expression of Fgf10. In Fgf10 null inner ears, we found that the initial development of semicircular, vestibular, and cochlear divisions is roughly normal, after which there are abnormalities of semicircular canal/cristae and vestibular development. The mutant semicircular disks remain without canal formation by the perinatal stage. To elucidate regulation of the Fgf10 expression during inner ear development, we isolated a 6.6‐kb fragment of its 5′‐upstream region and examined its transcriptional activity with transgenic mice, using a lacZ‐reporter system. From comparison of the mouse sequences of the 6.6‐kb fragment with corresponding sequences of the human and chicken Fgf10, we identified a 0.4‐kb enhancer sequence that drives Fgf10 expression in the developing inner ear. The enhancer sequences have motifs for many homeodomain‐containing proteins (e.g., Prx, Hox, Nkx), in addition to POU‐domain factors (e.g., Brn3), zinc‐finger transcription factors (e.g., GATA‐binding factors), TCF/LEF‐1, and a SMAD‐interacting protein. Thus, FGF10 signaling is dispensable for specification of otic compartment identity but is required for hollowing the semicircular disk. Furthermore, the analysis of a putative inner ear enhancer of Fgf10 has disclosed a complicated regulation of Fgf10 during inner ear development by numerous transcription factors and signaling pathways. Developmental Dynamics 233:177–187, 2005.

A non-mammalian type opsin 5 functions dually in the photoreceptive and non-photoreceptive organs of birds.

A mammalian type opsin 5 (neuropsin) is a recently identified ultraviolet (UV)-sensitive pigment of the retina and other photosensitive organs in birds. Two other opsin 5-related molecules have been found in the genomes of non-mammalian vertebrates. However, their functions have not been examined as yet. Here, we identify the molecular properties of a second avian opsin 5, cOpn5L2 (chicken opsin 5-like 2), and its localization in the post-hatch chicken. Spectrophotometric analysis and radionucleotide-binding assay have revealed that cOpn5L2 is a UV-sensitive bistable pigment that couples with the Gi subtype of guanine nucleotide-binding protein (G protein). As a bistable pigment, it also shows the direct binding ability to agonist all-trans-retinal to activate G protein. The absorption maxima of UV-light-absorbing and visible light-absorbing forms were 350 and 521 nm, respectively. Expression analysis showed relatively high expression of cOpn5L2 mRNA in the adrenal gland, which is not photoreceptive but an endocrine organ, while lower expression was found in the brain and retina. At the protein level, cOpn5L2 immunoreactive cells were present in the chromaffin cells of the adrenal gland. In the brain, cOpn5L2 immunoreactive cells were found in the paraventricular and supraoptic nuclei of the anterior hypothalamus, known for photoreceptive deep brain areas. In the retina, cOpn5L2 protein was localized to subsets of cells in the ganglion cell layer and the inner nuclear layer. These results suggest that the non-mammalian type opsin 5 (Opn5L2) functions as a second UV sensor in the photoreceptive organs, while it might function as chemosensor using its direct binding ability to agonist all-trans-retinal in non-photoreceptive organs such as the adrenal gland of birds.

Evolution of Mammalian Opn5 as a Specialized UV-absorbing Pigment by a Single Amino Acid Mutation

Background: Opn5 is considered to regulate nonvisual photoreception in the retina and brain of animals. Results: Mouse and primate UV-sensitive Opn5 along with retinoid isomerase are localized in the preoptic hypothalamus. Conclusion: Mammalian Opn5 can function as a high sensitivity photosensor in the deep brain with the assistance of 11-cis-retinal supplying system. Significance: Mammals, including humans, may detect short wavelength light within the brain via Opn5. Opn5 is one of the recently identified opsin groups that is responsible for nonvisual photoreception in animals. We previously showed that a chicken homolog of mammalian Opn5 (Opn5m) is a Gi-coupled UV sensor having molecular properties typical of bistable pigments. Here we demonstrated that mammalian Opn5m evolved to be a more specialized photosensor by losing one of the characteristics of bistable pigments, direct binding of all-trans-retinal. We first confirmed that Opn5m proteins in zebrafish, Xenopus tropicalis, mouse, and human are also UV-sensitive pigments. Then we found that only mammalian Opn5m proteins lack the ability to directly bind all-trans-retinal. Mutational analysis showed that these characteristics were acquired by a single amino acid replacement at position 168. By comparing the expression patterns of Opn5m between mammals and chicken, we found that, like chicken Opn5m, mammalian Opn5m was localized in the ganglion cell layer and inner nuclear layer of the retina. However, the mouse and primate (common marmoset) opsins were distributed not in the posterior hypothalamus (including the region along the third ventricle) where chicken Opn5m is localized, but in the preoptic hypothalamus. Interestingly, RPE65, an essential enzyme for forming 11-cis-retinal in the visual cycle is expressed near the preoptic hypothalamus of the mouse and common marmoset brain but not near the region of the chicken brain where chicken Opn5m is expressed. Therefore, mammalian Opn5m may work exclusively as a short wavelength sensor in the brain as well as in the retina with the assistance of an 11-cis-retinal-supplying system.

Expression patterns of the opsin 5–related genes in the developing chicken retina

The opsin gene family encodes G protein–coupled seven‐transmembrane proteins that bind to a retinaldehyde chromophore for photoreception. It has been reported that opsin 5 is expressed in mammalian neural tissue, but its function has been elusive. As a first step to understand the function for opsin 5 in the developing eye, we searched for chicken opsin 5‐related genes in the genome by a bioinformatic approach and isolated opsin 5 cDNA fragments from the embryonic retina by RT‐PCR. We found that there are three opsin 5–related genes, designated cOpn5m (chicken opsin 5, mammalian type), cOpn5L1 (chicken opsin 5‐like 1), and cOpn5L2 (chicken opsin 5‐like 2), in the chicken genome. Quantitative PCR analysis has revealed that cOpn5m is the most abundant in the developing and early posthatching neural retina. In situ hybridization analysis has shown that cOpn5m is specifically expressed in subsets of differentiating ganglion cells and amacrine cells. These results suggest that the mammalian type opsin 5 may contribute to the development of these retinal cells in the chicken. Developmental Dynamics 237:1910–1922, 2008.

Lhx1 in the proximal region of the optic vesicle permits neural retina development in the chicken.

Summary How the eye forms has been one of the fundamental issues in developmental biology. The retinal anlage first appears as the optic vesicle (OV) evaginating from the forebrain. Subsequently, its distal portion invaginates to form the two-walled optic cup, which develops into the outer pigmented and inner neurosensory layers of the retina. Recent work has shown that this optic-cup morphogenesis proceeds as a self-organizing activity without any extrinsic molecules. However, intrinsic factors that regulate this process have not been elucidated. Here we show that a LIM-homeobox gene, Lhx1, normally expressed in the proximal region of the nascent OV, induces a second neurosensory retina formation from the outer pigmented retina when overexpressed in the chicken OV. Lhx2, another LIM-homeobox gene supposed to be involved in early OV formation, could not substitute this function of Lhx1, while Lhx5, closely related to Lhx1, could replace it. Conversely, knockdown of Lhx1 expression by RNA interference resulted in the formation of a small or pigmented vesicle. These results suggest that the proximal region demarcated by Lhx1 expression permits OV development, eventually dividing the two retinal domains.

Autotaxin controls caudal diencephalon-mesencephalon development in the chick

The diencephalon is the embryonic anlagen of the higher integration centers of the brain. Recent studies have elucidated how the cells in the rostral diencephalon acquire their regional identities. However, the understanding of the mechanisms under which the caudal diencephalon is formed is still limited. Here we focus on the role of Autotaxin (ATX), a lysophospholipid‐generating exoenzyme, whose mRNA is detected in the caudal diencephalon. RNA interference against ATX altered the expression pattern of Pax6‐regualted genes, Tcf4, Lim1, and En1, implying that ATX is required for the maintenance of the regional identity of the caudal diencephalon and the diencephalon‐mesencephalon boundary (DMB). Furthermore, ATX‐RNAi inhibited neuroepithelial cell proliferation on both sides of the DMB. We propose a dual role of ATX in chick brain development, in which ATX not only contributes to the formation of caudal diencephalon as a short‐range signal, but also regulates the growth of mesencephalon as a long‐range signal. Developmental Dynamics 239:2647–2658, 2010.

Two Opsin 3-Related Proteins in the Chicken Retina and Brain: A TMT-Type Opsin 3 Is a Blue-Light Sensor in Retinal Horizontal Cells, Hypothalamus, and Cerebellum.

Opsin family genes encode G protein-coupled seven-transmembrane proteins that bind a retinaldehyde chromophore in photoreception. Here, we sought potential as yet undescribed avian retinal photoreceptors, focusing on Opsin 3 homologs in the chicken. We found two Opsin 3-related genes in the chicken genome: one corresponding to encephalopsin/panopsin (Opn3) in mammals, and the other belonging to the teleost multiple tissue opsin (TMT) 2 group. Bioluminescence imaging and G protein activation assays demonstrated that the chicken TMT opsin (cTMT) functions as a blue light sensor when forced-expressed in mammalian cultured cells. We did not detect evidence of light sensitivity for the chicken Opn3 (cOpn3). In situ hybridization demonstrated expression of cTMT in subsets of differentiating cells in the inner retina and, as development progressed, predominant localization to retinal horizontal cells (HCs). Immunohistochemistry (IHC) revealed cTMT in HCs as well as in small numbers of cells in the ganglion and inner nuclear layers of the post-hatch chicken retina. In contrast, cOpn3-IR cells were found in distinct subsets of cells in the inner nuclear layer. cTMT-IR cells were also found in subsets of cells in the hypothalamus. Finally, we found differential distribution of cOpn3 and cTMT proteins in specific cells of the cerebellum. The present results suggest that a novel TMT-type opsin 3 may function as a photoreceptor in the chicken retina and brain.