Origin of the Vertebrate Visual Cycle: III. Distinct Distribution of RPE65 and [beta]-carotene 15,15′-Monooxygenase Homologues in Ciona intestinalis[dagger]
We previously identified three genes that encode putative visual cycle proteins that are homologues of retinal G-protein coupled receptor (Ci-opsin3), cellular retinaldebyde-binding protein (Ci-CRALBP) and β-carotene 15,15′-monooxygenase (Ci-BCO) in the ascidian Ciona intestinalis. Ci-opsin3 and Ci-CRALBP are localized in both ocellus photoreceptor cells and surrounding non-photoreceptor cells in the brain vesicle of the larva. In the present study, we investigated the possible role and evolutionary origin of the BCO/RPE65 family in the visual cycle by analyzing Ci-BCO localization by immunohistochemistry and by identifying a novel gene that encodes a homologue of retinal pigment epithelium-specific 65 kDa protein (Ci-RPE65) in C. intestinalis. In situ hybridization and expressed sequence tag (EST) profiles consistently suggest that Ci-RPE65 is not significantly expressed in the ocellus and brain vesicle of the larva. Ci-RPE65 is expressed in the neural complex, a photoreceptor organ of the adult ascidian, at a level comparable to that of Ci-opsin3 and Ci-CRALBP. Ci-RPE65 is also expressed in various adult tissues, including the gill, body wall and intestine, suggesting that Ci-RPE65 plays a role in addition to that in the visual cycle. In contrast, Ci-BCO is predominantly localized in ocellus photoreceptor cells of the larva. The larval visual cycle seems to use Ci-opsin3 as a photoisomerase. Our results also suggest that the RPE65-dependent visual cycle is used in the adult photoreceptors of a primitive chordate.
Rhodopsin consists of opsin and the chromophore, 11-cis retinal. In mammalian rod photoreceptors, 11-cis retinal photoisomerizes to an all-trans form and is released from opsin. In order to regenerate rhodopsin, the released all-trans retinoid isomerizes to 11-cis retinoid in the adjacent retinal pigment epithelium (RPE). RPE cells in the vertebrate eye are highly active in the metabolism of retinoids. Many specialized enzymes and retinoid-binding proteins expressed in RPE are believed to play critical roles in the conversion of all-trans retinoids to 11-cis retinoids.
Recently there have been advances in our understanding of the functions of molecular components in the visual cycle mechanism in the RPE (1). Upon entering RPE cells, all-trans retinol is esterified to all-trans-retinyl ester by the lecithin retinol acyl transferase (LRAT) (2). Retinal pigment epithelium-specific 65 kDa protein (RPE65), a highly expressed protein in RPE, was recently confirmed to be the isomerohydrolase that converts all-trans-retinyl ester to 11-cis retinol in a light-independent manner (3-5). Cellular retinaldehyde-binding protein (CRALBP) is of importance as an acceptor of 11-cis retinoid after the isomerization reaction in the visual cycle (6-9). In addition to RPE65, there is another isomerase, retinal G-protein coupled receptor (RGR), in RPE (10-12). RGR is bound to all-trans retinal, and is capable of operating as the photoisomerase that generates 11-cis retinal in a light-dependent manner. β-Carotene 15,15′-monooxygenase (BCO) in RPE supplies all-trans retinal to the visual cycle through central cleavage of β-carotene (13-15). BCO and RPE65 are similar in amino acid sequence, having evolved from a common ancestor (16). Mammalian BCO and RPE65 are both expressed in RPE (13,17,18). The presence of RPE65 in cone photoreceptor cells is controversial (19,20). Müller cells that flank photoreceptor cells are the sites of the cone visual cycle, where CRALBP plays a crucial role in all-trans to 11-cis retinoid isomerization (21).
Ascidians are lower chordates, and their tadpole-like larvae share a basic body plan with vertebrates. The ascidian larva has a remarkably simple central nervous system with about 100 neurons (22). The brain contains a simple eyespot (ocellus), which is responsible for characteristic photo responses in the swimming behavior of the tadpole larva as demonstrated by laser ablation experiments (23,24). The adult ascidian also responds to light. It has been suggested that the cerebral ganglion of the neural complex contains photoreceptor cells (25,26). The simple and primitive features of the ascidian larva provide us with a unique opportunity to study the evolution and basic mechanisms of the visual cycle of vertebrates.
The ocellus of the ascidian Ciona intestinalis larva uses a vertebrate-type visual pigment, Ci-opsin1, as its photoreceptor molecule (27,28). Knockdown of Ci-opsin1 with antisense morpholino oligonucleotide inhibits the photic behavior of the ascidian larva (28). Genes that encode putative visual cycle proteins, homologues of RGR (Ci-opsin3), CRALBP (Ci-CRALBP), and BCO (Ci-BCO), have been identified in C. intestinatis (29,30). Ci-opsin3 is a photoisomerase that converts 11-cis retinal to all-trans retinal upon absorption of light (29). The localization of both mRNA and protein encoded by Ci-opsin3 and Ci-CRALBP are restricted to the brain vesicle and the visceral ganglion in the larva (29,30). In the brain vesicle, Ci-opsin3 and Ci-CRALBP are present both in photoreceptor cells of the ocellus and surrounding non-photoreceptor cells, suggesting that the re-isomerization of all-trans to 11-cis retinoids occurs in photoreceptor cells of the ascidian larva. The transcripts of Ci-BCO are also localized in the brain vesicle and visceral ganglion (29). Because of the lack of specific antibodies against Ci-BCO, however, localization of Ci-BCO in the larva has been previously unexamined. Although Ci-BCO exhibits sequence homology to both BCO and RPE65 in vertebrates, it is more closely related to BCO than RPE65 (29). An ascidian orthologue of RPE65 has not been identified in previous studies.
In this article we report the RPE65 orthologue in C. intestinalis, Ci-RPE65. We also report the localization of Ci-BCO as determined by immunohistochemistry. Photoreceptor cells of the larval ocellus express Ci-BCO but not Ci-RPE65, whereas distinct expression of Ci-RPE65 was found in the adult neural complex. Our results suggest that the larval visual cycle is RPE65-independent and uses Ci-opsin3 as a photoisomerase. Based on these results, we discuss the possible roles and the evolutionary origin of the BCO/ RPE65 family in the chordate visual cycle.
MATERIALS AND METHODS
Animals and embryos. Mature adults of C. intestinalis were collected from harbors in Murotsu and Aioi, Hyogo, Japan, and maintained in indoor tanks of artificial seawater (Marine Art BR, Senju Seiyaku, Osaka, Japan) at 18°C. Embryos and larvae were prepared as described previously (31).
Identification of the C. intestinalis RPE65 orthologue. The Ci-RPE65 gene was found in the C. intestinalis genome database (32) by TBlastn (Translating Basic Local Alignment Search Tool) searches using the amino acid sequence of human RPE65 as a query. Ci-RPE65 encompasses two gene models of different scaffolds (ci0100147220 of scaffold_777 and ci0100136709 of scaffold_448) in the JGI C. intestinalis Genome Database v. 1.0 (http://genome.jgi-psf/org/ciona4/ciona4.home.html). Total RNA was isolated from embryos as described previously (27). First-strand complementary DNA (cDNA) was synthesized from 5 µg total RNA with Super-Script III reverse transcriptase (Invitrogen Corp., Carlsbad, CA) with an oligo(dT) primer. First-strand cDNA was used as the template for polymerase chain reaction (PCR) using Taq DNA polymerase (Ex Taq; Takara Bio, Otsu, Japan) with the primers RPE65-F1 (5′-CAATACAGAGGCGCCCATT-3′) and RPE65-R1 (5′-CTTAATAAATGGTCACGATTATAAACTGC-3′). Nested PCR was performed with the primers RPE65-F1 and RPE65-R2 (5′-CGTCGCCTGGTTAGAAGTATATTAG-3′), and a cDNA fragment of 1762 base pairs (bp) was obtained. The resultant cDNA was cloned into the pBluescript II vector (Stratagene, La Jolla, CA). The entire nucleotide sequence of the cDNA clone was determined on both strands by the cycle sequencing method with an Applied Biosystems 3100 Genetic Analyzer (Applied Biosystems, Foster City, CA).
Molecular phylogenetic analysis. The deduced amino acid sequence of Ci-RPE65 was aligned with amino acid sequences of RPE65/BCO/BCDO2 family proteins from various animals using the ClustalW program (33). Sites with gaps were excluded from analysis. Neighbor joining (NJ) trees were constructed using the ClustalW program. Maximum parsimony (MP) trees were reconstructed using the PROTPARS program of PHYLIP version 3.573c (34). One thousand bootstrap replicates were performed for NJ and MP analyses. Maximum likelihood (ML) trees were reconstructed by the quartet puzzling algorithm using the TREE-PUZZLE program (35). The reliability value for each internal branch indicates in percent how often the corresponding cluster was found among the 1000 intermediate trees. Sequences used were: Homo sapiens BCO AF294900, Gallus gallus BCO AJ271386, Danio rerio BCO AJ290390, C. intestinalis BCO AK116061, Drosophila melanogaster BCO AJ276682, H. sapiens RPE65 U18991, G. gallus RPE65 AB017594, Ambystoma tigrinum RPE65 AF047465, H. sapiens BCDO2 CAC27994, Mus musculus BCDO2 NP_573480, Xenopus tropicalis BCDO2 NP_001006739 and D. rerio BCDO2 CAC37567.
Fluorescent in situ hybridization (FISH). We used cDNA clones for Ciopsin3, Ci-CRALBP, Ci-BCO and Ci-RPE65 as templates for the synthesis of digoxigenin (DIG)-labeled antisense RNA probes using a DIG RNA labeling kit (Roche Diagnostics, Indianapolis, IN). In situ hybridization of whole-mount specimens was carried out as described in Nakashima et al. (29) with the following modification: hybridization signals were visualized by fluorescence using a 2-hydroxy-3-naphtoic acid-2′-phenylanilide phosphate (HNPP) Fluorescent Detection Set (Roche Diagnostics).
Expression profile analysis. Developmental expression profiles of visual cycle genes were analyzed with expressed sequence tag (EST) clones found in unamplified stage-specific cDNA libraries (36).
Reverse transcriptase (RT)-PCR analysis. Total RNA was isolated from larvae and adult tissues as described previously (27). One microgram of total RNA was used as the template to synthesize first-strand cDNA using an oligo(dT) primer according to the manufacturer’s protocol (SuperScript III First Strand Synthesis System for RT-PCR; Invitrogen Corp.). cDNA fragments of Ci-opsin3 (29), Ci-RPE65, Ci-BCO (29), Ci-CRALBP (29) or cytoskeletal actin (37), which was used as a control, were amplified by PCR from first-strand cDNA. The primer sets used were as follows: Ci-opsin3 (nucleotides [nt] 40-355), 5′-CAAGATGGAAGTTAATGACAAGCGA-3′ and 5′-GTAATATTTGTCCTTGGCAACGAGA-3′; Ci-RPE65 (nt 1338-1647), 5′-GGATCAATTATGACGGCTATAACGG-3′ and 5′-GGGTAGGACATCTTGGTCTCA-3′; Ci-BCO (nt 37-391), 5′-CAAGTCCCATAGTAATGGATTTCCC-3′ and 5′-CCAAGAAATCCTTCAGAGTGAGTAG-3′; Ci-CRALBP (nt 771-1008), 5′-AGCAAAACTCTTTGAAAGGATCCAC-3′ and 5′-CGTGTAGGGTGGAAATAACGCTT-3′; cylosketetal actin (nt 1282-1380). 5′-AATCGAGGCTGCGTGTTACTTAA-3′ and 5′-GAATTGGAATGATAAGCACTGCAT-3′. After denaturation at 94°C for 5 min, PCR was performed for 28 (cytoskelelal actin), 30 (Ci-opsin3 and Ci-RPE65) or 35 (Ci-BCO and Ci-CRALBP] cycles (94°C for 30 s, 56°C [58°C for cytoskelelal actin] for 30 s and 72°C for 30 s) followed by a final extension at 72°C for 5 min using 2% (0.4 µL) of the cDNA reaction mixture as a template, 10 pmol of each primer, and 1.0 U of Taq DNA polymerase (Ex Taq; Takara Bio). To eliminate the possibility of PCR amplification from contaminating genomic DNA, 35 cycles of amplification were carried out without reverse transcriptase in a control experiment. PCR products were separated on a 3% NuSieve/1% SeaKem GTG agarose (FMC, Philadelphia, PA) gel (for cytosketetal actin) or on a 2% agarose gel (for Ci-opsin3, Ci-CRALBP, Ci-BCO and Ci-RPE65) and stained with ethidium bromide.
Antibody preparation. Production of polyclonal antibodies against C. intestinalis arrestin (Ci-Arr), Ci-opsin3 and Ci-CRALBP was previously reported (30). For preparation of the Ci-BCO-specific antibody, a cDNA fragment that encodes the carboxyl (C)-terminal amino acids R1433-R1717 of Ci-BCO was amplified by PCR (using the primer pair 5′-TTAGATCTCCCACCAGGAGAAATGTTG-3′ and 5′-TCAAGCTTTAGGGACAAATACTCCGTG-3′) and cloned into the pQE40 vector (Qiagen GmbH, Hilden, Germany). The plasmid was introduced into the Escherichia coli strain XL1 Blue (Stratagene, La Jolla, CA). The C-terminal region of Ci-BCO was produced as a fusion protein with a dihydrofolate reductase and histidine tag, isolated and used to immunize mice. Antisera were prepared according to a standard method.
Immunostaining. C. intestinalis larvae were fixed with 10% formalin in artificial seawater for 3 h at 4°C. After fixation, larvae were washed with PBS containing 0.1% Triton X-100 (T-PBS) and treated with 10% goat serum in T-PBS (blocking buffer) for 3 h. Larvae were then incubated overnight with the primary antiserum diluted 1000-fold with blocking buffer, and washed with T-PBS for 8 h at 4°C. Specimens were then incubated with an Alexa 488-conjugated anti-mouse or anti-rabbit IgG goat antibody (Molecular Probes, Inc., Eugene, OR). For double labeling, an Alexa 594-conjugated secondary antibody (Molecular Probes, Inc.) was used. After rinsing several times with T-PBS, larvae were mounted in 50% glycerol and observed with a confocal microscope (LSM 510; Cart Zeiss, Jena, Germany).
Identification of the RPE65 orthologue in C. intestinalis
We previously reported a C. intestinalis gene that encodes a BCO/ RPE65 family protein. Although this gene was named Ci-BCO/ RPE65, molecular phylogenetic analysis suggested that its encoded amino acid sequence was more closely related to BCO than to RPE65 (29). Therefore, we reexamined the BCO/RPE65 family in the C. intestinalis genome by a TBlastn search with amino acid sequences of three human representatives of this protein family, RPE65, BCO and β-carotene-9′,10′-dioxygenase (BCDO2) as the queries. In addition to the previously identified gene, we detected a novel gene that belongs to the BCO/RPE65 family (Fig. 1). Based on the molecular phylogenetic analyses described in detail below, Ci-BCO/RPE65 was renamed Ci-BCO, and the newly identified gene was named Ci-KPE65, The deduced amino acid sequence of Ci-RPE65 contains 553 amino acids. The amino acid identity of Ci-RPE65 is 36.5% to human RPE65 and 36.3% to human BCO (Fig. 1).
To investigate the evolutionary relationships among Ci-BCO, Ci-RPE65 and vertebrate BCO/RPE65 family proteins, we performed molecular phylogenetic analyses of amino acid sequences with three different algorithms. Phylogenetic trees were constructed using the NJ (Fig. 2a), MP (not shown) and ML (Fig. 2b) methods. Each phylogenetic tree strongly supports the close relationship of Ci-BCO to vertebrate BCO. The close relationship of Ci-RPE65 to vertebrate RPE65 is also supported by high reliability values (92% in the ML analysis). In contrast, none of the trees support the possibility that Ci-BCO and Ci-R PE65 arose by gene duplication of a common ancestral gene after the divergence of ascidians and vertebrates. These results suggest that Ci-BCO and Ci-RPE65 are orthologous to vertebrate BCO and RPE65, respectively. Our analysis also suggests that there is no BCDO2 orthologue in the C. intestinalis genome. The nucleotide sequence of Ci-RPE65 cDNA will appear in the DNA Data Bank of Japan (DDBJ), European Molecular Biology Laboratory (EMBL) and GenBank nucleotide sequence databases under the accession number AB246321.
Spatial and temporal expression patterns of Ci-RPE65 and other visual cycle genes in C. intestinalis
The expression patterns of Ci-opsin3, Ci-CRALBP, Ci-BCO and Ci-RPE65 were examined in larva by FISH (Fig. 3). Both Ci-opsin3 and Ci-CRALBP are specifically expressed in the brain vesicle and visceral ganglion (Fig. 3a,b); their expression levels are much higher in the brain vesicle than in the visceral ganglion. In the brain vesicle, they are expressed both in ocellus photoreceptor cells and some non-photoreceptor cells. Expression of Ci-BCO is more restricted to the ocellus than Ci-opsin3 and Ci-CRALBP (Fig. 3c); it is strongly expressed in ocellus photoreceptor cells, but much more weakly in non-photoreceptor cells of the brain vesicle. Only very faint expression was observed for Ci-BCO in the visceral ganglion. In contrast to Ci-opsin3, Ci-CRALBP and Ci-BCO. Ci-RPE65 expression was not detected in the larva.
The low level of expression of Ci-RPE65 in the larva was further confirmed by developmental profiles of EST distribution in unamplified stage-specific C. intestinalis libraries (Table 1). For Ci-opsin3, Ci-CRALBP and Ci-BCO, multiple EST clones were present in the cDNA library of larvae. Clones of Ci-opsin3 and Ci-BCO were also frequently found in the cDNA library of tailbud embryos. For example, 20 and 10 clones were found for Ci-BCO in the library of tailbud embryos and larvae, respectively. In contrast, EST clones for Ci-RPE65 were only found in the adult library; no clones were present in embryonic and larval stage libraries (Table 1).
The above results suggest that Ci-RPE65 is expressed in adult photoreceptor organs. We have previously shown that the cerebral ganglion, which is a part of the neural complex of the adult ascidian, contains photoreceptor cells (25). Therefore, we examined expression of visual cycle genes in the neural complex and other adult tissues by RT-PCR (Fig. 4). Ci-RPE65 was expressed along with other visual cycle protein genes in the neural complex. Transcripts of Ci-RPE65 were also detected in the body wall, gill and intestine. Interestingly, the expression levels of Ci-opsin3, Ci-CRALBP and Ci-BCO in the neural complex were much lower than in larvae, whereas Ci-RPE65 was expressed at a higher level in the neural complex than in larvae. Thus, in contrast to the situation in larvae, Ci-RPE65 is highly expressed in the neural complex of the adult ascidian. It is noteworthy that each visual cycle gene was also expressed in various adult tissues, including the body wall, gill and intestine.
Distribution of visual cycle proteins in the C. intestinalis larva
By immunohistochemical analysis using specific antibodies, we previously showed that Ci-opsin3 and Ci-CRALBP are localized in both ocellus photoreceptor cells and surrounding non-photoreceptor cells in the brain vesicle (Fig. 5) (30). Since antibodies against Ci-BCO were not available, localization of Ci-BCO was not determined. Here, we raised polyclonal antiserum against Ci-BCO and used it to compare localization of Ci-BCO with that of Ci-opsin3 and Ci-CRALBP in ascidian larvae by whole-mount immunohistochemistry (Fig. 5). The results are similar to those obtained by FISH. Ci-BCO was found in the brain vesicle, including the photoreceptor cells, which were specifically labeled with anti-Ci-Arr antibody. Compared with Ci-opsin3 and Ci-CRALBP, the positive cells of which are distributed broadly in the brain vesicle, localization of Ci-BCO was restricted to specific populations of cells in the brain vesicle. Only photoreceptor cells and cells at the anterior peripheral region of the brain vesicle were positive for anti-Ci-BCO. Ci-opsin3 and Ci-CRALBP were also found in the visceral ganglion, while no Ci-BCO staining was observed in the visceral ganglion.
In this article, we describe the localization of the BCO and RPE65 orthologues in the visual cycle system of larvae and adults of a primitive chordate, C. intestinalis, as examined by RT-PCR, FISH and whole-mount immunohistochemistry. Our results show that the visual cycle proteins Ci-opsin3, Ci-CRALBP and Ci-BCO are strongly and specifically localized to the brain vesicle of the ascidian larva, as are their mRNAs, with each presenting distinct expression patterns. On the other hand, Ci-RPE65 expression is scarce in the larva but is relatively abundant in the adult neural complex.
Among the visual cycle proteins, Ci-BCO demonstrates especially restricted expression to ocellus photoreceptor cells, suggesting that BCO plays a specific role in the visual system of the ascidian larva. In mice, among tissues from various organs, the testis, small intestine, kidney and liver demonstrate clear BCO expression by Northern analysis (13). BCO is also expressed in retina and RPE in monkey, but its low and variable expression indicate its unimportance in the visual cycle system (38). Elimination of the BCO gene in zebrafish and D. melanogaster causes developmental defects and photoreceptor degeneration (14,39), suggesting that â-carotene-derived retinoids are necessary for normal development. However, knockdown of Ci-BCO with antisense morpholino oligonucleotide causes neither morphological defects nor abnormality in photic behavior of the ascidian larva (N. Takimoto, T. Kusakabe and M. Tsuda, unpublished). Therefore, the role of Ci-BCO in ascidian photoreceptor cells is unclear at this moment.
We found that Ci-RPE6S, the RPE65 orthologue, is scarcely expressed in the larva while predominantly expressed in the neural complex, a photoreceptor organ of the adult ascidian. In contrast. Ci-opsin3. the RGR homolog, is abundant in ocellus photoreceptor cells of the larva. Both Ci-RPE6S and Ci-opsin3 are expressed with Ci-CRALBP and Ci-BCO at each dominant expression site. The localization patterns of visual cycle proteins suggest that the visual cycle of the ascidian larva uses Ci-opsin3 as a photoisomerase. It is also suggested that the visual cycle in the adult neural complex is RPE65 dependent. Therefore, the RPE65-dependent visual cycle seems to have a deep evolutionary origin and may have arisen before the divergence of tunicates and vertebrates.
It was shown that RGR is involved in the formation of 11-cis retinal in mice and functions as the isomerase in a light-dependent pathway of the rod visual cycle (11). RGR also enhances isomerohydrolase activity, independent of light (40). However, knockout mice that lack the RGR gene demonstrate only a slight decrease in recovery of 11-cis retinal after intense bleaching, suggesting that RGR is not essential in the visual cycle (41). Since our results suggest that ascidians use Ci-opsin3 as a major isomerase in the larval visual cycle, the common ancestor of chordates probably used an RGR-like opsin to photoisomerize all-trans retinal to 11-cis retinal in the visual cycle.
Amino acid sequences that are well conserved between RPE65 and BCO suggest their related functions (16). Both enzymes have iron-dependent activities. Four hisiidine residues (H180, H241, H313 and H527) and one glutamate residue (E469) are essential to constitute the iron-binding pocket in both enzymes (5,42). These four histidine residues are conserved at the corresponding positions in both Ci-RPE65 and Ci-BCO, and E469 is conserved in Ci-RPE65. The presence of distinct orthologues of RPE65 and BCO in ascidians suggests that functional divergence of RPE65 and BCO occurred before the emergence of the last common ancestor of chordates. RPE65 and BCO constitute a protein family with BCDO2, another enzyme involved in carotenoid metabolism (16,43). We failed to find a BCDO2 orthologue in the C. intestinalis genome. Therefore, BCDO2 might have arisen by gene duplication of either ancestral BCO or RPE65 after divergence from the common ancestor of ascidians and vertebrates. Alternatively, ascidians might have lost BCDO2 during evolution. Although CiRPE65 and Ci-BCO are structurally similar to vertebrate RPE65 and BCO, respectively, biochemical assessment of their functions would be of great importance in elucidating the functional evolutionary history of the RPE65/BCO/BCDO2 family.
In addition to their expression in the neural complex, each of the visual cycle genes is expressed in various adult tissues. Transcripts of Ci-KPE6S and Ci-CRALBP are found in the gill, body wall and intestine. Ci-opsin3 and Ci-BCO are also expressed in the body wall and intestine. There are two possible scenarios, which are not necessarily mutually exclusive, to explain this situation: First, cells with a photosensing ability are widely distributed across various tissues; and second, visual cycle proteins have a function that is not related to photoreception. Future studies on the function of visual cycle proteins in ascidians may provide clues to help unravel unknown functions of their vertebrate counterparts.
In conclusion, ascidians have a repertoire of visual cycle proteins similar to that of vertebrates. Together with our previous finding that the ascidian larval ocellus uses a vertebrate-type visual pigment Ci-opsin1 (27,28), the present study demonstrates that ascidians share characteristics of the visual system with vertebrates. Despite such a high degree of similarity with vertebrates, however, ascidians are unique in that their larval visual cycle lacks RPE65. The detailed distributions of visual cycle proteins in the larval brain and adult neural complex remain to be examined. Future targets include the identification of the function of each visual cycle protein. The identification of functions of Ci-BCO and Ci-RPE65 in ascidians will be beneficial to our further understanding of the origin of the vertebrate visual cycle.
Acknowledgements-This work was supported in part by Grants-in-Aid for Scientific Research from the MEXT to M.T. (16370075) and T.K. (No. 17018018), and by a grant from Japan Space Forum to M.T. (h160179). Takeo Horie is a recipient of a Japan Society for the Promotion of Science (JSPS) Predoctoral Fellowship.[dagger] This paper is dedicated to Professor Thomas Ebrey on the occasion of his retirement from the University of Washington.
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Noriko Takimoto, Takehiro Kusakabe, Takeo Horie, Yuki Miyamoto and Motoyuki Tsuda*
Department of Life Science, Graduate School of Life Science, University of Hyogo, 3-2-1 Kouto, Kamigori, Ako-gun, Hyogo 678-1297, Japan
Received 14 January 2006; Accepted 07 March 2006; published online 17 March 2006 DOI: 10.1562/2006-01-14-RA-775
* Corresponding author email: email@example.com (Motoyuki Tsuda)
Copyright American Society for Photobiology Nov/Dec 2006
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