Ž . Molecular Brain Research 73 1999 110–118 www.elsevier.comrlocaterbres Research report Exo-rhodopsin: a novel rhodopsin expressed in the zebrafish pineal gland Hiroaki Mano a,b , Daisuke Kojima a,b , Yoshitaka Fukada a,b, ) a Department of Biophysics and Biochemistry, Graduate School of Science, The UniÕersity of Tokyo, Hongo 7-3-1, Bunkyo-ku, Tokyo 113-0033, Japan b CREST, JST, Japan Accepted 27 July 1999 Abstract The zebrafish, a useful animal model for genetic studies, has a photosensitive pineal gland, which has an endogenous circadian w Ž . x 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, Ž . 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 ex tra-o cular 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. q 1999 Elsevier Science B.V. All rights reserved. Ž . Keywords: Exo-rhodopsin; Rhodopsin; Opsin; Pineal gland; Circadian rhythm; Zebrafish Danio rerio 1. Introduction The pineal glands of many non-mammalian vertebrates are photoreceptive organs, and the light-signal captured in such extraocular tissues plays important roles for a variety of physiological functions. For example, a photoreceptive molecule in the chicken pineal gland mediates photoen- trainment of the endogenous circadian clock which drives w x rhythmic production of melatonin 7,8 . A candidate for w x such a photoreceptive molecule is pinopsin 18,25 , a member of the opsin family. Pinopsin was identified first w x in the chicken pineal gland 18,25 , and later in the pigeon w x w x 13,26 and American chameleon 14 , but the physio- logical role of pinopsin has not been fully evaluated because genetic approaches are not feasible in these ani- Ž . mals. In this respect, the zebrafish Danio rerio is a AbbreÕiations: ORF, open reading frame; Rh, rhodopsin ) Corresponding author. Department of Biophysics and Biochemistry, Graduate School of Science, The University of Tokyo, Hongo 7-3-1, Bunkyo-ku, Tokyo 113-0033, Japan. Fax: q81-3-5802-8871; e-mail: sfukada@mail.ecc.u-tokyo.ac.jp useful animal model for studying the function of pineal opsins by forward and reverse genetics. It has been demon- strated that the zebrafish pineal gland contains both the Ž. circadian pacemaker and photoreceptive molecule s re- wx sponsible for the entrainment of the clock phase 6 . How- ever, the identity of the pineal photoreceptive molecule has been an open question in spite of the presence of rhodopsin-immunoreactivities in the teleost pineal gland w x 37,38 . We have undertaken identification of the zebrafish pineal photoreceptor, and found a novel pineal opsin show- ing the highest similarity in amino acid sequence to retinal rhodopsins. This novel rhodopsin gene was also found in other teleosts, suggesting that it is an opsin common to teleosts. 2. Materials and methods 2.1. Isolation of zebrafish exo-rhodopsin cDNA A total RNA fraction isolated from the adult zebrafish brain including the pineal gland was used for the synthesis 0169-328Xr99r$ - see front matter q 1999 Elsevier Science B.V. All rights reserved. Ž . PII: S0169-328X 99 00242-9