Encephalopsin: a Novel Mammalian Extraretinal Opsin Discretely Localized in the Brain

The Journal of Neuroscience, May 15, 1999, 19(10):3681–3690 Encephalopsin: A Novel Mammalian Extraretinal Opsin Discretely Localized in the Brain Seth Blackshaw and Solomon H. Snyder The Johns Hopkins University, School of Medicine, Departments of Neuroscience, Pharmacology and Molecular Sciences, and Psychiatry, Baltimore, Maryland 21205 We have identified a mammalian opsin, encephalopsin, that in the ventral horn of the spinal cord. Rostrocaudal gradients of shows strong and specific expression in the brain. Encepha- encephalopsin expression are present in the cortex, cerebel- lopsin defines a new family of opsins and shows highest ho- lum, and striatum. Radial stripes of encephalopsin expression mology to vertebrate retinal and pineal opsins. Encephalopsin are seen in the cerebellum. In the cortex and cerebellum, is highly expressed in the preoptic area and paraventricular encephalopsin expression is considerably higher and more nucleus of the hypothalamus, both regions implicated in ence- highly patterned in the adult than in the neonate. Encephalopsin phalic photoreception in nonmammalian vertebrates. In addi- is the first putative extraocular opsin identified in mammals and tion, encephalopsin shows highly patterned expression in other may play a role in encephalic photoreception. regions of the brain, being enriched in selected regions of the Key words: photoreceptor; circadian; photopigment; pineal; cerebral cortex, cerebellar Purkinje cells, a subset of striatal retina; in situ hybridization; Purkinje; stripes; cerebellum; hypo- neurons, selected thalamic nuclei, and a subset of interneurons thalamus; deep brain Vertebrate phototransduction serves multiple purposes. Visual ditionally been thought to lack extraretinal photoreceptors (Fos- phototransduction has received the great bulk of attention and ter, 1998), given the fact that intact eyes are needed to entrain has been extensively characterized at a molecular level (Palcze- adult mammalian circadian behavioral rhythms. Recent work, wski, 1994). The molecular mechanism of nonvisual phototrans- however, has raised the possibility that mammals may indeed duction, geared toward detecting changes in ambient light inten- possess extraretinal photoreceptors. In humans there is evidence sity and entrainment of circadian and other biological rhythms, of circadian entrainment mediated by light applied to the backs of has been generally much less well studied. Although retinal pho- the knees (Campbell and Murphy, 1998). Moreover, free-running toreceptors mediate visual phototransduction, the identity of the circadian rhythms have been identified in a variety of cultured photoreceptors mediating circadian photoentrainment is un- mammalian cells (Balsalobre et al., 1998) and tissues (Zylka et al., known (Foster, 1998; Green, 1998). The photoreceptors mediat- 1998), although it is not yet clear whether light can directly ing circadian photoreception may include both the rods and cones entrain rhythms at these sites. of the retina and other as yet unidentified sites (Provencio and The molecular identity of the photopigments involved in non- Foster, 1995; Menaker et al., 1997). Cryptochromes, homologs of visual phototransduction is not fully known. The past few years the blue-light receptor from plants, have been shown recently to have seen the identification of a variety of novel opsins in non- play a role in light entrainment of circadian rhythms in both mammals and Drosophila (Thresher et al., 1998). However, it is mammalian vertebrates, including the pineal complex-specific pi- not yet clear whether they are actual photoreceptors or part of the nopsin (Okano et al., 1994; Max et al., 1995) and parapinopsin molecular machinery coupling phototransduction to the circadian (Blackshaw and Snyder, 1997b), melanopsin, which is expressed clock. in melanophores and retinal horizontal cells (Provencio et al., Although visual phototransduction is specifically retinal, non- 1998), and vertebrate ancient (VA) opsin, which is expressed in visual phototransduction occurs in many extraretinal sites retinal horizontal and amacrine cells (Soni et al., 1998). The (Menaker et al., 1997). These include the pineal complex, the molecular characteristics of the encephalic photoreceptor system brain, the iris, melanocytes of the skin, and possibly other sites. are unknown, although in amphibians and birds (Provencio et al., Although extraretinal phototransduction has been extensively 1998; Wada et al., 1998; Yoshikawa et al., 1998) expression of studied in nonmammalian vertebrates, adult mammals have tra- either rhodopsin, pinopsin, or melanopsin has been reported in the diencephalon, depending on the species examined. Molecular Received Jan. 12, 1999; accepted March 2, 1999. evidence of extraretinal photoreceptors in mammals has been This work was supported by United States Public Health Service Grant DA-00268 lacking, however, except for the expression of visual opsins in the and Research Scientist Award DA-00074 to S.H.S. We thank H. Sun and D. Krautwurst for assistance with the biochemical reconstitution of encephalopsin, J. pineal gland (Blackshaw and Snyder, 1997a) and cryptochromes, Nathans and K.-W. Yau for comments on this manuscript, and J. Pevsner, D. which are expressed in a wide range of neuronal and peripheral Linden, and C. Riley for helpful discussions. Correspondence should be addressed to Dr. Solomon H. Snyder, The Johns tissues (Miyamoto and Sancar, 1998). Hopkins University, School of Medicine, Department of Neuroscience, 725 North We now describe the identification, cloning, and characteriza- Wolfe Street, Baltimore, MD 21205. tion of a novel mammalian opsin, encephalopsin, which is highly Dr. Blackshaw’s present address: Department of Genetics, Harvard Medical School, 200 Longwood Avenue, Boston, MA 02115. concentrated in the brain and testes and localized to discrete Copyright © 1999 Society for Neuroscience 0270-6474/99/193681-10$05.00/0 areas of the brain. 3682 J. Neurosci., May 15, 1999, 19(10):3681–3690 Blackshaw and Snyder • Encephalopsin MATERIALS AND METHODS formed essentially as described elsewhere (Vassar et al., 1994), except 7 33 Cloning of encephalopsin. A 180 bp fragment of a novel opsin from that 10 cpm of P-labeled sense and antisense probe was used per slide. Xenopus laevis covering transmembrane domains 6 and 7 (S. Blackshaw Slides were exposed to Kodak MR-2 film for 4 d. and S. H. Snyder, unpublished observations) was used to screen the database of expressed sequence tags (dbESTs) using TBLASTX. A RESULTS homologous clone from mouse (GenBank accession number AA276584) was identified. This was then used as a query to search the dbESTs, and Molecular cloning of encephalopsin several highly homologous human ESTs (GenBank accession numbers In the course of previous work, we have identified several novel T83438, AA088574, AA367654, R70620, and R76620) were identified. After the completion of one round of 59 rapid amplification of cDNA putative extraretinal opsins from cold-blooded vertebrates via ends (RACE), which extended the mouse encephalopsin cDNA to 335 PCR with primers designed to regions of homology shared by bp from the start of the coding sequence, another human EST (accession pinopsin and parapinopsin but not retinal opsins (Blackshaw and number AA297478) was identified that extended as far as the third Snyder, unpublished observations). We used one of these novel transmembrane domain of the encephalopsin-coding sequence. These sequences were used to design 59-RACE primers for amplifi- partial-length cDNAs isolated from Xenopus as a query in a cation of full-length cDNAs (Frohman et al., 1988). Human and mouse search for related genes in the dbESTs. We identified novel cerebellar and testes RNA was used as first-strand cDNA for RACE, transcripts from human and mouse with high homology to a with GC-MELT (Clontech, Palo Alto, CA) added to the final round of variety of retinal and extra retinal opsins, although they did not RACE to obtain the GC-rich 59 end. Human RNA was purchased from Clontech, whereas mouse total RNA was isolated via RNeasy (Qiagen, prove to be true homologs of the novel Xenopus opsin used as a Hilden, Germany). Three successive rounds of 59 RACE were performed query sequence. to obtain the mouse 59 end, whereas two rounds were required to obtain To clone encephalopsin, we obtained full-length cDNAs by 9 the human 5 end. DNA sequence was confirmed by sequencing multiple RACE from human and mouse brain. The 39-end sequence was independent cDNAs and via genomic sequencing, which was also used to obtained by sequencing multiple independent ESTs. Full-length confirm the position of intron–exon junctions. 59-RACE primers for mouse encephalopsin covered residues 261–285, 471–498, and 1180– clones were reconstructed by PCR. The mouse and human se- 1207, as numbered from the start of the cDNA-coding sequence. Human quences have been deposited in GenBank under accession num- 59-RACE primers covered residues 241–260 and 525–554. 39 sequences bers AF140241 and AF140242, respectively. for mouse and human encephalopsin were obtained via sequencing Analysis of the human and mouse encephalopsin sequences multiple partial-length ESTs and, in the mouse, via genomic DNA sequencing. reveals high homology to other members of the opsin family (Fig. Genomic clones for mouse encephalopsin were isolated via a combi- 1). A number of molecular features establish that encephalopsin nation of genomic library screening and inverse PCR. A RACE product fulfills the criteria expected

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