Naturwissenschaften (2006) 93: 379–385 DOI 10.1007/s00114-006-0119-9 ORIGINAL ARTICLE Elena Frigato . Daniela Vallone . Cristiano Bertolucci . Nicholas S. Foulkes Isolation and characterization of melanopsin and pinopsin expression within photoreceptive sites of reptiles Received: 7 November 2005 / Accepted: 9 April 2006 / Published online: 11 May 2006 # Springer-Verlag 2006 Abstract Non-mammalian vertebrates have multiple extra- ocular photoreceptors, mainly localised in the pineal complex and the brain, to mediate irradiance detection. In this study, we report the full-length cDNA cloning of ruin lizard melanopsin and pinopsin. The high level of identity with opsins in both the transmembrane regions, where the chromophore binding site is located, and the intracellular loops, where the G-proteins interact, suggests that both melanopsin and pinopsin should be able to generate a stable photopigment, capable of triggering a transduction cascade mediated by G-proteins. Phylogenetic analysis showed that both opsins are located on the expected branches of the corresponding sequences of ortholog proteins. Subse- quently, using RT-PCR and RPA analysis, we verified the expression of ruin lizard melanopsin and pinopsin in directly photosensitive organs, such as the lateral eye, brain, pineal gland and parietal eye. Melanopsin expression was detected in the lateral eye and all major regions of the brain. However, different from the situation in Xenopus and chicken, melanopsin is not expressed in the ruin lizard pineal. Pinopsin mRNA expression was only detected in the pineal complex. As a result of their phylogenetic position and ecology, reptiles provide the circadian field with some of the most interesting models for understanding the evolution of the vertebrate circadian timing system and its response to light. This characterization of melanopsin and pinopsin expression in the ruin lizard will be important for future studies aimed at understanding the molecular basis of circadian light detection in reptiles. Introduction Circadian clocks drive the rhythmic expression of genes involved in physiology and behaviour. To be useful, these clocks must be entrained by environmental time cues (zeitgebers). The primary environmental zeitgeber is light, and the regular daily change in light intensity at dawn or dusk seems to determine the circadian photoentrainment (Roenneberg and Foster 1997). Vertebrate photoentrainment is mediated by specialised non-image forming photorecep- tors that in mammals are exclusively located within the eyes (Foster and Hankins 2002). Differently, non-mammalian vertebrates have multiple extraocular photoreceptors, mainly localised in the pineal complex and the brain, to mediate irradiance detection tasks such as circadian entrainment and behavioural orientation (Bertolucci and Foà 2004). Several non-visual opsin genes have been isolated from extraocular photoreceptive sites in non-mammalian verte- brates (Bertolucci and Foà 2004). The first non-visual opsin to be cloned was pinopsin from the chicken pineal; it possesses the characteristic features of a visual opsin with a rhodopsin-like heptahelical structure and an 11-cis-retinal chromophore (Okano et al. 1994). Representatives of the pinopsin class have subsequently been cloned from the pineal of the pigeon, the iguanid lizard Anolis carolinensis and the toad Bufo japonicus (Kawamura and Yokoyama 1996, 1997; Yoshikawa et al. 1998). Curiously, whilst the pinopsin gene has been encountered in amphibians, birds and reptiles, to date, mammalian and teleost orthologs have not been found. Additional vertebrate non-visual opsin genes have been characterised. For example, melanopsin was first cloned from melanophores of Xenopus laevis and subsequently, it was found to be expressed in the retina as well as hypothalamic neurons (Provencio et al. 1998). In mam- mals, expression of melanopsin is restricted to the amacrine and ganglion cell layers of the retina, which project into the suprachiasmatic nucleus (SCN) of the hypothalamus, the site of the primary circadian pacemaker (Klein et al. 1991; Provencio et al. 2000; Hattar et al. 2003). Current evidence points to melanopsin functioning as the cardinal photopig- E. Frigato . C. Bertolucci (*) Dipartimento di Biologia and Centro di Neuroscienze, Università di Ferrara, Via Luigi Borsari 46, 44100 Ferrara, Italia e-mail: bru@unife.it Tel.: +39-0532-291485 Fax: +39-0532-207143 D. Vallone . N. S. Foulkes Max-Planck-Institut für Entwicklungsbiologie, Tübingen, Germany