Teleost multiple tissue (tmt) opsin: a candidate photopigment regulating the peripheral clocks of zebrafish?

Abstract

Isolated organs and cell lines from zebrafish exhibit circadian oscillations in clock gene expression that can be entrained to a 24-h light/dark cycle. The mechanism underlying this cellular photosensitivity is unknown. We report the identification of a novel opsin family, tmt-opsin, that has a genomic structure characteristic of vertebrate photopigments, an amino acid identity equivalent to the known photopigment opsins, and the essential residues required for photopigment function. Significantly, tmt-opsin is expressed in a wide variety of neural and non-neural tissues, including a zebrafish embryonic cell line that exhibits a light entrainable clock. Collectively the data suggest that tmt-opsin is a strong candidate for the photic regulation of zebrafish peripheral clocks.

Introduction

The primary role of the circadian system is to anticipate change so that physiology can be ‘fine-tuned’ in advance of a periodic environment. The ability to anticipate environmental changes enables an organism to exploit the new circumstances without the delay inherent in a reactive response. For the circadian system to have any survival value, however, it must remain synchronised or entrained to the solar day, and the systematic daily change in the irradiance of light at dawn or dusk provides the most reliable indicator of the phase of the day. As a result, most organisms have evolved to use the twilight transition as the primary zeitgeber to adjust circadian phase (photoentrainment) [47]. In mammals, and probably all vertebrates, the mechanism for the generation of a 24-h oscillation appears to involve a core set of clock genes (per’s and crys’s), which code for proteins that feedback to inhibit their own transcription [48]. Photoentrainment involves the alignment of this molecular oscillation to dawn and dusk, and in all vertebrates appears to be effected by specialised non-image forming photoreceptors. For example, in mammals specialised non-rod, non-cone photoreceptors and photopigment within the eye provide the dominant light input to the SCN (for full discussion see [5], [19], [23], [24], [31]). Whilst in non-mammals, specialised non-image forming photopigments within the eye, pineal and brain have been either shown or implicated in entrainment [18], [51].

Until recently, circadian clocks were thought to reside exclusively within discrete regions of the central nervous system, such as the suprachiasmatic nuclei (SCN) of mammals [44] or the pineal of non-mammals [55]. However, since 1998 results have accumulated showing that a variety of tissues can show rhythmic patterns of clock gene expression, even when isolated from central clocks such as the SCN or pineal. The first of these studies involved an investigation of an immortalised cell line of rat fibroblast and hepatocyte. When these cells were ‘shocked’ by exposure to 50% horse serum in vitro they exhibited oscillations in clock gene expression for several cycles [2]. In more recent experiments rhythmic clock gene expression was monitored in organ culture using period-luciferase transgenic rats [53]. Several 24-h cycles of clock gene expression were observed in these isolated organs before rhythms damped and disappeared. In these studies, light was incapable of entraining the patterns of clock gene expression [53], supporting the notion that mammalian photoentrainment is mediated exclusively by ocular photoreceptors [16]. Entrainment of these peripheral clocks is thought to be via the SCN utilising both humoral and non-humoral pathways [2], [53]. Thus the SCN clock does not appear to drive rhythmicity in peripheral tissues, but rather acts to entrain and sustain the rhythmicity of molecular oscillators in the periphery (for reviews see [10]).

Parallel studies in zebrafish have shown a more advanced pattern of circadian decentralisation. Individual organs and tissues show robust 24-h patterns of clock gene expression, and unlike mammalian tissues, these rhythms will continue for many cycles in vitro without damping out [57]. Perhaps more surprising, however, is that these rhythms in clock gene expression appear to be entrained by light [39], [57]. As a result, peripheral organs, tissues and cells must contain a photopigment and signal transduction cascade that is capable of mediating these effects of light on the molecular machinery of the clock. Several novel opsin gene families have been isolated from the non-mammalian vertebrates. These genes include pinopsin (P-opsin) [37], VA opsin [51], and melanopsin [6], [42]. All of these genes have been localised to eyes and brain, and have been variously implicated in circadian entrainment, but none are known to be expressed outside the central nervous system.

In an effort to characterise the peripheral photoreceptors from teleost fish, we screened genomic libraries from both Fugu (Fugu rubripes) and zebrafish (Danio rerio). These screens identified a novel opsin expressed in a wide variety of tissues, and we have tentatively named this new gene family the teleost multiple tissue (tmt) opsins. The expression of this gene is found throughout most tissues, including embryonic cell lines. Significantly, this gene is expressed in cell lines that possess a light entrainable clock. Collectively, the patterns of tmt-opsin tissue expression, the deduced structural features of the protein, and the structure of the tmt-opsin gene, make this a strong candidate for the photic regulation of the peripheral clocks in zebrafish.