Expression of cMOP4, a putative clock component in retina, pineal gland and peripheral tissues

Abstract

Purpose: The avian circadian system is regulated by a multi-oscillatory system consisting of clocks in the retina, the pineal gland and the hypothalamus. In chickens, retinal and pineal circadian clocks govern the daily rhythmic secretion of melatonin via clock- dependent expression of arylalkylamine N-acetyltransferase (AANAT). In order to further characterize the avian circadian pacemaking system, we have examined the temporal expression patterns of the putative clock gene, cMOP4, in the retina, pineal gland and peripheral tissues. Methods: One-day old chicks were housed for two weeks on a 12 h light/12 h dark (LD) cycle, with lights on at zeitgeber time (ZT) 0. Following this, the animals were transferred to constant (24 h/day) light (LL) or darkness (DD). The animals were sacrificed at the times indicated in the figures. A partial fragment of cMOP4 (approx. 1.2 Kb) was amplified using degenerate-PCR and was used for northern hybridization. Total RNA was reverse transcribed using M-MLV reverse transcriptase. cDNA from each sample was amplified with specific primers for cMOP4 using quantitative real time RT-PCR. Results: Real time RT-PCR and northern blot analyses indicate that cMOP4 is widely expressed in neuronal and peripheral tissues. Retina expresses two transcripts of cMOP4, with the sizes of 4.7 Kb and 7.0 Kb. Retina and pineal gland express robust rhythms of cMOP4 mRNA with peak levels at ZT 12 in LD, LL and DD. cMOP4 mRNA is also rhythmically expressed in liver and heart. However, the peak of cMOP4 mRNA is 4 h delayed in liver and 4 h advanced in heart as compared to the retina and pineal gland. Conclusions: Our results indicate that cMOP4 expression is controlled as a circadian rhythm. cMOP4 may serve as a component of the central oscillator in retinal and pineal clocks and/or as an output mechanism, coupling the circadian clocks to rhythmic physiology