Purpose: Circadian rhythms are generated in pacemaker cells and are entrained by environmental cues such as the light:dark (LD) cycle. The chicken retina and pineal gland contain endogenous clocks that govern the rhythmic synthesis of melatonin. The dynamic circadian control of melatonin production occurs in part through clock-controlled regulation of arylalkylamine N-acetyltransferase (AANAT), the penultimate enzyme in the melatonin biosynthetic pathway. The transcription of the chicken AANAT gene is regulated directly by the interaction of circadian transcription factors with the AANAT promoter. The aim of the present study was to examine the temporal expression pattern of Bmal1, one of the clock genes implicated in AANAT induction, in chick retina, pineal gland and peripheral tissues.
Methods: Newborn chickens were housed for two weeks on a 12 hr light/12 hr dark (LD) cycle with light on at zeitgeber time (ZT) 0. A fragment of cBmal1 cDNA (1.9 kb) was used as a probe for northern hybridization. Northern blot and quantitative real-time RT-PCR analyses were performed to investigate the temporal expression patterns of Bmal1 in LD, in constant light (LL), and in constant darkness (DD). Tissue distribution of chicken Bmal1 splice variants was studied by RT-PCR and Southern blot analysis.
Results: Northern blot and RT-PCR analyses showed that cBmal1 mRNA is expressed in a circadian manner in the retina, with high levels during early subjective night (ZT12) in both LL and DD. The phase of the circadian rhythm of cBmal1 mRNA in DD was reversed by reversing the prior LD cycle, indicative of photoentrainment. The expression of the Bmal1 is also rhythmic in the pineal gland, with a similar phase relationship to that observed in retina. Rhythmic expression of Bmal1 was also observed in heart and liver, but the peaks of Bmal1 mRNA are 8 h delayed in the liver and 4 h advanced in the heart relative to the Bmal1 peaks in retina and pineal gland. Alternatively spliced variants of cBmal1, with Bmal1b' the primary splice variant, are expressed in retina and other tissues.
Conclusions:Our results indicate that cBMAL1 is widely expressed in the chick and may function as a dimeric partner of MOP4 and CLOCK for control of clock-related physiology in central and peripheral tissues.