Role of BMAL1 and CLOCK in regulating the secretion of melatonin in chick retina under monochromatic green light

As the circadian pacemaker of birds, the retina possesses the ability to receive light information, generate circadian oscillation, and secrete melatonin. Previous studies have confirmed that monochromatic green light can accelerate the circadian rhythmic expression of clock genes in the chick retina, thereby increasing cAanat mRNA level and melatonin secretion. However, as the core components of the transcriptional-translational negative feedback loop, the role that cBmal1 and cClock plays in the regulation of the retinal molecular clock system and melatonin secretion under monochromatic green light is unknown. To explore their in these processes, embryonic chick retinal cells at six embryo ages were isolated and cultured under light-dark (LD) 12:12 monochromatic green light with, and the role of cBmal1 and cClock in the regulation of the retinal molecular clock and melatonin secretion in the chick retina was explored by siRNA interference and overexpression. The results showed siRNA interference and overexpression of cBmal1 obliterated the circadian rhythm of cCry1, cPer2, cPer3, cAanat, and melatonin secretion. Moreover, the siRNA interference of cBmal1 significantly reduced the average expression levels of the positive clock genes cBmal2 and cClock, positive clock protein CLOCK, negative clock genes cCry1, cCry2, cPer2, cPer3, as well as cAanat and retinal melatonin. The over-expression of cBmal1 increased the average levels of the above-detected targets. However, siRNA interference and overexpression of cClock did not change the rhythm of all of the clock genes, clock proteins, cAanat, and melatonin secretion, while it only affected the circadian mesors (24 h time series means), amplitudes, and acrophases (peak times) of cCry1, cPer2, cPer3, cAanat, and melatonin, as well as the average levels of arrhythmic cBmal2 and cCry2. Moreover, interference and overexpression of cClock did not affect cBmal1 mRNA level and BMAL1 protein expression. The above results reveal interference and overexpression of cBmal1 completely abolished the molecular circadian oscillation and the rhythm of melatonin output signal of chick retinal cells, indicating that cBmal1 is on the top of the avian retinal molecular clock feedback loop and regulates the downstream molecular clock oscillation and output under monochromatic green light. cClock plays a subordinate role in maintaining the circadian oscillation of the molecular clock and melatonin secretion in retinal cells, and it has a stabilizing and amplifying effect on molecular clock oscillation.