The Development and Aging of the Circadian Clock in Drosophila melanogaster

Abstract

The circadian clock is the endogenous pacemaker that controls daily rhythms in behaviour and physiology, and it is driven by the auto-regulated transcription-translation feedback loops of clock genes. The molecular mechanism can be found in almost every cell, which largely constitutes the peripheral oscillators aside from the specialized central clock in the brain. Development is the series of changes that organisms undergo in their passage from the embryonic state to maturity. Aging is the process of time-related deterioration of the physiological functions necessary for survival. We hypothesize that the circadian clock develops, matures and ages in alignment with the changes of the whole body throughout lifespan, in a systemic, interactive, and hierarchical manner. By using Drosophila melanogaster as the animal model, we aimed to study when the molecular circadian clock and its light sensitivity develop, how the aged clock changes intrinsically under constant conditions as well as under cyclic entrainment, and how the clock reacts immediately preceding death. Transgenic luciferase-reporter fruit flies were used to measure the real-time expression of two key clock genes period and timeless in vivo. We found that first, PERIOD expression in the presumptive central clock dorsal neurons started to oscillate in the embryos, while PERIOD in the peripheral tissues increased during the embryonic stage but only started to oscillate in the adult stage. Secondly, PERIOD expression in the central clock neurons stayed robust in aged fruit flies, however, rhythms of PERIOD and TIMELESS in the peripheral tissues throughout the body showed reduction in both expression level and rhythmicity. Thirdly, in the days prior to death TIMELESS expression increased and lost circadian rhythmicity. These findings prove that the central clock is already functional during embryogenesis, while the peripheral clocks develop later, maturing only after eclosion when cyclic and synchronized expression of PERIOD throughout the animal commences. Significantly, we reveal that cyclic clock gene expression, presumably in precursors of dorsal clock neurons occurs during the embryonic stage, which is earlier than previously thought. Equally important, when the animals grow old, the aged molecular clock still functions well at the central level but declines gradually at the peripheral level. It indicates that the peripheral clocks are more seriously damaged under aging that they should be considered as the potential target for further exploration and possible intervention. Furthermore, we report a novel marker of imminent death in the expression of the clock gene TIMELESS. It is of importance that this marker in the expression of TIMELESS is not age dependent and predicts death equally well in fruit flies of different ages and under different circumstances. Here we show a dynamic network system of the circadian clock changing throughout lifespan, spatially from the centre to the periphery and temporally from the birth to the death. In summary, the circadian clock develops gradually during early stages, declines with age and breaks down days before death, showing distinguishing characteristics between central and peripheral oscillators.