Entrainment and generation of circadian rhythms in Drosophila

Circadian rhythms are controlled by an endogenous clock in which circadian oscillations of clock gene products are thought to provide time-of-day signals. In Drosophila RNA and protein levels of the period and timeless genes oscillate and the proteins autoregulate their transcription. The circadian clock can be reset by environmental cues such as light which induces rapid degradation of TIMELESS. This work addresses the mechanisms that transmit light to the clock and the role of clock gene oscillations in providing time keeping cues. Finally, the isolation of novel molecules that appear to affect circadian rhythms is described. To dissect the role of the visual transduction pathway in circadian photoentrainment, we assayed light-induced TIMELESS degradation as well as the behavioral response in wild type and several visually mutant strains. We showed that the TIMELESS response to light correlates with the effect of light on the behavioral rhythm. The visually blind mutants displayed subtle or no circadian entrainment phenotype. These results supported the hypothesis that circadian entrainment does not rely on the visual system and likely involves a dedicated pathway for photoreception. To test the relevance of the molecular oscillations in generating behavioral rhythms, we expressed period and timeless under control of constitutively active promoters. Constitutive expression of either RNA supported protein cycling and behavioral rhythms in the respective null mutant although constitutive timeless was less effective than constitutive period. Constitutive expression of both genes drove cyclic expression of the clock-controlled RNA, vrille, and restored behavioral rhythms although these rhythms showed deficits in photic resetting. Overexpression of either period or timeless, but especially timeless , attenuated behavioral rhythmicity and protein cycling in lateral neurons in wild type flies. We propose that the two proteins must cycle to drive rhythmic expression of downstream genes. To identify additional circadian related components, we conducted an over-/mis-expression gain-of-function screen, as opposed to the conventional genetic screens that usually isolate loss-of-function mutants. Several positive lines were identified and characterized.