Isolation of new genes involved in temperature synchronization of the circadian clock of Drosophila melanogaster

Abstract

Circadian clocks regulate behaviour and physiology of many organisms and keep them in synchrony with the environment. Drosophila's circadian clock is mainly synchronized by natural light-dark cycles and temperature fluctuations, both at molecular and behavioural levels. The mechanisms underlying temperature entrainment are poorly understood, but previous studies have shown that this process can be genetically dissected. In this work, I isolated several mutants which interfere with the temperature synchronization of Drosophila's circadian clock. Three variants were isolated in a chemical EMS-mutagenesis screen monitoring putative second- and thirdchromosomal mutations. The mutants behave normal in light-dark cycles suggesting that they specifically interfere with temperature entrainment. In a different, RNAi-based screen, a Forkhead-domain transcription factor encoding gene was isolated, which shows defective circadian activity of per expression and PER accumulation in temperature-entrainment condition, when down-regulated. Finally, a candidate approach led me to identify three genes encoding proteins belonging to the TRP family of ion channels. Mutations in the pyrexia, trpM and trpA1 genes show abnormal temperature synchronization of locomotor behaviour, similar to our EMS-candidates. The isolation and analysis of those mutations are described, as well as a behavioural analysis of the already-known "temperature-mutant" nocte. In particular, I discuss the involvement of chordotonal organs as structures required for temperature entrainment of the clock and the role of nocte for signalling the temperature information from the periphery to the brain. The rest-activity pattern is a well-studied circadian output behaviour; the pupal emergence, named eclosion, is another behaviour strictly regulated by the circadian clock. Here we show that genes important for entrainment of adult locomotor behaviour to temperature do not play the same role in regulating the synchronization of eclosion. To gain insight into the synchronization mechanisms of eclosion, I studied how different entrainment conditions affect the phase and free-running period of eclosion.