Circadian and non-circadian functions of timeless2 in Drosophila melanogaster

timeless2 (tim2 or timeout), the Drosophila melanogaster orthologue of mammalian Timeless (mTim), is a member of an evolutionary conserved family of genes with essential functions. mTim has a critical role for survival and may influence the circadian clock mechanism. Here, we show that Drosophila tim2 is required for both viability and synchronization of circadian behavior. A unique isoform of tim2 mRNA is expressed throughout development and in adult flies. Expression levels are very high in embryos and third instar larvae and low during pupal life. In adults, tim2 is strongly expressed in the tracheal system of brains and the peripheral head structures (eyes and cuticle). Early induction of stable tim2 knockdown (KD; via dsRNAi driven by Actin-Gal4) caused lethality at late pupal stages, with defects in head development. Similar results were obtained driving RNAi specifically in the tracheal system (via btl-Gal4). We also characterized 7 tim2 mutant alleles recently generated by PiggyBac insertional mutagenesis. Animals homozygous for 6 of these alleles die few hours after puparium formation. Cytological analysis of larval brains from these tim2 lethal mutants revealed the presence of chromosome aberrations in approximately 30% of metaphases. To investigate the role of Tim2 in the adult circadian clock, we induced Tim2 depletion via dsRNAi triggered by neuronal or circadian drivers (elav-, tim1-, pdf-Gal4), and evaluated the effects on locomotor activity under different environmental regimes. We also analysed the locomotor behaviour of heterozygous tim2/+ adult flies. Our data indicate that the behavioural periodicity is not affected in either tim2 KD or tim2/+ flies. In contrast, the responses to light-pulses, delivered at night, seemed to be altered in both tim2/+ and tim2 KD flies, but only when Tim2 depletion was induced by the pan-neuronal elav-Gal4 driver. These results suggest a possible function for Tim2 in the synchronization of circadian behaviour.