Functional characterization of timeless2 in Drosophila melanogaster

Drosophila melanogaster has been shown to possess two timeless genes (tim1 and tim2 or timeout; Myers et al., 1995; Benna et al., 2000; Gotter et al., 2000). While the circadian role of tim1 is well demonstrated, the role of its paralogue tim2 is poorly understood. tim2 maps on the third chromosome and extends for 75 Kb. It is organized in 18 exons and 17 introns and codes for a 1384 aa putative protein . To investigate the role of tim2 during Drosophila development, the tim2 expression profile at different developmental stages was studied. RT- and Real Time PCR analyses revealed a unique isoform of tim2 mRNA expressed at variable levels throughout development. In particular, tim2 expression appeared to be high in embryonic and 3rd instar larval stages, decreasing during pupal life. In the adult, tim2 was strongly expressed in the head. To study the effects of a tim2 knockdown (KD), different transgenic lines, carrying a tim2-specific dsRNAi construct, were generated. Early induction of dsRNAi caused lethality at late pupal stages, with mainly head development defects. Recently a tim2 mutant strain has been generated. Preliminary analyses showed that homozygous tim2- flies die during development, at earlier stages with respect to flies carrying the dsRNAi construct. Moreover, cytological analysis of homozygous tim2- larval brains revealed the presence of chromosome breaks and deletions in ~ 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-, pdfGal4) and evaluated the effects on locomotor activity under different environmental regimes. We analysed also the locomotor behavior of heterozygous tim2- /+ adult flies. Our data indicated that the behavioral periodicity was not affected in either tim2 KD lines or tim2- /+ flies. On the contrary, the responses to light-pulses, given during the night, seemed to be altered in tim2 KD lines, only directed by the pan-neuronal elavGal4 driver. These data suggest a possible function for TIM2 in non canonical clock neurons, important for the synchronization of circadian behavior to environmental cue