Genetic Analysis of Ectopic Circadian Clock Induction in Drosophila

Circadian rhythms of behavior and physiology derive from cell-based clocks (Hastings et al., 2008). Clock cells show circadian molecular oscillations in core clock gene RNAs, proteins, phosphorylation states, and their cellular location. Cellular oscillations are synchronized to one another and to the environment by cycles of light and other cues, zeitgebers, either directly or through neural/hormonal signaling. In flies and mammals the most familiar circadian behavior, locomotion, is largely controlled by discrete groups of brain pacemaker neurons that continue to oscillate synchronously in constant conditions (Nitabach and Taghert, 2008; Okamura, 2007). Clock genes also oscillate in other neural and non-neural tissues (Glossop and Hardin, 2002). These peripheral clocks can entrain to light in flies, but in mammals they respond to SCN signals and other zeitgebers (Plautz et al., 1997; Schibler, 2009). The molecular-genetic basis of the clockworks is based on well-conserved cell-autonomous transcriptional feedback loops. In Drosophila the heterodimeric transcription factor composed of CLOCK and CYCLE drives the transcription of period (per) and timeless (tim) (Allada et al., 1998; Rutila et al., 1998). PER and TIM themselves encode transcription factors that heterodimerize and accumulate in the cytoplasm. After timed phosphorylation PER and TIM enter the nucleus, likely separately, where PER homodimers and/or PER/TIM heterodimers bind to CLK-CYC and thereby reduce their own transcription (Darlington et al., 1998; Landskron et al., 2009; Lee et al., 1998; Meyer, 2006). In this way their repression of CLK-CYC is self-limiting and its relief initiates the next daily cycle of CLK-CYC activation. Many, although not all, clock cells are intrinsically light sensitive through the photopigment CRYPTOCHROME (CRY) (Stanewsky et al., 1998). PER/TIM interactions with CRY provide one route of light entrainment for the fly clock (Ceriani et al., 1999; Rosato et al., 2001). cry may also have other functions in flies, as a component of the oscillator or in temperature-response pathways (Collins et al., 2006; Kaushik et al., 2007; Krishnan et al., 2001). In mammals it functions as one mediator of negative feedback (Kume et al., 1999). Other zeitgebers can also entrain fly clocks, most notably temperature (Stanewsky et al., 1998; Wheeler et al., 1993). CLK-CYC also activates expression of a bHLH repressor, clockwork orange (cwo), that feeds back and represses and/or activates CLK-CYC-mediated activation (Kadener et al., 2007; Lim et al., 2007; Matsumoto et al., 2007; Richier et al., 2008). CLK-CYC activates another feedback loop including the bZIP transcriptional regulators Par domain protein 1 (Pdp1) and vrille (vri) (Blau and Young, 1999; Cyran et al., 2003; Glossop et al., 2003). VRI represses Clk transcription perhaps by antagonizing activation by PDP1 to drive cycling transcription of Clk (Cyran et al., 2003). Because CLK-CYC activates the components of multiple loops that feed back to regulate CLK, this suggests that CLK may act as a master regulator. Consistent with this hypothesis, we showed that ectopic expression of Clk was able to induce circadian tim and cry oscillations in putatively naive cells (Zhao et al., 2003). CLK expression was also found to precede that of PER during late embryonic development (Houl et al., 2008). We suggested Clk expression might be a critical organizer of clock formation, potentially serving as a master regulator capable of inducing the clock program in any cell (Zhao et al., 2003). Here we set out to determine minimal requirements for circadian clock induction. We found Clk uniquely able among clock genes to induce the core molecular feedback loop. Ectopic clocks share cardinal features with endogenous peripheral clocks including genetic requirements, cycling induction of core loop components, and daily rhythmicity. Adult-restricted Clk expression can also induce oscillations in ectopic locations, but such expression must be persistent to sustain clocks. Clocks are induced broadly in the brain; however, in some ectopic locations Clk induces noncycling PER, consistent with a requirement for other factors. Despite the likely role of other contributing factors in sustaining and entraining ectopic clocks, these results indicate that persistent Clock induction is uniquely capable of broadly inducing ectopic rhythms even in adults, consistent with a special role for Clk at the top of a clock gene hierarchy.