The Drosophila circadian system

Drosophila, similar to N.crassa, represents one of the most successful systems in advancing the molecular dissection of circadian oscillators. In Drosophila, the clock controls a number of rhythmic outputs, including adult emergence (eclosion), locomotor activity, and olfactory physiology (Figure 10). Thus, the circadian clock gates the emergence of adults from their pupal state to dawn. Indeed in the wild, at this time of the day, the atmosphere is cool and moist. This minimizes the risk of desiccation as the emerging fly expands its folded wings and hardens its cuticle. Once emerged, adult flies restrict flying, foraging and mating activities to the day, while they tend to rest during the night. In contrast, their olfactory responses are highest at night.

The Drosophila circadian timing system serves as a good model for clocks in other animals, given that many of the components have been conserved. The molecular-genetic

Figure 10. Drosophila activity/rest cycle and per::luciferase in tissue explant. Circadian cycle of locomotor activity (monitored by infra-red beams) of a Drosophila melanogaster kept in continuous darkness (left). Activity occurs during anticipated day (grey bar), around dawn and dusk. This fly’s intrinsic period is slightly less than 24 hours and so activity drifts to the left. In period::luciferase transgenic flies light-sensitive, autonomous local pacemakers can be monitored as cycles of bioluminescence (right), here recorded from an isolated wing, initially synchronized to LD cycles, and then in continuous darkness. This Figure is issued from (Hastings et al., 2008).

study of circadian rhythms was initiated with the identification of the period (dper) mutant gene, based on its perturbation of the timing of fly eclosion and activity rhythms (Konopka and Benzer, 1971). Since then, several additional clock genes have been isolated and characterized (Williams and Sehgal, 2001).

The Drosophila circadian oscillator is based on two feedback loops in gene expression: a per/tim loop and a dClk loop. Transcriptional and post-transcriptional events are required for the connection and function of both loops (Figure 11). The first clues for the per/tim feedback loop came from the work of Hardin and co-workers in which mutations in per were shown to alter the period and phase, not only of the fly’s daily behavior, but also of per mRNA accumulation. Introduction of an active per gene rescued cycling of mutated per RNA as well as rhythmic locomotor activity, suggesting the implication of the active PER protein in an autoregulatory feedback loop mechanism (Hardin et al., 1990). This finding occurred before the discovery of the tim mutation, which was isolated in 1994 and shown to produce also arrhythmia of eclosion and locomotor activity (Sehgal et al., 1994). It is now known that to initiate the per/tim feedback loop, dCLK–CYC heterodimers bind E-box regulatory elements with a CACGTG consensus sequence on per and tim promoters, thereby activating the transcription of these genes (Taylor and Hardin, 2008). Mutations in either dClk or Cyc reduce levels of per and tim RNA as well as protein and also render flies arrhythmic for rest/activity and eclosion (Allada et al., 1998; Rutila et al., 1998). dCLK and CYC heterodimerize via helix-loop-helix motives and PAS domains. The latter domains also contribute to the dimerization of PER and the N.crassa proteins WC-1 and WC-2 (see above).

However, while WC-1 and WC-2 use Zinc fingers to bind DNA, dCLK and CYC bind DNA via bHLH (basic helix-loop-helix) domains (Darlington et al., 1998). The levels of per and tim transcripts and proteins are highly rhythmic, however, there is a lag in protein accumulation of ~5h with regard to mRNA accumulation. This delay is the result of

phosphorylation-dependent destabilization of PER by DBT (doubletime, the homolog of mammalian casein kinase 1ε), and possibly also CK2, followed by stabilization of phosphorylated PER by TIM binding (Lin et al., 2002; Nawathean and Rosbash, 2004). Loss-of-function mutations in the DBT kinase slow or stop the oscillator because of delayed PER degradation and hypophosphorylated PER accumulation (Price et al., 1998). The nuclear translocation of the PER/TIM heterodimer is regulated by the phorphorylation of TIM by SGG/GSK-3 (shaggy, glycogen synthase kinase 3 ortolog). Reducing SGG/GSK-3 activity lengthens the period, because of a delayed nuclear entry of PER/TIM (Martinek et al., 2001).

DBT remains bound to PER to form DBT/PER/TIM complexes that can enter the nucleus. In the nucleus, PER is thought to repress dCLK/CYC dependent transcription by binding to dCLK and inhibiting the DNA binding activity of CLK/CYC dimers (Chang and Reppert, 2003; Lee et al., 1999). Recent analysis also suggests that DBT-dependent phosphorylation destabilizes dCLK (Yu et al., 2009). Thus PER and TIM inhibit their own transcription as well as the transcription of other dCLK/CYC-dependent clock controlled genes, and the approximately 24h period is in part due to the lag in PER and TIM protein accumulation prior

Figure 11. A scheme illustrating current knowledge about the molecular machinery of the Drosophila circadian clock. See text for details. This Figure is taken from (Tomioka and Matsumoto, 2010).

to nuclear entry (Figure 11).

The second loop drives the circadian oscillation of dClk expression. Besides per and tim, dCLK/CYC activates the transcription of vrille (vri) and PAR domain protein 1ε (Pdp1ε).

The vri mRNA is translated into its product protein VRI, which enters the nucleus and inhibits transcription of dClk by binding to a promoter region, V/P-box. The translation of Pdp1ε occurs in a rather delayed manner. PDP1ε is thought to bind to V/P-box competitively with VRI and activate the transcription of dClk (Cyran et al., 2003). While Cyc is constantly expressed, dClk expression is thus highly rhythmic, with an accumulation profile that is antiphasic to that of per and tim. The dClk feedback loop contributes to sustaining a high-amplitude circadian oscillation (Figure 11).

In addition, the transcriptional repressor CWO (clockwork orange) was recently shown to inhibit not only its own transcription, but also the one of other clock genes, such as per and tim, through E-box elements. CWO belongs to the bHLH ORANGE family (proteins that contain bHLH and Orange domains) and is rhythmically expressed under the regulation by CLK/CYC (Matsumoto et al., 2007). CWO protein is a structural ortholog of DEC1 and DEC2 (deleted in esophageal cancer 1 and 2) proteins in mammals. It might be part of a third feedback loop participating in the generation of high-amplitude oscillations (Figure 11).

In Drosophila, the circadian system is divided into a central clock, which resides in the brain and is known to control rhythms in locomotor activity, and peripheral clocks, which comprise all other clock tissues and which are thought to control other rhythmic outputs. In the brain, about 150 neurons express clock genes. These neurons are divided into seven groups distributed around the central brain; three groups (DN1, DN2, and DN3) are located in the dorsal region, and the remaining four (LNd, l-LNv, s-LNv, and LPN) are located laterally.

l-LNv and s-LNv (for large- and small-lateral-ventral neurons) groups are close to the optic lobes and are suggested to represent the Drosophila counterpart of mammalian

suprachiasmatic nucleus (SCN) (Figure 12). Their importance for overt activity rhythm generation was revealed by various surgical and electrophysiological experiments (Tomioka and Matsumoto, 2010). Similar to the SCN in mammals, LNvs receive light input from retinal photoreceptors in the compound eyes and extra-retinal photoreceptors within the brain;

however, they can also be entrained directly by light that penetrates the cuticle.

The pathways through which overt rhythms are regulated are not well understood.

Pigment-dispersing factor (PDF) is the best characterized output molecule of the Drosophila circadian clock system. It is thought to be the principal neurotransmitter implicated in the coordination of circadian behavior and metabolism throughout the fly’s body. Pdf is expressed in the l-LNv and s-LNv groups of neurons close to the optic lobes. Flies in which

Figure 12. Circadian oscillators in Drosophila heads. In Drosophila, there are seven main groups of circadian neurons (left). DN1, DN2, and DN3, dorsal neurons; LNd, lateral-dorsal-neurons; l-LNv and s-LNv, large- and smallateraventraneurons; and LPN, lateraposterior-neurons. The s-LNv and l-LNv express PDF neuropeptide. l-l-LNvs have their processes in the optic lobe and send their axonal projection to the contralateral optic lobe through the posterior optic tract (POT). s-LNvs have axonal projection to the dorsomedial region of the protocerebrum. External structures containing circadian oscillators (right). A frontal view of a Drosophila head is shown. OC, ocelli; CE, compound eyes;

AN2, second antennal segment; AN3, third antennal segment; MP, maxillary palps; PR, proboscis.

These Figures are issued from (Tomioka and Matsumoto, 2010) and (Hardin, 2005) respectively.

LNv neurons have been selectively ablated are arrhythmic under constant conditions. The same phenotype is observed in pdf mutant animals (Renn et al., 1999). However, in both case, some aspect of rhythmicity remains, revealing the presence of additional timekeeping mechanisms in the other groups of clock neurons and/or in peripheral tissues. dClk and vri independently contribute to pdf regulation, with vri affecting a posttranscriptional stage of pdf expression. While pdf mRNA levels do not cycle, rhythmic outputs are thus likely the result of cyclic PDF protein activity (Blau and Young, 1999). Pdp1ε and takeout (to) have also been located downstream of the circadian clock in Drosophila. PDP1ε seems to drive behavioral rhythms when some specific genes, in addition to dClk, are controlled by its binding to the V/P- box (Benito et al., 2007). To mutant flies show aberrant locomotor activity and die quickly upon starvation, indicating that this clock-controlled output gene may control feeding behavior (Sarov-Blat et al., 2000). Recent work suggests that in fact, PDP1ε is a regulator of to (Benito et al., 2010). In addition, the levels of mRNAs of hundreds of other genes have been shown to oscillate (Matsumoto, 2006).

A crucial discovery was that circadian oscillators exist in almost every part of the body. Transgenic Drosophila with the luciferase expressed under the control of the per promoter have shown that per is rhythmically expressed in cells of the eyes, antennae, proboscis, wings and legs, and that this rhythm can be reset by light. This supports that every cell in the body possesses a functional oscillator and a system to transduce photic signals to this oscillator (Plautz et al., 1997). One remarkable difference between the molecular machinery of the clock in peripheral tissues and in the central brain pacemaker is the blue light photoreceptor Cryptochrome (CRY)’s function (see below). A Drosophila cry mutant, cry^b, leaves circadian oscillator function intact in the central circadian pacemaker neurons, but renders peripheral circadian oscillators largely arrhythmic (Krishnan et al., 2001). Therefore, in addition of transducing light signal in the central and peripheral clock cells, CRY also acts

as a core component necessary for cell autonomous circadian oscillator function in some peripheral tissues (Hao et al., 2008; Ivanchenko et al., 2001). Another important evidence for the functional importance of peripheral clocks comes from an experiment in which the expression of the essential clock gene per was restricted to the LNvs. Such flies exhibited a rhythmic behavior, but had lost any rhythm in electrophysiological responses of antennae to olfactory stimuli. This showed that, in contrast to the rhythm of rest-activity cycles, the circadian rhythm of olfactory responses requires functional oscillators outside the central clock (Krishnan et al., 1999). All together, this suggests that fly peripheral oscillators depend less on the central clocks than do their mammalian counterparts on the SCN (see below). How much influence the central clocks have over fly peripheral oscillators is not known, given that light penetrating through the cuticle and perceived by the blue light receptor CRY can entrain peripheral clocks. The scheme of a “master clock” required to synchronize other oscillators in the fly thus has to be reconsidered, without ruling out the possibility that there may be communication between central and peripheral oscillators.

The Drosophila clock can be entrained by environmental light and temperature. The link between light signals and circadian oscillators is mediated by both rhodopsin and the blue light photoreceptor Cryptochrome (CRY). Photic organs (compound eyes, ocelli, H-B eyelet that is a remnant of larval eye) perceive light via rhodopsin photoreceptors and then synchronize circadian neurons (Figure 12) (Ashmore and Sehgal, 2003). The exact neural pathways toward circadian neurons and the molecular mechanisms for synchronizing them are not yet elucidated. Blind flies (i.e. flies lacking signaling components of visual phototransduction system in the retina) are less sensitive to light synchronization, but their circadian response is not eliminated. This shows that although the visual system plays a role in entrainment, there must be other photoreceptive pathways that entrain circadian rhythms (Suri et al., 1998; Yang et al., 1998). Drosophila indeed uses a parallel extra-ocular pathway, by which light signals are perceived across the cuticle by CRY intracellular photoreceptor,

which directly influence the molecular oscillator in clock neurons and in most tissues of the fly. CRY binds directly to TIM in a light-dependent manner and triggers degradation of TIM.

TIM degradation exposes PER to phosphorylation and subsequent proteasomal degradation, which results in the resetting of the circadian clock in a phase-dependent manner (Ceriani et al., 1999; Lee et al., 1996). This posttranslational mechanism is in sharp contrast to the pacemaker in N.crassa, where light evokes rapid changes in the transcriptional profile of frq clock gene via a WCC conformational change. The level cry RNA cycles in both LD cycles and in constant darkness, indicating that it is clock controlled. The level of CRY protein also cycles, however, unlike TIM, which continues to cycle in DD, it rather accumulates at constant levels in DD. This indicates that rhythmicity of CRY level is light driven and not clock driven (Emery et al., 1998).

Temperature is also an important timing cue to synchronize the circadian Drosophila clock. Although the fly clock is temperature-compensated over a wide range of constant physiological temperatures, it has already been shown that eclosion and locomotor activity can entrain to cycling temperature changes (Wheeler et al., 1993; Zimmerman et al., 1968).

Temperature cycles, as well as temperature steps can adjust the phase of the clock. In constant light, where Drosophila rhythms are normally disrupted, a single temperature depression by 10C°, or temperature cycles can induce circadian rhythms at both the molecular and behavioral level. Whereas, a single step up (10°C) induced a single locomotor activity peak circa 9 h after the temperature transition (Yoshii et al., 2007). The temperature sensing system is still largely unclear, but it might involve the recently characterized nocte gene (no circadian temperature entrainment) and PLC (phospholipase C), mutations of which specifically abolish/attenuate synchronization of molecular and behavioral rhythms to temperature cycles but not light-dark cycles (Glaser and Stanewsky, 2005; Sehadova et al., 2009). The function of nocte is not known. However, PLC was shown to be involved in the temperature regulation of alternative splicing in the 3’ untranslated region of per mRNA. This

splicing exhibits daily fluctuations and may influence transcript stability. At low temperatures, relatively more of the spliced variant (type B’) is present as compared to the unspliced variant (type A). Type B’ per transcripts lead to a rapid PER protein accumulation.

The constitutively high level of type B’ over A type per transcipt in norpA (which encodes phospholipase C) flies suggests that PLC has a role in downregulating B’ splicing (Majercak et al., 2004). Parallel findings show that short photoperiods enhance the efficiency of B’

splicing (Collins et al., 2004; Majercak et al., 2004). In addition, at cold but not warm temperatures, light was shown to acutely stimulate expression of tim in a temporally gated manner (Chen et al., 2006). Thus, the dual photoperiodic and temperature regulation of tim and per transcripts accumulation work in concert to modulate the timing of activity in response to daily and seasonal changes.