Clock

DEVELOPMENTAL BIOLOGY

Embryonic

Clock mRNA is expressed widely, being found in head, body and appendage fractions. Unlike per and tim, Clock mRNA oscillates in a bimodal fashion. It peaks at zeitgeber time 5 (ZT5; five hours after lights on) and at ZT23 (near the end of lights off), whereas PER and TIM peak at ZT16. Preliminary experiments indicate that the drop at ZT1 occurs even if the animals are kept in darkness. Whether this profile is reflected at the protein level is not known. To explore the possible significance of splice variant B, in which the coding region goes out of frame after the bHLH domain, its level of expression was measured. Splice variant B is expressed weakly at all times of the day, and it cycles in phase with the full-length form (Darlington, 1998).

Clock (Clk) is a core component of the transcriptional feedback loops that comprise the circadian timekeeping mechanism in Drosophila. As a heterodimer with Cycle (Cyc), Clk binds E-boxes to activate the transcription of rhythmically expressed genes within and downstream of the circadian clock, but this activation unexpectedly occurs at times when Clk is at its lowest levels on Western blots. Recent studies demonstrate that Clk also regulates nonrhythmic gene expression and behaviors. Despite the critical roles Clk plays within and outside the circadian clock, its spatial expression pattern has not been characterized. Using a newly developed Clk antibody, Clk is shown to be coexpressed with Period (Per) in canonical oscillator cells throughout the head and body. In contrast to Per, however, the levels of Clk immunoreactivity do not cycle in intensity, Clk is detected primarily in the nucleus throughout the circadian cycle, and Clk is expressed in non-oscillator cells within the lateral and dorsal brain, including Kenyon cells, which mediate various forms of learning and memory. These results indicate that constitutive levels of nuclear Clk regulate rhythmic transcription in circadian oscillator cells and suggest that Clk contributes to other behavioral processes by regulating gene expression in non-oscillator cells (Houl, 2006).

Adult

Mechanisms composing Drosophila's clock are conserved within the animal kingdom. To learn how such clocks influence behavioral and physiological rhythms, the complement of circadian transcripts in adult Drosophila heads was determined. High-density oligonucleotide arrays were used to collect data in the form of three 12-point time course experiments spanning a total of 6 days. Analyses of 24 hr Fourier components of the expression patterns revealed significant oscillations for ~400 transcripts. Based on secondary filters and experimental verifications, a subset of 158 genes showed particularly robust cycling and many oscillatory phases. Circadian expression is associated with genes involved in diverse biological processes, including learning and memory/synapse function, vision, olfaction, locomotion, detoxification, and areas of metabolism. Data collected from three different clock mutants (per₀, tim₀₁, and Clk_Jrk), are consistent with both known and novel regulatory mechanisms controlling circadian transcription (Claridge-Chang, 2001).

A genome-wide expression analysis was performed aimed at identifying all transcripts from the fruit fly head that exhibit circadian oscillations in their expression. By taking time points every 4 hr, a data set was obtained that has a high enough sampling rate to reliably extract 24 hr Fourier components. Time course experiments spanning a day of entrainment followed by a day of free-running were performed to take advantage of both the self-sustaining property of circadian patterns and the improved amplitude and synchrony of circadian patterns found during entrainment. 36 RNA isolates from wild-type adult fruit fly heads, representing three 2 day time courses, were analyzed on high-density oligonucleotide arrays. Each array contained 14,010 probe sets (each composed of 14 pairs of oligonucleotide features) including ~13,600 genes annotated from complete sequence determination of the Drosophila genome. To identify different regulatory patterns underlying circadian transcript oscillations, four-point time course data was colleced from three strains of mutant flies with defects in clock genes (per⁰, tim⁰¹, and Clk^Jrk) during a single day of entrainment. Because all previously known clock-controlled genes cease to oscillate in these mutants but exhibit changes in their average absolute expression levels, the analysis of the mutant data was focused on changes in absolute expression levels rather than on evaluations of periodicity (Claridge-Chang, 2001).

To organize the 158 statistically significant circadian transcripts in a way that was informed by the data, hierarchical clustering was performed. Both the log ratio wild-type data (normalized per experiment) and the log ratios for each of the three clock mutants (normalized to the entire data set) were included to achieve clusters that have both a more or less uniform phase and a uniform pattern of responses to defects in the circadian clock. One of the most interesting clusters generated by this organization is the per cluster. This cluster contains genes that have an expression peak around ZT16 and a tendency to be reduced in expression in the Clk^Jrk mutant. Strikingly, all genes previously known to show this pattern of oscillation (per, tim, vri) are found in this cluster (Claridge-Chang, 2001).

The genes in a second cluster (Clock cluster are primarily grouped together based on their peak phase (average phase ZT2). By virtue of the mutant expression data, several subclusters within this phase group can be identified. The known circadian genes Clock and takeout (to) are part of this cluster. Clk is found in a clustered pair with the leucyl aminopeptidase gene CG9285. In terms of chromosomal organization, to, CG11891, and CG10513 map closely together on chromosome 3R. Two additional circadian genes in this chromosomal region (CG11852, CG10553). Interestingly, the Clk cluster contains three pairs of homologous genes with very similar expression patterns: the UDP-glycosyl transferase genes Ugt35a and Ugt35b, the enteropeptidase genes CG9645 and CG9649, and the long-chain fatty acid transporter genes CG6178 and CG11407. In the first two cases, the homologous genes are also directly adjacent to each other on the chromosome. An overview of the map positions of all circadian genes in this study is available as supplemental information online (http://www.neuron.org/cgi/content/full/32/4/657/DC1). Apart from Ugt35a and Ugt35b, several other genes with a predicted function in detoxification are members of the Clk cluster (CG17524, CG8993, CG3174, Cyp6a21). It may also be noteworthy that the genes for three oxidoreductases found in this group [Photoreceptor dehydrogenase (Pdh), CG15093, CG12116] have almost identical phases (ZT3) (Claridge-Chang, 2001).

Circadian clocks in antennal neurons are necessary and sufficient for olfaction rhythms in Drosophila

The Drosophila circadian clock is controlled by interlocked transcriptional feedback loops that operate in many neuronal and nonneuronal tissues. These clocks are roughly 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 are thought to control other rhythmic outputs. Peripheral oscillators are required to mediate rhythmic olfactory responses in the antenna, but the identity and relative autonomy of these peripheral oscillators has not been defined. Targeted ablation of lateral neurons by using apoptosis-promoting factors and targeted clock disruption in antennal neurons with newly developed dominant-negative versions of Clock and Cycle show that antennal neurons, but not central clock cells, are necessary for olfactory rhythms. Targeted rescue of antennal neuron oscillators in cyc⁰¹ flies, using wild-type cycle, shows that these neurons are also sufficient for olfaction rhythms. It is concluded that antennal neurons are both necessary and sufficient for olfaction rhythms, which demonstrates for the first time that a peripheral tissue can function as an autonomous pacemaker in Drosophila. These results reveal fundamental differences in the function and organization of circadian oscillators in Drosophila and mammals and suggest that components of the olfactory signal transduction cascade could be targets of circadian regulation (Tanoue, 2004).

Circadian clocks control daily rhythms in physiology, metabolism, and behavior in a wide array of organisms. These clocks maintain circadian time via interlocked feedback loops in gene expression. In Drosophila, the operation of these feedback loops is dependent on the heterodimeric bHLH-PAS transcription factors Clock and Cycle, which bind E box elements to activate the transcriptional feedback regulators vrille, PAR domain protein 1, period, and timeless. Vri and Pdp1 feedback to control the inhibition and subsequent activation of Clk transcription, respectively, while Per and Tim feedback as a heterotrimeric complex with Doubletime kinase to inhibit Clk-Cyc-dependent transcription. These transcriptional regulatory events, along with posttranslational control of protein accumulation, subcellular localization, and degradation maintain circadian cycles in gene expression that control rhythmic outputs (Tanoue, 2004 and references therein).

Circadian oscillators are present in a variety of tissues throughout the head, thorax, and abdomen. One rhythmic output that has been described in adult flies is a rhythm in olfactory responses (Krishnan, 1999). This rhythm is measured by using an assay for odor-induced electrophysiological responses in antennae called the electroantennogram (EAG). EAG responses to a food odorant, ethyl acetate, are lowest during the late night and early day, begin to increase at the end of the day, and ultimately reach a peak in the middle of the night. This 2-fold rhythm in EAG responses is associated with an 100-fold difference in threshold sensitivity to odor at the peak and trough time points. This pattern is seen in wild-type flies during either LD or DD conditions and is controlled by a per/tim dependent clock that is similar, but not identical, to the one that controls locomotor activity rhythms. However, the sLN_vs that mediate locomotor activity rhythms are not sufficient for EAG rhythms; thus oscillators in peripheral tissues are necessary for EAG rhythms (Tanoue, 2004).

Although the central sLNv oscillators are not sufficient for EAG rhythms, a role for these oscillators cannot be excluded. For instance, these rhythms might require oscillator function in both peripheral tissues and sLN_vs. Alternatively, EAG rhythms may be regulated solely by oscillators in one or more peripheral tissues, such as nonneuronal accessory cells in the antennae, antennal neurons, both neurons and accessory cells in the antenna, or even neurons in the antennal lobe. Ablating central oscillator cells is shown to have no affect on EAG rhythms, eliminating circadian oscillator function in antennal neurons by using newly developed Clk and Cyc dominant-negative transgenes abolishes rhythms in EAG responses, and rescuing oscillator function in antennal neurons restores EAG rhythms. These results demonstrate that EAG rhythms are controlled by local oscillators in antennal neurons that function independently of the central sLN_v oscillator, which indicates that the circadian systems of Drosophila and mammals are organized differently and that the circadian clock may control EAG rhythms via some component of the olfaction signal transduction cascade (Tanoue, 2004).

A set of ten bilaterally symmetric LN_vs function to control locomotor activity rhythms in Drosophila and are therefore roughly equivalent to the central pacemaker cells in the mammalian SCN that also control activity rhythms. The SCN also functions to control oscillators in peripheral tissues and, by extension, any rhythmic processes that those tissues control. Although oscillators in peripheral tissues of adult Drosophila are not dependent on the LN_vs, it is possible that as in mammals, the LN_v 'central oscillator' is required to control rhythmic outputs emanating from peripheral tissues. This issue was addressed by defining the oscillators that are necessary and sufficient for olfaction rhythms, which emanate from a peripheral tissue (i.e., the antenna) that itself contains a circadian oscillator. LN_v oscillators are not required for olfaction rhythms but oscillators within antennal neurons are both necessary and sufficient for olfaction rhythms. The ability of these antennal neuron oscillators to control olfaction rhythms indicates that they function as autonomous circadian pacemakers. The control of olfaction rhythms by local oscillators in antennal neurons, independent of the LN_v oscillators, suggests that in adult Drosophila the circadian system is organized as a set of independent oscillators that autonomously control rhythmic outputs as opposed to the hierarchical organization of the mammalian circadian system (Tanoue, 2004).

A distributed set of independent oscillators may best benefit an animal like Drosophila that is diurnally active and has oscillators which, as a whole, directly see and entrain to light. These attributes are not characteristic of mammals, but they are found in some vertebrates; zebrafish are diurnally active and their peripheral organs (e.g., heart, kidney) are directly entrainable by light. Other nonmammalian vertebrates (i.e., Xenopus, birds, and lizards) also have peripheral tissues that harbor autonomous light-entrainable clocks, but these are restricted to photoreceptive tissues such as the pineal gland, the retina, and the parietal eye. Just as a hierarchy is not the rule for circadian organization in vertebrates, a distributed organization is not the rule for circadian organization in insects. The pupal prothoracic gland and LNs are both required, but neither are sufficient, for eclosion rhythms in Drosophila. This corequirement for central and peripheral oscillators could simply be a characteristic of earlier developmental stages, though other examples of this mode of oscillator organization may be found in adults as more rhythmic outputs are discovered. Regardless, it is clear that oscillator organization can differ within a species. Rhythmic EAG responses in antennae of the cockroach Leucophaea maderae were recently described, indicating that circadian control of olfaction is conserved in two distantly related insects (Page, 2003). However, these EAG rhythms are dependent on the central pacemaker cells in the optic lobe, and photic entrainment is dependent on the eye (Page, 2003). This result indicates that the circadian system of this nocturnal insect is hierarchically organized, further demonstrating that circadian organization is not an evolutionarily conserved characteristic (Tanoue, 2004).

Identifying the oscillator cells that control different rhythmic phenomena is a fundamental neuroanatomical question. Knowledge of the cells required for behavioral, physiological, or metabolic rhythms is often necessary before delving into the molecular mechanisms that regulate these phenomena. One method that is often used to define cells required for rhythmic output is to ablate cells by using cell death genes. This method has been useful for defining cells necessary for circadian behavior but is not useful for defining cells required for physiological or metabolic rhythms that may emanate from the oscillator cells themselves or rhythms in cells required for viability. For these sorts of rhythmic outputs and cell types, the only method available for eliminating clock function in specific cells has been to overexpress Per. High levels of Per can block Clk-Cyc activity, thereby abolishing molecular oscillations, but is difficult to achieve in practice because molecular and behavioral rhythmicity persists unless Per is drastically overexpressed. The availability of cell specific drivers that can produce enough per to effectively eliminate clock function may be somewhat limited (Tanoue, 2004).

As an alternative to per overexpression, a method has been developed for eliminating oscillator function that employs dominant-negative forms of Clk and Cyc. Such a strategy has been used to block circadian oscillator function in Xenopus photoreceptors, but in this case the dominant-negative was generated by removing the activation domain of xCLOCK. Regardless of which dominant-negative (i.e., ClkΔ or CycΔ) is expressed in clock cells, clock function is effectively abolished at both the molecular and physiological/behavioral levels. Not only did ClkΔ and CycΔ block clock function when expressed from the relatively strong tim promoter, but also when the Or, pdf, and Clk promoters were used. Since the expression of ClkΔ and CycΔ inhibits CLK-CYC-dependent transcription, these dominant-negatives would also be expected to reduce expression from the tim-Gal and pdf-Gal drivers and, consequently, the levels of ClkΔ and CycΔ. Apparently the relative stability of GAL4, together with continued low-level expression from the tim-Gal4 and pdf-Gal4 drivers, produces enough ClkΔ and CycΔ to effectively repress Clk-Cyc dependent genes (Tanoue, 2004).

It is interesting that ClkΔ and CycΔ eliminated clock function so effectively given that Cyc is substantially more abundant than Clk, Per, or Tim in fly heads. The expression pattern of Cyc has not been characterized, and it is possible that the majority of Cyc expression could be in nonclock cells. If so, Cyc could be relatively low in abundance in clock cells, thus promoting more effective competition of CycΔ for Clk binding and ClkΔ for Cyc binding. Alternatively, ClkΔ-Cyc and CycΔ-Clk heterodimers may be more stable than the natural Clk-Cyc heterodimer. Clk was recently found to be quite labile when bound to BMAL1 in mice. Given the effectiveness of ClkΔ and CycΔ at blocking clock function, these dominant-negatives will be generally useful for mapping cells required for rhythmic outputs. A similar strategy may also be useful for mapping neuroanatomical pathways that mediate clock outputs in mammals (Tanoue, 2004).

The finding that circadian oscillators in ORNs are sufficient for EAG rhythms implies that not only the clock, but also the EAG rhythm output pathway is contained within these cells. Colocalization of a circadian oscillator and a rhythmic output in these neurons suggests a number of potential targets for clock regulation. Olfactory signal transduction in Drosophila is initiated by G protein-coupled odorant receptors, involves Gq- and IP₃-mediated signal transduction, and ultimately controls ion channels including the paralytic (para) Na⁺ channel and the ether-a-go-go (eag) K⁺ channel. The clock could modulate the sensitivity of the olfactory system by decreasing OR signaling. For instance, GPCRs such as the ORs can be desensitized through action of G protein-coupled receptor kinases and arrestins, thus making these two types of regulators potential targets for clock control. Likewise, the generation of IP₃ and the modulation of its receptor and downstream effector proteins also provide a rich source of targets for clock control. Activity of the eag and para ion channels can also be modulated via CaMKII-dependent phosphorylation and interaction with TipE protein, respectively, and could serve as targets for clock control. In fact, the circadian clock is known or suspected to regulate the activity of several types of ion channels. In chickens, activity of the ILOT cation channel is under circadian control in the pineal gland, and a cGMP gated cationic channel in the retina is regulated, at least indirectly, by ERK- and CaMKII-dependent phosphorylation. In Drosophila, the abundance of Slowpoke (Slo), a Ca⁺² dependent K⁺ channel protein, is controlled by the circadian clock, and levels of mRNA encoding a slo regulatory protein, called slo binding protein (slob), cycle in a circadian manner (Tanoue, 2004).

Irrespective of which or how many olfaction signal-transduction components are under clock control, it is important to understand the mechanism by which the clock imposes circadian regulation. Several components of the circadian timekeeping mechanism are controlled at the transcriptional level. Microrarray analysis has identified more than 100 additional transcripts (i.e., those that do not encode components of the timekeeping mechanism) that are under circadian clock control; this suggests that transcription may be a major mechanism by which the clock controls rhythmic outputs. Although minor compared to transcriptional regulation, posttranscriptional regulation also contributes to per mRNA cycling, and thus may control circadian outputs as well. The circadian clock also controls the abundance and subcellular localization of core circadian oscillator proteins through posttranslational mechanisms. For instance, Dbt- and CkII-dependent phosphorylation of Per increases it's degradation, while Sgg-dependent phosphorylation of Tim promotes the nuclear localization of Per-Tim heterodimers. From these examples, the circadian clock could regulate olfactory signal transduction components at several different levels. A comprehensive understanding of how the clock regulates olfaction will ultimately enable the tracing of an output pathway from the timekeeping mechanism to physiology (Tanoue, 2004).

CLOCK expression identifies developing circadian oscillator neurons in the brains of Drosophila embryos

The Drosophila circadian oscillator is composed of transcriptional feedback loops in which CLOCK-CYCLE (CLK-CYC) heterodimers activate their feedback regulators period (per) and timeless (tim) via E-box mediated transcription. These feedback loop oscillators are present in distinct clusters of dorsal and lateral neurons in the adult brain, but how this pattern of expression is established during development is not known. Since CLK is required to initiate feedback loop function, defining the pattern of CLK expression in embryos and larvae will shed light on oscillator neuron development (Houl, 2008).

A novel CLK antiserum is used to show that CLK expression in the larval CNS and adult brain is limited to circadian oscillator cells. CLK is initially expressed in presumptive small ventral lateral neurons (s-LN_vs), dorsal neurons 2 s (DN₂s), and dorsal neuron 1 s (DN₁s) at embryonic stage (ES) 16, and this CLK expression pattern persists through larval development. PER then accumulates in all CLK-expressing cells except presumptive DN₂s during late ES 16 and ES 17, consistent with the delayed accumulation of PER in adult oscillator neurons and antiphase cycling of PER in larval DN₂s. PER is also expressed in non-CLK-expressing cells in the embryonic CNS starting at ES 12. Although PER expression in CLK-negative cells continues in Clk^Jrk embryos, PER expression in cells that co-express PER and CLK is eliminated. These data demonstrate that brain oscillator neurons begin development during embryogenesis, that PER expression in non-oscillator cells is CLK-independent, and that oscillator phase is an intrinsic characteristic of brain oscillator neurons. These results define the temporal and spatial coordinates of factors that initiate Clk expression, imply that circadian photoreceptors are not activated until the end of embryogenesis, and suggest that PER functions in a different capacity before oscillator cell development is initiated (Houl, 2008).

Effects of Mutation and Ectopic Expression

To identify novel genes involved in Drosophila circadian rhythms, chemically mutagenized flies were screened for mutants that alter or abolish circadian locomotor activity rhythms. Specifically, treated sibs were mated to homozygose third chromosomes, permitting the identification of recessive mutations on this chromosome. More than 6000 lines were screened, approximately 50% of which were free of recessive lethal mutations on the third chromosome; the remainder of the lines were tested as heterozygotes. One novel semidominant third chromosome arrhythmic mutant, Jrk, was identified as a homozygous mutant with completely arrhythmic locomotor behavior in constant darkness. As heterozygotes, approximately half of all Jrk flies are arrhythmic, and those flies that do manifest a rhythm have a slightly longer period than wild-type controls. In addition to abolishing rhythmicity under free running conditions, homozygous Jrk flies manifested unusual locomotor activity even under 12 hr light:12 hr dark (LD) conditions. The percentage of flies with a measurable rhythm in LD fell from 64% for heterozygotes to only 14% for Jrk homozygotes. Although examination of raw activity counts indicates that wild-type, per⁰¹, and Jrk flies have similar levels of activity, their diurnal activity patterns differ. Under these standard LD conditions, wild-type flies anticipate the lights on and lights off transitions by slowly increasing activity, reflecting internal clock function. Thus, it is not surprising that arrhythmic Jrk flies do not anticipate the L-to-D or D-to-L transitions. But wild-type flies as well as per⁰¹ and tim⁰¹ also react to the light transitions with an acute activity burst that quickly dissipates. Like wild-type flies, some Jrk flies still react to the lights-off transition, but they all fail to react to the lights-on transition. Optomotor testing of Jrk flies reveals no abnormality of the conventional visual system, suggesting that the mutation may also impair some feature of the circadian visual response (Allada, 1998).

Although Jrk flies display no obvious morphologic or general behavioral defects, the absence of rhythms could be due to effects on the locomotor output pathway rather than on the clock itself. To address this question, an independent manifestation of circadian output, adult emergence, was assayed. Homozygous Jrk mutant flies are also arrhythmic for this eclosion phenotype. To measure more directly clock function, the fluctuations of the clock proteins, Per and Tim were assayed in wild-type, heterozygous, and homozygous Jrk flies under LD conditions. Both Per and Tim levels are extremely low and noncycling in homozygous Jrk flies, approximately equivalent to trough levels of wild-type flies. Per and Tim protein levels cycle well in Jrk heterozygotes, but the amplitude is reduced approximately 50%, consistent with the clear effects on behavioral rhythmicity in these flies (Allada, 1998).

Because of the semidominance of the Jrk behavioral phenotype and the general weakness of many deletion stocks, the behavioral results were verified by analyzing Per and Tim expression. The heterozygous deletion Df(3L)D1 still manifests Tim protein cycling. However, when heterozygous with Jrk, Tim levels fail to cycle and are comparable to those of Jrk homozygotes, i.e., a clear failure to complement. Similar results (low constitutive levels) are observed for Per protein. Clock is epistatic to per: Tim levels are very low in a per0;Clk arrhythmic strain, suggesting that Clock acts upstream of per (Allada, 1998).

Cryptochrome is a major Drosophila photoreceptor dedicated to the resetting of circadian rhythms. How is Cryptochrome mRNA cycling affected by mutations in four clock genes implicated in gene regulation: per, tim, Clock, and cycle? In all single mutants and double mutant combinations, little or no mRNA cycling is found, indicating that cycling requires a functional pacemaker and is not merely light driven. cry mRNA levels are a function of the specific mutant or mutant combination. They are relatively low in the per or tim null mutants as well as in the per;tim double mutant combination, whereas they are relatively high in the Clock and cycle mutants. The double mutants per;Clock and per;cycle also show high cry mRNA levels, indicating an epistatic effect of Clock and cycle over per. Thus, CRY mRNA levels are low in per and tim null mutants, the opposite of what is observed for autoregulation of PER and TIM mRNA levels. CRY mRNA levels are high in clock or cycle mutants, contrary to the low PER and TIM mRNA levels found in these novel clock mutants (Emery 1998 and references).

The circadian clock consists of a feedback loop in which clock genes are rhythmically expressed, giving rise to cycling levels of RNA and proteins. Four of the five circadian genes identified to date influence responsiveness to freebase cocaine in the fruit fly, Drosophila melanogaster. Sensitization to repeated cocaine exposures, a phenomenon also seen in humans and animal models and associated with enhanced drug craving, is eliminated in flies mutant for period, clock, cycle, and doubletime, but not in flies lacking the gene timeless. Flies that do not sensitize owing to lack of these genes do not show the induction of tyrosine decarboxylase normally seen after cocaine exposure. These findings indicate unexpected roles for these genes in regulating cocaine sensitization and indicate that they function as regulators of tyrosine decarboxylase (Andretic, 1999).

A recessive mutant of Drosophila Clock reveals a role in circadian rhythm amplitude

The transcription factor Clock (Clk) plays a critical role in animal circadian rhythms. Genetic studies defining its function have relied on two dominant negative alleles, one in Drosophila and one in mice. A novel recessive allele is described of Drosophila Clock, Clk^ar. Homozygous Clk^ar flies are viable and behaviorally arrhythmic. The Clk^ar phenotype is caused by a splice site mutation that severely disrupts splicing and reduces Clk activity. Despite the behavioral arrhythmicity, molecular oscillations are still detectable in Clk^ar flies. Transcription analysis indicates potent effects of Clk^ar on levels and amplitude of transcriptional oscillations. Taken together with other data, it is proposed that Clk makes a major contribution to the strength and amplitude of circadian rhythms (Allada, 2003).

This study describes the first recessive allele of Clock. The only previously reported allele, Clk^Jrk, is semi-dominant and behaves as a dominant negative. As a result, it is possible that Clk^Jrk may exert its effects through a gain-of-function effect. The strongest genetic evidence for a Clk requirement for normal rhythms is the long period of flies heterozygous for a deletion of the Clk locus. However, homozygous D1 flies do not live to adulthood, and behavioral rhythms therefore cannot be assessed in Clk-null animals. Even if they were arrhythmic, an essential contribution of Clk to rhythms would remain uncertain because the D1 deletion removes several genes. These most likely include PAR-domain protein 1 (Pdp1) and Henna (Hn), which are candidate circadian rhythm genes and are located within 40 kb (right) and 10 kb (left) of Clk, respectively. The D1 deletion was initially identified by its failure to complement the eye pigment phenotype of Hn, which lies just to the left of Clk. Furthermore, D1 fails to complement mutant loci to the right of Pdp1 and is therefore likely to remove this gene as well. The heterozygous phenotype of the D1 deletion may therefore be due to the absence of one or more of these genes. A similar ambiguity exists in mammals, since there is only a single semi-dominant allele and no hypomorphic or null alleles of the mouse Clock locus. All of these considerations make this new recessive arrhythmic Clk allele important and indicate that Clk is indeed required for behavioral rhythmicity (Allada, 2003).

Part of the proof that Clk^ar is truly a Clk mutant comes from the rescue with heterozygous combinations of crygal4 and UASClk. It was not possible, however, to effect successful rescue with other rhythm-relevant gal4 drivers: timgal4 and pergal4 were lethal in combination with UASClk, and pdfgal4 had no effect on the arrhythmic Clk^ar phenotype. The pdfgal4 driver was also unable to rescue the arrhythmic Clk^Jrk background. The rescuing ability of pdfgal4 was also tested in flies trans-heterozygous for the Clk mutants and the Clk deletion, D1: in these genetic backgrounds, pdfgal4 also failed to rescue either Clk^Jrk or Clk^ar. The most obvious difference between crygal4 and pdfgal4 is a more widespread expression pattern in the case of crygal4, which extends to another more dorsal group of neurons, the LNds. Locomotor activity rhythms may therefore require wild-type Clk expression in more than just the restricted pdf locations (Allada, 2003).

Another possible difference between the cry and pdf drivers is RNA cycling: cry mRNA cycles with a roughly similar amplitude and phase to Clk, whereas the pdf gene undergoes no substantial transcriptional fluctuations. Although Clk and cry mRNA cycling may contribute to circadian function, this explanation is not favored. The stability of GAL4 should prevent significant oscillations in protein levels, even if mRNA levels undergo robust cycling. This suggests that rhythmic transcription of Clk is unnecessary for behavioral rhythmicity and is consistent with other experiments indicating that the timing and levels of Clk oscillation can be substantially altered without strong effects on circadian behavior. This conclusion is also consistent with the notion that the Clk transcriptional loop is less important than the original per-tim loop for behavioral rhythms (Allada, 2003).

It is noted, however, that GAL4-mediated rescue of Clk is only partial, with 60% rhythmicity and shortened periods. This is comparable to the GAL4-mediated rescue of per⁰; tim⁰ double mutants in which rhythmicity is 40%-50% and periods range from 24-27 h. The similarly poor percent rhythmicity values may reflect comparable contributions of mRNA cycling -- per and tim in one case and Clk in the other. Alternatively, mRNA cycling may be largely irrelevant to penetrance; the poor rescue may result from inappropriate GAL4 levels (too high or too low) or from some other inadequate feature of GAL4-mediated expression (Allada, 2003).

The molecular assays in Clk^ar indicate bona fide rhythms with a predominant effect on circadian rhythm amplitude and no more than a modest effect on phase or period. With circadian per and tim enhancers, reduced enhancer activity and a reduced cycling amplitude are observed in a Clk^ar background, consistent with the role of Clk in regulating these enhancers. Nonetheless, the phase of oscillating bioluminescence is similar to that of wild-type flies. The presence of molecular rhythms contrasts with the absence of detectable behavioral rhythms. The notion is favored that this reflects a level or amplitude reduction below a critical threshold for behavioral rhythmicity. The absence of anticipation of LD transitions makes it very unlikely that an effect restricted to the lateral neurons, the absence of the neuropeptide PDF for example, is primarily responsible for the phenotype. This is also because LD behavioral rhythms are largely normal in flies devoid of PDF or the pacemaker lateral neurons. Moreover, both large and small PDF-expressing lateral pacemaker neurons are present in Clk^ar. However, a reduction was observed in pdf expression in the small lateral neurons that may contribute to Clk^ar arrhythmicity in constant darkness (Allada, 2003).

Previous results with Clk^Jrk also support a role for Clk in defining rhythmic amplitude. Clk^Jrk heterozygotes reveal a dominant reduction in the amplitude of molecular rhythms with little apparent change in phase. These heterozygotes also exhibit reductions in rhythmic behavior with only slightly long periods. Indeed, Clk overexpression results in a selective increase in the amplitude of per RNA oscillations. This modest effect on period or phase of varying Clk activity is similar to the phenotype of transgenic strains missing the per promoter or expressing per and tim from constitutive promoters. These strains also have reasonable periods (22-26 h) with poor rhythm amplitudes, as evidenced by the poor penetrance of rhythmicity. One argument for a role for Clk in period control is the phenotype of the D1 heterozygote (~25.5 h period). However, even this altered period is within the limited range of altering per or tim transcription. Taken together, these data suggest that substantial changes in Clock gene transcription have limited effects on circadian period. Separate control of circadian rhythm amplitude in one case, and period (or phase) in the other, is also consistent with anatomical experiments in both the fly and mammalian system (Allada, 2003).

It is proposed that the post-transcriptional phosphorylation turnover feedback loop involving several Clock components (e.g. per, tim, the protein kinase Dbt) is predominantly responsible for period determination. Excluding null alleles that are either arrhythmic or lethal, FlyBase lists mutant alleles of per, tim and Dbt that exhibit period alterations ranging from 16-30 h for per (8 mutant alleles), 21-33 h for tim (8 mutant alleles) and 18-29 h for Dbt (5 mutant alleles). Indeed, the only Dbt allele that fails to exhibit rhythmicity as a homozygote, displays a potent period-altering phenotype as a heterozygote. More recent additions to this list are the protein kinases shaggy and CK2. Indeed, one mutant allele of CK2alpha, CK2alpha^Tik, exhibits one of the strongest dominant period effects of any rhythm mutant. These large period effects contrast with the transcription factor mutants (Clk and cyc). Their phenotypes indicate that near-normal periods are maintained despite large protein level changes (Allada, 2003).

Drosophila CLOCK protein is under posttranscriptional control and influences light-induced activity

In the Drosophila circadian clock, daily cycles in the RNA levels of Clock (Clk) are antiphase to those of Period (Per). The timing/levels of Clk expression were altered by generating transgenic flies whereby per circadian regulatory sequences were used to drive rhythmic transcription of Clk. In this manner, Clk expression was regulated from both the endogenous wild-type gene and from a transgene that was activated during the mid day, compared to its normal mid night induction. The results indicate that posttranscriptional mechanisms make substantial contributions to the temporal changes in the abundance of the Clk protein. Circadian regulation is largely unaffected in the transgenic per-Clk flies despite higher mean levels of Clk protein. However, in per-Clk flies the duration of morning activity is lengthened in light-dark cycles and light pulses evoke longer lasting bouts of activity. These findings suggest that, in addition to a role in generating circadian rhythms, Clk modulates the direct effects of light on locomotion (Kim, 2002).

Large increases in the timing/levels of Clk expression do not result in gross alterations in the daily cycles of the RNA and protein products from several key clock genes. Activity rhythms measured in constant dark conditions were normal, indicating that canonical properties of clock function are not significantly influenced by large increases in the levels of Clk. Normal pacemaker function in flies harboring the per-dClk transgene (ARK flies) is probably achieved because alterations in the temporal regulation of Clk expression are 'neutralized' by the posttranscriptional regulation of Clk levels and the normal timing of Per and Tim interactions with Clk. Nonetheless, the results strongly suggest that the levels of Clk are an important variable in modulating the duration of light-induced bouts of activity in Drosophila (Kim, 2002).

Despite relatively large increases in the mean levels of Clk protein the circadian oscillator is only modestly affected. The near normal timing of interactions between Clk and the other clock proteins analyzed (i.e., Per, Tim, and Cyc) likely underlies the observation that the phases of the per, tim, and endogenous Clk RNA rhythms are relatively similar to those in the control situation. The main differences are in the amplitudes of the protein and RNA rhythms, which are generally higher in the ARK flies. Similar increases in the amplitudes of Clock protein and RNA rhythms are also observed in the tim-dClk transgenic flies (Kim, 2002).

Why do large increases in the levels of Clk only yield subtle effects on the circadian timing system? A likely explanation is that, despite higher overall levels of Clk in ARK flies, the values remain within a concentration range that can still be efficiently inhibited by Per and Tim. Indeed, Per and Tim interacted with Clk in ARK flies at higher levels than the counterpart situation in wild-type flies. Presumably, in a situation where the levels of Clk reach molar concentrations far exceeding those of Per and Tim, this would result in constitutively high expression of per and tim, blunted cycles in the amounts of Per and Tim proteins and weaker behavioral rhythms. Indeed, dramatic changes in one clock factor without compensatory alterations in other components can impair or abolish clock function. For example, high-level expression of per from a constitutive promoter abolishes rhythms in clock gene expression. These considerations suggest that normal clock function is highly dependent on not only the presence of sufficient levels of clock proteins but also in maintaining appropriate relative molar concentrations (Kim, 2002).

Clk produced from the per-dClk transgene cycles with a low amplitude that reaches peak values during the late night and trough amounts during the day. This profile is very different from the rhythm in the abundance of per-Clk RNA, which attains maximal values in the mid day and steadily declines thereafter. These results indicate that posttranscriptional mechanisms make a large contribution to the temporal regulation of Clk abundance. Similar findings were also obtained with transgenic flies bearing a tim-dClk transgene (Kim, 2002).

The posttranscriptional mechanism(s) contributing to temporal changes in the amounts of Clk is not known. A likely possibility is phosphorylation-induced changes in stability. Highly phosphorylated isoforms are more abundant during the late night/early morning. This raises the possibility that during the night Clk is preferentially phosphorylated. Alternatively, extensively phosphorylated isoforms of Clk might be more stable during the night. In the normal situation, the levels of Clk RNA accumulate during the late night, which would act to reinforce a putative stabilizing effect of nighttime Clk phosphorylation. The maintenance of a robust Clk phosphorylation program in per-dClk flies might underlie the quasi-normal abundance profile of Clk. In ARK flies, misalignment between the daytime accumulation of per-Clk RNA and nighttime Clk phosphorylation could account for the more blunted Clk abundance cycle. Variations in RNA cycles could also explain the different temporal profiles in the abundance of Clk from per-dClk and tim-dClk flies. Dual control by mRNA cycles and posttranslational mechanisms likely increase the dynamic range available for regulating clock protein abundance as a function of time (Kim, 2002).

Drosophila Clock can generate ectopic circadian clocks

Circadian rhythms of behavior, physiology, and gene expression are present in diverse tissues and organisms. The function of the transcriptional activator Clock is necessary in both Drosophila and mammals for the expression of many core clock components. In Drosophila, misexpression of Clock in naive brain regions induces circadian gene expression. This includes major components of the pacemaker program, since Clock also activates the rhythmic expression of cryptochrome, a gene that Clock normally represses. Moreover, this ectopic clock expression has potent effects on behavior, radically altering locomotor activity patterns. It is proposed that Clock is uniquely able to induce and organize the core elements of interdependent feedback loops necessary for circadian rhythms (Zhao, 2003).

To characterize the consequences of Clk misexpression, the GAL4/UAS system was used. Binding sites for the yeast transcription factor GAL4 (upstream activating sequence; UAS) were fused upstream of Clk cDNA (UASClk). The pattern of UASClk expression in transgenic flies is then determined by the spatial and temporal expression pattern of the GAL4 driver. In combination with numerous GAL4 drivers, UASClk results in developmental lethality. However, viable adult progeny were generated with three clock-relevant drivers: the pdf promoter (pdfGAL4), a previously described cry promoter also containing the large cry first intron (crypiGAL4; p = promoter; i = intron) and a cry promoter without the first intron (crypGAL4 (Zhao, 2003).

To characterize the GAL4 expression, these lines were crossed to a UAS-EGFP strain and the adult progeny assayed for brain GFP expression. pdfGAL4 and crypiGAL4 (line 13; crypiGAL4-13) expression is limited to a small number of adult neurons. pdfGAL4 expression is restricted to two cell groups whose morphology and position is consistent with the LNvS and LNvL. The crypiGAL4 appears to be expressed predominantly in the LNvL. This driver is also expressed in the LNd and the LNvS in addition to the LNvL. These three cell groups are a substantial subset of neuronal clock gene expression in the brain. The limited expression from these GAL4 lines contrasts markedly with the broader expression of two independent inserts of crypGAL4, crypGAL4-24, and crypGAL4-16. In addition to the canonical circadian cells, expression is observed in other areas, such as the ellipsoid body (EB). Based on their characteristic morphology, many of these cells appear to be neuronal. Interestingly, differences were observed between the two inserts. The most salient features of crypGAL4-16 relative to crypGAL4-24 were more prominent diffuse glial expression and much less (or absent) expression in the antennal neuropils (AN) as well as in the DNs and in the LNv axons. A third insert (crypGAL4-17) did not exhibit any detectable GAL4-driven GFP expression, further indicating that the crypGAL4 expression pattern is dependent on insert location. The ectopic expression patterns do not correspond with the pattern of any known circadian gene; the patterns are distinct from those observed for timeless promoter-GAL4 (timGAL4 (Zhao, 2003).

Misexpression of Clock induces ectopic clocks. These ectopic clocks are evident by measurements of clock gene expression under light-dark and constant darkness conditions. The basic result is not dependent on a cry-derived GAL4 driver, since the independent noncircadian GAL4 line MJ162a induces ectopic clocks in distinct brain regions. Furthermore, it is likely that Clk is inducing major components of the clock gene program since it also induces rhythmic expression of cry, a gene that the CLK-CYC complex normally represses. The ectopic clocks appear to have potent effects on the LD behavioral program (Zhao, 2003).

Several lines of evidence now place Clk at the top of a genetic hierarchy controlling circadian clock gene expression. Intact Clk is necessary for multiple aspects of the fly and mouse circadian phenotype. In both systems, there is strong genetic and biochemical evidence that Clk and its partner Cyc (BMAL1 in mammals) form a heterodimeric complex and directly activate transcription of several important clock genes. In Drosophila, these genes, per, tim, vri, and Pdp1 comprise the core elements of interdependent circadian feedback loops essential for rhythmic gene expression. Moreover, microarray analyses in both flies and mice indicate that all detectable rhythmic gene expression is dependent on Clk. The abnormal pacemaker neuronal morphology in the fly mutant is consistent with an additional role in regulating circadian neuronal development. All of these data suggest that the Clk gene may be necessary for many if not most aspects of clock cell specification as well as function (Zhao, 2003).

The ectopic clocks appear to strongly influence diurnal behavior, implying that these new clocks make functional connections with locomotor output pathways. There is an excellent correlation between the altered diurnal behavior and ectopic tim expression, for example in male versus female cry24 flies. In contrast, enhanced expression in the pacemaker lateral neurons with pdfGAL4 and crypiGAL4-13 has little or no effect on behavior under LD conditions. Consistent with this notion, the potent effects of Clk on LD behavior are not blocked in a pdf⁰¹ background (Zhao, 2003).

Although a role for increased expression in the lateral neurons or other known circadian cells cannot be completely excluded, the notion is favored that new clock cells are responsible for the altered LD behavior. One of the prominent regions of crypGAL4 driven gene expression is the ellipsoid body, a brain region previously implicated in the higher order control of locomotor activity. As such, Clk-driven expression here might be expected to influence locomotor activity. Interestingly, the MJ162a line does not drive detectable expression in the ellipsoid body, nor does it have prominent behavioral effects in combination with UASClk. Differences between cry16 and cry24 flies further suggest that other neurons or even glia may mediate some of the ectopic Clk behavioral effects. One possibility is that the transgenic strains manifest a dramatically suppressed evening activity peak, which is normally tightly regulated by the circadian clock. This suggests that light and these new clocks may collaborate to antagonize positive factors (such as PDF), which are normally released by canonical clock cells in a temporally gated fashion. The failure of Clk to induce PDF in the ectopic locations is consistent with the view that other humoral factors or perhaps even new neural connections are involved in the behavioral changes. Alternatively, the ectopic clocks may alter the coupling between the central pacemaker and outputs under LD conditions (Zhao, 2003).

To examine the mechanism of ectopic clock formation, it was first considered that Clk might induce new clocks only in cells that are highly predisposed to expressing rhythmicity. In this case, Clk expression would induce one or only a few missing clock genes necessary for molecular oscillator properties. An analogous case from mammals may be that of cultured rat-1 fibroblasts, which mimic the behavior of peripheral clocks such as the liver. Exposure of the rat-1 cells to high concentrations of serum (serum shock) can induce rhythmicity in cells that otherwise exhibit no apparent rhythmicity. The predisposition of these cells is reflected in their substantial level of clock gene expression. In contrast, it was found that tim and cry expression is undetectable in the ectopic cells without Clk expression. This expression analysis is consistent with prior reports indicating that there is no detectable per and tim protein in adult brain neurons outside of the LNs and DNs. The possibility was considered that tim is expressed in these ectopic locations in wild-type flies but that tim mRNA levels are simply below the level of detection. Consistent with this possibility, tim promoter gal4-driven GFP can be visualized in neurons without detectable tim expression. Similarly, broader expression of the per gene has been observed with artificial lacZ fusion proteins and in certain mutant backgrounds, suggesting some low level per expression in other brain regions. The functional relevance of these transgene expression patterns without detectable per or tim expression remains unclear. Moreover, it is not even certain that the expression of these reporters is Clk-dependent. Nonetheless, a comparison of the ectopic rhythmic cells with the timGAL4:UASEGFP pattern indicates that they are two distinct cell populations. The failure to express tim in the absence of Clk induction is consistent with the notion that these cells are not fully preprogrammed for rhythmicity. It will be of interest also to compare expression of per-lacZ fusion proteins that reveal potential cryptic per expression with the ectopic clock locations shown here (Zhao, 2003).

Most compelling perhaps is the absence of cry expression in these ectopic locations. If these cells were simply missing Clk, they should behave as Clk mutants and express high levels of cry mRNA. However, no cry expression was detected in these brain regions, arguing against the hypothesis that these cells are largely programmed for circadian rhythmicity. In addition, it was possible to induce ectopic rhythms in distinct locations using a noncircadian GAL4 line, MJ162a (Zhao, 2003).

This analysis raises some intriguing parallels between Clk and eyeless, a gene involved in the induction of eye morphogenesis. Like Clk for circadian rhythms, ectopic expression of eyeless can induce the formation of ectopic eyes. Both eyeless and Clk function in terminally differentiated neurons to control highly specialized gene expression: opsins in the case of eyeless and rhythm genes in the case of Clk. It has been proposed that activation of a photoreceptor gene was the original function of eyeless and that its morphogenetic role was acquired much later in evolution. Similarly, it is proposed that the original role of Clk was to activate expression of a clock gene ancestor, and its ability to direct the formation of temporally regulated feedback loops was a more recent acquisition (Zhao, 2003).

Despite its reported function as a 'master control gene', not all cells are substrates for eyeless-induced ectopic eye formation. Similarly, the presence of other rhythm factors, such as Cyc, are likely required for Clk expression to induce functional clocks. Furthermore, it was not possible to induce rhythmic gene expression with transfected Clk in Cyc-expressing S2 cells, suggesting that still other factors might be necessary. Future experiments assessing ectopic clock formation in different circadian mutant backgrounds and tissues should address this general issue. Despite the similarities between eyeless and Clk, there is reason to suspect that Clk may have more far reaching functions. For example, eyeless-induced ectopic eyes have never been shown to be functional, whereas substantial evidence is presented that ectopic clocks can alter behavior. Taken together with the Clk mutant effects on LNv anatomy, normal Clk expression may even contribute to pacemaker cell wiring properties. Given the similarities between the fly and mammalian clock systems, it is suggested that the mammalian orthologs of Clk and cyc may play similar roles (Zhao, 2003).

Genome-wide expression analysis in Drosophila reveals genes controlling circadian behavior

In Drosophila, a number of key processes such as emergence from the pupal case, locomotor activity, feeding, olfaction, and aspects of mating behavior are under circadian regulation. To identify clock-controlled output genes, an oligonucleotide-based high-density array was used that interrogates gene expression changes on a whole genome level. Genes regulating various physiological processes were found to be under circadian transcriptional regulation, ranging from protein stability and degradation, signal transduction, heme metabolism, detoxification, and immunity. By comparing rhythmically expressed genes in the fly head and body, it was found that the clock has adapted its output functions to the needs of each particular tissue, implying that tissue-specific regulation is superimposed on clock control of gene expression. Finally, taking full advantage of the fly as a model system, a cycling potassium channel protein has been identified as a key step in linking the transcriptional feedback loop to rhythmic locomotor behavior (Ceriani, 2002).

The availability of a more complete description of clock-controlled genes enabled the selection of several candidates for the control of locomotor behavior. One of these candidates was Slowpoke binding protein (Slob), which binds to the Ca²⁺-dependent voltage-gated potassium channel Slowpoke (Slo). A mutation in the gene coding for this channel causes behavioral defects and an altered mating song, also a hallmark of certain clock components. slowpoke participates in the repolarization of the action potential in flight muscles and in motoneurons. Slob has been shown to modulate Slo activity per se, and through the formation of a complex with the zeta isoform of 14-3-3 protein that acts downstream in several signaling pathways (Ceriani, 2002).

slob mRNA cycles robustly in fly heads in LD and DD. This pattern was lost in the y w;;Clk^jrk mutant background. Although slo was not detected as cycling by COSOPT because of its low level of expression, it was noticed that slo appears to cycle in phase with slob in both LD and DD. The cycling of slo was investigated by RT-PCR analysis, and the protein was shown to cycle and peak at ZT20 by Western blot. The slo spatial expression pattern has been studied extensively; slo mRNA is widely expressed in the adult brain. Furthermore, Slo protein has been localized both to neuronal cell bodies as well as to the neuronal projections (Ceriani, 2002).

Prompted by the speculation that Slo might be involved in circadian control of activity, the locomotor activity was examined in two slo mutants, slo I and slo 4. Wild-type flies show increased locomotor activity near dawn and dusk and remain quiescent the rest of the day. These bursts of activity do not merely follow the next temporal transition, but instead anticipate it. per^o and Clk^jrk mutants, which have defects in core clock components, behave differently from wild-type under entrained conditions. Although per^o flies still look mostly rhythmic in LD, Clk^jrk is often not. This apparent rhythmicity in per^o flies is caused by the so-called 'startle effect,' an immediate behavioral response to the light/dark transitions. Most of the slo 4 mutant flies display weak rhythms (defined as lacking a consolidated peak in the periodogram analysis) or no rhythms at all in LD. As expected, the lack of rhythmicity persists under free-running conditions. Surprisingly, this arrythmicity is comparable to, if not worse than, the one displayed by Clk^jrk (Ceriani, 2002).

slo I mutants, in contrast, display a milder phenotype, with only 40%-55% of rhythmic flies in LD and DD, respectively, which is commensurate with a hypomorphic slo mutation (as opposed to a true null, as is the case for slo 4). Given the nature of the slo 4 mutation and the difference in the strength of the phenotype observed between slo I and slo 4 mutants, slo 4/slo I trans-heterozygotes were tested to rule out the possibility that other loci (also affected by the chromosomal inversion) could be contributing to the observed phenotype. The slo 4/slo I mutants show a somewhat intermediate phenotype (especially obvious in DD) between that of slo 4 and slo I. A small number of slo4 heterozygotes (slo 4/+) were tested; most were either strongly or weakly rhythmic. No arrhythmic flies were found. This argues against an effect exerted by the other putative loci (Ceriani, 2002).

To determine whether this mutation causes a general decrease in motility, which by itself could result in arrhythmicity, the total locomotor activity displayed by the different genotypes under LD and DD conditions was quantified. Although wild-type flies appear to be slightly more active under constant darkness, both slo mutants are impervious to the lighting regimen. More importantly, the overall levels of activity are not different from those of the wild-type flies. The actograms of wild-type, slo 4, and slo I mutant flies were superimposed because the average activity plots are known to reveal features not apparent when individual flies are inspected. This analysis revealed that the most striking difference is the impaired anticipation of the transitions in the slo 4 (null) mutant flies, indicating that the temporal gating that consolidates behavior around dawn and dusk is absent in flies lacking slo function (Ceriani, 2002).

Microarray experiments are extremely powerful in their scope and should be taken as a starting point to delve into the specifics of different aspects of physiology that appear to be under control of the clock. Several genes were identified potentially linked to behavior. Follow-up of one of them, slo, implicates it as a central regulator of locomotor activity, because a null mutation (slo 4) in this locus results in behavioral arrhymicity without a major change in total activity levels. Several scenarios could account for these observations. A mutation in slo could cause arrhythmicity if it directly affects the output pathway controlling behavior by affecting the excitability of the neurons that control it, although if such were the case, hyperkinetic or hypokinetic flies would be expected. Alternatively, the mutation could act at the level of the pacemaker neurons by reducing the synchronous firing between the lateral neurons, which would also cause the observed lack of behavioral rhythmicity. slowpoke could also be 'gating' fly locomotor activity that would be regulated by additional unidentified components. The observation that slo 4 mutants lack the consolidation of behavior around dawn and dusk clearly favors this hypothesis, although additional work will be required to rule out other plausible scenarios, such as its involvement in the light input pathway that conveys environmental information to the clock or the core oscillator itself (Ceriani, 2002).

The notion that a potassium channel is involved in the generation of rhythmic activity was proposed a number of years ago after the analysis of membrane conductance changes in isolated retinal neurons of the mollusk Bulla. This observation, together with the finding that potassium currents are under circadian regulation in the mouse and that expression cycles in Kcnma1, the slowpoke mouse ortholog (Panda, 2002) strongly suggests that this mechanism of control of rhythmic activity could play a role in more complex organisms as well (Ceriani, 2002).

Sex- and clock-controlled expression of the neuropeptide F gene in Drosophila

Drosophila neuropeptide F (NPF), a homolog of vertebrate neuropeptide Y, functions in feeding and coordination of behavioral changes in larvae and in modulation of alcohol sensitivity in adults, suggesting diverse roles for this peptide. To gain more insight into adult-specific NPF neuronal functions, how npf expression is regulated in the adult brain was studied. npf expression is regulated in both sex-nonspecific and male-specific manners. The data show that male-specific npf (ms-npf) expression is controlled by the transformer (tra)-dependent sex-determination pathway. Furthermore, fruitless, one of the major genes functioning downstream of tra, is apparently an upstream regulator of ms-npf transcription. Males lacking ms-npf expression (through tra^F-mediated feminization) or npf-ablated male flies display significantly reduced male courtship activity, suggesting that one function of ms-npf neurons is to modulate fruitless-regulated sexual behavior. Interestingly, one of the ms-npf neuronal groups belongs to the previously defined clock-controlling dorsolateral neurons. Such ms-npf expression in the dorsolateral neurons is absent in arrhythmic Clock^Jrk and cycle⁰² mutants, suggesting that npf is under dual regulation by circadian and sex-determining factors. Based on these data, it is proposed that NPF also plays a role in clock-controlled sexual dimorphism in adult Drosophila (Lee, 2006).

Sexual dimorphism of brain structure and function generates differential neural circuitries, ultimately leading to the production of gender-specific behaviors. In Drosophila, fru is an essential neural sex determinant responsible for male courtship behavior. Because fru-encoded FRU^M protein is a BTB-Zn-finger transcription factor, FRU^M likely regulates expression of an array of genes to establish neural substrates controlling male behavior. However, such downstream targets of the FRU^M are poorly known, hampering understanding of the molecular mechanisms underlying fru-controlled establishment of male-specific neural circuitry. Intriguingly, these studies identified npf as an at least indirect target of the FRU^M, suggesting that NPF is a neurochemical factor mediating FRU^M functions (Lee, 2006).

Recent studies on the expression of sex-specific fru transcripts and of reporter expression driven by fru-gal4 suggest that fru acts in establishing sexually dimorphic anatomical differences and in rendering male-specific functions to neurons that are commonly present in both. Thus, one important question is whether ms-npf is due to the lack of corresponding neurons in the female brains or to cell-specific transcriptional activation of npf by FRU^M in males. The data support the argument that the latter is the case, at least for L1-s neurons, because a comparable number of such neurons independently marked by anti-TIM was observed in both males and females, and because FRU^M is persistently present in well differentiated ms-npf neurons. Therefore, it is suggested that one way of masculizing neurons directed by fru is to establish sex-specific production of neurosignaling molecules, which are likely to deliver male-specific neuronal functions. In line with this suggestion, it is notable that male-specific serotonin production in a group of eight neurons in the abdominal ganglion is also controlled by fru, and such serotonergic neurons innervate male reproductive organs to control appropriate male mating activities. These data indicate that ms-NPF is another neurosignaling molecule for fru-controlled male courtship. Although the neuronal targets of ms-npf are unknown, prolonged courtship-initiation latencies and general attenuation of courtship activities caused by the absence of ms-npf suggest that ms-NPF is involved in the central processing of courtship-activating stimuli or in an 'output pathway' that mediates courtship actions (Lee, 2006).

Sexually dimorphic NPY expression has been described in the rat hypothalamus. Large populations of hypothalamic NPY mRNA-producing cells are localized within the arcuate nucleus. Interestingly, the caudal region of the arcuate nucleus contains significantly more NPY cells in males than in females. Further studies suggested that the male gonadal hormone testosterone is a positive regulator of male-biased NPY expression. Similar sexually dimorphic NPY/NPF expression in the brains of distantly related species suggests that these neuropeptides play conserved roles associated with male-specific CNS functions that underlie sexual behavior (Lee, 2006).

Dual regulation of npf by sex and clock factors within a subset of male LN_d neurons suggests that NPF is associated with clock-controlled sexually dimorphic behavioral performance. In light/dark cycles, the circadian timing system directs bimodal daily peaks of locomotion, occurring at lights-on (morning) and at lights-off (evening) transitions in WT flies. Interestingly, a distinct sexual difference was observed in the peak of the morning locomotion, which occurs ~1 h earlier in males than in females. However, this male-specific phase of the morning activity was unaffected by npf-ablation, suggesting that npf is not associated with this aspect of sexual dimorphism (Lee, 2006).

The brain-behavioral system is also capable of anticipating photic transitions, as demonstrated by the gradual increase in activity levels before lights-on or lights-off. Among six groups of clock neurons defined in the Drosophila adult brain, clock-relevant functions are relatively well studied for the s-LN_vs and the LN_ds. In the former cell type, clock-controlled pigment-dispersing factor production is essential for circadian locomotor activity rhythms as well as lights-on anticipation, whereas the LN_ds are required for anticipation of lights-off. The data implicate NPF as a neuromodulatory substance within a subset of the LN_ds, involved in this 'late-day' component of the locomotor cycle in males (Lee, 2006).

Although the biological meaning of LN_d-regulated lights-off anticipation remains unknown, it is notable that the flies’ increasing locomotion at dusk is temporally coincident with especially vigorous mating activities of fruit flies. This finding, then, raises the possibility that anticipatory activity at dusk is causally connected with maximum mating propensity. In this respect, that there are sex- and clock-controlled npf expressions in the LN_ds provides the first glimpse of this neuropeptide as a putative output factor, which would participate in certain aspects of the clock-controlled reproductive behavior. These actions are intimately connected to evolutionary fitness, which may be one reason for the circadian system of Drosophila to have evolved and been refined (Lee, 2006).

Circadian regulation in the ability of Drosophila to combat pathogenic infections

This study sought to determine if the innate immune response is under circadian regulation and whether this impacts overall health status. To this end, infection of Drosophila with the human opportunistic pathogenic bacteria Pseudomonas aeruginosa was used as a model system. The survival rates of wild-type flies vary as a function of when, during the day, they are infected, peaking in the middle of the night. Although this rhythm is abolished in clock mutant flies, those with an inactive period gene are highly susceptible to infection, whereas mutants with impairment in other core clock genes exhibit enhanced survival. After an initial phase of strong suppression, the kinetics of bacterial growth correlate highly with time of day and clock mutant effects on survival. Expression profiling revealed that nighttime infection leads to a clock-regulated transient burst in the expression of a limited number of innate immunity genes. Circadian modulation of survival also was observed with another pathogen, Staphylococcus aureus. These findings suggest that medical intervention strategies incorporating chronobiological considerations could enhance the innate immune response, boosting the efficacy of combating pathogenic infections (Lee, 2008).

Together, these findings suggest the following scenario for how the clock in Drosophila might influence the progression of an infection with P. aeruginosa. Early during the infection a robust immune response (perhaps both cellular and humoral) is mounted, which is effective in pathogen clearance irrespective of when during a daily cycle the flies are infected, as indicated by the rapid drop in bacterial titer during the first 5 hr postinfection. However, the clock regulates the induced levels of a limited subset of innate immunity players, such as PGRP-SA and drc, whereby infections in the middle of the night result in a transient burst early during the infection. Higher levels of a few key immune players over a certain threshold may contribute to keeping the titer of pathogenic bacteria low after the initial rapid-declining phase. By prolonging the suppression of bacterial growth during a critical window of the infection, this might provide an opportunity to mount or recruit additional host defenses in addition to AMPs, resulting in improved survival. Thus, the results suggest that the clock modulates the strength or responsiveness of immune activation in a time-of-day-dependent manner but only during a critical early phase of the infection process that has physiological consequences on the ability of the host to survive pathogenic infections. Indeed, it is noteworthy that P. aeruginosa eludes host defenses by the early suppression of antimicrobial peptide gene expression. It will be of interest to determine why the postinfection expression profiles of only certain immune response genes exhibit circadian regulation and how this is apparently restricted to a particular phase of the immune response (Lee, 2008).

Although the time-of-day differences in the levels of induced peptidoglycan recognition protein PGRP-SA and drosocin (drc) are clearly consistent with the survival rates of wild-type flies infected at different times of day, this is not the case for the per⁰¹ and Clk^Jrk mutants in which the overall levels of drc are much lower in Clk^Jrk compared to per⁰¹ flies; a trend observed for other AMP genes surveyed. Although seemingly paradoxical, this is not unanticipated as there are precedents in the literature showing that flies can be more susceptible to bacterial infection despite elevated levels of AMP expression, indicating that excessive or inappropriate immune activation can be deleterious. In this context it is important to consider that besides the production of AMPs, innate immunity in adult Drosophila includes a proteolytic cascade leading to melanization and a cellular immune response characterized by phagocytosis. It is possible that inactivation of per might affect other host defense mechanisms that cannot be compensated by a potentially hypersensitive humoral immune response. Conversely, Clk^Jrk and other clock mutant flies might have a heightened activity of cellular immunity. Presently, the results, which are based on probing the expression profiles of several immune response genes, would seem to demand that the molecular mechanisms governing the time-of-day differences in survival for flies with functional clocks are different from those affecting survival rates in per⁰¹ and Clk^Jrk flies. Otherwise stated, it does not appear likely that the survival rates of per⁰¹ and Clk^Jrk flies are due simply to their clocks being pegged or held at a phase that is similar to either ZT/CT5 or ZT/CT17 in wild-type flies, respectively. Although future work will be required to resolve the molecular underpinnings governing the differential clock mutant effects on survival, these considerations suggest that core clock genes have 'non-circadian' related roles (i.e., roles not solely limited to their functions in timekeeping) in fighting microbial infections. Indeed, these findings add to a growing list of physiological and behavioral pathways that are differentially regulated in different clock mutants; e.g., mutations in per but not tim, Clk, or cyc play a key role in long-term memory formation in Drosophila (Lee, 2008).

If the ability to evoke a stronger response at night enhances the efficacy of fighting a microbial infection, why restrict it to the night? It is widely thought that maintaining an optimal immune system is metabolically costly, competing for limited metabolic resources with other energetically demanding activities such as foraging or mating. Within this framework it is suggested that the clock might function as a temporal sieve to ensure the proper allocation of metabolic resources at biologically desirable times. From a more medical perspective, the results suggest that the innate immune system is a prime target for interventions based on chronobiological considerations in the hopes of boosting the ability to combat pathogenic infections (Lee, 2008).

Circadian-clock-dependent rhythms in GPRK2 abundance control the rhythmic accumulation of ORs in OSN dendrites, which in turn control rhythms in olfactory responses

The Drosophila circadian clock controls rhythms in the amplitude of odor-induced electrophysiological responses that peak during the middle of night. These rhythms are dependent on clocks in olfactory sensory neurons (OSNs), suggesting that odorant receptors (ORs) or OR-dependent processes are under clock control. Because responses to odors are initiated by heteromeric OR complexes that form odor-gated and cyclic-nucleotide-activated cation channels, whether regulators of ORs were under circadian-clock control was tested. The levels of G protein-coupled receptor kinase 2 (Gprk2) messenger RNA and protein cycle in a circadian-clock-dependent manner with a peak around the middle of the night in antennae. Gprk2 overexpression in OSNs from wild-type or cyc⁰¹ flies elicits constant high-amplitude electroantennogram (EAG) responses to ethyl acetate, whereas Gprk2 mutants produce constant low-amplitude EAG responses. ORs accumulate to high levels in the dendrites of OSNs around the middle of the night, and this dendritic localization of ORs is enhanced by GPRK2 overexpression at times when ORs are primarily localized in the cell body. These results support a model in which circadian-clock-dependent rhythms in GPRK2 abundance control the rhythmic accumulation of ORs in OSN dendrites, which in turn control rhythms in olfactory responses. The enhancement of OR function by GPRK2 contrasts with the traditional role of GPRKs in desensitizing activated receptors and suggests that GPRK2 functions through a fundamentally different mechanism to modulate OR activity (Tanoue, 2008).

Many sensory systems are regulated by the circadian clock. Various insects including flies, moths, and cockroaches show circadian rhythms in odor-dependent electrophysiological and behavioral responses. In mammals, the firing rate of isolated mouse olfactory bulb neurons is regulated by the circadian clock, as are odor-evoked brain activity waves (e.g., event-related potentials [ERPs]) in humans. Daily rhythms in neuronal activity or sensitivity have been reported for other sensory systems, such as the visual and auditory systems (Tanoue, 2008).

The circadian clock modulates olfactory responses in Drosophila: Robust electroantennogram (EAG) responses are seen during the middle of the night, and weak EAG responses are seen during the middle of the day (Krishnan, 1999). These rhythms in EAG responses are controlled by the olfactory sensory neurons (OSNs) in Drosophila, which act as independent peripheral circadian oscillators (Tanoue, 2004). Colocalization of the circadian oscillator and a rhythmic output to the OSNs indicates that the abundance and/or activity of odorant receptors (ORs) and/or OR-dependent processes are under clock control. Drosophila ORs are seven-transmembrane-domain proteins that share some structural similarities with G protein-coupled receptors (GPCRs). However, recent studies demonstrate that Drosophila ORs have an inverted membrane topology compared to canonical GPCRs and function as odor-gated and cyclic-nucleotide-activated cation channels. To understand how the clock modulates odor-dependent responses, it was determined whether factors that modulate ORs were regulated by the circadian clock (Tanoue, 2008).

G protein-coupled receptor kinases (GPRKs) and arrestins act to terminate GPCR signaling in mammals, thereby protecting cells from receptor overstimulation. GPRK-phosophorylated GPCRs are internalized by arrestin and subsequently degraded or recycled. Two Gprk genes, Gprk1 and Gprk2, have been reported in Drosophila. Gprk1 messenger RNA (mRNA) is enriched in photoreceptor cells, and expression of a Gprk1 dominant-negative mutant in photoreceptors increases the amplitude of electroretinogram (ERG) responses (Lee, 2004). Gprk2 is required for egg and wing morphogenesis, as well as embryogenesis in Drosophila (Molnar, 2007; Schneider, 1997). In mammals, seven Gprk genes are divided into three subfamilies on the basis of sequence homology: the rhodopsin kinase or visual Gprk subfamily (Gprk1 and Gprk7), the β-adrenergic receptor kinase subfamily (Gprk2 and Gprk3), and the Gprk4 subfamily (Gprk4, Gprk5, and Gprk6). Gprk3 knockout mice are unable to mediate odor-induced desensitization of odorant receptors (Peppel, 1997). In contrast, loss of Gprk2 function in C. elegans olfactory sensory neurons results in reduced chemosensory behavior, suggesting that Gprk2 is necessary for GPCR signaling (Fukuto, 2004). These results suggest that GPRKs play different roles in vertebrate and invertebrate olfaction (Tanoue, 2008).

This study reports that Gprk2 expression is regulated by circadian clocks in antennae and that GPRK2 drives circadian rhythms in olfactory responses by enhancing OR accumulation in the dendrites of basiconic sensilla. Gprk2 mRNA and protein expression levels were high around the middle of the night, coincident with the peak of olfactory responses. Flies that overexpress Gprk2 in OSNs show constant high EAG responses to ethyl acetate during 12 hr light:12 hr dark (LD) cycles and accumulate high levels of ORs in OSN dendrites, whereas hypomorphic Gprk2 mutants show constant low EAG responses to ethyl acetate during LD. On the basis of these results, it is proposed that GPRK2 mediates cycles of OR accumulation in OSN dendrites to generate rhythms in EAG responses (Tanoue, 2008).

Gprk2 is a circadian output gene whose mRNA and protein peak during the middle of the night in antennae. This phase of mRNA expression is similar to that of per, tim, and other genes driven directly by CLK-CYC binding to E-box regulatory sequences. However, CLK-CYC-dependent genes are expressed at constitutively high levels in per⁰¹ and tim⁰¹ mutants and constitutively low levels in Clk^Jrk and cyc⁰¹ mutants, whereas Gprk2 is expressed at low levels in per⁰¹, tim⁰¹, and cyc⁰¹ mutants. Several rhythmically expressed transcripts identified by microarray analysis of heads have low levels of expression in per⁰¹ and Clk^Jrk mutants, but the mechanism governing their rhythmic expression has not been explored (Tanoue, 2008).

Analysis of arrhythmic clock mutants indicates that cycling levels of Gprk2 mRNA give rise to rhythms in GPRK2 protein. The levels of GPRK2 protein cycle in phase with Gprk2 mRNA and correspond to rhythms in EAG responses. Other kinases such as Double-time (Dbt), Shaggy (Sgg)/GSK3, and Casein kinase 2 (CK2) in Drosophila are constitutively expressed proteins, whereas GPRK2 is the first example of a rhythmically expressed kinase. However, other kinases such as Erk-MAP kinase and Calcium/calmodulin-dependent protein kinase II in the chicken retina are rhythmically activated due to phosphorylation and control cGMP-gated ion channels in cone photoreceptors (Tanoue, 2008).

Cycling levels of GPRK2 are coincident with rhythms in EAG responses; GPRK2 levels and EAG responses peak around the middle of the night and are at their lowest levels during the middle of the day. When GPRK2 levels are constitutively low, as in per⁰¹, tim⁰¹, and cyc⁰¹ mutants, EAG responses are also low. In addition, levels of GPRK2 are at or below the normal wild-type trough in Gprk2⁶⁹³⁶ and Gprk2^EY09213 mutants and generate EAG responses that are at or below those at the wild-type trough. GPRK2 levels are barely detectable in Gprk2^pj1 antennae. These results suggest that Gprk2^pj1 is not a null allele and raise the possibility that a Gprk2 null mutant will lack EAG responses altogether. Such a result would demonstrate that Gprk2 is required for olfactory responses per se. In contrast, constitutive overexpression of Gprk2 produces constant high EAG responses in both wild-type and cyc⁰¹ flies, demonstrating that high levels of GPRK2 can effect high amplitude EAG responses independent of other clock-dependent factors. Taken together, these results argue that Gprk2 levels control the amplitude of EAG responses. If so, this would imply that low levels of GPRK2 present in the Gprk2⁶⁹³⁶ mutant do not cycle in abundance (Tanoue, 2008).

Given that GPRK2 levels regulate the amplitude of EAG responses, what is the mechanism through which GPRK2 controls EAG response amplitude? The traditional targets of GPRKs are GPCRs. In the mammalian olfactory system, GPRK3 desensitizes ORs by triggering their internalization. These results suggest that Drosophila Gprk2 is necessary for EAG responses, and, taken together with C. elegans Gprk2 function, they indicate that GPRKs play a different role in invertebrate olfaction than in vertebrate olfaction. The subcellular localization of ORs is high in dendrites of basiconic sensilla at ZT17 and low at ZT5, but the abundance of ORs in these dendrites at ZT5 can be driven to high levels by increasing GPRK2 expression. These results support a model in which the circadian clock generates a rhythm of Gprk2 expression, which in turn generates rhythms in the amplitude of EAG responses by promoting OR accumulation (and consequently odor-gated cation-channel formation) in OSN dendrites from basiconic sensillae. GPRK2-dependent rhythms in the amplitude of spontaneous spikes are also seen in OSNs, thus demonstrating that the clock controls basic (i.e., odor-independent) properties of the OSN membrane. It is possible that the rhythmic localization of odor-gated cation channels to OSN dendrites accounts for rhythms in the amplitude of spontaneous spikes. The results can't exclude the possibility that cyclic expression of other genes also contribute to rhythms in EAG responses. mRNA cycling was tested for several genes that could potentially modulate EAG responses, including arrestin 2, Gprk1, and kurtz arrestin; arrestin 2 mRNA levels cycle, but neither Gprk1 or kurtz arrestin mRNA levels cycle. Given that microarray analysis was done on fly heads depleted of antennae, microarray analysis of antennae may reveal other rhythmically expressed genes that contribute to EAG rhythms (Tanoue, 2008).

Myc-tagged ORs did not accumulate to high levels in the dendrites of trichoid sensilla at ZT17. Trichoid sensilla have different functions than basiconic sensilla; T1 trichoid sensilla detect the pheromone 11-cis-vaccenyl acetate (cVA), whereas the basiconic sensilla recognize food and plant odors. It could be that the circadian clock regulates OSN activity differently in basiconic sensilla and trichoid sensilla, although the possibility cannot be excluded that detection of Myc-tagged ORs in dendrites failed because of low expression levels in trichoid sensillae, poor permeability of Myc antibody into trichoid sensilla, or the long, thin geometry of trichoid sensillae (Tanoue, 2008).

In summary, Drosophila Gprk2 mRNA and protein expression is under clock control in antennae. The levels of GPRK2 protein determine the amplitude of EAG responses to ethyl acetate in basiconic sensillae; high levels generate high-amplitude EAGs, and low levels produce low-amplitude EAGs. This result suggests that GPRK2 directly or indirectly enhances OR activity, in contrast to the inhibition of olfactory signaling by Gprk3 in mammals. Given that the most severe Drosophila Gprk2 mutant still produces low-amplitude EAG responses, a complete loss of Gprk2 function may lack EAG responses altogether and be required for olfaction. High levels of GPRK2 enhance OR localization to dendrites of basiconic sensillae and support a model in which rhythms in GPRK2 levels drive rhythms in OR localization to dendrites that ultimately mediates rhythms in EAG responses (Tanoue, 2008).

Adult circadian behavior in Drosophila requires developmental expression of cycle, but not period

Circadian clocks have evolved as internal time keeping mechanisms that allow anticipation of daily environmental changes and organization of a daily program of physiological and behavioral rhythms. To better examine the mechanisms underlying circadian clocks in animals and to ask whether clock gene expression and function during development affected subsequent daily time keeping in the adult, the genetic tools available in Drosophila were used to conditionally manipulate the function of the Cycle component of the positive regulator Clock/Cycle (Clk/Cyc) or its negative feedback inhibitor Period (Per). Differential manipulation of clock function during development and in adulthood indicated that there is no developmental requirement for either a running clock mechanism or expression of per. However, conditional suppression of Clk/Cyc activity either via per over-expression or cyc depletion during metamorphosis resulted in persistent arrhythmic behavior in the adult. Two distinct mechanisms were identified that may contribute to this developmental function of Clk/Cyc and both involve the ventral lateral clock neurons (LN_vs) that are crucial to circadian control of locomotor behavior: (1) selective depletion of cyc expression in the LN_vs resulted in abnormal peptidergic small-LN_v dorsal projections, and (2) Per expression rhythms in the adult LN_vs appeared to be affected by developmental inhibition of Clk/Cyc activity. Given the conservation of clock genes and circuits among animals, this study provides a rationale for investigating a possible similar developmental role of the homologous mammalian CLOCK/BMAL1 complex (Goda, 2011).

This study created transgenic flies with conditional clock function, in which expression of the essential clock components Cyc and Per was induced or repressed in relevant spatiotemporal patterns. In per⁰¹ [timP>per]^ts flies, which conditionally rescue the per⁰¹ mutation, clock function was conditional and readily reversible. Moreover, adult circadian behavior was restored in flies raised under restrictive conditions. In earlier studies conducted by Ewer widespread transgenic expression of per under control of a heat-shock protein 70 (hsp70) promoter was shown to partially rescue the per⁰¹ mutation resulting in restoration of behavioral rhythms at an abnormally long period length. These long period rhythms could be generated in a conditional manner even when induction was restricted to the adult phase(Ewer, 1999). the present study targeted expression of transgenic per specifically to clock-bearing cells and achieved a more complete conditional rescue of the per⁰¹ phenotype that did not require developmental per expression (Goda, 2011).

Although circadian behavior of per⁰¹ [timP>per]^ts flies at 25°C showed rhythmicity comparable to that observed for wild-type flies, period lengths were at least 2 h longer than those of wild-type flies and molecular rhythms showed a relatively low amplitude. One key difference between the molecular clock circuits in per⁰¹ [timP>per]^ts at the permissive temperature and those of wild-type flies is the constitutively high level of per mRNA expression in the transgenically rescued flies, which could contribute to the increased circadian period length and blunted molecular rhythms in per⁰¹ [timP>per]^ts flies. Wild-type flies exhibit a trough in per transcript levels in the early morning that may facilitate subsequent down-regulation of Per protein levels and optimal induction of CLK/Cyc-regulated. The lack of a trough in per mRNA expression in the conditionally rescued flies could account for a delay in the turnover of Per protein in the morning and, therefore, a lengthened period and blunted CLK/Cyc activity. This hypothesis also explains apparent discrepancies with previous reports, in which increased per gene dosage was associated with a shortened circadian period length and decreased per dosage or expression resulted in longer circadian period lengths. As long as per expression shows strong circadian regulation the timing of Per nuclear entry and Per-mediated transcriptional repression is predicted to be advanced by the introduction of one or two additional copies of the wild-type per gene and delayed by a reduction in per dosage, while neither manipulation is predicted to strongly affect subsequent Per turnover (Goda, 2011).

Adult circadian behavior was also conditional and reversible in [timP>per]^ts flies, which exhibit temperature-dependent over-expression of per. However, developmental over-expression of per during metamorphosis was associated with irreversible behavioral arrhythmia in adults. Likewise, depletion of cyc expression during the metamorphosis in cyc⁰¹ [elav>cyc]^ts flies resulted in disruption of adult circadian locomotor behavior under permissive conditions. Both increased levels of Per and decreased levels of Cyc negatively regulate CLK/Cyc activity. The Clk/Cyc heterodimer functions as the central transcriptional regulator in the Drosophila clock and its activity critically depends on the presence of both Clk and Cyc. Loss of functional cyc expression in the cyc⁰¹ mutant results in both molecular and circadian arrhythmia and constitutively low expression levels for Clk/Cyc-regulated target genes, whereas Per acts as a negative regulator of Clk/Cyc activity by binding and inactivating the Clk/Cyc complex. The arrhythmic locomotor behavior and molecular arrhythmia in the clock neurons observed as a result of per over-expression are, therefore, interpreted to result from constitutive inhibition of Clk/Cyc (Goda, 2011).

Adult behavioral arrhythmia in [timP>per]^ts or cyc⁰¹ [elav>cyc]^ts flies raised under permissive conditions was reversible. However, exposures to restrictive conditions of comparable duration resulted in long-term after-effects only when they occurred during development and, particularly, during the pupal and pharate adult stages. Therefore the effects of circadian arrests during development in [timP>per]^ts or cyc⁰¹ [elav>cyc]^ts flies on adult circadian behavior are attributed to a developmental requirement for Clk/Cyc function beyond its immediate role in maintaining daily time keeping. The requirement for Clk/Cyc activity, but not clock function per se may indicate that one or more transcriptional Clk/Cyc targets play a role in enabling adult circadian locomotor behavior. Such targets would likely be expressed constitutively along with other Clk/Cyc-regulated genes in conditionally arrested per⁰¹ [timP>per]^ts flies, but constitutively down-regulated in circadian arrests due to low Clk/Cyc activity (Goda, 2011).

A central question that remains is what mechanism links developmental Clk/Cyc activity to adult circadian behavior. The experiments indicate that both clock neuron anatomy and the molecular oscillator itself may be involved. Previously published studies of constitutively arrhythmic alleles of the Clk and cyc genes have documented a reduction in PDF expression as well as neuro-anatomical defects in the LN_vs that could be associated with a developmental role for the Clk/Cyc transcription factor. By selectively blocking transgenic rescue of cyc⁰¹ in the PDF-expressing clock neurons this study shows that the reduction of PDF expression and PDF-positive dorsal projections from the s-LN_vs is a cell-type specific phenotype. PDF is known to play an important role in mediating clock-controlled behavior in both LD and DD conditions. The PDF-producing s-LN_vs project towards the dorsal protocerebrum as do DN1, DN2, DN3, and LN_d clock neurons, suggesting that the dorsal s-LN_v projections may play an important part in signaling across the neural clock circuits. In this context, it may be relevant that expression of the PDF Receptor in 'E' cells, a subset of clock neurons including DN1s and LN_ds, has been associated with circadian control of locomotor activity. Moreover, the axonal terminals of the dorsal s-LNv projections undergo clock-controlled rhythms in remodeling that may play a role in circadian signaling. Nevertheless, the observed developmental requirement for Clk/Cyc activity also appears to involve mechanisms other than PDF-mediated signaling for the following reasons. First, developmental over-expression of Per resulted in persistent adult arrhythmia, but did not lead to a loss of PDF-positive dorsal projections from the s-LN_vs. Second, while developmental suppression of Clk/Cyc activity uniformly affected the behavior of adult flies constitutive depletion of Cyc from the PDF-expressing neurons resulted in a variable phenotype in the s-LN_v dorsal projections. Third, the light/dark activity pattern of cyc⁰¹ (elav-Pdf)>cyc flies was strikingly different from that of Pdf⁰¹ flies or flies from which the PDF-expressing cells have been ablated, suggesting that PDF signaling persisted in cyc-depleted LN_vs in spite of the defects in PDF-positive dorsal projections (Goda, 2011).

In principle, neuro-anatomical defects affecting intercellular connectivity rather than cell-autonomous clock function could lead to behavioral phenotypes due to asynchrony among the clock neurons or the loss of output signals. Indeed, apparent separation of molecular and behavioral phenotypes has been reported previously for genetic manipulation of Clk/Cyc function. It may be particularly relevant that rescue of the per⁰¹ phenotype in the PDF-expressing clock neurons restores rhythmic behavior, while rescue of cyc⁰¹ in the same cells restores molecular, but not behavioral rhythms. However, the experimental results also provide support for developmental phenotypes at the level of the adult molecular clock circuits. Immunofluorescence expression analyses indicated that the molecular clock circuits in the adult PDF-expressing clock neurons were affected by developmental over-expression of Per. PDF-expressing LN_vs in adults that were behaviorally arrhythmic due to developmental Per over-expression exhibited adult Per expression with an altered daily profile, but not necessarily at excessively high levels. Future studies may determine the degree to which neuro-anatomical and molecular phenotypes are linked and help determine the effect of intercellular connectivity in the neural clock circuit on the function of molecular circadian rhythms in individual clock neurons (Goda, 2011).

Dopamine acts through Cryptochrome to promote acute arousal in Drosophila

The fruit fly, Drosophila melanogaster, is generally diurnal, but a few mutant strains, such as the circadian clock mutant Clk^Jrk, have been described as nocturnal. This study reports that increased nighttime activity of Clk mutants is mediated by high levels of the circadian photoreceptor Cryptochrome (Cry) in large ventral lateral neurons (l-LN_vs). Cry expression is also required for nighttime activity in mutants that have high dopamine signaling. In fact, dopamine signaling is elevated in Clk^Jrk mutants and acts through Cry to promote the nocturnal activity of this mutant. Notably, dopamine and Cry are required for acute arousal upon sensory stimulation. Because dopamine signaling and Cry levels are typically high at night, this may explain why a chronic increase in levels of these molecules produces sustained nighttime activity. It is proposed that Cry has a distinct role in acute responses to sensory stimuli: (1) circadian responses to light, as previously reported, and (2) noncircadian effects on arousal, as shown in this study (Kumar, 2012).

Both dopamine and Cry are required for acute arousal at night. An arousal-promoting role for dopamine is supported by earlier studies. Dopamine transporter mutants were shown to exhibit a decreased arousal threshold, whereas the pale mutants exhibit an increased arousal threshold. This effect on arousal reflects a novel role for dopamine in sensory responses at night. Cry has not been implicated in arousal, although it promotes neural activity in a light-dependent manner. As in the case of the neural activity assay, this study found that arousal in response to sensory stimuli is reduced but not eliminated by the cryb mutant, indicating that the mechanism is distinct from the circadian response that is eliminated by cryb. Both neural activity and behavioral arousal responses are eliminated by the cry0 mutant, suggesting that the neural response underlies the behavioral effect. It is proposed that Cry is required at multiple levels for acute responses to sensory stimuli. In the case of circadian photoreception, it is absolutely required for phase-shifting in response to pulses of light, although not for entrainment to LD cycles. In the case of responses to sensory stimuli, again it is required for the startle response. Any effects of Cry on light-induced activity (physiological or behavioral) are likely to be acute, since Cry gets degraded with increased light treatment. Interestingly, in two different species of Bactrocera, cry mRNA levels are positively correlated with the timing of mating, which is also indicative of a regulated response required for a specific purpose. A chronic effect is seen only in the case of Drosophila Clk mutants, where levels of Cry are considerably higher than normal, and dopamine signaling is also elevated. It is hypothesized that Cry only promotes nocturnal activity in flies with chronically elevated dopamine signaling because dopamine acts as a trigger to activate Cry. However, this activation may be different from activation in a circadian context, given that different mechanisms appear to underlie the circadian and arousal-promoting roles of Cry. Dopamine- and Cry-mediated locomotor activity is restricted largely to the night because of light-induced Cry degradation and light-induced inhibition of dopamine signaling (Kumar, 2012).

At night, animals sleep, and the arousal threshold is increased. However, they still need to be able to respond in case of sudden events. It is speculated that dopamine and Cry are essential for this. In the case of Cry, it may arouse the animal and also reset the clock. For instance, the immediate response of an animal to a pulse of light at night is to wake up, which may be driven by the arousal-promoting role of Cry. In addition, the circadian clock must be reset, which requires the circadian function of Cry. Whether or not these roles of Cry are conserved, it is speculated that dopamine functions similarly in mammals. Interestingly, melanopsin, which is the circadian photoreceptor in mammals (analogous to Cry in flies), is regulated by dopamine in intrinsically photosensitive retinal ganglion cells (ipRGCs). Like Cry, melanopsin is also required for acute behavioral responses to light, specifically for sleep induction in nocturnal animals during the day. These ipRGCs have been proposed as functionally similar to l-LNvs, so a conserved function for the relevant molecules is intriguing. Finally, it is noted that elevated dopamine has been linked to increased nighttime activity in humans, which are, of course, diurnal like Drosophila. People with Sundown syndrome or nocturnal delirium show increased agitation and sleep disturbances in the early evening, which can be treated with anti-psychotic medications that target dopamine signaling (Kumar, 2012).

Role for circadian clock genes in seasonal timing: testing the bunning hypothesis

A major question in chronobiology focuses around the 'Bunning hypothesis' which implicates the circadian clock in photoperiodic (day-length) measurement and is supported in some systems (e.g. plants) but disputed in others. This study used the seasonally-regulated thermotolerance of Drosophila melanogaster to test the role of various clock genes in day-length measurement. In Drosophila, freezing temperatures induce reversible chill coma, a narcosis-like state. Previous observations were have corroborated that wild-type flies developing under short photoperiods (winter-like) exhibit significantly shorter chill-coma recovery times (CCRt) than flies that were raised under long (summer-like) photoperiods. Arrhythmic mutant strains, per⁰¹, tim⁰¹ and Clk^Jrk, as well as variants that speed up or slow down the circadian period, disrupt the photoperiodic component of CCRt. The results support an underlying circadian function mediating seasonal daylength measurement and indicate that clock genes are tightly involved in photo- and thermo-periodic measurements (Pegoraro, 2014: PubMed).

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