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).

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).

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