Spatio-temporal pattern of cells expressing the clock genes period and timeless and the lineages of period expressing neurons in the embryonic CNS of Drosophila

The initial steps towards the generation of cell diversity in the central nervous system of Drosophila take place during early phases of embryonic development when a stereotypic population of neural progenitor cells (neuroblasts and midline precursors) is formed in a precise spatial and temporal pattern, and subsequently expresses a particular sequence of genes. The clarification of the positional, temporal and molecular features of the individual progenitor cells in the nerve cord and brain as well as of their specific types of neuronal and/or glial progeny cells forms an essential basis to understand the mechanisms controlling their development. The present study contributes to this effort by tracing the expression of period and timeless, two genes that encode transcription factors with a key role in the molecular mechanism of the biological clock. Using a combination of genetic markers and immunocytochemistry with antibodies specific for Period and Timeless this study defines the number, location, origin and lineage of period cells in the nerve cord throughout embryogenesis. This study also provides the first description of the expression of timeless in the embryonic central nervous system. A major transformation in the number and types of cells that express period and timeless takes place between embryonic and larval life (Ruiz, 2011).

By using a combination of cell lineage tracing and molecular markers this study uncover the neuroblast or midline precursor origin of the neurons that express per in the nerve cord. In the embryonic brain three populations of cells were found regarding expression of tim and per. One of them comprises 130 cells expressing per, other comprises 160 cells expressing tim and the third is a group of about 20 cells that co-express both genes. Cells expressing either tim, or per, or both exist also in the larval brain, although in much smaller numbers, and part of them initiate expression of per or tim first after some hours of larval life. Considering their lack of expression and their number, the 20 cells that co-express per and tim in the brain at embryonic stage 16 could be the same 20 cells that co-express these genes in the larval brain and function as clock neurons. This is reinforced by their approximate distribution within the protocerebrum and the detection of oscillator features in a similar, probably identical array of per and clock expressing brain neurons at the same stage. Since synaptic activity in central neurons develops in Drosophila during the same phase of development (from late stage 16-17), it is possible that a network of clock neurons becomes functional at this time or shortly thereafter (Ruiz, 2011).

The number of per and tim-expressing cells in the brain increases again during metamorphosis and reaches values in the adult similar to those reported here for the embryo, suggesting that such a large population of cells expressing these clock genes is required twice during life. This is in accordance with the identification, in a careful study of the global profile of gene expression during the life cycle of Drosophila, of a group of genes with expression peaks late in embryogenesis and pupal life (Ruiz, 2011).

Another considerable difference between embryonic and other life stages regards the expression of per and tim in glial cells. Hundreds of glia of different types express per and/or tim in the adult but none or few in the larval brain. This study found that per is not expressed by glial cells in the embryonic CNS, indicating that either per expression is turned on later in some of these cells, or in glial cells born during postembryonic stages. On the contrary, this study found that embryonic glial cells do express tim, although at apparently lower levels than in neurons. The approximate number of cells that was found to co-express repo and tim in the embryo is smaller than the total number of repo-positive cells at this stage, defining two new subpopulations of glial cells. The correlation found in this study between weak anti-Tim fluorescence in Repo-positive cells and strong anti-Tim fluorescence in Repo-negative cells in both the brain and the nerve cord suggests a common regulation of the expression of both genes within CNS cells. In summary, these results indicate that during the few hours from late stage 16 to shortly after larval eclosion there is a significant change in the transcriptional profile of CNS cells, turning off the expression of per and tim in neurons and the expression of tim in glia or eliminating these cells through programmed cell death (Ruiz, 2011).

In the nerve cord the per and tim neurons defined in this stdy are located in close vicinity but are generated by many different NBs. In each NB lineage, only one, or very few cells turn on the expression of either tim or per. Thus, expression of these genes is not inherited by lineage, but needs to be induced in individual postmitotic progeny cells. As suggested by the occurrence of only one positive cell in some of the lineages this is also true for sibling cells derived from individual ganglion mother cells. Differential cell fate among such sibling cells may result form asymmetric division induced by Notch signaling as previously demonstrated (Ruiz, 2011).

A clear, although not complete segregation of per and tim cells was found along the CNS. Within the nerve cord some of the most posterior per cells were located in the engrailed domain (the posterior compartment of the segment) and almost all tim cells were anterior to the engrailed domain. The same relationship was found in the brain, although without the clear segmental pattern of the nerve cord (i.e. lacking correlation with the small engrailed domains of the brain). Instead, the distribution of per and tim cells in the brain can roughly be described as two large fields with abundant overlap, with the majority of the tim cells located in the more anterior field i.e. the same relative position with regards to per cells as observed in the nerve cord (Ruiz, 2011).

The best known function of Per and Tim is as transcriptional regulators acting within molecular feedback loops responsible for the timekeeping mechanism of the biological clock. Within this conceptual frame, the 'traditional' function of Per and Tim in the CNS is restricted to 'oscillator cells' or clock neurons, in which the cycling of gene expression, mRNA and protein, as well as the shuttling of Per and Tim between cytoplasm and nucleus has been documented with several methods. Thus, the embryonic expression of per and tim in cells other than the clock neurons, as the glia, interneurons and motorneurons identified in this study in the nerve cord, suggests that these proteins might have other functions, perhaps in neuronal development (Ruiz, 2011).


The circadian timekeeping system of Drosophila functions from the first larval instar (L1) onward. The phase of eclosion and of adult activity rhythms can be set by light pulses delivered to animals entering or progressing through L1. period and timeless are rhythmically expressed in several groups of neurons in the larval CNS both in light/dark cycles and in constant dark conditions. There are a total of six to eight per-expressing cells in the nervous system of L3. These cells are identified as neurons. Among the clock gene-expressing neurons there is a subset of the putative pacemaker neurons, the "lateral neurons" (LNs), that have been analyzed mainly in adult flies. Like the adult LNs, the larval ones are also immunoreactive to a peptide called pigment-dispersing hormone. Neurons not cycling Period protein are also identified and consist of three to five clusters by position. The LNs are the only larval cells that maintain a strong cycling in PER from L1 onward, throughout metamorphosis and into adulthood. The phase of Per cycling is similar to that found in the adult, with a peak at 23 hours and a trough at 6 to 12 hours. In these LNs, it is Tim (which also cycles) that is found to be both nuclear and cytoplasmic. Therefore, they are the best candidates for being pacemaker neurons responsible for the larval "time memory" (inferred from previous experiments). Putative dendritic trees of LNs are found to be in close proximity to the terminals of the larval optic nerve Bolwig's nerve, possibly receiving photic input from the larval eyes. LNs also express Pigment dispersing hormone (PDH). In addition to the LNs, a subset of the larval dorsal neurons (DNLs) expresses per and tim. These two groups of neurons, identified as "cycling" neurons expressing per are termed Dorsal neurons-1Larval and Dorsal neurons-2Larval (DNL). Intriguingly, two neurons of this DNL group cycle in PER and TIM immunoreactively, almost in antiphase to the other DNLs and to the LNs. These DNLs also express PDH. Thus, the temporal expression of per and tim are regulated differentially in different neurons. Furthermore, the light sensitivity associated with levels of the TIM protein is different from that in the heads of adult Drosophila (Kaneko, 1997).

Circadian control of eclosion: Interaction between a central and peripheral clock

Drosophila displays overt circadian rhythms in rest:activity behavior and eclosion. These rhythms have an endogenous period of approximately 24 hr and can adjust or 'entrain' to environmental inputs such as light. Circadian rhythms depend upon a functioning molecular clock that includes the core clock genes period and timeless. Although a clock in the lateral neurons (LNs) of the brain controls rest:activity rhythms, the cellular basis of eclosion rhythms is less well understood. The LN clock has been shown to be insufficient to drive eclosion rhythms. The prothoracic gland (PG), a tissue required for fly development, contains a functional clock at the time of eclosion. This clock is required for normal eclosion rhythms. However, both the PG clock function and eclosion rhythms require the presence of LNs. In addition, it is demonstrated that pigment-dispersing factor (PDF), a neuropeptide secreted from LNs, is necessary for the PG clock and eclosion rhythms. Unlike other clocks in the fly periphery, the PG is similar to mammalian peripheral oscillators because it depends upon input, including PDF, from central pacemaker cells. This is the first report of a peripheral clock necessary for a circadian event (Myers, 2003).

Lateral neurons (LNs) are considered the central circadian pacemaker. These LNs are required for rest:activity rhythms and are most likely required for controlling the timing of eclosion (adult emergence from the pupal case). Eclosion is considered to be under the control of the circadian system because its timing is gated such that it is restricted to the hours surrounding dawn each day, even for flies that are developmentally ready hours earlier. Because eclosion occurs once in a single fly's lifetime, the multiple events that occur over several days within a population are considered a rhythm. This gating is absent in flies mutant for the clock gene period (per) or timeless (tim) and is also absent in disconnected flies that lack LNs (Myers, 2003).

Neuronal clocks (including those in the LNs) are sufficient to drive rest:activity rhythms, but perhaps not eclosion rhythms. It was of interest to determine whether these clocks would be sufficient for eclosion gating by using fly lines in which the molecular clock functions in neurons only. The gal4-UAS binary system was used to express Tim only in neurons by using the elavc155-gal4 driver and a UAS-tim transgene in an arrhythmic tim null background. This manipulation does not rescue eclosion rhythms as it does locomotor rhythms in adults (Myers, 2003).

Another circadian mutant line, one that also displays rhythmic rest:activity behavior, was also arrhythmic for eclosion. This fly line, cryb, is mutant for a circadian photoreceptor, Cryptochrome (Cry). Cryb affects the sensitivity of the LN molecular clock to pulses of light but does not affect its endogenous rhythm. Emerging evidence now suggests that Cry is a central clock gene in peripheral clocks. Besides demonstrating that the LN clock is insufficient to drive eclosion rhythms, the eclosion data suggest that Cry may also be required within a relevant peripheral clock mechanism (Myers, 2003).

From these data, it is concluded that a LN molecular clock, which can drive rest:activity rhythms, is not sufficient to restore eclosion rhythms. A peripheral clock, then, is necessary to maintain eclosion rhythms, even in the presence of a functioning LN clock (Myers, 2003).

The prothoracic gland (PG), is part of an endocrine structure known as the ring gland. This structure surrounds the heart just anterior to the cardia and is present during all stages of life except adulthood. The PG secretes ecdysteroids, which when converted to the active form of 20-hydroxyecdysone bind to their nuclear hormone receptor (ecdysone receptor, EcR) and affect gene transcription. These alterations in gene expression cause tissue metamorphosis over the course of development. Levels of ecdysteroids peak at the beginning of larval and pupal stages but, during the two days just prior to eclosion, drop to nearly undetectable levels. In Manduca sexta (tobacco hornworm), this drop in ecdysteroid titer is necessary for eclosion to proceed normally (Myers, 2003).

Previous studies suggest that there is some circadian control over PG function. Notably, ecdysone titers cycle in a circadian fashion in Rhodnius prolixus. In Drosophila, Per is present and oscillates in central brain-PG cultures taken from white prepupae. It is not known, however, whether both Per and Tim oscillate in this tissue under free-running conditions (in constant darkness and temperature) immediately preceding eclosion. Presumably, these conditions should be met before one considers the PG a true clock tissue and an appropriate candidate clock tissue involved in the control of eclosion gating (Myers, 2003).

Clock function was assessed in the PG by quantitating Per and Tim levels over the course of the day in intact pupae. Both Per and Tim levels change over the course of the LD cycle, with a significant difference between the peak (late night) and the trough (late day) values. The peak of Per expression is slightly later when compared to Tim. Both expression profiles, though, match those seen in the LNs. Per and Tim still show significant differences in daily expression in constant darkness (DD), although the difference between the peak and trough values is, as in other fly tissues, smaller. Per expression in DD, although significantly different throughout the course of the day, does not match its LD profile. This effect is similar to the delay in Per degradation in DD versus LD seen in head extracts, although the delay is more pronounced within the PG (Myers, 2003).

It is concluded that there is a molecular clock inside cells of the PG at the time when pupae are developmentally ready for imminent eclosion. Because differences between peak and trough levels of Per are smaller, the profiles of daily Tim expression in DD were used to report PG clock function (or clock synchrony within the population) in subsequent experiments (Myers, 2003).

Does circadian output of the PG clock gate eclosion? Because lesions of the PG are lethal, the necessity of the PG clock was established by assaying eclosion rhythms in fly lines in which genetic manipulation had disrupted the clock inside the PG. To disrupt the molecular clock inside the PG, Tim (UAS-tim2-1) was expressed at all times of day specifically in PG cells by using the Mai60-gal4 driver. Expressing Tim in this manner disrupts eclosion rhythms. Peaks are present in the UAS-tim2-1; Mai60-gal4 eclosion profile, but a rhythm and gating are absent. In this line, adult locomotor behavior remains rhythmic (80.3% of adults had a significant circadian period to their rest:activity behavior in constant light. Consistent with a role for the PG in eclosion gating, it was found that there are no significant differences in the daily expression of Tim in the PG of the arrhythmic cryb flies (Myers, 2003).

Although the clock inside the LNs is not sufficient for eclosion rhythms, the cells still appear to be required for eclosion gating. It is likely that the LNs could control PG clocks, much like the suprachiasmatic nucleus (SCN) of the hypothalamus is believed to drive peripheral clocks in mammals. Anatomical evidence does suggest that the LN axons (containing pigment-dispersing factor, PDF) indirectly innervate the PG (Myers, 2003).

To determine whether LNs are necessary, tests were carried out for the presence of both eclosion rhythms, and a PG clock in a fly line that lacked LN cells. A fly line was used in which LNs were ablated without lesioning many other neurons. This is a more focused disruption than that caused by the disconnected (disco) mutation. LN cells were ablated by driving a cell death gene, head-involution defective (hid), with pdf-gal4. These pdf-gal4 X UAS-hid flies are also arrhythmic for rest:activity behavior as adults. In the PG of pdf-gal4 × UAS-hid and in disco01 flies, there is no longer any significant difference in Tim expression over the course of the day. Although eclosion gating in the pdf-gal4 X UAS-hid line appears to be present during the first two days in constant darkness, rhythms do not persist. It is speculated that the flies emerging during the first two days of this assay are gated because their exposure to the entraining LD cycle persists until relatively late in development. This may result in limited and short-lived synchrony through unknown mechanisms. Clearly, though, LNs are required to maintain eclosion rhythms (Myers, 2003).

To determine whether a functioning molecular clock inside the LNs is necessary for their influence on the peripheral clock and on eclosion, the effect of disrupting this clock was examined. Tim (UAS-tim3-1) was expressed in neurons of wild-type flies at all times of day by using an elavc155-gal4 driver, all in a wild-type background. This perturbation of Tim expression is sufficient to disrupt locomotor rhythms in the adult fly. Although the molecular clock in the LNs is disrupted, there are still daily changes of Tim in the PG, and eclosion remains rhythmic (Myers, 2003).

It is possible that the role of the LNs is to provide, via PDF, a signal to the PG clock. In the LN axons that project to the dorsal brain, PDF expression cycles, with PDF release believed to occur during subjective night. The rhythmicity of PDF release and eclosion correlate well. For instance, in per0 and tim0 flies, PDF is no longer released in a rhythmic fashion from these dorsal, LN projections, and eclosion is arrhythmic as well. To determine whether PDF is part of the LN output pathway to the PG or involved in eclosion gating, both PG clock function and eclosion were assayed in flies with no functional PDF protein (pdf01). These flies are arrhythmic for locomotor behavior as well as for eclosion. In the PG, Tim levels were significantly different over the course of the day in the presence of an LD cycle, but neither Per in LD nor Tim in DD showed significant differences in their daily expression profile. These data indicate that the endogenous clock inside the PG cannot function (or entrain) without PDF in the fly. PDF overexpression and anatomical studies suggest that the LNs are the best candidates for a source of PDF relevant to eclosion behavior and the PG clock. PDF, though, is also expressed in a small subset of neurons in the central brain (LNvs and two to four tritocerebral cells) and in four to six abdominal cells. In addition, there may be other inputs to the PG. For instance, the Tim cycling seen in LD conditions is most likely due to an acute light response, suggesting the presence of photic input to the PG. Supporting this hypothesis are data from R. proxilus, whose PG clock (its presence is inferred from cyclic release of ecdysteroids in culture) is also directly photosensitive (Myers, 2003 and references therein).

Thus, the PG molecular clock is under control of the central clock. This is unlike other Drosophila peripheral clocks, such as those in the antenna, which can operate autonomously. It is also unexpected when one considers data from studies in which PG clock function is directly or indirectly assayed in culture and determined to be independent of the central brain (or independent of any tetrodotoxin-dependent output from the brain, in the case of Drosophila). The PG clock, in fact, is more similar to peripheral clocks in the mammalian circadian system. In addition, it is important to note that edysteroid synthesis (and presumably PG clock function) in the cockroach Periplaneta americana also depends upon input from the central brain. Perhaps, then, the mechanisms controlling peripheral clock function are not the same in different tissues in the same insect or in the same tissue in different insects (Myers, 2003).

From these data, a model for the circadian gating of eclosion emerges. The LNs secrete PDF into the anterior protocerebrum, where it acts on neurons that innervate the PG. Appropriate regulation of PDF levels is critical. Just as the absence of PDF disrupts eclosion, so can excess levels of it in the dorsal brain. However, the mechanisms underlying this PDF overexpression phenotype are unknown (Myers, 2003).

This study introduces a new set of clock cells necessary for the regulation of eclosion rhythms. It is not known, however, whether the LN and PG clocks together are sufficient to control eclosion gating. The current hypothesis holds that, in the ventral nervous system of Drosophila, cells containing Crustacean Cardioactive Peptide (see Cardioacceleratory peptide) are the most likely sites for control of eclosion gating, as indicated by two lines of evidence. The first is that CCAP, in response to eclosion hormone (EH), can activate ecdysis within minutes in Manduca sexta. However, the circadian gate of eclosion cannot be regulated solely by EH or CCAP because flies without either of these sets of neurons still eclose within a circadian gate. The second line of evidence is that some CCAP cells in Drosophila also express LARK, an RNA binding protein that regulates eclosion rhythms. LARK oscillates in a clock-dependent manner inside these cells. Interestingly, PG cells also contain LARK, although there have been no reports of cycling LARK outside of the CCAP cells. The exact mechanism for how and where development and circadian inputs are coordinated to control eclosion gating are still important and open questions (Myers, 2003 and references therein).


TIM protein accumulates during the dark cycle in photoreceptor nuclei of eyes and in lateral neuron pacemaker cells of the brain. Lower levels of TIM are found in cells dispersed throughout the optic lobes. TIM is found in the cytoplasm, but not the nucleus in per mutants reared in constant darkness. TIM nuclear localization depends on PER (Myers, 1996).

Per and Tim are both expressed in the Malpighian tubules of Drosophila. Per and Tim show a clear rhythm in level in both intact and decapitated flies, indicating that an endogenous rhythm in protein levels is independent of the brain. Per and Tim are present in the nuclei of all secretory cells toward the end of the dark period, but the proteins are not found toward the end of the light period. The cycling of Per and Tim in tubules persists after decapitation; when decapitated flies are exposed to a 12 hour shift in photoperiod, the Per and Tim rhythms in their tubules is also reset by 12 hours, just as in the intact fly. Thus, tubules exhibit free-running cycling of clock proteins, the rhythms of which can be reset by light, in the absence of both brain and eyes. What is the function of the Malpighian tubule clock? Adult flies have behavioral rhythms that may require periods of higher excretory activity (associated with increased metabolic rates). Fluid excretion in fly tubules is driven by transmembrane ion movements. Ion pumps, such as vacuolar ATPase, operate in this tissue, so a circadian clock in the tubules may affect the rates of ion movement across the cell membranes - as in other circadian systems (Giebultowicz, 1997).

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 ClkJrk), 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 ClkJrk) 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 ClkJrk mutant. Strikingly, all genes previously known to show this pattern of oscillation (per, tim, vri) are found in this cluster. In fact, the tim gene, which has multiple representations on the oligonucleotide arrays, has two independent representations in this cluster. Together with the novel oscillator CG5798, per, tim, and vri form a subcluster (average phase ZT14) that shows upregulation in both the per0 and tim01 mutants. The fact that per, tim, and vri all function in the central circadian clock raises the possibility that several other genes from this cluster, including the ubiquitin thiolesterase gene CG5798 and the gene coding for the channel modulator Slowpoke binding protein (Slob) may function in the circadian clock or directly downstream of it (Claridge-Chang, 2001).

Disruption of Cryptochrome partially restores circadian rhythmicity to the arrhythmic period mutant of Drosophila: a period-independent role for timeless in the Drosophila circadian pacemaker

The Drosophila circadian clock is generated by interlocked feedback loops, and null mutations in core genes such as period and timeless generate behavioral arrhythmicity in constant darkness. In light-dark cycles, the elevation in locomotor activity that usually anticipates the light on or off signals is severely compromised in these mutants. Light transduction pathways mediated by the rhodopsins and the dedicated circadian blue light photoreceptor cryptochrome are also critical in providing the circadian clock with entraining light signals from the environment. The cryb mutation reduces the light sensitivity of the fly's clock, yet locomotor activity rhythms in constant darkness or light-dark cycles are relatively normal, because the rhodopsins compensate for the lack of cryptochrome function. Remarkably, when a period-null mutation was combinded with cryb, circadian rhythmicity in locomotor behavior in light-dark cycles was restored, as measured by a number of different criteria. This effect was significantly reduced in timeless-null mutant backgrounds. Circadian rhythmicity in constant darkness was not restored, and Tim protein did not exhibit oscillations in level or localize to the nuclei of brain neurons known to be essential for circadian locomotor activity. Therefore, this study uncovered residual rhythmicity in the absence of period gene function that may be mediated by a previously undescribed period-independent role for timeless in the Drosophila circadian pacemaker. Although a molecular correlate for these apparently iconoclastic observations is not available, a systems explanation for these results is provided, based on differential sensitivities of subsets of circadian pacemaker neurons to light (Collins, 2005).

This study has revealed a surprising and intriguing restoration of circadian rhythmicity in LD cycles in per01; cryb flies. This partial rescue can even be extended to the adaptive thermal change in locomotor behavior mediated by 3' UTR splicing of the per transcript (Collins, 2004: Majercak, 2004; Majercak, 1997). A number of criteria have been used to dissect rhythmic behavior, including phase shifting in response to light pulses in LD and the use of T cycles to suggest that a residual oscillation, rather than an hourglass, underlies the behavior of the double mutant. The phase shifting of the per01; cryb oscillator is particularly informative because per01 is effectively rescuing this phenotype in cryb. This can be understood in terms of the robust, high-amplitude oscillator in cryb, being less 'perturbable' by light as Cry photoreception is lost, whereas the damped oscillator in per01; cryb is more sensitive to the environmental stimulus, precisely because of its low amplitude. The damped oscillation in the per01; cryb double mutant can be eliminated by removing tim function, but this is temperature dependent, so tim cannot supply the full explanation for these residual cycles. Although these experiments have focused on the 'evening' oscillator, of related interest is that the residual 'morning' oscillator that anticipates the lights-on signal in per01 was also observed. It is clear that both of these studies raise again the possibility of an underlying rhythmicity in per01 flies that was initially suggested from statistical analyses of mutant locomotor records (Collins, 2005).

The entrainment of a frequency-less oscillator in Neurospora crassa has been the subject of some recent debate, and the parallels with a residual rhythmicity in per-null Drosophila are striking. Furthermore, the rescue of per01 behavior by cryb would appear, at least superficially, to be similar to the situation in mammals in which a Cry mutation restores free-running rhythms to the arrhythmic mPer2 mutant mouse; this has been explained in terms of the freeing up in the double mutant of other mPer and Cry paralogues to interact and restore the original behavior. Since the fly does not have paralogues of per and cry, an explanation must be sought elsewhere. The only other genotypes identified so far with an anticipatory locomotor activity peak in LD and loss of rhythmicity in DD are disconnected (disco) and Pdf0. Neither mutation affects the molecular core of the circadian clock, rather the network of pacemaker neurons is disrupted. PDF is required for the functional integration of several clock neuronal groups within the brain, suggesting that disruption of interneuronal signaling causes arrhythmic behavioral output in the absence of synchronizing cues. In arrhythmic disco mutants, the clock gene expressing lateral neurons (LNvs and LNds) are usually absent, whereas the dorsal neurons are still present, thus indicating that the former are necessary for self-sustained rhythmicity, whereas the latter can only mediate rhythmic behavior under LD conditions (Collins, 2005).

This networking of clock neurons provides a basis for possible models to explain LD behavioral anticipation in the absence of Per, based on functional differences between the three groups of clock genes expressing LNs. Of these, only the small ventral LNs (sLNvs) and dorsal LNs (LNds) have a self-sustaining molecular clock when initially released into DD, although the latter depends on the former for synchronization. The third group, the large ventral LNs (l-LNvs) do not have a self-sustaining clock, although after a few days, tim mRNA again begins to accumulate rhythmically in these cells. Furthermore, rhythmic Tim expression is more sensitive to disruption by cry mutations in the l-LNvs, than in the s-LNvs or the LNds under LD conditions, suggesting that rhythmic output from the l-LNvs are compromised in a cryb background. In turn, this may contribute to the peculiar defects of cryb that includes robust entrainment to LD cycles, but significantly reduces behavioral phase shifts to brief light pulses, and, unlike wild-type, the maintenance of rhythmic behavior in constant light (Collins, 2005).

In the favored model, the robust s-LNv and LNd oscillators in cryb 'resist' the effects of brief light pulses, because of the impaired light input that is relayed to the s-LNvs, and from the s-LNvs to the LNds, by the more light-relevant l-LNvs. In per01, the molecular clock is severely dampened in all clock neurons, more so in the s-LNvs and LNds that have an endogenous cycle than the l-LNvs that do not. Thus, the light-mediated input from the l-LNv neurons into the s-LNvs, and indirectly to the LNds, is no longer resisted, and now overwhelms the residual damped per01 oscillator in these neurons, stimulating light-induced non-rhythmic locomotor behavioral output. However, when cryb and per01 are combined, the weak oscillator of per01 is no longer overcome by the light input because it is attenuated by cryb and mediated via the l-LNvs. Thus, rhythmic behavior is observed in LD cycles, providing a glimpse of the residual Per-independent, partly Tim-regulated clock. This model is preferred over a simpler one in which only the s-LNvs are involved, because previous studies have shown that the only direct photoreceptive input into these neurons is from the Hofbauer-Buchner eyelet, which is a very weak photoreceptor at best and it cannot, in the absence of other photoreceptors, entrain the fly's behavior (Helfrich-Forster, 2002). Thus, it is difficult to see how light information would be received by the s-LNvs to entrain the per01; cryb double mutant so effectively, unless it is transmitted from another neuronal source: the l-LNvs (Collins, 2005 and references therein).

In support of the model, there appears to be both direct and indirect neural connections between the compound eyes and the l-LNvs, suggesting that the l-LNvs may act as the light 'amplifier'. This study extends earlier observations by showing that photoreceptor cells expressing the rhodopsin genes, Rh3 and Rh5, send their axons through the medulla terminating in close proximity to the general region where the l-LNvs likely extend their dendritic arborizations..Although not definitive, these results support earlier claims that the photoreceptors may directly (or indirectly) synapse with the l-LNvs. As stated above, these molecular and proposed functional differences between s- and l-LNvs may also contribute to explaining the loss of light responsiveness in cryb mutant flies, which are blind to constant light and brief light pulses, despite retaining light input from the canonical visual transduction pathway. Thus Cryptochrome, aside from being a photoreceptor in its own right, also appears to control a gateway for rhodopsin-mediated light input into the clock (Collins, 2005).

Although the disruption of neural networks in this way probably explains the light responses of the clock in per01; cryb, it offers no molecular basis for the observed behavior. The loss of anticipation in tim-null-bearing genotypes suggests that Tim may play a key role. Although no significant nuclear Tim was observed in the LNvs or LNds of per01; cryb, the latter neurons being particularly relevant for providing the evening peak of locomotor activity present in the double mutants, it is suspected that Tim is shuttling continually in and out of the nucleus because Tim can enter the nucleus alone, but requires Per for nuclear retention, at least in larval clock neurons. Once in the nucleus, Tim is presumably interacting with as yet unidentified protein(s) in a light-dependent manner, generating behavioral rhythms in the double mutants. A microarray study found that 18 of the 72 genes that cycled in LD in wild-type also cycle in per01. Any one or more of these light-controlled proteins could therefore interact with Tim, contributing to the light-dependent oscillator of per01; cryb. In fact, it has been noted by others that a glutamine-rich transcriptional activator domain found within Tim may allow it to regulate other genes in a Per-independent manner (Collins, 2005).

A resetting signal between Drosophila pacemakers synchronizes morning and evening activity

The biochemical machinery that underlies circadian rhythms is conserved among animal species and drives self-sustained molecular oscillations and functions, even within individual asynchronous tissue-culture cells. Yet the rhythm-generating neural centres of higher eukaryotes are usually composed of interconnected cellular networks, which contribute to robustness and synchrony as well as other complex features of rhythmic behaviour. In mammals, little is known about how individual brain oscillators are organized to orchestrate a complex behavioural pattern. Drosophila is arguably more advanced from this point of view: a group of adult brain clock neurons expresses the neuropeptide PDF and controls morning activity (small LNv cells; M-cells), whereas another group of clock neurons controls evening activity (CRY+, PDF- cells; E-cells). Transgenic mosaic animals were generated with different circadian periods in morning and evening cells. This study shows by behavioural and molecular assays, that the six canonical groups of clock neurons are organized into two separate neuronal circuits. One has no apparent effect on locomotor rhythmicity in darkness, but within the second circuit the molecular and behavioural timing of the evening cells is determined by morning-cell properties. This is due to a daily resetting signal from the morning to the evening cells, which run at their genetically programmed pace between consecutive signals. This neural circuit and oscillator-coupling mechanism ensures a proper relationship between the timing of morning and evening locomotor activity (Stoleru, 2005).

Overexpression of the Tim kinase Shaggy (Sgg; Drosophila GSK3) shortens the period by 3-4 h. Sgg expression was driven in all clock cells by crossing tim-GAL4 with flies carrying an EP element inserted at the Sgg locus (EP1576, referred to as UAS-Sgg). The locomotor activity rhythm of tim-GAL4/UAS-Sgg (timSgg) flies in constant darkness (DD) confirmed previous results, in that the period was about 3 h shorter than that of control flies (Stoleru, 2005).

Sgg was expressed exclusively in LNv cells by constructing a Pdf-GAL4/UAS-Sgg genotype. The Pdf-GAL4 driver is well characterized and drives gene expression only in two clock-cell groups: the PDF+ small LNv (s-LNv) cells (that is, M-cells) and the PDF+ large LNv (l-LNv) cells. The driver is inactive in the CRY+PDF- evening cells. Pdf-GAL4/UAS-Sgg (PdfSgg) flies also manifested a short period. The period shortening was less than that of timSgg flies, probably because of weaker expression from Pdf-GAL4 driver in LNv cells. Sgg expression from an even weaker driver, cry13-GAL4, did not affect behavioural period (Stoleru, 2005).

A close inspection of the behavioural actograms revealed that the period of evening activity is significantly shorter in PdfSgg flies (with a daily advance of about 2 h). This indicates that the pace of E-cells was accelerated, although the period manipulation was restricted to M-cells. An advanced evening peak, without an increase in E-cell Sgg expression, indicates that the faster M oscillator might be setting the E-cell pace. It is therefore proposed that the PDF+ cells influence molecular circadian events within E-cells (Stoleru, 2005).

To investigate this possibility, the molecular period (cycle duration) of each clock-cell group was estimated in these different genotypes: UAS-Sgg (control), timSgg and PdfSgg. Fly brains were analysed by in situ hybridization for tim RNA expression pattern after 4 days in DD, so that a barely detectable daily advance by 2-3 h would result in an aggregate advance of 8-12 h on DD4 (fourth day of DD). Indeed, Sgg overexpression in all clock neurons (timSgg) markedly shifted the interval of high tim mRNA expression on DD4 by about 12 h, from between CT10 and CT18 to before CT6. (CT is the circadian time within a constant-darkness experiment; CT0 is the hour of the last lights-on event.) All neurons expressing clock genes showed a similar temporal pattern, consistent with the expected Sgg-induced period shortening in all clock cells, and with a deterministic relationship between the molecular period and the locomotor activity period (Stoleru, 2005).

However, the PdfSgg tim RNA profiles were strikingly different and unexpected. Whereas the s-LNv cells showed a roughly 8 h advance in DD4, expected from a period shortening of 2 h per day, the l-LNv cells showed no appreciable change from those in control flies; that is, their molecular program is apparently unaffected by Sgg overexpression within these cells. Also surprising were the DN1 and DN3 profiles, which showed a roughly 8 h advance, as were the LNd cells, which were advanced by about 6 h relative to those in control flies. Since PdfSgg flies do not overexpress Sgg in these three cell groups, their molecular programs behave in a non-cell-autonomous manner. Because the E-cells are included within these groups and because the s-LNv cells (the M-cells) are the only cells with a cell-autonomous program that match the behavioural period of the flies, the M-cells apparently determine the clock pace of these other neuronal groups, including the E-cells (Stoleru, 2005).

The l-LNv cells and DN2 cells emerged as the only clock-gene-expressing neurons that evaded control of the M-cells and maintained a wild-type-like phase of tim RNA cycling in PdfSgg flies. Because DN2 cells are genotypically wild type in these flies, it is inferred that they oscillate with cell-autonomous properties and are the best candidates for determining the non-cell-autonomous wild-type-like characteristics of the l-LNv cells. As a consequence there are at least two parallel clock-cell circuits in the Drosophila brain in constant darkness: the M-E circuit controls locomotor activity rhythms and is driven by the M-cells (s-LNv cells), whereas the DN2-l-LNv circuit has as yet unknown functions and is driven by the DN2 cells (Stoleru, 2005).

To verify and extend these concepts, a genotype was generated in which the E-cells should run faster than M-cells. By adding the previously described Pdf-GAL80 repressor construct to the tim-GAL4;UAS-Sgg background, Sgg was expected to be overexpressed in all clock neurons with the exception of PDF-expressing cells. As these include the M-cells (s-LNv cells), they should run more slowly (24 h) than the E-cells (about 21 h). A 'faster takes all' rule predicts that the short-period E-cells will dominate over the normal 24 h M-cells in this genotype and generate a behavioural rhythm of about 21 h. Alternatively, dominant M-cells will give rise to a behavioural period of 24 h despite the faster endogenous oscillator in the E-cells (Stoleru, 2005).

Consistent with a dominant M-cell model was the observation that timSgg/PdfGAL80 flies had an almost wild-type period in DD. The molecular analysis is also consistent, since the s-LNv cells manifested a wild-type-like program: tim mRNA peaked between CT12 and CT20 on DD4. Despite Sgg overexpression, the LNd cells, DN1 cells and DN3 cells had a similar and wild-type-like pattern of tim expression. As described above, this indicates that all three cell groups behave non-autonomously and are probably driven by the s-LNv cells. This result is supported by the anatomical pattern of s-LNv neuronal processes, which project towards the brain regions populated by LNd, DN1 and DN3 cells. DN2 cells were again the only Sgg-overexpressing cells in which the phase of tim RNA oscillation corresponded to the predicted accelerated pace. The l-LNv cells, despite lacking Sgg overexpression (because of the PdfGAL80 transgene), also showed a comparable advance of tim expression. These timSgg/PdfGAL80 results confirm that the s-LNv cells determine the phase of LNd, DN1 and DN3 cells and that an independent cellular network includes the DN2 and l-LNv cells. Because the behavioural period was wild-type-like and paralleled the molecular clock within the s-LNv cells, the results confirm that these M-cells assign the circadian period in the absence of light cues (Stoleru, 2005).

To confirm the lack of a contribution of DN2/l-LNv to the E-M network function and to locomotor rhythms, the timSgg/cryGAL80 genotype was also examined. It is similar to the timSgg/PdfGAL80 genotype described above, except that Sgg overexpression is repressed in a wider group of cells. These include most if not all of the E-cells and l-LNv cells as well as the M-cells. Since DN2 cells are the only clock cells in which cry promoter-driven expression was not detected, it is expected that the faster clock in timSgg/cryGAL80 would be limited to CRY- cells, including the apparently cell-autonomous DN2 cells (Stoleru, 2005).

Indeed, tim hybridization in situ confirmed that the period of DN2 rhythm was shortened by about 2-3 h per day. The l-LNv neurons were shifted to about the same extent, which is consistent with the notion that they behave non-cell-autonomously and follow the pace of the DN2 clock program. All other clock cells maintained a pattern similar to that of control flies. Because timSgg/cryGAL80 flies had a normal behavioural period, these results confirm that l-LNv and DN2 cells do not contribute detectably to locomotor activity rhythms. This conclusion is in agreement with previous results showing that wild-type flies have persistent DD behavioural rhythms, despite protein oscillation idiosyncrasies of the l-LNv and DN2 cells (Stoleru, 2005).

How does the M-cell (s-LNv) clock determine the period of E-cells (LNd cells/DN cells)? Although previous work indicated possible oscillator coupling and a direct effect of LNv on the transcriptional oscillations of other clock cells, it was difficult to envision how the M-cells could override the intrinsic molecular timing of the E-cells. A second possibility is therefore considered, namely that the E-cells maintain an unaltered intrinsic clock program but receive a daily resetting signal from the M-cells. This model predicts that the timing of the evening activity within every cycle (between two consecutive mornings) reflects the status of the endogenous clock of E-cells, whereas the overall period exhibited by the evening peaks reflects the pace of the M-cell resetting signal (Stoleru, 2005).

To examine this possibility, the different transgenic strains were assayed for their average evening activity phase within a cycle, by using the leading morning peak as a reference and then measuring the average time until the subsequent evening peak. The overall DD period correlated with the genotype of M-cells as expected, but the length of the subjective day (M-E interval) correlated only with the genotype of the E-cells. In control flies with a period of about 24 h, the subjective day was roughly 12 h, similar to the duration of subjective day of PdfSgg. The latter strain features a wild-type-like E-oscillator but a fast, Sgg-expressing M-oscillator and a period of about 22 h. In contrast, timSgg flies express Sgg in both E-cells and M-cells, and both the average length of subjective day and the period (M-M) are reduced. The results indicate that the E-cells run an autonomous clock program whose starting (or ending) points are determined by daily resetting signals from the M-cells (Stoleru, 2005).

A DD unidirectional M ---> E resetting mechanism also predicts that a slower (24 h) M-cell clock and a faster E-cell clock will have a normal morning peak phase but an advanced evening peak phase. To test this prediction, the behavioural outputs of timSgg/PdfGAL80 and timSgg/cryGAL80 flies, which differ only in the genotypes of their E-cells, were compared. Both strains have periods of about 24 h, but the former should give rise to a fast E-cell molecular program, whereas the latter should have an E-clock of 24 h as a result of suppression of Sgg expression (Stoleru, 2005).

Indeed, the evening phase of timSgg/cryGAL80 is similar to that of control flies, and it always occurs about 2.5 h later than that of timSgg/PdfGAL80. The evening phase of timSgg/PdfGAL80 is more similar to that of timSgg, although the latter genotype has a much shorter period than the former. The length of subjective day of timSgg/PdfGAL80 flies further confirms that the evening phase within each cycle is a reflection of the endogenous E-cell rhythm, whereas the period of the cycle (M-M) correlates with the intrinsic M-cell clock (Stoleru, 2005).

These comparisons indicate that the circadian network is modulated by intercellular communication signals, which achieve and maintain circadian coherence -- the proper relationship between morning and evening activity. The dominant M-clock determines the period of the entire system by providing a daily reset signal to the E-clock in darkness and is therefore a true cellular Zeitgeber. Because the M-cells can delay as well as advance E-cells, the resetting signal may be required for E-cell oscillations. The usual candidate for this signal is the M-cell-specific neuropeptide PDF. It contributes to the normal synchrony and/or rhythmicity in constant darkness, with a striking similarity to the mammalian neuropeptide VIP. Moreover, injecting PDF into the cockroach brain causes circadian phase delays. Other principles and/or molecules may also be relevant to the M-E subnetwork, because E-cells can drive clockless M-cells to manifest cyclical behavioural outputs under 12 h light/12 h dark (LD) conditions (Stoleru, 2005).

The l-LNv and DN2 cells are the two neuronal groups that escape the M-cell reset signal in DD. They constitute a second circadian subnetwork with no apparent effect on locomotor activity rhythms and no known function. The DN2 cells are among the few clock-gene-expressing brain cells in larvae and are also the only clock cells that do not change their morphology after eclosion. Larval DN2 cells are apparently devoid of CRY and manifest anti-phase oscillations of Tim and PER. It is therefore likely that both the DN2 cells and the l-LNv cells impart circadian regulation to unknown physiological functions relevant to both larvae and adults. More generally, it is expected that the organizational principles of the two subnetworks described in this study will also be relevant to mammalian neuronal networks with important behavioural functions, for example the relationship between different oscillators in the SCN (Stoleru, 2005).

Neurotoxic protein expression reveals connections between the circadian clock and mating behavior in Drosophila

To investigate the functions of circadian neurons, two strategies were added to the standard Drosophila behavioral genetics repertoire. The first was to express a polyglutamine-expanded neurotoxic protein (MJDtr78Q; MJD, Machado-Joseph disease) in the major timeless (tim)-expressing cells of the adult brain. These Tim-MJD flies were viable, in contrast to the use of cell-death gene expression for tim neuron inactivation. Moreover, they were more arrhythmic than flies expressing other neurotoxins and had low but detectable tim mRNA levels. The second extended standard microarray technology from fly heads to dissected fly brains. By combining the two approaches, a population of Tim-MJD-affected mRNAs was identified. Some had been previously identified as sex-specific and relevant to courtship, including mRNAs localized to brain-proximal fat-body tissue and brain courtship centers. Finally, a decrease was found in the number of neurons that expressed male-specific forms of the Fruitless protein in the laterodorsal region of the brain. The decrease was not a consequence of toxic protein expression within these specialized cells but a likely effect of communication with neighboring TIM-expressing neurons. The data suggest a functional interaction between adjacent circadian and mating circuits within the fly brain, as well as an interaction between circadian circuits and brain-proximal fat body (Kadener, 2006; full text of article).

This study combined two strategies, neurotoxic protein expression and brain microarrays, to investigate circadian clock gene expression and behavior. Some Tim-MJD-affected mRNAs had been previously identified as cycling, whereas others are sex-specific and relevant to courtship. Because Tim-MJD flies also exhibit a mating defect, it is suggested that this phenotype reflects circadian neuron inactivation as well as an important courtship-relevant connection between these neurons and cells affecting reproductive behavior (Kadener, 2006).

The goal was to examine the behavioral and molecular phenotype in the absence of circadian neuron function. This had not yet been achieved, because the proapoptotic UAS genes hid and reaper are embryonic lethal in combination with per or tim drivers. Importantly, Ddc-gal4 is also lethal in combination with UAS-hid. Other neuronal inactivation tools, e.g., UAS-tetanus toxin and UAS-modified K+ channels, are rhythmic with per and tim drivers in LD cycles. Although these UAS transgenes might cause arrhythmicity in combination with stronger circadian drivers, the fully arrhythmic phenotype of the same tim driver with UAS-MJDtr78Q indicates it is a stronger reagent (Kadener, 2006).

Young flies expressing neurotoxic proteins under the control of the tim-gal4 driver not only were viable but also had similar morphology, fertility, and locomotor activity levels compared with young control flies. The only striking phenotypes were complete arrhythmicity under all conditions, mating defects, and a shortened lifespan. Several lines of evidence suggest that the arrhythmic phenotypes are related to ablation/inactivation of the clock in tim-expressing fly brain neurons. It is possible that MJDtr78Q expression in tim-expressing glial and fat-body cells contributes to the shortened lifespan. Indeed, it was recently shown that MJDtr78Q expression in glial cells causes a shortened lifespan. However, no specific changes in gene expression of glial-specific transcripts were found in Tim-MJD flies, suggesting that these cells are not strongly or universally affected by the tim driver (Kadener, 2006).

Are Tim-MJD behavioral and molecular phenotypes due to clock-neuron death? Two independent observations indicate that MJDtr78Q expression predominantly leads to circadian transcriptional misregulation, at least in young flies. First, the gene expression changes in Tim-MJD resemble those observed in the clock transcription factor mutant ClkJrk. Of 552 genes down-regulated in heads of Tim-MJD, 104 (19%) were reported as down-regulated in ClkJrk, and only 27 (4.9%) were up-regulated in ClkJrk. A comparison of genes up-regulated in Tim-MJD heads (368 genes) with genes up-regulated in ClkJrk has a similar proportion of identical transcripts (19%), making it unlikely that this down–down and up–up relationship between the two strains is fortuitous. Perhaps CLK is recruited to inclusions, and circadian transcription is inhibited because it is the only core pacemaker protein with a clear polyQ region (Kadener, 2006).

Second, the Tim-MJD effect on the LNv cell population is remarkably similar to that observed in another transcription factor mutant cycle02 as well as in ClkJrk. Because large LNvs are born much later than small LNvs (sLNvs), the apparent Tim-MJD selectivity for sLNvs may reflect longer neurotoxic protein exposure. Nonetheless, sLNvs are still present in young adult flies, suggesting that some circadian neurons survive MJDtr78Q expression from first larval instar to adulthood. Persistent adult expression may then explain the short lifespan (Kadener, 2006).

Despite the presence of Pdf gene expression, disruption of per and tim transcription by Tim-MJD expression seems complete in the ~75 pairs of pacemaker neurons. This contrasts with the more modest effect (50%-70% decrease) on the levels of timeless mRNA and of other clock-related mRNAs from whole heads or brains. Moreover, molecular oscillations still persist in Tim-MJD brains, suggesting that cell-autonomous molecular oscillations continue outside of the pacemaker cells in Tim-MJD flies, at least under these LD conditions. If the remaining 30%-50% of clock gene expression derives from a much larger number of extra-pacemaker clock cells, these must be low-expressing brain clock cells, which explains the failure to detect tim or per expression by immunostaining or in situ hybridization in Tim-MJD brains. Lower expression levels per cell would also explain the likely persistence of these neurons in Tim-MJD flies, i.e., expression levels would be below a toxicity threshold. Combined with relatively late tim expression during eye development, low expression levels might also contribute to the lack of a Tim-MJD rough eye phenotype (Kadener, 2006).

Some cycling-head mRNAs come from the fat body as well as the eye and brain, and some fat-body mRNAs are strongly affected in Tim-MJD flies. Strikingly, the MJD effect is dramatic in brain RNA but less strong or absent in head RNA for most of these fat-body transcripts. This is reminiscent of a previously reported difference in gene expression between brain-proximal and canonical fat-body cells; the former should constitute a more prominent source of fat-body signal in the dissected-brain samples than in the total head samples. That yolk-protein mRNAs are among the Tim-MJD-affected brain transcript population suggests this fat-body subset is not restricted to behavioral function (Kadener, 2006).

Although courtship and mating are mainly controlled by fruitless-expressing regions of the central nervous system, recent evidence suggests that the circadian clock, as well as the fat body, contributes to these behaviors. Therefore, the mating phenotype could be a direct effect of disrupting the clock mechanism in this tissue. However, because brain FRUM expression in some neuronal groups is implicated in the early steps of male courtship, the decrease in FRUM-expressing neurons in Tim-MJD might also be relevant to the mating phenotype. This decrease might impact not only courtship-relevant targets within the brain but also brain-proximal fat-body gene expression. In this view, FRUM contributes to transmitting circadian information from brain clock centers to mating-relevant peripheral tissues such as the fat body. In any case, it is suggested that circadian neurons affect courtship and mating behavior by communicating with brain courtship centers as well as peripheral tissues like the fat body (Kadener, 2006).

Temperature-dependent resetting of the molecular circadian oscillator in Drosophila

Circadian clocks responsible for daily time keeping in a wide range of organisms synchronize to daily temperature cycles via pathways that remain poorly understood. To address this problem from the perspective of the molecular oscillator, temperature-dependent resetting of four of its core components was monitored in Drosophila: the transcripts and proteins for the clock genes period (per) and timeless (tim). The molecular circadian cycle in adult heads exhibited parallel responses to temperature-mediated resetting at the levels of per transcript, tim transcript and Tim protein. Early phase adjustment specific to per transcript rhythms was explained by clock-independent temperature-driven transcription of per. The cold-induced expression of Drosophila per contrasts with the previously reported heat-induced regulation of mammalian Period 2. An altered and more readily re-entrainable temperature-synchronized circadian oscillator that featured temperature-driven per transcript rhythms and phase-shifted Tim and Per protein rhythms was found for flies of the 'Tim 4' genotype, which lacked daily tim transcript oscillations but maintained post-transcriptional temperature entrainment of tim expression. The accelerated molecular and behavioural temperature entrainment observed for Tim 4 flies indicates that clock-controlled tim expression constrains the rate of temperature cycle-mediated circadian resetting (Goda, 2014).

Effects of light interruption on sleep and viability of Drosophila melanogaster

Light is a very important regulator of the daily sleep rhythm. This study investigated the influence of nocturnal light stimulation on Drosophila sleep. Results showed that total daytime sleep was reduced due to a decrease in daytime sleep episode duration caused by discontinuous light stimulation, but sleep was not strongly impacted at nighttime although the discontinuous light stimulation occurred during the dark phase of the cycle, the scotophase. During a subsequent recovery period without light interruption, the sleep quality of nighttime sleep was improved and of daytime sleep reduced, indicating flies have a persistent response to nocturnal light stimulation. Further studies showed that the discontinuous light stimulation damped the daily rhythm of a circadian light-sensitive protein Cryptochrome both at the mRNA and protein levels, which subsequently caused disappearance of circadian rhythm of the core oscillator timeless and decrease of Timeless protein at nighttime. These data indicate that the nocturnal light interruption plays an important role in sleep through core proteins Cryptochrome and Timeless, Moreover, interruption of sleep further impacted reproduction and viability (Liu, 2014; 25148297).

Effects of Mutation or Deletion

Mutations in tim produce arhythmic behavior and suppress the circadian oscillation of PER transcripts (Vosshall, 1994). There is altered eclosion rhythm accompanied by night-emerging and day-emerging adults, and defects in locomotor activity in which flies become arhythmic when the entraining light-dark cues are missing. There are no detectable defects in the nervous system, visual system or brain (Sehgal, 1994).

Animals bearing the tim mutation produce very low levels of endogenous PER which does not cycle. tim mutation appears to disrupt the normal circadian regulation of PER phosphorylation (Price, 1995).

In tim mutants, PER cannot be detected in the nucleus (Vosshall, 1994).

To identify new components of the Drosophila circadian clock, chemically mutagenized flies were screened for suppressors or enhancers of the long periods characteristic of the period mutant allele per. A mutant maps at the timeless gene. This novel allele, timSL, alters the temporal pattern of per protein nuclear localization and restores temperature compensation to per mutant flies. perL mutants exhibit a lengthening period of locomoter activity from 24 to 29 hours, and lack the ability to compensate photoperiod response to substantial changes in temperature. timSL (tim suppressor of perL mutation) more generally manifests specific interactions with different per alleles. The identification of this first period-altering timSL allele (previous tim mutants have been null) provides further evidence that TIM is a major component of the clock, and the allele-specific interactions with PER provide evidence that the PER/TIM heterodimer is a unit of circadian function. timSL alters the TIM phosphorylation pattern during the late night. The effects of timSL on TIM phosphorylation (TIM-SL is phosphorylated to a greater extent than wild-type TIM) suggest that timSL functions as a partial bypass suppressor of per and provide evidence that the TIM phosphorylation program contributes to the circadian timekeeping mechanism (Rutila, 1996).

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).

Regulation of the Drosophila pigment-dispersing factor (pdf) gene products was analyzed in wild-type and clock mutants. Mutations in the transcription factors Clock and Cycle severely diminish pdf RNA and neuropeptide (PDF) levels in a single cluster of clock-gene-expressing brain cells, called small ventrolateral neurons (s-LNvs). This clock-gene regulation of specific cells does not operate through an E-box found within pdf regulatory sequences. PDF immunoreactivity exhibits daily cycling, but only within terminals of axons projecting from the s-LNvs. This posttranslational rhythm is eliminated by period or timeless null mutations, which do not affect PDF staining in cell bodies or pdf mRNA levels. Therefore, within these chronobiologically important neurons, separate elements of the central pacemaking machinery regulate pdf or its product in novel and different ways. Coupled with contemporary results showing a pdf-null mutant to be severely defective in its behavioral rhythmicity, the present results reveal PDF as an important circadian mediator whose expression and function are downstream of the clockworks (Park, 2000).

To assess the effects of clock mutations on pdf expression, the normal cellular distribution of the Drosophila gene's native products were examined. By in situ hybridization, the expression pattern of pdf mRNA has been shown to be similar to that determined with anti-crab-PDH. There are four positive cells in each larval brain hemisphere; these persist into adulthood and become the small ventrolateral neurons (s-LNvs), whose neurites project into a dorsal region of the adult brain. Four large ventrolateral neurons (l-LNvs) also express pdf; these emerge during metamorphosis and send projections into the optic lobe and across the brain midline. Larvae and adults also contain pdf mRNA in the posterior extremity of the CNS (Park, 2000).

Northern blots reveal no daily rhythm of pdf mRNA abundance, but they could have failed to detect pdf mRNA cycling in a subset of the cells. Thus temporal in situ hybridizations were performed; neither category of pdf-expressing neurons exhibit systematic fluctuations in signal intensities. Therefore, there is no pdf mRNA rhythm for clock mutations to affect (Park, 2000).

No effect of a period-null mutation on pdf mRNA levels had been detectable in previous Northern blottings. Neither per01 nor a timeless-null mutation affects the RNA's abundance, by Northern blottings and by in situ hybridizations. To search further for regulation by per or tim, adult brains were stained with anti-PDH at different times of day and night. Strikingly, nerve terminals in a dorsal region of the central brain exhibit rhythms of anti-PDH-mediated staining. The neurites that terminate in this region project from the s-LNv cells. In an LD cycle, the peak and trough times for the nerve-terminal cycling are 1 h after lights-on and lights-off, respectively. Staining levels in the perikarya of s-LNvs exhibit some fluctuations but no regular pattern. The adult-specific, larger PDF neurons also exhibit no appreciable cycling of anti-PDH-mediated staining, either in l-LNv cell bodies or in the termini of their neurites that ramify over the surface of the medulla optic lobe (Park, 2000).

The dorsal-brain, nerve-terminal cycling persists in constant darkness with an ~24-h period in wild type. In that condition the cycle duration is shortened to ~20 h by the perS mutation, which causes behavioral periodicities to be about 5 h shorter than normal. In the dorsal brains of the per01 null mutant, nerve-terminal cycling is abolished, and the signal strengths are very low. However, the immunohistochemical procedure performed on these brain sections is not very sensitive. Therefore, a quantitative fluorescence method was used, the better to judge PDF staining intensities in whole-mounted brains. At the peak and trough time-points, nerve-terminal signals in wild type are again higher in the early morning compared with the early night. This temporal difference is not observed in the dorsal brains of the arrhythmic per01 and tim01 mutants. In per01, the staining intensities at both times are nearly identical and at levels intermediate between the per+ peaks and troughs. In tim01, the PDF terminal signals are also the same at the two time-points but significantly higher than in tim+ and. The mutational effects of these clock genes on daily fluctuations of PDF abundance at certain nerve terminals indicate that an aspect of this peptide's regulation is, in one way clock controlled, and in another was posttranslationally regulated (Park, 2000).

A Timeless-independent function for Period proteins in the Drosophila clock is described. The mutation timelessUL (UL for ultralong) generates 33 hr rhythms, prolonged nuclear localization of Period/TimelessUL protein complexes, and protracted derepression of period and timeless transcription. Light induced elimination of TimUL from nuclear Per/TimUL complexes gives strong downregulation of per and tim expression. Thus, in the absence of Tim, nuclear Per can function as a potent negative transcriptional regulator. Two additional studies support this role for Per: (1) Drosophila expressing Per that constitutively localizes to nuclei produce dominant behavioral arrhythmicity, and (2) constitutively nuclear Per represses Clock/Cycle-mediated transcription of per in cultured cells without Tim. Conversion of Per/Tim heterodimers to nuclear Per proteins appears to be required to complete transcriptional repression and terminate each circadian molecular cycle (Rothenfluh, 2000a).

What is the relevance of controlling the step from Per/Tim complex to Per? Toward the end of each molecular cycle, nuclear Per that is released from Per/Tim complexes becomes increasingly phosphorylated in a fashion dependent on the kinase Doubletime. When this phosphorylation is suppressed by dbt mutants, nuclear Per shows greatly increased stability. Thus, phosphorylation should regulate the duration of repression by nuclear Per. Since both Per/Tim complexes and nuclear Per can repress per and tim transcription, but only phosphorylated Per proteins are significantly degraded, termination of each molecular cycle should be triggered by the conversion of Per/Tim complexes to Per. While periodic degradation of Tim will be precisely set by an LD cycle, sustained molecular oscillations and behavioral rhythmicity close to 24 hr must be set in DD by light-independent turnover of Tim. This specific downregulation of Tim can be seen to occur in DD several hours before a corresponding diminution in the level of Per. Thus, a light-independent mechanism effecting nuclear Tim degradation should be a key determinant of period length. timUL may affect this mechanism (Rothenfluh, 2000a).

These studies raise the possibility that nuclear Per and the Per/Tim complex can perform distinct functions. For example, the observation that per and tim expression is decreased by removing Tim from Per/TimUL complexes indicates that quantitative or qualitative differences distinguish the activities of Tim-independent and Tim-associated forms of Per in vivo. This also suggests that, in vivo, full repression of per and tim expression requires the activity of nuclear Per at the end of each molecular cycle. There may also be different contributions to the regulation of Drosophila Clk expression. Clk protein negatively regulates CLK RNA accumulation, which cycles with a phase distinct from that of per and tim. Per and Tim block this Clk activity, such that Per/Tim nuclear translocation is associated with increased CLK RNA synthesis. However, CLK RNA levels fall at dawn, suggesting that the conversion of Per/Tim dimers to nuclear Per restores the autoregulatory activity of Clk. Accordingly, Per may regulate per and tim expression, while Per/Tim complexes control transcription of per, tim, and Clk. Such a mechanism could provide a general basis for establishing molecular oscillations with a variety of phases from a single clock (Rothenfluh, 2000a).

A model is proposed for the roles of Per and the Per/Tim Complex in transcriptional regulation. per and tim transcription promotes accumulation, with a delay, of heterodimeric complexes of Per and Tim proteins. The Per/Tim complex then translocates to the nucleus, initiates repression of per and tim transcription, and derepresses Clock. Per/Tim complexes are stable; however, specific degradation of Tim releases nuclear Per. In the absence of Tim, nuclear Per shows further repression of per and tim transcription, bringing PER and TIM RNA pools to their lowest levels. Phosphorylation of nuclear Per, regulated by Dbt, leads to Per degradation, and the cycle starts anew. Phosphorylation of nuclear Per may also promote its repressor function in the absence of Tim. In this model, no role for Tim without Per is proposed because Per-independent Tim proteins have not been observed in wild-type nuclei (Rothenfluh, 2000a).

In genetic screens for Drosophila mutations affecting circadian locomotion rhythms, six new alleles of the timeless gene have been isolated. Two of these mutations cause short-period rhythms of 21-22 hr in constant darkness, and four result in long-period cycles of 26-28 hr. All alleles are semidominant. Studies of the genetic interactions of some of the tim alleles with period-altering period mutations indicate that these interactions are close to multiplicative: a given allele changes the period length of the genetic background by a fixed percentage, rather than by a fixed number of hours. The timL1 allele was studied in molecular detail. The long behavioral period of timL1 is reflected in a lengthened molecular oscillation of per and tim RNA and protein levels. The lengthened period is partly caused by delayed nuclear translocation of TIML1 protein, shown directly by immunocytochemistry and indirectly by an analysis of the phase response curve of timL1 flies (Rothenfluh, 2000b).

Circadian rhythms of female mating activity governed by clock genes in Drosophila

Wild-type Drosophila melanogaster displays a robust circadian rhythm in the mating activity, and these rhythms are abolished in period- or timeless-null mutant flies (per01 and tim01). Circadian rhythms are lost when rhythm mutant females are paired with wild-type males, demonstrating that female mating activity is governed by clock genes. Furthermore, an antiphasic relationship in the circadian rhythms of mating activity was detected between D. melanogaster and its sibling species Drosophila simulans. Female- and species-specific circadian rhythms in the mating activity of Drosophila seem to cause reproductive isolation (Sakai, 2001).

To determine whether mating activities differ between day and night, the mating frequencies of D. melanogaster maintained in 12:12 LD cycles were measured. The mating activities of 3-day-old flies do not significantly differ between day (ZT3) and night (ZT12). However, the mating activities of 5-, 7-, and 9-day-old flies significantly differ between day and night (Sakai, 2001).

To determine whether the mating activity of D. melanogaster fluctuates over the day, the mean mating frequencies of two strains (Canton-S and OGS-4) at 9 days of age were measured. The mating activities of both D. melanogaster strains are similar over the day under LD cycles (lights on at 9:00 and lights off at 21:00). The mating activities of both strains at ZT12 are significantly lower than at other times. The rhythms of 7-day-old Canton-S flies were the same under the same LD conditions. Furthermore, the rhythms of 7-day-old flies that are kept under different LD cycles (lights on at 6:00 and lights off at 18:00) after eclosion are similar to those of 7- and 9-day-old flies kept under LD cycles with lights on at 9:00. These results indicate that the rhythms of Drosophila mating activity become synchronized (entrained) to daily LD cycles. To determine whether these rhythms are controlled by an endogenous clock, the mating activities of flies on day 2 of DD after 7 days of entrainment in LD cycles were measured. The reduction of mating activity at circadian time (CT) 12 remains intact in both strains under DD as well as in LD. These results indicate that the mating activity of D. melanogaster is under the restricted control of an endogenous clock (Sakai, 2001).

To know whether the mating-activity rhythms of Drosophila are controlled by circadian clock genes, the mating activity was measured in per01and tim01 flies that lack rhythms in adult emergence and locomotor activity. In contrast to the two wild-type strains under LD cycles, these mutant flies do not recover mating activity within 3 to 6 h from lights off. When the flies are allowed to mate for 15 min, mating activities in these rhythm mutants are not reduced at CT12 in DD. When per01 flies are allowed to mate for 25 min, mating activity over the day is high (37%-50%) and not reduced at CT12. These results indicate that the circadian clock genes, per and tim, affect the circadian rhythm of Drosophila mating activity. Mating activities of the per01 and tim01 mutants are elevated in the morning. However, mating activity is not elevated in the two mutants under DD. These results indicate that light signals also directly affect mating activity in the morning (Sakai, 2001).

In D. melanogaster, the specific neurons of the optic lobe seem to play a major role as pacemakers for locomotor activity rhythms, because a transgenic line in which per expression is restricted to the lateral neurons has rhythmic locomotor activity. Further evidence is provided by studies of disconnected (disco) mutants that have a severe defect in the optic lobe and are missing lateral neurons. Both locomotor activity and eclosion of the disco mutant are arrhythmic under DD. The present study found that mating activities of disco mutants, like those of per01 and tim01 mutants, are not reduced at CT12. Taken together, these results suggest that arrhythmicity in the mating activities of disco mutants is also caused by defective lateral neurons (Sakai, 2001).

The specific role of sex that may be involved in the circadian rhythm of mating activity can be investigated in rhythm mutants. To determine whether the robust circadian rhythm in mating activity shown in the wild-type is caused specifically by males, females, or a combination of both sexes, Canton-S females were paired with either tim01 or Canton-S males, and tim01 females were paired with either tim01 or Canton-S males. Mating-activity rhythm was abolished in tim01 females crossed with Canton-S males. In contrast, mating-activity rhythm was undetectable regardless of which females were mated with tim01 males. The mating activity of such pairs was very low over the day, suggesting that the tim gene or the genetic background of the tim01 mutant is responsible for low courtship activity of the mutant males. Other experiments demonstrate that females are responsible for generating the circadian rhythm of mating activity in Drosophila. The findings suggested that females need lateral neurons to generate these rhythms and that a female-specific circadian clock suppresses mating activity at CT12 (Sakai, 2001).

Significant differences exist in the mating activities of 5-, 7-, and 9-day-old flies between day and night. To determine whether these differences are caused specifically by males, females, or both sexes, 9- and 3-day-old males were paired with 3- and 9-day-old females, respectively. When males are paired with 9-day-old females, day/night activities clearly differ. In contrast, these differences are absent when 3-day-old females are paired with 3- and 9-day-old males. Thus, Drosophila females contribute to the day/night differences in the mating activities; the experiments suggest that a female-specific circadian clock drives mating activities at least after 5 days of age. In 3-day-old females, the mechanisms to modulate the mating activity may be undeveloped. Alternatively, some sort of mating drive may overwhelm clock control in the youngest females (Sakai, 2001).

To know whether the per gene affects the reduction of mating activities at CT12, the mating activity in combinations of both types of hsp-per females and Canton-S males was examined. No differences in the mating activities between CT12 and CT18 were detected in the HS- experiment as well as in the per01 mutant. However, mating activities at CT12 are significantly lower than those at CT18 in the HS+ experiment. These results are similar to those from the wild-type, suggesting that per gene expression causes the reduction in mating activity at CT12, and that arrhythmicity in the mating activity of the per01 mutant is caused by the per mutation of female flies rather than by the genetic background of the mutants (Sakai, 2001).

The results of the present study demonstrate that mating activity is driven by two mechanisms in Drosophila. One is a circadian pacemaker consisting of clock genes and the other is the direct effect of light. The mating-activity rhythm of D. melanogaster females is under the restricted control of circadian clock genes, and the lateral neurons might be essential to generate the rhythm. Flies, especially males, use olfactory cues for mating, and the circadian rhythm of the olfactory response is robust in Drosophila . Olfactory responses of the wild-type are elevated in the middle of the night in LD cycles, but mating activities are decreased during the early part of the night. Furthermore, the lateral neurons are insufficient to sustain olfactory rhythm but the optic lobe, including the lateral neurons, seems to be essential for mating-activity rhythm according to these results. Thus, the mechanism that generates the mating-activity rhythms might be independent of that which generates olfactory rhythms. A female sex pheromone attracts male courtship in Drosophila, and the sound produced by male wing vibration, referred to as courtship song, affects female receptivity. One explanation for the generation of female mating activity in Drosophila is that females show circadian rhythms in pheromone release and/or responses to auditory signals (Sakai, 2001).

Loss of circadian clock function decreases reproductive fitness in males of Drosophila melanogaster

Circadian coordination of life functions is believed to contribute to an organism's fitness; however, such contributions have not been convincingly demonstrated in any animal. The most significant measure of fitness is the reproductive output of the individual and species. The consequences of loss of clock function on reproductive fitness in Drosophila have been examined with mutated period (per0), timeless (tim0), cycle (cyc0), and Clock (ClkJrk) genes. Single mating among couples with clock-deficient phenotypes results in ~40% fewer progeny compared with wild-type flies, because of a decreased number of eggs laid and a greater rate of unfertilized eggs. Male contribution to this phenotype was demonstrated by a decrease in reproductive capacity among per0 and tim0 males mated with wild-type females. The important role of clock genes for reproductive fitness was confirmed by reversal of the low-fertility phenotype in flies with rescued per or tim function. Males lacking a functional clock show a significant decline in the quantity of sperm released from the testes to seminal vesicles, and these tissues displayed rhythmic and autonomous expression of clock genes. By combining molecular and physiological approaches, a circadian clock was identified in the reproductive system and its role in the sperm release that promotes reproductive fitness in D. melanogaster was defined (Beaver, 2002).

To determine whether the low-sperm phenotype is correlated with clock function in the reproductive system, the activity of clock genes in these tissues was studied. Spatial expression of per and tim was evaluated in flies that carry a per-lacZ reporter construct or express green fluorescent protein under control of the tim promoter. Both reporters exhibited strong activity in the lower testes and SVs, weak activity in the ejaculatory duct and the upper testes, and no activity in the paragonial (accessory) glands. Immunocytochemical analysis shows rhythmic expression of PER and TIM proteins limited to the lower testes and the SVs. PER and TIM were not detected at ZT 8, whereas both proteins are ubiquitously expressed in the nuclei of the epithelial cells forming the lower testes and the SV at ZT 20. PER and TIM proteins are absent in per01, per04, and tim01 flies. However, similar to wild-type, distribution of both proteins in the SV and lower testes was observed in the reproductive system of per01 mutants rescued with a per+ construct and tim01 mutants transformed with a tim+ construct. Nuclear localization of PER and TIM was evident in those flies at ZT 20 (Beaver, 2002).

Because the functional clock of the fruit fly involves out-of-phase cycling of per and Clk mRNAs, in situ hybridization of the male reproductive system was performed with antisense probes for both genes. Both per and Clk mRNA were detected in the lower-testes-SV epithelium. The level of per mRNA was low at ZT 4 and high at ZT 16, whereas Clk mRNA shows cycling in the opposite phase with high levels at ZT 4 and low levels at ZT 16. Taken together, these results demonstrate cycling of clock components that is similar to patterns observed in fly brains and is consistent with the existence of a circadian clock in the male reproductive system (Beaver, 2002).

To elucidate the autonomy of the testes-SV circadian system, BG-luc (reporting per) or tim-luc transgenic flies were used. Testes-SV complexes were dissected from these flies and individually cultured in vitro in LD followed by dark/dark (DD) cycles and a return to LD. Isolated organs show clear, high-amplitude cycling of BG-luc and tim-luc activity during the initial LD cycles with peak expression during the night. Quantitative analysis of the data reveals that 59% of testes-SVs from BG-luc flies and 76% of same organs from tim-luc flies are rhythmic in vitro in the circadian range. On transfer to DD, cycling continues in 36% of both BG-luc and tim-luc organs with reduced amplitude. When the free-running cultures are returned to LD cycles, the amplitude increased for both constructs, demonstrating direct light responsiveness of the testes-SV circadian system. When both constructs were crossed into genetic backgrounds carrying a loss-of-function mutation for the respective clock gene (BG-luc into per01 and tim-luc into tim01), circadian oscillations were eliminated, indicating that wild-type alleles of the two clock genes are needed to support reporter-gene cycling (Beaver, 2002).

Stress response genes protect against lethal effects of sleep deprivation in Drosophila

Sleep is controlled by two processes: a homeostatic drive that increases during waking and dissipates during sleep, and a circadian pacemaker that controls its timing. Although these two systems can operate independently, recent studies indicate a more intimate relationship. To study the interaction between homeostatic and circadian processes in Drosophila, homeostasis was examined in the canonical loss-of-function clock mutants period (per01), timeless (tim01), clock (Clkjrk) and cycle (cyc01). cyc01 mutants show a disproportionately large sleep rebound and die after 10 hours of sleep deprivation, although they are more resistant than other clock mutants to various stressors. Unlike other clock mutants, cyc01 flies show a reduced expression of heat-shock genes after sleep loss. However, activating heat-shock genes before sleep deprivation rescues cyc01 flies from its lethal effects. Consistent with the protective effect of heat-shock genes, is the observation that flies carrying a mutation for the heat-shock protein Hsp83 (Hsp8308445) show exaggerated homeostatic response and die after sleep deprivation. These data represent the first step in identifying the molecular mechanisms that constitute the sleep homeostat (Shaw, 2002).

A sleep-like state has been described in Drosophila on the basis of its similarities to mammalian sleep. This state is characterized by increased arousal thresholds and is regulated homeostatically. Like mammalian sleep, it is abundant in young flies, decreases in older animals and is modulated by stimulants and hypnotics. Perhaps the most important similarity between mammals and flies is homeostatic regulation: when flies are kept awake, they show a large compensatory increase in sleep the next day (Shaw, 2002).

In mammals, the circadian pacemaker alternately promotes and maintains both wakefulness and sleep. Although the circadian pacemaker and the sleep homeostat can interact, little is known about the underlying mechanisms. To evaluate this relationship, homeostasis was evaluated in clock mutants maintained in constant darkness (DD) and deprived of sleep for 3, 6, 9 and 12 h. Under these conditions, sleep is evenly distributed across the day. Upon release from sleep deprivation, wild-type Canton-S flies recover ~30%-40% of the sleep that they lost within 12 h. per01 and Clkjrk show a more prominent sleep rebound, reclaiming ~100% of lost sleep within 12 h. tim01 flies did not show a homeostatic response after 3–6 h of sleep deprivation but displayed a sleep rebound similar to that of per01 and Clkjrk flies after 7, 9 and 12 h of sleep deprivation (Shaw, 2002).

To determine whether death in cyc01 flies is due specifically to sleep deprivation or to hypersensitivity to any environmental challenge, per01, tim01, Clkjrk, cyc01 and Canton-S flies were subjected to several stressors including heat stress, oxidative stress, starvation, desiccation and physical stress. cyc01 flies were as sensitive, but no more so than other genotypes to desiccation and vortex-mixing and survived longer than per01, tim01 and Clkjrk flies when challenged with heat, oxidative stress and starvation. Canton-S flies, which have an intact clock, were more resistant to starvation and desiccation than tim01, Clkjrk and cyc01 flies. These data indicate that cyc01 mutants are vulnerable to prolonged wakefulness in itself and are not merely hypersensitive to non-related stressors (Shaw, 2002).

Regulation of Copulation Duration by period and timeless in Drosophila

The circadian clock involves several clock genes encoding interacting transcriptional regulators. Mutations in the Drosophila clock genes period, timeless, Clock and cycle produce multiple phenotypes associated with physiology, behavior, development, and morphology. It is not clear whether these genes always work as clock components or may also act in some unknown pleiotropic fashion. per and tim are shown to be involved in a novel, male-specific phenotype that affects behavioral timing on the order of minutes. Males lacking per or tim copulate significantly longer than males with normal per or tim function, while females do not show this effect. No correlation between fertility and extended copulation duration was found. Several lines of evidence suggest that the time in copula (TIC) is not regulated by the known clock mechanism: (1) the period of free-running clock oscillations does not appear to affect this phenotype; (2) constant light, which abolishes the clock function, does not alter TIC (3) mutations in the positively acting clock transcription factors, Clk and cyc, do not affect TIC. This study extends the repertoire of behavioral functions involving per and tim genes and uncovers another time scale over which these genes may act (Beaver, 2004).

The genetic basis for copulation duration in Drosophila was first demonstrated through the directional selection of flies for short and long copulation durations over subsequent generations. Since that time, only a handful of genes have been identified that affect copulation duration. Most of these genes appear to affect the development of physical structures or neuronal circuits necessary for successful copulation. Consequently, male flies may display difficulties with the physical interaction of copulation, such as withdrawing genitalia and dismounting from females. In contrast, males observed in this study had no apparent defects of this kind; they terminated prolonged copulations in a manner similar to wild-type males. This suggests that per and tim are somehow involved in measuring the duration of mating behavior as part of their broad functions related to the timing of biological processes on different time scales ranging from seconds to days (Beaver, 2004).

There are two possible mechanisms by which per and tim could participate in determining copulation duration. (1) per and tim may exert pleiotropic effects related to their involvement in development of the fly. It is known that per and tim are expressed during embryonic, larval, and pupal stages; therefore, these genes could affect the development of the CNS, peripheral sense organs, and muscles, leading to altered behavior in adults. (2) per and tim may regulate copulation duration via their expression in sexually mature males. These genes are expressed in the male reproductive system as key components of peripheral circadian oscillators. However, this clock-related expression is not likely to contribute to the extended TIC phenotype because TIM expressed in the male reproductive organs is light sensitive, and constant light does not produce an extended TIC phenotype. These results suggest that the TIC phenotype may involve tissues in adult males where per and tim are expressed in constant light and are not regulated by cyc and Clk. Such unorthodox behavior of per and tim has in fact been reported in several studies. For example, expression of TIM and PER in the fly ovary persists in constant light and does not depend on cyc and Clk; a similar situation could conceivably occur in some as yet unidentified male peripheral tissues. With regard to the nervous system, it has been shown that certain subsets of larval and adult brain neurons show high levels of Tim and Per during the light phase of an LD cycle. Moreover, both proteins were detected in certain brain neurons in cyc and Clk loss-of-function mutants. Such putative neural sites where levels of Tim and Per would be light insensitive and independent of Clk and cyc function may be involved in regulating duration of copulation (Beaver, 2004).

Previous studies have shown that per and tim play significant roles in fly reproductive fitness as regulators of fertility in both male and female flies. This current study further extends the range of per and tim functions in reproduction by demonstrating their interesting role as key modulators of an important reproductive behavior (Beaver, 2004).

The novel Drosophila timblind mutation affects behavioral rhythms but not periodic eclosion

Circadian clock function depends on the tightly regulated exclusion or presence of clock proteins within the nucleus. A newly induced long-period timeless mutant, timblind, encodes a constitutively hypophosphorylated Tim protein. The mutant protein is not properly degraded by light, and timblind flies show abnormal behavioral responses to light pulses. This is probably caused by impaired nuclear accumulation of TimBLIND protein, that is observed in brain pacemaker neurons and photoreceptor cells of the compound eye. timblind encodes two closely spaced amino acid changes compared to the wild-type Tim protein; one of them is within a putative nuclear export signal of Tim. Under constant conditions, timblind flies exhibit 26-hr free-running locomotor rhythms, which are not correlated with a period lengthening of eclosion rhythms and period-luciferase reporter-gene oscillations. Therefore it seems possible that Tim -- in addition to its well-established role as core clock factor -- functions as a clock output factor, involved in determining the period length of adult locomotor rhythms (Wulbeck, 2005).

Evidence is presented for the importance of Tim nuclear accumulation for the proper regulation of locomotor behavior. A chemically induced mutation within the tim gene (timblind) was isolated whose encoded mutant protein is constitutively hypophosphorylated, is partially resistant to light-induced degradation, and fails to accumulate in photoreceptor cell and neuronal nuclei. The TimBLIND protein contains two amino acid changes in the C-terminal part of the protein, one of which (Leu1131 to Met) is part of a potential NES (Wulbeck, 2005).

Although a combinatorial action of the two changes cannot be ruled out, the results suggest that a defect in the proposed NES accounts for the observed timblind phenotypes. (1) The Ala1128-to-Val change is conservative and occurs in a region of the protein that is not part of any known target or signal sequence and that is not involved in any known protein interactions (except for the large CRY interaction domain, which consists of almost the entire Tim protein). Therefore, this amino acid substitution most likely does not interfere with either structure or function of Tim. (2) There is no substantial accumulation of mutant TimBLIND protein in the nucleus at any time during the circadian cycle. This is due to either impaired nuclear entry or enhanced nuclear export. Given that the Leu1131 to Met change occurs in a putative NES, it is possible that TimBLIND is now tightly and constitutively bound to nuclear export factors. This would also explain why it was not possible to detect substantial amounts of hyperphosphorylated forms of TimBLIND in the second part of the night when Tim is normally nuclear. Perhaps it is this nuclear phosphorylation of Tim that blocks nuclear export in wild-type flies. In this view it is also not surprising that overexpression of GSK-3 kinase, known to phosphorylate Tim, is not able to shift mutant hypophosphorylated forms of TimBLIND to hyperphosphorylated forms. Either GSK-3 is not the responsible enzyme for nuclear phosphorylation of Tim or there is just not enough Tim substrate because of constant nuclear depletion of TimBLIND (Wulbeck, 2005).

What are the consequences of the faulty Tim phosphorylation observed in timblind flies? They largely seem to be restricted to Tim itself, because cyclic expression, nuclear accumulation, as well as temporal mobility changes of PER are affected to a lesser extent. In contrast, the TimBLIND protein shows drastic defects in nuclear accumulation and almost no abundance fluctuation during the circadian cycle, in addition to the phosphorylation defects described above. This is another example of a newly emerging picture that Per and Tim can function independently of each other (Wulbeck, 2005).

If Per expression and function is not strongly affected by timblind, how is it that the mutant flies free-run with a 26-hr period? Although the free-running period of Per oscillations in the behavior controlling clock neurons has not been recorded directly, recordings of luminescence rhythms of flies expressing a PER-LUC fusion protein predominantly in clock neurons of the dorsal brain suggest that the circadian clock in timblind flies may tick with a 24-hr period and not with a 26-hr one, as would be predicted from their behavioral rhythm. Moreover, eclosion rhythms free-run with a 24-hr period in timblind flies. This is intriguing, because other tim alleles increase the period length of locomotor rhythms and eclosion to a similar extent. Both pupal and adult brains contain the same set of pacemaker neurons: the ventrally located small and large lateral neurons (LNv's, the more dorsally located LNd's, and three groups of neurons in the dorsal brain (DN1-3). Nevertheless, eclosion and adult locomotion could be controlled by different subsets of pacemaker neurons, because clock gene cycling has not been determined in all of these groups under free-running conditions. Therefore, it is possible that some of these cells indeed show a period of 26 hr (Wulbeck, 2005).

Alternatively, the discrepancy between the apparently normal clock function and lengthened free-running period of locomotor rhythms of timblind flies could be explained by a novel function of Tim in the nucleus in addition to its well-established role as crucial clock factor. If the circadian clock in timblind flies runs in a globally slow manner, all clock outputs would have longer-than-normal periods. But this is clearly not the case, given the normal eclosion rhythms and PER-LUC oscillations observed in the mutant flies. Therefore, it seems that the timblind defect is not at the level of the central oscillator but rather at the interface between the pacemaker and the output mediating locomotor rhythms. Although Tim alone is not able to function as a repressor of Clk/Cyc-activated transcription in vitro, it is possible that Tim acts alone or together with other proteins to regulate clock-controlled-genes (CCGs) downstream of the core molecular clock. A Per-independent function of Tim in regulating CCGs has been inferred from in vitro studies in which high levels of Tim (without Per) resulted in the activation (rather than the expected suppression) of E-box-driven reporter-gene expression. That this might indeed be the case is also indicated by the distinct effects of timblind on per-luc vs. tim-luc expression. Whereas per transcription occurs with an advanced peak and reduced cycling amplitude compared with control flies, tim expression levels are drastically increased, and the cycling amplitude is also blunted. This indicates differences in the regulation of the per and tim promoters and different functions for Tim in the feedback regulation acting on these regulatory sequences (Wulbeck, 2005).

Control of daily transcript oscillations in Drosophila by light and the circadian clock

The transcriptional circuits of circadian clocks control physiological and behavioral rhythms. Light may affect such overt rhythms in two ways: (1) by entraining the clock circuits and (2) via clock-independent molecular pathways. In this study the relationship between autonomous transcript oscillations and light-driven transcript responses were examined. Transcript profiles of wild-type and arrhythmic mutant Drosophila were recorded both in the presence of an environmental photocycle and in constant darkness. Systematic autonomous oscillations in the 12- to 48-h period range were detectable only in wild-type flies and occurred preferentially at the circadian period length. However, an extensive program of light-driven expression was confirmed in arrhythmic mutant flies. Many light-responsive transcripts are preferentially expressed in the compound eyes and the phospholipase C component of phototransduction, NORPA (no receptor potential), is required for their light-dependent regulation. Although there is evidence for the existence of multiple molecular clock circuits in cyanobacteria, protists, plants, and fungi, Drosophila appears to possess only one such system. The sustained photic expression responses identified here are partially coupled to the circadian clock and may reflect a mechanism for flies to modulate functions such as visual sensitivity and synaptic transmission in response to seasonal changes in photoperiod (Wijnen, 2006).

In recent years, five different sets of circadian transcripts have been proposed for the Drosophila head. Unfortunately, the overlap between these transcript sets is very poor (seven transcripts), and it falsely excludes numerous confirmed circadian transcript oscillations. These recent genome-wide surveys for rhythmic transcription have defined groups of circadian transcripts based on empirical ranking and filtering approaches, often using necessarily arbitrary cut-offs. To complement these studies a method was developed for examining periodic expression at the systems level, allowing pursuit of a number of new investigations. This new strategy enabled description of the programs of circadian and light-driven transcription in the adult fly head. Because this method emphasizes uniformity in period length and peak phase while tolerating inter-experimental variability in amplitude, it is particularly successful at measuring oscillatory trends across different independent experiments. Integrative analysis of all available microarray time-series data allowed detection and ranking of oscillatory transcript profiles with improved resolution and revealed a circadian expression program that is much more substantial than the apparent consensus (or lack thereof) between different published studies indicates. Some of the best described and strongest circadian oscillations (per, Clk, Pdp1, cry, and to) were missed in one or more of the previously published studies, but all of these rank high in the current integrative analysis. Although there are relatively few genes (~50) that show the same level of circadian regulation as the oscillating components in the core clock circuits (per, tim, Clk, cry, vri, and Pdp1), the results provide evidence for a substantially broader circadian expression program downstream of the core oscillator. This suggests that the circadian clock is responsible for both the purely circadian expression patterns of a limited set of genes and the partial circadian regulation of a much greater group (Wijnen, 2006).

Whereas many of the genes composing the Drosophila clock are expressed with a circadian rhythm in wild-type flies, all known clock gene oscillations cease if just one of them is lost by mutation. It was reasoned that all of the circadian oscillations in gene expression that were identified in this study should stop in tim01 mutants if these were truly devoid of a circadian clock. Alternatively, rhythmicity could theoretically persist in a subset of the genes if their expression depended on a parallel, novel circadian clock. The distribution analyses allowed addressing of these two alternative possibilities. No alternative systems of oscillatory expression are detectable for the 12-48-h range of period lengths. In the absence of tim-dependent clock circuits, no circadian patterns of gene expression were detected. This latter result, from microarray and Northern analyses, is in agreement with earlier observations, with limited sampling of individual circadian transcripts. Moreover, the absence of detectable molecular circadian rhythms fits well with the abolition of circadian eclosion and locomotor rhythms in tim01 flies. Thus, Drosophila appears to possess only one, tim-dependent, circadian clock. This observation contrasts with results from cyanobacteria, protists, fungi, and plants that suggest the presence of multiple oscillators, sometimes even in the same cell. Although there is no compelling evidence supporting the existence of alternative circadian clocks in Drosophila that are not entrainable to light or independent from transcriptional rhythms, this study does not disprove these possibilities. The results complement and extend previous microarray and differential display analyses using different arrhythmic mutants (per0 or Clkjrk) in which few or apparently no daily transcript oscillations persisted in the mutant context (Wijnen, 2006).

Comparative analysis of data collected from wild-type and arrhythmic mutant flies in the presence or absence of an environmental photocycle allowed identification of a program of light-driven regulation. The tim01 mutant flies used for these experiments do not just have a defective circadian clock, but because TIM degradation is a major mechanism of clock re-setting, they have also lost the main photic input pathway that entrains the clock circuits to light. In a wild-type context, light can directly entrain clock-bearing tissues in a cell-autonomous manner by activating the circadian photoreceptor CRY, or it can entrain the pacemaker neurons in the brain via phototransduction in the visual organs. TIM is the target for CRY's effect on the clock circuits, and it may also play a role in mediating entrainment via the visual organs. In spite of their defective clock circuits and circadian entrainment pathways, tim01 mutant flies retain an extensive set of daily transcript oscillations in the presence of an environmental photocycle. By comparing the light-driven expression signature that was found for tim01 with the microarray analysis for per0 LD and with confirmatory northern analyses, it was established that many light-driven transcripts show the same expression profiles in per0 and tim01 arrhythmic mutants. Moreover, the light-driven expression response found in a combined per0 and tim01 LD microarray dataset is comparable in size to the clock-dependent circadian expression program (Wijnen, 2006).

Light-regulated genes fall into two classes, a clock-independent class, and a group of genes that are also clock-controlled. That there are clock-independent patterns of light-regulated gene expression suggests that coordinate clock- and light-control can be disadvantageous in some circumstances. For example, although the clock carries phase information about the photocycle, it may not be able to carry information about day length and sunlight intensity, and some photoprotective functions might be better linked to acute light activation so that they are delivered only when needed. Such a case might be made for ultraviolet-induced melanogenesis in human skin. In contrast, it is suspected that many genes controlled by light and the clock contribute to processes that require both daily anticipation of changes in light and light responsiveness (Wijnen, 2006).

A survey of published expression studies for the selection of light-regulated genes indicates that many of them are prominently expressed in the adult compound eyes (trpl, CdsA, Pkc53E, dlg1, Slob, CG17352, CG5798, CG7077, CdsA, dlg1, Slob, and trpl). Indeed, comparative transcript profiling studies of wild-type and eya2 mutant flies predict expression in the adult compound eyes for 22 of the 27 light-dependent transcripts (Wijnen, 2006).

Two of the confirmed light-regulated transcripts (trpl and CdsA) encode known regulators of phototransduction. Daily oscillations in the transcript levels have been observed for trpl, which encodes a light-activated calcium channel. Although some effects on light-activated conductance have been observed in a trpl null mutant, the major light-dependent cation channel in Drosophila appears to be encoded by its homolog trp (transient receptor potential). Instead, the TRPL protein may have a specific function in phototransduction during extended illuminations and for adaptation of the light response to dim background light. The effect of TRPL on long-term adaptation is thought to be mediated via light-dependent subcellular translocation of TRPL protein, resulting in a preferred localization at the photoreceptor membranes in the dark and in the cell-bodies in the light. Experiments in the blowfly Calliphora vicina indicate that this translocation does not require regulation at the transcript level, but it is possible that the daily evening peaks of the trpl transcript in Drosophila facilitate efficient accumulation of TRPL protein at the rhabdomeres around dusk. Daily fluctuations are also exhibited by the transcript for CdsA (CDP diglyceride synthetase). The CDSA protein is localized to photoreceptor neurons and catalyzes the synthesis of CDP-diacyl glycerol from phosphatidic acid and CTP071. This enzymatic function helps generate the signaling compound phosphatidyl inositol 4,5-bisphosphate, which is consumed during phototransduction by the phospholipase C NORPA. Studies of CdsA loss-of-function and gain-of-function mutants indicate that by controlling availability of phosphatidyl inositol 4,5-bisphosphate, CDSA expression levels affect the gain of the phototransduction response. Periodic variation of CdsA expression under influence of the environmental photocycle could, therefore, be hypothesized to promote daily variations in visual sensitivity (Wijnen, 2006).

Two other light-driven transcripts, dlg1 and Slob, are associated with the regulation of synaptic transmission. The dlg1 (discs large 1) gene has roles in control of cell growth and differentiation as well as synaptic function. DLG1 spatial expression pattern includes synaptic sites in the adult brain and the outer membrane of photoreceptors, where it localizes Sh (Shaker) potassium channels (Wijnen, 2006).

Slob is negatively regulated by light in a clock-independent manner in addition to being one of the most robustly oscillating circadian transcripts in the adult head. The clock-dependent and light-dependent fluctuations that were uncovered for the Slob transcript are reflected in the SLOB protein levels observed in photoreceptor cells and whole heads. A number of findings point to a possible role for SLOB in mediating overt behavioral rhythms. SLOB protein is thought to bind the SLO and EAG potassium channels, and can directly enhance SLO activity, as well as mediate the inhibitory effect of 14-3-3ζ on SLO. slo mutants have altered potassium channel currents and reported defects in flight, male courtship, and circadian locomotor behavior, whereas mutations of eag display hyperactivity, and affect potassium currents and courtship behavior (Wijnen, 2006).

As mentioned above, circadian rhythms in adult Drosophila can be entrained to a LD cycle via either opsin-mediated photoreception in the light-sensing organs (compound eyes, ocelli, and eyelets) or cell-autonomous activation of the circadian blue-light photoreceptor CRY. Interestingly, the contribution of visual photo-transduction to circadian photo-entrainment is apparently restricted to a few pacemaker neurons in the brain, a situation reminiscent of photo-entrainment of the clock circuits in the mammalian brain via the retina and the retino-hypothalamic tract. In contrast, Drosophila CRY contributes to photo-entrainment in many more clock-bearing tissues, including the visual organs. CRY mediates the light-dependent degradation of TIM, which in turn affects CLK/CYC transcriptional activity in a manner that depends on the phase of the circadian cycle (Wijnen, 2006).

The light-driven transcript responses identified in this study resemble circadian responses in amplitude and duration in the context of a photocycle, and are found for a number of genes with a verified circadian expression profile. It was, therefore, asked whether these light-driven transcript responses depend on the same light sensors as the circadian system. For the most part light-driven regulation was found not to require CRY. Given TIM's status as a target for CRY-mediated light responses, it is perhaps not surprising that light-driven expression responses that do not require TIM function also persist in the absence of CRY. There is one interesting exception to this rule: The light-mediated repression of the Slob transcript apparently requires CRY, but not TIM. If this observation indeed represents a previously unappreciated function for CRY, it may share this role with the phospholipase C enzyme NORPA, since norpA mutants similarly affect the Slob transcript (Wijnen, 2006).

In contrast with CRY, it was found that NORPA phototransduction mediates many if not all of the other clock-independent light responses identified in this study. Based on the overlapping expression of both NORPA and its target transcripts in the adult compound eyes and NORPA's well-documented role in phototransduction, the simplest interpretation of these observations would be that light-driven expression responses are mediated by visual phototransduction. Nevertheless, NORPA is known to be expressed outside of the visual organs, and it has been reported to affect functions unrelated to phototransduction, such as olfaction and temperature-controlled clock gene oscillations. Additionally, norpA loss-of-function mutants show a number of defects in circadian locomotor behavior. Their activity profiles reveal an advanced evening activity peak under LD conditions and a shortened intrinsic period length under DD conditions, and they are slow to adjust their behavior to shifting cycles of light and dark. One possible interpretation of these observations is that NORPA plays a role in seasonal photoperiodic control of locomotor behavior. The norpA mutant phenotype partially mimics the effect of a shortened photoperiod, which also leads to advanced evening activity peaks and shortened period lengths. Recent studies provide further evidence connecting norpA to seasonal control of daily locomotor activity patterns. norpA mutants show abnormally high levels of splicing in the 3' untranslated region of per mRNA. Increased splicing of per transcripts at this site has been shown to contribute to the advanced accumulation of PER protein and the advanced timing of evening locomotor activity that is observed for shorter photoperiods and lower temperatures. Thus, NORPA's effect on splicing of per may be an important determinant of the 'short day' locomotor behavior phenotype of norpA mutants. The sustained photic expression responses that are identified here may reflect yet another mechanism for flies to translate a seasonal environmental signal (photoperiod) into a set of molecular signals. Photoperiodic control of transcripts associated with functions in visual sensitivity (trpl and CdsA) and synaptic transmission (Slob and dlg1) may be relevant to adaptive responses in the visual system and the brain. NORPA's involvement in both regulating per splicing and mediating photoresponses at the transcript level raises questions as to if and how these two molecular functions are connected. One possibility is that both reflect NORPA-dependent selective regulation of mRNA stability that takes place in the compound eyes (and perhaps also the brain). Whether or not NORPA's function in circadian locomotor behavior involves some of the light-dependent expression responses that have been identified could be examined by targeted misexpression studies. The subset of transcripts that have been independently confirmed to exhibit both NORPA-dependent light responses and strong clock-dependent circadian regulation might be particularly relevant to these experiments (Wijnen, 2006).

This paper has reported a new strategy for analyzing oscillatory patterns in microarrray data that allowed answer general questions about oscillatory gene systems in the fly head. By applying this strategy to 17 d of data, it was conclusively demonstrated that there are more than a hundred circadian transcript oscillations in the fly head. Additionally, in a search for rhythmic gene activity over a wide range of periods (from 12 to 48 h), it was established that 24-h periodicity constitutes the only broad program of transcriptional oscillation. It was further found that the tim-dependent clock is the sole transcriptional circadian clock in Drosophila. Thus, the fly appears to differ from cyanobacteria, protists, plants, and fungi, which are thought to possess multiple circadian clocks. Lastly, a novel, light-regulated system of gene regulation was found in Drosophila that is largely dependent on norpA-mediated phototransduction. This system regulates about the same number of genes as the clock, including a number of circadian genes. This study defines three types of transcripts that oscillate in wild-type flies: those from purely clock-regulated genes, those that are purely photocycle-regulated, and those expressed by genes that respond to both inputs (Wijnen, 2006).

Veela defines a molecular link between Cryptochrome and Timeless in the light-input pathway to Drosophila's circadian clock

Organisms use the daily cycles of light and darkness to synchronize their internal circadian clocks with the environment. Because they optimize physiological processes and behavior, properly synchronized circadian clocks are thought to be important for the overall fitness. In Drosophila, the circadian clock is synchronized with the natural environment by light-dependent degradation of the clock protein Timeless, mediated by the blue-light photoreceptor Cryptochrome (Cry). This paper report identification of a genetic variant, Veela, which severely disrupts this process, because these genetically altered flies maintain behavioral and molecular rhythmicity under constant-light conditions that usually stop the clock. The Veela strain carries a natural timeless allele (ls-tim), which encodes a less-light-sensitive form of Timeless in combination with a mutant variant of the F-box protein Jetlag. However, neither the ls-tim nor the jetlag genetic variant alone is sufficient to disrupt light input into the central pacemaker. A strong interaction between Veela and cryptochrome genetic variants, demonstrating that the Jetlag, Timeless, and Cry proteins function in the same pathway. Veela also reveals a function for the two natural variants of timeless, which differ in their sensitivity to light. In combination with the complex array of retinal and extraretinal photoreceptors known to signal light to the pacemaker, this previously undescribed molecular component of photic sensitivity mediated by the two Timeless proteins reveals that an unexpectedly rich complexity underlies modulation of this process (Peschel, 2006).

Veela is abnormally rhythmic in constant light, similar to mutations affecting the blue-light photoreceptor Cry. Veela's phenotype is due to the simultaneous presence of the ls-tim allele (encoding a less-sensitive form of Tim) and the jetc variant encoding a mutant form of the F-box protein Jet. Veela genetically and molecularly interacts with cryb, indicating that Tim, Jet, and Cry function in the same circadian light-synchronization pathway. These findings show that additional factors are necessary to elicit the phenotypes previously associated with jet variants. In particular, only when jetc is linked to the ls-tim allele, which encodes a less-light-sensitive form of Tim, can abnormal behavioral rhythmicity in LL be observed. The importance of the Jet protein per se in the light-entrainment process remains unclear, also when considering certain aspects of the original jet study in conjunction with the findings presented in his study. All control flies used by Koh (2006) came from a y w genetic background, which carries the s-tim allele. Contrarily, all jetc or jetr mutant flies carried the ls-tim allele (necessarily; otherwise, they would have behaved like WT). It follows that behavioral and molecular differences between control and mutant flies reported by Koh in fact reflect the combined effects of ls-tim (vs. s-tim) and jetc (vs. jet +). In conjunction with Western blot data showing an increased jet-independent stability of the larger Tim form compared with the smaller one, it seems that the effects on Tim degradation previously attributed to jet variants are mainly a reflection of the different features of the two Tim proteins. This may also explain why Koh saw only very subtle effects of their mutant Jet proteins on Tim degradation in vitro (Peschel, 2006).

Nevertheless, it is clear that jet influences the light-input pathway of the circadian clock; WT flies behave arrhythmically in LL, even though they carry ls-tim. Moreover, Veela strongly interacts with Cry, a crucial protein for circadian light input in flies. Importantly, these findings reveal that, with the current knowledge, an in vivo function for jet's F-box protein can be demonstrated only when the available jet variants are combined with ls-tim. To ultimately resolve the specific function of the Jet protein in the light-input pathway, loss-of-function jet mutants (Debruyne, 2006) or specific RNAi transgenics need to be generated and analyzed chronobiologically (Peschel, 2006).

Characterization of Veela also led to the assignment of a biological function for the two natural tim variants that were identified many years ago. This study has show that Tim encoded by the ls-tim allele is more stable after light exposure, and that this increased stability has behavioral consequences when flies are exposed to constant light; if the ls-tim allele is linked to jetc, these flies behave abnormally rhythmically in LL. If jetc is linked to s-tim, the flies behave like WT and become arrhythmic in LL. Therefore, the less-light-sensitive Tim form encoded by ls-tim is necessary and sufficient to block light input into the circadian clock of jetc flies. In nature, the natural polymorphism at the tim (and perhaps jet) locus might be used to fine-tune the light sensitivity of Drosophila's circadian clock on a purely molecular level. In conjunction with various anatomical light-input routes that are known to send light to Drosophila's circadian pacemaker, these findings reveal a glimpse of the potential complexity of this process. The frequent and random occurrence of tim and jet variants in currently used laboratory strains also speaks to a more cautious strain selection and genotyping in all studies concerning light-input pathways to the circadian clock (Peschel, 2006).

Natural selection favors a newly derived timeless allele in Drosophila melanogaster

Circadian and other natural clock-like endogenous rhythms may have evolved to anticipate regular temporal changes in the environment. A mutation in the circadian clock gene timeless in Drosophila melanogaster has arisen and spread by natural selection relatively recently in Europe. When introduced into different genetic backgrounds, natural and artificial alleles of the timeless gene affect the incidence of ovarian diapause, arrested ovarian development, in response to changes in light and temperature. The natural mutant allele alters an important life history trait that may enhance the fly's adaptation to seasonal conditions (Tauber, 2007).

Although polymorphism at a single locus may sustain adaptive variation in nature for both behavioral and morphological phenotypes, there are few well-documented examples where a new, naturally arising adaptive mutation has spread through a population. In Drosophila, circadian behavior is generated by regulatory interactions among a number of canonical clock genes. One of these genes, timeless (tim), encodes a light-responsive component that has two allelic forms, ls-tim and s-tim (Rosato, 1997). The ls-tim allele generates both full-length L-TIM1421 and truncated S-TIM1398 products from an upstream initiating methionine codon and a second ATG 23 codons downstream. In s-tim, deletion of the G nucleotide at position 294 interrupts the upstream reading frame with a stop codon, generating S-TIM1398, from the downstream ATG. These variants were identified initially in laboratory strains; whether this polymorphism was present in nature was investigated (Tauber, 2007).

Drosophila melanogaster isofemale lines were established from natural populations collected from southern Italy to Sweden. A polymerase chain reaction-based strategy identified the status of the two 5' tim haplotypes in flies from isofemale lines. The frequency of ls-tim was plotted against latitude; regression analysis and subsequent spatial autocorrelation statistics revealed a significant latitudinal cline, with high frequencies of ls-tim in southern Europe. Phylogenetic analyses of tim alleles showed that all ls-tim haplotypes, irrespective of geographical location, clustered at the top of the trees, which suggests that this is the derived allele produced by the insertion of the G nucleotide. Assuming that the split between D. melanogaster and D. simulans occurred 2 million to 2.5 million years ago, it is calculated that the ls-tim allele originated ~8000 to 10,000 years ago, coinciding with the postglacial period and subsequent colonization of the Eurasian continent by D. melanogaster (Tauber, 2007).

The frequencies were examined of the derived ls-tim allele southward from the putative site of origin, Novoli, Italy, which has the highest ls-tim frequency. Isofemale lines were established from populations in Crete, Israel, and Africa (Kenya and Zimbabwe). The frequency of ls-tim was 0.138 in Crete, 0.318 in Israel, and zero in sub-Saharan Africa. When the Cretan and Israeli populations were added to the analyses, the data did not conform to the latitudinal cline. However, when all the ls-tim frequencies were replotted against direct distance from Novoli, the regression was highly significant and was further enhanced when realistic and predominantly land-based distances between Novoli and all locations were used (Tauber, 2007).

The results support the view that the derived ls-tim allele arose in southern Italy about 8000 to 10,000 years ago and has spread, perhaps quite recently, in all directions as a result of directional selection. Alternatively, a recent selective sweep might not have allowed enough time for the accumulation of genetic variation around the polymorphic tim site, and consequently balancing selection might be difficult to detect with neutrality tests. Under such a balancing scenario, ls-tim would be particularly well adapted to southern Italy but would be less advantageous farther north or farther south (Tauber, 2007).

To investigate phenotypes that might provide the substrate for selection, tests were performed to see whether temperature compensation -- the ability of the clock to maintain a circadian 24-hour period during fluctuations in temperature -- is driving the observed directional selection. Polymorphism in another clock gene, period, is maintained by balancing selection, possibly by differential circadian temperature compensation, and shows a robust latitudinal distribution in Europe. However, replicate homozygous natural lines of s-tim and ls-tim and two laboratory strains carrying ls-tim showed similar temperature compensation. To avoid complications with genetic background and to study the effect of the L-TIM isoform, four independent transgenic lines were generated for two tim transgenes, P[L-tim] and P[S-tim], which generated one or the other isoform, respectively, with the available tim promoter sequences. All lines rescued circadian locomotor rhythms in arrhythmic tim01 hosts, with no effect of genotype on temperature compensation (Tauber, 2007). D. melanogaster survive unfavorable seasons by entering a reproductive adult diapause that is mediated in part by a response to short days and long nights at low temperatures. This combined response can be diagnosed in individual females by the lack of eggs in their ovaries caused by an arrest in oogenesis. Isofemale lines were establised from recently captured natural populations, two from southern Italy (Bitetto and Salice Salento) and one from the Netherlands (Houten). Analysis of diapause in homozygous ls-tim and s-tim females within these populations revealed highly significant effects for population, genotype, photoperiod, and population x genotype interaction. The ls-tim females showed reproductive arrest more readily than s-tim females in all three populations. The photoperiodic curves for the two genotypes were largely parallel for the Salice and Houten populations, where even at the longest photoperiod, ls-tim females were more prone than s-tim females to diapause. In contrast, for Bitteto, significant genotype differences emerged only as photoperiods grew shorter [14 hours light/10 hours dark (LD14:10). Latitude also had a significant effect, with northern s-tim females showing significantly higher levels of diapause than southern s-tim females. These results reveal that the ovarian diapause of European D. melanogaster is enhanced at shorter day lengths, at northern latitudes, and by the derived ls-tim allele relative to the ancestral s-tim variant (Tauber, 2007).

Diapause was examined in the tim01 hosts transformed with P[S-tim], P[L-tim],and P[LS-tim], a corresponding transformant line that carries the ls-tim sequence. Highly significant genotype and photoperiod effects were observed, with an enhancement in the diapause responses of females carrying the P[L-tim] and P[LS-tim] transgenes relative to those carrying P[S-tim]. In addition, an unexpectedly low diapause response was observed at the shortest 10-hour photoperiod (LD10:14), in contrast to the data from natural strains. Similar results have been observed in shorter photoperiods with long-standing laboratory stocks, so this may reflect the genetic background on which the transgenes are expressed, or limitations of the 5' tim promoter used to drive the transgenes. Nonetheless, these results reveal that irrespective of genetic background, ls-tim females show a significantly higher level of diapause than s-tim females, and that this is due to the tim locus itself. If these findings are extrapolated to nature, ls-tim (and P[L-tim] and P[LS-tim]) females might be expected to enter diapause earlier than s-tim (and P[S-tim]) females in response to the oncoming European winter (Tauber, 2007).

Whether the circadian arrhythmic tim01null mutant would affect the ovarian phenotype was examined. tim01 was compared to a wild type (ls-tim) after minimizing differences in genetic background, and significantly higher levels of diapause were observed in the mutant, but without a significant response to photoperiod. A similar result was obtained with hemizygous P[LS-tim] transformants compared to tim01 at two photoperiods (LD8:16 and LD16:8) on a different genetic background. Consequently, variation in tim itself, not genetic background, is responsible for these changes in the incidence of diapause (Tauber, 2007).

A latitudinal cline in the incidence of diapause was observed in natural D. melanogaster populations in the eastern United States, with a higher incidence at northern latitudes. Within a single temperate population, genotypes that show higher levels of diapause are stress-resistant and have enhanced fitness under such unfavorable conditions, demonstrating that in temperate habitats with strong seasonality, enhanced diapause in D. melanogaster has adaptive value. The higher levels of diapause observed with ls-tim genotypes may have similar adaptive value in the European environment. Natural strains also show a higher incidence of diapause in carriers of the ancestral s-tim allele in northern populations than in southern populations; this result is consistent with findings that in arthropods, the higher the latitude or altitude, the more readily diapause is induced. Consequently, there is genetic variation other than in tim that is causing this latitudinal change within the s-tim genotype. A candidate locus is the insulin-regulated PI3 kinase gene, which determines diapause levels in two D. melanogaster populations from North America (Williams, 2006). If diapause contributes to the enhanced adaptive value of ls-tim, it is difficult to envisage a balancing scenario where ls-tim would be highly favored in a small region of southern Italy but less favored farther north or south. It is proposed that an origin of the derived ls-tim allele in southern Europe, followed by its subsequent spread by directional selection, provides, counterintuitively, a more compelling model for understanding the elevated frequencies of ls-tim in this geographical region (Tauber, 2007).

A molecular basis for natural selection at the timeless locus in Drosophila melanogaster

Diapause is a protective response to unfavorable environments that results in a suspension of insect development and is most often associated with the onset of winter. The ls-tim mutation in the Drosophila clock gene timeless has spread in Europe over the past 10,000 years, possibly because it enhances diapause. The mutant allele attenuates the photosensitivity of the circadian clock and causes decreased dimerization of the mutant Timeless protein isoform to Cryptochrome, the circadian photoreceptor. This interaction results in a more stable Timeless product. These findings reveal a molecular link between diapause and circadian photoreception (Sandrelli, 2007).

Wild European populations of Drosophila melanogaster have two major alleles of the timeless (tim) gene, ls-tim and s-tim. These alleles differ in their use of two alternative translational starts to generate longer (L-TIM1421) and/or shorter (S-TIM1398) isoforms. The ls-tim allele is derived from the s-tim allele, and directional selection is thought to have created a latitudinal gradient of ls-tim frequency within the past 10,000 years, perhaps due to an enhanced fitness of ls-tim individuals in temperate environments. TIM is a cardinal component of the circadian clock, and its light sensitivity via its physical interaction with the circadian photoreceptor cryptochrome (CRY) mediates the fly's circadian responses to light. This photoresponse can be quantified at the behavioral level by studying the fly's locomotor response to brief light pulses delivered at zeitgeber time 15 (ZT15), three hours into the night phase of a light/dark [12 hours of light alternating with 12 hours of darkness (LD12:12)] cycle that generates a phase delay of a few hours; the same light stimulus administered late at night (ZT21) generates a phase advance (Sandrelli, 2007).

The enhanced stability of L-TIM might be expected to contribute to the higher levels of TIM observed in natural ls-tim flies and to reduced circadian photoresponsiveness. Circadian light responses in Drosophila are mediated both by the canonical visual pathway, which uses rhodopsins, and by CRY. After stimulation by light, CRY can physically interact with TIM and/or PERIOD in yeast, in Drosophila S2 cells, and in vivo. These PER/TIM/CRY interactions lead to TIM degradation and subsequent PER instability, which releases the negative autoregulation of PER on the per and tim genes. Therefore the physical interaction of the L-TIM and S-TIM isoforms with CRY was examined in the yeast two-hybrid system. No interactions between TIM and CRY occurred in the dark, and the level of interaction between CRY and L-TIM in light was weaker than that between CRY and S-TIM in both plate and liquid assays. As a control, the interaction was examined of L-TIM and S-TIM with the large fragment of PER (residues 233 to 685) that is stable in yeast, but these PER/TIM interactions were not significantly different. These results indicate that the differences in interaction between the two TIM isoforms and CRY are a specific effect due to the additional N-terminal 23 residues in L-TIM, which interfere with the light-dependent dimerization of CRY (Sandrelli, 2007).

A reduced L-TIM/CRY interaction may explain the differences in the fly's circadian photoresponsiveness and the enhanced L-TIM stability. The observation that ls-tim females are more prone to ovarian diapause at any day length (Tauber, 2007) is also consistent with the results presented in this study. As in the corresponding diapause profiles (Tauber, 2007), the transformants conclusively reveal that the circadian photoresponsive phenotypes of natural tim variants are not due to linkage disequilibrium between tim and a nearby locus, but they are attributable to tim itself. Furthermore, the similarity in behavior of natural s-tim variants and P[S-TIM] transformants suggests that the residual putative truncated N-terminal 19-residue TIM product from the s-tim allele does not play any major role in the studied phenotypes (Sandrelli, 2007).

It has been argued that the light sensitivity of the circadian clock needs to be abated in temperate zones because of the dramatic increase in summer day lengths in northern latitudes. One mechanism for this process involves a reduced sensitivity to light-induced disturbance by having a higher pacemaker amplitude. However, the amplitude of TIM cycling in DD was not significantly different between the two variants, nor were there any significant differences in amplitude or phase of the tim mRNA cycle between the s-tim and ls-tim genotypes. Another way to attenuate circadian photoresponsiveness in temperate zones may be by filtering light input into the clock. The molecular changes to the L-TIM protein may buffer the circadian response to light in ls-tim individuals, even in the presence of S-TIM, and may contribute to the positive Darwinian selection observed for ls-tim in the European seasonal environment (Sandrelli, 2007).

Contribution of visual and circadian neural circuits to memory for prolonged mating induced by rivals

Rival exposure causes Drosophila melanogaster males to prolong mating. Longer mating duration (LMD) may enhance reproductive success, but its underlying mechanism is currently unknown. This study found that LMD is context dependent and can be induced solely via visual stimuli. In addition, it was found that LMD involves neural circuits that are important for visual memory, including central neurons in the ellipsoid body, but not the mushroom bodies or the fan-shaped bodies, and may rely on the rival exposure memory lasting for several hours. LMD is affected by a subset of learning and memory mutants. LMD depends on the circadian clock genes timeless and period, but not Clock or cycle, and persists in many arrhythmic conditions. Moreover, LMD critically depends on a subset of pigment dispersing factor neurons rather than the entire circadian neural circuit. This study thus delineates parts of the molecular and cellular basis for LMD, a plastic social behavior elicited by visual cues (Kim, 2012).

These findings provide evidence that males retain the memory of rival exposure, based primarily on visual stimuli, for several hours and lengthen mating duration accordingly. Indeed, LMD could be induced by allowing a male to view flies of either sex through a transparent partition, flies of different species or images of themselves in a mirror, indicating that LMD could be generated by visual stimuli without chemical communication. Not only could the LMD defects of per and tim mutants be rescued by the expression of PER or TIM via the nonrhythmic GAL4 driver, expression of PER in PDF neurons was sufficient to restore LMD to per mutants. Moreover, LMD requires electrical activity in lateral neurons, but not some of the dorsal neurons that are important for circadian rhythm. It therefore seems unlikely that circadian rhythm regulation is crucial for LMD. LMD involves the memory of rival exposure that lasts for several hours and is resistant to anesthesia and that it requires the rut function in the ellipsoid body. Finally, it was found that LMD generation depends on the activity of the compound eye, the PDF neurons and a subset of neurons in the ellipsoid body (Kim, 2012).

Recent studies of social experience-mediated and context-dependent sexual behaviors of the fruit fly implicate chemical communication of males via pheromones as being important. This study found that vision in a social setting is also important for generating LMD. Although a recent report found that males use multiple redundant cues to detect mating rivals, this study found that LMD can be elicited by visual cues because rearing flies in constant darkness eliminated LMD, blind mutants and males with defective vision showed no LMD, and LMD can be generated simply by placing a mirror to allow a singly reared male fruit fly to see his reflection for 5 d. The visual stimulus for LMD likely derives from the red compound eye in motion because LMD can be induced by males of different species or females visible through a transparent film, but not by mutant males without red pigment in their compound eyes (Kim, 2012).

LMD provides a new method for studying visual memory. To date, learning and memory studies in flies have focused primarily on the memory circuits in mushroom bodies; however, LMD requires a subset of neurons in the ellipsoid body rather than mushroom bodies. The ellipsoid body is the central brain region required for visual learning and memory, whereas mushroom bodies are not required for memory formation in visual learning in a flight simulator. Given that the mating duration assay is simpler than the flight simulator for the investigation of visual memory, it can be useful for large-scale genetic screens to identify mutants with altered visual memory (Kim, 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 ClkJrk, 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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timeless: Biological Overview | Evolutionary Homologs | Regulation | Protein Interactions and Post-transcriptional Regulation | Developmental Biology | Effects of Mutation

date revised: 10 April 2017

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