timeless is an essential component of the circadian clock in a primitive insect, the firebrat Thermobia domestica

Recent studies show that the timeless (tim) gene is not an essential component of the circadian clock in some insects. In the present study, we have investigated whether the tim gene was originally involved in the insect clock or acquired as a clock component later during the course of evolution using an apterygote insect, Thermobia domestica. A cDNA of the clock gene tim (Td'tim) was cloned, and its structural analysis showed that Td'TIM includes 4 defined functional domains, that is, 2 regions for dimerization with PERIOD (PER-1, PER-2), nuclear localization signal (NLS), and cytoplasmic localization domain (CLD), like Drosophila TIM. Td'tim exhibited rhythmic expression in its mRNA levels with a peak during late day to early night in LD, and the rhythm persisted in DD. A single injection of double-stranded RNA (dsRNA) of Td'tim (dstim) into the abdomen of adult firebrats effectively knocked down mRNA levels of Td'tim and abolished its rhythmic expression. Most dsRNA-injected firebrats lost their circadian locomotor rhythm in DD up to 30 days after injection. DsRNA of cycle (cyc) and Clock genes also abolished the rhythmic expression of Td'tim mRNA by knocking down Td'tim mRNA to its basal level of intact firebrats, suggesting that the underlying molecular clock of firebrats resembles that of Drosophila. Interestingly, however, dstim also reduced cyc mRNA to its basal level of intact animals and eliminated its rhythmic expression, suggesting the involvement of Td'tim in the regulation of cyc expression. These results suggest that tim is an essential component of the circadian clock of the primitive insect T. domestica; thus, it might have been involved in the clock machinery from a very early stage of insect evolution, but its role might be different from that in Drosophila.     

Circadian regulation of egg-laying behavior in fruit flies Drosophila melanogaster

Significant progress has been made in our understanding of the neurogenetics of circadian clocks in fruit flies Drosophila melanogaster. Several pacemaker neurons and clock genes have now been identified and their roles in the cellular and molecular clockwork established. Some recent findings suggest that the basic architecture of the clock is multi-oscillatory; the clock mechanisms in the ventral lateral neurons (LN(v)s) of the fly brain govern locomotor activity and adult emergence rhythms, while the peripheral oscillators located in antennal cells regulate olfactory rhythm. Among circadian phenomena exhibited by Drosophila, the egg-laying rhythm is unique in many ways: (i) this rhythm persists under constant light (LL), while locomotor activity and adult emergence become arrhythmic, (ii) its circadian periodicity is much longer than 24h, and (iii) while egg-laying is rhythmic under constant darkness, the expression of two core clock genes period (per) and timeless (tim), is non-oscillatory in the ovaries. In this paper, we review our current knowledge of the circadian regulation of egg-laying behavior in Drosophila, and provide some possible explanations for its self-sustained nature. We conclude by discussing the existing limitations in our understanding of the regulatory mechanisms and propose few approaches to address them.     

Circadian changes in Drosophila motor terminals

In Drosophila melanogaster, as in most other higher organisms, a circadian clock controls the rhythmic distribution of rest/sleep and locomotor activity. Here we report that the morphology of Drosophila flight neuromuscular terminals changes between day and night, with a rhythm in synaptic bouton size that continues in constant darkness, but is abolished during aging. Furthermore, arrhythmic mutations in the clock genes timeless and period also disrupt this circadian rhythm. Finally, these clock mutants also have an opposing effect on the nonrhythmic phenotype of neuronal branching, with tim mutants showing a dramatic hyperbranching morphology and per mutants having fewer branches than wild-type flies. These unexpected results reveal further circadian as well as nonclock related pleiotropic effects for these classic behavioral mutants.     

Transplanted Drosophila excretory tubules maintain circadian clock cycling out of phase with the host

Circadian rhythms in behaviors and physiological processes are driven by conserved molecular mechanisms involving the rhythmic expression of clock genes in the brains of animals [1]. The persistence of similar molecular rhythms in peripheral tissues in vitro [2] [3] suggests that these tissues contain self-sustained circadian clocks that may be linked to rhythmic physiological functions. It is not known how brain and peripheral clocks are organized into a synchronized timing system; however, it has been assumed that peripheral clocks submit to a master clock in the brain. To address this matter we examined the expression of two clock genes, period (per) and timeless (tim), in host and transplanted abdominal organs of Drosophila. We found that excretory organs in tissue culture display free-running, light-sensitive oscillations in per and tim gene activity indicating that they house self-sustained circadian clocks. To test for humoral factors, we monitored cycling of the TIM protein in excretory tubules transplanted into host flies entrained to an opposite light-dark cycle. We show that the clock protein in the donor tubules cycled out of phase with that in the host tubules, indicating that different organs may cycle independently, despite sharing the same hormonal milieu. We suggest that one way to achieve circadian coordination of physiological sub-systems in higher animals may be through the direct entrainment of light-sensitive clocks by environmental signals.     

RNA interference of the clock gene period disrupts circadian rhythms in the cricket Gryllus bimaculatus

Periodic expression of so-called clock genes is an essential part of the circadian clock. In Drosophila melanogaster the cyclic expression of per and tim through an autoregulatory feedback loop is believed to play a central role in circadian rhythm generation. However, it is still elusive whether this hypothesis is applicable to other insect species. Here it is shown that per gene plays a key role in the rhythm generation in the cricket Gryllus bimaculatus. Measurement of per mRNA levels in the optic lobe revealed the rhythmic expression of per in light cycles with a peak in the late day to early night, persisting in constant darkness. A single injection of per double-stranded RNA (dsRNA) into the abdomen of the final instar nymphs effectively knocked down the mRNA levels as adult to about 50% of control animals. Most of the per dsRNA-injected crickets completely lost the circadian locomotor activity rhythm in constant darkness up to 50 days after the injection, whereas those injected with DsRed2 dsRNA as a negative control clearly maintained it. The electrical activity of optic lobe efferents also became arrhythmic in the per dsRNA-injected crickets. These results not only suggest that per plays an important role in the circadian rhythm generation also in the cricket but also show that RNA interference is a powerful tool to dissect the molecular machinery of the cricket circadian clock.     

A fly's eye view of circadian entrainment

The Drosophila circadian clock is an ideal model system for teasing out the molecular mechanisms of circadian behavior and the means by which animals synchronize to day-night cycles. The clock that drives behavioral rhythms, located in the lateral neurons in the central brain, consists of a feedback loop of the circadian genes period (per) and timeless (tim). The molecular cycle, roughly 24 h long, is constantly reset by the environment. This review focuses on the main input pathways of the dominant circadian zeitgeber, light. Light acts directly on the clock primarily through cryptochrome (cry), a deep brain blue-light photoreceptor. CRY activation causes rapid TIM degradation, which is a predicted means of resetting the clock both on a daily basis at dawn and on an acute basis following an entraining light pulse during the night hours. In the absence of cry, the clock can still be driven by photic input through the visual system, though the mechanisms underlying this entrainment are unclear. Temperature can also entrain the clock, although the mechanisms by which this occurs are also unclear.     

An antibody to the Drosophila period protein recognizes circadian pacemaker neurons in Aplysia and Bulla

The molecular mechanisms of the pacemakers underlying circadian rhythms are not well understood. One molecule that presumably functions in the circadian clock of Drosophila is the product of the period (per) gene, which dramatically affects biological rhythms when mutated. An antibody specific for the per protein labels putative circadian pacemaker neurons and fibers in eyes of two marine gastropods, Aplysia and Bulla. As was found for the Drosophila per protein, there is a daily rhythm in the levels of the per-like antigen in Aplysia eyes. Thus, certain molecular features of the per protein, as well as aspects of the temporal regulation of its expression, may be conserved in circadian pacemakers of widely divergent species.     

Time, love and species

Physiological and behavioral phenomena of many animals are restricted to certain times of the day. Many organisms show daily rhythms in their mating. The daily fluctuation in mating activity of a few insects is controlled by an endogenous clock. The fruitfly, Drosophila, is the most suitable material to characterize the genetic basis of circadian rhythms of mating because some mutants with defective core oscillator mechanism, feedback loops, have been isolated. D. melanogaster wild-type display a robust circadian rhythm in the mating activity, and the rhythms are abolished in period or timeless null mutant flies (per(01) and tim(01)), the rhythms are generated by females but not males. Disconnected (disco) mutants which have a severe defect in the optic lobe and are missing lateral neurons show arrhythmicity in mating activities. Thus, the lateral neurons seem to be essential for the circadian rhythm in mating activity of Drosophila. Furthermore, an anti-phasic relation in circadian rhythms of the mating activity was detected between D. melanogaster and their sibling species D. simulans. The Queensland fruit flies or wild gypsy moth also show species-specific mating rhythm, suggesting that species-specific circadian rhythms in mating activity of insect appear to cause a reproductive isolation.     

Flies, clocks and evolution.

The negative feedback model for gene regulation of the circadian mechanism is described for the fruitfly, Drosophila melanogaster. The conservation of function of clock molecules is illustrated by comparison with the mammalian circadian system, and the apparent swapping of roles between various canonical clock gene components is highlighted. The role of clock gene duplications and divergence of function is introduced via the timeless gene. The impressive similarities in clock gene regulation between flies and mammals could suggest that variation between more closely related species within insects might be minimal. However, this is not borne out because the expression of clock molecules in the brain of the giant silk moth, Antheraea pernyi, is not easy to reconcile with the negative feedback roles of the period and timeless genes. Variation in clock gene sequences between and within fly species is examined and the role of co-evolution between and within clock molecules is described, particularly with reference to adaptive functions of the circadian phenotype.     

Involvement of the period gene in developmental time-memory: effect of the perShort mutation on phase shifts induced by light pulses delivered to Drosophila larvae

Phases of circadian locomotor activity rhythms of adult Drosophila reared in constant darkness have been shown to be set by a light stimulus delivered as early as the first-instar larval stage. This implies that a circadian clock functions continuously throughout postembryonic development. The clock genes period (per) and timeless (tim) are expressed cyclically in the larval central nervous system of Drosophila, and daily oscillations of per expression persist throughout metamorphosis in a group of cells, which gives rise to the pacemaker cells underlying locomotor activity rhythms of adults. Therefore, PER and TIM cyclings in these neurons may be responsible for the phenomenon of "larval time-memory." In the absence of any evidence for the involvement of these genes in such a developmental clock, and because circadian-pacemaker functions are underanalyzed in terms of the functions during development, the authors tested the time-memory of a fast-clock period mutant. They show that dark-reared perS mutant individuals as well as wild-type flies can be entrained as larvae and that a brief light pulse given to such entrained larvae can induce phase shifts in animals of either genotype. However, the direction and magnitude of phase shifts were different between wild type and perS, suggesting that a clock under the control of period gene participates in the regulation of developmental time-memory. The authors show that the relevant clock can be entrained by two light input pathways, one involving the phospholipase C encoded by the norpA gene, the other mediated by the blue-light receptor cryptochrome. Phase shifts of molecular oscillations during the larval stage were smaller than those measured by adult behavior, suggesting molecularly transient responses during development.     

Drosophila olfactory response rhythms require clock genes but not pigment dispersing factor or lateral neurons

Odors elicit a number of behavioral responses, including attraction and repulsion in Drosophila. In this study, the authors used a T-maze apparatus to show that wild-type Drosophila melanogaster exhibit a robust circadian rhythm in the olfactory attractive and repulsive responses. These responses were lower during the day and began to rise at early night, peaking at about the middle of the night and then declining thereafter. They were also independent of locomotor activity. The olfactory response rhythms were lost in period or timeless mutant flies (per0, tim0), indicating that clock genes control circadian rhythms of olfactory behavior. The rhythms in olfactory response persisted in the absence of the pigment-dispersing factor neuropeptide or the central pacemaker lateral neurons known to drive circadian patterns of locomotion and eclosion. These results indicate that the circadian rhythms in olfactory behavior in Drosophila are driven by pacemakers that do not control the rest-activity cycle and are likely in the antennae.     

Phosphorylation of period is influenced by cycling physical associations of double-time, period, and timeless in the Drosophila clock.

The clock gene double-time (dbt) encodes an ortholog of casein kinase Iepsilon that promotes phosphorylation and turnover of the PERIOD protein. Whereas the period (per), timeless (tim), and dClock (dClk) genes of Drosophila each contribute cycling mRNA and protein to a circadian clock, dbt RNA and DBT protein are constitutively expressed. Robust circadian changes in DBT subcellular localization are nevertheless observed in clock-containing cells of the fly head. These localization rhythms accompany formation of protein complexes that include PER, TIM, and DBT, and reflect periodic redistribution between the nucleus and the cytoplasm. Nuclear phosphorylation of PER is strongly enhanced when TIM is removed from PER/TIM/DBT complexes. The varying associations of PER, DBT and TIM appear to determine the onset and duration of nuclear PER function within the Drosophila clock.