Noncircadian regulation and function of clock genes period and timeless in oogenesis of Drosophila melanogaster

Circadian clock genes are ubiquitously expressed in the nervous system and peripheral tissues of complex animals. While clock genes in the brain are essential for behavioral rhythms, the physiological roles of these genes in the periphery are not well understood. Constitutive expression of the clock gene period was reported in the ovaries of Drosophila melanogaster; however, its molecular interactions and functional significance remained unknown. This study demonstrates that period (per) and timeless (tim) are involved in a novel noncircadian function in the ovary. PER and TIM are constantly expressed in the follicle cells enveloping young oocytes. Genetic evidence suggests that PER and TIM interact in these cells, yet they do not translocate to the nucleus. The levels of TIM and PER in the ovary are affected neither by light nor by the lack of clock-positive elements Clock (Clk) and cycle (cyc). Taken together, these data suggest that per and tim are regulated differently in follicle cells than in clock cells. Experimental evidence suggests that a novel fitness-related phenotype may be linked to noncircadian expression of clock genes in the ovaries. Mated females lacking either per or tim show nearly a 50% decline in progeny, and virgin females show a similar decline in the production of mature oocytes. Disruption of circadian mechanism by either the depletion of TIM via constant light treatment or continuous expression of PER via GAL4/UAS expression system has no adverse effect on the production of mature oocytes.     

Conserved regions of the timeless (tim) clock gene in Drosophila analyzed through phylogenetic and functional studies.

Circadian (approximately 24-hr) rhythms in Drosophila melanogaster depend upon cyclic expression of the period (per) and timeless (tim) genes, which encode interacting components of the endogenous clock. The per gene has been isolated from other insects and, more recently, a per ortholog was found in mammals where its expression oscillates in a circadian fashion. We report here the complete sequence of a tim gene from another species, Drosophila virilis. TIM is better conserved than the PER protein is between these two species (76 vs. 54% overall amino acid identity), and putative functional domains, such as the PER interaction domains and the nuclear localization signal, are highly conserved. The acidic domain and the cytoplasmic localization domain, however, are within the least conserved regions. In addition, the initiating methionine in the D. virilis gene lies downstream of the proposed translation start for the original D. melanogaster tim cDNA and corresponds to the one used by D. simulans and D. yakuba. Among the most conserved parts of TIM is a region of unknown function near the N terminus. We show here that deletion of a 32 amino acid segment within this region affects rescue of rhythms in arrhythmic tim01 flies. Flies carrying a full-length tim transgene displayed rhythms with approximately 24-hr periods, indicating that a fully functional clock can be restored in tim01 flies through expression of a tim transgene. Deletion of the segment mentioned above resulted in very long activity rhythms with periods ranging from 30.5 to 48 hr.     

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

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

Modeling the molecular regulatory mechanism of circadian rhythms in Drosophila

Thanks to genetic and biochemical advances on the molecular mechanism of circadian rhythms in Drosophila, theoretical models closely related to experimental observations can be considered for the regulatory mechanism of the circadian clock in this organism. Modeling is based on the autoregulatory negative feedback exerted by a complex between PER and TIM proteins on the expression of per and tim genes. The model predicts the occurrence of sustained circadian oscillations in continuous darkness. When incorporating light-induced TIM degradation, the model accounts for damping of oscillations in constant light, entrainment of the rhythm by light-dark cycles of varying period or photoperiod, and phase shifting by light pulses. The model further provides a molecular dynamical explanation for the permanent or transient suppression of circadian rhythmicity triggered in a variety of organisms by a critical pulse of light. Finally, the model shows that to produce a robust rhythm the various clock genes must be expressed at the appropriate levels since sustained oscillations only occur in a precise range of parameter values. BioEssays 22:84-93, 2000.     

Ectopic CRYPTOCHROME renders TIM light sensitive in the Drosophila ovary

The period (per) and timeless (tim) genes play a central role in the Drosophila circadian clock mechanism. PERIOD (PER) and TIMELESS (TIM) proteins periodically accumulate in the nuclei of pace-making cells in the fly brain and many cells in peripheral organs. In contrast, TIM and PER in the ovarian follicle cells remain cytoplasmic and do not show daily oscillations in their levels. Moreover, TIM is not light sensitive in the ovary, while it is highly sensitive to this input in circadian tissues. The mechanism underlying this intriguing difference is addressed here. It is demonstrated that the circadian photoreceptor CRYPTOCHROME (CRY) is not expressed in ovarian tissues. Remarkably, ectopic cry expression in the ovary is sufficient to cause degradation of TIM after exposure to light. In addition, PER levels are reduced in response to light when CRY is present, as observed in circadian cells. Hence, CRY is the key component of the light input pathway missing in the ovary. However, the factors regulating PER and TIM levels downstream of light/cry action appear to be present in this non-circadian organ.     

Serotonin modulates circadian entrainment in Drosophila

Entrainment of the Drosophila circadian clock to light involves the light-induced degradation of the clock protein timeless (TIM). We show here that this entrainment mechanism is inhibited by serotonin, acting through the Drosophila serotonin receptor 1B (d5-HT1B). d5-HT1B is expressed in clock neurons, and alterations of its levels affect molecular and behavioral responses of the clock to light. Effects of d5-HT1B are synergistic with a mutation in the circadian photoreceptor cryptochrome (CRY) and are mediated by SHAGGY (SGG), Drosophila glycogen synthase kinase 3beta (GSK3beta), which phosphorylates TIM. Levels of serotonin are decreased in flies maintained in extended constant darkness, suggesting that modulation of the clock by serotonin may vary under different environmental conditions. These data identify a molecular connection between serotonin signaling and the central clock component TIM and suggest a homeostatic mechanism for the regulation of circadian photosensitivity in Drosophila.     

A key temporal delay in the circadian cycle of Drosophila is mediated by a nuclear localization signal in the timeless protein

Regulated nuclear entry of the Period (PER) and Timeless (TIM) proteins, two components of the Drosophila circadian clock, is essential for the generation and maintenance of circadian behavior. PER and TIM shift from the cytoplasm to the nucleus daily, and the length of time that PER and TIM reside in the cytoplasm is an important determinant of the period length of the circadian rhythm. Here we identify a TIM nuclear localization signal (NLS) that is required for appropriately timed nuclear accumulation of both TIM and PER. Transgenic flies with a mutated TIM NLS produced circadian rhythms with a period of ～30 hr. In pacemaker cells of the brain, PER and TIM proteins rise to abnormally high levels in the cytoplasm of tim(ΔNLS) mutants, but show substantially reduced nuclear accumulation. In cultured S2 cells, the mutant TIM(ΔNLS) protein significantly delays nuclear accumulation of both TIM and wild-type PER proteins. These studies confirm that TIM is required for the nuclear localization of PER and point to a key role for the TIM NLS in the regulated nuclear accumulation of both proteins.     

Hofbauer-Buchner eyelet affects circadian photosensitivity and coordinates TIM and PER expression in Drosophila clock neurons

Extraretinal photoreception is a common input route for light resetting signals into the circadian clock of animals. In Drosophila melanogaster, substantial circadian light inputs are mediated via the blue light photoreceptor CRYPTOCHROME (CRY) expressed in clock neurons within the brain. The current model predicts that, upon light activation, CRY interacts with the clock proteins TIMELESS (TIM) and PERIOD (PER), thereby inducing their degradation, which in turn leads to a resetting of the molecular oscillations within the circadian clock. Here the authors investigate the function of another putative extraretinal circadian photoreceptor, the Hofbauer-Buchner eyelet (H-B eyelet), located between the retina and the medulla in the fly optic lobes. Blocking synaptic transmission between the H-B eyelet and its potential target cells, the ventral circadian pacemaker neurons, impaired the flies' ability to resynchronize their behavior under jet-lag conditions in the context of nonfunctional retinal photoreception and a mutation in the CRY-encoding gene. The same manipulation also affected synchronized expression of the clock proteins TIM and PER in different subsets of the clock neurons. This shows that synaptic communication between the H-B eyelet and clock neurons contributes to synchronization of molecular and behavioral rhythms and confirms that the H-B eyelet functions as a circadian photoreceptor. Blockage of synaptic transmission from the H-B eyelet in the presence of functional compound eyes and the absence of CRY also results in increased numbers of flies that are unable to synchronize to extreme photoperiods, supplying independent proof for the role of the H-B eyelet as a circadian photoreceptor.     

Kinetics of doubletime kinase-dependent degradation of the Drosophila period protein

Robust circadian oscillations of the proteins PERIOD (PER) and TIMELESS (TIM) are hallmarks of a functional clock in the fruit fly Drosophila melanogaster. Early morning phosphorylation of PER by the kinase Doubletime (DBT) and subsequent PER turnover is an essential step in the functioning of the Drosophila circadian clock. Here using time-lapse fluorescence microscopy we study PER stability in the presence of DBT and its short, long, arrhythmic, and inactive mutants in S2 cells. We observe robust PER degradation in a DBT allele-specific manner. With the exception of doubletime-short (DBT(S)), all mutants produce differential PER degradation profiles that show direct correspondence with their respective Drosophila behavioral phenotypes. The kinetics of PER degradation with DBT(S) in cell culture resembles that with wild-type DBT and posits that, in flies DBT(S) likely does not modulate the clock by simply affecting PER degradation kinetics. For all the other tested DBT alleles, the study provides a simple model in which the changes in Drosophila behavioral rhythms can be explained solely by changes in the rate of PER degradation.