Circadian Rhythms In Drosophila Research Articles

Amy Kiger: Abuzz on cell biology

From flattened epithelial cells to elongated neurons, cell morphology is heavily intertwined with cell growth and function. Understanding the genetic factors that govern cell shape might therefore provide insights into the processes underlying development and disease. Figure 1 Amy Kiger Amy Kiger has focused on these and other fundamental questions of cell biology in Drosophila throughout her career. She first worked on circadian rhythm in Drosophila as a research technician in a laboratory at The Rockefeller University, then studied male germ line stem cell biology in flies as a graduate student at Stanford (1, 2). Her postdoctoral studies in Norbert Perrimon's laboratory at Harvard on the genetic basis of cell morphology and growth in Drosophila have yielded several novel insights (3–5). With the results of these massive genome-wide screens (5), Kiger is now striking out on her own at the University of California, San Diego (UCSD), investigating the genetic basis for cell shape in her favorite model system—the fruit fly. She discussed with us the dizzying possibilities presented by her work.

G Protein-Coupled Receptor Kinase 2 Is Required for Rhythmic Olfactory Responses in Drosophila

The Drosophila circadian clock controls rhythms in the amplitude of odor-induced electrophysiological responses that peak during the middle of night. These rhythms are dependent on clocks in olfactory sensory neurons (OSNs), suggesting that odorant receptors (ORs) or OR-dependent processes are under clock control. Because responses to odors are initiated by heteromeric OR complexes that form odor-gated and cyclic-nucleotide-activated cation channels, we tested whether regulators of ORs were under circadian-clock control. The levels of G protein-coupled receptor kinase 2 (Gprk2) messenger RNA and protein cycle in a circadian-clock-dependent manner with a peak around the middle of the night in antennae. Gprk2 overexpression in OSNs from wild-type or cyc(01) flies elicits constant high-amplitude electroantennogram (EAG) responses to ethyl acetate, whereas Gprk2 mutants produce constant low-amplitude EAG responses. ORs accumulate to high levels in the dendrites of OSNs around the middle of the night, and this dendritic localization of ORs is enhanced by GPRK2 overexpression at times when ORs are primarily localized in the cell body. These results support a model in which circadian-clock-dependent rhythms in GPRK2 abundance control the rhythmic accumulation of ORs in OSN dendrites, which in turn control rhythms in olfactory responses. The enhancement of OR function by GPRK2 contrasts with the traditional role of GPRKs in desensitizing activated receptors and suggests that GPRK2 functions through a fundamentally different mechanism to modulate OR activity.

Pigment Dispersing Factor-Dependent and -Independent Circadian Locomotor Behavioral Rhythms

Circadian pacemaker circuits consist of ensembles of neurons, each expressing molecular oscillations, but how circuit-wide coordination of multiple oscillators regulates rhythmic physiological and behavioral outputs remains an open question. To investigate the relationship between the pattern of oscillator phase throughout the circadian pacemaker circuit and locomotor activity rhythms in Drosophila, we perturbed the electrical activity and pigment dispersing factor (PDF) levels of the lateral ventral neurons (LNv) and assayed their combinatorial effect on molecular oscillations in different parts of the circuit and on locomotor activity behavior. Altered electrical activity of PDF-expressing LNv causes initial behavioral arrhythmicity followed by gradual long-term emergence of two concurrent short- and long-period circadian behavioral activity bouts in approximately 60% of flies. Initial desynchrony of circuit-wide molecular oscillations is followed by the emergence of a novel pattern of period (PER) synchrony whereby two subgroups of dorsal neurons (DN1 and DN2) exhibit PER oscillation peaks coinciding with two activity bouts, whereas other neuronal subgroups exhibit a single PER peak coinciding with one of the two activity bouts. The emergence of this novel pattern of circuit-wide oscillator synchrony is not accompanied by concurrent change in the electrical activity of the LNv. In PDF-null flies, altered electrical activity of LNv drives a short-period circadian activity bout only, indicating that PDF-independent factors underlie the short-period circadian activity component and that the long-period circadian component is PDF-dependent. Thus, polyrhythmic behavioral patterns in electrically manipulated flies are regulated by circuit-wide coordination of molecular oscillations and electrical activity of LNv via PDF-dependent and -independent factors.

Modelling Circadian Rhythms in Drosophila and Investigation of VRI and PDP1 Feedback Loops Using a New Mathematical Model

We present a brief review of molecular biological basis and mathematical mod- elling of circadian rhythms in Drosophila. We discuss pertinent aspects of a new model that incorporates the transcriptional feedback loops revealed so far in the network of the circadian clock (PER/TIM and VRI/PDP1 loops). Conventional Hill functions are not used to describe the regulation of genes, instead the explicit reactions of binding and unbinding processes of transcription factors to promoters are probabilistically modelled. The model is described by a set of ordinary difierential equations, and the parameters are estimated from the in vitro experimental data of the clocks' components. The model is robust over a wide range of parameter variations. Through the sensitivity analysis of the model, roles of VRI and PDP1 feedback loops are investigated, and it is proposed that they increase the robustness of the clock.

PERIOD–TIMELESS Interval Timer May Require an Additional Feedback Loop

In this study we present a detailed, mechanism-based mathematical framework of Drosophila circadian rhythms. This framework facilitates a more systematic approach to understanding circadian rhythms using a comprehensive representation of the network underlying this phenomenon. The possible mechanisms underlying the cytoplasmic “interval timer” created by PERIOD–TIMELESS association are investigated, suggesting a novel positive feedback regulatory structure. Incorporation of this additional feedback into a full circadian model produced results that are consistent with previous experimental observations of wild-type protein profiles and numerous mutant phenotypes.

Phase Sensitivity Analysis of Circadian Rhythm Entrainment

As a biological clock, circadian rhythms evolve to accomplish a stable (robust) entrainment to environmental cycles, of which light is the most obvious. The mechanism of photic entrainment is not known, but two models of entrainment have been proposed based on whether light has a continuous (parametric) or discrete (nonparametric) effect on the circadian pacemaker. A novel sensitivity analysis is developed to study the circadian entrainment in silico based on a limit cycle approach and applied to a model of Drosophila circadian rhythm. The comparative analyses of complete and skeleton photoperiods suggest a trade-off between the contribution of period modulation (parametric effect) and phase shift (nonparametric effect) in Drosophila circadian entrainment. The results also give suggestions for an experimental study to (in)validate the two models of entrainment.

Even a stopped clock tells the right time twice a day: circadian timekeeping in Drosophila

"Even a stopped clock tells the right time twice a day, and for once I'm inclined to believe Withnail is right. We are indeed drifting into the arena of the unwell... What we need is harmony. Fresh air. Stuff like that" "Bruce Robinson (1986, ref. 1)". Although a stopped Drosophila clock probably does not tell the right time even once a day, recent findings have demonstrated that accurate circadian time-keeping is dependent on harmony between groups of clock neurons within the brain. Furthermore, when harmony between the environment and the endogenous clock is lost, as during jet lag, we definitely feel unwell. In this review, we provide an overview of the current understanding of circadian rhythms in Drosophila, focussing on recent discoveries that demonstrate how approximately 100 neurons within the Drosophila brain control the behaviour of the whole fly, and how these rhythms respond to the environment.

A PER/TIM/DBT Interval Timer forDrosophila's Circadian Clock

Circadian rhythms in Drosophila are supported by a negative feedback loop, in which PERIOD (PER) and Timeless (TIM) shut down their own transcription as they translocate once a day from the cytoplasm of clock-containing cells to the nucleus. Period length is partially determined by an interval of cytoplasmic retention of the TIM and PER proteins. To study this process, we examined PER/TIM/Doubletime (DBT) physical interactions and nuclear translocation by imaging individual cultured Drosophila cells. Using live cell video microscopy and green fluorescent protein (GFP) tags, we observed dynamic patterns of stability and localization for DBT, PER, and TIM that resembled those previously found in vivo. These studies suggest that a cytoplasmic interval timer regulates nuclear translocation of these proteins. The cultured cell assay provides a potent system to study interactions among new and known genes involved in the generation of circadian behavior.

Transcriptional Feedback and Definition of the Circadian Pacemaker inDrosophilaand Animals

The modern era of Drosophila circadian rhythms began with the landmark Benzer and Konopka paper and its definition of the period gene. The recombinant DNA revolution then led to the cloning and sequencing of this gene. This work did not result in a coherent view of circadian rhythm biochemistry, but experiments eventually gave rise to a transcription-centric view of circadian rhythm generation. Although these circadian transcription-translation feedback loops are still important, their contribution to core timekeeping is under challenge. Indeed, kinases and posttranslational regulation may be more important, based in part on recent in vitro work from cyanobacteria. In addition, kinase mutants or suspected kinase substrate mutants have unusually large period effects in Drosophila. This chapter discusses our recent experiments, which indicate that circadian transcription does indeed contribute to period determination in this system. We propose that cyanobacteria and animal clocks reflect two independent origins of circadian rhythms.

Neuronal mechanisms for photic and thermoperiodic entrainment of Drosophila circadian locomotor rhythms

Neuronal mechanisms for photic and thermoperiodic entrainment of Drosophila circadian locomotor rhythms

Entrainment of Drosophila circadian rhythms by temperature cycles

The fruit fly, Drosophila melanogaster, has been a good organism for elucidating the molecular and cellular bases of circadian behavioral rhythms. The fly shows a bimodal locomotor rhythm with a period slightly different from 24-h. The rhythm synchronizes with the environmental cycle using light as a powerful zeitgeber. It is now believed that this rhythm is generated by two interlocked transcriptional feedback loops consisting of so-called clock genes. Light resets the clock loop through degradation of a clock protein, TIMELESS. Temperature is another powerful zeitgeber that can entrain the rhythm but the cellular and molecular basis for temperature entrainment is still under investigation. This review will try to give an overview of temperature entrainment, considering multiple clocks with different responsiveness to light and temperature.

Isochron-Based Phase Response Analysis of Circadian Rhythms

Circadian rhythms possess the ability to robustly entrain to the environmental cycles. This ability relies on the phase synchronization of circadian rhythm gene regulation to different environmental cues, of which light is the most obvious and important. The elucidation of the mechanism of circadian entrainment requires an understanding of circadian phase behavior. This article presents two phase analyses of oscillatory systems for infinitesimal and finite perturbations based on isochrons as a phase metric of a limit cycle. The phase response curve of circadian rhythm can be computed from the results of the analyses. The application to a mechanistic Drosophila circadian rhythm model gives experimentally testable hypotheses for the control mechanisms of circadian phase responses and evidence for the role of phase and period modulations in circadian photic entrainment.

Amplitude of circadian oscillations entrained by 24-h light–dark cycles

An intriguing property of circadian clocks is that their free-running period is not exactly 24 h. Using models for circadian rhythms in Neurospora and Drosophila, we determine how the entrainment of these rhythms is affected by the free-running period and by the amplitude of the external light–dark cycle. We first consider the model for Neurospora, in which light acts by inducing the expression of a clock gene. We show that the amplitude of the oscillations of the clock protein entrained by light–dark cycles is maximized when the free-running period is smaller than 24 h. Moreover, if the amplitude of the light–dark cycle is very strong, complex oscillations occur when the free-running period is close to 24 h. In the model for circadian rhythms in Drosophila, light acts by enhancing the degradation of a clock protein. We show that while the amplitude of circadian oscillations entrained by light–dark cycles is also maximized if the free-running period is smaller than 24 h, the range of entrainment is centered around 24 h in this model. We discuss the physiological relevance of these results in regard to the setting of the free-running period of the circadian clock.

Model Reduction and Physical Understanding of Slowly Oscillating Processes: The Circadian Cycle

A differential system that models the circadian rhythm in Drosophila is analyzed with the computational singular perturbation (CSP) algorithm. Reduced nonstiff models of prespecified accuracy are constructed, the form and size of which are time-dependent. When compared with conventional asymptotic analysis, CSP exhibits superior performance in constructing reduced models, since it can algorithmically identify and apply all the required order of magnitude estimates and algebraic manipulations. A similar performance is demonstrated by CSP in generating data that allow for the acquisition of physical understanding. It is shown that the processes driving the circadian cycle are (i) mRNA translation into monomer protein, and monomer protein destruction by phosphorylation and degradation (along the largest portion of the cycle); and (ii) mRNA synthesis (along a short portion of the cycle). These are slow processes. Their action in driving the cycle is allowed by the equilibration of the fastest processes; (1) the monomer dimerization with the dimer dissociation (along the largest portion of the cycle); and (2) the net production of monomer+dimmer proteins with that of mRNA (along the short portion of the cycle). Additional results (regarding the time scales of the established equilibria, their origin, the rate limiting steps, the couplings among themore » variables, etc.) highlight the utility of CSP for automated identification of the important underlying dynamical features, otherwise accessible only for simple systems whose various suitable simplifications can easily be recognized.« less

Identification and expression pattern ofBmlark, a homolog of theDrosophilagenelarkinBombyx mori†

Two Bombyx mori isoforms of the gene lark, which is shown to play an important role in Drosophila circadian rhythms, were identified and named Bmlark-PA and Bmlark-PB, respectively. Bmlark-PA consists of 5 exons and encodes a protein of 343 amino acid residues which contains 3 functional domains: two RRM (RNA recognization motif) domains and an RTZF (retroviral-type zinc finger) and shares 72% identity with the Drosophila gene lark at the amino acid level. Bmlark-PB lacks the sequence between 118 and 791 nt of Bmlark-PA and codes for a protein of 68 amino acid residues, which contains no distinct functional domains. Alignments of the cDNAs of Bmlark to the genomic draft sequence of B. mori showed that the gene Bmlark had a single copy in the genome, suggesting that an alternative splicing mechanism occurs in the gene Bmlark. RT-PCR analysis indicated that Bmlark-PA was expressed only in late pupae and adult but Bmlark-PB was broadly expressed in many tissues and throughout the developmental stages from embryo to adult.

Coupling of Human Circadian and Cell Cycles by the Timeless Protein

The Timeless protein is essential for circadian rhythm in Drosophila. The Timeless orthologue in mice is essential for viability and appears to be required for the maintenance of a robust circadian rhythm as well. We have found that the human Timeless protein interacts with both the circadian clock protein cryptochrome 2 and with the cell cycle checkpoint proteins Chk1 and the ATR-ATRIP complex and plays an important role in the DNA damage checkpoint response. Down-regulation of Timeless in human cells seriously compromises replication and intra-S checkpoints, indicating an intimate connection between the circadian cycle and the DNA damage checkpoints that is in part mediated by the Timeless protein.

Bio-Object, a stochastic simulator for post-transcriptional regulation

Recently, biologists learnt that the transport and degradation of transcribed mRNA and protein present critically important steps for the regulation of gene expression through extensive studies of RNA interference, none-sense mediated decay and ubiquitination. However, adequate consideration of these factors has not been done in the past in in silico analysis compared with transcriptional regulations. We have developed a bio-system simulator 'Bio-Object' and assessed the contribution of numerous factors including movements, stability and interactions of both mRNAs and proteins in the virtual cell space to the Drosophila circadian rhythm. The oscillations of period (per), timeless (tim) and Drosophila Clock (dClk) mRNAs and proteins predicted by the simulations agreed with the observed data in Drosophila and were lost with the knock-out of either the per or the dClk gene as observed experimentally. Bio-Object predicts that (1) the stability of dClk mRNA, (2) the stability of dCLK and (3) the affinity of the PER-TIM complex are determinants of the circadian duration. The source code is available for download from http://www.tmd.ac.jp/mri/mri-end/bio-object/download/

My tryst with the bats of Madurai

I have often been asked by people how I came to work with bats. The story may be well worth recounting, weaving into my account, for the benefit of younger readers, the minor hurdles, the adventure, excitement and thrill of working out in the open at night and inside caves with bats. My affair with the bats of Madurai had lasted two decades and five of my students wrote their PhD theses on the biology, ecology, behaviour and biological clocks of bats. Today the Department of Animal Behaviour & Physiology, Madurai Kamaraj University, has the biggest data base on the biology and behaviour of bats anywhere. In this article I wish to show that first-rate work on animal behaviour can be done in our backyards in India. J B S Haldane felt that the study of animal behaviour was the area in which we are likely to do entirely original research and R.Gadagkar has belaboured this point and, of course, set an example himself with his own researches on the paper wasp. My enterprising first student R Subbaraj had begun field work on bats a year before I had even returned to India from Germany and joined the School of Biological Sciences at the Madurai Kamaraj University in the summer of 1975. Bats were far from my mind, having until then worked for a whole decade on rather abstract concepts such as phase response curves, kinetics of phase shifts of circadian clocks, transient and limit cycles, singularities and light and energy relations of the circadian rhythms in Drosophila. I told Subbaraj that if he wanted to work for a PhD it would have to be based on laboratory work on the biological clock of bats. Subbaraj was intelligent, bright eyed, quick in grasping problems, eager to learn and was a natural talent. He was thrilled that I was opening up a new world, the world of chronobiology, for him. I was glad he was introducing me reciprocally to the exotic world of bats. I told him "The farmer does not eat what the farmer does not know. So let me get to know your bats properly".

Simulation of Drosophila Circadian Oscillations, Mutations, and Light Responses by a Model with VRI, PDP-1, and CLK

A model of Drosophila circadian rhythm generation was developed to represent feedback loops based on transcriptional regulation of per, Clk (dclock), Pdp-1, and vri (vrille). The model postulates that histone acetylation kinetics make transcriptional activation a nonlinear function of [CLK]. Such a nonlinearity is essential to simulate robust circadian oscillations of transcription in our model and in previous models. Simulations suggest that two positive feedback loops involving Clk are not essential for oscillations, because oscillations of [PER] were preserved when Clk, vri, or Pdp-1 expression was fixed. However, eliminating positive feedback by fixing vri expression altered the oscillation period. Eliminating the negative feedback loop in which PER represses per expression abolished oscillations. Simulations of per or Clk null mutations, of per overexpression, and of vri, Clk, or Pdp-1 heterozygous null mutations altered model behavior in ways similar to experimental data. The model simulated a photic phase-response curve resembling experimental curves, and oscillations entrained to simulated light-dark cycles. Temperature compensation of oscillation period could be simulated if temperature elevation slowed PER nuclear entry or PER phosphorylation. The model makes experimental predictions, some of which could be tested in transgenic Drosophila.

A single nucleotide polymorphism in glycogen synthase kinase 3-β promoter gene influences onset of illness in patients affected by bipolar disorder

Genetic studies in medicine exploited age of onset as a criterion to delineate subgroups of illness. Bipolar patients stratified with this criterion were shown to share clinical characteristics and patterns of inheritance of illness. The molecular mechanisms driving the biological clock in the suprachiasmatic nucleus of the hypothalamus may play a role in mood disorders. A single nucleotide polymorphism (SNP) (−50 T/C) falling into the effective promoter region (nt −171 to +29) of the gene coding for glycogen synthase kinase 3-β (GSK3-β) has been identified. GSK3-β codes for an enzyme which is a target for the action of lithium and which is also known to regulate circadian rhythms in Drosophila. We studied the effect of this polymorphism on the age at onset of bipolar disorder type I. A homogeneous sample of 185 Italian patients affected by bipolar disorder was genotyped. Age at onset was retrospectively ascertained with best estimation procedures. No association was detected between GSK3-β −50 T/C SNP and the presence of bipolar illness. Homozygotes for the wild variant (T/T) showed an earlier age at onset than carriers of the mutant allele ( F=5.53, d.f.=2,182, P=0.0047). Results warrant interest for the variants of genes pertaining to the molecular clock as possible endophenotypes of bipolar disorder, but caution ought to be taken in interpreting these preliminary results and future replication studies must be awaited.