Genetic interactions between clock mutations in Neurospora crassa: can they help us to understand complexity?

Recent work on circadian clocks in Neurospora has primarily focused on the frequency (frq) and white-collar (wc) loci. However, a number of other genes are known that affect either the period or temperature compensation of the rhythm. These include the period (no relationship to the period gene of Drosophila) genes and a number of genes that affect cellular metabolism. How these other loci fit into the circadian system is not known, and metabolic effects on the clock are typically not considered in single-oscillator models. Recent evidence has pointed to multiple oscillators in Neurospora, at least one of which is predicted to incorporate metabolic processes. Here, the Neurospora clock-affecting mutations will be reviewed and their genetic interactions discussed in the context of a more complex clock model involving two coupled oscillators: a FRQ/WC-based oscillator and a 'frq-less' oscillator that may involve metabolic components.     

Modeling Temperature Compensation in Chemical and Biological Oscillators

All physicochemical and biological oscillators maintain a balance between destabilizing reactions (as, for example, intrinsic autocatalytic or amplifying reactions) and stabilizing processes. These two groups of processes tend to influence the period in opposite directions and may lead to temperature compensation whenever their overall influence balances. This principle of “antagonistic balance” has been tested for several chemical and biological oscillators. The Goodwin negative feedback oscillator appears of particular interest for modeling the circadian clocks in Neurospora and Drosophila and their temperature compensation. Remarkably, the Goodwin oscillator not only gives qualitative, correct phase response curves for temperature steps and temperature pulses, but also simulates the temperature behavior of Neurospora frq and Drosophila per mutants almost quantitatively. The Goodwin oscillator predicts that circadian periods are strongly dependent on the turnover of the clock mRNA or clock protein. A more rapid turnover of clock mRNA or clock protein results, in short, a slower turnover in longer period lengths. (Chronobiology International, 14(5), 499–510, 1997)     

Circadian rhythms in Neurospora: a new measurement, the reset zone

The authors define a new feature of a circadian rhythm, the reset zone, and point out its usefulness for predictions concerning oscillator behavior. The reset zone measures the responses of a circadian system to resetting pulses. It can be easily determined from a phase transition curve (PTC), which is simply a phase response curve (PRC) replotted as new phase versus old phase (Winfree's format). The reset zone is the range of new phases seen in such a plot and has two potentially useful characteristics: its size and its midpoint. A series of experiments with Neurospora involving temperature pulses indicated that the size of the reset zone changed in a nonlinear way in response to both the duration of 40 degrees C pulses and to the magnitude of temperature change for 3-h pulses. Other existing data are replotted to show how the reset zone size varies with growth temperature and with the period of different clock mutants. Employing exclusively reset zone data within the framework of a limit cycle displacement model, an equation is formulated that predicts the relative changes in the values of state variables of the oscillator for changes in any given environmental condition, such as temperature. Examples are also drawn from other organisms, such as hamsters, Gonyalaux, Kalanchoe, and Drosophila, illustrating the usefulness of the reset zone measurement. It can be used as a numerical scale for assessing the strength of a pulse, for comparing the relative effects of a given pulse applied to different organisms or mutants, for determining the directionality of the changes in state variables produced by various types of pulses, and possibly for measuring clock amplitude.     

Timing and consolidation of human sleep, wakefulness, and performance by a symphony of oscillators

Daily rhythms in sleep and waking performance are generated by the interplay of multiple external and internal oscillators. These include the light-dark and social cycles, a circadian hypothalamic oscillator oscillating virtually independently of behavior, and a homeostatic oscillator driven primarily by sleep-wake behavior. Both internal oscillators contribute to variation in many aspects of sleep and wakefulness (e.g., sleep timing and duration, REM sleep, non-REM sleep, REM density, sleep spindles, slow-wave sleep, electroencephalographic oscillations during wakefulness and sleep, and performance parameters, including attention and memory). The relative contribution of the oscillators varies greatly between these variables. Sleep and performance cannot be predicted by either oscillator independently but critically depend on their phase relationship and amplitude. The homeostatic oscillator feeds back onto the central pacemaker or its outputs. Thus, the amplitude of observed circadian variation in sleep and performance depends on how long we have been asleep or awake. During entrainment to external 24-h cycles, the opposing interplay between circadian and homeostatic changes in sleep propensity consolidates sleep and wakefulness. Some physiological correlates and mediators of both the circadian process (e.g., melatonin and hypocretin rhythms) and the homeostat (e.g., EEG, slow-wave activity, and adenosine release) have been established, offering targets for the development of countermeasures for circadian sleep and performance disorders. Interindividual differences in sleep timing, duration, and morning or evening preference are associated with changes of circadian or sleep homeostatic processes or both. Molecular genetic correlates, including polymorphisms in clock genes, of some of these interindividual differences are emerging.     

Molecular mechanism of suppression of circadian rhythms by a critical stimulus

Circadian singularity behavior (also called suppression of circadian rhythms) is a phenomenon characterized by the abolishment of circadian rhythmicities by a critical stimulus. Here we demonstrate that both temperature step up and light pulse, stimuli that activate the expression of the Neurospora circadian clock gene frequency (frq), can trigger singularity behavior in this organism. The arrhythmicity is transient and is followed by the resumption of rhythm in randomly distributed phases. In addition, we show that induction of FRQ expression alone can trigger singularity behavior, indicating that FRQ is a state variable of the Neurospora circadian oscillator. Furthermore, mutations of frq lead to changes in the amplitude of FRQ oscillation, which determines the sensitivity of the clock to phase-resetting cues. Our results further suggest that the singularity behavior is due to the loss of rhythm in all cells. Together, these data suggest that the singularity behavior is due to a circadian negative feedback loop driven to a steady state after the critical treatment. After the initial arrhythmicity, cell populations are then desynchronized.     

Temperature effects on circadian clocks

Periodic temperature changes represent one of the most effective entraining (Zeitgeber) signals for circadian clocks in many organisms. Different constant temperatures affect the circadian amplitude and ultimately the expression of circadian clocks, while the circadian period length (tau) remains approximately constant (temperature compensation). Experimental results and theoretical models are presented that may serve to explain these effects. After introducing the physico-chemical basis of temperature on enzyme-catalyzed and physiological reactions, and after describing mechanisms for temperature adaptation of physiological reactions to different thermal environments, general effects of temperature on chemical and biological oscillators are described. Kinetic models for circadian clocks and temperature compensation are presented and compared with experimental results. Special attention is given to the question how constant but different temperature levels affect clock amplitude, period length and phase. Influences of single and periodic temperature variations (steps or pulses) on circadian clocks are presented together with models which may explain the resulting phase response curves and entrainment patterns. Because temperature compensation is only one aspect of a general homeostatic mechanism that keeps the circadian period rather constant, the influence of other environmental variables and their relationship to temperature are discussed.     

Phase resetting of the Neurospora crassa circadian oscillator: effects of inositol depletion on sensitivity to light.

The input pathway between the blue-light photoreceptor and the circadian oscillator of Neurospora crassa has not yet been identified. To test the hypothesis that an inositol phospholipid signaling system might be involved in blue-light signal transduction, phase resetting by light was assayed in the inositol-requiring inl strain under conditions of inositol depletion. Phase-resetting curves and dose-response curves indicated that cultures maintained on low inositol (25 microM) were several orders of magnitude more sensitive to light than those maintained on high inositol (250 microM). This difference in light sensitivity was a property of inositol auxotrophy and was not seen in the wild type or in an inositol-independent inl+ revertant. Phase resetting by temperature was not affected by inositol depletion, indicating that the effect on light resetting is specific to the light input pathway and is not the result of a change in the amplitude of the oscillator itself. The results indicate an indirect role for inositol metabolites in the light input pathway--one that is not likely to involve direct participation of an inositol phospholipid signal transduction mechanism.     

Two circadian timing circuits in Neurospora crassa cells share components and regulate distinct rhythmic processes

In Neurospora crassa, FRQ, WC-1, and WC-2 proteins comprise the core circadian FRQ-based oscillator that is directly responsive to light and drives daily rhythms in spore development and gene expression. However, physiological and biochemical studies have demonstrated the existence of additional oscillators in the cell that function in the absence of FRQ (collectively termed FRQ-less oscillators [FLOs]). Whether or not these represent temperature-compensated, entrainable circadian oscillators is not known. The authors previously identified an evening-peaking gene, W06H2 (now called clock-controlled gene 16 [ccg-16]), which is expressed with a robust daily rhythm in cells that lack FRQ protein, suggesting that ccg-16 is regulated by a FLO. In this study, the authors provide evidence that the FLO driving ccg-16 rhythmicity is a circadian oscillator. They find that ccg-16 rhythms are generated by a temperature-responsive, temperature-compensated circadian FLO that, similar to the FRQ-based oscillator, requires functional WC-1 and WC-2 proteins for activity. They also find that FRQ is not essential for rhythmic WC-1 protein levels, raising the possibility that this WCFLO is involved in the generation of WC-1 rhythms. The results are consistent with the presence of 2 circadian oscillators within Neurospora cells, which the authors speculate may interact with each other through the shared WC proteins.     

Alteration of period and amplitude of circadian rhythms in shift workers. With special reference to temperature, right and left hand grip strength

48 male shift workers in various industries volunteered to document circadian rhythms in sleeping and working, oral temperature, grip strength of both hands, peak expiratory flow and heart rate. All physiological variables were self-measured 4 to 5 times a day for 2 to 4 weeks. Individual time series were analyzed according to several statistical methods (power spectrum, cosinor, chi squares, ANOVA, correlation, etc.) in order to estimate rhythm parameters such as circadian period (tau) and amplitude (A), and to evaluate subgroup differences with regard to tolerance to shift work, age, duration of shift work, speed of rotation and type of industry. The present study confirms for oral temperature and extends to other variables (grip strength of both hands, heart rate) that intolerance to shift work is frequently associated with both internal desynchronization and small circadian amplitude. The internal desynchronization among several circadian rhythms supports the hypothesis that these latter are driven by several oscillators. Many differences were observed between circadian rhythms in right and left hand grip strength: circadian tau in oral temperature was correlated with that in the grip strength of the dominant hand but not with that of the other hand; changes in tau s of the non-dominant hand were age-related but did not correlate with temperature tau; only the circadian A of the non-dominant hand was associated with a desynchronization. Thus, circadian rhythms in oral temperature and dominant hand grip strength may be driven by the same oscillator while that of the non-dominant hand may be governed by a different one. Internal desynchronization between both hand grip rhythms as well as desynchronization of performance rhythms reported by others provide indirect evidence that circadian oscillator(s) may be located in the human cerebral cortex.     

Natural allelic variation in the temperature-compensation mechanisms of the Arabidopsis thaliana circadian clock

Temperature compensation is a defining feature of circadian oscillators, yet no components contributing to the phenomenon have been identified in plants. We tested 27 accessions of Arabidopsis thaliana for circadian leaf movement at a range of constant temperatures. The accessions showed varying patterns of temperature compensation, but no clear associations to the geographic origin of the accessions could be made. Quantitative trait loci (QTL) were mapped for period and amplitude of leaf movement in the Columbia by Landsberg erecta (CoL) and Cape Verde Islands by Landsberg erecta (CvL) recombinant inbred lines (RILs) at 12 degrees , 22 degrees , and 27 degrees . Six CvL and three CoL QTL were located for circadian period. All of the period QTL were temperature specific, suggesting that they may be involved in temperature compensation. The flowering-time gene GIGANTEA and F-box protein ZEITLUPE were identified as strong candidates for two of the QTL on the basis of mapping in near isogenic lines (NILs) and sequence comparison. The identity of these and other candidates suggests that temperature compensation is not wholly determined by the intrinsic properties of the central clock proteins in Arabidopsis, but rather by other genes that act in trans to alter the regulation of these core proteins.     

Circadian Clock-Regulated Expression of Phytochrome and Cryptochrome Genes in Arabidopsis

Many physiological and biochemical processes in plants exhibit endogenous rhythms with a period of about 24 h. Endogenous oscillators called circadian clocks regulate these rhythms. The circadian clocks are synchronized to the periodic environmental changes (e.g. day/night cycles) by specific stimuli; among these, the most important is the light. Photoreceptors, phytochromes, and cryptochromes are involved in setting the clock by transducing the light signal to the central oscillator. In this work, we analyzed the spatial, temporal, and long-term light-regulated expression patterns of the Arabidopsis phytochrome (PHYA to PHYE) and cryptochrome (CRY1 and CRY2) promoters fused to the luciferase (LUC(+)) reporter gene. The results revealed new details of the tissue-specific expression and light regulation of the PHYC and CRY1 and 2 promoters. More importantly, the data obtained demonstrate that the activities of the promoter::LUC(+) constructs, with the exception of PHYC::LUC(+), display circadian oscillations under constant conditions. In addition, it is shown by measuring the mRNA abundance of PHY and CRY genes under constant light conditions that the circadian control is also maintained at the level of mRNA accumulation. These observations indicate that the plant circadian clock controls the expression of these photoreceptors, revealing the formation of a new regulatory loop that could modulate gating and resetting of the circadian clock.     

A phase dynamics model of human circadian rhythms

Nonphotic entrainment of an overt sleep-wake rhythm and a circadian pacemaker-driving temperature/melatonin rhythm suggests existence of feedback mechanisms in the human circadian system. In this study, the authors constructed a phase dynamics model that consisted of two oscillators driving temperature/melatonin and sleep-wake rhythms, and an additional oscillator generating an overt sleep-wake rhythm. The feedback mechanism was implemented by modifying couplings between the constituent oscillators according to the history of correlations between them. The model successfully simulated the behavior of human circadian rhythms in response to forced rest-activity schedules under free-run situations: the sleep-wake rhythm is reentrained with the circadian pacemaker after release from the schedule, there is a critical period for the schedule to fully entrain the sleep-wake rhythm, and the forced rest-activity schedule can entrain the circadian pacemaker with the aid of exercise. The behavior of human circadian rhythms was reproduced with variations in only a few model parameters. Because conventional models are unable to reproduce the experimental results concerned here, it was suggested that the feedback mechanisms included in this model underlie nonphotic entrainment of human circadian rhythms.     

Photic resetting of the circadian clock is correlated with photic habitat in <Emphasis Type="Italic">Anolis</Emphasis> lizards

Circadian rhythms are regulated by an internal clock, which is itself synchronized to environmental cues such as light and temperature. It is widely assumed that the circadian system is adapted to local cues, which vary enormously across habitats, yet the comparative data necessary for testing this idea are lacking. We examined photic and thermal resetting of the circadian clock in five species of Anolis lizards whose microhabitats differ in the amounts of sun and shade. The primary circadian oscillator in Anolis is the pineal gland, which produces the hormone melatonin. A flow-through culture system was employed to measure rhythmic melatonin output from individually cultured pineal glands. All species showed temperature-compensated circadian rhythms of pineal melatonin. Light caused significant phase delays of the melatonin rhythm, and this effect varied among species. Controlling for phylogenetic differences, the results indicate that the pineal glands of shade-dwelling species are more sensitive to photic resetting than species living in more brightly illuminated habitats. The differences were not due to variation in free-running period, but may be due to variation in oscillator phase and/or robustness. Surprisingly, thermal resetting was not statistically significant. Overall, the results suggest that the Anolis circadian system is adapted to photic habitat.     

Transient and steady state phase response curves of limit cycle oscillators

The experiment of phase shifts resulting from discrete perturbations of stable biological rhythms has been carried out to study entrainment behavior of oscillators. There are two kinds of phase response curves, which are measured in experiments, according to as one measures the phase shifts immediately or long after the perturbation. The former is the first transient phase response curve and the latter is the steady state phase response curve. We redefine both curves within the framework of dynamical system theory and homotopy theory. Several topological properties of both curves are clarified. Consequently, it is shown that we must compare the shapes of both two phase response curves to investigate the inner structures of biological oscillators. Moreover, we prove that a single limit cycle oscillator involving only two variables cannot simulate transient resetting behavior reported by Pittendrigh and Minis (1964). In other words, the circadian oscillator of Drosophila pseudoobscura does not consist of a single oscillator of two variables. Finally we show that a model which consists of two limit cycle oscillators is able to simulate qualitatively the phase response curves of Drosophila.     

The Neurospora circadian clock: simple or complex?

The fungus Neurospora crassa is being used by a number of research groups as a model organism to investigate circadian (daily) rhythmicity. In this review we concentrate on recent work relating to the complexity of the circadian system in this organism. We discuss: the advantages of Neurospora as a model system for clock studies; the frequency (frq), white collar-1 and white collar-2 genes and their roles in rhythmicity; the phenomenon of rhythmicity in null frq mutants and its implications for clock mechanisms; the study of output pathways using clock-controlled genes; other rhythms in fungi; mathematical modelling of the Neurospora circadian system; and the application of new technologies to the study of Neurospora rhythmicity. We conclude that there may be many gene products involved in the clock mechanism, there may be multiple interacting oscillators comprising the clock mechanism, there may be feedback from output pathways onto the oscillator(s) and from the oscillator(s) onto input pathways, and there may be several independent clocks coexisting in one organism. Thus even a relatively simple lower eukaryote can be used to address questions about a complex, networked circadian system.