Night length measurements by the circadian clock controlling diapause induction in the mosquito Aedes atropalpus

Night interruption experiments were used to investigate the behavior of the clock controlling diapause induction in the mosquito, Aedes atropalpus. The data from these experiments indicated that the clock included a circadian oscillator which was phase set at dusk. Following this event the oscillator proceeded to drive a nightly rhythm of sensitivity to light. This rhythm included a photoinducible phase where light interruption inhibited diapause. The photoinducible phase was fixed, occurring 7 to 9 hr after dusk in all photoperiod regimens tested. The photoinducible phase was followed by a refractory phase, which continued until dawn. During the refractory period light did not inhibit diapause. These observations indicated that the circadian clock behaved like an interval timer which was set at dusk. The rhythm of sensitivity to light, an inherited time scale, limited the induction of diapause to seasonal periods when nights were longer than 9 hr. As a result, diapause was induced only when the daylength dropped below the critical photoperiod of L15:D9 (hours of light:hours of dark).A ‘T’ experimental design was used to confirm the importance of the length of the night in clock controlled induction of diapause in this mosquito.     

Day-length perception and the photoperiodic regulation of flowering in Arabidopsis

The flowering of Arabidopsis plants is accelerated by long-day photoperiods, and recent genetic studies have identified elements of the photoperiodic timing mechanism. These elements comprise genes that regulate the function of the circadian clock, photoreceptors, and downstream components of light signaling pathways. These results provide evidence for the role of the circadian clock in photoperiodic time measurement and suggest that photoperiod perception may follow Pittendrigh's external coincidence model. T-cycle experiments indicated that changes in the timing of circadian rhythms, relative to dawn and dusk, correlated with altered flowering time. Thus, the perception of photoperiod maybe mediated by adjustments in the phase of the circadian cycle that arise upon re-entrainment to a different light-dark cycle. The nature of the rhythm underlying the floral response is not known, but candidate molecules have been identified.     

Robust circadian rhythmicity of Per1 and Per2 mutant mice in constant light,and dynamics of Per1 and Per2 gene expression under long and short photoperiods

The Per1 and Per2 genes are components of the mammalian circadian clock. Mutations in these genes alter phase resetting in response to a nocturnal light pulse, and Per2 mutant mice are known to become arrhythmic in constant darkness. We show that under constant light conditions, Per2 mutant mice exhibit robust activity rhythms as well as body temperature rhythms with a period length that is less than 24 h. In Per1 mutants, the period length of both activity and body temperature rhythms is longer than 24 h in constant light. Per1 mutants prolong their period length (tao) when illuminance is increased, whereas Per2 mutants shorten their endogenous period. Additionally, the authors show that the circadian pattern of Per1 and Per2 gene expression in mice is modified under different photoperiods and that there is a mutual influence of these genes on their timing of expression. We propose that, in mice, the phase relationship between Per1 and Per2 gene expression might be critical for transducing day length information to the organism. Per1 could be part of a morning oscillator tracking dawn, and Per2 could be part of an evening oscillator tracking dusk.     

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.     

Influence of the estrous cycle on clock gene expression in reproductive tissues: Effects of fluctuating ovarian steroid hormone levels

Circadian rhythms in physiology and behavior are known to be influenced by the estrous cycle in female rodents. The clock genes responsible for the generation of circadian oscillations are widely expressed both within the central nervous system and peripheral tissues, including those that comprise the reproductive system. To address whether the estrous cycle affects rhythms of clock gene expression in peripheral tissues, we first examined rhythms of clock gene expression (Per1, Per2, Bmal1) in reproductive (uterus, ovary) and non-reproductive (liver) tissues of cycling rats using quantitative real-time PCR (in vivo) and luminescent recording methods to measure circadian rhythms of PER2 expression in tissue explant cultures from cycling PER2::LUCIFERASE (PER2::LUC) knockin mice (ex vivo). We found significant estrous variations of clock gene expression in all three tissues in vivo, and in the uterus ex vivo. We also found that exogenous application of estrogen and progesterone altered rhythms of PER2::LUC expression in the uterus. In addition, we measured the effects of ovarian steroids on clock gene expression in a human breast cancer cell line (MCF-7 cells) as a model for endocrine cells that contain both the steroid hormone receptors and clock genes. We found that progesterone, but not estrogen, acutely up-regulated Per1, Per2, and Bmal1 expression in MCF-7 cells. Together, our findings demonstrate that the timing of the circadian clock in reproductive tissues is influenced by the estrous cycle and suggest that fluctuating steroid hormone levels may be responsible, in part, through direct effects on the timing of clock gene expression.     

Photoreception in decapitated larvae of silkworm Bombyx mori

To investigate the photoreception that controls daily oscillations at the periphery in insects, we decapitated larvae of the silkworm Bombyx mori (Lepidoptera: Bombycidae) by ligature, and observed rhythms in their peripheral tissues under several light conditions. We measured the mRNA expression of period (per) and timeless (tim), which are homologues of Drosophila clock genes that function in the core oscillator of the circadian clock system. The expression of both per and tim significantly changed in the midgut, Malpighian tubules and silk glands of decapitated larvae exposed to photophase and scotophase that were reversed from the original daily light–dark cycle under which the larvae were housed. Under constant darkness, the daily expression of tim mRNA persisted for at least one cycle in the midgut and silk gland. In addition, an appropriate light stimulus under constant darkness induced a significant phase shift in the endogenous timing system (probably a circadian clock) that determined peak levels of tim mRNA expression in the midgut and silk glands of decapitated larvae. Since light regulated the gene expression rhythm in peripheral tissues of decapitated silkworm larvae, neither the brain nor eyes were essential for photoreception to control daily oscillations in these tissues. Thus, peripheral tissues in insects might directly use light even at the larval stage.     

sn-1,2-diacylglycerol levels in the fungus Neurospora crassa display circadian rhythmicity

The fungus Neurospora crassa is a model organism for investigating the biochemical mechanism of circadian (daily) rhythmicity. When a choline-requiring strain (chol-1) is depleted of choline, the period of the conidiation rhythm lengthens. We have found that the levels of sn-1,2-diacylglycerol (DAG) increase in proportion to the increase in period. Other clock mutations that change the period do not affect the levels of DAG. Membrane-permeant DAGs and inhibitors of DAG kinase were found to further lengthen the period of choline-depleted cultures. The level of DAG oscillates with a period comparable to the rhythm of conidiation in wild-type strains, choline-depleted cultures, and frq mutants, including a null frq strain. The DAG rhythm is present at the growing margin and also persists in older areas that have completed development. The phase of the DAG rhythm can be set by the light-to-dark transition, but the level of DAG is not immediately affected by light. Our results indicate that rhythms in DAG levels in Neurospora are driven by a light-sensitive circadian oscillator that does not require the frq gene product. High levels of DAG may feed back on that oscillator to lengthen its period.     

Cycling of clock genes entrained to the solar rhythm enables plants to tell time: data from arabidopsis

Background and Aims An endogenous rhythm synchronized to dawn cannot time photosynthesis-linked genes to peak consistently at noon since the interval between sunrise and noon changes seasonally. In this study, a solar clock model that circumvents this limitation is proposed using two daily timing references synchronized to noon and midnight. Other rhythmic genes that are not directly linked to photosynthesis, and which peak at other times, also find an adaptive advantage in entrainment to the solar rhythm.Methods Fourteen datasets extracted from three published papers were used in a meta-analysis to examine the cyclic behaviour of the Arabidopsis thaliana photosynthesis-related gene CAB2 and the clock oscillator genes TOC1 and LHY in T cycles and N–H cycles.Key Results Changes in the rhythms of CAB2, TOC1 and LHY in plants subjected to non-24-h light:dark cycles matched the hypothesized changes in their behaviour as predicted by the solar clock model, thus validating it. The analysis further showed that TOC1 expression peaked ∼5·5 h after mid-day, CAB2 peaked close to noon, while LHY peaked ∼7·5 h after midnight, regardless of the cycle period, the photoperiod or the light:dark period ratio. The solar clock model correctly predicted the zeitgeber timing of these genes under 11 different lighting regimes comprising combinations of seven light periods, nine dark periods, four cycle periods and four light:dark period ratios. In short cycles that terminated before LHY could be expressed, the solar clock correctly predicted zeitgeber timing of its expression in the following cycle.Conclusions Regulation of gene phases by the solar clock enables the plant to tell the time, by which means a large number of genes are regulated. This facilitates the initiation of gene expression even before the arrival of sunrise, sunset or noon, thus allowing the plant to ‘anticipate’ dawn, dusk or mid-day respectively, independently of the photoperiod.     

Expression of the circadian clock gene Period2 in the hippocampus: possible implications for synaptic plasticity and learned behaviour

Genes responsible for generating circadian oscillations are expressed in a variety of brain regions not typically associated with circadian timing. The functions of this clock gene expression are largely unknown, and in the present study we sought to explore the role of the Per2 (Period 2) gene in hippocampal physiology and learned behaviour. We found that PER2 protein is highly expressed in hippocampal pyramidal cell layers and that the expression of both protein and mRNA varies with a circadian rhythm. The peaks of these rhythms occur in the late night or early morning and are almost 180° out-of-phase with the expression rhythms measured from the suprachiasmatic nucleus of the same animals. The rhythms in Per2 expression are autonomous as they are present in isolated hippocampal slices maintained in culture. Physiologically, Per2-mutant mice exhibit abnormal long-term potentiation. The underlying mechanism is suggested by the finding that levels of phosphorylated cAMP-response-element-binding protein, but not phosphorylated extracellular-signal-regulated kinase, are reduced in hippocampal tissue from mutant mice. Finally, Per2-mutant mice exhibit deficits in the recall of trace, but not cued, fear conditioning. Taken together, these results provide evidence that hippocampal cells contain an autonomous circadian clock. Furthermore, the clock gene Per2 may play a role in the regulation of long-term potentiation and in the recall of some forms of learned behaviour.