SIK3-HDAC4 in the suprachiasmatic nucleus regulates the timing of arousal at the dark onset and circadian period in mice.

Mammals exhibit circadian cycles of sleep and wakefulness under the control of the suprachiasmatic nucleus (SCN), such as the strong arousal phase-locked to the beginning of the dark phase in laboratory mice. Here, we demonstrate that salt-inducible kinase 3 (SIK3) deficiency in gamma-aminobutyric acid (GABA)-ergic neurons or neuromedin S (NMS)-producing neurons delayed the arousal peak phase and lengthened the behavioral circadian cycle under both 12-h light:12-h dark condition (LD) and constant dark condition (DD) without changing daily sleep amounts. In contrast, the induction of a gain-of-function mutant allele of Sik3 in GABAergic neurons exhibited advanced activity onset and a shorter circadian period. Loss of SIK3 in arginine vasopressin (AVP)-producing neurons lengthened the circadian cycle, but the arousal peak phase was similar to that in control mice. Heterozygous deficiency of histone deacetylase (HDAC) 4, a SIK3 substrate, shortened the circadian cycle, whereas mice with HDAC4 S245A, which is resistant to phosphorylation by SIK3, delayed the arousal peak phase. Phase-delayed core clock gene expressions were detected in the liver of mice lacking SIK3 in GABAergic neurons. These results suggest that the SIK3-HDAC4 pathway regulates the circadian period length and the timing of arousal through NMS-positive neurons in the SCN.

duper is a null mutation of Cryptochrome 1 in Syrian hamsters.

The duper mutation is a recessive mutation that shortens the period length of the circadian rhythm in Syrian hamsters. These animals show a large phase shift when responding to light pulses. Limited genetic resources for the Syrian hamster (Mesocricetus auratus) presented a major obstacle to cloning duper. This caused the duper mutation to remain unknown for over a decade. In this study, we did a de novo genome assembly of Syrian hamsters with long-read sequencing data from two different platforms, Pacific Biosciences and Oxford Nanopore Technologies. Using two distinct ecotypes and a fast homozygosity mapping strategy, we identified duper as an early nonsense allele of Cryptochrome 1 (Cry1) leading to a short, unstable protein. CRY1 is known as a highly conserved component of the repressive limb of the core circadian clock. The genome assembly and other genomic datasets generated in this study will facilitate the use of the Syrian hamster in biomedical research.

The Function, Regulation, and Mechanism of Protein Turnover in Circadian Systems in Neurospora and Other Species.

Circadian clocks drive a large array of physiological and behavioral activities. At the molecular level, circadian clocks are composed of positive and negative elements that form core oscillators generating the basic circadian rhythms. Over the course of the circadian period, circadian negative proteins undergo progressive hyperphosphorylation and eventually degrade, and their stability is finely controlled by complex post-translational pathways, including protein modifications, genetic codon preference, protein-protein interactions, chaperon-dependent conformation maintenance, degradation, etc. The effects of phosphorylation on the stability of circadian clock proteins are crucial for precisely determining protein function and turnover, and it has been proposed that the phosphorylation of core circadian clock proteins is tightly correlated with the circadian period. Nonetheless, recent studies have challenged this view. In this review, we summarize the research progress regarding the function, regulation, and mechanism of protein stability in the circadian clock systems of multiple model organisms, with an emphasis on Neurospora crassa, in which circadian mechanisms have been extensively investigated. Elucidation of the highly complex and dynamic regulation of protein stability in circadian clock networks would greatly benefit the integrated understanding of the function, regulation, and mechanism of protein stability in a wide spectrum of other biological processes.

ZmCCT regulates photoperiod-dependent flowering and response to stresses in maize.

BACKGROUND: Appropriate flowering time is very important to the success of modern agriculture. Maize (Zea mays L.) is a major cereal crop, originated in tropical areas, with photoperiod sensitivity. Which is an important obstacle to the utilization of tropical/subtropical germplasm resources in temperate regions. However, the study on the regulation mechanism of photoperiod sensitivity of maize is still in the early stage. Although it has been previously reported that ZmCCT is involved in the photoperiod response and delays maize flowering time under long-day conditions, the underlying mechanism remains unclear. RESULTS: Here, we showed that ZmCCT overexpression delays flowering time and confers maize drought tolerance under LD conditions. Implementing the Gal4-LexA/UAS system identified that ZmCCT has a transcriptional inhibitory activity, while the yeast system showed that ZmCCT has a transcriptional activation activity. DAP-Seq analysis and EMSA indicated that ZmCCT mainly binds to promoters containing the novel motifs CAAAAATC and AAATGGTC. DAP-Seq and RNA-Seq analysis showed that ZmCCT could directly repress the expression of ZmPRR5 and ZmCOL9, and promote the expression of ZmRVE6 to delay flowering under long-day conditions. Moreover, we also demonstrated that ZmCCT directly binds to the promoters of ZmHY5, ZmMPK3, ZmVOZ1 and ZmARR16 and promotes the expression of ZmHY5 and ZmMPK3, but represses ZmVOZ1 and ZmARR16 to enhance stress resistance. Additionally, ZmCCT regulates a set of genes associated with plant development. CONCLUSIONS: ZmCCT has dual functions in regulating maize flowering time and stress response under LD conditions. ZmCCT negatively regulates flowering time and enhances maize drought tolerance under LD conditions. ZmCCT represses most flowering time genes to delay flowering while promotes most stress response genes to enhance stress tolerance. Our data contribute to a comprehensive understanding of the regulatory mechanism of ZmCCT in controlling maize flowering time and stress response.

Genomic footprints of repeated evolution of CAM photosynthesis in a Neotropical species radiation.

The adaptive radiation of Bromeliaceae (pineapple family) is one of the most diverse among Neotropical flowering plants. Diversification in this group was facilitated by shifts in several adaptive traits or "key innovations" including the transition from C3 to CAM photosynthesis associated with xeric (heat/drought) adaptation. We used phylogenomic approaches, complemented by differential gene expression (RNA-seq) and targeted metabolite profiling, to address the mechanisms of C3 /CAM evolution in the extremely species-rich bromeliad genus, Tillandsia, and related taxa. Evolutionary analyses of whole-genome sequencing and RNA-seq data suggest that evolution of CAM is associated with coincident changes to different pathways mediating xeric adaptation in this group. At the molecular level, C3 /CAM shifts were accompanied by gene expansion of XAP5 CIRCADIAN TIMEKEEPER homologs, a regulator involved in sugar- and light-dependent regulation of growth and development. Our analyses also support the re-programming of abscisic acid-related gene expression via differential expression of ABF2/ABF3 transcription factor homologs, and adaptive sequence evolution of an ENO2/LOS2 enolase homolog, effectively tying carbohydrate flux to abscisic acid-mediated abiotic stress response. By pinpointing different regulators of overlapping molecular responses, our results suggest plausible mechanistic explanations for the repeated evolution of correlated adaptive traits seen in a textbook example of an adaptive radiation.

Early doors (Edo) mutant mouse reveals the importance of period 2 (PER2) PAS domain structure for circadian pacemaking.

The suprachiasmatic nucleus (SCN) defines 24 h of time via a transcriptional/posttranslational feedback loop in which transactivation of Per (period) and Cry (cryptochrome) genes by BMAL1-CLOCK complexes is suppressed by PER-CRY complexes. The molecular/structural basis of how circadian protein complexes function is poorly understood. We describe a novel N-ethyl-N-nitrosourea (ENU)-induced mutation, early doors (Edo), in the PER-ARNT-SIM (PAS) domain dimerization region of period 2 (PER2) (I324N) that accelerates the circadian clock of Per2(Edo/Edo) mice by 1.5 h. Structural and biophysical analyses revealed that Edo alters the packing of the highly conserved interdomain linker of the PER2 PAS core such that, although PER2(Edo) complexes with clock proteins, its vulnerability to degradation mediated by casein kinase 1ε (CSNK1E) is increased. The functional relevance of this mutation is revealed by the ultrashort (<19 h) but robust circadian rhythms in Per2(Edo/Edo); Csnk1e(Tau/Tau) mice and the SCN. These periods are unprecedented in mice. Thus, Per2(Edo) reveals a direct causal link between the molecular structure of the PER2 PAS core and the pace of SCN circadian timekeeping.

Artificial light at night shifts daily activity patterns but not the internal clock in the great tit (Parus major).

Artificial light at night has shown a dramatic increase over the last decades and continues to increase. Light at night can have strong effects on the behaviour and physiology of species, which includes changes in the daily timing of activity; a clear example is the advance in dawn song onset in songbirds by low levels of light at night. Although such effects are often referred to as changes in circadian timing, i.e. changes to the internal clock, two alternative mechanisms are possible. First, light at night can change the timing of clock controlled activity, without any change to the clock itself; e.g. by a change in the phase relation between the circadian clock and expression of activity. Second, changes in daily activity can be a direct response to light ('masking'), without any involvement of the circadian system. Here, we studied whether the advance in onset of activity by dim light at night in great tits (Parus major) is indeed attributable to a phase shift of the internal clock. We entrained birds to a normal light/dark (LD) cycle with bright light during daytime and darkness at night, and to a comparable (LDim) schedule with dim light at night. The dim light at night strongly advanced the onset of activity of the birds. After at least six days in LD or LDim, we kept birds in constant darkness (DD) by leaving off all lights so birds would revert to their endogenous, circadian system controlled timing of activity. We found that the timing of onset in DD was not dependent on whether the birds were kept at LD or LDim before the measurement. Thus, the advance of activity under light at night is caused by a direct effect of light rather than a phase shift of the internal clock. This demonstrates that birds are capable of changing their daily activity to low levels of light at night directly, without the need to alter their internal clock.

Temporal pathways between circadian rhythm, depression and anxiety in the transition from adolescence to early adulthood.

BACKGROUND: Sleep and circadian rhythm problems intertwine with affective disorders. Adolescents are particularly vulnerable to developing sleep and affective problems. Yet, the temporal pathways between circadian rhythm, depression and anxiety in the transition phase from adolescence to early adulthood are not fully understood. METHODS: 233 adolescents (76 % females) participated at two time points (T1 and T2) at an interval of 19-months (aged 16.8 and 18.4 years). We used The Beck Depression Inventory-II, Generalized Anxiety Disorder Assessment, GENEActiv actigraphy across 8 days (delayed sleep phase (DSP), sleep duration, midpoint, and regularity), and iButton 1922L thermologgers across 3 days (intrinsic circadian period length, amplitude, and mesor). RESULTS: A shorter sleep duration at T1 associated with an increase in affective problems at T2, and affective problems at T1 associated with an increase in sleep irregularity at T2. A longer circadian period at T1 associated with an increase in males' affective problems at T2. Moderate to severe depression and anxiety at T1 associated with a 2.69-fold risk (95 % CI 1.38-5.26, p = 0.004) and 2.11-fold risk (95 % CI 1.04-4.25, p = 0.038) of poor sleep quality at T2. Moderate to severe generalized anxiety associated with a 3.17-fold risk (95 % CI 1.35-7.41, p = 0.008) of DSP at T2. LIMITATIONS: The follow-up period is short. CONCLUSIONS: The results revealed bidirectional temporal links between sleep and affective problems. Novel observations include a heightened risk of future DSP following a current anxiety disorder and a heightened risk of affective problems following a longer circadian period measured from the 24-hour temperature variation in males.

XAP5 CIRCADIAN TIMEKEEPER specifically modulates 3' splice site recognition and is important for circadian clock regulation partly by alternative splicing of LHY and TIC.

Pre-mRNA splicing is an essential step during gene expression, which takes place in the spliceosome, a large dynamic ribonucleoprotein complex assembled in a stepwise manner. During the last decade, several spliceosomal mutants were functionally identified to cause a lengthened circadian period by introducing intron retention defects into circadian clock genes in Arabidopsis. However, the spliceosomal components that play opposite roles in the circadian period via alternative 3' splice site (Alt 3'ss) are largely unknown. Here, we demonstrated that XCT (XAP5 CIRCADIAN TIMEKEEPER) is a key spliceosomal component associated with multiple splicing factors. Moreover, genome-wide analysis revealed that inactivation of XCT particularly results in defects in Alt 3'ss recognition by RNA sequencing. Further analysis indicated that a strong alteration in the 3' splice sites of LHY and TIC partly accounts for the shortened circadian period of the xct mutant. Therefore, our results demonstrated that mutations in XCT shortened the circadian period partly by alternative splicing of LHY and TIC particularly in 3' splice site recognition, which provides new insight into the link between alternative splicing and the circadian clock.

Identification of Genes Contributing to a Long Circadian Period in Drosophila Melanogaster.

The endogenous circadian period of animals and humans is typically very close to 24 h. Individuals with much longer circadian periods have been observed, however, and in the case of humans, these deviations have health implications. Previously, we observed a line of Drosophila with a very long average period of 31.3 h for locomotor activity behavior. Preliminary mapping indicated that the long period did not map to known canonical clock genes but instead mapped to multiple chromosomes. Using RNA-Seq, we surveyed the whole transcriptome of fly heads from this line across time and compared it with a wild-type control. A three-way generalized linear model revealed that approximately two-thirds of the genes were expressed differentially among the two genotypes, while only one quarter of the genes varied across time. Using these results, we applied algorithms to search for genes that oscillated over 24 h, identifying genes not previously known to cycle. We identified 166 differentially expressed genes that overlapped with a previous Genome-wide Association Study (GWAS) of circadian behavior, strongly implicating them in the long-period phenotype. We tested mutations in 45 of these genes for their effect on the circadian period. Mutations in Alk, alph, CG10089, CG42540, CG6034, Kairos (CG6123), CG8768, klg, Lar, sick, and tinc had significant effects on the circadian period, with seven of these mutations increasing the circadian period of locomotor activity behavior. Genetic rescue of mutant Kairos restored the circadian period to wild-type levels, suggesting it has a critical role in determining period length in constant darkness.

Mice held at an environmental photic cycle oscillating at their tau-like period length do not show the high-fat diet-induced obesity that develops under the 24-hour photic cycle.

Circadian disruptions precede high-fat diet (HFD)-induced obesity (DIO). Deviation of the endogenous circadian rhythm period length (tau) from 24 hours correlates with mice inter-strain DIO under the 24-hour light-dark cycle (T-cycle). Additionally, entrainment to a tau-resembling T-cycle attenuates DIO, to some extent, in muted mice. These phenomena suggest that entrainment to a 24-hour T-cycle promotes DIO beyond that expected from the HFD-induced metabolic disruptions. However, the hypothesis that entrainment to a tau-resembling T-cycle attenuates DIO has not been tested in wild-type mice. Therefore, we examined, in newborn female FVB/N mice, whether DIO found under their 'regular' 24-hour T-cycle is attenuated under a T-cycle oscillating at their tau-resembling period of 23.7 h, which is diverted from 24 hours by only 0.3 h. Compared to mice fed a low-fat diet, those fed an HFD under the 24-hour T-cycle showed a disrupted pattern of circadian locomotor activity prior to DIO onset. Both these phenomena were absent under the tau-like T-cycle. DIO developed under the 24-hour T-cycle despite similar caloric intake, and was associated with the lower locomotor activity of HFD-fed mice compared to the other mouse groups. These results demonstrated that DIO is secondary to HFD-induced circadian disruptions that are not harmonized by the strongest Zeitgeber (light-dark cycle) when oscillating at a period that diverts by as little as ca. 0.3-h from tau. More importantly, imposing a light-dark cycle oscillating at a tau-like period length, which enhances entrainment and presumably reinforces endogenous circadian rhythms, prevented HFD-induced circadian disruptions and enabled tighter control of energy homeostasis, as manifested by the absence of DIO, even under ad-lib HFD feeding. These results support the identification of tau-related biomarkers, which may be considered as risk-factors for DIO. Moreover, these findings may promote the development of clock-related pharmaceutical interventions that will reduce the gap between tau and 24 hours, and increase the robustness of the endogenous and entrained circadian rhythms. This will enable reducing DIO, even without caloric restriction or time-restricted feeding.

Workshop report. Circadian rhythm sleep-wake disorders: gaps and opportunities.

This White Paper presents the results from a workshop cosponsored by the Sleep Research Society (SRS) and the Society for Research on Biological Rhythms (SRBR) whose goals were to bring together sleep clinicians and sleep and circadian rhythm researchers to identify existing gaps in diagnosis and treatment and areas of high-priority research in circadian rhythm sleep-wake disorders (CRSWD). CRSWD are a distinct class of sleep disorders caused by alterations of the circadian time-keeping system, its entrainment mechanisms, or a misalignment of the endogenous circadian rhythm and the external environment. In these disorders, the timing of the primary sleep episode is either earlier or later than desired, irregular from day-to-day, and/or sleep occurs at the wrong circadian time. While there are incomplete and insufficient prevalence data, CRSWD likely affect at least 800,000 and perhaps as many as 3 million individuals in the United States, and if Shift Work Disorder and Jet Lag are included, then many millions more are impacted. The SRS Advocacy Taskforce has identified CRSWD as a class of sleep disorders for which additional high-quality research could have a significant impact to improve patient care. Participants were selected for their expertise and were assigned to one of three working groups: Phase Disorders, Entrainment Disorders, and Other. Each working group presented a summary of the current state of the science for their specific CRSWD area, followed by discussion from all participants. The outcome of those presentations and discussions are presented here.

Attenuated TOR signaling lengthens circadian period in Arabidopsis.

By timing many diel rhythmic events, circadian clock provides an adaptive advantage for higher plants. Meanwhile, circadian clock displays plasticity and can be entrained by the external environmental cues and internal factors. However, whether cellular energy status can regulate circadian clock is largely unknown in higher plants. The evolutionarily conserved TOR (target of rapamycin) signaling among eukaryotic organisms has been implicated as an integrator for cellular nutrient and energy status. Here, we demonstrated that chemically blocking electron transport chain of mitochondrial can lengthen the circadian period. Similarly, chemical inhibition of TOR activity by Torin 1, a specific inhibitor for TOR kinase, and knockdown of TOR transcript levels significantly elongate the circadian period as well. Our findings imply that TOR signaling may mediate energy status-regulated circadian clock in plants, and the reciprocal regulation between the circadian clock and TOR signaling might be an evolutionary mechanism for fitness and adaptation in plants.

Populations Are Differentiated in Biological Rhythms without Explicit Elevational Clines in the Plant Mimulus laciniatus.

Environmental variation along an elevational gradient can yield phenotypic differentiation resulting from varying selection pressures on plant traits related to seasonal responses. Thus, genetic clines can evolve in a suite of traits, including the circadian clock, that drives daily cycling in varied traits and that shares its genetic background with adaptation to seasonality. We used populations of annual Mimulus laciniatus from different elevations in the Sierra Nevada in California to explore among-population differentiation in the circadian clock, flowering responses to photoperiod, and phenological traits (days to cotyledon emergence, days to flowering, and days to seed ripening) in controlled common-garden conditions. Further, we examined correlations of these traits with environmental variables related to temperature and precipitation. We observed that the circadian period in leaf movement was differentiated among populations sampled within about 100 km, with population means varying by 1.6 h. Significant local genetic variation occurred within 2 populations in which circadian period among families varied by up to 1.8 h. Replicated treatments with variable ecologically relevant photoperiods revealed marked population differentiation in critical day length for flowering that ranged from 11.0 to 14.1 h, corresponding to the time period between late February and mid-May in the wild. Flowering time varied among populations in a 14-h photoperiod. Regardless of this substantial population-level diversity, obvious linear clinality in trait variability across elevations could not be determined based on our genotypic sample; it is possible that more complex spatial patterns of variation arise in complex terrains such as those in the Sierra Nevada. Moreover, we did not find statistically significant bivariate correlations between population means of different traits. Our research contributes to the understanding of genetic variation in the circadian clock and in seasonal responses in natural populations, highlighting the need for more comprehensive investigations on the association between the clock and other adaptive traits in plants.

High-throughput sleep phenotyping produces robust and heritable traits in Diversity Outbred mice and their founder strains.

STUDY OBJECTIVES: This study describes high-throughput phenotyping strategies for sleep and circadian behavior in mice, including examinations of robustness, reliability, and heritability among Diversity Outbred (DO) mice and their eight founder strains. METHODS: We performed high-throughput sleep and circadian phenotyping in male mice from the DO population (n = 338) and their eight founder strains: A/J (n = 6), C57BL/6J (n = 14), 129S1/SvlmJ (n = 6), NOD/LtJ (n = 6), NZO/H1LtJ (n = 6), CAST/EiJ (n = 8), PWK/PhJ (n = 8), and WSB/EiJ (n = 6). Using infrared beam break systems, we defined sleep as at least 40 s of continuous inactivity and quantified sleep-wake amounts and bout characteristics. We developed assays to measure sleep latency in a new environment and during a modified Murine Multiple Sleep Latency Test, and estimated circadian period from wheel-running experiments. For each trait, broad-sense heritability (proportion of variability explained by all genetic factors) was derived in founder strains, while narrow-sense heritability (proportion of variability explained by additive genetic effects) was calculated in DO mice. RESULTS: Phenotypes were robust to different inactivity durations to define sleep. Differences across founder strains and moderate/high broad-sense heritability were observed for most traits. There was large phenotypic variability among DO mice, and phenotypes were reliable, although estimates of heritability were lower than in founder mice. This likely reflects important nonadditive genetic effects. CONCLUSIONS: A high-throughput phenotyping strategy in mice, based primarily on monitoring of activity patterns, provides reliable and heritable estimates of sleep and circadian traits. This approach is suitable for discovery analyses in DO mice, where genetic factors explain some proportion of phenotypic variation.

A high-throughput delayed fluorescence method reveals underlying differences in the control of circadian rhythms in Triticum aestivum and Brassica napus.

BACKGROUND: A robust circadian clock has been implicated in plant resilience, resource-use efficiency, competitive growth and yield. A huge number of physiological processes are under circadian control in plants including: responses to biotic and abiotic stresses; flowering time; plant metabolism; and mineral uptake. Understanding how the clock functions in crops such as Triticum aestivum (bread wheat) and Brassica napus (oilseed rape) therefore has great agricultural potential. Delayed fluorescence (DF) imaging has been shown to be applicable to a wide range of plant species and requires no genetic transformation. Although DF has been used to measure period length of both mutants and wild ecotypes of Arabidopsis, this assay has never been systematically optimised for crop plants. The physical size of both B. napus and T. aestivum led us to develop a representative sampling strategy which enables high-throughput imaging of these crops. RESULTS: In this study, we describe the plant-specific optimisation of DF imaging to obtain reliable circadian phenotypes with the robustness and reproducibility to detect diverging periods between cultivars of the same species. We find that the age of plant material, light regime and temperature conditions all significantly effect DF rhythms and describe the optimal conditions for measuring robust rhythms in each species. We also show that sections of leaf can be used to obtain period estimates with improved throughput for larger sample size experiments. CONCLUSIONS: We present an optimized protocol for high-throughput phenotyping of circadian period specific to two economically valuable crop plants. Application of this method revealed significant differences between the periods of several widely grown elite cultivars. This method also identified intriguing differential responses of circadian rhythms in T. aestivum compared to B. napus; specifically the dramatic change to rhythm robustness when plants were imaged under constant light versus constant darkness. This points towards diverging networks underlying circadian control in these two species.

Idiopathic Hypersomnia Patients Revealed Longer Circadian Period Length in Peripheral Skin Fibroblasts.

The vast majority of living organisms have evolved a circadian rhythm of roughly 24 h in adaptation to ever-changing environmental conditions, such as the cycle of light and darkness. In some sleep disorders like idiopathic hypersomnia (IH) this adaptation is defective. As the etiology of this disease is largely unknown, we examined the in vitro circadian period length of patients suffering from IH. The patients were diagnosed according to the ICSD3-criteria by clinical history, polysomnography (PSG), and multiple sleep latency testing (MSLT). In order to gain insight into the molecular mechanism of this sleep disorder we collected fibroblasts from skin biopsies of IH patients and healthy subjects. We determined the circadian period length of the primary fibroblast cells by lentiviral infection with a construct expressing a luciferase gene under the control of a BMAL1 promoter. The group of IH patients revealed on average a prolonged circadian period length. In comparison to the group of healthy controls (HC) the mean period length was estimated to be 0.82 h (95%-CI 0.44-1.20 h) longer in the patient group. This finding further stresses a disturbed regulation of the circadian rhythm in IH patients as part of the pathophysiology of this complex and poorly understood primary sleep disorder.

Circadian phase, circadian period and chronotype are reproducible over months.

The timing of the circadian clock, circadian period and chronotype varies among individuals. To date, not much is known about how these parameters vary over time in an individual. We performed an analysis of the following five common circadian clock and chronotype measures: 1) the dim light melatonin onset (DLMO, a measure of circadian phase), 2) phase angle of entrainment (the phase the circadian clock assumes within the 24-h day, measured here as the interval between DLMO and bedtime/dark onset), 3) free-running circadian period (tau) from an ultradian forced desynchrony protocol (tau influences circadian phase and phase angle of entrainment), 4) mid-sleep on work-free days (MSF from the Munich ChronoType Questionnaire; MCTQ) and 5) the score from the Morningness-Eveningness Questionnaire (MEQ). The first three are objective physiological measures, and the last two are measures of chronotype obtained from questionnaires. These data were collected from 18 individuals (10 men, eight women, ages 21-44 years) who participated in two studies with identical protocols for the first 10 days. We show how much these circadian rhythm and chronotype measures changed from the first to the second study. The time between the two studies ranged from 9 months to almost 3 years, depending on the individual. Since the full experiment required living in the laboratory for 14 days, participants were unemployed, had part-time jobs or were freelance workers with flexible hours. Thus, they did not have many constraints on their sleep schedules before the studies. The DLMO was measured on the first night in the lab, after free-sleeping at home and also after sleeping in the lab on fixed 8-h sleep schedules (loosely tailored to their sleep times before entering the laboratory) for four nights. Graphs with lines of unity (when the value from the first study is identical to the value from the second study) showed how much each variable changed from the first to the second study. The DLMO did not change more than 2 h from the first to the second study, except for two participants whose sleep schedules changed the most between studies, a change in sleep times of 3 h. Phase angle did not change by more than 2 h regardless of changes in the sleep schedule. Circadian period did not change more than 0.2 h, except for one participant. MSF did not change more than 1 h, except for two participants. MEQ did not change more than 10 points and the categories (e.g. M-type) did not change. Pearson's correlations for the DLMO between the first and second studies increased after participants slept in the lab on their individually timed fixed 8-h sleep schedules for four nights. A longer time between the two studies did not increase the difference between any of the variables from the first to the second study. This analysis shows that the circadian clock and chronotype measures were fairly reproducible, even after many months between the two studies.

The cell adhesion molecule EphA4 is involved in circadian clock functions.

Circadian (∼24 h) rhythms of cellular network plasticity in the central circadian clock, the suprachiasmatic nucleus (SCN), have been described. The neuronal network in the SCN regulates photic resetting of the circadian clock as well as stability of the circadian system during both entrained and constant conditions. EphA4, a cell adhesion molecule regulating synaptic plasticity by controlling connections of neurons and astrocytes, is expressed in the SCN. To address whether EphA4 plays a role in circadian photoreception and influences the neuronal network of the SCN, we have analyzed circadian wheel-running behavior of EphA4 knockout (EphA4-/- ) mice under different light conditions and upon photic resetting, as well as their light-induced protein response in the SCN. EphA4-/- mice exhibited reduced wheel-running activity, longer endogenous periods under constant darkness and shorter periods under constant light conditions, suggesting an effect of EphA4 on SCN function. Moreover, EphA4-/- mice exhibited suppressed phase delays of their wheel-running activity following a light pulse during the beginning of the subjective night (CT15). Accordingly, light-induced c-FOS (FBJ murine osteosarcoma viral oncogene homolog) expression was diminished. Our results suggest a circadian role for EphA4 in the SCN neuronal network, affecting the circadian system and contributing to the circadian response to light.

Geographic Variation of Plant Circadian Clock Function in Natural and Agricultural Settings.

The increasing demand for improved agricultural production will require more efficient breeding for traits that maintain yield under heterogeneous environments. The internal circadian oscillator is essential for perceiving and coordinating environmental cues such as day length, temperature, and abiotic stress responses within physiological processes. To investigate the contribution of the circadian clock to local adaptability, we have analyzed circadian period by leaf movement in natural populations of Mimulus guttatus and domesticated cultivars of Glycine max. We detected consistent variation in circadian period along a latitudinal gradient in annual populations of the wild plant and the selectively bred crop, and this provides novel evidence of natural and artificial selection for circadian performance. These findings provide new support that the circadian clock acts as a central regulator of plant adaptability and further highlight the potential of applying circadian clock gene variation to marker-assisted breeding programs in crops.