Individual Clock Research Articles

Association of sleep and generalized anxiety disorder in Korean adolescents

BackgroundGeneralized anxiety disorder (GAD) is a common anxiety disorder among adolescents, significantly impacting their concentration and learning capabilities. The connection between emotional well-being and sleep is well-established, and Korean adolescents are particularly prone to inadequate sleep. This study aimed to determine the association between sleep duration and GAD in Korean adolescents.MethodsThis study was conducted using data from 106,513 adolescents aged 12–18 years. Data from the 2020–2022 Korea Youth Risk Behavior Survey were used. Sleep duration was classified into five groups, based on an average sleep duration of 7–7.9 h in adolescents. Social jet lag was defined as a misalignment between an individual's biological and social clocks. Differences in sleep duration between weekdays and weekends, social jet lag, and bedtime were each classified into three categories. Multiple logistic regression analysis was performed to evaluate the association between sleep duration and GAD.ResultsComparing the five groups classified based on sleep duration, adolescents in the groups that slept less experienced a significant increase in the odds of developing GAD (adjusted odds ratio [aOR]: boys: 1.10 in the 6.0–6.9-h group, 1.14 in the 5.0–5.9-h group, and 1.23 in the ≤ 4.9-h group; girls: 1.05 in the 6.0–6.9-h group, 1.19 in the 5.0–5.9-h group, 1.22 in the ≤ 4.9-h group). Adolescents with poor sleep quality experienced more frequent instances of inadequate sleep (aOR: boys: 2.51; girls: 2.43).ConclusionsGAD is strongly associated with insufficient sleep. Consequently, it is imperative to assess and address GAD in adolescents with irregular sleep patterns.

Are circadian amplitudes and periods correlated? A new twist in the story

Three parameters are important to characterize a circadian and in general any biological clock: period, phase and amplitude. While circadian periods have been shown to correlate with entrainment phases, and clock amplitude influences the phase response of an oscillator to pulse-like zeitgeber signals, the co-modulations of amplitude and periods, which we term twist, have not been studied in detail. In this paper we define two concepts: parametric twist refers to amplitude-period correlations arising in ensembles of self-sustained, limit cycle clocks in the absence of external inputs, and phase space twist refers to the co-modulation of an individual clock’s amplitude and period in response to external zeitgebers. Our findings show that twist influences the interaction of oscillators with the environment, facilitating entrainment, speeding upfastening recovery to pulse-like perturbations or modifying the response of an individual clock to coupling. This theoretical framework might be applied to understand the emerging properties of other oscillating systems.

Steve Brown's legacy: Tools to study the individual human molecular circadian clock and its regulation.

Since the discovery of the genetic origin of the circadian clock in Drosophila melanogaster by Konopka and Benzer in 1971, most of the research about the regulation of the molecular circadian clock relies on laboratory models. Additional models such as Cyanobacteria, Neurospora crassa, Arabidopsis and rodents helped chronobiologists to describe the species-specific molecular clocks and their regulation. However, the lack of tools and the difficulty to access biological samples somehow excluded human from this research landscape outside behavioural research. Among many other impressive achievements, Steve Brown provided to the community of chronobiologists new tools and strategies to study the individual human circadian clock and its regulation.

Lunar synchrony, geography, and individual clocks shape autumn migration timing in an avian migrant.

Timing programs in animal migrants have been selected to synchronize movements that coincide with predictable resources on the breeding and nonbreeding grounds. Migrants face potential temporal conflicts if their migration schedules benefit from synchrony to conflicting rhythms associated with annual biogeographical (circannual) cues, lunar (circalunar) cues, or individually repeatable internal clocks. We repeat-tracked individuals of an avian lunaphilic species, Eastern Whip-poor-will (Antrostomus vociferus), for two to three successive autumn migrations to determine the influence of the lunar cycle, breeding location, and individual repeatability on migration timing. Almost all birds avoided departing for migration during a full moon, likely to take advantage of the bright moonlight to facilitate visual foraging and enhance pre-migration fattening. However, groups from two latitudinally distant sampling areas adjusted their autumn departure timing differently relative to the timing of the September full moon, presumably due to differences in seasonal prey availability. Individual repeatability increased throughout autumn migration, suggesting that the factors responsible for shaping migration timing may differ for different migration stages. Our results, that lunar synchrony, local climate, and individual internal clocks appeared to account for much of the variation in migration timing in whip-poor-wills, underscore the value of measuring potentially interacting factors that shape migratory behavior at species, group, and individual levels. It remains unclear if, or how, maintaining individually repeatable annual migration schedules provides an adaptive benefit for whip-poor-wills or other lunaphilic migrants. Further clarifying the reasons for phenotypic variation in whip-poor-will migration timing will improve predictions of their abilities to adjust migratory movements under changing environmental conditions.

Using digital assessment technology to detect neuropsychological problems in primary care settings.

Screening for neurocognitive impairment and psychological distress in ambulatory primary and specialty care medical settings is an increasing necessity. The Core Cognitive Evaluation™ (CCE) is administered/scored using an iPad, requires approximately 8 min, assesses 3- word free recall and clock drawing to command and copy, asks questions about lifestyle and health, and queries for psychological distress. This information is linked with patients' self- reported concerns about memory and their cardiovascular risks. A total of 199 ambulatory patients were screened with the CCE as part of their routine medical care. The CCE provides several summary indices, and scores on 44 individual digital clock variables across command and copy tests conditions. Subjective memory concerns were endorsed by 41% of participants. Approximately 31% of participants reported psychological distress involving loneliness, anxiety, or depression. Patients with self-reported memory concerns scored lower on a combined delay 3- word/ clock drawing index (p < 0.016), the total summary clock drawing command/ copy score (p < 0.050), and clock drawing to command Drawing Efficiency (p < 0.036) and Simple and Complex Motor (p < 0.029) indices. Patients treated for diabetes and atherosclerotic cardiovascular disease (ASCVD) scored lower on selected CCE outcome measures (p < 0.035). Factor analyses suggest that approximately 10 underlying variables can explain digital clock drawing performance. The CCE is a powerful neurocognitive assessment tool that is sensitive to patient's subjective concerns about possible decline in memory, mood symptoms, possible cognitive impairment, and cardiovascular risk. iPad administration ensures total reliability for test administration and scoring. The CCE is easily deployable in outpatient ambulatory primary care settings.

Differential Expression of Circadian Clock Genes in the Bovine Neuroendocrine Adrenal System.

Knowledge of circadian rhythm clock gene expression outside the suprachiasmatic nucleus is increasing. The purpose of this study was to determine whether expression of circadian clock genes differed within or among the bovine stress axis tissues (e.g., amygdala, hypothalamus, pituitary, adrenal cortex, and adrenal medulla). Tissues were obtained at an abattoir from eight mature nonpregnant Brahman cows that had been maintained in the same pasture and nutritional conditions. Sample tissues were stored in RNase-free sterile cryovials at -80 °C until the total RNA was extracted, quantified, assessed, and sequenced (NovaSeq 6000 system; paired-end 150 bp cycles). The trimmed reads were then mapped to a Bos taurus (B. taurus) reference genome (Umd3.1). Further analysis used the edgeR package. Raw gene count tables were read into RStudio, and low-expression genes were filtered out using the criteria of three minimum reads per gene in at least five samples. Normalization factors were then calculated using the trimmed mean of M values method to produce normalized gene counts within each sample tissue. The normalized gene counts important for a circadian rhythm were analyzed within and between each tissue of the stress axis using the GLM and CORR procedures of the Statistical Analysis System (SAS). The relative expression profiles of circadian clock genes differed (p < 0.01) within each tissue, with neuronal PAS domain protein 2 (NPAS2) having greater expression in the amygdala (p < 0.01) and period circadian regulator (PER1) having greater expression in all other tissues (p < 0.01). The expression among tissues also differed (p < 0.01) for individual circadian clock genes, with circadian locomotor output cycles protein kaput (CLOCK) expression being greater within the adrenal tissues and nuclear receptor subfamily 1 group D member 1 (NR1D1) expression being greater within the other tissues (p < 0.01). Overall, the results indicate that within each tissue, the various circadian clock genes were differentially expressed, in addition to being differentially expressed among the stress tissues of mature Brahman cows. Future use of these findings may assist in improving livestock husbandry and welfare by understanding interactions of the environment, stress responsiveness, and peripheral circadian rhythms.

Joint Synchronization and Range Estimation With Correlated Arrival Times in Molecular Communication Networks

Time synchronization is needed in information exchange and collaboration among nanomachines for molecular communication networks. The time of arrival-based synchronization method requires the range information between the transmitter nanomachine (TN) and the receiver nanomachine (RN) in order to improve the accuracy of clock synchronization. The range information between the nanomachines helps to choose the optimal transmission rate. In contrast to the existing methods, this article proposes a joint clock offset and range estimation with correlated arrival times based on the maximum likelihood estimator. The RN uses the molecules' transmission and correlated arrival times to estimate the clock offset and range between the TN and RN. Even with correlated arrival times, the individual clock offset estimation performance is comparable to independent arrival times performance. Notably, the joint estimation problem with correlated arrival times is generic and realistic for molecular applications.

Are circadian amplitudes and periods correlated? A new twist in the story.

Three parameters are important to characterize a circadian and in general any biological clock: period, phase and amplitude. While circadian periods have been shown to correlate with entrainment phases, and clock amplitude influences the phase response of an oscillator to pulse-like zeitgeber signals, the co-modulations of amplitude and periods, which we term twist, have not been studied in detail. In this paper we define two concepts: parametric twist refers to amplitude-period correlations arising in ensembles of self-sustained, limit cycle clocks in the absence of external inputs, and phase space twist refers to the co-modulation of an individual clock's amplitude and period in response to external zeitgebers. Our findings show that twist influences the interaction of oscillators with the environment, facilitating entrainment, speeding upfastening recovery to pulse-like perturbations or modifying the response of an individual clock to coupling. This theoretical framework might be applied to understand the emerging properties of other oscillating systems.

Detection of Alzheimer's Disease Using Logistic Regression and Clock Drawing Errors.

Alzheimer's disease is an incurable disorder that accounts for up to 70% of all dementia cases. While the prevalence of Alzheimer's disease and other types of dementia has increased by more than 160% in the last 30 years, the rates of undetected cases remain critically high. The present work aims to address the underdetection of Alzheimer's disease by proposing four logistic regression models that can be used as a foundation for community-based screening tools that do not require the participation of medical professionals. Our models make use of individual clock drawing errors as well as complementary patient data that is highly available and easily collectible. All models were controlled for age, education, and gender. The discriminative ability of the models was evaluated by area under the receiver operating characteristic curve (AUC), the Hosmer-Lemeshow test, and calibration plots were used to assess calibration. Finally, decision curve analysis was used to quantify clinical utility. We found that among 10 possible CDT errors, only 3 were informative for the detection of Alzheimer's disease. Our base regression model, containing only control variables and clock drawing errors, produced an AUC of 0.825. The other three models were built as extensions of the base model with the step-wise addition of three groups of complementary data, namely cognitive features (semantic fluency score), genetic predisposition (family history of dementia), and cardio-vascular features (BMI, blood pressure). The addition of verbal fluency scores significantly improved the AUC compared to the base model (0.91 AUC). However, further additions did not make a notable difference in discriminatory power. All models showed good calibration. In terms of clinical utility, the derived models scored similarly and greatly outperformed the base model. Our results suggest that the combination of clock symmetry and clock time errors plus verbal fluency scores may be a suitable candidate for developing accessible screening tools for Alzheimer's disease. However, future work should validate our findings in larger and more diverse datasets.

Protocols for in vitro reconstitution of the cyanobacterial circadian clock.

Circadian clocks are intracellular systems that orchestrate metabolic processes in anticipation of sunrise and sunset by providing an internal representation of local time. Because the ~24-h metabolic rhythms they produce are important to health across diverse life forms there is growing interest in their mechanisms. However, mechanistic studies are challenging in vivo due to the complex, that is, poorly defined, milieu of live cells. Recently, we reconstituted the intact circadian clock of cyanobacteria in vitro. It oscillates autonomously and remains phase coherent for many days with a fluorescence-based readout that enables real-time observation of individual clock proteins and promoter DNA simultaneously under defined conditions without user intervention. We found that reproducibility of the reactions required strict adherence to the quality of each recombinant clock protein purified from Escherichia coli. Here, we provide protocols for preparing in vitro clock samples so that other labs can ask questions about how changing environments, like temperature, metabolites, and protein levels are reflected in the core oscillator and propagated to regulation of transcription, providing deeper mechanistic insights into clock biology.

Liver and muscle circadian clocks cooperate to support glucose tolerance in mice

Physiology is regulated by interconnected cell and tissue circadian clocks. Disruption of the rhythms generated by the concerted activity of these clocks is associated with metabolic disease. Here we tested the interactions between clocks in two critical components of organismal metabolism, liver and skeletal muscle, by rescuing clock function either in each organ separately or in both organs simultaneously in otherwise clock-less mice. Experiments showed that individual clocks are partially sufficient for tissue glucose metabolism, yet the connections between both tissue clocks coupled to daily feeding rhythms support systemic glucose tolerance. This synergy relies in part on local transcriptional control of the glucose machinery, feeding-responsive signals such as insulin, and metabolic cycles that connect the muscle and liver. We posit that spatiotemporal mechanisms of muscle and liver play an essential role in the maintenance of systemic glucose homeostasis and that disrupting this diurnal coordination can contribute to metabolic disease.

Circadian clock genes and photoperiodic diapause in the moth Sesamia nonagrioides.

Insects, like most organisms, have an internal circadian clock that oscillates with a daily rhythmicity, and a timing mechanism (photoperiodic clock) that mediates seasonal events, including diapause. It has been argued that there is a connection between the two clocks. The Mediterranean corn stalk borer moth, Sesamia nonagrioides, undergoes facultative diapause governed by photoperiod. To obtain clues to the link between the molecular mechanism of circadian and photoperiod clocks, we cloned and investigated the expression profiles of the clock genes Snper, Sntim, Sncyc and Sncry1 in the aforementioned moth species. Our previous results suggested that these genes might be implicated in the regulation of the diapause programming in S. nonagrioides. Here we studied the expression patterns of these four clock genes in larvae reared under abnormal non-24 h light-dark cycles (L10:D62 and L10:D14:L10:D62) in order to assess whether disruption of circadian clock would have any effect in the photoperiodic regulation of diapause. In the L10:D14:L10:D62 cycle abnormal expression patterns of the Sntim/Sncry1 and Snper/Sncyc, pairs were found, compared to normal 24 h light-dark photoperiods suggesting that individual clock genes are acting independently in the molecular diapause program of S. nonagrioides. Photoperiod therefore appears to be the crucial signal for the regulation of these four genes.

Abstract A027: Defining the role of PER1 and circadian rhythm dysregulation in KRAS/LKB1-mutant lung adenocarcinoma

Abstract KRAS and LKB1 are frequently co-mutated in lung adenocarcinoma and characterize a particularly aggressive, metastatic, and treatment-resistant subtype of this disease. We recently published that the outputs of biological rhythm pathways downstream of the AMPK kinase are altered in KRAS/LKB1-mutant tumors. In a live-cell phenotyping screen we used RNAseq to identify the circadian clock genes PER1 and PER2 as uniquely upregulated in partially transformed and invasive KRAS/LKB1-mutant human bronchial epithelial cells. We have analyzed expression of individual circadian clock genes in KRAS-mutant and KRAS/LKB1-mutant patient tumors and found that expression of the PER genes, particularly PER1, is increased in the KRAS/LKB1 subtype of lung adenocarcinoma. At the protein level, PER1 expression is increased in KRAS/LKB1-mutant HBECs as compared to KRAS-mutant cells. Addback of wild-type LKB1 in A549 cells, which are KRAS/LKB1-mutant, results in a decrease in PER1 protein levels, confirming that LKB1 has a role in controlling PER1 levels. Multiple phosphorylation events lead to PER1 nuclear translocation to control its role as a transcriptional repressor in the core clock. In circadian-synced HBECs, PER1 enters the nucleus in KRAS-mutant cells, but remains cytosolic in KRAS/LKB1-mutant cells. In circadian-synced A549 cells, we also observed a lack of nuclear PER1 localization. Addback of wild-type LKB1 restored nuclear localization of PER1, as did expression of kinase-dead LKB1, suggesting that LKB1’s regulation of PER1 localization is kinase-independent. Our current and future experiments aim to determine how clock-controlled gene expression may be altered in KRAS/LKB1-mutant cells, what the functional impact of altered PER1 expression and localization is on growth of KRAS/LKB1-mutant tumors, and whether pharmacological manipulation of LKB1-related signaling pathways can restore nuclear localization of PER1. Citation Format: Rebecca E. Parker, Junghui Koo, Bhakti Dwivedi, Adam I. Marcus, Melissa Gilbert-Ross. Defining the role of PER1 and circadian rhythm dysregulation in KRAS/LKB1-mutant lung adenocarcinoma [abstract]. In: Proceedings of the AACR Special Conference: Targeting RAS; 2023 Mar 5-8; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Res 2023;21(5_Suppl):Abstract nr A027.

TimeTeller for timing health: The potential of circadian medicine to improve performance, prevent disease and optimize treatment.

Circadian medicine, the study of the effects of time on health and disease has seen an uprising in recent years as a means to enhance health and performance, and optimize treatment timing. Our endogenous time generating system -the circadian clock- regulates behavioural, physiological and cellular processes. Disruptions of the clock, via external factors like shift work or jet lag, or internal perturbations such as genetic alterations, are linked to an increased risk of various diseases like obesity, diabetes, cardiovascular diseases and cancer. By aligning an individual's circadian clock with optimal times for performing daily routines, physical and mental performance, and also the effectiveness of certain therapies can be improved. Despite the benefits of circadian medicine, the lack of non-invasive tools for characterizing the clock limits the potential of the field. TimeTeller is a non-invasive molecular/digital tool for the characterization of circadian rhythms and prediction of daily routines, including treatment timing, to unlock the potential of circadian medicine and implementing it in various settings. Given the multiple known and potentially yet unknown dependent health factors of individual circadian rhythms, the utility of this emerging biomarker is best exploited in data driven, personalized medicine use cases, using health information across lifestyle, care, and research settings.

Generalised Kuramoto models with time-delayed phase-resetting for [formula omitted]-dimensional clocks

We consider a class of Kuramoto models, with an array of N individual k-dimensional clocks (k>1), coupled within a directed, range dependent, network. For each directed connection, a signal triggered at the sending clock incurs a (real valued) time delay before arriving at the receiving clock, where it induces an instantaneous phase reset affecting all k-phases. Instantaneous phase resetting maps (PRMs) for k-dimensional clocks have received little attention. The system may be treated as open and subject to periodic, or other types of, PRM forcing at any individual clock, as a result of external forcing stimuli. We show how the full system, with Nk phase variables, responds to such stimuli, as the impact spreads across the entire network. Beyond simulations, we employ methods to reverse engineer the dynamical behaviour of the whole: estimating the intrinsic dimensions of the responses to different experiments; and by analysing pairwise comparisons between those responses. This shows that the system’s responses are governed by a hierarchy of internal dynamical modes, existing across both the Nk phases and over time.We argue that this Kuramoto system is a model for the human cortex, where each k-dimensional clock models the dynamics of a single neural column, which contains 10,000 densely inter-connected neurons. The Kuramoto model exploits the natural network of networks architecture of the human cortex. An array of N=1M such columns/clocks is at the 10B neuron scale of the human cortex. However its simulation is far more accessible than very large scale (VLS) simulations of neuron-to-neuron systems on supercomputers. The latent modes may have important implications for cognition (information processing) and for consciousness (the existence of internal phenomenological experiences). We argue that the existence of the latter plays a key role in preconditioning the former, reducing the decision sets and the cognitive load, and thus enabling a fast-thinking evolutionary advantage.This is the first time that systems of k-dimensional clocks (k> 1), coupled via time-lagged PRMs, within range dependent networks, have been deployed to demonstrate the basic internal phenomenological elements (of consciousness) and their potential role within immediate cognition.Statement of Significance: We argue that this Kuramoto system is a model for the human cortex, where each of 1M k-dimensional clocks models the dynamics of a single neural column (containing 10,000 densely inter-connected neurons). This Kuramoto model exploits the natural network of networks architecture of the human cortex. Large scale human cortex simulations, with 10B neutrons, usually require a super computer. We show that similar results, using this model, can be obtained on a laptop. In particular we show that such dynamical can support internal phenomenological elements (of conscious experience) and we discuss their potential role in preconditioning immediate cognition, furnishing a “fast thinking” evolutionary advantage to the human brain.

Asymptotic Gradient Clock Synchronization in Wireless Sensor Networks for UWB Localization

Time-of-flight-based localization requires high-accurate synchronization among the nodes in a network. Gradient clock synchronization (GCS) is a class of distributed algorithms capable of providing the demanded accuracy. Global clock rates defined by GCS algorithms are susceptible to drift relative to the individual hardware clock rates. This is due to the lack of a hard tie to a physical clock rate and is identified as the chaotic clock rate phenomenon. This scales range of measurements and can lead to stability issues in the network. This article presents a novel GCS algorithm for ultrawideband (UWB) ranging networks, addressing the chaotic global clock rate phenomenon. A correction term is introduced in the generic GCS algorithm so that the global clock rate is guaranteed to be converging into the average of individual clock rates. This also achieves asymptotic stability in the clock rate error state. The stability of the generic GCS and the proposed method for time-invariant hardware clock rates are compared using eigenvalue analysis in the clock error state space. A Kalman filter-based technique is used to precisely estimate the interanchor clock dynamics and ranges, which are then used in the asymptotic GCS (AGCS) update rule to calculate synchronization parameters. Simulations and experiments are conducted to evaluate the stability and synchronization accuracy of the proposed algorithm. The localization accuracy is evaluated for an indoor quadcopter localization task, which uses a range-assisted inertial navigation system (INS), resulting in rms position errors in the order of 0.2 m.

Adaptive Clock Management of HLS-generated Circuits on FPGAs

In this article, we present Syncopation , a performance-boosting fine-grained timing analysis and adaptive clock management technique for High-Level Synthesis-generated circuits implemented on Field-Programmable Gate Arrays. The key idea is to use the HLS scheduling information along with the placement and routing results to determine the worst-case timing path for individual clock cycles. By adjusting the clock period on a cycle-by-cycle basis, we can increase performance of an HLS-generated circuit. Our experiments show that Syncopation improves performance by 3.2% (geomean) across all benchmarks (up to 47%). In addition, by employing targeted synthesis techniques along with Syncopation, we can achieve 10.3% performance improvement (geomean) across all benchmarks (up to 50%). Syncopation instrumentation is implemented entirely in soft logic without requiring alterations to the HLS-synthesis toolchain or changes to the FPGA, and has been validated on real hardware.

Streamlined single-molecule RNA-FISH of core clock mRNAs in clock neurons in whole mount Drosophila brains.

Circadian clocks are ∼24-h timekeepers that control rhythms in almost all aspects of our behavior and physiology. While it is well known that subcellular localization of core clock proteins plays a critical role in circadian regulation, very little is known about the spatiotemporal organization of core clock mRNAs and its role in generating ∼24-h circadian rhythms. Here we describe a streamlined single molecule Fluorescence In Situ Hybridization (smFISH) protocol and a fully automated analysis pipeline to precisely quantify the number and subcellular location of mRNAs of Clock, a core circadian transcription factor, in individual clock neurons in whole mount Drosophila adult brains. Specifically, we used ∼48 fluorescent oligonucleotide probes that can bind to an individual Clock mRNA molecule, which can then be detected as a diffraction-limited spot. Further, we developed a machine learning-based approach for 3-D cell segmentation, based on a pretrained encoder-decoder convolutional neural network, to automatically identify the cytoplasm and nuclei of clock neurons. We combined our segmentation model with a spot counting algorithm to detect Clock mRNA spots in individual clock neurons. Our results demonstrate that the number of Clock mRNA molecules cycle in large ventral lateral clock neurons (lLNvs) with peak levels at ZT4 (4h after lights are turned on) with ∼80 molecules/neuron and trough levels at ZT16 with ∼30 molecules/neuron. Our streamlined smFISH protocol and deep learning-based analysis pipeline can be employed to quantify the number and subcellular location of any mRNA in individual clock neurons in Drosophila brains. Further, this method can open mechanistic and functional studies into how spatiotemporal localization of clock mRNAs affect circadian rhythms.

Real time, in vivo measurement of neuronal and peripheral clocks in Drosophila melanogaster.

Circadian clocks are highly conserved transcriptional regulators that control ~24 hr oscillations in gene expression, physiological function, and behavior. Circadian clocks exist in almost every tissue and are thought to control tissue-specific gene expression and function, synchronized by the brain clock. Many disease states are associated with loss of circadian regulation. How and when circadian clocks fail during pathogenesis remains largely unknown because it is currently difficult to monitor tissue-specific clock function in intact organisms. Here, we developed a method to directly measure the transcriptional oscillation of distinct neuronal and peripheral clocks in live, intact Drosophila, which we term Locally Activatable BioLuminescence, or LABL. Using this method, we observed that specific neuronal and peripheral clocks exhibit distinct transcriptional properties. Loss of the receptor for PDF, a circadian neurotransmitter critical for the function of the brain clock, disrupts circadian locomotor activity but not all tissue-specific circadian clocks. We found that, while peripheral clocks in non-neuronal tissues were less stable after the loss of PDF signaling, they continued to oscillate. We also demonstrate that distinct clocks exhibit differences in their loss of oscillatory amplitude or their change in period, depending on their anatomical location, mutation, or fly age. Our results demonstrate that LABL is an effective tool that allows rapid, affordable, and direct real-time monitoring of individual clocks in vivo.

Knockouts of positive and negative elements of the circadian clock disrupt photoperiodic diapause induction in the silkworm, Bombyx mori

Diapause is one of the most important traits that have sustained insects to thrive. To survive harsh seasons, most insects can arrest their development and enter diapause. The photoperiod is the signal that indicates insects the proper timing to enter diapause. Circadian clock genes are shown to be involved in photoperiodic diapause induction in various insect species. The silkworm, Bombyx mori, enters diapause at the embryonic stage. In bivoltine strains, diapause determination is under maternal control and affected by temperature and photoperiodic conditions that mothers experienced during embryonic and larval stages. Two independent studies showed that knocking out the core clock gene, period, perturb photoperiodic diapause induction in B. mori. However, whether the circadian clock as whole or individual clock genes are responsible for the photoperiodic diapause induction remains unknown. In this study, using CRISPR/Cas9 we knocked out negative (period and timeless) and positive elements (Clock and cycle) in p50T, a bivoltine strain which exhibits photoperiodic diapause induction during both embryonic and larval stages. The temporal expression patterns of clock genes changed in each core clock gene knockout strain, suggesting disruption of normal feedback loops produced by circadian clock genes. Furthermore, the ability of female moths to appropriately produce diapause or non-diapause eggs in response to photoperiod in both embryonic and larval stages was lost in all knockout strains. Our results indicate the involvement of circadian clock in photoperiodic diapause induction in B. mori.