Individual Clock Research Articles

Generation of dispersed presomitic mesoderm cell cultures for imaging of the zebrafish segmentation clock in single cells.

Segmentation is a periodic and sequential morphogenetic process in vertebrates. This rhythmic formation of blocks of tissue called somites along the body axis is evidence of a genetic oscillator patterning the developing embryo. In zebrafish, the intracellular clock driving segmentation is comprised of members of the Her/Hes transcription factor family organized into negative feedback loops. We have recently generated transgenic fluorescent reporter lines for the cyclic gene her1 that recapitulate the spatio-temporal pattern of oscillations in the presomitic mesoderm (PSM). Using these lines, we developed an in vitro culture system that allows real-time analysis of segmentation clock oscillations within single, isolated PSM cells. By removing PSM tissue from transgenic embryos and then dispersing cells from oscillating regions onto glass-bottom dishes, we generated cultures suitable for time-lapse imaging of fluorescence signal from individual clock cells. This approach provides an experimental and conceptual framework for direct manipulation of the segmentation clock with unprecedented single-cell resolution, allowing its cell-autonomous and tissue-level properties to be distinguished and dissected.

Generation of Dispersed Presomitic Mesoderm Cell Cultures for Imaging of the Zebrafish Segmentation Clock in Single Cells

Segmentation is a periodic and sequential morphogenetic process in vertebrates. This rhythmic formation of blocks of tissue called somites along the body axis is evidence of a genetic oscillator patterning the developing embryo. In zebrafish, the intracellular clock driving segmentation is comprised of members of the Her/Hes transcription factor family organized into negative feedback loops. We have recently generated transgenic fluorescent reporter lines for the cyclic gene her1 that recapitulate the spatio-temporal pattern of oscillations in the presomitic mesoderm (PSM). Using these lines, we developed an in vitro culture system that allows real-time analysis of segmentation clock oscillations within single, isolated PSM cells. By removing PSM tissue from transgenic embryos and then dispersing cells from oscillating regions onto glass-bottom dishes, we generated cultures suitable for time-lapse imaging of fluorescence signal from individual clock cells. This approach provides an experimental and conceptual framework for direct manipulation of the segmentation clock with unprecedented single-cell resolution, allowing its cell-autonomous and tissue-level properties to be distinguished and dissected.

Staging Glaucoma Using Stratus OCT in a U.S. Veteran Population

This study examines the ability of time domain optical coherence tomography (Stratus OCT) of the retinal nerve fiber layer (RNFL) to discriminate between normal patients and patients with three stages of glaucomatous vision loss in a US veteran population. A review of consecutive patients who underwent automated perimetry and Stratus OCT Fast RNFL scanning within a 6-month period was conducted. Patients with nonglaucomatous ocular disease that might affect the RNFL or perimetry results were excluded. Glaucomatous eyes were staged using the Hodapp-Parrish-Anderson grading system. Average, quadrant, and individual clock hour RNFL thicknesses and normative results between all groups were compared. A total of 247 normal subjects and 157 glaucoma subjects were included. Significant differences in RNFL thickness measurements were observed between all groups (p < 0.05). Receiver operating curve analysis of highest area under the curve showed average RNFL for normal versus mild glaucoma (0.86), inferior quadrant for mild versus moderate glaucoma (0.80), and superior quadrant for moderate versus severe disease (0.86). Normative results for individual parameters demonstrated high specificity but low sensitivity for mild disease versus normal control subjects with increasing sensitivity and decreasing specificity in subsequent stages of disease. The number of OCT parameters classified as borderline or abnormal increased with advancing disease, but a combination of high sensitivity and specificity was not detected for any stage. Optical coherence tomography RNFL thickness measurements, individual parameter normative results, and the number of parameters classified as borderline or abnormal poorly distinguished between severities of disease. Significant RNFL thickness overlap and lack of normative parameters demonstrating both high sensitivity and specificity between consecutive stages of disease highlighted the wide variability of structural findings using time domain OCT in the staging of glaucoma.

Social Modulation of Circadian Synchronization and Temporal Perception in Complete Isolation

Social Modulation of Circadian Synchronization and Temporal Perception in Complete Isolation

Development and entrainment of the colonic circadian clock during ontogenesis

Colonic morphology and function change significantly during ontogenesis. In mammals, many colonic physiological functions are temporally controlled by the circadian clock in the colon, which is entrained by the central circadian clock in the suprachiasmatic nuclei (SCN). The aim of this present study was to ascertain when and how the circadian clock in the colon develops during the perinatal period and whether maternal cues and/or the developing pup SCN may influence the ontogenesis of the colonic clock. Daily profiles of clock genes Per1, Per2, Cry1, Cry2, Rev-erbα, Bmal1, and Clock expression in the colon underwent significant modifications since embryonic day 20 (E20) through postnatal days (P) 2, 10, 20, and 30 via changes in the mutual phasing among the individual clock gene expression rhythms, their relative phasing to the light-dark regime, and their amplitudes. An adult-like state was achieved around P20. The foster study revealed that during the prenatal period, the maternal circadian phase may partially modulate development of the colonic clock. Postnatally, the absence and/or presence of rhythmic maternal care affected the phasing of the clock gene expression profiles in pups at P10 and P20. A reversal in the colonic clock phase between P10 and P20 occurred in the absence of rhythmic signals from the pup SCN. The data demonstrate ontogenetic maturation of the colonic clock and stress the importance of prenatal and postnatal maternal rhythmic signals for its development. These data may contribute to the understanding of colonic function-related diseases in newborn children.

Global But Not Gonadotrope-Specific Disruption of Bmal1 Abolishes the Luteinizing Hormone Surge Without Affecting Ovulation

Although there is evidence for a circadian regulation of the preovulatory LH surge, the contributions of individual tissue clocks to this process remain unclear. We studied female mice deficient in the Bmal1 gene (Bmal1(-/-)), which is essential for circadian clock function, and found that they lack the proestrous LH surge. However, spontaneous ovulation on the day of estrus was unaffected in these animals. Bmal1(-/-) females were also deficient in the proestrous FSH surge, which, like the LH surge, is GnRH-dependent. In the absence of circadian or external timing cues, Bmal1(-/-) females continued to cycle in constant darkness albeit with increased cycle length and time spent in estrus. Because pituitary gonadotropes are the source of circulating LH and FSH, we assessed hypophyseal circadian clock function and found that female pituitaries rhythmically express clock components throughout all cycle stages. To determine the role of the gonadotrope clock in the preovulatory LH and FSH surge process, we generated mice that specifically lack BMAL1 in gonadotropes (GBmal1KO). GBmal1KO females exhibited a modest elevation in both proestrous and baseline LH levels across all estrous stages. BMAL1 elimination from gonadotropes also led to increased variability in estrous cycle length, yet GBmal1KO animals were otherwise reproductively normal. Together our data suggest that the intrinsic clock in gonadotropes is dispensable for LH surge regulation but contributes to estrous cycle robustness. Thus, clocks in the suprachiasmatic nucleus or elsewhere must be involved in the generation of the LH surge, which, surprisingly, is not required for spontaneous ovulation.

Evolutionary Links Between Circadian Clocks and Photoperiodic Diapause in Insects

In this article, we explore links between circadian clocks and the clock involved in photoperiodic regulation of diapause in insects. Classical resonance (Nanda-Hamner) and night interruption (Bünsow) experiments suggest a circadian basis for the diapause response in nearly all insects that have been studied. Neuroanatomical studies reveal physical connections between circadian clock cells and centers controlling the photoperiodic diapause response, and both mutations and knockdown of clock genes with RNA interference (RNAi) point to a connection between the clock genes and photoperiodic induction of diapause. We discuss the challenges of determining whether the clock, as a functioning module, or individual clock genes acting pleiotropically are responsible for the photoperiodic regulation of diapause, and how a stable, central circadian clock could be linked to plastic photoperiodic responses without compromising the clock's essential functions. Although we still lack an understanding of the exact mechanisms whereby insects measure day/night length, continued classical and neuroanatomical approaches, as well as forward and reverse genetic experiments, are highly complementary and should enable us to decipher the diverse ways in which circadian clocks have been involved in the evolution of photoperiodic induction of diapause in insects. The components of circadian clocks vary among insect species, and diapause appears to have evolved independently numerous times, thus, we anticipate that not all photoperiodic clocks of insects will interact with circadian clocks in the same fashion.

Circadian events in human diseases and in cytochrome P450‐related drug metabolism and therapy

The biochemical basis of the mammalian circadian clock can be described by transcriptional-translational feedback loops with a period of about 24 h. Crucial endogenous factors are under circadian control (i.e., body temperature, blood pressure, hormone secretion and metabolite levels). Also, drug metabolism, including phases I-III and the drug-responsive nuclear receptors, is controlled by the clock. Disturbances in circadian rhythm in humans can lead to pathologies, which is exemplified by increased cancer risk in long-term shift workers. On the other hand, best tolerability of drugs with minimum side effects can be achieved if the timing of drug treatment is synchronized with the patients' individual clock. The aim of this review is to underline the importance of accepting the individuals' endogenous clock which can contribute to personalized, patient-friendly optimization of drug therapies.

Analysis of the molecular pathophysiology of sleep disorders relevant to a disturbed biological clock

Genetic studies have revealed several clock gene variations/mutations involved in the manifestation of sleep disorders or interindividual differences in sleep-wake patterns, but only part of the genetic risk can be explained by the gene variations/mutations identified to date. Recent progress in research into circadian rhythm generation has provided efficient tools for eliciting the molecular basis of clock-relevant sleep disorders, complementing traditional genetic analysis. While the human master clock resides in the suprachiasmatic nucleus of the hypothalamus (central clock), peripheral tissue cells also generate self-sustained circadian oscillations of clock gene expression (peripheral clock), enabling estimation of individual human clock properties through a single collection of skin fibroblasts or venous blood cells. Some of the established cell lines exhibit autonomous circadian oscillations of clock gene expression, and introduction of clock gene variations into these cell lines by gene targeting makes it possible to investigate changes in the circadian phenotype induced by these variations/mutations without the need for generating transgenic animals. Estimation of human clock properties using peripheral tissue cells, in addition to genetic analysis, will facilitate comprehensive explication of the genetic risk of a variety of disorders relevant to biological clock disturbances, including sleep disorders, mood disorders, and metabolic diseases.

Connecting cellular metabolism to circadian clocks

The circadian clock is a cellular timekeeping mechanism that helps organisms to organize their behaviour and physiology around daily alternations of days and nights. In humans, misalignment of an individual's internal clock with its environment is associated with adverse health consequences, including metabolic disorders and cancers. In current models of the eukaryotic circadian oscillator, transcription/translation feedback loops (TTFLs) are considered the prime mechanism sustaining intracellular rhythms. The discovery of many cytosolic loops has extended the TTFL model by embedding it in cellular physiology. Recently, however, several studies have revealed metabolic rhythms that are independent of transcription, questioning the TTFL model as the sole cellular timekeeping mechanism. Thus, the time has come to carefully reassess these models of the clockwork in a broad cellular context to integrate its genetic, cytosolic, and metabolic components.

Divergent roles of clock genes in retinal and suprachiasmatic nucleus circadian oscillators.

The retina is both a sensory organ and a self-sustained circadian clock. Gene targeting studies have revealed that mammalian circadian clocks generate molecular circadian rhythms through coupled transcription/translation feedback loops which involve 6 core clock genes, namely Period (Per) 1 and 2, Cryptochrome (Cry) 1 and 2, Clock, and Bmal1 and that the roles of individual clock genes in rhythms generation are tissue-specific. However, the mechanisms of molecular circadian rhythms in the mammalian retina are incompletely understood and the extent to which retinal neural clocks share mechanisms with the suprachiasmatic nucleus (SCN), the central neural clock, is unclear. In the present study, we examined the rhythmic amplitude and period of real-time bioluminescence rhythms in explants of retina from Per1-, Per2-, Per3-, Cry1-, Cry2-, and Clock-deficient mice that carried transgenic PERIOD2::LUCIFERASE (PER2::LUC) or Period1::luciferase (Per1::luc) circadian reporters. Per1-, Cry1- and Clock-deficient retinal and SCN explants showed weakened or disrupted rhythms, with stronger effects in retina compared to SCN. Per2, Per3, and Cry2 were individually dispensable for sustained rhythms in both tissues. Retinal and SCN explants from double knockouts of Cry1 and Cry2 were arrhythmic. Gene effects on period were divergent with reduction in the number of Per1 alleles shortening circadian period in retina, but lengthening it in SCN, and knockout of Per3 substantially shortening retinal clock period, but leaving SCN unaffected. Thus, the retinal neural clock has a unique pattern of clock gene dependence at the tissue level that it is similar in pattern, but more severe in degree, than the SCN neural clock, with divergent clock gene regulation of rhythmic period.

Balance of Activity between LNvs and Glutamatergic Dorsal Clock Neurons Promotes Robust Circadian Rhythms in Drosophila

Circadian rhythms offer an excellent opportunity to dissect the neural circuits underlying innate behavior because the genes and neurons involved are relatively well understood. We first sought to understand how Drosophila clock neurons interact in the simple circuit that generates circadian rhythms in larval light avoidance. We used genetics to manipulate two groups of clock neurons, increasing or reducing excitability, stopping their molecular clocks, and blocking neurotransmitter release and reception. Our results revealed that lateral neurons (LN(v)s) promote and dorsal clock neurons (DN(1)s) inhibit light avoidance, these neurons probably signal at different times of day, and both signals are required for rhythmic behavior. We found that similar principles apply in the more complex adult circadian circuit that generates locomotor rhythms. Thus, the changing balance in activity between clock neurons with opposing behavioral effects generates robust circadian behavior and probably helps organisms transition between discrete behavioral states, such as sleep and wakefulness.

Entrainment of peripheral clock genes by cortisol

Circadian rhythmicity in mammals is primarily driven by the suprachiasmatic nucleus (SCN), often called the central pacemaker, which converts the photic information of light and dark cycles into neuronal and hormonal signals in the periphery of the body. Cells of peripheral tissues respond to these centrally mediated cues by adjusting their molecular function to optimize organism performance. Numerous systemic cues orchestrate peripheral rhythmicity, such as feeding, body temperature, the autonomic nervous system, and hormones. We propose a semimechanistic model for the entrainment of peripheral clock genes by cortisol as a representative entrainer of peripheral cells. This model demonstrates the importance of entrainer's characteristics in terms of the synchronization and entrainment of peripheral clock genes, and predicts the loss of intercellular synchrony when cortisol moves out of its homeostatic amplitude and frequency range, as has been observed clinically in chronic stress and cancer. The model also predicts a dynamic regime of entrainment, when cortisol has a slightly decreased amplitude rhythm, where individual clock genes remain relatively synchronized among themselves but are phase shifted in relation to the entrainer. The model illustrates how the loss of communication between the SCN and peripheral tissues could result in desynchronization of peripheral clocks.

Modeling the Emergence of Circadian Rhythms in a Clock Neuron Network

Circadian rhythms in pacemaker cells persist for weeks in constant darkness, while in other types of cells the molecular oscillations that underlie circadian rhythms damp rapidly under the same conditions. Although much progress has been made in understanding the biochemical and cellular basis of circadian rhythms, the mechanisms leading to damped or self-sustained oscillations remain largely unknown. There exist many mathematical models that reproduce the circadian rhythms in the case of a single cell of the Drosophila fly. However, not much is known about the mechanisms leading to coherent circadian oscillation in clock neuron networks. In this work we have implemented a model for a network of interacting clock neurons to describe the emergence (or damping) of circadian rhythms in Drosophila fly, in the absence of zeitgebers. Our model consists of an array of pacemakers that interact through the modulation of some parameters by a network feedback. The individual pacemakers are described by a well-known biochemical model for circadian oscillation, to which we have added degradation of PER protein by light and multiplicative noise. The network feedback is the PER protein level averaged over the whole network. In particular, we have investigated the effect of modulation of the parameters associated with (i) the control of net entrance of PER into the nucleus and (ii) the non-photic degradation of PER. Our results indicate that the modulation of PER entrance into the nucleus allows the synchronization of clock neurons, leading to coherent circadian oscillations under constant dark condition. On the other hand, the modulation of non-photic degradation cannot reset the phases of individual clocks subjected to intrinsic biochemical noise.

Peripheral circadian rhythms and their regulatory mechanism in insects and some other arthropods: a review

Many physiological functions of insects show a rhythmic change to adapt to daily environmental cycles. These rhythms are controlled by a multi-clock system. A principal clock located in the brain usually organizes the overall behavioral rhythms, so that it is called the "central clock". However, the rhythms observed in a variety of peripheral tissues are often driven by clocks that reside in those tissues. Such autonomous rhythms can be found in sensory organs, digestive and reproductive systems. Using Drosophila melanogaster as a model organism, researchers have revealed that the peripheral clocks are self-sustained oscillators with a molecular machinery slightly different from that of the central clock. However, individual clocks normally run in harmony with each other to keep a coordinated temporal structure within an animal. How can this be achieved? What is the molecular mechanism underlying the oscillation? Also how are the peripheral clocks entrained by light-dark cycles? There are still many questions remaining in this research field. In the last several years, molecular techniques have become available in non-model insects so that the molecular oscillatory mechanisms are comparatively investigated among different insects, which give us more hints to understand the essential regulatory mechanism of the multi-oscillatory system across insects and other arthropods. Here we review current knowledge on arthropod's peripheral clocks and discuss their physiological roles and molecular mechanisms.

Entrainment of breast (cancer) epithelial cells detects distinct circadian oscillation patterns for clock and hormone receptor genes

Most physiological and biological processes are regulated by endogenous circadian rhythms under the control of both a master clock, which acts systemically and individual cellular clocks, which act at the single cell level. The cellular clock is based on a network of core clock genes, which drive the circadian expression of non-clock genes involved in many cellular processes. Circadian deregulation of gene expression has emerged to be as important as deregulation of estrogen signaling in breast tumorigenesis. Whether there is a mutual deregulation of circadian and hormone signaling is the question that we address in this study. Here we show that, upon entrainment by serum shock, cultured human mammary epithelial cells maintain an inner circadian oscillator, with key clock genes oscillating in a circadian fashion. In the same cells, the expression of the estrogen receptor α (ER A) gene also oscillates in a circadian fashion. In contrast, ER A-positive and -negative breast cancer epithelial cells show disruption of the inner clock. Further, ER A-positive breast cancer cells do not display circadian oscillation of ER A expression. Our findings suggest that estrogen signaling could be affected not only in ER A-negative breast cancer, but also in ER A-positive breast cancer due to lack of circadian availability of ER A. Entrainment of the inner clock of breast epithelial cells, by taking into consideration the biological time component, provides a novel tool to test mechanistically whether defective circadian mechanisms can affect hormone signaling relevant to breast cancer.

地域在住高齢者の認知機能スクリーニングのための時計描画テスト―定量的および定性的評価による検討―

The purpose of this study was to evaluate whether the clock drawing test (CDT) is useful to assess the cognitive function of community-dwelling elderly people. We evaluated the CDT as a tool to measure cognitive function by qualitative and quantitative analyses. A total of 14,949 community-dwelling elderly were invited by mail to undergo cognitive screening by CDT. Of these, 8,815 responded, of which 8,684 were eligible for enrollment. We were also able to determine the educational background of 7,404 of these. There were 3,525 men (age: 73.05±6.20 [mean±standard deviation] years old, duration of education: 11.40±2.81 years) and 3,879 women (73.67±6.66, 10.34±2.19) . The drawn clocks were evaluated using the Freedman method, and those clocks drawn with obvious errors such as no circle, numbers, or hands were recorded and analyzed. In addition, any vertical deviation from the center points was also evaluated. The recorded percentages of the subjects who correctly completed the individual clock drawing test components varied. The mean total scores were 14.16±1.67 in men and 14.40±1.36 in women. The percentages of subjects with total scores of less than 13 were 16.09% in men and 11.7% in women. The percentage of subjects who made obvious errors was 3.24%, whose total points were significantly lower than those of the subjects who did not. Approximately half of all subjects showed vertical deviation from the center of the clock, and the percentage of upper deviation was greater than that of lower deviation. CDT is useful to assess the cognitive function of community-dwelling elderly people, and it is also helpful to determine subjects with a potential risk of cognitive impairments.

The circadian clock genes affect reproductive capacity in the desert locust Schistocerca gregaria

The circadian clocks govern many metabolic and behavioral processes in an organism. In insects, these clocks and their molecular machinery have been found to influence reproduction in many different ways. Reproductive behavior including courtship, copulation and egg deposition, is under strong influence of the daily rhythm. At the molecular level, the individual clock components also have their role in normal progress of oogenesis and spermatogenesis. In this study on the desert locust Schistocerca gregaria, three circadian clock genes were identified and their expression profiles were determined. High expression was predominantly found in reproductive tissues. Similar daily expression profiles were found for period ( per) and timeless ( tim), while the clock ( clk) mRNA level is higher 12 h before the first per and tim peak. A knockdown of either per or tim resulted in a significant decrease in the progeny produced by dsRNA treated females confirming the role of clock genes in reproduction and providing evidence that both PER and TIM are needed in the ovaries for egg development. Since the knockdown of clk is lethal for the desert locust, its function remains yet to be elucidated.

Evaluation of Retinal Nerve Fiber Layer Progression in Glaucoma: A Comparison between the Fast and the Regular Retinal Nerve Fiber Layer Scans

To compare the performance of the fast (256 A-scans in each scan circle) and the regular (512 A-scans in each scan circle) retinal nerve fiber layer (RNFL) scan protocols for detection of glaucoma progression using the Stratus optical coherence tomography (OCT) device (Carl Zeiss Meditec, Dublin, CA). Retrospective, longitudinal study. One hundred twenty-nine eyes from 72 glaucoma patients. All patients had been followed up for 2.9 to 6.1 years with a median follow-up of 4 months. All eyes had at least 4 serial RNFL measurements obtained with both the fast and the regular RNFL scans. Visual field (VF) assessment was performed on the same day as RNFL imaging. Retinal nerve fiber layer thickness and VF progression were evaluated with linear regression analysis against age. The mean rate of average RNFL thickness reduction was estimated with linear mixed modeling. The agreement of progression detection and the rate of change of RNFL thicknesses. A total of 1373 fast and 1373 regular RNFL scans and 1236 VF tests were analyzed. With reference to the average RNFL thickness, the fast RNFL scan detected more eyes with progression (21 eyes from 19 patients vs. 15 eyes from 13 patients) than the regular scan at a comparable level of specificity (96.9% vs. 96.1%). More eyes were found to have increasing RNFL thickness with age at individual clock hours (except for 3, 5, 6, and 11 o'clock) when the measurements were obtained with the regular scan. The agreement between the fast and the regular scan for detection of RNFL progression was fair to moderate, with κ values ranging between 0.14 and 0.49. The rate of average RNFL thickness progression was -1.01 μm per year for the fast RNFL scan and -0.77 μm per year for the regular scan. The choice of scan protocols in the Stratus OCT has a significant impact in the evaluation of RNFL progression. The fast RNFL scan seems to be preferable to follow RNFL damage in glaucoma.

Circadian clock genes, ovarian development and diapause

Insects, like most organisms, have an internal circadian clock that oscillates with a daily rhythmicity, and a timing mechanism that mediates seasonal events, including diapause. In research published in BMC Biology, Ikeno et al. show that downregulation of the circadian clock genes period and cycle affects expression of ovarian diapause in the insect Riptortus pedestris. They interpret these important results as support for Erwin Bünning's (1936) hypothesis that the circadian clock constitutes the basis of photoperiodism. However, their observations could also be the result of pleiotropic effects of the individual clock genes.See research article http://www.biomedcentral.com/1741-7007/8/116