Coupling Controls the Synchrony of Clock Cells in Development and Knockouts.
ABSTRACT: In mammals, a network of coupled neurons within the hypothalamus coordinates physiological rhythms with daily changes in the environment. In each neuron, delayed negative transcriptional feedbacks generate oscillations, albeit noisy and unreliable ones. Coupling mediated by diffusible neuropeptides lends precision and robustness to circadian rhythms. The double knockout of Cryptochrome Cry turns adult mice arrhythmic. But, remarkably, double knockout neonates continue to show robust oscillation much like wild-type neonates and appear to lose rhythmicity with development. We study quantitatively dispersed neurons and brain slices from wild-type and Cry double knockout mice to understand the links between single cell rhythmicity and intercellular coupling. We quantify oscillator properties of dispersed cells using nonlinear regression and study bifurcations diagrams of network models. We find that varying just three parameters-oscillator strength, strength of coupling, and timing of coupling-can reproduce experimentally observed features. In particular, modeling reveals that minor changes in timing of coupling can destroy synchronization as observed in adult slices from knockout mice.
Project description:Circadian clocks are autonomous oscillators driving daily rhythms in physiology and behavior. In mammals, a network of coupled neurons in the suprachiasmatic nucleus (SCN) is entrained to environmental light-dark cycles and orchestrates the timing of peripheral organs. In each neuron, transcriptional feedbacks generate noisy oscillations. Coupling mediated by neuropeptides such as VIP and AVP lends precision and robustness to circadian rhythms. The detailed coupling mechanisms between SCN neurons are debated. We analyze organotypic SCN slices from neonatal and adult mice in wild-type and multiple knockout conditions. Different degrees of rhythmicity are quantified by pixel-level analysis of bioluminescence data. We use empirical orthogonal functions (EOFs) to characterize spatio-temporal patterns. Simulations of coupled stochastic single cell oscillators can reproduce the diversity of observed patterns. Our combination of data analysis and modeling provides deeper insight into the enormous complexity of the data: (1) Neonatal slices are typically stronger oscillators than adult slices pointing to developmental changes of coupling. (2) Wild-type slices are completely synchronized and exhibit specific spatio-temporal patterns of phases. (3) Some slices of Cry double knockouts obey impaired synchrony that can lead to co-existing rhythms ("splitting"). (4) The loss of VIP-coupling leads to desynchronized rhythms with few residual local clusters. Additional information was extracted from co-culturing slices with rhythmic neonatal wild-type SCNs. These co-culturing experiments were simulated using external forcing terms representing VIP and AVP signaling. The rescue of rhythmicity via co-culturing lead to surprising results, since a cocktail of AVP-antagonists improved synchrony. Our modeling suggests that these counter-intuitive observations are pointing to an antagonistic action of VIP and AVP coupling. Our systematic theoretical and experimental study shows that dual coupling mechanisms can explain the astonishing complexity of spatio-temporal patterns in SCN slices.
Project description:Molecular mechanisms of the mammalian circadian clock have been studied primarily by genetic perturbation and behavioral analysis. Here, we used bioluminescence imaging to monitor Per2 gene expression in tissues and cells from clock mutant mice. We discovered that Per1 and Cry1 are required for sustained rhythms in peripheral tissues and cells, and in neurons dissociated from the suprachiasmatic nuclei (SCN). Per2 is also required for sustained rhythms, whereas Cry2 and Per3 deficiencies cause only period length defects. However, oscillator network interactions in the SCN can compensate for Per1 or Cry1 deficiency, preserving sustained rhythmicity in mutant SCN slices and behavior. Thus, behavior does not necessarily reflect cell-autonomous clock phenotypes. Our studies reveal previously unappreciated requirements for Per1, Per2, and Cry1 in sustaining cellular circadian rhythmicity and demonstrate that SCN intercellular coupling is essential not only to synchronize component cellular oscillators but also for robustness against genetic perturbations.
Project description:Modern imaging techniques allow the monitoring of circadian rhythms of single cells. Coupling between these single cellular circadian oscillators can generate coherent periodic signals on the tissue level that subsequently orchestrate physiological outputs. The strength of coupling in such systems of oscillators is often unclear. In particular, effects on coupling strength by varying cell densities, by knockouts, and by inhibitor applications are debated. In this study, we suggest to quantify the relative coupling strength via analyzing period, phase, and amplitude distributions in ensembles of individual circadian oscillators. Simulations of different oscillator networks show that period and phase distributions become narrower with increasing coupling strength. Moreover, amplitudes can increase due to resonance effects. Variances of periods and phases decay monotonically with coupling strength, and can serve therefore as measures of relative coupling strength. Our theoretical predictions are confirmed by studying recently published experimental data from PERIOD2 expression in slices of the suprachiasmatic nucleus during and after the application of tetrodotoxin (TTX). On analyzing the corresponding period, phase, and amplitude distributions, we can show that treatment with TTX can be associated with a reduced coupling strength in the system of coupled oscillators. Analysis of an oscillator network derived directly from the data confirms our conclusions. We suggest that our approach is also applicable to quantify coupling in fibroblast cultures and hepatocyte networks, and for social synchronization of circadian rhythmicity in rodents, flies, and bees.
Project description:Circadian clocks are endogenous oscillators essential for orchestrating daily rhythms in physiology, metabolism and behavior. While mouse models have been instrumental to elucidate the molecular mechanism of circadian rhythm generation, our knowledge about the molecular makeup of circadian oscillators in humans is still limited. Here, we used duplex CRISPR/Cas9 technology to generate three cellular models for studying human circadian clocks: CRY1 knockout cells, CRY2 knockout cells as well as CRY1/CRY2 double knockout cells. Duplex CRISPR/Cas9 technology efficiently removed whole exons of CRY genes by using two guide RNAs targeting exon-flanking intron regions of human osteosarcoma cells (U-2 OS). Resulting cell clones did not express CRY proteins and showed short period, low-amplitude rhythms (for CRY1 knockout), long period rhythms (for CRY2 knockout) or were arrhythmic (for CRY1/CRY2 double knockout) similar to circadian phenotypes of cells derived from classical knockout mouse models.
Project description:In Neurospora crassa, the interactions between products of the frequency (frq), frequency-interacting RNA helicase (frh), white collar-1 (wc-1), and white collar-2 (wc-2) genes establish a molecular circadian clockwork, called the FRQ-WC-Oscillator (FWO), which is required for the generation of molecular and overt circadian rhythmicity. In strains carrying nonfunctional frq alleles, circadian rhythms in asexual spore development (conidiation) are abolished in constant conditions, yet conidiation remains rhythmic in temperature cycles. Certain characteristics of these temperature-synchronized rhythms have been attributed to the activity of a FRQ-less oscillator (FLO). The molecular components of this FLO are as yet unknown. To test whether the FLO depends on other circadian clock components, we created a strain that carries deletions in the frq, wc-1, wc-2, and vivid (vvd) genes. Conidiation in this ?FWO strain was still synchronized to cyclic temperature programs, but temperature-induced rhythmicity was distinct from that seen in single frq knockout strains. These results and other evidence presented indicate that components of the FWO are part of the temperature-induced FLO.
Project description:Animal circadian clocks are based on multiple oscillators whose interactions allow the daily control of complex behaviors. The Drosophila brain contains a circadian clock that controls rest-activity rhythms and relies upon different groups of PERIOD (PER)-expressing neurons. Two distinct oscillators have been functionally characterized under light-dark cycles. Lateral neurons (LNs) that express the pigment-dispersing factor (PDF) drive morning activity, whereas PDF-negative LNs are required for the evening activity. In constant darkness, several lines of evidence indicate that the LN morning oscillator (LN-MO) drives the activity rhythms, whereas the LN evening oscillator (LN-EO) does not. Since mutants devoid of functional CRYPTOCHROME (CRY), as opposed to wild-type flies, are rhythmic in constant light, we analyzed transgenic flies expressing PER or CRY in the LN-MO or LN-EO. We show that, under constant light conditions and reduced CRY function, the LN evening oscillator drives robust activity rhythms, whereas the LN morning oscillator does not. Remarkably, light acts by inhibiting the LN-MO behavioral output and activating the LN-EO behavioral output. Finally, we show that PDF signaling is not required for robust activity rhythms in constant light as opposed to its requirement in constant darkness, further supporting the minor contribution of the morning cells to the behavior in the presence of light. We therefore propose that day-night cycles alternatively activate behavioral outputs of the Drosophila evening and morning lateral neurons.
Project description:In mammals, the suprachiasmatic nucleus (SCN) is the central pacemaker organizing circadian rhythms of behavior and physiology. At the cellular level, the mammalian clock consists of autoregulatory feedback loops involving a set of "clock genes," including the Cryptochrome (Cry) genes, Cry1 and Cry2. Experimental evidence suggests that Cry1 and Cry2 play distinct roles in circadian clock function. In mice, Cry1 is required for sustained circadian rhythms in dissociated SCN neurons or fibroblasts but not in organotypic SCN slices or at the behavioral level, whereas Cry2 is not required at any of these levels. It has been argued that coupling among SCN cellular oscillators compensates for clock gene defects to preserve oscillatory function. Here we test this hypothesis in Cry1(-/-) mice by first disrupting intercellular coupling in vivo using constant light (resulting in behavioral arrhythmicity) and then examining circadian clock gene expression in SCN slices at the single cell level. In this manner, we were able to test the role of intercellular coupling without drugs and while preserving tissue organization, avoiding the confounding influences of more invasive manipulations. Cry1(-/-) mice (as well as control Cry2(-/-) mice) bearing the PER2::LUC knock-in reporter were transferred from a standard light:dark cycle to constant bright light (~650 lux) to induce arrhythmic locomotor patterns. In SCN slices from these animals, we used bioluminescence imaging to monitor PER2::LUC expression in single cells. We show that SCN slices from rhythmic Cry1(-/-) and Cry2(-/-) mice had similarly high percentages of functional single-cell oscillators. In contrast, SCN slices from arrhythmic Cry1(-/-) mice had significantly fewer rhythmic cells than SCN slices from arrhythmic Cry2(-/-) mice. Thus, constant light in vivo disrupted intercellular SCN coupling to reveal a cell-autonomous circadian defect in Cry1(-/-) cells that is normally compensated by intercellular coupling in vivo.
Project description:Insulinoma-associated protein (IA)-2 and IA-2? are transmembrane proteins involved in neurotransmitter secretion. Mice with targeted disruption of both IA-2 and IA-2? (double-knockout, or DKO mice) have numerous endocrine and physiological disruptions, including disruption of circadian and diurnal rhythms. In the present study, we have assessed the impact of disruption of IA-2 and IA-2? on molecular rhythms in the brain and peripheral oscillators. We used in situ hybridization to assess molecular rhythms in the hypothalamic suprachiasmatic nuclei (SCN) of wild-type (WT) and DKO mice. The results indicate significant disruption of molecular rhythmicity in the SCN, which serves as the central pacemaker regulating circadian behavior. We also used quantitative PCR to assess gene expression rhythms in peripheral tissues of DKO, single-knockout, and WT mice. The results indicate significant attenuation of gene expression rhythms in several peripheral tissues of DKO mice but not in either single knockout. To distinguish whether this reduction in rhythmicity reflects defective oscillatory function in peripheral tissues or lack of entrainment of peripheral tissues, animals were injected with dexamethasone daily for 15 days, and then molecular rhythms were assessed throughout the day after discontinuation of injections. Dexamethasone injections improved gene expression rhythms in liver and heart of DKO mice. These results are consistent with the hypothesis that peripheral tissues of DKO mice have a functioning circadian clockwork, but rhythmicity is greatly reduced in the absence of robust, rhythmic physiological signals originating from the SCN. Thus, IA-2 and IA-2? play an important role in the regulation of circadian rhythms, likely through their participation in neurochemical communication among SCN neurons.
Project description:Circadian rhythms in Per1, PER2 expression and intracellular Ca2+ were measured from a solitary SCN neuron or glial cell which was physically isolated from other cells. Dispersed cells were cultured on a platform of microisland (100-200??m in diameter) in a culture dish. Significant circadian rhythms were detected in 57.1% for Per1 and 70.0% for PER2 expression. When two neurons were located on the same island, the circadian rhythms showed desynchronization, indicating a lack of oscillatory coupling. Circadian rhythms were also detected in intracellular Ca2+ of solitary SCN neurons. The ratio of circadian positive neurons was significantly larger without co-habitant of glial cells (84.4%) than with it (25.0%). A relatively large fraction of SCN neurons generates the intrinsic circadian oscillation without neural or humoral networks. In addition, glial cells seem to interrupt the expression of the circadian rhythmicity of intracellular Ca2+ under these conditions.
Project description:Various physiological and behavioral processes exhibit circadian rhythmicity. These rhythms are usually maintained by negative feedback loops of core clock genes, namely, CLOCK, BMAL, PER, and CRY. Recently, dysfunction in the circadian clock has been recognized as an important foundation for the pathophysiology of lifestyle-related diseases, such as obesity, cardiovascular disease, and some cancers. We have reported that angiopoietin-like protein 2 (ANGPTL2) contributes to the pathogenesis of these lifestyle-related diseases by inducing chronic inflammation. However, molecular mechanisms underlying regulation of ANGPTL2 expression are poorly understood. Here, we assess circadian rhythmicity of ANGPTL2 expression in various mouse tissues. We observed that ANGPTL2 rhythmicity was similar to that of the PER2 gene, which is regulated by the CLOCK/BMAL1 complex. Promoter activity of the human ANGPTL2 gene was significantly induced by CLOCK and BMAL1, an induction markedly attenuated by CRY co-expression. We also identified functional E-boxes in the ANGPTL2 promoter and observed occupancy of these sites by endogenous CLOCK in human osteosarcoma cells. Furthermore, Cry-deficient mice exhibited arrhythmic Angptl2 expression. Taken together, these data suggest that periodic expression of ANGPTL2 is regulated by a molecular clock.