Analysis of core circadian feedback loop in suprachiasmatic nucleus of
            <i>mCry1-luc</i>
            transgenic reporter mouse

Maywood, Elizabeth S.; Drynan, Lesley; Chesham, Johanna E.; Edwards, Mathew D.; Dardente, Hugues; Fustin, Jean‐Michel; Hazlerigg, David G.; O’Neill, John S.; Codner, Gemma; Smyllie, Nicola J.; Brancaccio, Marco; Hastings, Michael H.

doi:10.1073/pnas.1220894110

Cited by 57 publications

(47 citation statements)

References 31 publications

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“…1). These data are consistent with those of previous studies demonstrating that Per1 transcriptionally suppresses Cry2 (33,36,37,39). Taken together, our observations lend support to a hypothetical model in which Per1 action on target genes may occur via a potential Cry2-Clock/Bmal1-dependent mechanism.…”

Section: Perspectives and Significancesupporting

confidence: 92%

Opposing actions of Per1 and Cry2 in the regulation of Per1 target gene expression in the liver and kidney

Richards

All

Skopis

et al. 2013

American Journal of Physiology-Regulatory, Integrative and Comp

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Mounting evidence suggests that the circadian clock plays an integral role in the regulation of many physiological processes including blood pressure, renal function, and metabolism. The canonical molecular clock functions via activation of circadian target genes by Clock/Bmal1 and repression of Clock/Bmal1 activity by Per1–3 and Cry1/2. However, we have previously shown that Per1 activates genes important for renal sodium reabsorption, which contradicts the canonical role of Per1 as a repressor. Moreover, Per1 knockout (KO) mice exhibit a lowered blood pressure and heavier body weight phenotype similar to Clock KO mice, and opposite that of Cry1/2 KO mice. Recent work has highlighted the potential role of Per1 in repression of Cry2. Therefore, we postulated that Per1 potentially activates target genes through a Cry2-Clock/Bmal1-dependent mechanism, in which Per1 antagonizes Cry2, preventing its repression of Clock/Bmal1. This hypothesis was tested in vitro and in vivo. The Per1 target genes αENaC and Fxyd5 were identified as Clock targets in mpkCCDc14 cells, a model of the renal cortical collecting duct. We identified PPARα and DEC1 as novel Per1 targets in the mouse hepatocyte cell line, AML12, and in the liver in vivo. Per1 knockdown resulted in upregulation of Cry2 in vitro, and this result was confirmed in vivo in mice with reduced expression of Per1. Importantly, siRNA-mediated knockdown of Cry2 and Per1 demonstrated opposing actions for Cry2 and Per1 on Per1 target genes, supporting the potential Cry2-Clock/Bmal1-dependent mechanism underlying Per1 action in the liver and kidney.

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Section: Perspectives and Significancesupporting

confidence: 92%

Opposing actions of Per1 and Cry2 in the regulation of Per1 target gene expression in the liver and kidney

Richards

All

Skopis

et al. 2013

American Journal of Physiology-Regulatory, Integrative and Comp

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“…Importantly, this is dependent on neuropeptide-mediated interneuronal communication (7)(8)(9). This property is embodied in an emergent spatiotemporal wave of gene expression that progresses daily across the SCN, observed in real-time recordings of Per-and Cry-driven bioluminescence in SCN slices (8,10). The principles that underpin the relationship between cell-autonomous and circuit-level properties are unknown.…”

mentioning

confidence: 99%

Temporally chimeric mice reveal flexibility of circadian period-setting in the suprachiasmatic nucleus

Smyllie

Chesham

Hamnett

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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The suprachiasmatic nucleus (SCN) is the master circadian clock controlling daily behavior in mammals. It consists of a heterogeneous network of neurons, in which cell-autonomous molecular feedback loops determine the period and amplitude of circadian oscillations of individual cells. In contrast, circuit-level properties of coherence, synchrony, and ensemble period are determined by intercellular signals and are embodied in a circadian wave of gene expression that progresses daily across the SCN. How cell-autonomous and circuit-level mechanisms interact in timekeeping is poorly understood. To explore this interaction, we used intersectional genetics to create temporally chimeric mice with SCN containing dopamine 1a receptor (Drd1a) cells with an intrinsic period of 24 h alongside nonDrd1a cells with 20-h clocks. Recording of circadian behavior in vivo alongside cellular molecular pacemaking in SCN slices in vitro demonstrated that such chimeric circuits form robust and resilient circadian clocks. It also showed that the computation of ensemble period is nonlinear. Moreover, the chimeric circuit sustained a wave of gene expression comparable to that of nonchimeric SCN, demonstrating that this circuit-level property is independent of differences in cell-intrinsic periods. The relative dominance of 24-h Drd1a and 20-h non-Drd1a neurons in setting ensemble period could be switched by exposure to resonant or nonresonant 24-h or 20-h lighting cycles. The chimeric circuit therefore reveals unanticipated principles of circuit-level operation underlying the emergent plasticity, resilience, and robustness of the SCN clock. The spontaneous and light-driven flexibility of period observed in chimeric mice provides a new perspective on the concept of SCN pacemaker cells.aily rhythms of behavior and physiology adapt organisms to the solar cycle. In mammals, these rhythms are coordinated by a central circadian pacemaker: the suprachiasmatic nucleus (SCN) of the hypothalamus (1). The SCN consists of 10,000 neurons that work together to ensure that rhythms are tuned appropriately to anticipate day and night. Circadian dysfunction is increasingly linked to a variety of psychological and metabolic disorders (2). The principal subdivisions of the SCN are the shell, characterized by neurons expressing arginine vasopressin (AVP), and the retinorecipient core, containing vasoactive intestinal peptide (VIP) and gastrin-releasing peptide (GRP) neurons. The core mediates entrainment to light after birth (3), whereas neurons expressing the dopamine 1a receptor (Drd1a) mediate transplacental dopaminergic entrainment of the SCN in utero (4). Drd1a cells are, therefore, a functionally distinct subpopulation that spans elements of both core and shell.Timekeeping in individual SCN neurons involves a molecular clockwork based on a transcriptional-translational feedback loop (TTFL) whereby PERIOD and CRYPTOCHROME proteins inhibit their own transactivation by CLOCK/BMAL1 heterodimers (5). Almost all of the cells in the body also have this TTFL, b...

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“…Cry1/2-null SCN were transduced with AAVs encoding the CRY1::EGFP fusion protein controlled by a minimal Cry1 promoter (pCry1) that exhibits circadian activation in the SCN (Fig. 1A) (9). Typically, ∼5 × 10 9 viral particles (1 μL of ∼5 × 10 12 GC/mL) were dropped directly onto each slice 7 d after dissection or after bioluminescence recordings, as previously reported (10).…”

Section: Resultsmentioning

confidence: 99%

Rhythmic expression of cryptochrome induces the circadian clock of arrhythmic suprachiasmatic nuclei through arginine vasopressin signaling

Edwards

Brancaccio

Chesham

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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Circadian rhythms in mammals are coordinated by the suprachiasmatic nucleus (SCN). SCN neurons define circadian time using transcriptional/ posttranslational feedback loops (TTFL) in which expression of Cryptochrome (Cry) and Period (Per) genes is inhibited by their protein products. Loss of Cry1 and Cry2 stops the SCN clock, whereas individual deletions accelerate and decelerate it, respectively. At the circuit level, neuronal interactions synchronize cellular TTFLs, creating a spatiotemporal wave of gene expression across the SCN that is lost in Cry1/2-deficient SCN. To interrogate the properties of CRY proteins required for circadian function, we expressed CRY in SCN of Cry-deficient mice using adeno-associated virus (AAV). Expression of CRY1::EGFP or CRY2:: EGFP under a minimal Cry1 promoter was circadian and rapidly induced PER2-dependent bioluminescence rhythms in previously arrhythmic Cry1/2-deficient SCN, with periods appropriate to each isoform. CRY1::EGFP appropriately lengthened the behavioral period in Cry1-deficient mice. Thus, determination of specific circadian periods reflects properties of the respective proteins, independently of their phase of expression. Phase of CRY1::EGFP expression was critical, however, because constitutive or phase-delayed promoters failed to sustain coherent rhythms. At the circuit level, CRY1::EGFP induced the spatiotemporal wave of PER2 expression in Cry1/2-deficient SCN. This was dependent on the neuropeptide arginine vasopressin (AVP) because it was prevented by pharmacological blockade of AVP receptors. Thus, our genetic complementation assay reveals acute, protein-specific induction of cell-autonomous and network-level circadian rhythmicity in SCN never previously exposed to CRY. Specifically, Cry expression must be circadian and appropriately phased to support rhythms, and AVP receptor signaling is required to impose circuit-level circadian function.he suprachiasmatic nucleus (SCN) is a precise biological clock that coordinates circadian (∼24 h) rhythms in mammals. Composed of ∼10,000 neurons, the SCN sits atop the optic chiasm, through which it receives retinal innervation that entrains its cellular clockwork to the light/dark cycle. In turn, the SCN entrains peripheral circadian clocks distributed across the organism through neural pathways incorporating neuropeptidergic and GABA-ergic signaling (1). At the molecular level, circadian timing in SCN neurons revolves around transcriptional/posttranslational feedback loops (TTFL), in which the positive components BMAL1 and CLOCK activate E/E′-box-mediated transcription of the negative components Period (Per1, Per2) and Cryptochrome (Cry1, Cry2). PER/CRY heterodimers then inhibit BMAL1/CLOCK activity and thus prevent their own transcription. Subsequent proteasomal degradation of PER and CRY proteins relieves this inhibition, allowing the molecular cycle to begin again. Genetic studies have established that CRY1 and CRY2 are essential for behavioral and molecular circadian rhythmicity, acting as core components of th...

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Analysis of core circadian feedback loop in suprachiasmatic nucleus of mCry1-luc transgenic reporter mouse

Cited by 57 publications

References 31 publications

Opposing actions of Per1 and Cry2 in the regulation of Per1 target gene expression in the liver and kidney

Opposing actions of Per1 and Cry2 in the regulation of Per1 target gene expression in the liver and kidney

Temporally chimeric mice reveal flexibility of circadian period-setting in the suprachiasmatic nucleus

Rhythmic expression of cryptochrome induces the circadian clock of arrhythmic suprachiasmatic nuclei through arginine vasopressin signaling

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