Fibroblast PER2 circadian rhythmicity depends on cell density.

Like neurons in the suprachiasmatic nucleus (SCN), the master circadian pacemaker in the brain, single fibroblasts can function as independent oscillators. In the SCN, synaptic and paracrine signaling among cells creates a robust, synchronized circadian oscillation, whereas there is no evidence for such integration in fibroblast cultures. However, interactions among single-cell fibroblast oscillators cannot be completely excluded, because fibroblasts were not isolated in previous work. In this study, we tested the autonomy of fibroblasts as single-cell circadian oscillators in high- and low-density culture, by single-cell imaging of cells from PER2::LUC circadian reporter mice. We found greatly reduced PER2::LUC rhythmicity in low-density cultures, which could result from lack of either constitutive or rhythmic paracrine signals from neighboring fibroblasts. To discriminate between these 2 possibilities, we mixed PER2::LUC wild-type (WT) cells with nonluminescent, nonrhythmic Bmal1-/- cells, so that density of rhythmic cells was low but overall cell density remained high. In this condition, WT cells showed clear rhythmicity similar to high-density cultures. We also mixed PER2::LUC WT cells with nonluminescent, long period Cry2-/- cells. In this condition, WT cells showed a period no different from cells cultured with rhythmic WT cells or nonrhythmic Bmal1-/- cells. In previous work, we found that low K⁺ suppresses fibroblast rhythmicity, and we and others have found that either low K⁺ or low Ca²⁺ suppresses SCN rhythmicity. Therefore, we attempted to rescue rhythmicity of low-density fibroblasts with high K⁺ (21 mM), high Ca²⁺ (3.6 mM), or conditioned medium. Conditioned medium from high-density fibroblast cultures rescued rhythmicity of low-density cultures, whereas high K⁺ or Ca²⁺ medium did not consistently rescue rhythmicity. These data suggest that fibroblasts require paracrine signals from adjacent cells for normal expression of rhythmicity, but that these signals do not have to be rhythmic, and that rhythmic signals from other cells do not affect the intrinsic periods of fibroblasts

NPAS2 Compensates for Loss of CLOCK in Peripheral Circadian Oscillators.

Heterodimers of CLOCK and BMAL1 are the major transcriptional activators of the mammalian circadian clock. Because the paralog NPAS2 can substitute for CLOCK in the suprachiasmatic nucleus (SCN), the master circadian pacemaker, CLOCK-deficient mice maintain circadian rhythms in behavior and in tissues in vivo. However, when isolated from the SCN, CLOCK-deficient peripheral tissues are reportedly arrhythmic, suggesting a fundamental difference in circadian clock function between SCN and peripheral tissues. Surprisingly, however, using luminometry and single-cell bioluminescence imaging of PER2 expression, we now find that CLOCK-deficient dispersed SCN neurons and peripheral cells exhibit similarly stable, autonomous circadian rhythms in vitro. In CLOCK-deficient fibroblasts, knockdown of Npas2 leads to arrhythmicity, suggesting that NPAS2 can compensate for loss of CLOCK in peripheral cells as well as in SCN. Our data overturn the notion of an SCN-specific role for NPAS2 in the molecular circadian clock, and instead indicate that, at the cellular level, the core loops of SCN neuron and peripheral cell circadian clocks are fundamentally similar

NPAS2 Compensates for Loss of CLOCK in Peripheral Circadian Oscillators

<div><p>Heterodimers of CLOCK and BMAL1 are the major transcriptional activators of the mammalian circadian clock. Because the paralog NPAS2 can substitute for CLOCK in the suprachiasmatic nucleus (SCN), the master circadian pacemaker, CLOCK-deficient mice maintain circadian rhythms in behavior and in tissues <i>in vivo</i>. However, when isolated from the SCN, CLOCK-deficient peripheral tissues are reportedly arrhythmic, suggesting a fundamental difference in circadian clock function between SCN and peripheral tissues. Surprisingly, however, using luminometry and single-cell bioluminescence imaging of PER2 expression, we now find that CLOCK-deficient dispersed SCN neurons and peripheral cells exhibit similarly stable, autonomous circadian rhythms <i>in vitro</i>. In CLOCK-deficient fibroblasts, knockdown of <i>Npas2</i> leads to arrhythmicity, suggesting that NPAS2 can compensate for loss of CLOCK in peripheral cells as well as in SCN. Our data overturn the notion of an SCN-specific role for NPAS2 in the molecular circadian clock, and instead indicate that, at the cellular level, the core loops of SCN neuron and peripheral cell circadian clocks are fundamentally similar.</p></div

Knockdown of <i>Npas2</i> expression suppresses circadian rhythms in <i>Clock</i>^<i>-/-</i> fibroblasts.

<p>(A) Fibroblasts dispersed from wild type and <i>Clock</i>^<i>-/-</i> mice were treated with lentiviral vectors carrying an <i>Npas2</i>-KD or scrambled DNA sequence, as well as a GFP reporter. Simultaneous fluorescence and bioluminescence images of a representative field show GFP expression marking transfected cells (left, green) and mPer2^Luc bioluminescence from both transfected and untransfected cells (middle, red). The overlay shows that circadian rhythms could be measured from both transfected (filled arrowheads) and untransfected (unfilled arrowheads) fibroblasts in the same culture dish. (B) Percentage of wild type (left) and <i>Clock</i>^<i>-/-</i> (right) fibroblasts treated with <i>Npas2</i>-KD lentiviruses (top) or scrambled lentiviruses (bottom) that were significantly rhythmic. ***<i>p</i>≤0.001 (Fisher’s exact test); number of cells are given in parentheses. (C) mPer2^Luc rhythms of representative individual dispersed untransfected (top) and transfected (bottom) <i>Clock</i>^<i>-/-</i> fibroblasts from the same culture dish.</p

Peripheral organs of <i>Clock</i>^<i>-/-</i> mice can exhibit circadian rhythms <i>in vitro</i>.

<p>(A) Two representative mPer2^Luc bioluminescence rhythms of rhythmic organotypic liver (black), lung (blue), kidney (red), and adrenal (green) slice cultures from wild type (left) and <i>Clock</i>^<i>-/-</i> (right) mice. After ~7 culture days, samples were treated with 10 μM forskolin (arrow). Y-axis scales are adjusted to amplitudes for better visualization of data. (B) Circadian mPer2^Luc rhythm period, amplitude, damping constant (days to reach 1/e of initial amplitude), and phase of first peak after forskolin treatment, and % of slices from wild type (unfilled bars) and <i>Clock</i>^<i>-/-</i> mice (filled bars) that were significantly rhythmic after forskolin treatment (culture days 8–14). Data are shown as mean ± SEM; *<i>p</i>≤0.05, **<i>p</i>≤0.01, ***<i>p</i>≤0.001 (student’s t-test); or % of slices rhythmic; n = 8.</p

Dispersed <i>Clock</i>^<i>-/-</i> fibroblasts show circadian rhythms comparable to those of dispersed <i>Clock</i>^<i>-/-</i> SCN neurons.

<p>(A) Raster plots of bioluminescence intensity of individual dispersed wild-type (left, n = 320) and <i>Clock</i>^<i>-/-</i> (right, n = 121) SCN cells. Data are presented as in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005882#pgen.1005882.g001" target="_blank">Fig 1C</a>. (B) Images of mPer2^Luc expression of representative rhythmic wild-type (left) and <i>Clock</i>^<i>-/-</i> (right) fibroblasts. (C) Two representative mPer2^Luc bioluminescence rhythms of individual rhythmic wild-type (left) and <i>Clock</i>^<i>-/-</i> (right) fibroblasts. (D) Circadian period, amplitude, and goodness of fit of mPer2^Luc rhythms, and % of cells that were significantly rhythmic, for individual wild type (left) and <i>Clock</i>^<i>-/-</i> (right) fibroblasts. Data are shown as mean ± SEM; *<i>p</i>≤0.05, ***<i>p</i>≤0.001 (Student’s t-test); or % of cells rhythmic; ***<i>p</i>≤0.001 (Fisher’s exact test); WT: n (rhythmic/total) = 320/321; <i>Clock</i>^<i>-/-</i>: n = 121/163.</p

Calcium Circadian Rhythmicity in the Suprachiasmatic Nucleus: Cell Autonomy and Network Modulation.

Circadian rhythms of mammalian physiology and behavior are coordinated by the suprachiasmatic nucleus (SCN) in the hypothalamus. Within SCN neurons, various aspects of cell physiology exhibit circadian oscillations, including circadian clock gene expression, levels of intracellular Ca2+ ([Ca2+]i), and neuronal firing rate. [Ca2+]i oscillates in SCN neurons even in the absence of neuronal firing. To determine the causal relationship between circadian clock gene expression and [Ca2+]i rhythms in the SCN, as well as the SCN neuronal network dependence of [Ca2+]i rhythms, we introduced GCaMP3, a genetically encoded fluorescent Ca2+ indicator, into SCN neurons from PER2::LUC knock-in reporter mice. Then, PER2 and [Ca2+]i were imaged in SCN dispersed and organotypic slice cultures. In dispersed cells, PER2 and [Ca2+]i both exhibited cell autonomous circadian rhythms, but [Ca2+]i rhythms were typically weaker than PER2 rhythms. This result matches the predictions of a detailed mathematical model in which clock gene rhythms drive [Ca2+]i rhythms. As predicted by the model, PER2 and [Ca2+]i rhythms were both stronger in SCN slices than in dispersed cells and were weakened by blocking neuronal firing in slices but not in dispersed cells. The phase relationship between [Ca2+]i and PER2 rhythms was more variable in cells within slices than in dispersed cells. Both PER2 and [Ca2+]i rhythms were abolished in SCN cells deficient in the essential clock gene Bmal1. These results suggest that the circadian rhythm of [Ca2+]i in SCN neurons is cell autonomous and dependent on clock gene rhythms, but reinforced and modulated by a synchronized SCN neuronal network

Calcium Circadian Rhythmicity in the Suprachiasmatic Nucleus: Cell Autonomy and Network Modulation.

Circadian rhythms of mammalian physiology and behavior are coordinated by the suprachiasmatic nucleus (SCN) in the hypothalamus. Within SCN neurons, various aspects of cell physiology exhibit circadian oscillations, including circadian clock gene expression, levels of intracellular Ca2+ ([Ca2+]i), and neuronal firing rate. [Ca2+]i oscillates in SCN neurons even in the absence of neuronal firing. To determine the causal relationship between circadian clock gene expression and [Ca2+]i rhythms in the SCN, as well as the SCN neuronal network dependence of [Ca2+]i rhythms, we introduced GCaMP3, a genetically encoded fluorescent Ca2+ indicator, into SCN neurons from PER2::LUC knock-in reporter mice. Then, PER2 and [Ca2+]i were imaged in SCN dispersed and organotypic slice cultures. In dispersed cells, PER2 and [Ca2+]i both exhibited cell autonomous circadian rhythms, but [Ca2+]i rhythms were typically weaker than PER2 rhythms. This result matches the predictions of a detailed mathematical model in which clock gene rhythms drive [Ca2+]i rhythms. As predicted by the model, PER2 and [Ca2+]i rhythms were both stronger in SCN slices than in dispersed cells and were weakened by blocking neuronal firing in slices but not in dispersed cells. The phase relationship between [Ca2+]i and PER2 rhythms was more variable in cells within slices than in dispersed cells. Both PER2 and [Ca2+]i rhythms were abolished in SCN cells deficient in the essential clock gene Bmal1. These results suggest that the circadian rhythm of [Ca2+]i in SCN neurons is cell autonomous and dependent on clock gene rhythms, but reinforced and modulated by a synchronized SCN neuronal network

The SCN oscillator network is responsible for stable rhythms in <i>Clock</i>^<i>-/-</i> SCN neurons.

<p>(A) Raster plots of mPer2^Luc bioluminescence intensity of individual dispersed wild-type (left, n = 246) and <i>Clock</i>^<i>-/-</i> (right, n = 161) SCN cells. Data are presented as in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005882#pgen.1005882.g001" target="_blank">Fig 1C</a>. (B) mPer2^Luc bioluminescence rhythms of two representative rhythmic individual dispersed SCN neurons from wild type (left) and <i>Clock</i>^<i>-/-</i> mice (right). (C) Circadian period, amplitude, and sine wave goodness-of-fit of cellular mPer2^Luc rhythms, and the percentage of rhythmic neurons in dispersed SCN cultures from wild type (white), <i>Clock</i>^<i>-/-</i> (black), and <i>Bmal1</i>^<i>-/-</i> (patterned) mice. <i>Bmal1</i>^<i>-/-</i> data are from Ko et al [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005882#pgen.1005882.ref018" target="_blank">18</a>]. Data are shown as mean ± SEM; ***<i>p</i>≤0.001 (student’s t-test); or as percentage of cells that are significantly rhythmic; WT: n (rhythmic/total) = 234/255; <i>Clock</i>^<i>-/-</i>: n = 138/208; <i>Bmal1</i>^<i>-/-</i>: n = 30/243.</p