Embryonic development and maternal regulation of murine circadian clock function

<div><p>The importance of circadian clocks in the regulation of adult physiology in mammals is well established. In contrast, the ontogenesis of the circadian system and its role in embryonic development are still poorly understood. Although there is experimental evidence that the clock machinery is present prior to birth, data on gestational clock functionality are inconsistent. Moreover, little is known about the dependence of embryonic rhythms on maternal and environmental time cues and the role of circadian oscillations for embryonic development. The aim of this study was to test if fetal mouse tissues from early embryonic stages are capable of expressing endogenous, self-sustained circadian rhythms and their contribution to embryogenesis. Starting on embryonic day 13, we collected precursor tissues for suprachiasmatic nucleus (SCN), liver and kidney from embryos carrying the circadian reporter gene <i>Per2::Luc</i> and investigated rhythmicity and circadian traits of these tissues <i>ex vivo</i>. We found that even before the respective organs were fully developed, embryonic tissues were capable of expressing circadian rhythms. Period and amplitude of which were determined very early during development and phases of liver and kidney explants are not influenced by tissue preparation, whereas SCN explants phasing is strongly dependent on preparation time. Embryonic circadian rhythms also developed in the absence of maternal and environmental time signals. Morphological and histological comparison of offspring from matings of <i>Clock-Δ19</i> mutant and wild-type mice revealed that both fetal and maternal clocks have distinct roles in embryogenesis. While genetic disruptions of maternal and embryonic clock function leads to increased fetal fat depots, abnormal ossification and organ development, <i>Clock</i> gene mutant newborns from mothers with a functional clock showed a larger body size compared to wild-type littermates. These data may contribute to the understanding of the ontogenesis of circadian clocks and the risk of disturbed maternal or embryonic circadian rhythms for embryonic development.</p></div

Circadian Clock Genes <em>Per1</em> and <em>Per2</em> Regulate the Response of Metabolism-Associated Transcripts to Sleep Disruption

<div><p>Human and animal studies demonstrate that short sleep or poor sleep quality, e.g. in night shift workers, promote the development of obesity and diabetes. Effects of sleep disruption on glucose homeostasis and liver physiology are well documented. However, changes in adipokine levels after sleep disruption suggest that adipocytes might be another important peripheral target of sleep. Circadian clocks regulate metabolic homeostasis and clock disruption can result in obesity and the metabolic syndrome. The finding that sleep and clock disruption have very similar metabolic effects prompted us to ask whether the circadian clock machinery may mediate the metabolic consequences of sleep disruption. To test this we analyzed energy homeostasis and adipocyte transcriptome regulation in a mouse model of shift work, in which we prevented mice from sleeping during the first six hours of their normal inactive phase for five consecutive days (<em>timed sleep restriction</em> – TSR). We compared the effects of TSR between wild-type and <em>Per1/2</em> double mutant mice with the prediction that the absence of a circadian clock in <em>Per1/2</em> mutants would result in a blunted metabolic response to TSR. In wild-types, TSR induces significant transcriptional reprogramming of white adipose tissue, suggestive of increased lipogenesis, together with increased secretion of the adipokine leptin and increased food intake, hallmarks of obesity and associated leptin resistance. Some of these changes persist for at least one week after the end of TSR, indicating that even short episodes of sleep disruption can induce prolonged physiological impairments. In contrast, <em>Per1/2</em> deficient mice show blunted effects of TSR on food intake, leptin levels and adipose transcription. We conclude that the absence of a functional clock in <em>Per1/2</em> double mutants protects these mice from TSR-induced metabolic reprogramming, suggesting a role of the circadian timing system in regulating the physiological effects of sleep disruption.</p> </div

Sustained and wild-type-specific induction of lipogenic and glycolytic genes in WAT.

<p>A–J) Expression of glycolytic and lipogenic genes in epididymal WAT at ZT6 and at ZT18 in control conditions, on the last day of TSR and on the 7^th day of recovery for wild-type and <i>Per1/2</i> mutant animals. Expression values are normalized to the mean of the wild-type control group at ZT6. Data are shown as mean ± SEM and data for each ZT are statistically compared using two-way ANOVAs (details are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052983#pone.0052983.s005" target="_blank">Table S1</a>), followed by Bonferroni post-tests, comparing control vs. TSR and control vs. recovery for each genotype. * p<0.05 in post-test. Post-tests comparing genotypes for each condition (control, TSR and recovery) are shown in Suppl. table ST1. K) Schematic overview of WAT glycolysis and lipogenesis pathways. Transcripts which were found to be changed by TSR are highlighted in red. Sample sizes were 3–4 per group.</p

Effects of TSR on plasma metabolite levels in wild-types and <i>Per1/2</i> mutants.

<p>Plasma metabolites were measured at ZT6 and ZT18 in control conditions, on the last day of TSR and on the 7^th day of recovery for wild-type and <i>Per1/2</i> mutant animals. Data are shown as mean +/− SEM. Sample sizes were 3–5 per group.</p>*<p>: p<0.05 compared to control conditions in the same genotype,</p>#<p>: p<0.05 compared to wild-type in the same condition using 2-way ANOVA and Bonferroni post-tests. Statistical details are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052983#pone.0052983.s005" target="_blank">Table S1</a>. NEFAs: non-esterified fatty acids.</p

TSR alters diurnal activity profiles, but not food intake or plasma leptin levels in <i>Per1/2</i> mutants.

<p>A) Representative double plotted activity recording during five days of control, five days of TSR and five days of recovery. TSR (ZT0–6) is highlighted by a red rectangle. Light and dark phases are indicated by white and grey boxes, respectively. B) Mean diurnal activity profiles (n = 5) were generated by plotting the relative locomotor activity for every 30 min bin as percentage of total daily activity. Light and dark phases are indicated in white and grey, respectively. Data are plotted as mean ± SEM (dotted lines). C) Relative activity during the second half of the night (ZT 18–24). Activity is expressed relative to the average activity during the same time in the control week (in %). *: p<0.001 control vs. TSR, two-way ANOVA with Bonferroni post-test, see also Suppl. Table ST1. D) Food intake during one day of control, during the last day of TSR and during the 7^th day of recovery. Two-way ANOVA with Bonferroni post-test not significant, see also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052983#pone.0052983.s005" target="_blank">Table S1</a>. E) Body weight gain per day of control, TSR and recovery. *: p<0.001 control vs. TSR, p<0.05 control vs. recovery, two-way ANOVA with Bonferroni post-test, see also Suppl. Table ST1. F) Plasma leptin levels at ZT18 measured on one day of control, the last day of TSR and the 7^th day of recovery. Two-way ANOVA with Bonferroni post-test, not significant, see also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052983#pone.0052983.s005" target="_blank">Table S1</a>. All data are shown as mean ± SEM. Sample sizes were 5 per group for activity, 4–8 per group for food intake, 17–33 per group for body weight and 4–5 per group for leptin.</p

TSR-induced transcriptional reprogramming of WAT in wild-type mice.

<p>A) Gene ontology analysis of microarray data of epididymal WAT at ZT18 on the last day of TSR compared to control WAT at ZT18. Only nodes (GO categories) with at least 10 regulated genes are shown. Significant overrepresentation of nodes is highlighted in red. B) Individual normalized log-transformed expression values for all genes involved in lipid metabolic pathways which are regulated at least 2 fold between control and TSR are plotted sorted for fold change. C) Individual normalized log-transformed expression values for all genes involved in glucose metabolic pathways which are regulated at least 2 fold between control and TSR are plotted ordered by fold change. D) Clock gene regulation by TSR in WAT at ZT18 sorted for fold change. Only clock genes with significant expression under control conditions are shown. With the exception of <i>Per2</i> and <i>Rora</i>, all clock genes were significantly regulated by TSR. Green represents low expression, red represents high expression. Sample sizes were 3 per group.</p