Glucocorticoids regulate a large part of the diurnal transcriptome.

<p>(A) <i>rx3</i> mutant phenotypes. Blue: <i>pomc</i> expression in larval head. sib: wild-type sibling. (B) Sampling schedules of the transcriptomics studies. Zebrafish embryos/larvae were raised under 12 h light (yellow) / 12 h dark (black) cycles. dpf: days post fertilization. ZT: <i>Zeitgeber</i> Time in hours after lights on (ZT0); lights off: ZT12. (C,D) Heatmaps of normalized mRNA expression levels of model 11 (C) and model 5-6-14-15 (D) genes. Red, high expression. Green, low expression. Expression levels normalized by the mean of each gene within each phenotype combination. (E) Overview of gene expression profiles in the different models. Black curved line: oscillation with same phase and amplitude as wild-type, red or green curved lines: oscillation with a phase or amplitude different from wild-type, black straight line: no oscillation. The green curved line indicates an oscillation with an amplitude and/or phase that is both different from wild-type (black) and from the other changed oscillation (red). (F) Model distribution among genes of different functional categories. (G) Tukey boxplots depicting amplitudes of gene expression oscillations in the indicated functional categories. Red stars, significant amplitude change (adjusted <i>p</i>-value = 2.2e-16).</p

Diurnal metabolite patterns show differential dependence on glucocorticoids.

<p>(A, B) Principal component analysis (PCA) score plots of the NMR spectra of untreated samples (A) and of DEX treated together with control samples (B). (C-E) Left: Temporal profiles of metabolite levels determined by UPLC-FLR for the indicated phenotype/treatment combinations. Lines fitted by linear regression if statistical models indicate rhythmicity for wild-type (black) and <i>rx3 strong</i> mutant samples (white). Rhythmic model fits are indicated as continuous lines, non-rhythmic fits as broken lines. Error bars represent SEM. Right: Tukey boxplots depicting metabolite levels at all examined time points in wild-type siblings (WT, white) and <i>rx3 strong</i> mutants (RX3, grey) in the absence or presence of DEX.</p

A module of E-box and GRE elements drives circadian expression under constant DEX treatment in <i>vitro</i> and <i>in vivo</i>.

<p>(A) Multiple alignment of the <i>pck1</i> promoters of the indicated species. Red boxes indicate GRE half sites. (B) Graph depicting p values for observed vs. random occurrence of E-boxes and GREs in the promoter regions of model 5-6-14-15 genes rescued (green) or not rescued (red) by DEX treatment. (C-H) Bioluminescence traces recorded from zebrafish cells expressing the indicated reporter constructs under different doses of DEX. G, GRE. E, E-box. P_min,TATA box. pA, polyadenylation site. (I) <i>In vivo</i> bioluminescence traces recorded from transgenic zebrafish larvae carrying the 1xE-box/GRE reporter in the indicated phenotype/treatment combinations. (J) Boxplots depicting amplitudes of the bioluminescence oscillations shown in D. *, <i>p</i> < 0.05, **, <i>p</i> < 0.01, ***, <i>p</i> < 0.001.</p

Glucocorticoid dependent alterations of diurnal TCA cycle function.

<p>(A) Schematic depicting glycolysis, gluconeogenesis, the TCA cycle, and glutaminolysis, indicating the steps where genes with dysregulated temporal mRNA expression in <i>rx3 strong</i> mutants are involved. Gene names are color-coded according to the models in C. Check marks in (A) and (C) indicate rescue of dysregulated expression in <i>rx3 strong</i> by DEX treatment. (B) Tukey boxplots showing fold changes of TCA cycle metabolites when comparing the indicated conditions. Statistically significant changes are shown in green for up-regulation and in red for down-regulation. (C) Heatmap of normalized mRNA expression levels of the genes shown in (A), classified according to their expression models. Red, high expression. Green, low expression. (D, E) Gene expression levels of <i>pck1</i> (D) and <i>gls2</i> (E) mRNA levels in 5 dpf larvae in the indicated conditions. Lines fitted by linear regression if statistical models indicate rhythmicity for wild-type (continuous) and <i>rx3 strong</i> mutant samples (broken).</p