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Figures
- Fig. 1.
Circadian rhythms in PER2::LUC jejunal explants. Representative jejunal segments displaying 1.5- to 2-fold circadian PER2 oscillations for up to 3 days following excision, with lengthening of the period of PER2 oscillations over time.
- Fig. 2.
Organotypic PER2::LUC jejunal organoids display circadian variation in PER2 and LUC expression. (A–C) Representative three-dimensional confocal image reconstruction of a PER2::LUC organoid with crypt, villous and lumen domains 42 hours after serum shock and staining with fluorescent antibodies against PER2 and LUC throughout. Comparison of single plane confocal images at (D–G) 28 hours following serum shock versus (H–K) 42 hours following serum shock reveals a time-dependent increase in PER2 and LUC staining.
- Fig. 3.
Robust circadian rhythms in PERIOD2::LUCIFERASE jejunal organoids. (A) PER2::LUC organoids display 1.5- to 3-fold circadian oscillations of PER2 abundance following serum shock (blue), as measured by luciferase bioluminescence. In the absence of serum shock (red), PER2::LUC organoids develop synchronized PER2 oscillations within 8 to 12 hours of recording (n=4 per group). (B) Representative traces of detrended luciferase bioluminescence of unshocked organoids that had been treated with the steroid antagonist RU486 (20 μM, red) or an ethanol vehicle control (black). Standard medium is shown in blue. (C) Averaged tracings of detrended luciferase bioluminescence in unshocked PER2::LUC jejunal enteroids that had been treated with either the steroid receptor antagonist RU486 (red) or an ethanol vehicle control (blue). Treatment with RU486 delays the initial peak of PER2 oscillations by a mean of 2.1 hours (*P<0.0001 by Mann–Whitney U test; n=9 samples per group). Numbers in boxes indicate the circadian time at which the peak PER2 oscillation is reached. (D) Representative tracings of detrended luciferase bioluminescence in shocked (blue) and unshocked (red) organoids in the absence of Matrigel™. PER2 oscillations appear more variable and lower in amplitude but maintain a clear circadian rhythm. (E) Replenishment of organoid medium and luciferin every 5 days enhances and extends PER2 oscillations beyond 2 weeks but resets oscillations after day 10 (representative detrended tracings).
- Fig. 4.
Effects of organoid growth factors on PER2 rhythms. (A) Doubling standard concentrations of EGF, Noggin and R-spondin in organoid medium increases the amplitude of PER2 oscillations (representative detrended tracings). (B) Subtraction of these growth factors individually from organoid medium 24 hours following serum shock does not dampen PER2 rhythms compared with standard medium (blue). Representative detrended tracings are shown. (C) Top panels, sequential micrographs (4× magnification) of serum-shocked organoids passaged into standard medium over 5 days. Middle panels, organoids with R-spondin washout at 24 hours following serum shock. Bottom three panels, organoids passaged immediately into R-spondin-negative medium following serum shock show loss of proliferation and death by day 5.
- Fig. 5.
Growth factors induce phase shifts in PER2 oscillations in unshocked organoids. (A,B) Serum-shocked organoids show no response to growth factors (red) pulsed at either an (A) upslope or (B) downslope of PER2 oscillations versus a PBS vehicle control (blue). By contrast, unshocked enteroids that were pulsed with growth factors (red) at an (C) upslope or (D) downslope of PER2 oscillations both show phase shifts by day 5 of recording (representative detrended tracings). CT, circadian time.
- Fig. 6.
Circadian oscillations of Per2 mRNA expression in PER2::LUC organoids and cell lines. (A) Per2 mRNA expression over 48 hours in mouse small intestinal organoids (n=3 per time point). (B) Per2mRNA expression over 48 hours in nontransformed mouse small intestinal epithelial (MSIE) cells under quiescent conditions (n=3 per time point). (C) Per2 mRNA expression over 48 hours in transformed human colorectal adenocarcinoma Caco-2 cells (n=3 per time point). Means±s.e.m. are shown.
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