Figure 5.

The intestinal epithelium has a clock. (A) Intestinal organoids were established from both BMAL1^+/+ and ^-/- intestines. Phase-contrast images are shown in brightfield. (B) BMAL1^+/+ organoids show circadian rhythms in clock gene expression: PER2 (F = 46.41; P < .0001), BMAL1 (F = 38.41; P < .0001), REV-ERBα (F = 46.10; P < .0001), and CRY1 (F = 15.96; P = .0009). The control gene TBP does not show such rhythms but rather a gradual decrease following dexamethasone synchronization. There are significant differences between genotypes in PER2 (F = 5.21; P = .0387), BMAL1 (F = 866.6; P < .0001), REV-ERBα (F = 556.8; P < .0001), and CRY1 (F = 32.17; P < .0001). (C) Organoids were established from 3 separate PER2-Luc intestines and luminescence readings show that rhythms in transcriptional clock activity persist for several days (1 representative tracing is shown). (D) Cellular markers in unsynchronized organoids quantified by real-time quantitative polymerase chain reaction show that BMAL1^-/- intestinal organoids have decreased expression of CDH1 (P = .0148), VIL1 (P = .0223), LYZ1 (P = .0498), and LGR5 (P = .0149) expression. (E) The number of viable organoid cells (CellTiter Glo assay) is equivalent between BMAL1^+/+ and BMAL1^-/- organoids, 4 days after varying doses of X-ray irradiation (as indicated in the graph). The intestinal epithelium has no significant BMAL1-dependent differences in sensitivity to radiation (P = .5310).