Figure 6.

The regenerative response influences the 24-hour rhythms of genes. (A) WEE1 shows diurnal variability in the undamaged intestine, with significant differences between BMAL1^+/+ and BMAL1^-/- (F = 8.04; P = .0005). CCND1 and CCL2 expression are similar in both genotypes, whereas TNF exhibits 24-hour variability in the undamaged BMAL1^+/+ intestine, but none in the BMAL1^-/- (F = 15.99; P = .0008). (B) During the gastrointestinal syndrome (IR), WEE1 is arrhythmic (P = .7382), but CCND1 displays high and low periods of expression over 24 hours. These are not likely a function of the circadian clock, but rather a response to environmental changes (eg, feeding), because the BMAL1^-/- mutant also shows the same pattern, albeit at a significantly lower level (F = 16.04; P = .0006). CCL2 exhibits a BMAL1-dependent circadian rhythm (F = 4.318; P = .0176), as does TNF (F = 4.204; P = .0221). There are significant differences between genotypes in CCL2 (F = 7.13; P = .0137) and TNF (F = 7.66; P = .0109). Increased levels of TNF are present in BMAL1^-/- mice at ZT4, whose levels are otherwise lower at all times compared with BMAL1^+/+. It is only at this time point that expression is elevated, suggesting that TNF production in the absence of BMAL1 may be a response to an environmental cue (eg, feeding). (C) BMAL1^+/+ organoids show no rhythms in WEE1 or CCL2 expression (compare with the control gene TBP in Figure 5B); however, TNF is expressed with a 24-hour rhythm in BMAL1^+/+ but not BMAL1^-/- organoids (F = 10.45; P = .0060). This suggests that only TNF is regulated by the clock in intestinal epithelial cells. IR, irradiation.