Figure 5.

Stem Cell Signaling Pathways Affect ISC Circadian Clock Activity

(A) Representative confocal z stacks of Clock^PER reporter for ISC/EB-specific pathway disruption at CT0. Dl+ and Pros+ are used to mark the ISC versus EE tumors, respectively. ISCs expressing esg in these tumors are outlined, showing the abnormal Dl+ cells caused by signaling pathway activation. CYC RNAi and N RNAi reduce GFP+, while APC RNAi and Yki overexpression increase it. Scale bar represents 10 μm.

(B) Analysis of Clock^PER and Clock^TIM GFP signals in single Dl+ cells from confocal sections at CT0. Activation of the Wnt or the Hippo pathway raises clock activity, and loss of the N pathway lowers clock activity. Data presented for n ≥ 15 cells in each group, error bars show ±SEM. One-way ANOVA for Clock^PER (F = 9.624, p < 0.0001); Clock^TIM (F = 12.99, p < 0.0001).

(C) Twenty-four-hour analysis of Clock^PER from esg>APC RNAi. Elevated Wnt pathway does not alter the phase of normal circadian clock activity in ISCs (compare with Figure 3D). Data presented for n = 25 cells in each time point, error bars show ±SEM (one-way ANOVA F = 9.153, p < 0.0001).

(D) The same analysis on esg>N RNAi. In this case, clock activity is not only lowered but becomes arrhythmic. Data presented for n = 25 cells in each time point, error bars show ±SEM (one-way ANOVA F = 0.6754, p = 0.5692).