Figure 1.

The Clock^PER Reporter Measures Intestinal Circadian Clock Activity

(A) Schematic of the circadian clock in Drosophila showing that CLK/CYC promotes transcription, and PER/TIM repress this activity. Light acts on CRY degrading TIM to entrain the Drosophila clock to photoperiod.

(B) Schematic of the Clock^PER reporter where 123bp PER promoter drives expression of destabilized GFP (dGFP) to report temporal changes in clock activity.

(C) Representative image of a Drosophila intestine at ZT3 showing Clock^PER (GFP) in the anterior (R2) region and in the posterior (R5) region. DAPI counterstains nuclei. A, the anterior region; P, posterior. Scale bar represents 500 μm.

(D) Representative images of Clock^PER (GFP) over a 24-hr timeline under LD photoperiod shows 24-hr changes in expression with a peak at ZT0. A, anterior region; P, posterior. Scale bar represents 500 μm.

(E) RT-qPCR expression of entire Clock^PER intestine for GFP, PER, and TIM shows that these have similar expression phases, hence the Clock^PER reporter reports endogenous CLK/CYC transcriptional activity. Each data point represents a signal obtained from n = 10 intestines. Results of additional qPCR experiments are shown in Figure S1.

(F) Graph of Clock^PER GFP signal normalized to DAPI under LD photoperiod, followed by 24 hr in DD for the entire intestine. Circadian rhythms of GFP are present, and the cyc⁰ mutant has no circadian transactivation and is thus negative at all times.

Data presented as mean of n ≥ 10 intestines, error bars show ±SEM (two-way ANOVA F = 9.552, p < 0.0001). See also Figures S1 and S2.