Rhythmic expression of aanat2 persists in constant conditions. Embryos were raised in a 14:10 h L/D cycle. At 71.5 hpf, ZT 23.5, a set of embryos was transferred to constant dark, constant temperature conditions. At 77.5 hpf, ZT 5.5 a set of embryos were transferred to constant light, constant temperature conditions. (A) Embryos were fixed at the indicated stages and ZT and processed for WISH for expression of aanat2. Note the time of peak expression is similar between the cyc embryos and their WT siblings. All images are dorsal views, anterior to the top. (B) Quantification of the WISH signal indicates that the amplitude of aanat2 gene expression is slightly lower in cyc mutants than in their WT siblings in constant conditions. Two-way ANOVA reveals that the differences between aanat2 signal is significantly different between cyc and WT (p < 0.01 for both constant light and constant dark, n≥10 larva per time point, images of 4 larva per each experimental condition and time point used for statistical analysis). For the samples in a L/D cycle, position within the photoperiod is indicated by ZT and light conditions by the white (light period) and black (dark period) bars. For the constant light samples, the original L/D cycle is indicated by the ZT, and the white (original light period) and light grey (original dark period) bars. For the constant dark experiment, the original L/D cycle is indicated by the ZT, and the black (original dark period) and grey (original light period) bars. Experiment was repeated two times with similar results, and representative images from one of the experiments are shown in A. Scale bars = 20 μm.