Rhythmic expression of pineal genes persists in cyc mutants. Embryos were raised in a 14:10 h L/D cycle and then sets of embryos were fixed at the indicated stages and ZT and processed for WISH for expression of (A) exorh or (B) aanat2. Note the time of peak (marked with closed arrowheads for exorh) and trough (marked with open arrowheads for exorh) expression is the same between both sets of embryos. (C, D) Quantification of the WISH signal indicates that the amplitude of aanat2 and exorh gene expression is slightly lower in cyc mutants than in their WT siblings. Two-way ANOVA reveals that the difference in the strength of the aanat2 signals are significantly different between cyc and WT (p < 0.01), while the difference in the strength of the exorh signals does not reach significance (p = 0.14). (E, F) In contrast, the expression of otx5, a gene expressed in all pineal cells, is comparable between WT and cyc mutants. (F) At 54 hpf, ZT6 (left graph) and 78 hpf, ZT6 (right graph), length of otx5 gene expression domain is similar between WT and cyc mutants, while the width of the pineal at each stage is slightly bigger in the mutants, perhaps due to changes in the morphology of the cyc head (54 hpf, pineal length, p = 0.11; 54 hpf, pineal width, p = 0.0045; 78 hpf, pineal length, p = 0.52; 78 hpf, pineal width, p = 0.14, n≥3 embryos per time point). In A, B, and E, light conditions are indicated by the white (light period) and black (dark period) bars. All images are dorsal views, anterior to the top. Each experiment was repeated three times, and representative images are shown. Scale bars = 20 μm.
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