avpl expression is absent in cyc mutants. Embryos were fixed at (A, B) 24 hours post-fertilization (hpf), (C-F) 46 hpf, and (G-I) 72 hpf and then processed for WISH with antisense probes for the indicated mRNAs. shh- expression in the ventral brain, including the hypothalamus (h, closed arrowhead) is (A) apparent in WT embryos but (B) absent in cyc mutants. (C, E, G, and I) avpl is expressed in the dorsal preoptic area (open arrowheads) and the ventral hypothalamus (closed arrowheads). (D, F, H, and J) Both avpl expression domains are eliminated in cyc mutants. (A-D, G, and H) are lateral views and (E, F, I, and J) are ventral views. The images in panels (C-J) are representative embryos of three independent experiments (n = 3 fish per experiment). avpl expression was also completely absent in cyc mutants at 47 hpf, Zeitgeber Time (ZT) 23.5; 52 hpf, ZT 5.5; 62 hpf, ZT 15.5, and 71.5 hpf, ZT 23.5. No circadian rhythm in avpl expression was detected in the WT larva processed in parallel (n> 4 larva per time point). Scale bars = 50 μm.