Generation of Tg(aanat2:EGFP-?CLK) fish.

(A) Schematic representation of the transgenic construct used for the generation of Tg(aanat2:EGFP-?CLK) fish. The pT2-aanat2:EGFP-2A-?clocka-5×MYC plasmid consists of two arms from the Tol2 transposable element (black), aanat2 regulatory regions (gray), EGFP coding sequence (CDS; green), 2A peptide sequence (orange), ?CLK CDS (blue), 5×Myc tags (purple) and SV40 poly(A) signal (yellow). (B and B') EGFP expression is restricted to the pineal gland of Tg(aanat2:EGFP-?CLK) larvae. Dorsal views of the head region of 7-day post-fertilization (dpf) larvae, anterior to the top; confocal z-stack projection (B) and a single confocal plane (B'). (C) Immunostaining with anti-Myc antibody confirms that ?CLK is specifically expressed in the pineal gland of Tg(aanat2:EGFP-?CLK) larvae. Dorsal view of the head region of a 5-dpf larva, anterior to the top; confocal z-stack projection.

Clock-controlled aanat2 mRNA rhythm is abolished in the pineal gland of Tg(aanat2:EGFP-?CLK) larvae.

(A) Tg(aanat2:EGFP-?CLK) larvae (?CLK) exhibit arrhythmic pineal aanat2 mRNA levels under DD compared with a robust rhythm in their WT siblings (p<0.0001, two-way ANOVA), indicating that the pineal gland molecular clock is disrupted. Each value represents the mean optical density ± SE of the pineal aanat2 whole-mount ISH signal (n = 10?15 larvae). Different letters represent statistically different values (p<0.05, Tukey's test). (B) Representative whole-mount ISH aanat2 signals (8 dpf, dorsal view) in the pineal glands of WT sibling larvae (upper panel) and Tg(aanat2:EGFP-?CLK) larvae (?CLK; bottom panel). Black and gray bars represent subjective night and day, respectively. CT, circadian time.