Daily profiles of zCry1aa, zCry1ab and zCry2 mRNA expression levels in central and peripheral tissues under white light/dark cycles. Each tissue was collected at ZT1, ZT7, ZT15 or ZT19 from zebrafish (n = 4) entrained to white light/dark cycles (14L:10D). Each mRNA level was estimated using zβ-actin as a reference gene because it was the most stably expressed in the examined tissues among the control genes. Data were analyzed by one-way ANOVA (Supplementary Table S1) and Tukey-Kramer post-hoc tests (Supplementary Tables S2–S31). Error bars represent ± SE. Peak times of cosine-fitting to the data as calculated by CircWave1.4 (red dashed curves; Supplementary Table S32) are shown. A down-pointing triangle in panel g denotes the sustained expression of zCry1ab transcripts at ZT15 in the eye.

Daily profiles of zCry1ba, zCry1bb and zCry4 mRNA expression levels in central and peripheral tissues under white light/dark cycles. The experimental conditions and data analyses were the same as those described in Fig. 1.

Maximum Cry expression in zebrafish tissues. Acrophases of Cry expression as estimated by cosinor analysis (Supplementary Table S32) are shown. Red up-pointing triangles, orange squares, yellow circles, green diamonds, dark-blue down-pointing triangles and light-blue crosses show estimated acrophases for zCry2, zCry1ab, zCry1aa, zCry4, zCry1ba and zCry1bb, respectively.

zCry mRNA levels under blue light/dark and dark conditions. Zebrafish were entrained to white light/dark cycles (14L:10D) and irradiated with blue (λ_max = 462 nm; λ_1/2 = 453 nm and 573 nm; blue circles) light or kept in darkness (black squares) at 28 °C for 14 h from ZT0 (Supplementary Fig. S1). Light intensities were set at 5.586 × 10¹⁴ photons s⁻¹ m⁻² that corresponds to 240 μW cm⁻². Tissues were collected from three (brain samples at ZT0 and ZT19, and eye samples at ZT7 and ZT19) or four (other samples) zebrafish at ZT0, ZT1, ZT3, ZT7, ZT14, ZT15, ZT19 or ZT0 (ZT24) (see Methods for details). Each mRNA level was estimated as a relative value to the geometric mean of mRNA levels of zβ-actin, zGapdh and zEf1α (arithmetic means of their CT values were used in the 2^−ΔΔCT method because they were stably expressed in the brain and eye). Data were analyzed by two-way ANOVA (Supplementary Table S33) and Tukey-Kramer post-hoc tests (Supplementary Tables S34–S57). Error bars represent ± SE.

Daily profiles of zCry1ab mRNA expression levels in the eye under different photoperiods. Zebrafish were entrained to 18L:6D extra-long-day (ELD; panel a), 14L:10D long-day (LD; panel b), 10L:14D short-day (SD; panel c) or 6L:18D extra-short-day (ESD; panel d) cycles. Eyes (n = 5, no mark; n = 4, +; n = 3, *) were collected to measure zCry1ab mRNA levels (see Methods for details) at the indicated time points from ZT1. Each mRNA level was estimated as a relative value to the geometric mean of mRNA levels of zβ-actin, zGapdh and zEf1α. Error bars represent ± SD. Light and dark periods are indicated in blue and grey, respectively. Data were analyzed by two-way ANOVA (Supplementary Table S58) and Tukey-Kramer post-hoc tests (Supplementary Tables S59–S63).

Double plots of 24-h profiles of relative zCry1ab expression levels in the eye under different photoperiods. Relative values of zCry1ab expression levels (Fig. 5; normalized by the peak) were double-plotted using ‘lights on’ (panel a), ‘lights off’ (panel b) or the middle of the night (panel c) as reference points. Data for two points at ZT1 (the start and end of sampling) were averaged. Evening peaks and increasing phases to morning (shoulder) peaks are indicated by E and M, respectively. Light conditions are indicated by bars with colored lines at the top of each panel.

zCry1ab mRNA levels in the eye under constant light (LL) or dark (DD) conditions after entrainment to different photoperiods. (a) Zebrafish were entrained to a 14L:10D long-day cycle and kept in constant blue light (LL; yellow up-pointing triangles) or in the dark (DD; black up-pointing triangles). (b) Zebrafish were entrained to a 10L:14D short-day cycle and kept in constant blue light (LL; blue down-pointing triangles) or in the dark (DD; black down-pointing triangles). Eyes (n = 4, no mark; n = 3, +; n = 2, *) were collected to measure zCry1ab mRNA levels (see Methods for details) at the indicated time points from ZT0 on the last day. Each mRNA level was estimated as a relative value to the geometric mean of mRNA levels of zβ-actin, zGapdh and zEf1α. Data were analyzed by two-way ANOVA (Supplementary Tables S64 and S68) and Tukey-Kramer post-hoc tests (Supplementary Tables S65–S67 and S69–S71). Error bars represent ± SD.

qRT-PCR analysis of zCry1ab mRNA levels in the zebrafish retinal cells captured by laser microdissection (LMD). Retinas were collected at ZT1 or ZT13 or ZT17 from zebrafish entrained to long-day cycles (14L10D). Photoreceptor cell layer (PRL) mainly containing outer nuclear layer (visual photoreceptors) and inner retinal layer (IRL) containing inner nuclear layer, inner plexiform layer and retinal ganglion cells were obtained from the retinal sections. (a) A retinal section (ZT1) showing PRL and IRL. (b) An example of retinal sections after the collection of PRL and IRL samples by LMD. (c) Levels of zCry1ab mRNA in PRL and IRL. The levels of zCry1ab mRNA and 18S rRNA were measured by qRT-PCR, and the zCry1ab mRNA levels relative to 18S rRNA levels were shown. Error bars represent ± SE. Data were analyzed by two-way ANOVA (Supplementary Table S72) and Tukey-Kramer post-hoc tests (Supplementary Table S73). Asterisks represent significant difference (p < 0.05).

Model of a possible zCry1ab regulatory circuit in the zebrafish eye. The zCry1ab mRNA level was regulated by morning, evening, light and day length signals. Based on their mRNA expression patterns (Figs. 1–3), Cry1aa/Cry2 and Cry1ba/Cry1bb were considered morning and evening oscillators, respectively. The morning signal induced the expression of zCry1ab at midnight, regardless of light and day length conditions. In addition to resetting the morning and evening clocks, the external light signal induced zCry1ab expression in the evening to form the evening peak, which may have been based on an integrated signal of light, day length and evening information. The morning and evening peaks were independent and additive. Shown on the right are schematic drawings of zCry1ab expression profiles under long-day and short-day cycles (modified from data shown in Figs. 4 and 7). Biological signal integration and regulation are represented using logic gates, with representative images of daily regulation patterns.