Fig. S1

prok2 and prokr2 expression levels do not oscillate in circadian manner, prok2 expression level is not affected by light, and changes in light do not activate prok2- expressing neurons in larval zebrafish. Related to Figure 1. (A, F) qRT-PCR analysis of prok2 and per3 (A), and prokr2 and per3 (F), mRNA is shown. Larvae were raised in 14:10 hr LD conditions and collected at the indicated times beginning at 6 dpf. Mean ± SEM for triplicate biological samples normalized to actin at each time point is shown. prok2 and prokr2 expression does not change significantly over 24 hr, while per3 expression oscillates with a 24 hr period. P values were calculated using one-way ANOVA. (B) 53 μm thick confocal projections showing prok2 FISH in larval zebrafish brains fixed at the indicated times starting at 6 dpf. Larvae were raised as described above. (C) Quantification of total prok2 fluorescence pixel intensity. prok2 expression does not oscillate (peak:trough ratio=1.24, p=0.40 by one-way ANOVA). Mean ± SEM for five brains at each time point is shown. a.u. = arbitrary units. (D) A 53 μm thick confocal projection showing prok2 FISH in brains of larvae raised in LL or DD and fixed at 6 dpf. (E) Mean ± SEM prok2 FISH fluorescence intensity for 5 brains in each condition, normalized to the LL condition. There is no significant difference in prok2 mRNA level between the two conditions (p=0.40 by two-tailed Student’s t test). (G, H) Larvae were raised until 6 dpf in either DD or LL, then exposed to light or dark, respectively. Samples were fixed at the indicated times after the change in lighting condition. Control larvae were maintained in the original lighting condition. qRT-PCR was performed to measure the level of prok2 (G) or prokr2 (H) mRNA. Mean ± SEM for triplicate biological samples normalized to actin at each time point is shown. Statistical significance was assessed by two-tailed Student’s t test. (I-L) prok2- expressing neurons do not express c-fos in response to changes in lighting conditions. Larvae raised in DD (I, J) or LL (K, L) until 5 dpf were transferred to light (I, J) or dark (K, L) conditions, and fixed 15 or 30 min later. Double FISH using prok2- and c-fos-specific probes showed that c-fos is not expressed in prok2-expressing neurons in either case. Scale bars: 10 μm.