Fig. 1-S3

npvf expression levels do not vary in a circadian manner and NPVF overexpression-induced sleep does not require entrained behavioral circadian rhythms.

(A,B) Larval zebrafish were raised on a 14:10 h light:dark cycle at 28.5°C with lights on at 9 a.m. and off at 11 p.m. (A) FISH using a npvf-specific probe in animals fixed at the indicated times revealed no obvious circadian pattern of npvf expression. Representative images are shown. (B) At the indicated time points, total RNA was collected from 20-pooled larvae, and npvf mRNA levels were quantified by qPCR normalized to the reference gene ribosomal protein l13a, and then normalized to the sample with the lowest npvf expression level. Results from six combined experiments are shown. There is no significant difference in npvf mRNA level at any time point (p>0.05 by One-way ANOVA). (C–I) Zebrafish larvae raised and tested in constant light (LL) do not exhibit circadian locomotor activity or sleep rhythms (C,F). NPVF overexpression decreased locomotor activity (C,D) and increased sleep (F,G). This phenotype was due to an increase in the number of sleep bouts (H) and was associated with a decrease in wake activity (E). (J–P) Zebrafish larvae raised and tested in constant dark (DD) do not exhibit circadian locomotor activity or sleep rhythms (J,M). NPVF overexpression decreased locomotor activity (J,K) and increased sleep (M,N). This phenotype was due to an increase in the length of sleep bouts (P) and was associated with a decrease in wake activity (L). Yellow bars indicate heat shock. White and black bars below line graphs indicate developmental time periods. Bar graphs represent mean ± SEM for twelve hours pre- and post-HS. n = number of animals. n.s. = not significant. *p<0.05, ***p<0.005 by Two-way ANOVA with Holm-Sidak post-hoc test.