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Light-Dependent Sleep Induction by Prok2 Is Correlated with Increased galn Expression and Requires galn
(A–D) 29-μm-thick projections from representative brains showing galn expression in the anterior hypothalamus. Larvae were raised in LD until 5 dpf and then were not heat shocked (A and A′), heat shocked from 3 to 4 p.m. and kept in light (B and B′), or heat shocked from 3 to 4 p.m. and kept in dark (C and C′) and then fixed at 8 p.m. (D and D′) WT larvae were treated with either DMSO vehicle or 20 μM melatonin at 4 p.m. and fixed at 8 p.m. Ventral views with rostral at top are shown.
(E) galn ISH in a 5-dpf brain. Red box indicates the area shown in (A)–(D) and quantified in (F).
(F) Mean ± SEM fluorescence intensity of galn ISH. ∗∗∗p < 0.001 by two-way ANOVA for pairwise comparisons of the mean of each control sample (blue) with its corresponding hsp:Prok2 or melatonin-treated sample (red), with Bonferroni correction for multiple comparisons. White numbers indicate number of animals. Scale bar in (A)–(D), 50 μm, and in (E), 100 μm.
(G) Amino acid sequences of WT and mutant zebrafish Galn (isoforms a and b). Blue and red boxes indicate Galn peptide and Galn message-associated peptide, respectively. Gray shading indicates altered amino acids in the mutant.
(H–K) Larval progeny from a Tg(hsp:Prok2)/+; galn+/− to galn+/− mating were heat shocked (gray bar in H and J) during the day. This resulted in a significant decrease in locomotor activity (H and I) and increase in sleep (J and K) in Tg(hsp:Prok2)/+; galn+/+ and Tg(hsp:Prok2)/+; galn+/− animals compared to their non-transgenic galn−/−, galn+/−, and galn+/+ siblings. This phenotype was significantly suppressed in Tg(hsp:Prok2)/+; galn−/− animals, particularly following 1 hr after heat shock, reaching non-transgenic control levels after 3 hr. Line and bar graphs show mean ± SEM from a representative experiment containing two plates of four experiments, with different heat-shock times that preclude combining datasets from different experiments. n.s., not significant, ∗p < 0.001, ∗∗p < 1 × 10−10 by one-way ANOVA with post hoc Tukey’s test.
See also Figure S8.
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