Individual RFRP peptides act synergistically to promote sleep. (A) Schematic diagram of the NPVF preproprotein and its three RFRP peptides. (B) Eight heat shock-inducible transgenes containing all possible combinations of one, two or three scrambled RFRP peptides tested. Colors correspond to those used in panels (C–U). (C) Amino acid sequences of zebrafish Tg(hs:NPVF) transgenes containing single or multiple scrambled RFRP peptides. RFRP peptide sequences are predicted based on conservation with peptides isolated in other species and on the location of glycine amidation signals and lysine or arginine endoproteolytic cleavage sites. The C-terminus of each predicted peptide contains a LPXRF motif, where X = L or Q. Gray shading indicates scrambled sequences. Daytime activity (D) and sleep (E) of transgenic animals normalized to their WT siblings following afternoon heat-shock-induced transgene overexpression are shown as mean ± SEM for two pooled experiments, with at least 64 animals per genotype. Examples of activity and sleep data for each transgene are shown in (F–U). Yellow bars indicate heat shock. White and black bars under behavioral traces indicate day (14 hr) and night, respectively. n = number of animals. n.s. = not significant, ***p<0.0001 by One-way ANOVA compared to scrambled NPVF peptide control (black) using Dunnett’s multiple comparison test.
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