Figure 4.

Figure 4. Mutation of the dmist-related gene fxyd1 causes reduced sleep at night.

(A) Schematic of zebrafish Dmist and Fxyd1 protein domains and alignments comparing human, mouse, and zebrafish Dmist and FXYD1 protein sequences. Black and grey shading indicate amino acid identity and similarity, respectively. The FXYD domain is indicated with a red line and the RRR motif in the C-terminus is indicated with a dark blue line. (B) CRISPR-Cas9 targeting of the third exon of fxyd1 created a 28 bp deletion, resulting in a predicted truncated protein. The start codon is marked by a cyan line. Guide RNA target sequence and PAM sequence are shown as black bars. The mutant deleted sequence is indicated with a red bar.(C) In situ hybridisation of fxyd1 at 24 hpf (whole animal) and 5 dpf brain (ventral view). Anterior is to the left. Scale bar = 0.5 mm (24 hpf); 0.1 mm (5 dpf). (D, E) Representative behavioural experiment showing fxyd1^Δ28 mutants have decreased night-time sleep (D) but normal waking activity at night (E). (F) Combining five independent experiments with a linear mixed effects model reveals fxyd1^{Δ28/ Δ28} larvae sleep significantly less at night due to shorter sleep bouts compared to fxyd1^+/+ siblings. Plotted are the genotype effect sizes (95% confidence interval) on each parameter relative to wild type. Shading indicates day (white) and night (grey). p-Values are assigned by an F-test on the fixed effects coefficients from the linear mixed effects model. *p<0.05, **p<0.01, ***p<0.001, ns p>0.05.