Fig 1: Generation of the disc1 mutant genetic model in zebrafish(A) Representation of the 15 exon disc1 gene. Red asterisk indicates the site targeted.(B) The CRISPR-Cas9 target site is highlighted in yellow, with the PAM underlined. A mutant line with a 20-base pair deletion (denoted by red dashes) was identified.(C) Depiction of the putative Disc1 protein domains. The red line indicates the predicted length of the truncated protein in disc1 mutants.(D) Western blot of Disc1 protein in lysates from disc1 WT and HOM larvae at 7 dpf (25 larvae per lane).(E) Ratio of genotypes in offspring generated by disc1 heterozygous incrosses (n = 451; age = 3 months).(F) Light micrographs of disc1 WT and HOM larvae at 7 dpf.(G) Diagram depicting the timeline of neurobehavioral assays conducted through lifespan.
Fig 2: disc1 mutant embryos and larvae display blunted behavioral responses to sensory stimuli(A) Overview of the photomotor response assay: Ten embryos (28 hpf) per well were loaded into 96-well plates and motion was measured using the ZebraBox.(B) Example motion index graph. For each well, basal motion was captured, followed by an excitation period induced by a light stimulus (denoted by a magenta vertical bar), and a refractory period in which a second light stimulus (denoted by a magenta vertical bar) did not elicit motion.(C) Box and whisker plot showing motion indexes for the basal, excitation, and refractory phases in WT (gray) and mutant (magenta) embryos (n = 48 wells per genotype). The box in the plot represents the 25th percentile to the 75th percentile, the line across the box represents the median, and the whiskers are the maximum and minimum data-point values.(D) Behavioral profile generated in 7 dpf larvae using a battery of sensory and acoustic stimuli demonstrating decreased motor output in disc1 mutants in response to auditory stimuli. Plot shows the average motion index of WT (gray) and disc1 mutant (magenta) larvae (8 larvae per well, 36 wells per genotype). The x-and y axes indicate time and the motion index, respectively. Behavioral assay names and their order in the run are indicated below the x axis. AS = auditory stimulus, VS. = visual stimulus and AV = auditory plus visual stimulus.
Fig 3: Adult female disc1 mutants do not exhibit decreased anxiogenic behavior and increased exploration behavior in a novel environment(A) Diagram of behavioral testing arena.(B) Spatiotemporal data (heat maps) depicting the amount of time spent at each location in the testing arena by a representative female WT sibling and female disc1 mutant (red indicates increased time and blue indicates decreased time).(C-J) Box and whisker plots of the (C) total time spent in the top zone, (D) distance traveled in the top zone, (E) distance traveled in the bottom zone, (F) latency time to first visit to the top zone, (G) the number of transitions between the top and bottom zone, (H) average time per visit in the top zone, (I) mean speed throughout the duration of the assay, and (J) total distance traveled in the top and bottom zone combined. WT (black, n = 16 females), mutant (red, n = 16 females). * = p < 0.01. The box in the plot represents the 25th percentile to the 75th percentile, the line across the box represents the median, and the whiskers are the maximum and minimum data-point values. * = p<0.05.
Fig 4: Adult disc1 mutants do not exhibit scototaxis in the light-dark preference test(A) Diagram of behavioral testing arena.(B-G) Total time spent in the light zone (open bars) and dark zone (black bars) by WT sibling and mutant (B) males (n = 16) and (C) females (n = 16). Latency to first entry into the light zone in (D) males and (E) females. Number of transitions into the light zone in (F) males and (G) females.(H and I) Average visit time in the light zone in (H) males and (I) females. ** = FDR significant, * = nominally significant.Data are presented as box and whisker plots. The box in the plot represents the 25th percentile to the 75th percentile, the line across the box represents the median, and the whiskers are the maximum and minimum data-point values.
Fig 5: Adult male disc1 mutants exhibit decreased anxiogenic behavior and increased exploration behavior in a novel environment(A) Diagram of behavioral testing arena.(B) Spatiotemporal data (heat maps) depicting the amount of time spent at each location in the testing arena by a representative male WT sibling and male disc1 mutant (red indicates increased time and blue indicates decreased time).(C-J) Box and whisker plots of the (C) total time spent in the top zone, (D) distance traveled in the top zone, (E) distance traveled in the bottom zone, (F) latency time to first visit to the top zone, (G) the number of transitions between the top and bottom zone, (H) average time per visit in the top zone, (I) mean speed throughout the duration of the assay, and (J) total distance traveled in the top and bottom zone combined. WTs (black, n = 16 males), HOMs (green, n = 16 males). * = p < 0.01. The box in the plot represents the 25th percentile to the 75th percentile, the line across the box represents the median, and the whiskers are the maximum and minimum data-point values. * = p<0.05.
Supplier Page from Abcam for Anti-DISC1 antibody