Figure 5

(A) Asymmetric expression pattern of kctd12.1 is right-isomerized in bsx homozygotes at five dpf. (B) Projections of Tg(lhx2a:gap-YFP) labeled olfactory mitral cells terminate bilaterally (open arrowheads) in the dHb of bsx^m1376 homozygous mutants at five dpf. (C) In bsx mutants, axons from both left (open arrowhead) and right dHb lratd2a neurons project to the same region of the vIPN. Scale bar, 50 μm. (D) Bilateral fos-expressing neurons in right-isomerized mutants. fos (blue) and lratd2a (brown) transcripts in the olfactory bulbs (upper panels) and dHb (bottom panels) of 10-month-old bsx^m1376 heterozygotes and homozygous mutants detected by RNA in situ hybridization 30 min after addition of cadaverine to the test tank. Brackets indicate fos-expressing cells. Scale bar, 100 μm. (E) Quantification of fos-expressing cells in the dHb after application of cadaverine in bsx^m1376/+ [9.47 ± 2.37 cells on the left and 15.41 ± 2.19 cells on the right, n = 17 sections from nine adults] and bsx^m1376/m1376 adults [17.64 ± 1.59 cells on the left and 18.5 ± 1.76 cells on the right, n = 14 sections from eight adults]. Two-way mixed ANOVA reveals significant effect of group [F_{(1, 16)} = 5.178, p = 0.037] and left vs. right [F_{(1, 16)} = 6.885, p = 0.0184], but no effect of interaction [F_{(1, 10)} = 3.85]. Post-hoc analysis by Bonferroni’s multiple comparisons. (F) Preference index for bsx adults for an average of 2 min before (white) and for each of 3 min after (gray) the addition of cadaverine. Both bsx homozygotes and heterozygotes showed reduced responsiveness to cadaverine. Two-way ANOVA reveals significant effect of interaction [F_{(3, 34)} = 5.483, p = 0.005], but no effect of time [F_{(3, 25)} = 0.987] and group [F_{(1, 14)} = 2.728]. Post-hoc analysis by Bonferroni’s multiple comparisons. (G) Swimming speed for 30 s before and after addition of alarm substance. In heterozygous adults, swimming speed was 1.22 ± 0.31 cm/s before and 3.52 ± 0.44 cm/sec after and, in homozygotes, 0.46 ± 0.08 cm/s before and 2.80 ± 0.37 cm/s after, n = 15 adults for each group. Two-way ANOVA reveals significant effect of time [F_{(1, 14)} = 113.4, p < 0.0001], but no effect of group [F_{(1, 14)} = 4.459] and interaction [F_{(1, 14)} = 0.0023]. Post-hoc analysis by Bonferroni’s multiple comparisons. (H) Duration in the upper half of the test tank prior to and after addition of alarm substance for bsx^m1376/m1376 adults was 194.86 ± 25.66 s and 51.89 ± 14.84 s and was 63.55 ± 10.11 s and 7.95 ± 3.24 s for bsx^m1376/+, n = 15 fish for each group. Two-way ANOVA reveals significant effect of time [F_{(1, 14)} = 44.35, p < 0.0001], group [F_{(1, 14)} = 22.45, p = 0.0003] and interaction [F_{(1, 14)} = 20.89, p = 0.0004]. Post-hoc analysis by Bonferroni’s multiple comparisons. (I) Onset of fast swimming after application of alarm substance was observed at 33.13 ± 19.19 s in bsx^m1376/+ and at 37.73 ± 4.27 s in bsx^m1376/m1376 fish [p = 0.816, unpaired t-test]. (J) Time interval between increased swimming speed and freezing behavior for bsx^m1376/+ (103.1 ± 30.75 s) and for bsx^m1376/m1376 (186.7 ± 28.23 s) [p = 0.055, unpaired t-test]. For E-J, all numbers represent the mean ± SEM.