FIGURE 1 (Continued)(A,B) Schematic illustration of the isl1:GFP-expressing neuron population in the forebrain of wildtype 48 hpf zebrafish embryos. (B) Heterozygous loss of ctnnd2b results in ectopic isl1:GFP-expressing cells in the ORR (arrows), not detected in control embryos. Wildtype: embryos obtained from an incross of Tg(isl1:GFP) line; ctnnd2a^+/+,ctnnd2b^+/+: non-carriers from the incross of the ctnnd2a^+/−,ctnnd2b^+/− line. (C) Quantification of Isl1:GFP-positive neurons in the ORR confirmed that heterozygous and homozygous loss of ctnnd2b result in ectopic Isl1-positive cells in the region. (D) In ctnnd2b^+/− embryos the ectopic Isl1:GFP-positive cells display a vertical rather than horizontal orientation (arrows, lateral view in D′) and the cluster seems more disorganized when compared to these cells in control (ctnnd2a^+/+,ctnnd2b^+/+) embryos. (E) Control and ctnnd2b^+/− 54 hpf embryos show no difference in nkx2.1 expression, investigated in lateral and rostral (inset) views. Dashed box: ORR; Tel: telencephalon; ORR: optic recess region; Hyp: hypothalamus; AC: anterior commissure; POC: post-optic commissure; SOT: supraoptic tract; TPOC: tract of the POC; ****p < 0.0001. Scale bar =10 μm.

(A) Heterozygous loss of ctnnd2b affects GABA neuronal specification in the ORR, resulting in a significant decrease in the number and organization of GABA-positive cells in the ORR (dashed box, arrowheads in A; C). (A,B) Isl:GFP-positive cells are negative for GABA (arrows). **p = 0.0017. Scale bar =10 μm.

Increase in Isl1-positive cells is likely due to misspecification rather than migration defects. 3D renders of timelapse imaged ctnnd2b^+/+(A-D) and ctnnd2b^+/− embryos (A’-D’) with highlighted ORR (insets). Embryos at 32 hpf, prior to appearance of neurons at ORR, are shown in (A,A’). Isl1:GFP-positive neurons start appearing at the ORR by 37 hpf (B,B’), and detected at 42.5 hpf (C,C’) and 48 hpf (D,D’). Insets show the ORR at the corresponding timepoints. By the end of recording only a few isl1:GFP-expressing cells are found in ctnnd2b^+/+ embryos compared to ctnnd2b^+/− embryos.

(A) Assessment of the swimming performance using the visual motor response test showed an increase in activity from ctnnd2b^+/− 5 dpf larvae compared to wildtype larvae when exposed to darkness (shaded time windows). (B) Quantification of total distance moved per light condition showed that mutant larvae moved significantly move in both light and dark environments. (C) Evaluation of swimming patterns during a dark iteration of the test showed a statistically significant increase preference to swimming in the outer zone of the arena, or thigmotaxis, from the ctnnd2b^+/− larvae compared to wildtype controls. **p = 0.0033, ***p = 0.0006, ****p < 0.0001.

Behavior of adult individuals was evaluated using the novel tank test. (A) Total distance moved for the duration of the test was similar between ctnnd2b^+/− and wildtype individuals. (B) Anxiety-like behavior was tested by defining a top and bottom zone of the tank used in the test. Quantification of the distance moved per minute per zone (C) and the number of bottom-to-top transitions during the test (D) showed no difference between genotypes. (E) While no difference in the distance moved per zone was found between females, heterozygous mutant males show a statistically significant increase in the distance moved in the top zone of the tank compared to wildtype individuals. Graphs: mean ± SEM, *p = 0.0252.