Expression of adap1 during early embryogenesis. (a–c) Embryos stained with the adap1 sense probe (a: Lateral view) and the adap1 antisense probe (b: Lateral view, c: Dorsal view) at 32 hpf. (d–f) Embryos stained with the adap1 sense probe (d: Lateral view) and the adap1 antisense probe (e: Lateral view, f: Dorsal view) at 48 hpf. (g–i) Embryos stained with the adap1 sense probe (g: Lateral view) and the adap1 antisense probe (h: Lateral view, i: Dorsal view) at 72 hpf. Expression of adap1 was widely detected in the forebrain, midbrain and hindbrain at 32 hpf. Expression of adap1 in the midbrain, and hindbrain was maintained at 48 hpf and 72 hpf. Scale bar, 200 μm (a).

Establishment of adap1 mutants. (a) Molecular structure of adap1. The ArfGAP, pleckstrin homology 1 (PH1) and PH2 domains in the Adap1 protein are indicated by the red, green, and blue rectangles, respectively. The adap1‐uy70 and adap1‐uy71 mutants contained premature stop codons after 24 missense amino acids starting at amino acid 125 and after 30 missense amino acids starting at amino acid 118, respectively. (b–d) Morphology of the wild‐type (b), adap1^uy70/uy70 mutant (c), and adap1^uy71/uy71 mutant (d) zebrafish. Scale bar, 500 μm (b). Genotyping of individual embryos was performed by genomic PCR.

Neural gene expression in wild‐type and adap1 mutant fish. (a, c, e, g, i, k) Embryos with the adap1 wild‐type allele at 24 hpf. (b, d, f, h, j, l) Embryos with the adap1 homozygous mutant alleles at 24 hpf. WISH with emx1 (a, b), mbx (c, d), islet1 (e, f), oligo2 (g, h), gfap (i, j) and huC (k, l). All the images show lateral views, with the anterior to the left. Scale bar, 200 μm (i). The expression of emx1, mbx, islet1, oligo2, gfap, and huC was comparable between the wild‐type and the adap1 mutant embryos at 24 hpf. Genotyping of individual embryos was performed by genomic PCR.

Open field test of wild‐type and adap1 mutants in the adult stage. (a) Schematic representation of the open field test used in this study. (b and c) The distance moved by zebrafish in chamber II and the time spent in each area (area 1 and 2) for each minute. The distance moved of adap1^uy70/WT, adap1^uy70/uy70, and adap1^uy71/uy71 was shorter than that of wild‐type. The adap1^uy70/uy70 and adap1^uy71/uy71 stayed longer in area 1 and both stayed less time in area 2. The numbers of zebrafish are Ν = 11 for WT/WT, Ν = 18 for WT/uy70, Ν = 15 for uy70/uy70, Ν = 10 for WT/WT, Ν = 12 for WT/uy71, Ν = 11 for uy71/uy71. *p < 0.05, **p < 0.01. (d and e) Heat map of the behavior of wild‐type and adap1 mutants. The numbers of zebrafish in each group are same in (b and c). A high number of color indicators indicates that zebrafish were frequently located at that position during the 20 min test period.

Social behavior analysis of wild‐type and adap1 mutants in the adult stage. (a) Schematic representation of the social behavior test used in this study. Areas 3 and 5 were set as the region near the stimulus exploration. One and three novel conspecifics (e.g., albino line) were placed in chamber I and III, respectively. (b and c) The distance moved by zebrafish in chamber II and the time spent in each area (areas 3, 4, and 5) for each minute. The adap1^uy70/WT and adap1^uy70/uy70 swam longer than the wild type (WT). The adap1^uy70/uy70 stayed longer than the wild type in area 3. The adap1^uy71/WT stayed shorter than the wild type in area 3. The adap1^uy70/WT, adap1^uy70/uy70, and adap1^uy71/WT stayed shorter than the wild type in area 4. The adap1^uy70/WT, adap1^uy70/uy70, and adap1^uy71/WT stayed longer than the wild type in area 5. The numbers of zebrafish are Ν = 16 for WT/WT, Ν = 19 for WT/uy70, Ν = 17 for uy70/uy70, Ν = 30 for WT/WT, Ν = 34 for WT/uy71, Ν = 29 for uy71/uy71. *p < 0.05, **p < 0.01. (d and e) Heat map of the behavior of wild‐type and adap1 mutants. The numbers of zebrafish in each group are same in (b and c). A high number of color indicators indicates that zebrafish were frequently located at that position during the 20 min test period.