Synteny analysis of c-fos genes. The synteny analysis was performed by searching genes flanking c-fos in genomes of human (Homo sapiens), mouse (Mus musculus), chicken (Gallus gallus), tropical clawed frog (Xenopus tropicalis), Vaquita (Phocoena sinus), West African lungfish (Protopterus annectens), Gray bichir (Polypterus senegalus), coelacanth (Latimeria chalumnae), zebrafish (Danio rerio), medaka (Oryzias latipes). The color of the font in the box was marked in red indicated that the change in gene location.

Sequence alignment of human (Homo sapiens, Hs), Vaquita (Phocoena sinus, Ps), mouse (Mus musculus, Mm), chicken (Gallus gallus, Gg), tropical clawed frog (Xenopus tropicalis, Xt), West African lungfish (Protopterus annectens, Pa), coelacanth (Latimeria chalumnae, Lc), Gray bichir (Polypterus senegalus, Ps), zebrafish (Danio rerio, Dr) and medaka (Oryzias latipes, Ol). Red with white letters represents fully conserved residues.

The Neighbor-joining tree based on the Fos proteins. The tree was built by the JTT model bootstrap method with 1,000 replications. The lines with the same color (red, blue or green) belong to one branch. The zebrafish fosaa and fosab were marked with a black dot.

Generation of fosaa^−/− and fosab^−/− zebrafish using CRISPR/Cas9. (A): Schematic representation of gene structure for fosaa and fosab. The open reading frame (ORF) of fosaa and fosab are respectively indicated by blue and green boxes. The black lines show untranslated introns. The write boxes show regions. The translation start site (ATG) is indicated by a broken line. Both fosaa gene and fosab gene have 4 exons and the target sites are located in exon 2 by black arrowhead. The black and red letters respectively represent the target region and protospacer adjacent motif (PAM). (B): Mutation pattern of fosaa and fosab knockout. The black and red boxes represent sgRNA target binding site and PAM respectively. The red letters and hyphens represent nucleotide insertion and deletion. (C): The fosaa and fosab deletions and insertions caused frame-shift early termination of translation, resulting in fosaa^−/− and fosab^−/− proteins with only 70 and 63 amino acids (aa) individually. (D): Left and right panels show PCR genotyping of fosaa and fosab knockout zebrafish. WT, ^+/− and ^−/− indicate wild-type, heterozygous and homozygous zebrafish, respectively. (E): Relative mRNA expression of fosaa and fosab in WT zebrafish, fosaa^−/− and fosab^−/− zebrafish. Data represent mean ± SEM (n = 6). Values marked with * is significantly different with the WT group (P < 0.05) and *** is significantly different with the WT group (P < 0.001).

Growth performance and brain weight were observed in fosaa^−/−, fosab^−/− and WT groups. (A): Body length and body weight of fosaa^−/−, fosab^−/− and WT groups. (B): Brain weight of fosaa^−/−, fosab^−/− and WT groups. The data in the figure were expressed as mean ± S.E.M. (n = 6), and values marked with * is significantly different with the WT group (P < 0.05) and *** is significantly different with the WT group (P < 0.001).

The average time required by zebrafish to complete trials in pre-training (2 days), discrimination learning training (16 days) and decision test (7 days). The data represented by mean ± SEM (n = 6). *, **, *** indicated significant differences (P < 0.05) (P < 0.01) (P < 0.001) compared with the WT group at the same session respectively.

The acetylcholinesterase (AChE) acetylcholine and transferase (AChT) activity in the brain of WT, fosaa^−/− and fosab^−/− zebrafish. (A): The acetylcholinesterase (AChE) acetylcholine in the brain of WT, fosaa^−/− and fosab^−/− zebrafish. (B): The transferase (AChT) activity in the brain of WT, fosaa^−/− and fosab^−/− zebrafish. The data in the figure were expressed as mean ± S.E.M. (n = 3).

Relative mRNA expression of t of bdnf and c-jun in the brain of fosaa^−/− and fosab^−/− zebrafish. Data represent mean ± SEM (n = 6). Values marked with * are significantly different with the WT group (P < 0.05).