Cyp19a1b mutant zebrafish characterization. (A) The position of the 10 deleted nucleotides is shown on the top (red sequence), and the corresponding truncated protein is indicated below the gene sequence. The restriction enzyme cut site used for genotyping is written in green (TauI). Primer sequences used for genotyping are highlighted in green (forward) and blue (reverse). (B) Sequencing of the cyp19a1b gene in WT and mutant zebrafish showing the nucleotide deletion in exon 2. (C) Periventricular hypothalamus sections of AroB immunoreactivity in WT and mutant fish. Scale bars: 50 μm.

Aromatase activity expressed in pmole per hour in the brain and the gonad of WT and mutant adult zebrafish, in males and females. n = 7 in each group. Mean ± SEM; **p < 0.001 versus WT group.

Non‐reproductive behavior in WT and mutant adult fish: Distance traveled in the shoaling test, the y‐maze, and the novel tank diving test (A). Boldness measured by the latency to exit the shelter area in the z‐maze (B). Aggressiveness test with the number of visits and the time spent in the mirror area (C). Sociability is measured by the number of visits and the time spent in the social area (D). Data are shown as mean ± SEM. *p < 0.05; **p < 0.01; ***p < 0.001; #p < 0.1 versus the WT group.

Quantification of the number of PCNA‐labeled cells in WT and mutant fish. (A) Olfactory bulbs (OB). (B) Medial zone of dorsal telencephalic area (Dm). (C) Lateral zone of dorsal telencephalic area (Di). (D) Ventral nucleus of ventral telencephalic area (Vv). (E) Parvocellular preoptic nucleus (PPa). (F) Caudal zone of periventricular hypothalamus (Hc). Red dots on schematic frontal sections indicate quantified areas. Mean ± SEM, *p < 0.05; **p < 0.001; ***p < 0.0001 versus WT group.

Quantification of TH‐positive cells in WT and mutant fish. (A) Olfactory bulbs (OB). (B) Ventral nucleus of ventral telencephalic area (Vv). (C) Parvocellular preoptic nucleus (PPa). (D) Thalamus. (E) Posterior tuberal nucleus (PTN). (F) Caudal zone of periventricular hypothalamus (Hc). Red plots represent regions of interest in which TH‐positive cells have been quantified. Mean ± SEM; the number of analyzed brains is indicated in each bar.

Expression of 5‐HT in positive cells in WT and mutant fish. (A) Paraventricular organ (PVO). (B) Posterior tuberal nucleus (PTN). (C) Caudal zone of periventricular hypothalamus (Hc). Red dots on frontal brain section drawings indicate areas of interest where expression is observed. Mean ± SEM.

Workflow for BRB‐Seq data processing and heatmap of differentially expressed genes (DEGs) in the three different parts of the brain (olfactory bulb, telencephalon, and hypothalamus) on male and female WT and mutant adult fish. (A) Filtration steps based on limit of detection, fold change cut‐off of 1.5, and statistics leading to 703 DEGs. Hypo, hypothalamus; OB, olfactory bulb; Tel, telencephalon. (B) Heatmap of all DEGs in each WT and mutant fish in the three areas of the brain. Genes are clustered in 13 distinct patterns: P1. 36 DEGs, P2. 31 DEGs, P3. 27 DEGs, P4. 213 DEGs, P5. 74 DEGs, P6. 43 DEGs, P7. 15 DEGs, P8. 66 DEGs, P9. 24 DEGs, P10. 23 DEGs, P11. 19 DEGs, P12. 120 DEGs, P13. 12 DEGs. On the right‐hand side of the heatmap, each dysregulated gene is represented as a tile, with a color intensity reflecting the average log2 fold change of samples for each comparison and brain region. Scale: Log2 fold change. Red = significantly upregulated expression, Blue = significantly downregulated expression, White = no significant change. Male: N = 6 WT and 5 mutants; Female: N = 6 WT and 4 mutants.

Functional interaction and annotation of genes from patterns (Figure 7B) P1, P2, P4, and P5 (indicated by the node border color) are significantly differentially expressed in mutants versus WT in the three brain areas, in males and/or in females (indicated by the node shape).

Cytoscape network visualization: Functional interaction (STRING functional score: 0.50) and annotation of genes from pattern of expression (described in Figure 7B and indicated by the node border color) P7, P8, P9, P12, and P13 (indicated by the node border color), significantly differentially expressed in female mutant versus WT, in the olfactory bulbs (OB).