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Fig. 2 Differential gene expression established by RNA-sequencing analysis. (A) Principal component analysis of gene expression data (transcripts per kilobase million) from all six replicates of the two genotypes. (B) Volcano plot of transcriptional differences between (stim2a;stim2b)−/− and (stim2a;stim2b)+/+ 5 dpf larvae presenting genes with the highest fold change with an increase in annexin A3a expression (encircled in black). Logarithms (base 2) of fold changes in individual genes (x-axis) are plotted against negative logarithms (base 10) of their adjusted p-values (y-axis). Positive log2 (fold change) values represent upregulation in (stim2a;stim2b)−/− mutant larvae compared with (stim2a;stim2b)+/+ larvae, and negative log2 (fold change) values represent downregulation. Points above the dotted line parallel to the x-axis represent differentially expressed genes in (stim2a;stim2b)−/− mutants with p < 0.05 after correction for multiple testing. (C) Selected terms from functional enrichment analysis, which were significantly enriched in significantly downregulated genes (adjusted p < 0.05), are shown. The color of the bar represents adjusted p-values (red to blue, with dark red being close to 0 and dark blue being close to 0.05). The length of the bar represents the ratio of the number of genes in the term, which were significantly downregulated (intersection size), and total number of genes in the term (term size). The term “sensory perception of light stimulus” is marked within a black rectangle. (D) Volcano plot from (B) with focus on the data-rich section. Downregulated genes that are related to the sensory perception of light stimuli in (stim2a;stim2b)−/− larvae are marked on the volcano plot (in black). Genes associated with inflammasome activation are marked in purple. (E, F) Increase in the number of Neutral Red-positive cells, likely microglia, in (stim2a;stim2b)−/− fish, imaged in vivo in the brain. Arrows (E) indicate the Neutral Red-positive cells. Analysis was carried out on 15 larvae of (stim2a;stim2b)+/+ and 13 of (stim2a;stim2b)−/−. The experiments were carried out in triplicate. ***p < 0.001.