scn8ab Regulates neuronal activity in the brain. (A) Representative section in situ hybridization of scn8ab transcripts in brain and 2 dpa fins of wild-type fish. Black arrows indicate scn8ab expression. Black arrowhead marks amputation plane. (B) RT-PCR showing scn8ab expression in the zebrafish brain but not in uninjured or regenerating (2 dpa) fins. huC/elavl3 Is used as a brain-specific gene; and4 is a representative gene for regenerating fins; and actb2 is used as a loading control. (C) RNA-seq genome-browser tracks of fin-specific genes (and4 and cldn1), brain-specific genes (elavl3 and elavl4), and scn8ab. Brain and uninjured and 36 h postamputation (hpa) fin profiles of controls were used. (D and E) Confocal, whole-mount, immuno-stained image against pan-Nav antibody of uninjured and 2 dpa wild-type fins. Dashed white lines mark amputation plane. (F) Representative images of brain phosphorylated ERK (p-ERK; red) and HuC/D (green) immunostaining in control (heterozygous siblings) and temca placed at 33 °C for 2 h prior to dissection. White arrows indicate p-ERK+/HuC/D+ double-stained neurons. (G) Quantification of p-ERK+ neurons in 300 × 300 µm2 dorsal telencephalon region (n = 6). Data are presented as mean ± SD. Student’s unpaired two-tailed t test. (H) RNA-seq volcano plot displaying significantly reduced genes in temca placed at 33 °C for 1 d before brain collection. (I) Top Gene Ontology terms for genes up-regulated and down-regulated in temca brains. (D–F) Scale bars, 50 µm. ac-Tub, acetylated α-tubulin; Adjust, adjusted.
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