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Zebrafish naa80 F0 KO larvae showed hearing loss-related phenotypes. (A) Schematic representation depicts sensory tissues in zebrafish larva at 5–6 dpf including inner ear and neuromasts. Five sensory patches of inner ear including anterior crista (Ac), lateral crista (Lc), posterior crista (Pc), anterior macula (am), and posterior macula (pm). Schematic representation of lateral crista at lower right corner. Kinocilia and stereocilia of hair cell can be revealed by immunohistochemistry using anti-acetylated tubulin Ac-tub and phalloidin (F-actin), respectively. (B, C)naa80 F0 KO larvae have no obvious morphological abnormalities but showed smaller otoliths compared with Cas9-injected control larvae. (D) RT-qPCR showed a down-regulated naa80 expression in F0 KO (n = 3). Expression levels were normalized to 18S housekeeping gene and compared with the Cas9-injected controls. (E, F, E′, F′, E″, F″) Immunohistochemistry of both anti-acetylated tubulin (magenta, (E, F)) and phalloidin (cyan, E′, F′), and merged (E″, F″). (G) Quantification of hair cell numbers in the lateral crista of control (n = 3 larvae) and KO (n = 4 larvae) larvae. (H) Stereocilia length measurement in lateral crista hair cells of control and KO larvae. n = 31 stereocilia from three larvae for each group. (I) Evaluation of acoustic startle response. n = 48 larvae of each group. (J, K) Representative fluorescent images of Cas9-injected control (n = 12 larvae) and naa80 F0 KO (n = 13 larvae) larvae after Yo-Pro-1 uptake. Left panel (J, K) was whole-mount images after invert color into black and white, anterior to the left and dorsal to the top. Right panel (J′ and K′) was enlarged single neuromast. (L) Quantification of Yo-Pro-1 positive cells per neuromast. Two-tailed unpaired t test with Welch’s correction. **P < 0.01 and ****P < 0.0001.
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