Comparison of her6 and her9 expression between wild-type sibling and mib1 ta52 b mutant zebrafish.In situ hybridization was conducted to assess the spatial expression of her6 and her9 mRNA in wild-type and mib1 ta52 b homozygous embryos. (A) her6 expression at the bud stage, (B) her6 expression at 24 h post-fertilization (hpf), (C) her9 expression at the bud stage, and (D) her9 expression at 24 hpf. Embryos are depicted in dorsal views with anterior to the top. To facilitate imaging, the yolk of 24 hpf embryos was removed and flat-mounted. Key anatomical features include the anp, anterior neural plate; e, eye; fb, forebrain; fmb, fore-midbrain boundary; hb, hindbrain; in, interneurons; ipd, inter-proneuronal domain; mb, midbrain; mhb, mid-hindbrain boundary; ml, midline; mn, motoneurons; op. otic placode; rb, rhombomeres; th, thalamus; sn, sensory neurons. The percentages in each panel indicate the proportion of embryos displaying the same phenotype as that shown in the photographs of the total embryos examined. The column charts at the bottom of each in situ hybridization photo represent the quantification of mRNA expression using qRT-PCR. Quantitative data are presented as mean ± standard error of mean (SEM); statistical analysis was performed by Student?s t-test. All reactions were performed in triplicate for each sample. *, p < 0.05; **, p < 0.01.

Expression of her3 in wild-type and mib1 ta52 b mutant zebrafish embryos. In situ hybridization demonstrates the expression of her3 in wild-type siblings (left panels) and mib1 ta52 b mutant embryos (right panels) at the bud stage (A) and 24 h post-fertilization (hpf) (B). Images depict dorsal views with anterior to the top. fmb, fore-midbrain boundary; hb, hindbrain; ipd, inter-proneuronal domain; mb, midbrain; mhb, mid-hindbrain boundary; scn, spinal cord neurons. The expression levels of her3 were validated using quantitative real-time polymerase chain reaction (qRT-PCR), displayed at the bottom panels. Quantitative data are presented as mean ± standard error of mean (SEM). Statistical analysis was performed by Student?s t-test. All reactions were performed in triplicate for each sample. **, p < 0.01.

Comparison of her4.1, her12, and her15.1 expression in wild-type and mib1 ta52 b mutant embryos. The expression of her4.1 (A,B), her12 (C,D), and her15.1 (E,F) was detected in wild-type sibling embryos (left panels) and mib1 ta52 b homozygous mutant zebrafish embryos (right panels) at the bud stage (A,C,E) and 24 hpf (B,D,F) using in situ hybridization. Dorsal views with anterior toward the top. Key anatomical features include the anp, anterior neural plate; e, eye; fb, forebrain; fmb, fore-midbrain boundary; hb, hindbrain; in, interneurons; mn, motor neurons; psm, presomitic mesoderm; r, retina; sn, sensory neurons; sc, spinal cord. The expression levels were further validated using quantitative real-time polymerase chain reaction (qRT-PCR). Quantitative data are presented as mean ± standard error of mean (SEM); statistical analysis was performed by Student?s t-test. All reactions were performed in triplicate for each sample. **, p < 0.01, ****, p < 0.0001.

Comparison of her8 a, her8.2, her13, and hes6 mRNA expression in wild-type and mib1 ta52 b mutant embryos by in situ hybridization. The expression of her8 a (A,B), her8.2 (C,D), her13 (E,F), and hes6 (G,H) were detected in wild-type siblings (left panels) and mib1 ta52 b homozygous mutant embryos (right panels) at the bud stage (A,C,E,G) and 24 hpf (B,D,F,H). Dorsal views with anterior toward the top. ad, anterior diencephalon; fb, forebrain; hb, hindbrain; in, interneurons; mb, midbrain; mhb, mid-hindbrain boundary; mn, motor neurons; psm, presomitic mesoderm, r, retina; rb, rhombomeres; rob, rhombomere boundaries, tc, telencephalon; sn, sensory neurons; sc, spinal cord. The expression levels were validated using quantitative real-time polymerase chain reaction (qRT-PCR). Quantitative data are presented as mean ± standard error of mean (SEM). Statistical analysis was performed by Student?s t-test. All reactions were performed in triplicate for each sample. **, p < 0.01; ***, p < 0.001; ****, p < 0.0001.

Expression of her5 and her11 in wild-type and mib1 ta52 b mutant embryos at the bud and 24 hpf stages. The expression of her5 (A,B) and her11 (C,D) was detected in wild-type siblings (left panels) and mib1 ta52 b homozygous mutants (right panels) at the bud (A,C) and 24 hpf (B,D) stages. Dorsal views with anterior toward the top. hb, hindbrain; mb, midbrain; mhb, mid-hindbrain boundary. qRT-PCR results show the quantification of expression levels. Quantitative data are presented as mean ± standard error of mean (SEM). Statistical analysis was performed by Student?s t-test. All reactions were performed in triplicate for each sample. *, p < 0.05; ***, p < 0.001.