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Morpholino knockdown of Sox10 selectively inhibits microvillous neurogenesis. (A) and (A′) Control morpholino-injected live embryos at 29 hpf have ciliated (red) and microvillous (green) neurons. (B)–(C′) Embryos with mild and moderate phenotypes (assayed histologically) display only slight changes in ciliated neurons, whereas microvillous neurons are significantly decreased in number and organization. (D)–(F′) Similar results are seen at 33 hpf, with almost no Sox10:eGFP+ (green) cells becoming microvillous (blue) neurons. Ciliated (red) neurons remain relatively unaffected. (G) and (H) Antibody staining with anti-GFP against TRPC2:Venus in fixed embryos demonstrates a persistent decrease in the number of microvillous cells and disorganization at 60 hpf in Sox10 morpholino-treated embryos (H) in comparison to control embryos (G). All images were captured at identical settings to facilitate direct comparison of control and experimental embryos. (I)–(K) All embryos (I) were divided into ‘high pigmentation′ (J) or ‘low pigmentation′ (K) to roughly correlate Sox10 levels (higher and lower, respectively) with degree of olfactory phenotype. As expected, all control morpholino-treated embryos have high pigmentation, and all Sox10 morpholino-treated embryos with high pigmentation have only a mild phenotype. In all cases (J and K), Sox10 morpholino treatment results in most embryos having either no microvillous neurons or a decreased number. Ratios correlate with degrees of pigmentation and phenotype. (A–C′, G, and H) TRPC2:Venus: green; OMP:RFP: red. (D–F′) Sox10:eGFP: green; OMP:RFP: red; TRPC2:Venus: blue. Orientation arrows: D: dorsal; V: ventral; L: lateral. Scale bars: 50 μm. *p<0.001. See also Figure 5—figure supplement 1.
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