Islet1 knock-down reduces Rohon-Beard cell number and expression of scn8aa in the dorsal spinal cord. (A-C) In 24 hours post-fertilization (hpf) Tg(scn8aa:gfp)ym1 embryos, Rohon-Beard (RB) and RB-like cells express green fluorescent protein (GFP) (dorsal views) . Islet1 knock-down reduces the number of GFP+ neurons (C versus A and B). (D) In E3 morphants, the number of GFP+ RB-like cells is 44% that of GFP+ RB cells in controls (*P < 0.001) (see Methods). (E-G) In uninjected (Uninj) and control 24 hpf double Tg(scn8aa:gfp,-3.4neurog1:dsRed) embryos, most RB neurons express both reporters (E, F) and few express only dsRed (arrowheads) (lateral views). In E3 morphants, many RB-like cells express only dsRed (G, arrowheads). (H) Compared to RBs, more RB-like cells express only dsRed (#P < 0.01). (I-K) Following Islet1 knock-down (K), less scn8aa mRNA (asterisks) is detected compared to controls (I) (lateral views). (J)The sense control scn8aa probe reveals little signal, indicating specificity of the antisense probe (I and K). (L-N) In 24 hpf double transgenic Tg(ssx-mini-ICP:egfp,-3.4neurog1:dsRed) embryos, most RB cells (asterisks) express both reporter proteins (N). Fewer ventral interneurons express GFP (L) versus dsRed (M) (arrowheads). (O) Quantitative reverse transcription PCR analysis (see Methods) shows a five-fold reduction in scn8aa expression in RB-like cells of E3 morphants (*P < 0.04). (P, Q) RBs express vglut2.1/2.2 mRNA (red) (lateral views). In uninjected and CtlMO injected embryos, vglut2.1/2.2 mRNA (red) colocalizes (white arrows) with GFP+ RBs (green). (R) In E3 morphants, vglut2.1/2.2 mRNA (red) co-localizes with GFP+ RB-like neurons (green; yellow arrow), indicating no obvious effect of Islet1 knock-down. (S, T) GABAergic neurons express gad65/67 mRNA (red). (U) Islet1 knock-down has no obvious effect on gad65/67 in the dorsal domain. Scale bars = 50 µm in A (for A to C), E (for E to G), I (for I to K), L (for L to N), P (for P to R) and S for (S to U).