Two primary spinal neuron types, RB cells and CaPs, expressed scn8a. (A) Hu immunoreactivity revealed RB somata in a 48 hpf embryo. (B) In situ hybridization indicated that dorsal spinal cord neurons contained scn8a transcripts. (C) Dual examination of Hu immunoreactivity and scn8a mRNA identified RB cells as the dorsal neurons that express scn8a. (D) GFP+ neurons (arrowheads) in Tg(flh:GFP) embryos projected their axons (arrows) to ventral muscle, indicating that they were CaPs. In addition, GFP+ neurons were ISL+ (arrowheads, yellow nuclei), as were RB cells (asterisks). In two of the three hemisegments shown, VaPs were present with adjacent CaPs. VaPs are essentially duplicated CaPs that do not extend their axons as far ventrally, because of competition (Eisen, 1992; Eisen and Melancon, 2001). (E) GFP+ neurons (arrowheads) in Tg(flh:GFP) embryos did not express isl1 mRNA (carats), a marker of MiP. In the middle hemisegment, both CaP and VaP were present. (F) GFP+ CaPs (arrowheads) in Tg(flh:GFP) embryos expressed scn8a, as did dorsal RB cells (asterisks). (G) Double in situ hybridization for isl2, a marker of CaP, and for scn8a revealed that both CaPs (arrowheads) as well as RB cells (asterisks) expressed scn8a. (A-C) Dorsal views, (D-G) lateral views, anterior is towards the left and dorsal is upwards. Scale bars: in C, 30 μm for A-C; in G, 30 μm in D-F; 15 μm in G.
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