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phox2b, but not ascl1, is indispensable for sympathetic neuronal differentiation. (A–F) Whole-mount ISH of 3-dpf embryos for th and dbh following ascl1 MO knockdown. Lateral views depict a minimal decrease in expression of th (A,B) and dbh (D,E) in ascl1 MO-injected embryos compared to control MO-injected (A,D) embryos. Simultaneous knockdown of both ascl1 and phox2b led to a significant decrease in expression of th and dbh in the SCG (C,F). (G) Relative intensity levels of dbh-staining in the SCG of embryos expressing ascl1 MO, phox2b MO or the combination of the two. Data are presented as means ± SEM (***P<0.001; n = 15 per group). (H–M) Whole-mount ISH for expression of ascl (H–J) and phox2b (K–M) in embryos in which ascl1 expression was abrogated by MO knockdown, either singly (I, L) or in combination with phox2b (J, M). (N,O) Whole-mount ISH for phox2a expression in 4-dpf embryos in which ascl1 expression was abrogated by MO knockdown. Arrows point to region of SCG.
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