Fig. 8.

HCN2 channels restore nicotine exposure induced mispatterning of brain markers during neural development. Stage 25 embryos as illustrated with the angle of view marked by the black arrow. Control (untreated/uninjected) embryos (a, e, i, l), embryos exposed to nicotine (0.1 mg/mL – stage 10 onwards) (b, f, j, n), and nicotine-exposed embryos microinjected with Hcn2-WT mRNA (0.75 ng/injection) in both blastomeres at 2-cell stage (c, g, k, o). In situ hybridization for Otx2 (a–d), Xbf1 (e–h), Emx (i–l), and Pax6 (m–p) show that nicotine exposure leads to significantly mispatterned expression (magenta arrows) of Otx2 [46% n = 24] and Xbf1 [35% n = 23], but has little effect on Emx [17%, n = 29] and Pax6 [12%, n = 33] in comparison to controls [10%, n = 28, 4.5%, n = 22, 0%, n = 28, and 3%, n = 29, respectively]. Nicotine-exposed embryos that were also microinjected with Hcn2-WT mRNA showed largely normal expression of Otx2 [4% n = 23], Xbf1 [4% n = 26], Emx [9% n = 23], and Pax6 [6% n = 34] in comparison to controls