Figure 6.

HCN2 donor tissue can rescue brain patterning in nicotine-exposed recipient embryos over long distances. (A) Xenopus animal cap transplant experimental setup. The excised animal caps were transplanted into age-matched sibling embryos. (B–G) Representative images of stage 45 tadpoles from transplant receiving host for indicated treatment conditions. Blue arrowheads indicate intact nostrils, orange brackets indicate intact forebrain (FB), yellow brackets indicate intact midbrain (MB), cyan brackets indicate intact hindbrain (HB), and magenta arrowheads indicate severe brain morphology defects. The red region in (E) and (G) is the tomato tracer indicating the transplanted graft. (H) Quantification of stage 45 tadpole brain morphology defects under indicated conditions. Percentage of tadpoles with brain defects for each experimental group are Controls—5%, Nicotine—58%, Nicotine+HCN2-WT-tomato—22%, Nicotine+tomato—59%, and Nicotine+DN-HCN2-tomato—65%. Data are mean ± SD, ***p < 0.001, n.s.: non-significant (one-way ANOVA with Tukey’s post hoc test for n = 3 experiments with N > 20 host embryos per treatment group per experiment). (I,J) Quantification of brain morphology defects in stage 45 tadpoles exposed to nicotine and receiving Hcn2-WT+tomato mRNA expressing tissue transplant. The tadpoles were either sorted by the size of the transplant (I) or location of transplant (J) based on the tomato signal (red) at stage 45. Percentage of tadpoles with brain defects for each experimental group are: Large—27%, Small—83%, Fin—81%, and Near neural—0%. Data pooled from n = 3 independent experiments are represented as mean with χ² squared test for differences in proportions, ****p < 0.0001, **p < 0.01.