Whole mount in situ hybridization analyses of TH or gad1 expression on stage 24 embryos following injection of mRNA and/or morpholinos (Mo), as indicated, in one blastomere at the two-cell stage. (A, C) Arrows point to ectopic TH or gad1 expression (anterior on the left, dorsal side up). Sibling embryos were also hybridized with the tubb2b probe as a positive control (data not shown). (B, D) Quantification of embryos displaying ectopic TH or gad1 expression. Note that Phox2a and Hand2 knockdown significantly reduces Ascl1 ability to induce TH expression but does not impair its GABAergic inducing activity (B), which specifically depends on Ptf1a (D). Total number of analyzed embryos per condition is indicated in each bar. Error bars represent 95% confidence intervals. P<0,001 (***), p<0,01 (**) (binomial test). Scale bar represents 500 μm.