Fig. 9

(A–F) Analysis of DA neurons in cnot8^m1061 mutant embryos combined with a mutation in the fgf3 locus (lia^t24152) or pharmacological suppression of FGF signaling by SU5402. Embryos were fixed at 4 dpf, stained by anti TH immunofluorescence and DA neurons documented by confocal microscopy. Shown are Z-projections of confocal stacks representing the ventral diencephalic DA groups 1 to 7 (dorsal views). Scale bar 50 µm. (A–D) DA neurons in WT, cnot8^m1061 or lia^t24152 single mutant, and cnot8^m1061, lia^t24152 double mutant embryos. Double mutant embryos show loss of cnot8^m1061-mediated increase of DC7 caudal hypothalamic DA neurons. (E, F) DA neurons in WT and cnot8^m1061 mutant embryos treated with SU5402 from 42 to 48 hpf. Inhibition of FGF signaling by SU5402 reduces the number of DC7 caudal hypothalamic DA neurons in cnot8^m1061 mutants below WT levels. (G) Quantification of effects on CA neurons by cell counting of forebrain DA neuronal clusters and locus coeruleus NA neurons in genetic and experimental conditions as indicated in the index at right. Each bar shows the average number of CA neurons in five independent embryos for each experimental condition. Error bars indicate standard deviation. Average DC7 cell numbers: cnot8+/+ lia +/+ 50.8 (WT control); cnot8-/- lia +/+ 103.2; cnot8-/- lia -/- 52.6; cnot8+/+ lia -/- (38.8); cnot8+/+ SU5402 20.6; cnot8-/-SU5402 56.2. Significance was evaluated by Mann-Whitney test. The cell count in single mutant cnot8^m1061 (-/-)lia^t24152 (+/+) is significantly different from single mutant cnot8^m1061 (+/+) lia^t24152 (-/-) embryos (p = 0.008). Comparison of cnot8^m1061, lia^t24152 double mutant and WT embryos reveal no significant difference (p = 0.45). For SU5402 treatments, the number of DC7 DA neurons differs significantly between WT controls and SU5402 treated WT (p = 0.008) and between cnot8^m1061 and SU5402-treated cnot8^m1061 embryos (p = 0.008). For all other catecholaminergic groups, no significant differences were observed when WT was compared to single mutants, double mutants, or SU5402-treated embryos.