Fig. 2. Loss of DA neurons in Ngn2 null mutant mice. An almost complete ablation of DA neurons was detected at E11.5 as assessed by Th (A-C) and Pitx3 (D,E) immunostaining. The expression of the pan-neuronal marker, ßIII tubulin, together with Toto nuclear labeling, revealed a complete loss of neuronal cell bodies in the midline region of Ngn2 mutant mice at E11.5 (F). Arrows point to Toto-labeled nuclei and asterisks denote the absence of cell bodies, with only ßIII tubulin⁺ processes present. At E14.5, Toto/Tuj1 and MAP2/Hoechst expression revealed a reduction of the size of the marginal zone in mutant embryos, indicating a severely reduced number of neurons (G,H). A severe reduction in number of Th⁺ cells (I-K) and in Pitx3 staining (L,M) was also detected. Note that Th⁺ cells in mutant embryos are localized lateral to the midline. ^***P<0.001 and ^**P<0.01, as assessed by Student's t-test with Welch's correction (n=4-5 in A,H). Scale bar: 100 µm in B-E,G,H,J-M; 10 µm in F.