PAX5 deficiency causes hypoplasia of the SN and VTA with loss of GABAergic neurons. (A) Left: Coronal MRI brain scans of the patient and a representative control. The SNr (blue), SNc (yellow), and VTA (red) are indicated. Right: Volumetric quantification of the three midbrain regions. (B) Left: Coronal MRI brain scans of adult Pax5^+/+ and Pax5^R31Q/− mice. Right: Quantification of the three midbrain regions. (C) Top: Anti-Pax5 staining (white) of Pax5^+/+, Pax5^+/−, and Pax5^R31Q/− adult mouse brain sections. Dotted lines delineate the SNr, SNc, VTA, and interpeduncular nucleus (IPN). Bottom: Quantification of Pax5-expressing cells. (D and E) smRNA-FISH staining. (D) SN- and VTA-containing section of an adult wild-type mouse with Pax5 (white), Rbfox3 (red), Gad1/Gad2 (green), and DAPI (blue) staining. Data are representative of three experiments. Dotted lines delineate cell boundaries demarcated by Gad1/Gad2 mRNA staining. (E) Top: Gad1/Gad2 mRNA (white) of Pax5^+/+, Pax5^+/−, and Pax5^R31Q/− adult mouse brains. Dotted lines delineate SNr, SNc, VTA, and IPN. Bottom: Quantification of Gad1/Gad2-expressing cells (n ≥ 3 mice per indicated genotype). (F) Immunofluorescent staining of coronal sections of an adult wild-type midbrain with antibodies detecting tyrosine hydroxylase (TH; green) and Pax5 (red) in combination with DAPI (blue). The data are representative of three experiments with different wild-type mice. Scale bars: 500 μm (C and E); 10 μm (D); 20 μm (F). RU, relative units. Data (A–C and E) are shown as mean values with SEM (n = 5 for controls in A, n ≥ 4 per genotype for B, and n ≥ 3 per genotype for C and E). Two-tailed one-sample t test with false discovery rate (FDR) correction (A), unpaired t test with FDR correction (B), ANOVA with Dunnett’s multiple comparisons test (C and E); *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001. For detailed statistical information (A–C and E), see Table S6. Each dot represents one individual (A) or a single mouse (B, C, and E).