Figure 7.

Midbrain specific deletion of miR-218. a, Schematic representation of miR-218-2^fl/fl allele generated through homologous recombination (for details, see Extended Data Fig. 7-1; Materials and Methods). b, Survival rate of conditional double KO mice (c-dKO2). c, TaqMan assay for the expression of miR-218 in the ventral midbrain of P39 WT, En1CRE, and miR-218 KO models. Data are normalized to the average of the reference sno-202 and represented as 2^−ΔCt values from 4 distinct animals. Median, interquartile range, and minimum and maximum are represented. *p < 0.05 with respect to Ctrl samples (Kruskal–Wallis test plus Dunn's multiple comparison correction). d, e, qPCR for midbrain expression of Slit2 (b) and Slit3 (c) in the ventral midbrain of P39 WT, En1^CRE, and miR-218 KO models. Data are normalized to the average of the reference Hprt and represent the mean ± SEM and all values of the 2^−ΔCt values from at least 4 animals. f–h, The expression of the dopaminergic markers TH, VMAT2, and DAT in the ventral midbrain of P39 WT and KO mice was evaluated by qPCR. Data are normalized to the average of the reference Hprt and represent the median ± 2nd and 3rd quartile of 2^−ΔCt values from at least 3 different animals from three independent litters. i, Representative images of TH⁺ neurons in the ventral midbrain from control (En1^CRE) and En1^CRE c-KO2 adult brain. Scale bar, 250 µm. j–l, Quantification for the number of TH⁺ cells (j), TH fluorescent density (k), and pixel intensity (l) does not show significant differences. m, TH protein expression in the ventral midbrain of P39 WT, En1^CRE, and miR-218 KO models analyzed by Western blot. Data are normalized to the average of β-ACTIN and represent the mean ± SEM. n = 3. WT: miR-218-1^+/±; miR-218-2^+/±. KO1: miR-218-1^–/–; miR-218-2^+/±. KO2: miR-218-1^+/±; miR-218-2^–/–. dKO: miR-218-1^–/–; miR-218-2^–/–.