Fig 2: Sema7a/Plxnc1 interaction regulates mDA axon guidance. a, b Schematic representations of coronal views of the axonal projections in the striatum showing that Plxnc1 mDA axons (in red) are more numerous in the dorso-posterior striatal region of Sema7a KO mice (b) compared to control animals (a). c, d Confocal images of TH and Plxnc1 immunostained striatal sections from control and Sema7a KO mice. e–g Quantification of TH relative optical density at different striatum levels (n = 3, two-tailed unpaired t test, p (Ant D) = 0.2855, p (Ant V) = 0.5355, p (Cent D) = 0.7461, p (Cent V) = 0.3933, p (Post D) = 0.0491, p (Post V) = 0.9514, p (Ant D/V) = 0.0023, p (Cent D/V) = 0.4418, p (Post D/V) = 0.0023). h Schematic representation of coronal section of mouse brain at the midbrain level. Dashed line indicates the boundaries of the pictures shown in i. i Representative confocal images of the VTA and SNpc for Control and Sema7a KO mice. j Stereological counts of mDA neurons in control and Sema7a KO mice at P1 (n = 3, two-tailed unpaired t test, p = 0.4843). k Schematic representation of the injection site of the AAV-retro-GFP in the dorsal striatum. l Representative confocal images of the injection site in the dorsal striatum. m–p Representative confocal images of the retrogradely labeled cells in control (m, n) and in Sema7a KO mice (o, p) 17 days after injection in the dorsal striatum at P30 (TH in red, AAV-Retro-GFP in green and DAPI in blue). n, p Higher magnifications in the VTA as indicated by the dashed boxes in m, o. q Quantification of the percentage of retrogradely labeled neurons in VTA (GFP+ TH+ in VTA on total GFP+ TH+; n = 4 for controls and n = 7 for Sema7a KO mice; two-tailed unpaired t test, p = 0.0245). r, s Schematic and confocal images of TH labeling in Plxnc1 overexpression mice showing a loss of DA innervation in dorso-posterior striatum. t RT-qPCR quantification of Plxnc1 from the ventral midbrain section of control and Plxnc1 overexpression mice at P1 (n = 3, one-tailed unpaired t test, p = 0.0322). u Stereological counts of mDA neurons in control and Plxnc1 overexpression mice at P1 (n = 3, two-tailed unpaired t test, p = 0.5431). v, w Schematic and confocal images of TH and td-Tomato in the VTA and SNpc for one hemisphere. The reporter tdTomato indicates that all mDA neurons express the transgene (Plxnc1-ires-tdTomato). x Optical density measurements of TH-positive axons in the striatum at three antero–posterior levels (n = 4, two-tailed unpaired t test, n = 4, Mann–Whitney U, p (Ant D/V) = 0.6857, two-tailed unpaired t test, p (Cent D/V) = 0.1945, p (Post D/V) = 0.0019). Scale bars: 200 µm except n, p, 100 µm

Fig 3: Otx2 and Lmx1a/b control Plxnc1 expression in the VTA. a, b Confocal images of Otx2 immunostaining in TH-positive cells in coronal midbrain sections of control and Lmx1a/b cKO mutant mice showing ectopic Otx2 expression in SNpc mDA neurons. c Quantification of the number of mDA neurons in SNpc with ectopic expression of Otx2 (n = 3, two-tailed unpaired t test, p = 0.0015). d Rt-qPCR quantification of Otx2 in VTA and SNpc isolated from control and Lmx1a/b cKO with LCM (VTA: n = 3 for control and n = 5 for Lmx1a/b cKO, two-tailed unpaired t test, p = 0.9128; in SNpc n = 3, one-tailed Mann–Whitney U, p = 0.05). e Overexpression of Otx2 in primary cell cultures leads to an increase of Plxnc1 expression as quantified by RT-qPCR (n = 3 independent cultures; for Plxnc1: two-tailed unpaired t test, p = 0.03; for Otx2: Mann–Whitney U, p = 0.0286). f RT-qPCR quantification of Otx2 in mDA primary cell cultures following Lmx1a and Lmx1b overexpression (n = 8 for control, n = 7 for Lmx1a and n = 5 for Lmx1b; one-way ANOVA with Tukey’s multiple comparison test, not significant). g Representative images of TH and Otx2 immunolabeling from E15.5 Lmx1a/b cKO embryo at E15.5 showing SNpc neurons co-expressing Otx2 and TH (arrows) before and after LCM. Right panels are high magnification of the left panels. h RT-qPCR quantification of Plxnc1 expression from LCM isolated cells co-expressing Otx2 and TH in VTA and SNpc from control and Lmx1a/b cKO embryos. For control SNpc, TH-positive cells were isolated (n = 3, 5–8 cells per embryo, one-way ANOVA with Tukey’s multiple comparison test, not significant between groups expressing Plxnc1). Image on the right is a representative image of the gel showing the RT-qPCR product. Plxnc1 was not detected in mDA neurons of SNpc from control. Scale bars: a, 200 µm; b, 10 µm; g (left), 100 µm, (right), 10 µm