Figure 6

Cells labeled with Shh-GIFM at different time points have a changing potential to contribute to subpopulations of DA neurons. (A-H) Immunofluorescent staining for β-gal-positive fate-mapped cells (Shh-GIFM at E8.5 or E11.5) and DA neurons (TH) on coronal sections of the ventral midbrain (P21 to P30). The areas shown in (A-D) are indicated in (E-H). Arrows indicate double-labeled cells; arrowheads indicate β-gal-positive cells with astrocytic morphology. (E'-H') Representative schematics of the immunostained sections showing the distribution of TH-positive fate-mapped cells (red dots) and of fate-mapped cells with astrocytic morphology (yellow crosses). Rostral, Bregma -2.92; caudal, Bregma -3.40 [20]. If, interfascicular nucleus; Pn, paranigral nucleus; Snc, substantia nigra pars compacta; Snl, substantia nigra lateralis; Vta, VTA. Fate-mapped cells outside these areas are not represented. Cells with astrocytic morphology are not present with TM8.5. Scale bars: (A-D) 40 μm; (E-H) 200 μm. (I) Relative contribution of cells marked with Shh-GIFM between E8.5 and E11.5 to the SN (Snl + Snc), dorsal-lateral VTA (Vta) and ventral-medial VTA (Pn + If); see schematic in (L). For each animal (n ≥ 3), TH-positive fate-mapped cells were counted in the three indicated areas and normalized for the combined number of overlapping cells counted in these areas (in percent). Error bars indicate standard deviation. Significance (*P < 0.05; **P < 0.01; ***P < 0.001) was determined by ANOVA and LSD post-hoc analysis. (J) Distribution of Calbindin- and Girk2-positive cells. (K) Relative contribution of fate-mapped cells to Calbindin (VTA) versus Girk2 (SN) positive cells. Calbindin- or Girk2-positive fate-mapped cells were counted in three different rostral-caudal midbrain areas (n ≥ 3). The ratio of Calbindin-positive fate-mapped cells to Girk2-positive fate-mapped cells was determined. Significance (***P < 0.001) was determined by Student's t-test. (L) Schematic showing the SN, dlVTA and vmVTA. (M) Fate mapping strategy.