Figure 2
The effects of single and combined MIA and VITD on early mesencephalic dopamine (mesDA) cell number. (A–D) Fate mapping of developing mesDA cells in a medial coronal mesencephalic (MES) sections at gestational day (GD) 11. Markers are the nuclear dye DAPI (blue, A), mesDA specification factor Lmx1a (green, B), neuronal progenitor maker Sox2 (red, C), and a channel-merged image (D). (A’–D’) Higher magnification images of the dashed outlined boxes representing the floor plate (FP) from (A–D) accordingly. Lmx1a specifically marked mesDA cells in the FP dorsoventrally from ventricular zone (VZ), intermediate zone (IZ) to mantle zone (MZ). MesDA progenitors (Lmx1a+Sox2+) were primarily located in the proliferative VZ, in contrast, post-mitotic (Lmx1a+Sox2−) mesDAs were mostly located in IZ and MZ. (E) The cell number of these two mesDA subpopulations were counted using CellProfiler software at 60 µm intervals along the anterior-posterior (A–P) axis. The numbers of mesDA progenitors (Lmx1a+Sox2+) were reduced by all treatments relative to the control (CON-VEH) (p’s < 0.05). (F) Post-mitotic (Lmx1a+Sox2−) mesDA number was not altered by any treatment. (G–J) Fate mapping of post-mitotic mesDA subgroups that are characterized by Nurr1 and TH in medial coronal MES sections at GD11. Markers are the nuclear dye DAPI (blue, G), Nurr1 (magenta, H), TH (green, I), and a channel-merged image (J). (G’–J’) Higher magnification images of the dashed outlined boxes representing FP from (G–J) accordingly. Immature Nurr1+ mesDAs were primarily located in the IZ and MZ. Mature (Nurr1+ TH+) mesDAs were primarily located in MZ. (K) There were no significant differences of immature mesDA number among treatment groups (p’s > 0.05). (L) VITD treatment itself increased mature (Nurr1+ TH+) mesDA number compared to its vehicle (VEH) (p < 0.05). VITD treatment itself (CON-VITD) particularly increased mature mesDA number in the posterior MES compared to control (CON-VEH) (p’s < 0.05). All values were means ± SEM. *P < 0.05. n.s., represents not statistically significant. Scale bars: 100 µm (A–D and G–J); 50 µm (A’–D’ and G’–J’).
