Analysis of midbrain glutamatergic markers. (A-B) Brn3a expression is increased in the ventral midbrain of Uncx4.1−/− embryos compared to controls. (C-D) Nkx6.1 expression is normal in E12.5 embryos, indicating a proper establishment of glutamatergic progenitors. (E-F) and (K-L) The numbers of Pax6-expressing cells are normal at E11.5 and E12.5 in Uncx4.1-deficient embryos compared to control. (G-J) and (M-N) Immunohistochemistry (IHC) reveals increased numbers of Pax6-expressing cells at E13.5 and E17.5 in the absence of Uncx4.1. (U) Quantification of Pax6-expressing cells in the ventral midbrain of Uncx4.1−/− and control embryos. Error bars represent the standard deviation. P values <0.05 were assessed as statistically significant. (M-N) Co-labeling with Foxa2 revealed that Pax6 cells are extended more ventrally at E13.5 (white arrows). The dashed line marks the border in the Foxa2 expression domain. (O-T) Pax6 and green fluorescent protein (GFP) staining of Ngn2−/− embryos shows no difference in the numbers of Pax6-expressing cells in the basal plate of the ventral midbrain at E11.5 (O-Q) and E13.5 (R-T). E, embryonic day; vMB: ventral midbrain.