Analysis of early mDA markers in Uncx4.1 -deficient embryos showed that Ngn2-expression is reduced in mutant embryos. A detailed analysis of Nurr1 expression in early and late embryonic stages of Uncx4.1−/−embryos revealed that the loss of Nurr1 is consistent during midbrain dopaminergic neurons (mDA) development. (A-B) In situ hybridization (ISH) using Lmx1a reveals no difference in the expression of Lmx1a in Uncx4.1−/− embryos at E11.0. (C-F) Reduction of Ngn2 in the ventral (C-D) and dorsal (E-F) midbrain of Uncx4.1−/− embryos compared to control using ISH. (G-H) At E11.5 Mash1 expression is normal in Uncx4.1-deficient embryos compared to control. (I-L) Showing Nurr1 expression in control and Uncx4.1−/− embryos by immunohistochemistry (IHC). (O) Nurr1 is significantly reduced at E11.5 and E16.5 compared to wild-type (WT) animals. Error bars represent the standard deviation. P values <0.05 were assessed as statistically significant. At least three animals per stage and genotype were analyzed. (M, N) ISH using Lmx1b reveals no difference in Lmx1b expression in Uncx4.1−/− embryos. E, embryonic day; vMB, ventral midbrain.