Fig. 3

Overexpression of Zeb2 reduces NR4A2 and PITX3 levels and impairs neurogenesis in the embryonic midbrain floor plate. a, c Mouse embryos were in utero electroporated with either pCAG-Egfp (a) control vector or pCAG-Zeb2-Egfp (c) to overexpress mouse Zeb2 in the midbrain floor plate at E11.5. Boxed areas are magnified and each channel (NR4A2, magenta; CXCR4, turquoise; GFP, green) is shown. Dopaminergic linage was targeted by electroporation, as shown in (a, c), where full arrowheads indicate NR4A2⁺/GFP⁺ and open arrowheads indicate NR4A2⁻/GFP⁺ cells. b Quantification of NR4A2 immunoreactivity (IR) in GFP⁺ cells in pCAG-Egfp (n = 4; median 1.02, range 0.975–1.05) or pCAG-Zeb2-Egfp (n = 4; median 0.723, range 0.617–0.801) electroporated midbrain. d Quantification of the percentage of CXCR4⁺/GFP⁺ cells in total GFP⁺ cells in ventral midbrain dopaminergic domain in pCAG-Egfp (n = 4; median 41.9%, range 39.7–45.2%) or pCAG-Zeb2-Egfp (n = 4; median 7.71%, range 7.05–8.16%) electroporated midbrain. e, f Immunofluorescence images show the immunoreactivity of PITX3 (magenta) in GFP⁺ cells electroporated with pCAG-Egfp (e) or pCAG-Zeb2-Gfp (f). Boxed areas in e and f are magnified. White arrowheads indicate PITX3⁺/GFP⁺, whereas open arrowheads indicate PITX⁻/GFP⁺ cells. g Quantification of PITX3 IR in pCAG-Egfp (n = 4; median 1.04, range 1.02–1.06) or pCAG-Zeb2-Egfp (n = 4; median 0.0485, range 0.0453–0.0544) electroporated midbrain. h, j Immunofluorescence images show TH⁺/GFP⁺ cells in pCAG-Egfp (n = 4) or pCAG-Zeb2-Egfp (n = 4) electroporated midbrain. Boxed areas in h and i are magnified. White arrowheads indicate TH⁺/GFP⁺ while open arrowheads indicate TH⁻/GFP⁺ cells. j Quantification of the percentage of TH⁺/GFP⁺ cells in total GFP⁺ cells in ventral midbrain dopaminergic domain in pCAG-Egfp (n = 4; median 10.7%, range 10.3–11.3%) or pCAG-Zeb2-Egfp (n = 4; median 1.69%, range 1.41–2.44%) electroporated midbrain. Scale bars: 50 μm. Mann–Whitney U-test was used.