Fig. 5

MiR-200c is expressed in midbrain floor plate and represses ZEB2. a, b In situ hybridization shows the expression of primary miR-200c (pri-miR-200c) in the VZ and IZ of the midbrain floor plate at E12.5. b–d Higher magnification images of boxed area in (a. c, d) In situ hybridization of pri-miR-200c and immunofluorescence staining of CXCR4 (green) show their colocalization in IZ. e pCAG-Zeb2-Egfp plasmids were electroporated into the midbrain floor plate at E11.5 and in situ hybridization was conducted to show pri-miR-200c at E12.5. Besides endogenous pri-miR-200c (magenta) in IZ of floor plate, high level of signal was also detected in GFP⁺ cells (green). f, g Individual channels show pri-miR-200c (f; magenta) and GFP (green). h Immunofluorescence image show the overexpression of ZEB2 in GFP⁺ cells. i–p The midbrain floor plate was electroporated with control vector, pCAG-Egfp (i–l), or pMiR-200c-CAG-Egfp (m–p) at E11.5 and analyzed at E13.5. The effects of miR-200c overexpression on ZEB2 were examined in a field (boxed area in i and m), centered in the boundary between the VZ and the IZ (dashed line), at the midline level. Boxes in i and m are magnified in j–l and n–p, respectively. ZEB2 immunoreactivity is much lower in most of GFP⁺ (green) cells overexpressing miR-200c (n–p) compared with GFP⁺ cells in pCAG-Egfp electroporated floor plate (j–l). White arrowheads indicate ZEB⁺/GFP⁺ cells while open arrowheads indicate ZEB⁻/GFP⁺ ones in j–l and n–p. Scale bars: 50 μm. IZ, intermediate zone; MZ, marginal zone; VZ, ventricular zone.