Figure 5

Overexpression of En2 cDNA decreases mitotic labeling and increases neurite outgrowth of P7 mouse GNPs. P7 mouse GNPs were transfected for 5 h with GFP control or En2-GFP cDNA vectors and 24 h later following BrdU treatment were fixed and immunostained for BrdU. Transfection efficiency was approximately 10% for each vector, with similar total numbers of transfected cells. Cells are shown (400×) under phase (A, E) or fluorescence (B-D; F-H) microscopy, after transfection with GFP control vector (A-D) and En2-GFP vector (E-H), revealing GFP (green; B, F), BrdU (red; C, G) and double labeling (D, H). Arrows point to double-labeled cells in A-D, whereas arrowheads in E-H identify cells that express GFP only, separate from cells with BrdU signal. (I) Examples of GNPs from WT mice transfected with GFP control and En2-GFP vectors that extend neurites >2 cell somas exhibiting a range in size. (J) Examples of GNPs from KO mice transfected with GFP and En2-GFP vectors that extend neurites >2 cell somas. (K) En2 overexpression increased the proportion of GNPs exhibiting neuronal morphologies by 2.5-fold in WT cells as well as by two-fold in KO cells. n = 6-15, each vector, 3–5 experiments; *, significance between GFP and En2, p ≤ 0.05; **, p ≤ 0.01.