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Figure 4 | Neural Development

Figure 4

From: E-proteins orchestrate the progression of neural stem cell differentiation in the postnatal forebrain

Figure 4

E2-2 and E2A sequentially affect RGC differentiation and progenitor proliferation. (A) Representative illustration of a green fluorescence protein (GFP) plasmid electroporation into the lateral wall of the LV. (B) In vitro characterization of the shRNAs used in this study. shRNA against E2-2 or E2A decreased E2-2 or E2A expression (100 ± 18.9 vs. 17.2 ± 2.4 or 100 ± 20.2 vs. 20.9 ± 2.5, respectively) within neurosphere cultures, when co-nucleofected with E2-2 or E47 overexpression plasmids, respectively. (C) E2-2 and E47 overexpression constructs increased transition from RGCs (bipolar processes, apical connection to LV lumen) to non-RGCs (no processes, spherical morphologies, distant from the LV wall) at 2 dpe (100 ± 3.9 vs. 130.5 ± 3.0 or 139.7 ± 1.4, respectively). When shRNA plasmids against E2-2 or E2A were applied, most electroporated cells maintained a RGC morphology, as reflected by the reduced ratio of non-RGC cells compared to control conditions (100 ± 3 vs. 81 ± 6.4 or 82.9 ± 3.6, respectively). Confocal micrographs illustrate RGCs and non-RGCs coexisting in the neurogenic niche. Inserts show a schematic view of the RGC to non-RGC transition. (D) E2-2 and E47 overexpression constructs decreased Ki67 immunoreactivity within progenitors (non-RGC) at 2 dpe (100 ± 5.6 vs. 61 ± 3.5 or 64.7 ± 4.1, respectively). shRNA constructs against E2-2 and E2A-induced opposite effects (100 ± 8 vs. 216 ± 13.2 or 161.9 ± 12.7, respectively). Confocal micrographs illustrate representative non-RGCs positive or negative for Ki67, respectively. P values: *P <0.05; **P <0.01; ***P <0.001. All quantifications were normalized to control conditions. Scale bars: C, 20 μm; D, 10 μm.

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