Figure 1

In vitro and in vivo modulation of bHLH function regulates neurogenesis. (A) Ascl1 nucleofection in NS5 cells caused an increased Map2 and CD24 expression whilst conversely decreasing ABCG2 mRNA expression, as detected via RT-qPCR (100 ± 19.1 vs. 299.4 ± 8.4, 100 ± 19.6 vs. 392.1 ± 46.1, 100 ± 15.2 vs. 43.8 ± 2.5, respectively). Additionally, all E-proteins (E2-2, E47, HEB) further potentiated Ascl1-induced alteration in marker expression, when co-nucleofected with Ascl1 (Map2: 299.4 ± 8.4 vs. 349 ± 21.2, 345.7 ± 10, 378.3 ± 21; CD24: 392.1 ± 46.1 vs. 423.3 ± 39.7, 508.5 ± 40.2, 426.4 ± 11.7; ABCG2: 43.8 ± 2.5 vs. 35.7 ± 2.9, 29.7 ± 0.5, 38.8 ± 0.6, respectively). (B) Schematic illustration of the dominant-negative construct of E47 (dnE47), where the nuclear localization sequence is missing, therefore preventing its nuclear translocation and transcriptional activity of its dimerizing partners (F, insert). (C) Co-nucleofection of dnE47 reduced Ascl1-induced neurogenesis as revealed by Map2 RT-qPCR measurements (100 ± 4.2 vs. 66.8 ± 8.5). (D, E) Targeted in vivo electroporation of the dnE47-RFP construct rapidly reduced RGC differentiation, as revealed by the lower proportion of non-RGCs, when compared to an empty RFP control plasmid (100 ± 5.5 vs. 12.6 ± 2.7) 2 days post-electroporation. (F) Cycling progenitors (non-RGC) were maintained proliferating (Ki67⁺) following dnE47 induction (100 ± 9.6 vs. 182 ± 9.6). P values: *P <0.05; **P <0.01; ***P <0.001. All quantifications were normalized to control conditions. Scale bars: D, 20 μm.