Fig. 6. A dominant-active version of Hes5 upregulates Ngn2 but not E47 expression. (A) NICD and Hes5 repressed the activity of the Ngn2 reporter construct in 293 HEK cells, whereas this reporter was activated by Hes5^ct-VP16. Data are represented as a logarithmic scale where mock is set to one. (B-E) Within 5 hours, Hes5^ct-VP16 transfected cells had upregulated the expression of Ngn2 (B,C) but not that of E47 (D); ectopic expression of neuronal markers could be detected 12 hours after transfection (E). (F) Quantification of electroporated cells (GFP⁺) in G-J expressing Tuj1. Data are represented as mean±s.e.m. ^***P<0.001, relative to NICD electroporated cells, Student's t-test. (G) Forty-two hours after electroporation, a majority of cells expressing Hes5^ct-VP16 was terminally differentiated and expressed Tuj1. (H) Cells co-transfected with NICD together with Hes5^ct-VP16 cells remained undifferentiated. (I) Similarly, cells co-transfected with Ngn2, NICD and Hes5^ct-VP16 remained undifferentiated and failed to upregulate the expression of Tuj1. (J) Misexpression of E47, Hes5^ct-VP16 and NICD promoted cells to commit to neurogenesis. (K) Proposed molecular pathway regulating neurogenesis in the vertebrate CNS. The proneural bHLH protein Ngn2 acts together with the E-protein E47 to drive the differentiation of neural progenitor cells by promoting cell cycle exit and the upregulation of neuronal protein expression. Notch signaling maintains neural cells in an undifferentiated state via the activation of CSL. Activated CSL is, in turn, inducing the expression of Hes1/5 and an alternative repressor (designated X), which subsequently represses the expression of Ngn2 and E47, respectively. SoxB1 transcription factors are according to this model, maintaining progenitor cells in an undifferentiated state by activating the expression of progenitor features and, in addition, blocking the activity of Ngn2 and E47. Scale bars: 20 µm in D; 50 µm in G.