Fig. 2. Notch-mediated block of neurogenesis depends on intact SoxB1 function. (A-C,Q) Misexpression of NICD for 42 hours prevented the generation of neurons expressing NeuN (C,Q) but retained expression of the progenitor protein Sox3 (A) and the incorporation of BrdU (B). (D-F,Q) Misexpression of HMG^Sox3-EnR for 24 hours caused cells to downregulate Sox3 (D), exit the cell cycle (E) and upregulate the expression of NeuN (F,Q), even in the presence of NICD misexpression. (G-I,Q) Twenty-four hours after transfection, expression of a dominant-negative version of CSL (dnCSL), which is unable to bind DNA, had induced cells to downregulate Sox3 (G), exit the cell cycle (H) and upregulate the expression of NeuN (I,Q). (J-L,Q) Combined expression of Sox3 and dnCSL for 42 hours efficiently blocked the generation of NeuN⁺ cells (L,Q) and maintained cells in a self-renewing (K) and Sox1 expressing (J) state. Black and white representations of A-L are shown in Fig. S3 (see supplementary material). (M-P) The gamma secretase inhibitor DAPT acted in a concentration-dependent manner and caused neural cells to downregulate Sox3 expression (M), exit the cell cycle (P) and upregulate the expression of Tuj1 and NeuN (M,O). Neural explants transfected with Sox3 did not upregulate neuronal marker expression (N,O) or exit the cell cycle (P), regardless of the DAPT concentration. (Q) Statistical representation of NeuN-expressing cells in neural tubes transfected with EGFP, NICD, dnCSL, dnCSL/Sox3 or NICD/HMG^Sox3-EnR. Data are represented as mean±s.e.m. ^**P<0.01, ^***P<0.001, Student's t-test relative to 0 µm DAPT control in O and P and relative to EGFP-transfected control cells in Q. Student's t-test. Scale bars: 40 µm in L; 10 µm in N.