Figure 2.

Regulation of NSC self-renewal by Myc and p19^ARF. (A and B) Effects of retrovirus-mediated overexpression of Myc and p19^ARF on self-renewal. The frequency of neurosphere-forming cells among total virus-infected (GFP⁺) cells is compared using cells derived from different stages. Secondary (2nd) spheres were used for virus infection. Top insets show phase-contrast (left) and fluorescent (right) images of GFP virus–infected (arrows) and uninfected (arrowheads) neurospheres. Bottom insets show larger neurospheres formed by c-Myc–expressing cells (right) compared with those of control cells. (C–E) Effects of inactivation of c-Myc on self-renewal. In C, the frequency of neurosphere-forming cells in the forebrain is compared with c-Myc^+/+, c-Myc^+/−, and c-Myc^−/− mice. In D and E, c-Myc was conditionally inactivated either by infection with Cre viruses in vitro (D) or by crossing with Foxg1-Cre mice using c-Myc^flox/flox mice in vivo (E). The formation of neurospheres by cells with different genotypes is compared. (F) Effect of c-Myc inactivation on proliferation in monolayer. Forebrain neuroepithelial cells from Foxg1-Cre;c-Myc^flox/+ and Foxg1-Cre;c-Myc^flox/flox embryos were cultured in monolayer and labeled with BrdU for 48 h in the presence of FGF2 and EGF. The percentage of BrdU⁺ cells among Cre⁺ (c-Myc inactivated) and Cre⁻ (WT) cells was quantified. (G and H) Effects of inactivation in vivo (G) and acute down-regulation in vitro (H) of p19^ARF on self-renewal. In G, WT, p19^ARF−/−, and p19^ARF−/−;p16^INK4a−/− mice are compared. In H, short hairpin–p19-1 and -2 are retroviruses expressing shRNAs for p19^ARF, whereas short hairpin–Luc expresses shRNA for luciferase. *, P < 0.01 compared with control virus–infected culture (A, B, D, and H) or c-Myc^+/+ (C), Foxg1-Cre;c-Myc^flox/+ (E and F), and WT (G) mice. Error bars indicate mean + SD. Bars, 100 μm.