FIGURE 2. Bmi-1 deficiency reduces proliferation but does not increase cell death in CNS stem cell colonies. P0 SVZ cells were dissociated and plated in adherent cultures, and the number of cells per colony was counted after 4, 7 and 14 d (a).

All colonies were counted after 4 d, but only colonies that contained neurons and glia were counted after 7 and 14 d. At all time points, wild-type (WT) colonies contained significantly more cells than the Bmi-1^-/- colonies (*P < 0.01). Similar results were obtained at E14 and P30 (not shown). Only a few cells in adherent CNS stem cell colonies (b) or in the P0 SVZ (c) stained with antibodies against activated caspase-3 (a marker of cell death). Arrows and arrowheads indicate the SVZ and caspase-3-positive cells, respectively. Note that the choroid plexus in the lateral ventricle is highly autofluorescent (c). No statistically significant difference in the frequency of dying cells was observed between wild-type and Bmi-1^-/- CNS stem cell colonies on the basis of the frequency of condensed, fragmented nuclei identified by 4',6-diamidino-2-phenylindole dihydrochloride (DAPI) staining (d), or activated caspase-3 (not shown). Bmi-1 deficiency was associated with a significant decrease in the rate of BrdU incorporation into CNS stem cell colonies (e, f; *P < 0.01), indicating reduced proliferation. A similar reduction in proliferation was observed in Bmi-1^-/- NCSC colonies in culture (not shown).