Fig. 2. Precocious astroglial differentiation in methylation-deficient E11.5 and E15.5 mouse CNS cultures. (A) Wild-type E11.5 mouse cortical precursor cells from Balb/c wild-type mice were dissociated and cultured for 2 days (2 d), 4 days (4 d) and 7 days (7 d) in the absence (con) and presence of LIF (50 ng/ml). Cells were stained with antibodies against a neuronal marker MAP2 (green) and GFAP (red). (B) E11.5 CNS cells from control (con) and Dnmt1^–/– (mut) littermate embryos were cultured with or without LIF treatment for 2 days, and stained for GFAP (red) and a neural progenitor marker, nestin (green). Co-localization of nestin and GFAP (orange) in newly differentiated astrocytes in Dnmt1^–/–mutant cultures (mut-LIF) indicates precocious astrocyte differentiation. (C) Western blot analysis of GFAP protein in two pairs of 3-day-old E11.5 NPC cultures. ß-actin serves as a sample loading control. (D) S100ß staining (red) of E11.5 CNS cells that were cultured for 4 days with LIF treatment in the last 2 days. DAPI nuclear counterstaining (blue) indicates similar cell densities between control (con) and Dnmt1^–/– (mut) cultures. (E) 5'-methylcytosine (5'meC) antibody staining (red) of 3-day-old E11.5 control (con) and Dnmt1^–/– (mut) NPCs. Counterstaining with DAPI (blue). The nuclear staining pattern is distinct, with heterochromatic punctuates intensely positive for 5'meC. (F) NPCs were infected with a GFP-expressing retrovirus on the first day of culturing. Two- to 3 days later, the virally infected GFP cells were stained for GFAP or MAP2, and the percentage of cells differentiating into either neurons or glia was measured and plotted (n=4). (G) Four-day cultured E15.5 cortical cells from control (littermate) and Dnmt1^–/– mice in the presence and absence of LIF, were triple labeled with MAP2 (green), GFAP (red) and DAPI (blue). Scale bar: 32 µm.