Fig. 7. Reduction of cell proliferation and neurogenesis by persistent Hes1 expression. (A) The schematic structures of recombinant retroviruses. (B-G) Immunostaining for GFP. E11.5 telencephalic neural progenitors infected with CLIG or CLIG-Hes1 were examined at different time points. (H) Clonal sizes (with s.e.m.) of the virus-infected cells. Clonal sizes of CLIG-Hes1-infected cells are smaller than those of CLIG-infected cells. Experiments were repeated at least six times at each time point. (I) Ratios (with s.e.m.) of TUNEL-positive cells in the virus-infected cells at 72 hours after infection (at least 2,000 cells in three independent experiments were examined). Although there is a tendency for cell death to increase in CLIG-Hes1 infection compared to CLIG infection, the effect is not sufficient for the reduction of growth rates in CLIG-Hes1 infection. (J) Ratios (with s.e.m.) of proliferating cells in the virus-infected cells at 72 hours after infection (at least 500 cells in three independent experiments were examined). Ki67-positive cell ratios are similar between CLIG- and CLIG-Hes1-infected cells, whereas a ratio of cells positive for the G1 phase-specific cyclin D1 is higher in CLIG-Hes1 infection, suggesting that Hes1 overexpression prolongs G1 phase. (K-Z) Immunocytochemical staining of cells infected with CLIG (K-N,S-V) and CLIG-Hes1 (O-R,W-Z). After 5 days in culture, some CLIG-infected cells have differentiated into TuJ1-positive neurons (K-N) or GFAP-positive astrocytes (S-V) whereas virtually none of the CLIG-Hes1-infected cells (closed arrowheads) are neurons (O-R) or astrocytes (W-Z). Some neurons (TuJ1-positive cells) and astrocytes (GFAP-positive cells) are indicated by open arrowheads (R,V).