Fig. 3. Retroviral infection of heterogeneous primary cerebellar cultures is specific for proliferating CGNPs. (A,B) Cultures pretreated with Shh vehicle (Veh) alone prior to infection with GFP-carrying retroviruses showed only 5-10% infection levels (A), compared with 70% infection levels in cells pre-treated with Shh (B). Merge of GFP-immunolabeled cells and DAPI-stained fields (blue) are shown. (A) Several infected cells are highlighted (arrowheads). (C,D) CGNPs infected with GFP retroviruses that subsequently exit the cell cycle coexpress the neuronal marker, NeuN (FITC, green) (C), but not GFAP (FITC, green), an astroglial cell marker (D), indicating selective infection of neuronal precursors. After infection, cells were treated with vehicle alone for 48 hours prior to immunostaining. (C) Yellow pseudo-color indicates cells with overlapping expression (arrows); the inset shows infected cell with typical granule neuron morphology at higher power. (D) Arrowhead indicates a GFAP-positive cell. Note the paucity of GFAP-positive cells; we observed no astroglia that co-expressed GFP under our conditions of infection. (C',D') DAPI-staining (blue) of the fields shown in C,D.