Fig. 5. Flow cytometry analysis of cell cycle phase distribution of transfected cells. HH stage 11/12 embryos were electroporated with pCIG, pCIG+human BCL2, Gli3R and Gli3R+human BCL2-containing vectors. At 12 hours PE, neural tubes were dissected out, GFP-expressing cells separated by flow cytometry and cell cycle phases analyzed by Hoescht staining. (A-C) Representative examples of cell cycle profile of cells expressing pCIG (A), Gli3R (B) and Gli3R+human BCL2 (C). The sub-G1 cell population observed in Gli3R-electroporated cells corresponds to dead and dying cells (B). (D) Cell cycle phase distribution of transfected cells. At 12 hours PE, in Gli3R transfected embryos there was a 12% increase in the number of cells accumulating at G0/G1 phase of the cell cycle. (E,F) Gli3-R repressed transcription of genes involved on G1 transition. (E) In situ hybridization with a chick cyclin D1 probe, 12 hours PE of Gli3R+human BCL2. The ventral domain of cyclin D1 expression is lacking on the electroporated side of the neural tube. (F) In situ hybridization with a chick N-myc probe, 12 hours PE of Gli3R+human BCL2. N-myc expression is repressed on the electroporated side. (G) Summary of Shh/Gli activities on cell cycle progression of neuroepithelial cells.