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Fig. 4. Repressor forms of Gli (Gli3R) cause cell cycle arrest. HH stage 11/12 embryos electroporated with the indicated plasmids, were analyzed for cell proliferation by immunostaining with the mitosis marker pH3 and by incorporation of BrdU for 1 hour, in ovo. (A-C) Repressor forms of Gli (Gli3R) cause cell cycle arrest. (A) At 12 hours PE, the number of mitotic cells is reduced in the electroporated side. (B) Few electroporated cells can enter the S phase of the cell cycle, as revealed by the number of GFP-expressing cells (green) that can incorporate BrdU (red) and thus are double labeled (yellow). (C) At 24 hours PE, the electroporated side of the neural tube is reduced and there is a clear reduction in the number of GFP/BrdU double-labeled cells. (D,E) Embryos co-transfected with Gli3R+human BCL2 and analyzed 12 hours PE display reduced pH3 staining (D) and reduced BrdU incorporation (E). (F) Embryos transfected with Bcl2 have normal rates of proliferation. (G-I) Embryos transfected with Gli-ZnF have a normal sized neural tube, and a normal rate of proliferation. (G) At 12 hours PE, pH3 immunostaining is comparable on both sides of the neural tube. A high proportion of Gli-ZnF electroporated cells can incorporate BrdU, as assessed by double labeling, either at 12 hours PE (H) or 24 hours PE (I). (J-L) Blocking Gli-transcriptional activity rescues Ptc1^loop2-induced cell cycle arrest and the size reduction in the neural tube. At 12 hours PE of Ptc1^loop2+Gli-ZnF, pH3-immunostained cells are comparable on both sides of the neural tube (J). A high proportion of Ptc1^loop2+Gli-ZnF electroporated cells can incorporate BrdU, as assessed by double labeling, either at 12 hours PE (K) or at 24 hours PE (L). (M,N) Quantitative analysis of GFP-expressing cells (green) that have incorporated BrdU (red), after a 1 hour BrdU pulse. Analysis was carried out by separating dorsal and ventral halves of the neural tube. At 24 hours PE, the number of cells incorporating BrdU increased 15% in ventral regions and 50% in dorsal regions in Gli3A^HIGH transfected embryos. (O) Summary of Shh/Gli activities on proliferation of neuroepithelial cells. ^*P<0.05; ^**P<0.005; ^***P<0.0001 control versus treated.

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