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Fig. 1. Transfection of Ptc1^loop2 inhibits neural cell proliferation and survival. HH11/12 stage chick embryos were electroporated in ovo with a bi-cistronic vector containing Ptc1^loop2 and GFP, or with the empty vector (pCIG-GFP) as control, and assayed 12 and 24 hours post-electroporation (PE) for the indicated markers. Transfected side of the neural tube is to the right side of the figures. (A-D) 24 hours PE of Ptc1^loop2 caused reduction of pH3-immunostaining on the transfected side, compared with the control contralateral side (A). BrdU incorporation is reduced in GFP-positive cells. Electroporated GFP-positive cells that have entered S phase of the cell cycle are yellow (B). TUNEL staining (brown) is increased in Ptc1^loop2 (C) and activated caspase 3-immunostaining is also increased (D). (E-H) Ptc1^loop2 neural tubes 12 hours PE show a reduction in pH3 immunostaining (E). BrdU incorporation is reduced in GFP-positive cells (F). TUNEL (G) and caspase 3 immunostaining (H) are not increased on the electroporated side 12 hours PE. (I-L) Control embryos transfected with the empty vector (pCIG) identified by GFP (green) and analyzed 12 hours PE, show an equal number of pH3-immunostaining cells on the electroporated side compared with non-electroporated side (I), GFP-positive cells can incorporate BrdU, as indicated by the elevated proportion of double-labeled yellow cells (J). TUNEL staining (K) and activated caspase 3 immunostaining (L) are the same on transfected side and control contralateral side. (M) Quantitative analysis of pH3-immunostained cells in electroporated and non-electroporated sides of the neural tube. 12 hours PE there was no significant changes in pH3 immunostaining; however, 24 hours PE, the number of pH3 cells in Ptc1^loop2 EP embryos was significantly reduced on the transfected side of the neural tube. Error bars indicate s.d. (N) Quantitative analysis of Ptc1^loop2-GFP/BrdU double labeled cells in ventral and dorsal neural tubes, after a 4-hour BrdU pulse indicated a decrease of 15% in ventral regions and 50% in dorsal regions of the number of cells incorporating BrdU by 12 hours PE, and a 60% decrease in ventral regions and 40% decrease in dorsal regions by 24 hours PE. Error bars indicate s.d. (O) Quantitative analysis of activated caspase 3-positive cells in electroporated compared with non-electroporated side. 12 hours PE there was no increase in caspase 3 immunostaining, while 24 hours PE there was a 12 fold increase in caspase 3 immunostaining in neural tubes transfected with Ptc1^loop2. Error bars indicate s.d. (M-O) ^*P<0.05; ^**P<0.005; ^***P<0.0001 control versus treated. (P) Summary of the effect of Ptc1^loop2 on neuroepithelial cells. (Q) Schematic representation of the Shh pathway.

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