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Fig. 6. Gli3A^HIGH transfection induces cell fate changes and overproliferation. HH11/12 stage embryos were electroporated with Gli3A^HIGH and assayed 24 hours PE for the indicated markers. (A,B) Electroporation of Gli3A^HIGH causes cell-autonomous cell-fate changes, including repression of the dorsal marker Pax7 (A) and activation of the motoneuron marker MNR2 (B). (C,D) Electroporation of Gli3A^HIGH causes overgrowth of the electroporated side, together with increased pH3 staining (C) and increased BrdU incorporation (D). (E) Quantitative analysis of Gli3A^HIGH/BrdU double-labeled cells in ventral and dorsal neural tubes, after a 1 hour BrdU pulse. At 24 hours PE, the number of cells incorporating BrdU increased 58% in ventral regions and 75% in dorsal regions in Gli3A^HIGH transfected embryos. Error bars indicate s.d. ^*P<0.05; ^**P<0.005; ^***P<0.0001 control versus treated. (F-H) Flow cytometry analysis of cell cycle phase distribution of transfected cells. pCIG and Gli3A^HIGH vectors were electroporated in ovo. At 24 hours PE, neural tubes were dissected out, GFP-expressing cells separated by flow cytometry and cell cycle phases analyzed by Hoescht staining. (F,G) Representative examples of the cell cycle profile of cells expressing pCIG (F) or Gli3A^HIGH (G). (H) At 24 hours PE, in Gli3A^HIGH transfected embryos there was a 9% decrease in the number of cells accumulating at G0/G1 phase of the cell cycle. (I) In situ hybridization with a chick cyclin D1 probe in an embryo electroporated with Gli3A^HIGH, analyzed 24 hours PE. Electroporated side is to the right. cyclin D1 expression is upregulated. (J) Summary of Shh/Gli activities on cell cycle progression of neuroepithelial cells.

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