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Fig. 2. Gli transcriptional activation is required for neural cell survival. HH11/12 stage chick embryos were electroporated in ovo with a bi-cistronic vector containing the indicated plasmids and assayed, at the indicated times, for activated caspase 3 immunostaining as a marker for apoptotic cell death. (A-D) Repressor forms of Gli (Gli3R) cause high levels of apoptosis prior to inducing cell fate changes. (A,B) At 6 hours PE, Pax7 expression (red) is normal; however, caspase 3 immunostaining is highly activated on the electroporated side (B). (C,D) At 12 hours PE, Pax7 is ectopically expressed in ventral regions of the electroporated neural tube (C), together with an increased level of caspase 3 immunostaining (D). (E) Representation of Gli-variants transfected into the neural tube. Gli3A^HIGH acts as a constitutive transcriptional activator. Gli3R acts as a constitutive transcriptional repressor form. Gli-ZnF inhibits both Gli repression and activation. (F-H) Expression of Gli-ZnF results in lack of activation of Nkx2.2 (F) and normal expression of Pax7 (G); however, Gli-ZnF causes increased caspase 3 immunostaining (H). (I-K) Blocking Gli-transcriptional activity does not rescue Ptc1^loop2-induced apoptosis. Embryos were co-electroporated with Gli-ZnF/Myc and Ptc1^loop2/GFP. (I) Immunostaining with anti-Myc (red) and GFP fluorescence (green) revealed a high proportion of cells co-transfected with both plasmids. (J,K) At 24 hours PE of Gli-ZnF+ Ptc1^loop2, expression of Pax7 was normal (J); however, caspase 3 immunostaining was increased (K). (L) Gli-activator proteins induce expression of the survival gene Bcl2. At 12 hours PE of Gli3A^HIGH, there is increased Bcl2 immunostaining (red) in electroporated cells (green). (M,N) Gli-activator proteins rescue Ptc1^loop2-induced cell fate changes and apoptosis. (M) Embryos electroporated with Ptc1^loop2+Gli3A^HIGH demonstrate cell-autonomous downregulation of dorsal Pax7. Moreover, there is no increase in the level of activated caspase 3 (N). (O) Neural tubes electroporated with Gli3A^HIGH were collected 12 and 24 hours PE for western blot analysis. Low levels of Bcl2 protein (29 kDa) were detected in control sides (WT). Expression of Bcl2 was moderately increased by 12 hours PE (GliA). A twofold increase in protein levels was detected 24 hours PE (GliA). Uniformity of protein loading was confirmed by uniform levels of a 35 kDa marker band. Chemiluminiscence was quantified using a Biorad Analyzer. (P,Q) Transfection of human BCL2 rescues Gli3R induced apoptosis but not cell fate changes. Embryos co-transfected with human BCL2+Gli3R had ectopic ventral expression of Pax7 (P); however, the levels of activated caspase 3 were normal in human BCL2+Gli3R co-electroporated neural tubes (Q). (R) Summary of Shh/Gli activities on neuroepithelial cell survival. (S) Quantitative analysis of caspase 3-positive cells in electroporated versus non-electroporated sides of the neural tube. 12 hours PE there was a significant increase in apoptosis induced by Gli3R this was prevented by co-electroporation of BCL2. Ptc1^loop2 also significantly increase apoptosis at 12 hours PE which was prevented by the co-electroporation of either Gli-ZnF, Gli3A^HIGH or human BCL2. Error bars indicate s.d. ^*P<0.05; ^**P<0.005; ^***P<0.0001 control versus treated.

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