Fig. 4. Activity of Gli1, Gli2 and mutant Gli proteins in MNS70 cells. (A) Schematic of effector and reporter genes co-transfected into MNS70 cells. Different gli constructs were expressed under the control of a CMV promoter. Luciferase activity is induced in a reporter containing 8xGli protein binding sites from the mouse HNF3ß floor plate enhancer (see Sasaki et al., 1999). (B) pcDNA constructs encoding mouse Gli1 (mGli1) and mouse Gli2 (mGli2) both activate the luciferase reporter. A pcDNA construct encoding full-length zebrafish Gli1 (zfGli1) activates luciferase activity, while pcDNA constructs encoding zebrafish Gli2 (zfGli2) or the dtr/gli1 (tm276, te370, ts269) or yot/gli2 (ty119, ty17) mutations show no activation. When co-transfected with full-length gli1, dtr^tm276(but not dtr^te370or dtr^ts269) enhances reporter gene activation by wild-type Gli1. In contrast, co-transfection of gli1 with constructs encoding full-length Gli2 or the C-terminally truncated yot alleles result in the elimination of Gli1 mediated transcriptional activation. Transfection with a pJT4 plasmid encoding Shh activates luciferase activity. Co-transfection with pcDNA-zfgli1 and pJT4-shh has a roughly additive effect on luciferase activity. Co-transfection of pcDNA-gli2 with pJT4-shh reduces the luciferase activity induced by Shh alone. Averaged results of 2 experiments with standard errors. Relative luciferase activities are indicated by bars while protein schematics at top show the sites of the mutations encoded by each gli mutant construct. (C) Western analysis showing Gli proteins produced in cell culture. Asterisks indicate bands of predicted size for each transfected construct.