Fig. 3.

Targeting GLI2 to the ectoderm leads to the development of skin tumors. (a) Either 2 or 0.5 ng of GLI2 synthetic mRNA was injected along with tracer amounts of lacZ mRNA to mark injected cells. (b) An example of a loss-of-function allele (construct 4) where 5-bromo-4-chloro-3-indolyl β-d-galactoside-stained cells do not form tumors and show a pattern indistinguishable from that of lacZ mRNA-only injections (data not shown). (c) Histograms showing the percentage of affected embryos with overgrowths after misexpression of the different GLI2 alleles. Experiments were performed at two different concentrations, 0.5 ng (yellow) and 2.0 ng (green) per embryo. The 2-ng concentration was done once, and the 0.5-ng concentration was repeated three or four times per sample. The mean ± SEM is represented in the histogram. Each sample in each experiment had 20-70 embryos. All of the missense variants behaved similarly to WT, suggesting that they do not affect GLI2 function. On the other hand, allele 2 showed a less penetrant phenotype at low concentrations, indicating that it could be a hypomorph. This lower induction was significantly different from that of allele 6 (P < 0.05). Allele 8, which represents the more likely form, and allele 15 showed traces of activity, but the mean values were not significantly different from that of allele 4 (P > 0.5 for both), which had no activity. These alleles (nos. 8, 4, 15) plus alleles 5 and 16 are scored as loss-of-function in this assay. (d) Coinjection of WT GLI2 mRNA with each of the mutant constructs (in amounts of 0.5 ng of each) was assessed for synergy or antagonism. No evidence of dominant negative activity was demonstrable. Each allele was tested between 2 and 10 different times, and each sample had between 20 and 80 embryos. The bars represent the means ± SEM.