The 'specificity box' downstream of the homeodomain is necessary and sufficient for specific retinal action of XOTX2 and XOTX5b. (a-c) Results of in vivo lipofection of RPCs with wild-type Xotx2 and Xotx5b, and mutant Xotx2Mut3 constructs, showing the overall distribution of retinal cell types in clones lipofected with the different constructs, as indicated (a); PC, photoreceptor cells; HC, horizontal cells; BC, bipolar cells; AC, amacrine cells; GC, ganglion cells. The proportion of each cell type is represented as average ± standard error of the mean. Counted cells are indicated in the histogram (n), from 9 retinae for GFP, 10 retinae for Xotx5b, 9 retinae for Xotx2, and 14 retinae for Xotx2Mut3. Asterisks represent significant differences between Xotx constructs and GFP, as calculated by Tukey-Kramer test (*p < 0.05, **p <0.01, ***p <0.001). Sample sections are shown for retinae co-lipofected with GFP+Xotx2 (b) and GFP+Xotx2Mut3 (c); GCL, ganglion cell layer; INL, inner nuclear layer; ONL, outer nuclear layer. (d, e) In situ hybridization analyses showing examples of GFP-positive (green), Xotx2-lipofected bipolar cell expressing Xvsx1 (Fast Red detection) (d), and Xotx2Mut3-lipofected photoreceptor cell positive for IRBP probe (Fast Red detection) (e). (f) The RS box is required for the biological action of either XOTX2 or XOTX5b proteins: the histogram reports the overall distribution of retinal cell types in clones lipofected with the different constructs, as indicated. Counted cells are indicated in the histogram (n), from 11 retinae for GFP, 9 retinae for Xotx5b, 6 retinae for Xotx2, 8 retinae for Xotx2Δ, and 7 retinae for Xotx5Δ.