Fig. 6: Optimization of Dfd–Exd binding sites results in spatial mis-regulation of the AP2x-377 enhancer.

a Schematic representation of mutations inserted in the AP2x-377 to either disrupt (indicated by an asterisk) or optimize (indicated by an H) Dfd–Exd binding sites present in conserved domains 2 and 3. The mutated enhancers were used to generate transgenes driving the expression of GAL4. b The activity of the mutated AP2x-377 enhancers was determined by crossing flies carrying the different GAL4 transgenes with UAS-LacZ flies. c–h” AP2x-377 > LacZ (c-c”), AP2x-377-D3H > LacZ (d-d”), AP2x-377-D2H > LacZ (e-e”), AP2x-377-D23H > LacZ (f-f”), AP2x-377-D2mt-D3H > LacZ (g-g”) and AP2x-377-D2H-D3mt > LacZ (h-h”) stage 14 embryos were collected and stained for β−galactosidase (green in c, d, e, f, g, h, grey in c’, d’, e’, f’, g’, h’) to determine the activity of the different enhancers in the maxillary segment. Dfd staining labelled the maxillary segment (red in c, d, e, f, g, h, grey in c”, d”, e”, f”, g”, h”). The yellow lines in (c’, c”, d’, d”, e’, e”, f’ a, f”, g’ and g”) outline the domain of high Dfd expression in the maxillary segment. The asterisks in d’, f’ and g’ indicate the ectopic expression of the reporter gene. i Quantification of the activity of AP2x-377 (n = 8), AP2x-377-D2H (n = 6), AP2x-377-D3H (n = 10) and AP2x-377-D23H (n = 7) enhancers; n: number of embryos analysed. The activity was determined by measuring the β-galactosidase/Dfd fluorescence ratio in the posterior-ventral border cells of the maxillary segment. The plotted values indicate the mean and the corresponding standard error of the mean of the activity of each enhancer. Statistical relevance was tested with the one-way ANOVA test. *p-value = 0.0320, ns: non-significant. See also Supplementary Figs. 7, 8 and 9. Source files are provided in “Source-Data-File_values”.