Fig. 7: Dfd-Exd sites in the AP-2 enhancers respond to changes in Dfd levels. | Nature Communications

Fig. 7: Dfd-Exd sites in the AP-2 enhancers respond to changes in Dfd levels.

From: Specificity of the Hox member Deformed is determined by transcription factor levels and binding site affinities

Fig. 7

a Schematic outline of the experimental approach used to determine the effects of increased Dfd levels in the activity of AP2x-377-Luc:GFP (AP2x-377-GFP). To increase Dfd levels in the anterior part of the maxillary segment, the UAS-Dfd transgene was activated by means of the AP2x-377-D23H-GAL4 driver and the activity of AP2x-377-GFP was quantified. b–d” AP2x-377-GFP; + > Dfd (b-b”) and AP2x-377-GFP; AP2x-377-D23H > Dfd (c-d”) stage 14 embryos were stained for GFP (green in b, c, d; grey in b’, c’, d’) and Dfd (red in b, c, d; grey in b”, c”, d”). Yellow arrows mark maxillary anterior-ventral cells. Scale bar: 10 µm. (e-e”) AP2x-377-D23H > myrRFP embryos were stained for myrRFP (green in e; grey in e’) and Dfd (red in e; grey e”), highlighting the myrRFP expression induced by the AP2x-377-D23H-GAL4 driver. Stronger GFP expression in posterior and medial maxillary cells is not due to over-exposure of the images (c’, d’), as all images were taken at same settings, but due to increased Dfd expression resulting in stronger GFP induction. f Schematic outline of the experimental approach used to determine the effects of reducing Dfd levels in the activity of AP2x-377-D23H-Luc:GFP (AP2x-377-D23H-GFP). To reduce Dfd levels in anterior maxillary cells, the UAS-DfdRNAi transgene was activated by means of the ptc-GAL4 driver and the activity of AP2x-377-D23H-GFP was quantified. g-i” AP2x-377-D23H-GFP; + > DfdRNAi (g-g”) and AP2x-377-D23H-GFP; ptc > DfdRNAi (h-i”) stage 14 embryos were stained for GFP (green in g, h, i; grey in g’, h’, i’) and Dfd (red in g, h, i; grey in g”, h”, i”). The orange lines highlight anterior-ventral maxillary cells in which the ptc-GAL4 driver is active (j, j’). j-j” ptc > myrRFP stage 14 embryos were stained for myrRFP (green in j; grey in j’) and Dfd (red in j; grey j”), highlighting the myrRFP expression induced by the ptc-GAL4 driver. k, l Quantification of the expression levels of the GFP reporter and Dfd in the anterior-ventral region of the maxillary segment. In l, the area encircled by the orange line in g-g”, h-h” and i-i” was quantified. The plotted box plot represents the collected data shown in dots. Statistical relevance was tested with the two-sided t-test: (k) **p-values = 0.00037, ***p-values = 2.08E−5, (l) *p-value = 0.0103, Dfd: *p-value = 0.044 (tested embryos n > 8). See also Supplementary Figs. 10 and 11. Source files are provided in “Source-Data-File_values”.

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