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. 2004 Apr 12;101(16):6021–6026. doi: 10.1073/pnas.0401590101

Fig. 1.

Fig. 1.

Characterization of the sal wing blade-specific enhancer. (A) Phenotypic consequences of changes in sal and salr expression in adult wings. (Left) When both genes are absent (sal-, salr-), vein L2 does not form and L4 and L5 are fused. The intervein regions L2/3 and L4/5 disappear. (Right) When sal is expressed in the whole wing blade (UAS-sal/nub-Gal4), the veins L2 and L5 do not form, and the size of the wing is reduced. (B) Immunostaining showing Sal expression in a wild-type third-instar wing imaginal disc. Vein L2 forms in the anterior compartment in the region, where sal is expressed at low levels. The high levels of Sal coincide with the presumptive veins L3 and L4. In the posterior compartment, sal expression is adjacent to the presumptive vein L5. (C) Schematic representation of the antero/posterior axis of the wing blade. Phosphorylated Mad (pMad) colocalizes with high levels of Sal protein in the presumptive veins L3 and L4. pMad and sal are less abundant at the antero/posterior border. Brk overlaps with the regions of low levels of expression of sal. (D) Genomic region surrounding the sal transcript (gray boxes) according to Kühnlein et al. (21). Below, close-up of the 1.8-kb DNA fragment that contains the sal wing blade enhancer, indicating the relevant restriction enzymes used in our analysis. The oligonucleotides used in EMSA experiments are shown below, as well as the constructs sal328 and salE/Pv-Δ143–256. In the lower part, gray bars indicate the DNA fragments cloned in front of the reporter gene lacZ. EcoRI–PvuII corresponds to the salE/Pv enhancer. (E) Measurements of the areas of β-gal expression relative to the endogenous sal expression domain. Gray tones and numbers are according to D. Bars represent mean values and standard errors. Eliminating the EcoRI–SphI fragment increases the β-gal area by 21%. Eliminating the AseI–PvuII region also increases the β-gal area by 20%. (F) Third-instar wild-type wing imaginal discs stained with anti-Sal and anti-β-gal antibodies. The pictures show only the anti-β-gal single channels. Abbreviations for restriction enzymes: E, EcoRI; Pv, PvuII; R, RsaI; S, SalI; Xm, XmnI.