Figure 1.
Ahr mediates the cellular response to dioxin.
(A) Current core model of Ahr signalling in vertebrates. (B) Sequence conservation between Ahr and Ss. LBD, dioxin binding domain.
Figure 2.
Ahr fulfils ss functions in the absence of dioxin.
(A) Drosophila leg phenotypes; the numbers of tarsal segments present is indicated. From left to right: wild type, ss mutant, and ss mutant rescued by Ahr expression. (B) Larval salivary glands showing localisation of Ss-GFP and Ahr-GFP (green), Tgo (red) and DAPI (blue). Conditions as above the panels in A. Tgo is nuclear in the presence of either Ss-GFP or Ahr-GFP but not in their absence (middle panels). (C) Left, Ahr-GFP and Ss-GFP bind Tgo in in vivo co-immunoprecipitation assays. Ahr-GFP and Ss-GFP were precipitated using anti-GFP, Tgo was detected with anti-Tgo antibody. Right, anti-GFP reveals the presence of Ahr-GFP and Ss-GFP.
Figure 3.
Ahr activity is enhanced by dioxin or addition of Arnt or extra Tgo.
(A) Leg phenotypes. Genotypes and presence of dioxin are indicated above the panels. The number of tarsal segments present is indicated. Arrows point to proximal leg deformities. (B) Late third instar leg imaginal discs showing Dac (green) and Bar (red) expression; genotypes as above. The normal (arrowheads) and ectopic (arrows) repression of Dac and Bar is indicated. (C) Late third larval-instar leg imaginal discs showing Dac and Bar expression in a wild-type and ss mutant. (D) Wild-type Ss expression in leg imaginal disc during third larval instar. Notice the correlation between ss expression and repression of Dac and Bar (arrowheads). (E) Quantification of legs according to the number of tarsal segments displayed. From left to right: w; rn-Gal4 ssabr/sssta (ss mutant), w; UAS-ss/+; rn-Gal4 ssabr/sssta (ss rescue), w; UAS-Ahr/+; rn-Gal4 ssabr/sssta (Ahr rescue) and w; UAS-Ahr/+; rn-Gal4 ssabr/sssta fed with dioxin (Ahr+dioxin rescue). Notice that presence of five tarsal segments (red column) indicates full rescue of the mutant phenotype. A minimum of 80 legs, from at least 20 flies, were observed per sample (see Table S1).
Figure 4.
Hyperactivated Ahr produces abnormal eye development.
(A) SEM pictures of adult eyes taken at 500× (top panels) and 10000× (bottom panels). Genotypes and treatment are indicated above. Notice that UAS-ss, UAS-Ahr+Tgo and UAS-Ahr+dioxin produce fused ommatidia (red arrowheads) and enlarged bristles (red arrows). Compare with the wild-type morphology (Figure S1B) shown by UAS-Ahr, and the Wild-type exposed to dioxin. These phenotypes resemble those caused by mutations in tumour-supressor genes [28]. (B) Eye bristle size average and standard errors. The genotypes are: Oregon-R (wild-type), w; GMR-Gal4/+; UAS-ss/+ (UAS-Ss), w; GMR-Gal4/UAS-Ahr (UAS-Ahr), w; GMR-Gal4/UAS-Ahr UAS-tgo (UAS-Ahr UAS-tgo) and w; GMR-Gal4/UAS-Ahr fed with dioxin (UAS-Ahr+dioxin).
Figure 5.
Dioxin enhances Ahr transcriptional potency.
(A) Binding between Ahr and Tgo in absence or presence of dioxin in in vitro conditions. Ahr-35S is pulled-down with anti-Tgo conjugated beads. Concentrations of dioxin are indicated in nM. (B) Binding between Ahr and Arnt in absence and presence of dioxin. (C) Binding between Ss and Tgo in the absence of dioxin. (D) Left, the Bar XRE enhancer sequences and its conservation with the XRE from the mouse cytochrome P450 1a1 promoter and an in vitro probe that binds to Ahr::Arnt complexes [8] (the core XRE motif is in red and non conserved bases are in grey); right, lacZ reporter expression (red) driven by this enhancer (XRE-lacZ) in a wild-type background. (E) Repression of the XRE-lacZ reporter by Ss-GFP and Ahr-GFP in presence of dioxin, but not by Ahr-GFP alone. GFP is shown in green in the bottom panels. (F) Revised model of Ahr signalling. Our results corroborate that Ahr activity exists in the absence of dioxin and show that dioxin not only enhances the nuclear translocation of Ahr [29] but also enhances Ahr's transcriptional activity.