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Fig. 3. SO acts upon the soAE motif in vivo and in S2 cells. (A-C) 4xsoAE is
ectopically induced in wing discs by EYA and EYA+SO protein but not by SO
protein alone. (A) dpp-GAL4 driving UAS-so does not induce
4xsoAE-lacZ in wing discs. (B) Ectopic expression of
4xsoAE-lacZ is induced in spots along the AP boundary by
dpp-GAL4:UAS-eya. (C) Co-expression of so enhances
eya-mediated reporter gene activity. (D) Ocellus-specific expression
of so10-soAE is lost in so2 mutant flies (arrow).
so2 is a regulatory mutant that displays an ocelliless
phenotype. (E) EYA protein is detectable in the ocellar region of
so2 mutant flies (arrow). (F-I) so is necessary
for soAE activation: in so3 clones, 4xsoAE-lacZ
expression is lost although EYA is present in the cells. Clones are delimited
by a dashed line. (F) EYA expression is shown in red and marks the non-dying
cells within the clone. (G) so3 clones are negatively
marked by the absence of GFP expression (in green). (H) 4xsoAE-lacZ
reporter gene expression (blue) is lost in
so–/–cells. (I) overlay of G and H. (J)
ß-Galactosidase reporter assays in Drosophila S2 cells. Reporter
plasmids containing the lacZ gene under the control of different
enhancer fragments were transfected into the cells. Co-transfection of
so or eya alone together with the 10xsoAE-lacZ
reporter plasmid does not exceed basal activity (lanes 2, 3). By contrast,
co-expression of so+eya with the 5xsoAE or 10xsoAE reporter
leads to a strong induction of ß-galactosidase (lanes 4, 5). The mutated
versions of the 5xsoAE reporter (5xmutHD, 5xmutGATA) still show some amount of
induction when co-transfected with so+eya (lanes 6, 7).
ß-Galactosidase values were normalized by co-transfecting 5 ng of plasmid
expressing luciferase as an internal standard. The results represent an
average ß-galactosidase activity taken from transfections done in
triplicates (±s.d.) and are illustrated as the X-fold activation over
the basal activity found for the reporter plasmid alone.
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