Fig. 5. Box2 binds in vitro to AbdA and is sufficient to drive an AbdA-dependent expression pattern in the embryonic midgut. (A-D) Box2 responds in vivo to AbdA. Enhancer activity is visualized by immunohistochemistry using an anti-ß-galactosidase antibody. All panels show magnifications of lateral views of the midgut of stage 14 embryos. Arrowheads indicate PS8, the site of wg midgut expression. Arrows indicate ectopic enhancer activity. An oligomer consisting of three copies of Box2 drives lacZ expression in the posterior midgut in the AbdA expression domain. Expression is first detected posterior to the normal site of wg expression (A) and later in a domain that includes the third, and part of the fourth, midgut chambers (B). Box2 enhancer activity no longer occurs in abdA^JX2 homozygous mutants (C), and is induced ectopically in the entire midgut VM when AbdA is ubiquitously provided by a heat shock construct (D). (E) Gelshift experiments with AbdA, Exd and Hth proteins produced in vitro were performed on wild-type (lanes 1-10) and mutated (Box2m; lanes 11-14) forms of Box2. Box2m carries the same point mutations as those introduced in XC(Hox6/7). 3 µl of the programmed lysate were used for each protein and for the mock lysate (lanes 2 and 16). For the binding experiments combining Exd and Hth, the two proteins were simultaneously produced and 6 µl of the lysates were used. The anti-AbdA serum was used at a 1/20 dilution. The activity of the proteins were assayed on a DllR sequence (lanes 15-23), known to assemble an AbdA/Exd/Hth complex. The asterisk and the dot mark the position of the AbdA/Exd and AbdA/Exd/Hth complexes, respectively.

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