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Fig. 2. The Xvent2-BRE contains a motif that mediates Bmp
responsiveness in Drosophila. (A) The Drosophila
element contains Mad and Med sites (boxed) separated by a five-nucleotide
spacer that is required for recruitment of Shn. The sequence of Smad sites and
their relative spacing are maintained in the Xvent2-BRE with one
mismatch from the consensus (G
T) at position 4. Point mutations and
deletions in the Xvent2-BRE variants are marked in green. (B)
Mutant BREs were tested for their response to Bmp signaling in
Xenopus animal cap assays. Xvent2-BRE-luciferase reporters
(top) were microinjected into two- to four-cell stage embryos (bottom), animal
cap explants were dissected at stage 8-9 and cultured until siblings reached
early gastrula stages, then processed for luciferase assays. (C, left)
Mutant Xvent2-BREs respond identically in Xenopus and
Drosophila. Both the wild-type BRE and Xvent2-sub2, which
contains two transversions within the five-nucleotide spacer (see A), are
stimulated 
10- to 11-fold in response to CABR, compared with reporter
alone. By contrast, Xvent2-del2 bearing a two-nucleotide spacer
deletion (see A) fails to respond. In the experiment shown, 100 pg of CABR
mRNA/embryo was used to stimulate expression; however, the
Xvent2-del2 reporter fails to respond even at 2 ng. All results are
presented as fold activation relative to wild type in the absence of CABR. We
consistently observed higher luciferase counts for Xvent2-sub2
relative to wild type. (C, right) Wild type and Xvent2-sub2 respond
to endogenous Bmp signaling, whereas the Xvent2-del2 mutant does not.
Transgenic embryos containing BRE multimers driving GFP are at tailbud stage
31/32. In situ hybridization shows that wild type and Xvent2-sub2
mutants direct expression in a pattern similar to the endogenous
Xvent2 mRNA and Xvent2 transgenes described previously.
Transgenic Xvent2-del2 embryos had variable expression patterns that
are likely to reflect position effects due to random integration sites.
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