Figure 3. The msh CRM contains AE-resembling sites required for expression.

Embryos are viewed laterally (anterior to the left). ME deletions spanning 4 sequential blocks (D1-D4) contain distinct islands of conserved sequences on sequence conservation and binding site clusters. lacZ-reporter constructs carrying these deletions were then tested in vivo in transgenic embryos to identify additional potential BMP responsive sequences. (A) Wild-type ME depicting the location of putative AE sites in relation to SE sites and an Ind binding site. (B) Deletion of D2 where AE1 is located results in reduced lacZ reporter expression. (C) Deletion of D3, which contains the AE2 region results in severely reduced lacZ reporter expression. (D) Deletion of D4 results in ventrally expanded lacZ reporter expression consistent with an essential Ind repressor binding site being present [7]. (E) Site-directed mutagenesis of AE2 results in similarly reduced reporter expression as observed for the D3 deletion, demonstrating an essential role of AE2 site as an activation sequence. (F) A triple mutant comprised of point mutations in the SE1, SE2 and AE2 sites results in reduced reporter expression comparable to that observed in the AE2 mutant alone suggesting that this site does not mediate BMP-dependent activation. The AE consensus shown (GNCGNC(N)₆GNCV) is an expanded version based on the standard consensus indicated in the text (GGCGCCA(N)₄GNCV) and on our hand curation from the literature. In addition, sensitized embryos were tested for potential msh activation by particular doses of Dpp. (G) msh expression in an embryo lacking maternal Dorsal and lacking zygotic Dpp (dl- dpp-), msh (green) is ubiquitously expressed as previously reported [11]. (H) The addition of a single copy of a wild-type dpp gene under the control of the even-skipped stripe 2 CRM, which creates a Dpp gradient emanating from the zone of expression (red) [7] in dl- dpp- embryos abolishes most msh expression (green) throughout the embryo. These results suggest that Dpp does not have a positive role in msh regulation in the absence of Dorsal signaling in Drosophila at any dose.