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. 2014 Sep 11;10(9):e1004625. doi: 10.1371/journal.pgen.1004625

Figure 2. The msh CRM contains BMP Silencer Elements contributing to dorsal repression.

Figure 2

(A) Diagram of ME showing the location of Silencer Elements SE1 and SE2 within highly conserved regions. Conservation score (Cons) is based on alignment of the ME region in 12 Drosophila species [61]. (B) Autoradiograph of electrophoretic mobility shift assay gels with radiolabeled oligonucleotide probes incubated with extracts from S2 cells over-expressing activated Tkv (to induce BMP signaling), Med and Mad in the presence or absence of over-expressed C-terminal Schnurri (Shn). EMSA probe sequences depict the location of the GC-rich (red) and SBE (blue) motifs as well as the ind SE site [20]. Mutations relative to wild type sequences are indicated by lower case. SE1 and SE2 containing probes show higher molecular weight retardation when Shn is present versus Med and Mad alone (lanes 1–3 and 6–8). When the SBE motif of either SE1 or SE2 is mutated, probe retardation is no longer observed (lanes 4/5 and 9/10). When SE2 is mutated to conform to the canonical SE consensus, SE2*, the amount of probe retained appears higher compared to wild-type SE2 (lanes 11–13, see Fig. S1D for further verification of this effect in a competition assay). (C) Lateral and dorsal views (anterior to the left) of in situ hybridization detection of lacZ expression in ME-lacZ embryos demonstrating the in vivo effects of mutating SE1 and SE2 SBE motifs. ME: wild type embryos containing the intact ME-lacZ construct; dpp: dpp- mutant embryos show complete dorsal expansion of lacZ expression; D1: deletion of the first 100 bp of ME spanning the SE1 SE2 region leads to partial dorsal expansion accompanied by reduced levels of expression; SE1: mutating SE1 leads to modest dorsal expansion; SE2: mutating SE2 leads to significant dorsal expansion; SE1 SE2: mutating both SE1 and SE2 results in yet more pronounced dorsal expansion although not as extensive as observed for the wt-ME in a dpp- mutant background; SE2*: Converting SE2 to an optimal (ind-like) SE site results in reduced lacZ expression; SE2* dpp-: SE2* in a dpp- background shows complete dorsal expansion of lacZ.