Sequential Signalling at the Fng Boundary Figure 3. Spatial requirement of Ser activity at the ectopic FB during wing development. Clones are labelled by the absence of the GFP or RFP marker. (A, C, E, G) Ser clones abutting the posterior side of the A/P boundary do not affect the expression of Wg in early (A, G) as well as in late (C, E) stages. The arrowheads highlight the clone abutting the posterior side. (E) A disc in the late stages of third larval instar bearing a large Ser clone abutting the posterior boundary. (E’’) Magnification of the region highlighted in (E’) by the arrowhead. It reveals that the symmetric late phase of Wg expression is normally established indicated by the expression of Wg in the mutant territories in late stages (arrow in E’’). (B, D, F, H) Ser clones abutting the anterior side had variable effects. Arrowheads highlight the clones. A fraction of discs showed a strong shortening of the ectopic expression domain of Wg in the mutant area (B, D). In other cases the expression appears to be unaffected (F, H). (I) In these cases the expression in late stages remains restricted to ABCs. Thus, the symmetric phase is not established in the mutant region. doi:10.1371/journal.pone.0049007.g003 confirm this conclusion, we examined clones mutant for Dl in the wing anlage that were stained with an anti-Dl antibody (Fig. 5A– C). Previous work suggests that Dl is expressed at the D/V boundary, mainly in VBCs [12]. Additionally, diffuse weak expression is described either throughout the ventral or dorsal and ventral side [12,15]. However, it has never been tested, whether this is specific or background produced by the staining procedure. We found that the diffuse signal was strongly reduced in Dl mutant clones (Fig. 5B, C arrow). Furthermore, cells of the wild-type twin clones, which contained two copies of Dl, had higher levels of staining than their heterozygous neighbours (Fig. 5B, C, arrowhead). These observations indicate that Dl is expressed throughout the wing anlage at low levels. The ubiquitous expression of Dl provides the explanation for the ectopic activation of the Notch pathway at an ectopic FB. Furthermore, it reveals that the effect of Fng on the activity Dl at the ectopic FB is dependent on its location: In our experimental approach, Fng is ectopically expressed in a broad stripe within the wing pouch. However, activation of Wg expression is restricted to the FB. This indicates that Dl on Fng expressing cells appears to be insufficiently active to induce expression of Wg throughout the ptc domain despite the presence of Fng. Induction of Wg in Fng expressing cells requires Dl expressed on Fng non-expressing PBCs. Only Dl in Fng non-expressing cells generates a signal PLOS ONE | www.plosone.org 6 November 2012 | Volume 7 | Issue 11 | e49007