Sequential Signalling at the Fng Boundary Figure 4. Spatial requirement of Dl activity at the ectopic FB during wing development. (A–D) Dl clones abutting the posterior (A, C) and anterior (B, D) side of the A/P boundary. (A, C) Only clones abutting the posterior side (arrowheads) abolish the expression of Wg, indicating that Dl is required in PBCs. The expression of Wg is not recovered in later stages (C). Anterior clones do not exhibit any effect on Wg expression. (E, F) Dl Ser double mutant clones abutting the posterior (E) and anterior (F) side of the boundary abolish expression of Wg already during early stages. doi:10.1371/journal.pone.0049007.g004 beyond the threshold of Notch activation required to induce Wg expression in Fng expressing ABCs. Notch-signalling at the D/V Boundary We asked whether we could also observe sequential activation of Notch at the D/V boundary during early wing development. Therefore, we compared the initial expression of Wg at the D/V boundary with that of Ap (Fig. 5D–G). Indeed, we found that expression of Wg is mainly restricted to VBCs (Fig. 5D–G). This observation confirms a similar observation previously made by Miccheli et al. (1997) using wg-lacZ and suggests that the activation of Notch is initially asymmetric also at the D/V boundary. As has been previously reported, Wg becomes expressed in dorsal and ventral boundary cells at late stages [29] (data not shown). However, we observe an asymmetry in the expression of Wg also in later stages, since expression is restricted to the row of DBCs, but occurs in VBCs (Fig. 5H, I, arrowhead) and the adjacent ventral row of cells (Fig. 5H, I, arrows). In order to investigate the importance of asymmetric Notch signalling at the D/V boundary during early wing development, we performed several experiments. First, we exploited the observation that in contrast to mutants of other genes involved in Notch signalling, Su(H) mutant clones do not violate the D/ V boundary [10,27,28] (Fig. 6A, arrow). We induced Su(H) clones abutting both sides of the D/V boundary. The presence of a Ser- lacZ construct that captures the early Ap dependent expression of Ser (construct II-9, 5 in [34]), allowed us to determine the origin of the clone. As expected, clones that cross the D/V boundary interrupted expression of Wg along the D/V boundary (Fig. 6B– D, arrow). In contrast, clones abutting the dorsal side of the boundary resulted in the restriction of Wg expression to ventral boundary cells, but did not abolish it (Fig. 6D–I). The expression of Wg was abolished in regions where mutant clones abutted the ventral side of the D/V boundary in early and late discs (Fig. 6J– S). These findings indicate that the activation of Notch in ventral boundary cells is required to establish the expression of Wg. Secondly, we provided Notch activity selectively only on one side of the D/V boundary (Fig. 7). We provided Notch activity exclusively in dorsal cells by expressing an UAS Psn construct in Psn mutant discs with apGal4. We found that these discs fail to develop a proper wing anlage. Expression of Wg along the D/V boundary was absent and the diameters of the remaining ring-like expression domains of Wg were dramatically reduced as in Psn mutants (Fig. 7A–D). If UAS Psn was expressed at both sides of the boundary in Psn mutants, e.g. by dppGal4, expression of Wg at the D/V boundary was established (Fig. 7E, arrow). Thus, providing Notch activity exclusively in dorsal cells is not sufficient for wing development to occur. It was not possible to exclusively express UAS Psn in ventral cells, because of the lack of an appropriate Gal4 line. We therefore performed alternative experiments: Firstly, we provided active Notch ligands only in dorsal cells by expression of UAS mib1 in dorsal cells of mib1 mutant wing discs with apGal4 (Fig. 7F–H). Previous work has demonstrated that Mib1 is absolutely required PLOS ONE | www.plosone.org 7 November 2012 | Volume 7 | Issue 11 | e49007