Sequential Signalling at the Fng Boundary Figure 2. Spatial requirement of Notch activity at the ectopic FB at early and late stages of wing development. Clones are labelled by the absence of the GFP or RFP marker. (A–D) psn clones abutting the anterior side of the A/P boundary result in a loss of Wg expression in early (A, B) and late discs (C; D). (E–F) A psn clone abutting the posterior side of the boundary in a disc at late stages. The expression domain remains to be restricted to wildtype ABCs (arrows) in the region of the clone. In regions of wildtype PBCs the epression of Wg is symmetric and occurs in ABCs and PBCs (arrowheads). doi:10.1371/journal.pone.0049007.g002 the D/V boundary, Notch is activated ectopically, but not sufficiently enough to induce expression of Wg. Note, that the expression of Gbe+Su(H) covers the whole ptc domain. In late third instar discs the expression of Gbe+Su(H)-lacZ was lost (Fig. S3T). Induction of Wing Development in the Absence of Ser Function Hairless (H) and Su(H) are central members of the repressor complex that forms in absence of Notch signalling to silence the expression of target genes [32]. In the absence of H function the repressor complex fails to form and weak de-repression of some Notch target genes, among them the wing selector gene vg, can be observed [10]. However, expression of Wg is not induced. We previously found that, in H ap double mutants, this de-repression is sufficient for the formation of a wing pouch, which is absent in ap mutants [33]. Here, we found that the loss of H function enhances the residual activity of Notch observed in Ser mutants to a degree that wing development can continue (Fig. S4). SerRX106 HE31 double mutants developed to pharate adults, which had elaborated wings with margins, as well as halteres (Fig. S4A). In the corresponding wing discs, the expression of Gbe+Su(H)-lacZ and Wg along the DV boundary was restored, but restricted to ap- expressing DBCs, even in late third instar discs (Fig. S4B-E). Expression of Gbe+Su(H)-lacZ was abolished in ap; Ser H triple mutant wing imaginal discs (Fig. S4F), indicating that activation of Notch in Ser H mutant dorsal cells is probably dependent on the function of an Ap induced FB. Furthermore, Dl was expressed in Ser H double mutant discs (Fig. S4G), suggesting that it is responsible for the activation of Notch in Ser mutant wing discs. Thus, although the Dl signal is too weak to activate Wg in Ser mutants, it is sufficient in the sensitised Ser H double-mutant background. These results on the one hand confirm that signalling of the two ligands at the D/V boundary is to some extent independent of each other. On the other hand they suggest that Ser is required to achieve strong activation of Notch in cells at the D/V boundary that cannot be generated by Dl alone. We found that in Ser H double mutants, Wg expression at the ectopic FB is restricted to Fng expressing anterior cells, even in the late third instar stages (Fig. S4J-N). Hence, these results support the conclusion that Ser is required to establish the late symmetric phase of Notch activation at the ectopic FB. Note, that in contrast to H mutant discs, Wg and Gbe+Su(H) –lacZ are expressed throughout the fng domain in H Ser mutant discs. This suggests that Ser suppresses the activity of Notch throughout the fng domain during normal development. Delta is Expressed in All Cells of the Wing Anlage The presented results indicate that Dl initiates Notch signalling at the ectopic FB. Thus, Dl must be expressed throughout the ventral wing pouch at early stages of wing development. To PLOS ONE | www.plosone.org 5 November 2012 | Volume 7 | Issue 11 | e49007