Sequential Signalling at the Fng Boundary Figure 1. Asymmetric Notch-signalling at the ectopic FB during early wing development. (A–C) Ectopic expression of Wg in late third instar discs occurs in boundary cells at both sides of the boundary. Arrowheads highlight the expression of Wg in PBCs. The expression domain of ptcGal4 revealed by fluorescence of UAS GFP-GPi. Note, that ptcGal4 expression increases towards the A/P boundary and is highest in ABCs. It is not expressed in PBCs. (D–I) Ectopic activation of expression of Wg and wg-lacZ upon expression of UAS fng with ptcGal4 in early third instar wing imaginal discs. (D) The expression domain of ptcGal4 revealed by fluorescence of UAS GFP-GPi. (E–I) The induced ectopic expression of Wg (arrow in E) and wg-lacZ (F) is restricted to the GFP-positive Fng expressing ABCs (G, I). The arrowhead highlights the area where the expression of Wg along the D/V boundary is interrupted because of the ectopic expression of Fng. (H, I) The same disc as in (D–G) focussed on the expression of wg-lacZ. The merge shown in (I) reveals that the expression of wg-lacZ is restricted to anterior Fng expressing cells. Arrowhead in (H, I) highlight the A/B boundary. (J–P) Mapping of the ectopic expression domain of Wg using a clonal analysis. The A/P boundary was additionally revealed through the expression of UAS GFP, which labels the ptc expression domain. The arrowhead in (J) highlights a region containing RFP-homozygous clone abutting the anterior side of the A/P boundary, which is shown in (K-P) at higher magnification. The arrows in (K, M, O) point to the RFP homozygous clone. doi:10.1371/journal.pone.0049007.g001 A/P boundary, abolished expression of Wg at the ectopic FB at early stages (Fig. 2A, B) and late stages (Fig. 2C, D). This result confirms that Notch is asymmetrically activated in ABCs. It further indicates that the activation of Notch in ABCs at early stages is a prerequisite for the establishment of the late symmetric expression of Wg. Clones abutting the posterior side of the ectopic FB do not affect expression of Wg in ABCs, but prevents the establishment of the late symmetric expression in ABCs (Fig. 2E– H). This is indicated by the lack of its expression in mutant posterior boundary cells (PBCs) in discs in late stages (Fig. 2E–H). Thus, activation of Notch in PBCs is required for expression of Wg during the late symmetric phase of expression. The results also suggest that a Dl/Ser feedback loop established in boundary cells is not important during the early asymmetric phase to induce Wg expression at the ectopic FB. Requirement of Dl and Ser at the Ectopic FB In order to determine which ligand is required for the induction of the early asymmetric expression of Wg at the ectopic FB, we monitored Ser- and Dl-mutant clones abutting the A/P boundary (Fig. 3, 4). Please take in account that we observed that the ectopic domain of Wg expression at the end of the third larval instar is often shorter in Dl or Ser, but not Psn heterozygous wing discs (see Fig. S2). This effect is most pronounced for Dl. However, during early phases of the third instar stage, the ectopic induction of Wg expression is undistinguishable in all three genotypes and reaches until the ring-like domain of Wg expression (Fig. S2). In an initial round of experiments, we induced clones without revealing the A/ P boundary through co-expression of UAS GFP. However, in many cases we unable to unambiguously determine the compart- mental origin of the clones. Thus, we co-expressed UAS GFP in the subsequent experiments. Nevertheless, we included some of the clones of the initial experiments where the origin is clear in our analysis. We found that loss of Ser function in cells on the posterior side of the FB had little effect on wg-expression during early and late stages (Fig. 3A, C, E, G). Wg was expressed normally and symmetric expression was established (Fig. 3E). However, the loss on the anterior side had variable effects in early and late discs (Fig. 3B, D, F, H, I). In many discs, we observed a dramatic shortening of the ectopic domain of Wg expression in the clone regions (Fig. 3B, D). In other discs, the ectopic domain of Wg expression is present (Fig. 3F, H, I). In these cases, we never observed symmetric expression in late stages (Fig. 3I). These observations suggest that Ser contributes to activation of Wg expression in Fng expressing ABCs. They also indicate that it is required for the establishment of the symmetric expression of Wg in late stages. Thus Ser signals from ABCs to PBCs during late stages. We found that Dl-clones abutting the posterior side of the boundary abolished the expression of Wg in adjacent ABCs in early discs (Fig. 4A, C). This indicates that a Dl-mediated signal from PBCs to ABCs is required to establish the early asymmetric Wg expression. Posterior Dl-clones also prevented expression of Wg normally observed in late third instar wing discs (Fig. 4C). This reaffirms the conclusion that the early asymmetric phase of Notch activation is a prerequisite for the induction of the later symmetric one. Anterior abutting clones had no obvious effect on expression of Wg (Fig. 4B, D). Next, we tested the behaviour of Dl Ser double mutant clones in discs at early stages (Fig. 4E, F). As expected posterior abutting clones extinguished the expression of Wg in ABCs (Fig. 4E). However, anterior clones also completely extinguished the expression of Wg indicating that the activity of the Notch pathway is abolished (Fig. 4F). Anterior Ser clones dramatically reduced the expression of Wg in a fraction of discs, but never abolished it (see above). Hence, the result indicates that also Dl contributes to the activation of Notch within Fng expressing ABCs. Signalling at an Ectopic FB in Absence of Ser Function Loss of Ser function results in the loss of most wing structures including the margin and the pouch underlining its important function in wing development [30]. We have previously observed residual Notch activity in Ser94c/SerRX106 mutant wing imaginal discs during early third larval instar [19]. However, it is not clear whether this allelic combination represented a complete loss of function situation. During the course of our studies, we have generated a recombinant chromosome that bears the molecularly characterized allele SerRX106 and the Gbe+Su(H)-lacZ construct, which faithfully detects the activity of the Notch pathway in imaginal discs [31]. The recombination event appeared to remove a lethal second site mutation, since we observed that a small number of homozygous animals developed to the pharate adult stage. For a detailed description of the Ser phenotype, see Text S1. We found that Gbe+Su(H)-lacZ was expressed at the D/V boundary in wing imaginal discs of this mutant during early stages of third larval instar (Fig. S3B–D, G–Q). Interestingly, the expression was restricted to ap expressing dorsal wing cells and appears to have spread over the whole dorsal wing anlage (Fig. S3P, Q, arrowheads). Thus, weak activity of Notch, probably induced by Dl, is present in the wing primordium even in the complete absence of Ser function. However, activation of Notch appears to be too weak to activate expression of Wg. The finding indicates that the expression of Dl is to some extent independent of Ser function in early third instar wing discs. Expression of Gbe+Su(H)-lacZ is lost during later stages of wing development in the Ser mutant wing discs. This finding confirms that Ser has an additional function in maintenance of the activity of Notch. When we ectopically expressed Fng with ptcGal4 in SerRX106 mutants, we found that Gbe+Su(H)-lacZ, but not Wg was ectopically expressed in the ventral side of the remaining wing anlage (Fig. S3R, S, arrows). The expression was restricted to the ptcGal4 domain (Fig. S3R, S, arrows). This indicates that, like at PLOS ONE | www.plosone.org 4 November 2012 | Volume 7 | Issue 11 | e49007