that 1. Dl is expressed throughout the wing anlage in early discs and 2. Dl signals to DBCs at the D/V boundary in absence of Ser function. This holds true also for the ectopic FB: here Dl signals to the ABCs in the absence of Ser. However, in both cases Dl signalling is not strong enough to initiate Wg expression, which is a crucial event for wing development. Our results also reveal an unanticipated requirement of Ser in ABCs for the expression of Wg. This requirement could be explained through Ser signalling from the ABCs to PBCs to up- regulate Dl there, which in turn signals back to ABCs (Ser/Dl loop). This explanation would imply that the levels of Notch activation required for the induction of Dl expression are lower than that for Wg. Otherwise, the expression of Wg would not stay asymemmetric as it is observed. However, we found that Notch signalling is not required in PBCs during the early asymmetric phase. This excludes the mentioned explanation and suggests that Ser must activate Notch in the Fng expressing ABCs. This assumingly weak activation contributes to the total activity of Notch in these cells and guarantees levels of Notch signalling above the threshold required for expression of Wg. In the absence of Ser the activation by Dl from PBCs appears to fail to reach the threshold level in a fraction of discs. In agreement with this notion it has been shown that expression of Ser is broadly induced upon ectopic expression of Fng [14]. We further observed that concomitant loss of Ser and Dl function in ABCs always abolishes the expression of Wg at the ectopic FB. Hence, Dl must also signal in Fng expressing ABCs and contribute to Notch signalling in these cells. The results suggest that the total amount of Notch activity in ABCs is the sum of signalling by Dl and Ser in the Fng domain and Dl signalling from PBCs to ABCs. Whereby the signal from posteriors to anterior is the more important one, since its loss always abolishes expression of Wg. A similar requirement at on both sides of the boundary for Dl had been described for the D/V boundary [38]. Interestingly, our data indicate that Dl plays a similar role there as Ser at the ectopic FB. Another addition to the current model is that Fng has two antagonistic effects on each ligand. The modification of Notch by Fringe is known to polarise signalling at the D/V-boundary by enhancing Dl- and suppressing Ser-signalling [14]. Loss of function of fng during early stages of wing development abolishes expression of Wg at a time where it is solely dependent on Ser signalling. This observation indicates that Fng has a positive effect on Ser signalling in addition to its known negative one: it enhances Ser-signalling from Fng expressing dorsal to non-expressing ventral cells. This enhancement is required to activate expression of Wg in cells across the boundary. This finding is in good agreement with previous work that reports that Fng can enhance the ability of Ser to induce ectopic wing margins upon their co- overexpression [39]. It is a possibility that this positive influence on Ser is indirect: The modification of Notch mediated by Fng results in a decrease of binding Ser to Notch. As a consequence high levels of free Ser are available in Fng expressing boundary cells at the FB that can bind in trans to the unmodified Notch on the adjacent Fng non-expressing boundary cells. At the analysed ectopic FB, we found that Dl is required in Fng non-expressing PBCs to raise the activity of Notch signalling to a level that is sufficient for expression of Wg in Fng expressing anterior boundary cells, although Dl is expressed ubiquitously in early discs and thus, present in both cell populations at the same levels. This finding indicates that although Dl can induce activity of Notch in the Fng domain, this activity is insufficient to initiate the expression of Wg. The activity rises beyond the threshold only if the cells receive an additional Fng-enhanced Dl signal from non- Sequential Signalling at the Fng Boundary expressing. This suggests that Fng has a suppressing effect on Dl signalling within its domain. The opposing effects on the activity of each ligand have important implications for restricted Notch- signalling at the FB. Restricted expression of the ligands on opposite sides of the FB is not required to restrict Notch activation to the FB. Only Dl outside the Fng domain is sufficiently active to induce Wg expression in Fng expressing cells. In turn only Ser in Fng expressing cells is sufficiently active to induce Wg expression in Fng non-expressing cells. These conditions are only met at the FB. These properties reassure that expression of Wg is restricted to a FB even in a tissue where Dl or Ser are initially expressed uniformly, as it is the case for the ectopic FB. A difference between the ectopic FB and the D/V boundary is that the roles of the ligands are reversed: at the ectopic FB, Dl signalling is required for the establishment of the initial asymmetric phase and Ser to establish the symmetric phase and to maintain expression of Notch activity at a high level. In contrast, Ser signalling is required for the initial asymmetric phase at the D/V boundary and Dl for the establishment of the later symmetric phase and probably maintenance during later stages. Thus, activation of Notch-signalling at a FB can be initiated by both ligands. Another difference is the location of the asymmetric stripe of wg expression, which is located in Fng expressing cells at the ectopic FB, but in non-expressing cells at the DV boundary. We believe that the events observed at the ectopic FB represents the more general mode of interactions, since the interactions occur solely in the ventral pouch where the cells differ mainly with respect to the expression of Fng. In contrast, at the D/V boundary dorsal cells differ from ventral cells by expression the selector Ap, which might modify the outcome of the interactions. Interactions at a Boundary of Fng Expressing and Non- expressing Cells On the basis of our results, we first summarise the events at the ectopic boundary. During early stages of wing development, Dl is ubiquitously expressed throughout the wing anlage. Ectopic expression of fng with ptcGal4, creates a band-like Fng domain in the ventral pouch. During early stages Dl and Ser (probably induced by Dl) activate Notch signalling throughout the Fng domain at low levels that are not sufficient for activation of Wg. At the sharp posterior FB, Dl signalling from PBCs to ABCs, enhanced by Fng, raises the levels of Notch in ABCs beyond the threshold required for expression of Wg. The asymmetric phase is established. Dl signalling also up-regulates expression of Ser in ABCs. Over time Ser signalling from ABCs to PBCs (enhanced by Fng), induces Wg expression in PBCs. After the solid induction of symmetric expression of Wg, the Dl/Ser/Wg loop takes over and maintains Notch signalling at the D/V boundary. We observed that the expression of Gbe+Su(H)-lacZ is broader upon ectopic expression of Fng in Ser mutant early third instar discs. Moreover, Wg is ectopically expressed throughout the ventral ptc domain upon ectopic expression of Fng by with ptcGal4 in Ser H, but not in H mutant discs. Thus, Ser probably contributes to keeping the Notch activity in the Fng domain at low level. It is known that the expression of Ser can contribute to the suppression of Notch activity in a cell-autonomous manner through cis-inhibition [40]. Cis-inhibition has been discovered during analysis of wing development and appears to be involved in regulation and directional Notch signalling in several processes (Summarised in [41]. This mechanism causes strong Ser signalling only from Ser expressing to non-expressing cells. Since Ser expression is induced in the Fng domain, strong signalling occurs from ABCs to PBCs. Thus, it is likely that cis-inhibition contributes to the directional PLOS ONE | www.plosone.org 13 November 2012 | Volume 7 | Issue 11 | e49007