Notch with its ligands, such that it enhances Dl- and suppresses Ser-signalling during the early phase of signalling. The expression of Fng and Ser is activated by the selector protein for the dorsal fate, Apterous (Ap) during early stages of wing development [17,18,20]. Hence, the boundary of Ser- and Fng expressing and non-expressing cells coincides with the D/V boundary. Due to the presence of Fng in all cells that express Ser, Ser can activate Notch only in adjacent VBCs, which produce unmodified Notch. In contrast Dl, signals preferentially to DBCs, where Notch is modified. This polarization of signalling results in the symmetrical activation of Notch in DBCs and VBCs [24,25]. Hence, it is the boundary of Fng expressing and non-expressing cells (Fringe- boundary, FB) that regulates the activation of Notch signalling at the D/V boundary. It has to be noted here that the expression pattern of Dl during early stages is difficult to determine and it has never been tested whether the weak staining observed throughout the early third instar disc is specific. After establishment of the Wg expression domain, a second regulatory loop is initiated and maintains the activity of Notch in boundary cells [12,16]. The expression of Dl and Ser is now controlled by Wg (Dl/Ser/Wg loop). Wg, secreted by boundary cells, diffuses to adjacent dorsal and ventral pouch cells where it induces the expression of both ligands in a narrow dorsal and ventral band next to the Wg domain. [12,16]. Dl and Ser are thought to signal back from these cells to the boundary cells to maintain expression of Wg [12,16]. At this later phase, the expression pattern of Fng changes [20] and its contribution to the second loop has not been investigated. The current model suggests that Notch signalling at the D/V boundary respectively FB is symmetric throughout wing de- velopment [26]. However, this assumption has not been in- vestigated rigorously. Indeed, some observations suggest that signalling is initially asymmetric [16]. Moreover, Notch signalling is also required for the establishment and maintenance of the D/V compartment boundary. Consequently, loss of Notch signalling in boundary cells results in the loss of the compartment boundary and mutant clones cross over into the other compartment [27,28]. Thus, it is difficult to unambiguously determine the compartmen- tal origin of the clone, which is a prerequisite to determine the spatial requirements of components of the Notch pathway such as the ligands during signalling at this boundary using mosaic analysis. This might be a reason for the partial differences in the results obtained by the different groups [13,15–17]. The results of Couso et al. [17] and deCelis et al [13] indicate that Ser is required in DBCs, suggesting it mediates signalling from DBCs to VBCs. Miccheli et al. [16] reported cell clones mutant for each of the ligands occasionally induced loss of target genes. However, this loss was not reliable. Moreover, deCelis et al. [13] and Doherty et al. [15] reported that only Dl clones that contained dorsal and ventral cells caused extensive scalloping of the wing, while clones that abutted the ventral side of the boundary caused weak scalloping and dorsal clones had no effect. However, the later two groups analysed adult wings and it is not clear when during development the wing tissue is lost. In contrast Micccheli et al. analysed imaginal discs. This might account for the differences. In addition, the clonal analysis so far does not strictly take into account the existence of the two different feedback-loops and the importance of the ligand therein. The importance of each ligand might be different in the two regulatory loops. In order to further investigate the mechanism of Notch signalling at a FB, we analysed the function of Fng, Ser and Dl during early stages of wing development at an ectopic FB. With help of this information, we re-examined the situation at the D/V boundary. We find that Notch signalling is initiated in a sequential Sequential Signalling at the Fng Boundary fashion at both boundaries. During an initial asymmetric phase, only one ligand is required. The ligand required as well as the location of Notch activation relative to the Fng domain differs. Results Asymmetric Expression of Notch Target Genes at an Ectopic FB In order to generate an ectopic FB where the spatial requirement of Dl and Ser can be investigated, we ectopically expressed UAS fng with ptcGal4. ptcGal4 activates expression of UAS fng in a broad stripe of cells at the anterior side of the antero- posterior compartment boundary (A/P boundary) with increasing expression towards the posterior (see Fig. 1A, D and Fig. S1A–C). If expressed in this way, the sharp posterior expression boundary of UAS fng coincides with the antero-posterior (A/P) compartment boundary (arrow in Fig. 1A, D). Expression of Wg at the beginning of third larval instar stage is initially expressed throughout the ventral part of the wing imaginal disc (Fig. S1A, A’). The expression resolves in a domain along the D/V boundary and a ring-like domain, which surrounds the wing anlage (Fig. S1B, B’). The domain at the D/V boundary is induced by Notch signalling and is required for wing development. During later stages a second ring-like domain appears (Fig. S1C, C’). The ectopic expression of Fng results in a stripe of ectopic expression of Wg that straddles the sharp posterior FB in late third instar wing pouches in a similar manner as at the D/V-boundary ([29]; Fig. 1A–C). This symmetric expression of Wg is restricted to ventral pouch cells (arrowhead in Fig. 1A, arrow in Fig. 1E–I), since endogenous expression of Fng in all dorsal cells prevents the formation of a strong FB there. We initially focussed on wing discs of the early/mid third instar stage and monitored the expression of Wg and wg-lacZ as read-out for the activity of Notch. We found that during these stages, the expression of both markers map to cells of the anterior side of the A/P boundary (anterior boundary cells, ABCs), which express Fng (Fig. 1D–I). Cells of the anterior and posterior compartments never cross the A/P boundary. Instead they align along the boundary to form a straight border that can be visualised by lineage labelling techniques such as clonal analysis. Using clonal analysis, we confirmed that the ectopic stripe of Wg expression mapped to Fng expressing ABCs (Fig. 1J–P). These results suggest that Notch is activated asymmetrically at the ectopic FB only in Fng expressing ABCs during early stages of wing development, while it is activated symmetrically at both sides of the ectopic FB in late stages. In late third instar discs, the expression of Dl at the ectopic FB occurs in two stripe-like domains adjacent to the Wg expressing cells, indicating that the later operating feedback loop is established in a similar manner than at the D/V boundary (Fig. S1D–F). Requirement of Notch Activity at the Ectopic FB To further confirm the asymmetry in Notch activation, we determined on which side of the ectopic FB the activity of Notch is required by inducing clones mutant for psn abutting on each side of the boundary (Fig. 2). In contrast to the D/V boundary, establishment and maintenance of the A/P boundary is not dependent on the activity of the Notch pathway and clones mutant for genes encoding members of the pathway respect this boundary [27,28]. We co-expressed UAS GFP to label the A/P boundary and ABCs in the ptc domain. This enabled us to unambiguously determine the origin of the clone cells at the A/P boundary. We found that only Psn mutant clones abutting the anterior side of the PLOS ONE | www.plosone.org 2 November 2012 | Volume 7 | Issue 11 | e49007