Sequential Signalling at the Fng Boundary Figure 5. Dl is expressed throughout the wing primordium of early third instar wing imaginal discs. (A–C) Expression of Dl in an early third instar wing imaginal disc that express UAS fng with ptcGal4. (A) Expression of Dl in a disc without clones. Expression in enhanced at the D/V boundary and the ectopic FB (arrow). (B, C) Expression of Dl in a disc bearing Dlrev10 mutant clones. (B) The clones are labelled by the absence of GFP (arrowh in B, C). The arrowhead highlights a clone with cells homozygous for GFP and the Dl wildtype allele (GFP/GFP). (C) Expression of Dl in the same disc as in (B). The comparison of (B) and (C) reveals that the staining is strongly reduced in Dlrev10 mutant clones and enhanced in cell clones homozygous for the wildtype allele. (D–I) Asymmetric Notch signalling at the D/V boundary. (D–G) Expression of Wg and Ap in an early third larval instar wing imaginal disc. (F, G) is a magnification of the D/V region of the disc shown in (D, E). The domain of Wg along the D/V boundary is largely restricted to ventral boundary cells adjacent to the Ap domain (see merge in D). (H, I) In late stages Wg is expressed also in DBCs. However, while it is restricted to DBCs (arrowhead in I), it is expressed in VBCs and the adjacent row of ventral cells (arrows in I). doi:10.1371/journal.pone.0049007.g005 for the activity of Ser and Dl [35–37]. In the absence of mib1 function, the expression of Wg along the D/V boundary is absent [35–37]; (Fig. 7F). Dorsally restricted expression of UAS mib1 resulted in the establishment of Wg expression along the D/V boundary and wing development (Fig. 7G, H). Hence, a signal from dorsal to ventral boundary cells is sufficient for the initiation of expression of Wg and wing development. In the second experiment, we prevented Notch activation in dorsal cells by expression of UAS Su(H)-RNAi with apGal4. We found that these wing discs looked remarkably normal: the diameter of the two ring-like domains of wg expression is similar to wildtype and wg is expressed along the D/V boundary (Fig. 7I–M). A comparison with the ap expression domain revealed that Wg expression is restricted to VBCs also in late third instar discs (Fig. 7K–M). Hence, an active Notch pathway only in ventral cells appears to be sufficient to allow wing development to occur. Expression of UAS Su(H)-RNAi within DBCs and VBCs resulted in a Su(H) loss of function phenotype that is comparable to the allele Su(H)D47 (Fig. 7N, compare with Fig. 6A). This result underscores the efficiency and specificity of the RNAi line. We obtained similar results by suppression of Notch activity in dorsal cells through expression of UAS H with apGal4 (Fig. S5A–D). The flies depleted of Su(H) function developed until the adult stage and displayed the expected phenotypes. Flies where apGal4 was used had notae devoid of nearly all bristles, but had rather normal looking wings with broadened dorsal, but normal ventral wing veins and mild distal notches (Fig. S5F, G). At the margin, a few dorsal stout bristles form (not shown). This suggested the neural precursors of the dorsal bristles are induced, but probably fail to develop correctly due to the requirement of Notch throughout their lineage. This notion was confirmed by our finding that supernumerary sensory organ precursor cells de- veloped at the dorsal side of the prospective margin in late third instar discs (Fig. S5H). This indicates that the ventrally restricted Wg domain can still pattern the wing margin in a relatively normal manner. Animals where sdGal4 was used for expression had severely truncated wings that resembled that of Ser mutants (Fig. S5I, compare with Fig. S3A and Fig. S5E). We also analysed the effect of loss of fng function on expression of Notch target genes using clonal analysis (Fig. 8). Notably, we found that expression of Wg at the D/V boundary was interrupted in fng-mutant cell clones that cross the D/V boundary even during early stages of wing development (Fig. 8A–C). Apparently the loss of wg-expression cannot be compensated during later stages, since Wg expression was also absent in clones of older discs (Fig. 8D, E). Since Ser is required for initiation of wg expression in early discs, this observation suggests that Fng is required for Ser signalling to VBCs. Discussion During wing development, the activity of the Notch pathway is required to establish a stripe-like domain of expression of several genes along the D/V boundary that control wing growth and patterning, chief among them are wg and vg [11]. The D/V boundary is a FB, which provides an interface that is crucial for the activation of Notch and establishment of this organising centre. The current understanding is that Fng promotes Dl signalling and prevents Ser signalling through the modification of Notch. As a result, Dl signals strongly from ventral to dorsal and Ser from dorsal to ventral cells boundary cells [12,14]. The simultaneous signalling of the ligands in opposite direction establishes the expression of Notch target genes at both sides of the FB [12,14]. It is essential for this model to work at the D/V boundary that induction of expression of Ser is restricted to dorsal and that of Dl to ventral cells. If e.g. Dl expression could also be induced in dorsal cells by the Notch pathway, the activity of Notch would immediately spread throughout the dorsal half of the wing anlage. In agreement with this requirement, it has been observed that expression of Ser is restricted to dorsal cells upon expression of activated Notch in dorsal and ventral cells [14,19]. The combination of spatially restricted expression of the ligands and the Dl/Ser loop restricts the activation of Notch to the D/V boundary during early stages of wing development [14]. At the middle of the third larval instar stage the Dl/Ser/Wg loop takes over to maintain the activity of Notch. Thus, a critical step is the establishment of expression of Wg in boundary cells. Once this is achieved the second feedback-loop assures expression of Wg and Notch signalling throughout wing development. While this model can explain the events at the D/V boundary, it cannot explain the events at an ectopic FB, since differential expression of the ligands is unlikely to occur there. Nevertheless, the expression of Wg is also restricted at the ectopic FB [29]. The presented work provides further evidence for the current model of Fng action, but adds new details that enable it to explain also the events at an ectopic FB. One addition is the initial sequential establishment of the expression domain of Wg through asymmetric Notch signalling during early stages of wing development. We observed asymmetric expression of Wg only in VBCs at the D/V boundary, indicating that these cells achieve sufficiently high activation of Notch to initiate expression of Wg. To us the existence of the early asymmetric phase of Notch activation was surprising given the fact that activation of Notch results in the activation of the expression of Dl and Ser [14,15,19]. Consequent- ly, the activation of Notch in ventral cells should immediately lead to up-regulation of expression of Dl in ventral cells and back- signalling to dorsal cells. We therefore expected that if an asymmetric phase exists, it would be too short in time to be detected. Importantly, the initial asymmetric phase appears to be a general property for Notch signalling at a FB, since we observed on both analysed FBs. The existence of the asymmetric phase also indicates that a FB can be used to generate activity domains of the Notch pathway where the feedback-loop that regulates the expression of the ligands through Notch activation does not occur. So far the loop has been found only in the wing pouch. In the absence of the loop the asymmetric state would remain and thus, a defined stripe of high Notch activation would be generated in a field of cells that uniformly express Dl even in the absence of Ser. PLOS ONE | www.plosone.org 9 November 2012 | Volume 7 | Issue 11 | e49007