signalling of from ABCs to PBCs to induce the later symmetric phase. At the D/V boundary signalling is initiated differently. We have shown here that loss of the activity of the Notch pathway in dorsal cells does not prevent the establishment of expression of Wg along the D/V boundary, but restricts it to VBCs and allows wing development to proceed. Thus, the dorsal to ventral signal, which is mediated by Ser is most important. This notion is also in agreement with the phenotype of Ser (this work and [30]). Ser and Fng are initially expressed in all dorsal cells [20]. Fng enhances Ser signalling to VBCs, but suppresses signalling among dorsal cells. This strong polarised signalling results in the activation of Wg expression and up-regulation of Dl expression in VBCs (Fig. 7d). It is likely, that the cis- inhibitory effect of Ser [40] contributes to the suppression of the activation of Notch in dorsal cells through the initial phase of wing development, since we observe the expansion of the expression of Gbe+Su(H)–lacZ over the whole dorsal wing anlage in early Ser mutant discs. This is probably induced by the weak ubiquitous expression of Dl we observed in early wing discs. The analysis of the Ser and Ser H double mutants indicates that Dl can activate Notch signalling in absence of Ser function, but not strong enough to induce Wg expression, despite the presence of the FB. It appears that at the D/V boundary, Ser has to up-regulate the expression of Dl in VBCs over time beyond the threshold that is required to induce Wg expression in DBCs. Fng contributes to induction of Wg expression by enhancing Dl signalling from Fng non-expressing VBCs to expressing DBCs. The requirement for accumulation of Dl could contribute to the observed delay of the establish- ment of the symmetric phase of expression. After the initial asymmetric phase, the Ser/Dl/Wg loop is established to maintain Notch signalling and symmetric expression of Wg. Our results indicate that the initial Fng enhanced Ser signal is sufficiently strong to induce Wg in VBCs in a manner that enables also the establishment of the Ser/Dl/Wg loop. This is indicated by the observation that suppression of Notch activity in all dorsal cells throughout wing development does not prevent maintenance of expression of Wg in late stages of the third instar and even allows the development of adult wings with only minor patterning problems. Thus, the later signal from VBCs to DBCs is mainly required for positioning and patterning of the wing margin. Asymmetric signalling through the Hh pathway has been shown to establish the organising centre for the A/P axis at the anterior side of the boundary ([42]). It has been assumed that one difference in the establishment of the D/V organising centre is its symmetric placement on the D/V boundary. Our results suggest that at least during initial phases, the signalling at the D/V boundary is also asymmetric and that the established organising centre can also work if it is displaced ventrally. Thus, it appears that the signalling events at both boundaries are more similar than previously anticipated. We have previously observed that also mechanism for the establishment of both corresponding compart- ment boundaries is more similar than anticipated [10]. Materials and Methods Fly Stocks fng13 FRT80 [43], Dlrev10 FRT82B and SerVX82 FRT82B [16], PsnC1 FRT2A [44], mib11 and mib13 [36], Su(H)d47 P(B) FRT40A [45], SerRX106 is a 9k b deletion within the transcription unit that leads to a complete loss of expression [46], HE31 [47], apUG035 [48], Gbe+Su(H)-lacZ [31], vgBE-lacZ [49], Ser-lacZ [50]. Sequential Signalling at the Fng Boundary UAS fng [29], UAS Flp [51], UAS vg [21], UAS Ser [30], UAS Dl [19] UAS H [7], UAS mib1 [36], and UAS GFP [52], UAS GFP-GPi [53], UAS Su(H)-RNAi (DGRC 3497R-1), UAS Ser-RNAi (VDRC 27172), UAS Dcr2 [54]. The constructs were used in combination with sdGal4, ptcGal4, dppGal4, and apGal4. Clonal Analysis Clones were induced with an hsFLP construct during the first larval instar (24–48 h after egg laying). Histochemistry Following antibodies have been used: aNotch raised against the extracellular domain, ab-Gal, aWg, aDl and aSer. The aNotch, aWg, aDl and aHnt antibodies were obtained from the De- velopmental Studies Hybridoma Bank developed under the auspices of the NICHD and maintained by the University of Iowa, Department of Biological Sciences, Iowa City, IA 52242. ab-Gal was purchased from Cappel. aSer was a gift of E. Knust [46]. Staining was performed according to standard protocols. FITC, Alexa 488, Alexa 568, Alexa 647 and Texas Red conjugated secondary antibodies were purchased from Jackson Immuno Research and Invitrogen. Supporting Information Figure S1 (A–C) Expression of ptcGal4 and Wg throughout the third larval instar stage. (A) Wg is initially expressed in a ventral domain that defines the wing anlage. (B) This ventral domain results in a stripe-like domain along the D/V boundary (arrow) and a proximal ring-like domain (arrowhead). At late stages a second ring-like domain appears (arrowheads). ptcGal4 (green) is always expressed in a band-like domain in the centre of the wing anlage. (D–F) Expression of Dl in a wildtype disc (D, E) and a disc where fng is ectopically expressed with ptcGal4. The late expression pattern of Dl is shown. (D, E) Dl is expressed in two bands adjacent to the D/V boundary which is revealed in (E) through the expression of the notch target Gbe+Su(H)-lacZ. Similar bands are recognisable in the region of ectopic FB (arrowheads in F). (TIF) Figure S2 Range of the ectopic Wg expression domain induced by ptcGal4 UAS fng in different genetic backgrounds. (A-C) Early third instar discs. (D-F) Late third instar discs. For further explanation, see Text S1. (TIF) Figure S3 The Analysis of SerRX106 mutants.(A, A’) SerRX106 mutant flies lack a wing and halteres. They bear either a small wing rudiment (A) or a wing to notum duplication (A’). (B–D, G, H) Expression of Wg and Gbe+Su(H)-lacZ in a late (B–D) and early (G, H) third larval instar wing imaginal disc. (E, F) Expression of Wg in SerRX106 mutant wing discs. The arrows highlight the area of the wing anlage. In mutant discs either the outer ring-like domain of Wg is left (E) or a second band-like domain characteristic for the notum can be observed (F). Expression of Wg along the D/V boundary (arrows in G–I) is absent in early third instar disc mutant for SerRX106 (G–J). However, expression of Gbe+Su(H)-lacZ is present (see arrows in G–I, K). (M–Q) This situation is maintained also in later discs. (P, Q) A SerRX106 mutant discs of the mid third instar at higher magnification showing expression of Gbe+Su(H)-lacZ. Arrow- heads in (P, Q) mark the extents of the dorsal side of the rudimentary anlage. Note, that the expression of Gbe+Su(H)-lacZ in dorsal cells disappears in late discs (see panel (T)). (R–T) PLOS ONE | www.plosone.org 14 November 2012 | Volume 7 | Issue 11 | e49007