Sequential Notch Signalling at the Boundary of Fringe Expressing and Non-Expressing Cells Tobias Troost, Thomas Klein* Institut fu¨ r Genetik, Heinrich-Heine-Universita¨t Du¨ sseldorf, Universita¨tsstr.1, Duesseldorf, Germany Abstract Wing development in Drosophila requires the activation of Wingless (Wg) in a small stripe along the boundary of Fringe (Fng) expressing and non-expressing cells (FB), which coincides with the dorso-ventral (D/V) boundary of the wing imaginal disc. The expression of Wg is induced by interactions between dorsal and ventral cells mediated by the Notch signalling pathway. It appears that mutual signalling from dorsal to ventral and ventral to dorsal cells by the Notch ligands Serrate (Ser) and Delta (Dl) respectively establishes a symmetric domain of Wg that straddles the D/V boundary. The directional signalling of these ligands requires the modification of Notch in dorsal cells by the glycosyltransferase Fng and is based on the restricted expression of the ligands with Ser expression to the dorsal and that of Dl to the ventral side of the wing anlage. In order to further investigate the mechanism of Notch signalling at the FB, we analysed the function of Fng, Ser and Dl during wing development at an ectopic FB and at the D/V boundary. We find that Notch signalling is initiated in an asymmetric fashion on only one side of the FB. During this initial asymmetric phase, only one ligand is required, with Ser initiating Notch-signalling at the D/V and Dl at the ectopic FB. Furthermore, our analysis suggests that Fng has also a positive effect on Ser signalling. Because of these additional properties, differential expression of the ligands, which has been a prerequisite to restrict Notch activation to the FB in the current model, is not required to restrict Notch signalling to the FB. Citation: Troost T, Klein T (2012) Sequential Notch Signalling at the Boundary of Fringe Expressing and Non-Expressing Cells. PLoS ONE 7(11): e49007. doi:10.1371/journal.pone.0049007 Editor: Edward Giniger, National Institutes of Health (NIH), United States of America Received March 27, 2011; Accepted October 4, 2012; Published November 12, 2012 Copyright: 2012 Klein, Troost. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: The work was supported by the Deutsche Forschungs Gemeinschaft (DFG), through Sachbeihilfe KL 1028-3-1. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: Thomas.Klein@uni-duesseldorf.de Introduction The Notch signalling pathway is an evolutionary conserved short-range signalling pathway that is involved in numerous developmental processes and diseases [1–3]. The pathway consists of three core elements, a DSL (Delta/Serrate/Lag 2) ligand, the Notch receptor itself and a transcription factor of the CSL (CBF1/ Su(H)/Lag1) family. In Drosophila two ligands, Delta (Dl) and Serrate (Ser), exist together with one DNA binding CSL factor, Suppressor of Hairless (Su(H)). The binding of a ligand to Notch elicits a proteolytic cascade that results in the release of the intracellular domain of Notch (NICD). The cleavages are mediated by the metalloprotease Kuzbanian (Kuz)/ADAM10 and the c-secretase complex with Presenilin (Psn) at its catalytic centre [4–6]. The released NICD associates with Su(H) and activates the transcription of the target genes. In the absence of Notch signalling Su(H) is part of a repressor complex that silences the target genes. An important factor in this complex is Hairless (H), which connects the co-repressors Groucho and CtBP with Su(H) [7]. Consequently, loss of H or Su(H) function results in the weak de-repression of several target genes in the absence of Notch signalling in Drosophila [8–10]. During Drosophila wing development, the Notch pathway mediates interactions between cells across the dorso-ventral (D/ V) compartment boundary that activate and maintain the expression of several genes essential for the establishment, growth and patterning of the wing primordium, notably wingless (wg) and vestigial (vg) [11]. As a result of these interactions, the genes are eventually expressed in cells at both sides of the D/V boundary. This symmetrical expression at the D/V boundary is the result of signalling of Dl and Ser in opposite direction [12–14] and involves two regulatory loops. These loops operate at different times and depend on different expression patterns of the two Notch ligands during the third larval instar stage [12,14–16]. During early stages, Ser is expressed in all dorsal boundary cells (DBCs) [17,18], whereas Dl appears to be up-regulated in cells at the ventral side of the boundary (in ventral boundary cells (VBCs) [12,15]. During this phase, the high expression of both ligands at the boundary appears to be maintained by the activity of the Notch pathway. This notion is based on the observation that ectopic activation of the pathway leads to ectopic expression of the ligands [14,15,19]. During this time the expression of Ser appears to be restricted to the dorsal and that of Dl is up-regulated at the ventral side of the wing anlage. Their expression is maintained by Notch activity induced through the initial regulatory loop (Dl/Ser loop) at these sides [14]. The differential expression of the ligands entails that the activation of Notch stays restricted to the D/V boundary and does not spread from the D/V boundary into the nascent wing pouch [14]. The mutual directional signalling of the ligands requires the activity of the glycosyltransferase Fringe (Fng) [19–21]. Fng modifies several EGF modules in the extra-cellular domain of Notch [22,23]. This glycosylation regulates the interaction of PLOS ONE | www.plosone.org 1 November 2012 | Volume 7 | Issue 11 | e49007