123 Introduction The Notch signaling pathway serves as one of the cardinal means by which distinct fates are induced in adjacent cells during development. This is achieved by presentation of the ligands Delta (Dl) or Serrate (Ser) on the cell surface of the sending cell, and activation of the Notch receptor in the receiving cells. Given the transmembrane nature of the ligands, activation is executed only in cases of direct contact between the cells. Depending upon the context of the receiving cell and the convergence of other signaling pathways, the final outcome may be inhibition or induction of differentiation, proliferation or apoptosis (Artavanis-Tsakonas et al., 1999; Lai, 2004; Schweisguth, 2004). A sharp distinction between the sending and receiving cells is essential, because signaling is carried out only between neighboring cells. In some cases the Notch pathway itself provides the means for initiating the primary distinction between cells, in a seemingly homogeneous population of cells. It is thought that random fluctuations elevating the levels of Dl in a given cell embedded within a group of equivalent cells (e.g. a pro-neural field) will trigger a further elevation in Dl and a concomitant reduction in Notch levels. In parallel, activation of Notch in the adjacent (signal-receiving) cells leads to a reduction in Dl levels (Heitzler et al., 1996). In other instances the Notch pathway is dedicated to the refinement of an already established asymmetry between adjacent cell populations. A case in point is the definition of vein borders in the pupal wing. The epidermal growth factor receptor pathway is activated in the future veins, leading to induction of Dl expression. Localized expression of Dl activates Notch signaling in the adjacent cells, to inhibit the formation of veins in this territory (de Celis et al., 1997; Huppert et al., 1997). In this system Notch signaling also relies upon the simultaneous increase in Dl and decrease of Notch in the sending cells, and the elimination of Dl in the receiving cells. These responses are at the heart of maintaining a stable, unidirectional signaling by the Notch pathway. Which mechanisms contribute to changes in levels of Dl and Notch as a result of Notch signaling? Transcriptional repression of Dl, mediated by the Enhancer of split [E(Spl)] complex induced following Notch activation is a general and direct mechanism contributing to the reduction in Dl levels (Heitzler et al., 1996; Hinz et al., 1994; Kunisch et al., 1994). In other cases, induction of specific transcriptional repressors, such as Cut in the wing margin, by Notch activation leads to repression of Dl transcription (de Celis and Bray, 1997; Micchelli et al., 1997). Mechanisms for Notch protein modification also play a role in maintaining an asymmetric distribution or activity of Notch. In specific areas, such as the wing margin, modification of Notch by Fringe, a glycosyltransferase, renders it refractive to signaling by ligands such as Ser, or conversely more responsive to Dl expressed by the adjacent cells (Bruckner et al., 2000; Okajima and Irvine, 2002; Panin et al., 1997). In sensory-organ precursor cells, enhanced endocytosis of Notch by asymmetric segregation of Numb/α-Adaptin was shown to reduce Notch signaling (Berdnik et al., 2002; Frise et al., 1996). Enhanced endocytosis of Notch was also observed in Caenorhabditis elegans vulval development, in the cell that is induced to become the primary source for the Notch ligand (Shaye and Greenwald, 2002). In specific areas, such as the wing margin, high levels of Dl were shown to confer refractivity to Notch signaling, through an ill-defined dominant-negative effect of Dl (de Celis and Bray, 1997; Micchelli et al., 1997). Unidirectional signaling from cells expressing Delta (Dl) to cells expressing Notch is a key feature of many developmental processes. We demonstrate that the Drosophila ADAM metalloprotease Kuzbanian-like (Kul) plays a key role in promoting this asymmetry. Kul cleaves Dl efficiently both in cell culture and in flies, and has previously been shown not to be necessary for Notch processing during signaling. In the absence of Kul in the developing wing, the level of Dl in cells that normally receive the signal is elevated, and subsequent alterations in the directionality of Notch signaling lead to prominent phenotypic defects. Proteolytic cleavage of Dl by Kul represents a general mechanism for refining and maintaining the asymmetric distribution of Dl, in cases where transcriptional repression of Dl expression does not suffice to eliminate Dl protein. Key words: Notch, Delta, Serrate, ADAM metalloproteases, Drosophila, Wing development, Kuzbanian-like Summary Unidirectional Notch signaling depends on continuous cleavage of Delta Amir Sapir, Efrat Assa-Kunik, Rachel Tsruya, Eyal Schejter and Ben-Zion Shilo* Department of Molecular Genetics, Weizmann Institute of Science, Rehovot 76100, Israel *Author for correspondence (e-mail: benny.shilo@weizmann.ac.il) Accepted 26 October 2004 Development 132, 123-132 Published by The Company of Biologists 2005 doi:10.1242/dev.01546 Research article Development