Journal of Cell Science Genetic identification of intracellular trafficking regulators involved in Notch-dependent binary cell fate acquisition following asymmetric cell division Ste ´ phanie Le Bras 1,2, *, Christine Rondanino 1,2,` , Ge ´ raldine Kriegel-Taki 1,2 , Aurore Dussert 1,2,§ and Roland Le Borgne 1,2, * 1 CNRS, UMR 6061, Institut Ge ´ne ´tique et De ´ veloppement de Rennes, 35043 Rennes, France 2 Universite ´ Rennes 1, UEB, IFR 140, Faculte ´ de Me ´ decine, 35043 Rennes, France ` Present addresses: GReD Laboratory, CNRS UMR 6293, INSERM U1103, Clermont Universite ´ , 63177 Aubie ` re, France; Universite ´ d’Auvergne, Faculte ´ de Me ´ decine, 63000 Clermont-Ferrand, France § Present address: Department of Developmental and Regenerative Biology, Mount Sinai School of Medicine, New York, USA *Authors for correspondence (stephanie.lebras@univ-rennes1.fr; roland.leborgne@univ-rennes1.fr) Accepted 12 June 2012 Journal of Cell Science 125, 4886–4901 ß 2012. Published by The Company of Biologists Ltd doi: 10.1242/jcs.110171 Summary Notch signalling is involved in numerous cellular processes during development and throughout adult life. Although ligands and receptors are largely expressed in the whole organism, activation of Notch receptors only takes place in a subset of cells and/or tissues and is accurately regulated in time and space. Previous studies have demonstrated that endocytosis and recycling of both ligands and/or receptors are essential for this regulation. However, the precise endocytic routes, compartments and regulators involved in the spatiotemporal regulation are largely unknown. In order to identify intracellular trafficking regulators of Notch signalling, we have undertaken a tissue-specific dsRNA genetic screen of candidates potentially involved in endocytosis and recycling within the endolysosomal pathway. dsRNA against 418 genes was induced in the Drosophila melanogaster sensory organ lineage in which Notch signalling regulates binary cell fate acquisition. Gain or loss of Notch signalling phenotypes were observed in adult sensory organs for 113 of them. Furthermore, 26 genes were found to regulate the steady state localisation of Notch, Sanpodo, a Notch co-factor, and/or Delta in the pupal lineage. In particular, we identified 20 genes with previously unknown function in D. melanogaster intracellular trafficking. Among them, we identified CG2747 and we show that it regulates the localisation of clathrin adaptor AP-1 complex, a negative regulator of Notch signalling. Together, our results further demonstrate the essential function of intracellular trafficking in regulating Notch-signalling-dependent binary cell fate acquisition and constitute an additional step toward the elucidation of the routes followed by Notch receptor and ligands during signalling. Key words: Notch, Endocytosis, Recycling, Intracellular trafficking, AP-1 Introduction Notch cell-cell signalling is required in a vast majority of developmental processes and during the adult life of many organisms. It regulates cell fate specification as well as stem cell behaviour and defects can lead to numerous developmental pathologies and cancers underlying its crucial role (reviewed by Gridley, 2003; Miele et al., 2006). The challenging question is to understand the mechanisms allowing one cell to act as a signalling cell and the other one as the receiving cell, when both cells can potentially express both ligands and receptors. Although it can be performed through a spatial and temporal regulation of their expression, DSL (Delta, Serrate, Lag2) ligand and Notch receptor differential expression could not be sufficient to explain the subtle directionality of Notch signalling. In this context, regulation of the availability of both receptors and DSL ligands at the cell surface appears crucial to ensure a proper Notch signalling activation. Therefore ligand and receptor post- translational modifications and trafficking are emerging as crucial regulatory mechanisms. Several lines of evidence suggest that endocytic trafficking of DSL ligands enhances their signalling activity while receptor trafficking insures their steady state level at the cell surface thereby regulating their availability for ligand binding (reviewed by Bray, 2006; Fu ¨rthauer and Gonza ´lez-Gaita ´n, 2009; Kopan and Ilagan, 2009; Le Borgne, 2006; Weinmaster and Fischer, 2011; Yamamoto et al., 2010). Although recycling of DSL ligands is necessary to produce an active DSL ligand, the nature of this maturation is still poorly characterised and two models are actually favoured: endocytosis and pulling forces (Klueg and Muskavitch, 1999; Nichols et al., 2007; Windler and Bilder, 2010) versus endocytosis and recycling (Benhra et al., 2010; Emery et al., 2005; Jafar-Nejad et al., 2005; Le Borgne and Schweisguth, 2003; Rajan et al., 2009; Wang and Struhl, 2004). The cellular context dependence could account for these two non- mutually exclusive models and the Drosophila melanogaster sensory organ lineage, in which Notch unidirectional signalling is the only pathway involved (Heitzler and Simpson, 1991), represents an interesting study model in which the signal sending and receiving cells are easily distinguishable. Each sensory organ, present on the adult D. melanogaster notum, is derived from a single precursor cell (pI), which undergoes a stereotyped series of four asymmetric cell divisions 4886 Research Article