Journal of Cell Science Accepted manuscript

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1 2012. Published by The Company of Biologists Ltd. Journal of Cell Science Accepted manuscript Genetic identification of intracellular trafficking regulators involved in Notch dependent binary cell fate acquisition following asymmetric cell division. Stéphanie Le Bras 1,2, Christine Rondanino 1,2,3, Géraldine Kriegel-Taki 1,2, Aurore Dussert 1,2,4 and Roland Le Borgne 1,2 1. CNRS, UMR 6061, Institut Génétique et Développement de Rennes, F Rennes, France 2. Université Rennes 1, UEB, IFR 140, Faculté de Médecine, F Rennes, France 3. Present address: GReD Laboratory, CNRS UMR 6293, INSERM U1103, Clermont Université, F Aubière, France; Université d Auvergne, Faculté de Médecine, F Clermont-Ferrand, France 4. Present address: Department of Developmental & Regenerative Biology, Mount Sinai School of Medicine, New York, USA Corresponding authors: SLB stephanie.lebras@univ-rennes1.fr, and RLB roland.leborgne@univ-rennes1.fr Running title: Notch intracellular trafficking regulators Key words: Notch, endocytosis, recycling, intracellular trafficking, AP-1 JCS online publication date 23 July 2012

2 25 Summary Journal of Cell Science Accepted manuscript Notch signaling 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 spatio temporal regulation are largely unknown. In order to identify Notch signaling intracellular trafficking regulators, we have undertaken a tissue-specific dsrna genetic screen against candidates potentially involved in endocytosis and recycling within the endolysosomal pathway. dsrna against 418 genes was induced in Drosophila melanogaster sensory organ lineage in which Notch signaling regulates binary cell fate acquisition. Gain- or loss-of Notch signaling phenotypes were observed in adult sensory organs for 113 of them. Furthermore, 26 genes presented a change in the steady state localization 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 Drosophila melanogaster intracellular trafficking. Among them, we identified CG2747 and show that it regulates the localization of clathrin adaptor AP-1 complex, a negative regulator of Notch signaling. All together, our results further demonstrate the essential function of intracellular trafficking in regulating Notch signaling-dependent binary cell fate acquisition and constitute an additional step toward the elucidation of the routes followed by Notch receptor and ligands to signal.

3 Introduction Notch cell-cell signaling 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 behavior and defects can lead to numerous developmental pathologies and cancers underlying its crucial role (reviewed in (Gridley, 2003; Miele et al., 2006)). The challenging question is to understand the mechanisms allowing one cell to act as a signaling 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 ligand and Notch receptor differential expression could not be sufficient to explain the subtle directionality of Notch signaling. In this context, regulation of the availability of both receptors and DSL ligands (Delta, Serrate, Lag2) at the cell surface appears crucial to ensure a proper Notch signaling 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 signaling activity while receptor trafficking insures their steady state level at the cell surface thereby regulating their availability for ligand binding (reviewed in (Bray, 2006; Furthauer and Gonzalez-Gaitan, 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 characterized and two models are actually favored: 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 signaling 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 Drosophila melanogaster notum, is derived from a single precursor cell (pi), which undergoes a stereotyped series of four asymmetric cell divisions to generate five different cells, four composing the mechanosensory bristle and a glial cell (Fig. 1A,B). During each division, Notch signaling is involved in cell fate

4 acquisition. For example, Notch is inhibited in the pi daughter cell, which adopts the piib cell identity and eventually activates Notch signaling in the adjacent daughter cell becoming the piia cell. Although data from different laboratories have emphasized the role of intracellular trafficking in the uni-directionality of Notch signaling between these two daughter cells (Benhra et al., 2011; Benhra et al., 2010; Berdnik et al., 2002; Coumailleau et al., 2009; Couturier et al., 2012; Djiane et al., 2011; Emery et al., 2005; Gallagher and Knoblich, 2006; Hutterer and Knoblich, 2005; Jafar-Nejad et al., 2005; Langevin et al., 2005; Rajan et al., 2009; Roegiers et al., 2005; Tong et al., 2010), little is known and understood about the regulators and membrane compartments involved in this process during the pi mitosis and/or in each of its daughter cells. Nonetheless, some recent data have emphasized the importance of a pi daughter cell-specific intracellular trafficking of Delta, Notch and/or a Drosophila Notch co-factor, Sanpodo (Spdo, (O'Connor-Giles and Skeath, 2003)). In the signal sending piib cell, both basal to apical transcytosis of Delta mediated by Neur (see above and (Benhra et al., 2010)) and its trafficking toward an apical Actin Rich Structure (ARS) driven by WASp and the Arp2/3 complex (Rajan et al., 2009) are required for proper Notch signaling activation. While in the receiving cell, the clathrin adaptor complex AP-1 was genetically shown to be required for the correct localization of Notch and Spdo (Benhra et al., 2011). In order to identify novel regulators of the intracellular trafficking of Notch signaling major components, we have undertaken a tissue-specific double-strand RNA (dsrna) genetic screen against 418 genes potentially involved in endocytosis and/or recycling within the endolysosomal pathway. To validate our in vivo Notch-specific strategy, 50 previously known Notch signaling regulators were screened, including 24 for which the function has not yet been studied during sensory organ lineage development. We took advantage of the fact that the genetic impairment of Notch signaling directly affects the development of external sensory organs and therefore allows for adult phenotype screening (Hartenstein and Posakony, 1990). Among the 113 Notch regulators so-identified based on adult phenotype, 61 were screened for, and 26 presented a change in the steady state localization of Notch, Sanpodo and/or Delta, in the pupal sensory organ lineage. In particular, we identified genes with previously unknown function in intracellular trafficking in Drosophila melanogaster such as CG27247 a regulator of AP-1 localization, CG7787 putatively involved in the recycling pathway and members of the Tetraspanin family. Results

5 Principle and validation of gene silencing-inducible screen To screen specifically in the sensory organ lineage, we made use of a wellcharacterized and previously described dsrna in vivo strategy (Mummery-Widmer et al., 2009). Taking advantage of the GAL4-UAS binary expression system (Brand and Perrimon, 1993), we induced gene silencing of selected genes specifically in the notum where the sensory organs develop. To do so, transgenic females carrying a GAL4 under the control of a sensory organ promoter were crossed with males carrying an Upstream Activating Sequence (UAS)-dsRNA transgenic construct. In the F1 progeny, GAL4 specifically activates the UAS and eventually induced gene silencing in the fly notum during sensory organ development. For each cross, two experimenters analyzed at least 20 F1 progenies blindly. In order to identify specific regulators of Notch signaling, we scored for bristle phenotype on the notum (Fig. 1D-F). While a loss of bristle and/or double shafts without socket cell reflects a loss of Notch signaling in the sensory organ lineage (Fig. 1D,D ), an excess of socket cells and/or double shafts with socket cell is correlated with a gain of Notch signaling in the sensory organ lineage (Fig. 1F,F ). As Notch signaling is also involved in the process of pi specification, we could, additionally, score for an excess of sensory organs reflecting a loss of Notch signaling in lateral inhibition (Fig. 1E). Mummery-Widmer et al have previously performed a genomewide dsrna screen to identify regulators of Notch signaling in the sensory organs in which they used one sensory organ driver-gal4: pannier (pnr)-gal4 (Mummery-Widmer et al., 2009). In this previous screen, we noticed that the phenotype observed for 360 (86%) of our genes could not be assessed as expression of the dsrna induced either lethality or a morphological defect of the notum. This observation led us to modulate the strength of gene silencing by placing the F1 progenies at 18 C, 25 C or 29 C. As the efficiency of the GAL4- UAS system is partially temperature sensitive (Mondal et al., 2007), this allows inducing lower (at 18 C) or higher (at 29 C) dsrna expression. Additionally, to circumvent any technical GAL4-UAS biased-induced phenotype and further describe the Notch-like phenotype, we independently use two GAL4 transgenic constructs which both drive expression in the notum during development: apterous (ap)-gal4 (Calleja et al., 1996) and scabrous (sca)-gal4 (Mlodzik et al., 1990). In order to validate our Notch signaling-specific strategy (Knoblich, 2010), we choose to screen 50 known Notch signaling regulators and observe the same phenotype than previously described for 24 of them (see Table S1 in supplementary material). Although we could not

6 reproduce the Notch loss of function-like phenotype of only two known Notch regulators, aristaless (Kojima et al., 2005) and Liquid facets (Wang and Struhl, 2004), our data indicate that our strategy allows to specifically screen for Notch regulators in the sensory organ lineage as previously described (Mummery-Widmer et al., 2009). Interestingly, we also observed a bristle phenotype for 14 of the 24 known Notch regulators whose function in the sensory organ lineage had not been previously described. Not all the known Notch signaling regulators appear to be involved in the Drosophila sensory organ lineage, which further highlights the cellular context-dependence of Notch signaling in vivo as previously reported (Fuwa et al., 2006). For example, dsrna against Kurtz and Nedd4 did not induce an adult phenotype while they are negative regulators of Notch signaling in the Drosophila wing vein (Mukherjee et al., 2005; Sakata et al., 2004). Identification of Notch signaling traffic regulators To identify intracellular trafficking regulators of Notch signaling in Drosophila melanogaster sensory organs, we specifically screened for 368 genes from the endolysosomal pathway (see Table S2 in supplementary material). We selected these genes among members of intracellular trafficking regulator families mostly identified from yeast genetics and involved in different trafficking aspects such as: coat components (clathrin mediated endocytosis (Maldonado-Baez and Wendland, 2006)), lipid microdomain organization (nonclathrin mediated endocytosis (Simons and Gerl, 2010)), cytoskeleton (actin, myosin and/or microtubules (Hehnly and Stamnes, 2007)), small GTPases, ubiquitination/deubiquitination factors involved in vesicle targeting (Murphy et al., 2009; Wennerberg et al., 2005), Endosomal Sorting Complex Required for Transport (ESCRT) complexes (Henne et al., 2011), membrane recognition and/or fusion regulators (such as SNAP receptors, SNAREs, (Malsam et al., 2008), Exocyst (Hsu et al., 2004)) and ATPases (Forgac, 2007). We also based our selection on Gene Ontology (GO) annotation from Flybase (using the GO terms: endocytosis, endosomal sorting, secretion) and selected putative ortholog(s) of traffic regulators identified in a Caenorhabditis elegans genetic screen (Balklava et al., 2007) or mammal proteomic screens (Baust et al., 2008; Baust et al., 2006). Noteworthy, the molecular function of 54 of these genes has not yet been defined in Drosophila melanogaster ( Novel unknown function category in Table S2 in supplementary material). To perform our screen we used 716 dsrna lines, which represent the 368 candidates and 50 known Notch regulators, as we systematically screened with up to five different

7 dsrna lines, when available, in order to circumvent any effect due to the dsrna construct insertion site. Expression of 264/716 (36,8%) dsrna lines induced a bristle phenotype or lethality with either both or one of the GAL4 lines in our screen conditions. When two or more dsrna lines induced a bristle phenotype, it was consistently the same gain or loss of Notch signaling phenotype(s), depending on the candidate, as we never observed opposite bristle phenotype between different dsrna lines against the same candidate. In order to confirm and validate the results, we re-produced the screen, with all the positive and lethal dsrna hits and some negative ones as controls, using the same GAL4 lines and up to two additional GAL4 also driving expression in the notum: Eq-GAL4 (Pi et al., 2001) and/or pnr-gal4 (Calleja et al., 1996)). Among the dsrna lines individually crossed with these several GAL4 lines, we observed that 175/264 (66,3%) dsrna lines, representing 113 candidates, induced a reproducible bristle phenotype with one or more GAL4 (see Table S2 in supplementary material). To further validate our results, we had included 52 dsrna lines, obtained from the National Institute of Genetics Fly Stock Center (NIG-Fly) or the Transgenic RNAi Project (TRIP), which target different part of the candidate RNA sequence than the dsrna lines from the Vienna Drosophila RNAi Center (VDRC). In doing so, we confirmed the specific Notch-like bristle or lack of phenotype observed with the VDRC dsrna lines (see Table S2 in supplementary material). In fine, we firmly identified 113 Notch regulators in the sensory organ lineage (Table 1), which belong to the different screen categories that we initially defined (Fig. 1G). Specifically, we identified 77 previously unknown regulators of the Notch signaling pathway with a role in the sensory organ lineage. These regulators belong to all our initial screen categories, which cover various aspects of intracellular trafficking. The vast majority of the observed phenotypes resemble those of a loss of Notch signaling. Nevertheless, gain of Notch signaling-like phenotypes were observed for 20 genes from various screen categories including members of coat components (AP-1 and AP-2) or ESCRT complexes (-0, -I and III). Both AP-1 and AP-2 complexes had previously been identified as regulators of Spdo trafficking and eventually as negative regulators of Notch signaling pathway during binary cell fate decision (Benhra et al., 2011; Berdnik et al., 2002; Tong et al., 2010). Therefore, our genetic screen clearly led to the identification of potential intracellular trafficking regulators directly involved in the regulation of Notch signaling via its major components.

8 Notch, Sanpodo, Delta steady-state localization and cell fate identity A Notch-like sensory organ adult phenotype could be due to a defect in Notch signaling component traffic and/or induced by unrelated defects such as in cell fate determinant segregation, cell polarity, cell cycle control and/or general intracellular trafficking. Out of the 113 candidates that we genetically identified as Notch signaling regulators in the sensory organ lineage, we wanted to identify those involved in the intracellular trafficking of Notch signaling major components: Delta, Notch and its co-factor Spdo. This study is made feasible as they present a specific steady-state pattern of sub-cellular localization in the sensory organ pupal lineage during pi mitosis and at the pi daughter cellstage (Fig. 2A-F, also see (Benhra et al., 2010)). In a wild type lineage, while both apical Delta and Notch are mostly localized at the cortex (Fig. 2A -A,D -D ), basolateral Delta is found in vesicles in mitotic pi and piib/piia cells (Fig. 2B,C,E,F ). Spdo has a more dynamic pattern of localization: cytoplasmic in the mitotic pi (Fig. 2A -C ), its localization becomes asymmetric in the pi daughter cells. While Spdo is enriched along the apicobasal interface of pi daughter cells (Fig. 2D -F ), Spdo is mostly localized in vesicles in the anterior piib cell but at the basolateral plasma membrane in the posterior piia cell (Fig. 2E ). Changes in Notch, Spdo and/or Delta localization could either originate from an aberrant cell-fate identity acquisition in the lineage (two piib or piia-like cells) or reflect trafficking defect(s) causing a defective Notch signaling pathway. As a proof of principle, we recently demonstrated that the clathrin adaptor complex AP-1, identified in this screen, controls Spdo and Notch trafficking in the sensory organ lineage. In particular, a lack of AP-1 function induces Spdo and Notch sub-cellular localization changes and an adult gain of Notch signaling phenotype (Benhra et al., 2011). Similarly, loss of Neur, Sec15 or Arp2/3 functions induce changes in Spdo and/or Delta sub-cellular localization correlated with adult loss of Notch signaling phenotypes (Benhra et al., 2010; Jafar-Nejad et al., 2005; Le Borgne and Schweisguth, 2003; Rajan et al., 2009; Roegiers et al., 2005). Regulators of Notch, Sanpodo and/or Delta sub-cellular localization identified in the screen Among the 113 Notch regulators we identified, we decided to analyze those that were not previously known to cause sub-cellular localization changes and/or that do not have described function in cell polarity or in asymmetric cell division (see Table S3 in supplementary material). Among the 61 genes that we screened for a dsrna-induced change in Delta, Notch and/or Spdo steady-state localization (using one dsrna line for each), 32 did

9 not present any defect while three genes (gigas, CG31048 and CG8435) presented a lack of pi specification (revealed by an absence of Spdo staining, our sensory organ identity marker), which explains the observed adult bristle loss phenotype (see Table S3 in supplementary material). 26 genes presented a phenotype of Notch, Spdo and/or Delta mis-localization at the pi and/or pi daughter cell-stage (we used the threshold of at least three, out of 20 analyzed, sensory organs presenting the same phenotype on two different nota). Although a wide range of phenotypes was observed, they can be subdivided into three major categories (Table 2, Figs 3, 4, 5): 1- accumulation at pi daughter cell contact (Fig. 3): an excess of Spdo is seen subapically between pi daughter cells of CG2747 dsrna, Vacuolar protein sorting 28 (Vps28 dsrna ) and Chmp1 dsrna (arrowhead in Figs 3B, 6B and, data not shown). We also observed an accumulation of Spdo, Notch and Delta at the apical interface between the pi daughter cells in CG2747 dsrna, Signal transducing adaptor molecule (Stam dsrna ), Vps28 dsrna, Chmp1 dsrna, Vps2 dsrna and CG10341 dsrna (arrows in Figs 3A -A,C -C, D -D, 6A -A and, data not shown). Finally, we detected an accumulation of Spdo and Delta at the lateral membrane between pi daughter cells of CG7787 dsrna and Cullin-3 (Guftagu / Cul-3 dsrna ) (Fig. 3E - E,F -F arrows and, data not shown). Strikingly, accumulation of Spdo subapically and/or, with Notch and Delta, at the apical interface between pi daughter cells correlates with a Notch gain-of-function adult phenotype, while accumulation of Spdo and Delta at the lateral membrane between pi daughter cells is associated with Notch loss-of-function adult phenotype (Table 2). 2- vesicle excess (Fig. 4): a lineage reminiscent of two piib-like cells, in which Spdo is present in intracellular basal compartments in both daughter cells and absent from the cortex, is observed in specifically Rac1-associated protein 1 (Sra-1 dsrna ), Origin recognition complex subunit 6 (Orc-6 dsrna ), peanut (pnut dsrna ), Septin 5 (Sep5 dsrna ), Septin 2 (Sep2 dsrna ), Rab35 dsrna, Cul-3 dsrna, Vha16-1 dsrna and Vha16-2 dsrna (arrows in Figs 4B, 5C and, data not shown). This mislocalization is consistently associated with Notch loss-of-function phenotypes (Table 2). In a second group of phenotypes, we observed, in both pi daughter cells, enlarged basal vesicles that are positive for Spdo, Delta and Notch in CG7787 dsrna, Stam dsrna, Vps2 dsrna or Vacuolar protein sorting 4 (Vps4 dsrna ) (Fig. 4D -D arrows and, data not shown). Accumulation of Spdo, Delta and Notch in enlarged intracellular compartments is predominently associated with Notch gain-of-function phenotypes (Table 2).

10 Finally, an excess of Spdo and Delta positive basal compartments is observed in the anterior cell and towards the anterior in the posterior cell in Receptor mediated endocytosis 8 (Rme- 8 dsrna ), while only in the anterior cell in l(2)dtl dsrna (Fig. 4F -F arrows and data not shown) and correlates with Notch loss-of-function phenotype (Table 2). 3- apical and/or basolateral accumulation (Fig. 5): Spdo and Delta are found at the basolateral membrane or in close vicinity to the plasma membrane of both daughter cells in Stam dsrna, Syntaxin 7 (Syx7 dsrna ), Exo84 dsrna, Sec6 dsrna and Sec5 dsrna (Fig. 5A -A arrows and data not shown). This mislocalization is somewhat reminiscent of a two piia-like cells lineage and consistently associated with Notch gain-of-function phenotypes (Table 2). We also observed an accumulation of Spdo, and to a certain extent Delta and Notch, at the apical side of the anterior daughter cells of Sep2 dsrna and Rab35 dsrna (Fig. 5B -B arrowheads and, data not shown), associated with a Notch loss-of-function phenotype (Table 2). Apart from these sensory organ lineage-specific phenotypes, we observed an accumulation of Delta at the basolateral cortex of surrounding epidermal cells in O-fucosyltranferase 1 (Ofut1 dsrna, a known Notch trafficking regulator (Sasamura et al., 2007)), Tetraspanin 47F (Tsp47F dsrna ) and Tetraspanin 68C (Tsp68C dsrna ) (Fig. 5D arrowheads and, data not shown). In all cases, this phenotype is correlated with a lateral inhibition defect (excess of pi on the pupal notum based on Spdo staining, Table 1), which suggests a loss of Notch signaling during pi specification. This accumulation of Delta at the basolateral cortex could result from either a decrease in Delta internalization or an increase in Delta exocytosis to the basolateral membrane. To test if basolateral Delta endocytosis could be affected, we performed a 15 min pulse-chase labeling experiment (Benhra et al., 2010) to monitor Delta internalization in living pupae epidermal cells but did not observe any endocytosis failure (data not shown). These results raise the possibility that O-fut1, Tsp68C and Tsp47F could regulate, directly or not, Delta basolateral exocytosis. All together, our genetic and cellular results clearly validate the essential function of intracellular trafficking in regulating Notch signaling-dependent binary cell fate acquisition. Indeed, we identified 26 genes for which a Notch signaling adult phenotype is associated with a change in intracellular localization of major Notch signaling components after the first asymmetric cell division. CG2747 regulates clathrin adaptor AP-1 intracellular localization

11 Among the genes isolated in the genetic and cellular screen, CG2747 dsrna phenocopies the loss of AP-1 function (Benhra et al., 2011). Indeed, we observed a subapical accumulation of Spdo (Fig. 6B arrowhead) and an accumulation of Spdo, Delta and Notch at the apical interface between pi daughter cells (Fig. 6A-B arrows). To further validate the specificity of the phenotype induced by dsrna, we used two independent dsrna lines obtained from the NIG-Fly and TRIP centers (lines 2747R-3 and BL29322, respectively), which target different regions of the transcript (regions and of the transcript CG2747- RD, respectively), and observed the same phenotype (data not shown). We also observed an excess of socket cells and/or double shafts with socket cells with both dsrna lines (Fig. 6C arrows and, data not shown), which is reminiscent of Notch signaling gain-of-function phenotype observed in AP-1 loss of function (Benhra et al., 2011). Although CG2747 function has not yet been studied in Drosophila melanogaster, it belongs to the highly conserved HEAT repeat-containing protein 5 (HEATR5) family (Fernandez and Payne, 2006), previously shown to physically interact with the ear-domain of murine γ-adaptin subunit of the AP-1 complex (Lui et al., 2003). For instance CG2747 shares 49% of sequence identities with the homo sapiens HEATR5B and when we used the p200 antibody against the mammal HEATR5B (Hirst et al., 2005), we observed a staining in intracellular vesicles and at the junction of wild type notum epithelial cells (Fig. 6F-F ). In the area of a notum where CG2747 dsrna is induced, p200 staining is greatly reduced (Fig. 6E- E compared to 6F-F ), suggesting that the mammal antibody recognizes the Drosophila protein. Furthermore, it was demonstrated in Saccharomyces cerevisiae, that Laap1, sole member of the HEATR5 family, is necessary for proper AP-1 intracellular localization (Fernandez and Payne, 2006). To investigate whether CG2747 shares the same function in Drosophila melanogaster, we analyzed the localization of AP-1 on the pupal notum. AP-1γ staining is greatly reduced in the region of the notum where UAS-CG2747 dsrna expression is induced in comparison with the anterior head in which the ap-gal4 driver is not active (Fig. 6G). In the converse experiment, we observed that p200 staining is not affected in AP-47 (gene encoding the μ subunit of AP-1 complex) homozygote mutant cells (Fig. 6H-H ). Thus CG2747 localization is independent of AP-1 activity. We concluded that CG2747 dsrna reduces the level of CG2747 at a sufficient degree to prevent AP-1γ membrane localization and phenocopies the AP-1 cellular and adult phenotypes. All these results strongly support the

12 model in which CG2747 is necessary for AP-1 proper intracellular localization and function in Drosophila. Discussion Our dsrna genetic screen, performed in Drosophila melanogaster notum using the GAL4-UAS system, allowed us to specifically identify 113 Notch signaling trafficking regulators among 418 candidates chosen for previously described or suggested function in intracellular trafficking (Table 1). Importantly, up to 76% of the regulators we identified were not found in a similar genetic screen performed at a genome-wide level (Mummery-Widmer et al., 2009). Our study clearly shows that using multiple GAL4 drivers and three different temperatures increases the efficiency of such dsrna genetic screen as it allows identifying optimal dsrna technical conditions for each construct. In particular, using different GAL4 drivers limit the false positive or negative results observed when the driver itself induces morphological defects and/or when the expression of the dsrna induces lethality or notum morphological defects. Recovering almost 30% of positive hits in a screen is unusual. We interpret this high number first as a reflection of the tight interconnexion between membrane traffic and both Delta and Notch signaling to ultimately ensure the proper spatio temporal control of the pathway. Second, this high number is also explained by the fact that many regulators are acting as protein complexes (APs, ESCRTs, Exocysts, ATPases, Septins ). This property could be used to confirm the specificity of the dsrna effect, as inactivation of gene products belonging to the same protein complex is expected to give similar phenotype. If this prediction is fully fulfilled for AP-1, AP-2, septins and the ubiquitin ligase complex SCF, it is only partially fulfilled for the ESCRT and Exocyst complexes (see below). Although these results are novel and further validate the concept of a regulation of Notch signaling by intracellular trafficking (for review, see (Fortini and Bilder, 2009; Furthauer and Gonzalez-Gaitan, 2009; Kopan and Ilagan, 2009; Le Bras et al., 2011; Musse et al., 2012; Yamamoto et al., 2010)), we were aiming at identifying novel regulators of the subcellular localization of three major Notch signaling components: Notch, Spdo and Delta. Indeed, the observed adult phenotypes could result from various and multiple defects in

13 Notch signaling during pi asymmetric cell division and/or at the pi daughter cell-stage. Furthermore, our identified genes could regulate various molecular events such as cell fate determinants segregation, Delta, Spdo and/or Notch proper sub-cellular localization via endocytosis and/or recycling. Therefore, we decided to take advantage of the fact that Spdo, Delta and Notch localization are dynamic during pi division and at the pi daughter cell-stage in the pupal notum. We were able to observe localization changes for 26 out of the 61 genes that we studied to further understand the adult phenotypes induced by dsrna (Table 2). Three major categories of localization changes were identified at the pi daughter cell-stage : accumulation at pi daughter cell contact, vesicle excess (and in some cases, enlarged vesicles), and apical and/or basolateral accumulation. And, in each of these categories, we observed pupal lineages which correlate either with an adult Notch gain or loss-of-function like phenotype (as illustrated with Spdo localization in Figure S1 in supplementary material). For 10 genes, changes in Notch component localization can be a direct consequence of their invalidation or reflect a change in pi daughter cell fate acquisition. Indeed, we observed a pupal lineage somewhat reminiscent of two piia-like (Syx7, Exo84, Sec6, Sec5) or two piiblike (Sra-1, Orc6, pnut, Sep5, Vha16-1, Vha16-2) daughter cells (Fig. S1B,F, respectively in supplementary material). And these lineages correlate with the observed adult phenotypes i.e. Notch gain-of-function or loss-of-function, respectively. Although these results do not elucidate the function of these genes on Notch signaling regulation, they indicate that Notch signaling can be similarly regulated in various cellular contexts. For example, it was previously shown that the Vacuolar ATPase functions to control the acidification of endosomes required for Notch activation after binding by its ligand in the eye imaginal disc and ovaries (Vaccari et al., 2010; Yan et al., 2009). Surprisingly, our results suggest that part of the same complex might regulate different aspects of Notch signaling in the sensory organ lineage. Indeed, we observed a gain of Notch signaling-like adult phenotype for three Exocyst components (Exo84, Sec6 and Sec5) but also a loss of Notch signaling-like adult one for Exo84. These results can either reflect a bias in the RNAi silencing which might not be as effective for each Exocyst subunits and/or suggest that the Exocyst might have several functions during pi asymmetric cell division with opposite role in the regulation of Notch signaling. Further studies will be necessary to validate these results and to define if Exo84 could function with Sec15, another Exocyst component, which positively regulates Spdo and Delta post-endocytic trafficking in pi daughter cells (Jafar- Nejad et al., 2005).

14 For the first time, we identified members of the septin family (pnut and Sep5) and one of their regulator (Orc-6, (Chesnokov et al., 2003)) as putative regulators of Notch signaling. Septin complexes can act as scaffolds and/or diffusion barriers in various cellular events such as cytoskeleton organization, cytokinesis, membrane organization and vesicle targeting which could potentially regulate the pi asymmetric cell division (for review, see (Cao et al., 2009)). It is necessary to further decipher the pupal phenotype to define if these septins directly regulate Notch signaling traffic and/or pi cytokinesis (Founounou N and Le Borgne R, to be published elsewhere). While the pupal lineage is somewhat reminiscent to two piib-like daughter cells, we also observed an accumulation of Spdo, Notch and Delta at the apical side of the anterior pi daughter cell of Sep2 dsrna as well as of Rab35 dsrna (Fig. S1G, in supplementary material). Their common phenotype is not quite surprising knowing that human Rab35 was proposed to play an essential control on the terminal step of cytokinesis in part by controlling SEPT2 sub-cellular distribution during cell division (Kouranti et al., 2006). Although, it is not yet possible to decipher if this apical accumulation is a cause or a consequence of the Notch pupal and adult loss-of-function phenotype, this data identify two putative regulators of apical localization and confirm that Notch signaling major components are differentially trafficked between pi daughter cells. Invalidation of 14 genes led to localization changes pointing out multiple sub-cellular sites (plasma membrane, vesicular compartments, interface between the two daughter cells), which appear to be essential to the fine regulation of Notch signaling in the Drosophila sensory organ lineage. Indeed, subapical accumulation of Spdo, localization of Notch, Spdo and Delta at the apical interface between pi daughter cells and/or or in a excess of enlarged endosomes in both daughter cells are associated with an adult Notch gain-of-function like phenotypes (Fig. S1C, S1D and S1E, respectively in supplementary material). While accumulation of Spdo and Delta at the lateral membrane between pi daughter cells correlates with Notch signaling loss-of-function phenotype (Fig. S1H,I, in supplementary material). In a control situation, Notch accumulates transiently at the apical interface between piib and piia (Benhra et al., 2011; Couturier et al., 2012). Accumulation of Spdo together with Notch at this apical piib/piia interface has previously been observed in AP-1 loss of function mutant and correlated with a gain of Notch signaling (Benhra et al., 2011). Because, impairment of Delta trafficking towards the piib/piia interface in Arp2/3 mutants leads to a loss of Notch signaling (Rajan et al., 2009), it was proposed that Delta-Notch interaction resulting in Notch activation is taking place at the apical piib/piia interface that could

15 function as a signaling platform (Benhra et al, 2011). Nonetheless, this proposal awaits experimental demonstration. This proposal was recently challenged by F. Schweisguth and colleagues, who generated a functional Notch construct tagged with GFP and expressed at physiological level (NiGFP) (Couturier et al., 2012). Notch activation is reported to occur 15 to 45 min after cytokinesis and productive signaling is proposed to take place at the piia/piib interface of the cytokinetic furrow, where NiGFP accumulates in numb mutant background or when Dynamin-dependent endocytosis is blocked. Adult and sub-cellular AP-1 loss-of-function-type phenotypes are observed when two genes with previously unknown function in Drosophila, CG10341 and CG2747, were inactivated. Both of them had been identified as putative membrane trafficking regulators in a C. elegans screen (Balklava et al., 2007). CG10341 belongs to the Nuclear Assembly Factor 1 (NAF1) family involved in ribosome biogenesis, which suggests an indirect role, if so, in intracellular trafficking. On the contrary, CG2747 belongs to the HEATR5 family and we were able to show that CG2747 is required for the clathrin adaptor AP-1 complex sub-cellular localization, similarly to its putative ortholog of the Saccharomyces cerevisiae Laa1p (Fernandez and Payne, 2006). We observed that the human HEATR5B/p200 antibody staining is affected in CG2747 dsrna, which supports an evolutionary conservation of the HEATR5 function among metazoans. Nonetheless, the function of human HEATR5B/p200 remains unknown as no phenotype could be observed in p200-depleted cells maybe due to a poor silencing efficiency and/or a redundancy with the other human HEATR5 member, HEATR5A (Lui et al., 2003). Therefore, we identified a new regulator of Notch signaling that acts as a major regulator of the clathrin adaptor complex, AP-1. We also observed an accumulation of Notch, Spdo and Delta at the interface and/or in endosomes in both daughter cells, correlated with adult Notch gain-of-function phenotypes, when we inactivated members of the ESCRT complex (Stam, Vps28, Chmp1, Vps2 and Vps4). Accumulation at the interface and/or endosomes can result from a blockade in endosome maturation when ESCRT function is impaired and it has already been described that accumulation of Notch in endocytic compartments can result in an ectopic ligandindependent activation of Notch (Herz et al., 2009; Moberg et al., 2005; Thompson et al., 2005; Vaccari and Bilder, 2005; Vaccari et al., 2009). Additionally, ESCRT complexes are involved in various cellular mechanisms: cargo engagement and/or deubiquitination, Multi Vesicular Bodies maturation, vesicle budding and/or cytokinesis (for review, see (Henne et al., 2011)). In our screen, impairment of different ESCRT pathway components led to opposite adult phenotypes i.e. loss or gain of Notch signaling-like ones depending on the

16 complex and/or its subunit(s) depleted by dsrna (see Table 1). However, we didn t observe any Spdo, Delta or Notch localization changes associated with these adult loss-of-function phenotypes. Further studies are, therefore, necessary to elucidate our genetics results by identifying which sub-cellular mechanisms and which cargo(es) are regulated by the different so-identified ESCRT subunits to control Notch signaling. Accumulation of Spdo and Delta at the lateral membrane between pi daughter cells is correlated with adult Notch loss-of-function phenotypes in Cul-3 dsrna and CG7787 dsrna (Fig. S1H,I, respectively in supplementary material). This lateral membrane was previously named apical Actin Rich Structure stalk and identified as the lateral part of branched actin network present at the interface and through which endocytosed Delta traffic back (Rajan et al., 2009). Therefore, CG7787 and Cul-3 might regulate Spdo and/or Delta basolateral endocytosis and/or recycling required for Notch signaling as this accumulation phenotype is correlated with a loss of Notch signaling. While Cul-3 is a subunit of E3 ubiquitin ligase, CG7787 putatively encodes a Guanyl-nucleotide exchange factor. CG7787 belongs to the MSS4/DSS4 family proposed to function as chaperone for misfolded Rab proteins (Nuoffer et al, 1997) and in particular, Rabs associated with the Exocyst pathway (Itzen et al, 2006). Therefore, CG7787 might be involved in the same recycling pathway as Sec15 (see above) and positively regulates Notch signaling. Other data support the general idea that a recycling pathway positively regulates Notch signaling in the sensory organ lineage. Indeed, we observed an excess of Spdo, Notch and Delta positive vesicles either in the anterior or towards the anterior cell in Rme-8 dsrna and l(2)dtl dsrna, which is associated with an adult Notch loss-of-function phenotype. Although l(2)dtl function in intracellular trafficking is still unknown, Rme-8 was shown to regulate a recycling pathway (Shi et al., 2009). All those results confirm that Spdo, Notch and Delta transiently traffic through the lateral membrane and/or endosomes to ensure a proper Notch signaling. Finally, we observed a basolateral accumulation of Delta in epithelial cells of O- fut1 dsrna, Tsp47F dsrna and Tsp68C dsrna nota and that Delta endocytosis is not affected which suggest the existence of basolateral Delta exocytosis. In support of this hypothesis, it was already demonstrated that Delta can be fucosylated by mammalian O-fut1 (Panin et al., 2002) but this data remained to be demonstrated in vivo and in Drosophila. When confirmed by further studies in classical genetic mutants, these results will eventually highlight a function for Delta basolateral exocytosis and also a new role of the tetraspanins family on Notch signaling.

17 In conclusion, our screen led us to identify intracellular trafficking regulators of major Notch signaling actors. Although it is still up to debate to define if the sub-cellular localization changes observed are a cause or a consequence of the Notch signaling phenotype, the screen we performed led to the identification of 11 previously unknown regulators of Notch signaling (CG2747, Tsp47F, Orc-6, pnut, Sep2, Sep5, Rab35, CG7787, CG10341, l(2)dtl and Rme-8). Without any doubt, further analyses of our identified genes will bring a better understanding of their trafficking function in regulating Notch signaling-dependent binary cell fate acquisition, as well as of their putative molecular interactions. Journal of Cell Science Accepted manuscript

18 Material and methods Drosophila Stocks and Genetics Unless otherwise stated, fly stocks were obtained from the Bloomington Drosophila Stock Center. Driver-GAL4 stocks used in this study were: ap-gal4 (Calleja et al., 1996), sca- GAL4 (Mlodzik et al., 1990), Eq-GAL4 (Pi et al., 2001) and pnr-gal4 (Calleja et al., 1996). All dsrna transgenic lines were supplied by the Vienna Drosophila RNAi Center (VDRC, (Dietzl et al., 2007)); except lines (as indicated in Tables S1, S2, S3 in supplementary materials), which were obtained from the National Institute of Genetics Fly Stock Center (NIG-FLY) or the Transgenic RNAi Project (TRIP) via the Bloomington Drosophila Stock Center. For RNAi-induced phenotype study, crosses between UAS-hairpin RNAi males and driver-gal4 females were raised at 18 C, 25 C or shifted at 29 C during L2-L3 larval stages. For each cross in which the genotypes were blinded for objectivity purpose, two experimenters examined at least 20 flies sensory organ distribution and/or morphological phenotypes. w 1118 males were crossed with driver-gal4 females for control experiments. To obtain AP-47 SHE11 mitotic clones, we used the FLP-FRT technique and the stocks (1) y w hs- FLP; FRT82B, Ubi-GFP(S65T)nls and (2) FRT82B, AP-47 SHE11 /TM6 Tb Sb, as previously described (Benhra et al., 2011). Heat shocks were performed at L2 and L3 during 30 min. Immunocytochemistry Pupae were aged for 17h to 20h after puparium formation, dissected in 1X PBS, fixed in 4% paraformaldehyde and stained as previously described (Le Borgne and Schweisguth, 2003). Primary antibodies used were mouse anti-notch Extra Cellular Domain (NECD; DSHB, 1:100), rabbit anti-spdo (a kind gift from J. Skeath, 1:1000, (O'Connor-Giles and Skeath, 2003)), guinea pig anti-delta Extra Cellular Domain (GP582, a kind gift from M. Muskavitch, 1:2000), rabbit anti-heatr5b (p200; a kind gift from M. Robinson, 1:20, (Hirst et al., 2005)), rat anti-de-cad (DCAD2; DSHB 1:250) and mouse anti-ap-1γ (1:100, (Benhra et al., 2011)). Cy2-, Cy3- and Cy5- coupled secondary antibodies (1:500) were from Jackson s Laboratories. Delta 15 minutes internalization assays were performed with mouse anti-delta DSHB (1:100), as previously described (Benhra et al., 2011). Images were acquired on Leica SPE confocal microscope and noise-suppressed using the smooth function of ImageJ. In all figures, Notch (Cy5-) images were color balanced using ImageJ. Defect in lateral inhibition was acknowledged when more than four-five sensory

19 organs were systematically detected with a 63x lens-1.4n.a., zoom 3 on a notum. Acknowledgements We thank M. Muskavitch, M. Robinson, J. Skeath, the Bloomington Stock center, the Vienna Drosophila RNAi Center, the TRiP at Harvard Medical School (NIH/NIGMS R01- GM084947) and the National Institute of Genetics Fly Stock Center for providing antibodies or fly stocks, as well as the Microscopy Rennes Imaging Center. The monoclonal antibody generated by S. Artavanis Tsakonas (NECD) was obtained from the Developmental Studies Hybridoma Bank, generated under the auspices of the National Institute of Child Health and Human Development, and maintained by the University of Iowa Department of Biological Sciences. We thank members of the Le Borgne laboratory for helpful discussions. We thank A. Pacquelet and G. Michaux for critical reading of the manuscript. Special thanks to Amy Winehouse for her music that accompanied us, while we screened around flies. This work was supported in part by the Action Thématique Incitative Prioritaire programme CNRS, Région Bretagne (Programme Accueil de COMpétences en Bretagne Notasid n 2168), ARC n 4905, Fondation pour la Recherche Médicale, Rennes Métropole, and La Ligue contre le Cancer 35.

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24 Nichols, J. T., Miyamoto, A., Olsen, S. L., D'Souza, B., Yao, C., and Weinmaster, G. (2007). DSL ligand endocytosis physically dissociates Notch1 heterodimers before activating proteolysis can occur. J Cell Biol 176, Nuoffer, C., Wu, S. K., Dascher, C. and Balch, W. E. (1997). Mss4 does not function as an exchange factor for Rab in endoplasmic reticulum to Golgi transport. Mol Biol Cell 8, O'Connor-Giles, K. M., and Skeath, J. B. (2003). Numb inhibits membrane localization of Sanpodo, a four-pass transmembrane protein, to promote asymmetric divisions in Drosophila. Dev Cell 5, Panin, V. M., Shao, L., Lei, L., Moloney, D. J., Irvine, K. D., and Haltiwanger, R. S. (2002). Notch ligands are substrates for protein O-fucosyltransferase-1 and Fringe. J Biol Chem 277, Pi, H., Wu, H. J., and Chien, C. T. (2001). A dual function of phyllopod in Drosophila external sensory organ development: cell fate specification of sensory organ precursor and its progeny. Development 128, Rajan, A., Tien, A. C., Haueter, C. M., Schulze, K. L., and Bellen, H. J. (2009). The Arp2/3 complex and WASp are required for apical trafficking of Delta into microvilli during cell fate specification of sensory organ precursors. Nat Cell Biol 11, Roegiers, F., Jan, L. Y., and Jan, Y. N. (2005). Regulation of membrane localization of Sanpodo by lethal giant larvae and neuralized in asymmetrically dividing cells of Drosophila sensory organs. Mol Biol Cell 16, Sakata, T., Sakaguchi, H., Tsuda, L., Higashitani, A., Aigaki, T., Matsuno, K., and Hayashi, S. (2004). Drosophila Nedd4 regulates endocytosis of notch and suppresses its ligand-independent activation. Curr Biol 14, Sasamura, T., Ishikawa, H. O., Sasaki, N., Higashi, S., Kanai, M., Nakao, S., Ayukawa, T., Aigaki, T., Noda, K., Miyoshi, E., et al. (2007). The O-fucosyltransferase O-fut1 is an extracellular component that is essential for the constitutive endocytic trafficking of Notch in Drosophila. Development 134, Shi, A., Sun, L., Banerjee, R., Tobin, M., Zhang, Y., and Grant, B. D. (2009). Regulation of endosomal clathrin and retromer-mediated endosome to Golgi retrograde transport by the J-domain protein RME-8. Embo J 28, Simons, K., and Gerl, M.J. (2010). Revitalizing membrane rafts: new tools and insights. Nat Rev Mol Cell Biol 11,

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26 Table and figure legends Figure 1: Sensory organ lineage and screen results. A. Scheme of the adult sensory organ formed by two external cells (shaft and socket) and two internal cells (sheath and neuron). B. Scheme of the cell precursor pi pupal lineage leading to the specification of the adult sensory organ cells and one apoptotic glial cell after four asymmetric cell divisions. In A and B, blue nuclei represent Notch signaling responding cells and red nuclei represent Notch signaling sending cells. C-F. Examples of Notch-like bristle phenotype screened for, in the dsrna genetic screen induced in Drosophila melanogaster notum. D, F. Scheme of putative pi pupal lineages in case of a loss (D ) or gain (F ) of Notch signaling in all or some of the asymmetric cell divisions. G. Numbers of candidates with dsrna-induced adult phenotypes for each screen category (dark grey box: candidates with phenotype; light grey box: known Notch regulators with phenotype). Numbers into brackets indicate candidate genes / known Notch regulators screened in each category. Figure 2: Sanpodo, Delta and Notch specific steady-state pattern of localization. Localization of Sanpodo (Spdo, green), Delta (red) and Notch (blue) in wild-type pi dividing cell (A-C ) and at the pi daughter cell-stage (D-F ). (A-A ; D-D ) show apical and (B- B ; E-E ) basal confocal slices. (C-C ; F-F ) show orthogonal sections of cells from (A- B ; D-E ) respectively. The asymmetric localization of Spdo in endosomes in the anterior cell, and in endosomes and at the basolateral cortex in the posterior cell reflects the differential cell identity of the pi daughter cells (E ). In all panels, anterior is left and scale bar is 5 μm. Figure 3: Examples of accumulation at pi daughter cell contact. Localization of Sanpodo (Spdo, green), Delta (red) and Notch (blue) in pi daughter cells of Chmp dsrna (A-B ), CG10341 dsrna (C-D ) and Cullin-3 dsrna (Cul-3, E-F ). (A-A, C- C ) show apical and (E-E ) basal confocal slices. (B-B ), (D-D ) and (F-F ) show orthogonal sections of cells from (A-A ), (C-C ) and (E-E ) respectively. In Chmp dsrna, Spdo is accumulated subapically (B, arrowhead) and at the apical interface (A, arrow), between the two daughter cells, with Delta and Notch (A -A, arrows). In CG10341 dsrna, Spdo, Delta and Notch are accumulated at the apical interface between the two daughter cells (C -C and D -D, arrows). In Cullin-3 dsrna, Spdo and Delta are specifically accumulated at the lateral membrane between the pi daughter cells (E -E and F -F, arrows). In all

27 panels, anterior is left and scale bar is 5 μm. Figure 4: Examples of vesicles excess in pi daughter cells. Localization of Sanpodo (Spdo, green), Delta (red) and Notch (blue) in pi daughter cells of Vha16-2 dsrna (A-B ), Vps4 dsrna (C-D ) and l(2)dtl dsrna (E-F ). (A-A ), (C-C ), (E- E ) show apical and (B-B ), (D-D ), (F-F ) basal confocal slices. In Vha16-2 dsrna, Spdo is found in basal vesicles in both daughter cells (B, arrows), a lineage reminiscent of two piib-like cells. In Vps4 dsrna, Spdo, Delta and Notch colocalize in enlarged basal vesicles in both daughter cells (D -D, arrows). In l(2)dtl dsrna, an excess of Spdo, Delta and Notch positive basal compartments is observed in the the anterior cell (F -F, arrows). In all panels, anterior is left and scale bar is 5 μm. Figure 5: Examples of apical and/or basal accumulation. Localization of Sanpodo (green), Delta (red) and Notch (blue) in pi daughter cells of Exo84 dsrna (A-A ), Rab35 dsrna (B-C ) and Tsp68C dsrna (D-D ). (B-B ) show apical and (A-A ), (C-C ), (D-D ) basal confocal slices. In Exo84 dsrna, Spdo and Delta are found at the basolateral membrane or in close vicinity to the plasma membrane of both daughter cells (A -A, arrows), a lineage somewhat reminiscent of two piia-like cells. In Rab35 dsrna, Spdo, Delta and Notch are accumulated at the apical side of the anterior daughter cell (B -B, arrowheads). Spdo is also found in basal vesicles in both daughter cells (C, arrows), a lineage reminiscent of two piib-like cells. In Tsp68C dsrna, Delta can be found at the basolateral cortex of epidermal cells (D, arrowheads). In all panels, anterior is left and scale bar is 5 μm. Figure 6: CG2747 regulates clathrin adaptor AP-1 intracellular localization. (A-B ). Localization of Sanpodo (Spdo, green), Delta (red) and Notch (blue) in pi daughter cells of CG2747 dsrna. (A-A ) show apical confocal slice and (B-B ) show orthogonal section of cells from (A-A ). Spdo is accumulated subapically (B, arrowhead) and at the interface between the two daughter cells with Delta and Notch (A -A, arrows). (C). Excess of double shafts with sockets cells (arrows) observed on a CG2747 dsrna notum, adult phenotype reminiscent of gain of Notch signaling. (D-F ). Partial loss of HEATR5B staining (p200, green) in the median part of the notum where UAS-CG2747 dsrna is induced by ap- GAL4 (D-D left side, E-E ) compared to the posterior part of the notum where ap-gal4

28 does not drive UAS expression (D-D right side, star, F-F ). (E-F ) panels are higher magnifications of (D-D ) and taken at the level of adherens junctions (DE-CAD, red in E-E and F-F ). (G). Loss of AP-1γ staining in the median part of the notum where UAS- CG2747 dsrna is induced by ap-gal4 (right side) compared to the anterior head in which ap- GAL4 does not drive UAS expression (left side, star). (H-H ). HEATR5B staining is not affected in AP-47 SHE11 mutant cells (red, H and H, inside dotted lines). Mutant cells are identified by the absence of nls-gfp (H, blue, inside dotted line). H-H are confocal slices taken at the level of adherens junctions (DE-CAD, green in H and H ). In panels (A-B, D-H ), anterior is left. Scale bar is 5 μm in (A-B, G), 200 μm in (D-D ) and 15 μm in (E-F, H-H ). Table 1: Complete list of positive hits from the genetic screen. This table contains all positive genes identified in the genetic dsrna screen and listed in order of the screen category they have been initially selected in. The notum adult phenotypes are sub-divided into two major processes controlled by Notch signaling: lateral inhibition regulating the number of sensory organ precursors specified, and sensory organ lineage, which controls the morphology of the adult organs. The adult phenotypes are indicated as LOF pi (excess of organs resulting from a lateral inhibition defect), LOF (loss of Notch signaling-like phenotypes) and/or GOF (gain of Notch signaling-like phenotypes). n.o. : no phenotype observed. n.d. : phenotype could not be determined because of bald cuticle (phenotype of bristle loss). * : lateral inhibition defect detected on pupal notum by immunostaining Table 2: Complete list of genes with a defect in Notch, Sanpodo and/or Delta subcellular localization at the pi daughter cell-stage after dsrna induction. This table contains all positive genes with a Notch, Sanpodo and/or Delta localization changes and listed in order of the screen category they belong to.

29 A. B. Apoptotic glial cell Shaft Cuticle Socket Sheath pi piib piiib Neuron Sheath Shaft Neuron piia Socket C. Wild type D. Loss of bristles D. piib or Journal of Cell Science Accepted manuscript E. Excess of bristles F. Excess of socket cells G. Coat components and accessory proteins Lipid microdomain organization / Cytoskeleton, regulators or interactors Small GTPases, GEF/GAP or effectors Ubiquitination / Deubiquitination (8/6) ESCRT components and interactors (7/9) Membrane recognition and/or fusion (30/2) ATPases (17/1) Various (67/11) Cell polarity (13/2) Novel unknown function (54/4) F. 4 pi or pi or pi pi piib 5 piia piia piib piia 10 piib piia or or or 3 3 or or or or

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