Genetic identification of intracellular trafficking regulators involved in Notch-dependent binary...

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Genetic identification of intracellular traffickingregulators involved in Notch-dependent binary cell fateacquisition following asymmetric cell division

Stephanie Le Bras1,2,*, Christine Rondanino1,2,`, Geraldine Kriegel-Taki1,2, Aurore Dussert1,2,§ andRoland Le Borgne1,2,*1CNRS, UMR 6061, Institut Genetique et Developpement de Rennes, 35043 Rennes, France2Universite Rennes 1, UEB, IFR 140, Faculte de Medecine, 35043 Rennes, France`Present addresses: GReD Laboratory, CNRS UMR 6293, INSERM U1103, Clermont Universite, 63177 Aubiere, France; Universite d’Auvergne, Faculte de Medecine, 63000Clermont-Ferrand, France§Present address: Department of Developmental and Regenerative Biology, Mount Sinai School of Medicine, New York, USA*Authors for correspondence ([email protected]; [email protected])

Accepted 12 June 2012Journal of Cell Science 125, 4886–4901� 2012. Published by The Company of Biologists Ltddoi: 10.1242/jcs.110171

SummaryNotch signalling is involved in numerous cellular processes during development and throughout adult life. Although ligands andreceptors are largely expressed in the whole organism, activation of Notch receptors only takes place in a subset of cells and/or tissuesand 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 thespatiotemporal regulation are largely unknown. In order to identify intracellular trafficking regulators of Notch signalling, we haveundertaken 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 Notchsignalling regulates binary cell fate acquisition. Gain or loss of Notch signalling phenotypes were observed in adult sensory organs for113 of them. Furthermore, 26 genes were found to regulate the steady state localisation of Notch, Sanpodo, a Notch co-factor, and/orDelta 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, anegative regulator of Notch signalling. Together, our results further demonstrate the essential function of intracellular trafficking inregulating 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

IntroductionNotch cell-cell signalling is required in a vast majority ofdevelopmental processes and during the adult life of manyorganisms. It regulates cell fate specification as well as stem cell

behaviour and defects can lead to numerous developmentalpathologies and cancers underlying its crucial role (reviewed byGridley, 2003; Miele et al., 2006). The challenging question is to

understand the mechanisms allowing one cell to act as asignalling cell and the other one as the receiving cell, whenboth cells can potentially express both ligands and receptors.

Although it can be performed through a spatial and temporalregulation of their expression, DSL (Delta, Serrate, Lag2) ligandand Notch receptor differential expression could not be sufficient

to explain the subtle directionality of Notch signalling. In thiscontext, regulation of the availability of both receptors and DSLligands at the cell surface appears crucial to ensure a properNotch signalling activation. Therefore ligand and receptor post-

translational modifications and trafficking are emerging ascrucial regulatory mechanisms.

Several lines of evidence suggest that endocytic trafficking ofDSL ligands enhances their signalling activity while receptor

trafficking insures their steady state level at the cell surface

thereby regulating their availability for ligand binding (reviewedby Bray, 2006; Furthauer and Gonzalez-Gaitan, 2009; Kopan andIlagan, 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 thismaturation is still poorly characterised and two models areactually 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 isthe only pathway involved (Heitzler and Simpson, 1991),represents an interesting study model in which the signalsending and receiving cells are easily distinguishable.

Each sensory organ, present on the adult D. melanogaster

notum, is derived from a single precursor cell (pI), whichundergoes a stereotyped series of four asymmetric cell divisions

4886 Research Article

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to generate five different cells, four composing the

mechanosensory bristle and a glial cell (Fig. 1A,B). During each

division, Notch signalling is involved in cell fate acquisition. For

example, Notch is inhibited in the pI daughter cell, which adopts

the pIIb cell identity and eventually activates Notch signalling in

the adjacent daughter cell becoming the pIIa cell. Although data

from different laboratories have emphasised the role of

intracellular trafficking in the uni-directionality of Notch

signalling 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 emphasised 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

Neuralized (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 signalling activation. While in the receiving cell, the

clathrin adaptor complex AP-1 was genetically shown to be

required for the correct localisation of Notch and Spdo (Benhra

et al., 2011).

In order to identify novel regulators of the intracellular

trafficking of Notch signalling major components, we have

undertaken a tissue-specific double-strand RNA (dsRNA) genetic

screen of 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

signalling 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 signalling directly affects the development

of external sensory organs and therefore allows for adult

phenotype screening (Hartenstein and Posakony, 1990). Among

the 113 Notch regulators identified based on adult phenotype, 61

were screened for, and 26 were found to cause a change in the

steady state localisation 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

localisation, CG7787 putatively involved in the recycling

pathway and members of the Tetraspanin family.

Fig. 1. Sensory organ lineage and screen results. (A) Diagram

of the adult sensory organ composed of 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 indicate cells

responding to Notch signalling and red nuclei indicate cells

sending Notch signals. (C–F). Examples of Notch-like bristle

phenotype screened for, in the dsRNA genetic screen induced in the

Drosophila notum. (D9,F9) Scheme of putative pI pupal lineages in

case of a loss (D9) or gain (F9) of Notch signalling 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.

Notch intracellular trafficking regulators 4887

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ResultsPrinciple and validation of gene silencing-inducible screen

To screen specifically in the sensory organ lineage, we made use of

a well-characterised 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 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 induces gene

silencing in the fly notum during sensory organ development. For

each cross, two experimenters analysed at least 20 F1 progenies

blindly. In order to identify specific regulators of Notch signalling,

we scored for bristle phenotype on the notum (Fig. 1D–F). While a

loss of bristle and/or double shafts without a socket cell reflects a

loss of Notch signalling in the sensory organ lineage (Fig. 1D,D9),

an excess of socket cells and/or double shafts with socket cells is

correlated with a gain of Notch signalling in the sensory organ

lineage (Fig. 1F,F9). As Notch signalling is also involved in the

process of pI specification, we could, additionally, score for an

excess of sensory organs reflecting a loss of Notch signalling in

lateral inhibition (Fig. 1E). A genome-wide dsRNA screen was

previously performed to identify regulators of Notch signalling in

the sensory organs in which one sensory organ driver-GAL4 was

used: 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, 25 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 bias of GAL4–UAS-induced phenotype

and further describe the Notch-like phenotype, we independently

used 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 signalling-specific strategy

(Knoblich, 2010), we choose to screen 50 known Notch

signalling regulators and observe the same phenotype than

previously described for 24 of them (see supplementary material

Table S1). Although we could not 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 specific

screening 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 signalling regulators appear to be involved in the

Drosophila sensory organ lineage, which further highlights

the cellular context dependence of Notch signalling 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 signalling in the Drosophila

wing vein (Mukherjee et al., 2005; Sakata et al., 2004).

Identification of Notch signalling regulators

To identify intracellular trafficking regulators of Notch signallingin Drosophila melanogaster sensory organs, we specificallyscreened for 368 genes from the endolysosomal pathway (seesupplementary material Table S2). We selected these genes among

members of intracellular trafficking regulator families mostlyidentified from yeast genetics and involved in different traffickingaspects such as, coat components [clathrin mediated endocytosis

(Maldonado-Baez and Wendland, 2006)], lipid microdomainorganisation [non-clathrin mediated endocytosis (Simons andGerl, 2010)], cytoskeleton [actin, myosin and/or microtubules

(Hehnly and Stamnes, 2007)], small GTPases, ubiquitylation/deubiquitylation factors involved in vesicle targeting (Murphyet al., 2009; Wennerberg et al., 2005), Endosomal SortingComplex Required for Transport (ESCRT) complexes (Henne

et al., 2011), membrane recognition and/or fusion regulators [suchas SNAP receptors, SNAREs (Malsam et al., 2008), Exocyst (Hsuet al., 2004)] and ATPases (Forgac, 2007). We also based our

selection on gene ontology (GO) annotation from FlyBase (usingthe GO terms: endocytosis, endosomal sorting, secretion) andselected putative orthologue(s) of traffic regulators identified in a

Caenorhabditis elegans genetic screen (Balklava et al., 2007) ormammal 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 D. melanogaster (‘novel unknown function’category in supplementary material Table S2).

To perform our screen we used 716 dsRNA lines, whichrepresent the 368 candidates and 50 known Notch regulators, as we

systematically screened with up to five different dsRNA lines,when available, in order to circumvent any effect due to thedsRNA construct insertion site. Expression of 264/716 (36.8%)

dsRNA lines induced a bristle phenotype or lethality with eitherboth or one of the GAL4 lines in our screen conditions. When twoor more dsRNA lines induced a bristle phenotype, it wasconsistently the same gain or loss of Notch signalling

phenotype(s), depending on the candidate, as we never observedopposite bristle phenotype between different dsRNA lines againstthe same candidate.

In order to confirm and validate the results, we reproduced thescreen, with all the positive and lethal dsRNA hits and somenegative ones as controls, using the same GAL4 lines and up totwo 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 severalGAL4 lines, we observed that 175/264 (66.3%) dsRNA lines,

representing 113 candidates, induced a reproducible bristlephenotype with one or more GAL4 (see supplementarymaterial Table S2). To further validate our results, we had

included 52 dsRNA lines, obtained from the National Institute ofGenetics Fly Stock Center (NIG-Fly) or the Transgenic RNAiProject (TRIP), which target different part of the candidate RNA

sequence than the dsRNA lines from the Vienna DrosophilaRNAi Center (VDRC). In doing so, we confirmed the specificNotch-like bristle or lack of phenotype observed with the VDRCdsRNA lines (see supplementary material Table S2).

In the end, we firmly identified 113 Notch regulators in thesensory organ lineage (Table 1), which belong to the differentscreening categories that we initially defined (Fig. 1G).

Specifically, we identified 77 previously unknown regulators ofthe Notch signalling pathway with a role in the sensory organlineage. These regulators belong to all our initial screen categories,

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Table 1. Complete list of positive hits from the genetic dsRNA screen

Screen category/molecular function CG number Name

Notum adult phenotype

Excess of organs: lateralinhibition defect [Notch

signalling loss-of-function(LOF)-like]

Sensory organ defect: Notchsignalling gain-of-function(GOF)- or loss-of-function

(LOF)-like phenotypes

Coat components and accessory proteinsClathrin complex CG6948 Clathrin light chain LOF: excess of organs

(weak)n.o.

Clathrin binding CG2520 like-AP180 LOF: excess of organs(weak)

n.o.

Clathrin adaptor complex (AP-1) CG9113 AP-1c n.o. GOF: excess of socket cells,double shafts with socket cell

Clathrin adaptor complex (AP-1) CG5864 AP-1s n.o. GOF: excess of socket cells,double shafts with socket cell

Clathrin adaptor complex (AP-1) CG9388 AP-47 n.o. GOF: excess of socket cells,double shafts with socket cell

Clathrin adaptor complex (AP-2) CG7057 AP-50 n.o. GOF: excess of socket cells,double shafts with socket cell

Clathrin adaptor complex (AP-2) CG4260 a-adaptin n.o. GOF: excess of socket cells,double shafts with socket cell

Clathrin adaptor complex (AP-1 and AP-2) CG12532 b-adaptin n.o. GOF: excess of socket cellsUnknown (AP-1 accessory protein: putative

orthologue of S. cerevisiae Laa1p)CG2747 CG2747 n.o. GOF: excess of socket cells,

double shafts with socket cellLipid microdomain organization/interactors

Multispan membrane protein withunknown function

CG7740 prominin-like n.d. LOF: bristle loss

Tetraspanin CG10742 Tetraspanin 3A LOF: excess of organs(weak)

n.o.

Tetraspanin CG32136 Tetraspanin 68C LOF: excess of organs* LOF: bristle lossTetraspanin CG9496 Tetraspanin 29 Fb n.d. LOF: bristle lossTetraspanin CG12847 Tetraspanin 42Ec n.d. LOF: bristle lossTetraspanin CG12143 Tetraspanin 42Ej n.d. LOF: bristle lossTetraspanin CG12841 Tetraspanin 42Ek n.d. LOF: bristle lossTetraspanin CG9033 Tetraspanin 47F n.d.* LOF: bristle lossTetraspanin CG9494 Tetraspanin 29 Fa n.o. GOF: excess of socket cells

Cytoskeleton, regulators or interactorsActin binding CG5695 Jaguar n.d. GOF: excess of socket cellsActin binding CG1520 WASp n.d. LOF: bristle lossActin binding; microtubule-associated protein CG6450 lava lamp LOF: excess of organs n.o.WASp activation complex CG4931 specifically Rac1-associated

protein 1/CYFIPLOF: excess of organs* LOF: bristle loss

Arp2/3 actin binding complex CG4560 Arpc3A LOF: excess of organs n.o.Arp2/3 actin binding complex CG10954 Arc-p34 LOF: excess of organs LOF: bristle loss, double shaftsArp2/3 actin binding complex CG8978 Suppressor of profilin

2/Arpc1n.d. LOF: bristle loss

GTPase activity; actin binding,microtubule-associated protein

CG8705 peanut n.d. LOF: bristle loss

GTPase activity; microtubule-associated protein CG4173 Septin 2 n.d. LOF: bristle lossGTPase activity; microtubule-associated protein CG2916 Septin 5 n.d.* LOF: bristle lossDNA processing (replication) CG1584 Origin recognition

complex subunit 6n.d. LOF: bristle loss, double shafts

without socket cellRNA processing (Transcription factor) CG7238 Septin interacting protein 1 n.d. LOF: bristle lossDynein complex CG6998 cut up n.d. LOF: bristle lossEpsin-like CG42250 liquid facets-Related n.d.* LOF: bristle lossMicrotubule-associated protein CG3265 Eb1 n.d. LOF: bristle loss

Small GTPases, GEF/GAP or effectorsGTPase activity (Rab) CG17515 Rab21 LOF: excess of organs* LOF: bristle lossGTPase activity (Rab) CG9575 Rab35 LOF: excess of organs

(weak)LOF: bristle loss

GTPase activity (Rab) CG5771 Rab-protein 11 n.d. LOF: bristle lossGTPase activity (Ras) CG8416 Rho1 n.d. LOF: bristle lossGTPase activity (ARF) CG11027 ADP ribosylation factor 102F n.d.* LOF: bristle lossGTPase activity (ARF) CG7435 ADP ribosylation factor 84F n.o.* GOF: excess of socket cellsGTPase (Rap) activator activity (GAP) CG6975 gigas LOF: excess of organs

(weak)LOF: bristle loss

GTPase (Rho) activator activity (GAP) CG13345 tumbleweed n.d. LOF: bristle lossGuanyl-nucleotide exchange factor activity (GEF) CG7787 CG7787 LOF: excess of organs* LOF: bristle loss, double shaftsGuanyl-nucleotide exchange factor activity (GEF) CG15797 ric8a LOF: excess of organs n.o.

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Guanyl-nucleotide exchange factor activity (GEF) CG9491 Gef26 LOF: excess of organs(weak)

LOF: bristle loss

Guanyl-nucleotide exchange factor activity(GEF)

CG31048 CG31048 n.d. LOF: bristle loss


CG32239 Guanine nucleotide exchangefactor GEF64C

n.d. LOF: bristle loss


CG8114 Pebble n.d. LOF: bristle loss

GTPase (Rab5) effector protein CG8506 Rabenosyn-5 n.d. LOF: bristle lossGTPase (Ras) binding CG10255 Lap1 n.d. LOF: bristle loss

Ubiquitylation/deubiquitylationUbiquitin ligase complex (SCF); microtubule-

associated proteinCG16983 skpA n.d. LOF: bristle loss, double shafts

Ubiquitin ligase complex (SCF) CG8881 skpB n.d. LOF: bristle lossUbiquitin ligase complex (SCF) CG12700 skpD n.d. LOF: bristle lossUbiquitin ligase complex (SCF) CG11942 skpE n.d. LOF: bristle lossUbiquitin ligase (E3) CG5841 mind bomb 1 LOF: excess of organs n.o.Ubiquitin ligase (E3) CG11861 Cullin-3/Guftagu n.d.* LOF: bristle loss, double shaftsUbiquitin ligase (E3) CG11988 neuralized n.d. LOF: bristle loss, double shafts

ESCRT components and interactorsVacuolar protein sorting (Class E/ESCRT-0) CG2903 Hepatocyte growth factor

regulated tyrosine kinasesubstrate

n.d. LOF: bristle loss, double shafts

Vacuolar protein sorting (Class E/ESCRT-0) CG6521 Signal transducing adaptormolecule

LOF: excess of organs* GOF: excess of socket cells?

Vacuolar protein sorting (Class E/ESCRT-I) CG12770 Vacuolar protein sorting 28 n.o. GOF: excess of socket cellsVacuolar protein sorting (Class E/ESCRT-II) CG14750 Vacuolar protein sorting 25 LOF: excess of organs LOF: bristle lossVacuolar protein sorting (Class E/ESCRT-III) CG4071 Vacuolar protein sorting 20 LOF: excess of organs LOF: bristle lossVacuolar protein sorting (Class E/ESCRT-III) CG4108 Chmp1 LOF: excess of organs GOF: excess of socket cellsVacuolar protein sorting (Class E/ESCRT-III) CG14542 Vps2 LOF: excess of organs* GOF: excess of socket cells,

double shaftsATPase CG6842 Vacuolar protein sorting 4 n.o. GOF: excess of socket cells?

Membrane recognition and/or fusionExocyst complex CG6095 Exo84 LOF: excess of organs* LOF: bristle loss and GOF:

excess of socket cells, doubleshafts

Exocyst complex CG8843 Sec5 n.d.* GOF: double shafts with socketcell, loss of shaft cells

Exocyst complex CG5341 Sec6 n.d.* GOF: loss of shaft cellsExocyst complex CG7034 Sec15 LOF: excess of organs* LOF: bristle lossSNAP receptor activity (SNARE) CG1515 lethal (1) G0155 n.d.* LOF: bristle lossSNAP receptor activity (SNARE) CG4109 Syntaxin 8 n.d. LOF: bristle lossSNAP receptor activity (v-SNARE) CG12210 synaptobrevin n.d. LOF: bristle loss, double shaftsSNAP receptor activity (t-SNARE) CG5081 Syntaxin 7/Avalanche LOF: excess of organs GOF: excess of socket cells

ATPasesATPase CG9931 NEM-sensitive fusion protein 2 n.d.* LOF: bristle lossVacuolar ATPase (subunit c) CG3161 Vacuolar H+ ATPase subunit

16-1n.d. LOF: bristle loss

Vacuolar ATPase (subunit c) CG32089 Vacuolar H+ ATPase subunit16-2


Vacuolar ATPase (subunit c9) CG7007 Vacuolar H+ ATPase subunitPPA1-1

LOF: excess of organs LOF: bristle loss

Vacuolar ATPase (subunit A) CG3762 Vacuolar H+ ATPase subunit68-2


Vacuolar ATPase (subunit E) CG1088 Vacuolar H+ ATPase 26kD Esubunit


Vacuolar ATPase (subunit G) CG6213 Vacuolar H+ ATPase G-subunit n.d. LOF: bristle lossVarious

Calcium channel activity CG34146 bruchpilot n.d. LOF: bristle lossCalcium ion binding CG9126 Stromal interaction molecule n.d.* LOF: bristle loss, double shaftsCatalytic activity: Notch fucosyltransferase CG12366 O-fucosyltransferase 1 n.d.* LOF: bristle loss, double shaftsCatalytic activity: Notch glucosyltransferase CG31152 rumi n.d. LOF: bristle loss

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which cover various aspects of intracellular trafficking. The vast

majority of the observed phenotypes resemble those of a loss of

Notch signalling. Nevertheless, phenotypes similar to gain-of-

Notch signalling 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

signalling 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 signalling via its major components.

Steady-state localisation of Notch, Sanpodo, Delta and cell

fate identity

A Notch-like adult sensory organ phenotype could be due to a

defect in Notch signalling 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

signalling regulators in the sensory organ lineage, we wanted to

identify those involved in the intracellular trafficking of the major

components of Notch signalling: Delta, Notch and its co-factor

Spdo. This study was made feasible as they present a specific

steady-state pattern of subcellular localisation in the sensory organ

pupal lineage during pI mitosis and at the pI daughter cell stage







Catalytic activity: Notch c-secretase CG18803 presenilin n.d. LOF: bristle lossCatalytic activity: histone methyltransferase CG10955 Rtf1 n.d. LOF: bristle lossGuanylate protein kinase CG31349 polychaetoid LOF: excess of organs n.o.Serine/threonine protein kinase; chromosomal

passenger complex (CPC)CG6620 IplI-aurora-like kinase/Aurora B n.d. LOF: bristle loss

Protein binding: TGFb CG14026 thickveins LOF: excess of organs n.o.Protein binding CG9695 Disabled LOF: excess of organs n.o.Protein binding: Notch ligand activity CG3619 Delta LOF: excess of organs LOF: bristle lossProtein binding: Notch receptor activity CG3936 Notch n.d. LOF: bristle lossProtein binding: Notch CG31020 Sanpodo n.d. LOF: bristle loss, double shaftsProtein binding CG9834 endophilin B n.d. LOF: bristle lossProtein binding CG5820 Gp150 n.d. LOF: bristle loss

Cell polarityMyosin complex CG15792 zipper n.d. LOF: bristle lossMyosin binding CG2671 lethal (2) giant larvae n.o. GOF: excess of socket cellsProtein binding CG5055 bazooka n.d. LOF: bristle lossProtein binding CG6383 crumbs n.d. LOF: bristle lossProtein binding CG5884 par-6 n.o. GOF: excess of socket cellsSerine/threonine protein kinase CG3068 Aurora/aurora A LOF: excess of organs GOF: double shafts with socket

cellNovel unknown function

DNA processing (DNA helicase) CG18013 Psf2 n.d. LOF: bristle loss, double shaftswithout socket cell

Microtubule-associated protein CG8014 Receptor mediated endocytosis8


Protein binding CG6834 CG6834 n.d. LOF: bristle lossProtein binding: Notch CG2863 Notchless n.d. LOF: bristle lossRNA processing (snoRNA binding) CG10341 CG10341 n.o. GOF: double shafts with socket

cell, loss of shaft cellsRNA processing (transcription factor binding) CG7583 C-terminal binding protein n.o. GOF: excess of socket cellsRNA processing (transcription factor) CG5461 bunched n.d. LOF: bristle loss (weak)RNA processing (transcription factor) CG4882 CG4882 n.d. LOF: bristle lossRNA processing (transcription factor) CG4029 jumeau n.d. LOF: bristle loss, double shaftsRNA processing (translation elongation

factor); microtubule-associated proteinCG11901 Ef1c n.d. LOF: bristle loss

Unknown CG11295 lethal-(2)-denticleless n.d. LOF: bristle lossUnknown CG8435 CG8435 n.d.* LOF: bristle lossUnknown CG8639 Cirl n.d. LOF: bristle loss

Genes are listed in order of the screen category that they were initially selected in. The notum adult phenotypes are sub-divided into two major processescontrolled by Notch signalling: lateral inhibition regulating the number of sensory organ precursors specified, and sensory organ lineage, which controls themorphology of the adult organs. The adult phenotypes are indicated as LOF pI (excess of organs resulting from a lateral inhibition defect), LOF (loss of Notchsignalling-like phenotypes) and/or GOF (gain of Notch signalling-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 onpupal notum by immunostaining.

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(Fig. 2A–F90) (also see Benhra et al., 2010). In a wild-type lineage,

while both apical Delta and Notch are mostly localised at the

cortex (Fig. 2A0,A90, D0,D90), basolateral Delta is found in vesicles

in mitotic pI and pIIb/pIIa cells (Fig. 2B0,C0,E0,F0). Spdo has a

more dynamic pattern of localisation: cytoplasmic in the mitotic pI

(Fig. 2A9–C9), its localisation becomes asymmetric in the pI

daughter cells. While Spdo is enriched along the apicobasal

interface of pI daughter cells (Fig. 2D9–F9), Spdo is mostly

localised in vesicles in the anterior pIIb cell but at the basolateral

plasma membrane in the posterior pIIa cell (Fig. 2E9). Changes in

Notch, Spdo and/or Delta localisation 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 signalling 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 subcellular localisation changes and an adult gain of

Notch signalling phenotype (Benhra et al., 2011). Similarly, loss of

Neur, Sec15 or Arp2/3 functions induce changes in Spdo and/or

Delta subcellular localisation correlated with adult loss of Notch

signalling 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 subcellular

localisation identified in the screen

Among the 113 Notch regulators we identified, we decided to

analyse those that were not previously known to cause

subcellular localisation changes and/or that do not have

described function in cell polarity or in asymmetric cell

division (see supplementary material Table S3). Among the 61

genes that we screened for a dsRNA-induced change in Delta,

Notch and/or Spdo steady-state localisation (using one dsRNA

line for each), 32 did not cause any visible defect while three

genes (gigas, CG31048 and CG8435) caused 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 supplementary material Table

S3). Twenty-six genes caused a phenotype of Notch, Spdo and/or

Delta mis-localisation at the pI and/or pI daughter cell stage (we

used the threshold of at least three, out of 20 analysed, 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 3–5).

(1) Accumulation at pI daughter cell contact (Fig. 3). An excess

of Spdo is seen subapically between pI daughter cells of

CG2747dsRNA, Vacuolar protein sorting 28 (Vps28dsRNA) and

Chmp1dsRNA (Fig. 3B9, arrowhead; Fig. 6B9, arrowhead; and data

Fig. 2. The steady-state pattern of localisation of Sanpodo,

Delta and Notch. Localisation of Sanpodo (green), Delta (red)

and Notch (blue) in wild-type pI dividing cell (A–C90) and at

the pI daughter-cell stage (D–F90). A–A90, D–D90 show apical

and B–B90, E–E90 basal confocal slices. C–C90, F–F90 show

orthogonal sections of cells from A–B90 and D–E90,

respectively. The asymmetric localisation 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 (E9). In all panels, anterior

is left; scale bar: 5 mm.


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not shown). We also observed an accumulation of Spdo, Notch and

Delta at the apical interface between the pI daughter cells in

CG2747dsRNA, Signal transducing adaptor molecule (StamdsRNA),

Vps28dsRNA, Chmp1dsRNA, Vps2dsRNA and CG10341dsRNA (Fig. 3A9–

A90,C9–C90,D9–D90, arrows; Fig. 6A9–A90, arrows; and data not

shown). Finally, we detected an accumulation of Spdo and Delta at

the lateral membrane between pI daughter cells of CG7787dsRNA

and Cullin-3 (Guftagu/Cul-3dsRNA) (Fig. 3E9,E0,F9,F0, 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

Table 2. Complete list of genes that affect Notch, Sanpodo and/or Delta subcellular localisation at the pI daughter cell stage after

dsRNA induction

Screen category CandidateMolecularfunction

Adultsensoryorgan

phenotype2-cell stage localisation defects of Notch, Sanpodo and/or

Delta

Coat componentsand accessory proteins

CG2747 (putative orthologue ofSaccharomyces cerevisiae Laa1p)

AP-1 accessoryprotein

GOF Subapical accumulation of Spdo. Accumulationof Spdo, Notch and Delta at apical interface.

Lipid microdomainorganization

Tetraspanin 47F Tetraspanin LOF Cortex accumulation: Delta at basolateral cortex.

Tetraspanin 68C Tetraspanin LOF Cortex accumulation: Delta at basolateral cortex.Cytoskeleton,

regulators or interactorsspecifically Rac1-associated

protein 1 (Sra-1)/CYFIPWASp activation

complexLOF Lineage reminiscent to two pIIb-like cells.

Origin recognition complexsubunit 6 (Orc-6)

septin regulator LOF Lineage reminiscent to two pIIb-like cells.

peanut (pnut) septin LOF Lineage reminiscent to two pIIb-like cells.Septin 5 (Sep5) septin LOF Lineage reminiscent to two pIIb-like cells.Septin 2 (Sep2) septin LOF Lineage reminiscent to two pIIb-like cells. Cortex

accumulation: Spdo, Notch and Delta at apical cortex ofanterior cell.

Small GTPases,GEFs/GAPs

Rab35 Rab LOF Lineage reminiscent to two pIIb-like cells. Cortexaccumulation: Spdo, Notch and Delta at apical cortex ofanterior cell.

CG7787 RabGEF LOF Lateral accumulation of Spdo and Delta. Vesicle excess: largedonuts Spdo, Notch and Delta positive.

Ubiquitylation Cullin-3 (Cul-3)/Guftagu E3 ubiquitin ligase LOF Lateral accumulation of Spdo and Delta. Lineage reminiscentto two pIIb-like cells.

ESCRT componentsand interactors

Signal transducing adaptormolecule (Stam)

ESCRT O GOF ? Accumulation of Spdo, Notch and Delta at apical interface.Vesicle excess: Spdo, Notch and Delta positive. Lineagereminiscent to two pIIa-like cells.

Vacuolar proteinsorting 28 (Vps28)

ESCRT I GOF Subapical accumulation of Spdo. Accumulation of Spdo,Notch and Delta at apical interface. Weak vesicleexcess: Spdo, Notch and Delta positive.

Chmp1 ESCRT III GOF Subapical accumulation of Spdo. Accumulation of Spdo,Notch and Delta at apical interface. Weak vesicleexcess: Spdo, Notch and Delta positive.

Vps2 ESCRT III GOF Accumulation of Spdo, Notch and Delta at apicalinterface. Vesicle excess: large donuts Spdo, Notch andDelta positive.

Vacuolar protein sorting 4 (Vps4) ATPase GOF Vesicle excess: large donuts Spdo, Notch and Deltapositive.

Membrane recognitionand/or fusion

Syntaxin 7 (Syx7)/Avalanche t-SNARE GOF Lineage reminiscent to two pIIa-like cells.

Exo84 Exocyst complex LOF andGOF

Lineage reminiscent to two pIIa-like cells.

Sec6 Exocyst complex GOF Lineage reminiscent to two pIIa-like cells.Sec5 Exocyst complex GOF Lineage reminiscent to two pIIa-like cells.

Vacuolar ATPase Vacuolar H+ ATPasesubunit 16 -1 (Vha16-1)

vacuolar ATPase(Vo domain,subunit c)

LOF Lineage reminiscent to two pIIb-like cells.

Vacuolar H+ ATPasesubunit 16-2 (Vha16-2)

vacuolar ATPase(Vo domain,subunit c)

LOF Lineage reminiscent to two pIIb-like cells.

Various O-fucosyltransferase 1 Fucosyltransferase LOF Cortex accumulation: Delta at basolateral cortex.Novel unknown function CG10341 RNA biogenesis GOF Accumulation of Spdo, Notch and Delta at apical

interface.lethal-(2)-denticleless (l(2)dtl) unknown function LOF Vesicle excess in anterior cell (Spdo, Notch and Delta

positive).Recycling pathway,

unknown functionReceptor mediated

endocytosis 8 (Rme-8)Microtubule

associatedprotein

LOF Vesicle excess in anterior cell and toward the anterior inposterior cell (Spdo, Notch and Delta positive).

Genes are listed in order of the screen category they belong to.


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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-1dsRNA), Origin recognition complex subunit 6 (Orc-6dsRNA),

peanut (pnutdsRNA), Septin 5 (Sep5dsRNA), Septin 2 (Sep2dsRNA),

Rab35dsRNA, Cul-3dsRNA, Vha16-1dsRNA and Vha16-2dsRNA

(Fig. 4B9, arrows, Fig. 5C9, arrows, and data not shown). This

mislocalisation 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 CG7787dsRNA,

StamdsRNA, Vps2dsRNA or Vacuolar protein sorting 4 (Vps4dsRNA)

(Fig. 4D9–D90, arrows; and data not shown). Accumulation of

Spdo, Delta and Notch in enlarged intracellular compartments is

predominantly associated with Notch gain-of-function

phenotypes (Table 2). 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-8dsRNA), while only in the anterior cell in

l(2)dtldsRNA (Fig. 4F9–F90, 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 StamdsRNA,

Syntaxin 7 (Syx7dsRNA), Exo84dsRNA, Sec6dsRNA and Sec5dsRNA

(Fig. 5A9,A0, arrows; and data not shown). This mislocalisation

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 Sep2dsRNA and Rab35dsRNA (Fig. 5B9–B90,

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 [O-fut1dsRNA;

a known Notch trafficking regulator (Sasamura et al., 2007)],

Tetraspanin 47F (Tsp47FdsRNA) and Tetraspanin 68C

(Tsp68CdsRNA; Fig. 5D0, 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 signalling during pI specification.

This accumulation of Delta at the basolateral cortex could result

from either a decrease in Delta internalisation 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 labelling experiment (Benhra et al., 2010) to monitor Delta

internalisation 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

Fig. 3. Examples of accumulation at pI daughter cell

contacts. Localisation of Sanpodo (green), Delta (red) and Notch

(blue) in pI daughter cells of ChmpdsRNA (A–B90), CG10341dsRNA

(C–D90) and Cullin-3dsRNA (Cul-3; E–F90). A–A90, C–C90 show

apical, and E–E90 basal confocal slices. B–B90, D–D90 and F–F90

show orthogonal sections of cells from A–A90, C–C90 and E–E90,

respectively. In ChmpdsRNA, Spdo accumulates subapically

(B9, arrowhead) and at the apical interface (A9, arrow), between

the two daughter cells, with Delta and Notch (A0,A90, arrows). In

CG10341dsRNA, Spdo, Delta and Notch accumulate at the apical

interface between the two daughter cells (C9–C90 and D9–D90,

arrows). In Cullin-3dsRNA, Spdo and Delta specifically

accumulate at the lateral membrane between the pI daughter cells

(E9,E0 and F9,F0, arrows). In all panels, anterior is left; scale bar:

5 mm.


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Notch-signalling-dependent binary cell fate acquisition. Indeed,

we identified 26 genes for which a Notch signalling adult

phenotype is associated with a change in intracellular localisation

of major Notch signalling components after the first asymmetric

cell division.

CG2747 regulates clathrin adaptor AP-1 intracellular

localisation

Among the genes isolated in the genetic and cellular screen,

CG2747dsRNA phenocopies the loss of AP-1 function (Benhra

et al., 2011). Indeed, we observed a subapical accumulation of

Spdo (Fig. 6B9, arrowhead) and an accumulation of Spdo, Delta

and Notch at the apical interface between pI daughter cells

(Fig. 6A–B90, 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 1081–1612 and 407–906 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

signalling gain-of-function phenotype observed in AP-1 loss of

function (Benhra et al., 2011).

Although CG2747 function has not yet been studied in D.

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 c-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,F0). In the

area of a notum where CG2747dsRNA is induced, p200 staining is

greatly reduced (Fig. 6E,E0 compared to Fig. 6F,F0), suggesting

Fig. 4. Examples of excess vesicles in pI daughter cells.

Localisation of Sanpodo (green), Delta (red) and Notch (blue) in pI

daughter cells of Vha16-2dsRNA (A–B90), Vps4dsRNA (C–D90) and

l(2)dtldsRNA (E–F90). A–A90, C–C90, E–E90 show apical and B–B90,

D–D90 and F–F90 basal confocal slices. In Vha16-2dsRNA, Spdo is

found in basal vesicles in both daughter cells (B9, arrows), a lineage

reminiscent of two pIIb-like cells. In Vps4dsRNA, Spdo, Delta and

Notch colocalise in enlarged basal vesicles in both daughter cells

(D9–D90, arrows). In l(2)dtldsRNA, an excess of Spdo-, Delta- and

Notch-positive basal compartments is observed in the anterior cell

(F9–F90, arrows). In all panels, anterior is left; scale bar: 5 mm.


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that the mammal antibody recognises 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 localisation (Fernandez and Payne,

2006). To investigate whether CG2747 shares the same function

in D. melanogaster, we analysed the localisation of AP-1 on the

pupal notum. AP-1c staining is greatly reduced in the region of

the notum where UAS–CG2747dsRNA 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 mu

subunit of AP-1 complex) homozygote mutant cells (Fig. 6H–

H0). Thus CG2747 localisation is independent of AP-1 activity.

We concluded that CG2747dsRNA reduces the level of CG2747 at

a sufficient degree to prevent AP-1c membrane localisation and

phenocopies the AP-1 cellular and adult phenotypes. All these

results strongly support the model in which CG2747 is necessary

for AP-1 proper intracellular localisation and function in

Drosophila.

DiscussionOur dsRNA genetic screen, performed in D. melanogaster notum

using the GAL4–UAS system, allowed us to specifically identify

113 Notch signalling 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

interconnection between membrane traffic and both Delta and

Notch signalling to ultimately ensure the proper spatiotemporal

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 etc.). 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 signalling by intracellular

trafficking (for review, see Fortini and Bilder, 2009; Furthauer,

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

localisation of three major Notch signalling components:

Notch, Spdo and Delta. Indeed, the observed adult phenotypes

could result from various and multiple defects in Notch signalling

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 subcellular localisation via

Fig. 5. Examples of apical and/or basal accumulation.

Localisation of Sanpodo (green), Delta (red) and Notch (blue) in

pI daughter cells of Exo84dsRNA (A–A90), Rab35dsRNA (B–C90) and

Tsp68CdsRNA (D–D90). B–B90 show apical and A–A90, C–C90, D–

D90 basal confocal slices. In Exo84dsRNA, Spdo and Delta are

found at the basolateral membrane or in close proximity to the

plasma membrane of both daughter cells (A9–A0, arrows), a

lineage somewhat reminiscent of two pIIa-like cells. In

Rab35dsRNA, Spdo, Delta and Notch accumulate at the apical side

of the anterior daughter cell (B9–B90, arrowheads). Spdo is also

found in basal vesicles in both daughter cells (C9, arrows), a

lineage reminiscent of two pIIb-like cells. In Tsp68CdsRNA, Delta

can be found at the basolateral cortex of epidermal cells (D0,

arrowheads). In all panels, anterior is left; scale bar: 5 mm.


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endocytosis and/or recycling. Therefore, we decided to take

advantage of the fact that Spdo, Delta and Notch localisation are

dynamic during pI division and at the pI daughter cell stage in the

pupal notum. We were able to observe localisation 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 localisation 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 localisation in supplementary material Fig. S1).

For 10 genes, changes in Notch component localisation can be

a direct consequence of their inactivation 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 pIIb-like (Sra-1, Orc6, pnut, Sep5, Vha16-1,

Vha16-2) daughter cells (supplementary material Fig. S1B,F

respectively). 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 signalling regulation, they indicate that

Notch signalling 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 signalling in the

sensory organ lineage. Indeed, we observed a gain-of-Notch-

signalling-like adult phenotype for three Exocyst components

(Exo84, Sec6 and Sec5) but also a loss-of-Notch-signalling-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 signalling. 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).

For the first time, we identified members of the septin family

(pnut and Sep5) and one of their regulator [Orc-6 (Chesnokov

Fig. 6. CG2747 regulates clathrin adaptor AP-1 intracellular

localisation. (A–B90). Localisation of Sanpodo (green), Delta

(red) and Notch (blue) in pI daughter cells of CG2747dsRNA.

A–A90 show apical confocal slice and B–B90 show orthogonal

section of cells from A–A90. Spdo accumulates subapically

(B9, arrowhead) and at the interface between the two daughter

cells with Delta and Notch (A9–A90, arrows). (C) Excess of

double shafts with sockets cells (arrows) observed on a

CG2747dsRNA notum. This adult phenotype is reminiscent of gain

of Notch signalling. (D–F0). Partial loss of HEATR5B staining

(p200, green) in the median part of the notum where UAS–

CG2747dsRNA is induced by ap–GAL4 (D,D9 left side, E–E0)

compared with the posterior part of the notum where ap–GAL4

does not drive UAS expression (D,D9 right side, asterisk, F–F0).

E–F9 are higher magnifications of D–D9, taken at the level of

adherens junctions (DE-CAD, red in E,E9 and F,F9). (G) Loss of

AP-1c staining in the median part of the notum where UAS–

CG2747dsRNA is induced by ap–GAL4 (right side) compared with

the anterior head in which ap–GAL4 does not drive UAS

expression (left side, star). (H–H0) HEATR5B staining is not

affected in AP-47SHE11 mutant cells (red, H and H0, inside dotted

lines). Mutant cells are identified by the absence of nls–GFP (H,

blue, inside dotted line). H–H0 are confocal slices taken at the

level of adherens junctions (DE-CAD, green in H and H9). In

panels A–B90, D–H0, anterior is left. Scale bar: 5 mm (A–B90,G),

200 mm (D,D9) and 15 mm (E–F0,H–H0).


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et al., 2003)] as putative regulators of Notch signalling. Septincomplexes can act as scaffolds and/or diffusion barriers in

various cellular events such as cytoskeleton organisation,cytokinesis, membrane organisation and vesicle targeting whichcould potentially regulate the pI asymmetric cell division (for areview, see Cao et al., 2009). It is necessary to further decipher

the pupal phenotype to define if these septins directly regulateNotch signalling traffic and/or pI cytokinesis (N.B. Founounouand R.L.B. unpublished). While the pupal lineage is somewhat

reminiscent of two pIIb-like daughter cells, we also observed anaccumulation of Spdo, Notch and Delta at the apical side of theanterior pI daughter cell of Sep2dsRNA as well as of Rab35dsRNA

(supplementary material Fig. S1G). Their common phenotype isnot surprising knowing that human Rab35 was proposed to playan essential control on the terminal step of cytokinesis in part bycontrolling SEPT2 subcellular distribution during cell division

(Kouranti et al., 2006). Although it is not yet possible to decipherif this apical accumulation is a cause or a consequence of theNotch pupal and adult loss-of-function phenotype, this data

identify two putative regulators of apical localisation and confirmthat Notch signalling major components are differentiallytrafficked between pI daughter cells.

Inactivation of 14 genes led to localisation changes indicatingmultiple subcellular sites (plasma membrane, vesicularcompartments, interface between the two daughter cells), which

appear to be essential to the fine regulation of Notch signalling inthe Drosophila sensory organ lineage. Indeed, subapicalaccumulation of Spdo, localisation of Notch, Spdo and Delta atthe apical interface between pI daughter cells, and/or an excess of

enlarged endosomes in both daughter cells are associated withadult Notch gain-of-function-like phenotypes (supplementarymaterial Fig. S1C,D,E, respectively). However, accumulation of

Spdo and Delta at the lateral membrane between pI daughter cellscorrelates with Notch signalling loss-of-function phenotype(supplementary material Fig. S1H,I).

In a control situation, Notch accumulates transiently at theapical interface between pIIb and pIIa (Benhra et al., 2011;Couturier et al., 2012). Accumulation of Spdo together withNotch at this apical pIIb/pIIa interface has previously been

observed in AP-1 loss-of-function mutants and correlated with again of Notch signalling (Benhra et al., 2011). Because,impairment of Delta trafficking towards the pIIb/pIIa interface

in Arp2/3 mutants leads to a loss of Notch signalling (Rajan et al.,2009), it was proposed that Delta–Notch interaction resulting inNotch activation is taking place at the apical pIIb/pIIa interface

that could function as a signalling 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 taggedwith GFP and expressed at physiological level (NiGFP)(Couturier et al., 2012). Notch activation is reported to occur15–45 min after cytokinesis and productive signalling is

proposed to take place at the pIIa/pIIb interface of thecytokinetic furrow, where NiGFP accumulates in numb mutantbackground or when Dynamin-dependent endocytosis is blocked.

Adult and subcellular AP-1 loss-of-function-type phenotypesare observed when two genes with previously unknown functionin Drosophila, CG10341 and CG2747, were inactivated. Both of

them had been identified as putative membrane traffickingregulators 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 anindirect role, if so, in intracellular trafficking. On the contrary,

CG2747 belongs to the HEATR5 family and we were able toshow that CG2747 is required for the clathrin adaptor AP-1complex subcellular localisation, similarly to its putativeorthologue of the S. cerevisiae Laa1p (Fernandez and Payne,

2006). We observed that the human HEATR5B/p200 antibodystaining is affected in CG2747dsRNA, which supports anevolutionary conservation of the HEATR5 function among

metazoans. Nonetheless, the function of human HEATR5B/p200 remains unknown as no phenotype could be observed inp200-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 newregulator of Notch signalling that acts as a major regulator of theclathrin adaptor complex, AP-1.

We also observed an accumulation of Notch, Spdo and Delta atthe 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/orendosomes can result from a blockade in endosome maturation

when ESCRT function is impaired and it has already beendescribed that accumulation of Notch in endocytic compartmentscan result in an ectopic ligand-independent 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 deubiquitylation, maturation of multi

vesicular bodies, vesicle budding and/or cytokinesis (for areview, see Henne et al., 2011). In our screen, impairment ofdifferent ESCRT pathway components led to opposite adult

phenotypes i.e. loss- or gain-of-Notch-signalling-like onesdepending on the complex and/or its subunit(s) depleted bydsRNA (see Table 1). However, we did not observe any Spdo,

Delta or Notch localisation changes associated with these adultloss-of-function phenotypes. Further studies are, therefore,necessary to elucidate our genetics results by identifying whichsubcellular mechanisms and which cargo(es) are regulated by the

different identified ESCRT subunits to control Notch signalling.

Accumulation of Spdo and Delta at the lateral membranebetween pI daughter cells is correlated with adult Notch loss-of-

function phenotypes in Cul-3dsRNA and CG7787dsRNA

(supplementary material Fig. S1H,I respectively). This lateralmembrane was previously named apical actin-rich structure

‘stalk’ and identified as the lateral part of branched actin networkpresent at the interface and through which endocytosed Deltatraffic back (Rajan et al., 2009). Therefore, CG7787 and Cul-3might regulate Spdo and/or Delta basolateral endocytosis and/or

recycling required for Notch signalling as this accumulationphenotype is correlated with a loss of Notch signalling. WhileCul-3 is a subunit of E3 ubiquitin ligase, CG7787 putatively

encodes a guanyl-nucleotide exchange factor. CG7787 belongs tothe MSS4/DSS4 family proposed to function as chaperone formisfolded 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 recyclingpathway as Sec15 (see above) and positively regulates Notch

signalling. Other data support the general idea that a recyclingpathway positively regulates Notch signalling in the sensoryorgan lineage. Indeed, we observed an excess of Spdo-, Notch-


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and Delta-positive vesicles either in the anterior or towards the

anterior cell in Rme-8dsRNA and l(2)dtldsRNA, 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 signalling.

Finally, we observed a basolateral accumulation of Delta in

epithelial cells of O-fut1dsRNA, Tsp47FdsRNA and Tsp68CdsRNA

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 signalling.

In conclusion, our screen led us to identify intracellular

trafficking regulators of major Notch signalling actors. Although

it is still debatable whether the subcellular localisation changes

observed are a cause or a consequence of the Notch signalling

phenotype, the screen we performed led to the identification of

11 previously unknown regulators of Notch signalling (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-signalling-dependent

binary cell fate acquisition, as well as of their putative

molecular interactions.

Materials and MethodsDrosophila stocks and genetics

Unless otherwise stated, fly stocks were obtained from the Bloomington DrosophilaStock 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 ViennaDrosophila RNAi Center [VDRC, (Dietzl et al., 2007)]; except lines (as indicated insupplementary material Tables S1–S3), which were obtained from the National Instituteof Genetics Fly Stock Center (NIG-FLY) or the Transgenic RNAi Project (TRIP) via theBloomington Drosophila Stock Center. For RNAi-induced phenotype study, crossesbetween UAS-hairpin RNAi males and driver-GAL4 females were raised at 18 C, 25 Cor shifted at 29 C during L2–L3 larval stages. For each cross in which the genotypeswere blinded for objectivity purpose, two experimenters examined at least 20 fliessensory organ distribution and/or morphological phenotypes. w1118 males were crossedwith driver-GAL4 females for control experiments. To obtain AP-47SHE11 mitoticclones, we used the FLP-FRT technique and the stocks (1) y w hs-FLP; FRT82B, Ubi-

GFP(S65T)nls and (2) FRT82B, AP-47SHE11/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 17 h to 20 h after puparium formation, dissected in 16PBS,fixed in 4% paraformaldehyde and stained as previously described (Le Borgne andSchweisguth, 2003). Primary antibodies used were mouse anti-Notch ExtraCellular 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 ExtraCellular 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-1c [1:100 (Benhra et al.,2011)]. Cy2-, Cy3- and Cy5-coupled secondary antibodies (1:500) were fromJackson Laboratories. Delta 15 minutes internalisation assays were performed withmouse anti-Delta DSHB (1:100), as previously described (Benhra et al., 2011).

Images were acquired with a Leica SPE confocal microscope, which was noise-suppressed using the smooth function of ImageJ. In all figures, Notch (Cy5-)images were colour balanced using ImageJ. Defect in lateral inhibition wasacknowledged when more than four to five sensory organs were systematicallydetected with a 636 1.4 NA lens, zoom 3 on a notum.

AcknowledgementsWe thank M. Muskavitch, M. Robinson, J. Skeath, the BloomingtonStock Center, the Vienna Drosophila RNAi Center, the TRiP atHarvard Medical School (NIH/NIGMS R01-GM084947) and theNational Institute of Genetics Fly Stock Center for providingantibodies or fly stocks, as well as the Microscopy RennesImaging Center. The monoclonal antibody generated by S.Artavanis Tsakonas (NECD) was obtained from the DevelopmentalStudies Hybridoma Bank, generated under the auspices of theNational Institute of Child Health and Human Development, andmaintained by the University of Iowa Department of BiologicalSciences. We thank members of the Le Borgne laboratory for helpfuldiscussions. We thank A. Pacquelet and G. Michaux for criticalreading of the manuscript. Special thanks to Amy Winehouse for hermusic that accompanied us, while we screened around 100,000 flies.

FundingThis work was supported by the Action Thematique IncitativePrioritaire programme CNRS to R.L.B.; Region Bretagne(Programme Accueil de COMpetences en Bretagne ‘Notasid’[grant number 2168 to R.L.B.]; Fondation ARC pour la Recherchesur le Cancer [grant number 4905 to R.L.B.]; Fondation pour laRecherche Medicale to R.L.B., Rennes Metropole [grant ‘Aided’Installation Scientifique’ to S.L.B.]; and La Ligue contre le Cancer35 to R.L.B.

Supplementary material available online at

http://jcs.biologists.org/lookup/suppl/doi:10.1242/jcs.110171/-/DC1

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Genetic identification of intracellular trafficking regulators involved in Notch-dependent binary...

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