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Seminars in Cell & Developmental Biology 23 (2012) 437– 442

Contents lists available at SciVerse ScienceDirect

Seminars in Cell & Developmental Biology

jo u rn al hom epa ge: www.elsev ier .com/ locate /semcdb

eview

ndocytic routes to Notch activation

artin Baron ∗

niversity of Manchester, Faculty of Life Sciences, Michael Smith Building, Oxford Road, Manchester M13 9PT, UK

r t i c l e i n f o

rticle history:vailable online 23 January 2012

eywords:ignallingotchndocytosisbiquitineltexSCRT

a b s t r a c t

It is well established that Notch signalling is activated in response to ligand binding through a seriesof proteolytic cleavages that release the Notch intracellular domain, allowing it to translocate to thenucleus to regulate downstream target gene expression. However there is still much to learn aboutthe mechanisms that can bring about these proteolytic events in the numerous physiological contexts inwhich signal activation occurs. A number of studies have suggested that endocytosis of Notch contributesto the signal activation process, but the molecular details are unclear and controversial. There is conflictingdata as to whether endocytosis of the receptor is essential for ligand-induced signalling or supplements it.Other studies have revealed that Notch can be activated in the endosomal pathway, independently of itsligands, through the activity of Deltex, a Ring-domain Ubiquitin ligase that binds to the Notch intracellular

ysosome domain. However, it is unclear how the Deltex-activation mechanism relates to that of ligand-inducedsignalling, or to ectopic Notch signalling brought about by disruption of ESCRT complexes that affectmultivesicular body formation. This review will address these issues and argue that the data are bestreconciled by proposing distinct activation mechanisms in different cellular locations that contribute tothe cellular pool of the soluble Notch intracellular domain. The resulting signalling network may providedevelopmental robustness to environmental and genetic variation.

© 2012 Elsevier Ltd. All rights reserved.

ontents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4371.1. Structure–function of Deltex . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4381.2. Deltex regulates entry of Notch into a distinct late endosomal activation mechanism. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4381.3. Alternative trafficking destinations of Notch within the endocytic pathway are associated with signal up or down regulation. . . . . . . . . . 4391.4. Relationships between different Notch endocytic-dependent activation mechanisms. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4401.5. Endocytic-dependent activation of Notch in development . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4401.6. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 441Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 441References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 441

. Introduction

The evolutionary-conserved Notch receptor, first identified inrosophila through wing notching phenotypes, has numerousevelopmental functions [1] and in the adult is involved in stem cellaintenance and differentiation in many different tissues [2–4]. In

a series of proteolytic cleavages [6]. During trafficking to the surfaceNotch is cleaved by Furin protease at the S1 site in the extra-cellular domain [7]. Notch is localised to the cell surface as aprocessed heterodimer, although there is conflicting evidence asto whether this step is required in all cases [8–12]. At the cellsurface, binding of a membrane bound DSL (Delta/Serrate/Lag-

ome tissues, misactivation of Notch is oncogenic, while in oth-rs Notch signalling has a tumour suppressor function [5]. Strictegulation of the signalling network is therefore essential. In theanonical Notch signalling pathway, Notch is activated following

∗ Tel.: +44 161 275 5111; fax: +44 161 275 5082.E-mail address: mbaron@manchester.ac.uk

084-9521/$ – see front matter © 2012 Elsevier Ltd. All rights reserved.oi:10.1016/j.semcdb.2012.01.008

2) ligand to Notch initiates the metalloprotease-dependent S2site cleavage, producing the membrane tethered Notch intracel-lular domain known as NEXT [13–17]. The latter is constitutivelycleaved within its membrane-spanning region by the Presenilin-dependent �-secretase to release the Notch intracellular domain

(NICD), which traffics to the nucleus and activates Suppressor ofHairless-dependent transcription of specific target genes [18,19].Numerous regulatory processes, including protein ubiquitination

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nd endosomal trafficking, are superimposed on this core signallingechanism to allow context specific regulation and fine tuning of

he signalling output.Development is a remarkably robust process, involving intricate

etworks of control processes including feedback and feed-forwardoops, redundancy and modularity [20]. A simple example is theact that reducing the dosage of most genes by half normally hasittle effect on the developmental outcome. Notch signalling is aare exception to this generality since, in Drosophila, adult flieshat are heterozygous for loss of Notch or its ligand Delta areaploinsufficient for wing margin and vein development pheno-ypes. Nevertheless, despite the extraordinary dosage sensitivityf the adult fly wing phenotypes, all the essential developmen-al functions of Notch are accomplished perfectly well in theseeterozygotes. The deltex gene was originally identified by Mor-an [21] through its recessive viable phenotypes of wing veinhickening and margin notches, which resemble that of haploinsuf-cient Notch. The deltex mutation was later reisolated in a geneticcreen for suppressors of a lethal combination of gain of functionbruptex alleles of Notch. This together with analysis of variousenetic interactions of deltex indicated that in Drosophila it mainlycts a positive regulator of the signal [22,23]. Interestingly flieshat lack deltex show severe sensitivity to gene copy number. Forxample deltex mutations are lethal when combined with heterozy-ous Notch, Delta and Serrate [23,24]. Temperature insensitivity ofevelopment is another form of robustness that is poorly under-tood. The viable range of the lab fruit fly is between 18 ◦C and0 ◦C and though its development time from egg to adult over thisange varies considerably, the end result is extraordinarily repro-ucible. However in the absence of Deltex, the Notch signallingetwork loses its robustness to temperature variation. As tem-erature increases there is an increased penetrance of Notch-likehenotypes at various stages of embryonic and adult development,

ncluding embryonic neurogenesis [25]. Thus when flies are lackingeltex they are without a mechanism that allows the Notch sig-alling network to properly adjust to genetic and environmentalariation.

Recent work has shown that Deltex regulates the endocyto-is of Notch, and this determines entry of the receptor into a latendosomal-dependent mechanism of activation that bypasses theequirement for DSL-ligands [25–28]. Confusingly other work hasuggested that trafficking of Notch to the late endosome is normallyequired to prevent ectopic Notch signalling from occurring in thearly endosome [29–32]. In addition various studies have foundvidence for and against the involvement of Notch endocytosis inhe canonical ligand-induced form of signalling [17,33–36]. Theres thus considerable uncertainty as to the relationship betweenhese observations, the location of Notch endosomal activationnd to what extent endocytosis of the receptor is required forormal Notch signalling during development. This review williscuss the roles of endocytic sorting in both positive and nega-ive regulation of Notch and the possible relationships betweeneltex-induced Notch signalling and other means of Notch activa-

ion.

.1. Structure–function of Deltex

The structure of Deltex can be divided into three sections sep-rated by two stretches of polyglutamine repeat sequences [37].he N-terminus contains two WWE domains, which bind to thenkyrin repeat region of the Notch intracellular domain [38–42].he WWE domains are required for Deltex function and a mis-

ense mutation in this region produces a strong deltex phenotype inies [24]. The central section contains no recognisable domains but

ncludes a Proline-rich sequence that can bind to SH3 domain pro-eins such as Grb2 [40], although a functionally interacting partner

ental Biology 23 (2012) 437– 442

in vivo has not has been found. Deletion of this sequence gener-ates a dominant negative form of Deltex that, when expressed,down-regulates rather than activates Notch signalling [43]. The C-terminal region of Deltex contains a ring-H2 finger domain thatdefines Deltex as a member of the E3 ubiquitin ligase family pro-teins [43]. E3-ubiquitin ligases act as the final step in a transferreaction to facilitate the covalent attachment of ubiquitin to Lysineacceptor residues on proteins [44]. Ubiquitin may be attached in amonovalent form (monoubiquitination) or in polyubiquitin chainsof various branched configurations. Different polyubiquitin formsregulate a variety of outcomes including proteosome-dependentdegradation, endocytosis, endosomal sorting and lysosomal degra-dation. In addition altered protein activity can be brought aboutby conformation changes or recruitment of ubiquitin binding pro-teins [45]. Studies performed both in vivo and in cell cultureindicate that Deltex expression promotes Notch ubiquitination,although definitive evidence that Deltex acts directly on Notchas ubiquitin-ligase awaits formal demonstration with purifiedproteins [25,26,46]. Notch ubiquitination is correlated with Deltex-induced Notch endocytosis and signal regulation, but whether thisubiquitination is essential for its ability to activate Notch is notknown.

1.2. Deltex regulates entry of Notch into a distinct late endosomalactivation mechanism

Light was cast on the molecular function of Deltex when itsover-expression was found to promote Notch endocytosis andaccumulation of Notch in a late endosomal compartment [26]. Thesignalling induced by Deltex expression was found to be depen-dent on the endocytosis of Notch and could be blocked by theco-expression of a dominant negative Rab5, a critical componentof early endosomes. Importantly it was found that the canon-ical DSL (Delta/Serrate/Lag2)-ligands were not required for thisform of Notch activation. Genetic studies uncovered a numberof components required for Deltex-induced Notch signalling thatsupported the importance of trafficking of Notch to the late endo-some [25]. These included components of the Adaptor-protein-3(AP-3) complex, which selects cargo destined to be located in thelate-endosomal and lysosomal vesicle membranes [47]. In additionthe HOPS (Homotypic fusion and vacuole protein sorting) com-plex, which plays a role in both late endosome maturation andfusion to the lysosome [48] was also required. Further importantevidence came from manipulating the activity of the Rab5 andRab7 GTPases, which regulate early and late endosomal traffickingrespectively [48,49]. Rab5 is involved in early endosomal fusionand its over-expression produces enlarged early endosome com-partments. Notch strongly accumulated in these enlarged vesiclesbut Notch signalling was not altered. However, when coexpressedwith a constitutively active version of Rab7, Notch was relocalisedto late endosomes and Notch signalling up-regulated, in a HOPS-dependent manner [25].

The DSL ligand-independence of Deltex-induced signalling sug-gested that activation of Notch might be occurring by a differentmechanism to the canonical pathway. Indeed genetic evidence sup-ports this idea, as components of the AP-3 and HOPS complexeswere found to be necessary for Deltex-induced signalling but theirloss had no impact on DSL-ligand induced activation [25]. It is prob-able that the distinct requirements for Notch activation relates todiffering methods of removing the extracellular domain of Notch.This step is a prerequisite for the Presenilin dependent-cleavageand release of the soluble Notch intracellular domain that is essen-

tial for both DSL-ligand activated and Deltex-induced forms ofNotch signalling [18,19,25,28]. In the canonical activation mech-anism, endocytosis of the DSL-ligand into the signal-sending cell,while bound to the Notch extracellular domain, triggers the S2

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Fig. 1. Mechanisms of ligand-dependent and independent Notch activation. Notchis activated by sequential proteolytic cleavages (indicated by jagged arrows).Notch (N) signalling induced through binding to cell surface ligands Delta (Dl)or Serrate/Jagged (Ser) is depicted on left half of figure. Ligand binding inducesmetalloprotease-dependent S2 site cleavage at the cell surface and is associatedwith the endocytosis of the ligand along with the removed extracellular domain ofNotch (NECD). The resulting membrane bound intracellular domain product (NEXT)is further cleaved by Presenilin-dependent �-secretase to release the soluble Notchintracellular domain (NICD). This may occur at the cell surface but may also con-tinue after Dynamin-dependent endocytosis of NEXT into the early endosome (EE).The contribution of Notch activation from this endosomal location in physiologicalconditions is not clear and may vary in different developmental contexts. Notch canalso be activated independently of its ligands through the activity of Deltex (Dx).Dx binds to the intracellular domain of Notch and induces endocytosis of the full-length receptor through the Rab5 positive early endosome to the Rab7 positive lateendosome (LE). Routing of Notch to the late endosome depends on the activity ofthe HOPS complex, which regulates endosomal maturation. Depending on interac-tions with other regulatory factors like Suppressor of deltex (Su(dx)), Kurtz (Krz) andShrub, Notch can be sorted to internal vesicles of the multivesicular body resultingin its down-regulation, or to the limiting membrane of the late endosome where itcan be activated. The latter is dependent on S3 cleavage by Presenilin and most likelyoccurs after removal of NECD by an unknown mechanism. Activation of Notch bythis route depends on its trafficking to the late endosome and may further requireHOPS and Rab7-dependent fusion with the lysosome (LYS). Mutations in the ESCRTcomplex proteins prevent multivesicular body formation and Notch activation canalso occur, independently of Dl/Ser ligands, within the resultant aberrant endosomes(not depicted). NICD released by these different mechanisms is shown contributingto a common pool, which can be transported to the nucleus to regulate gene tran-scription. However it cannot be ruled out that there may be subtle differences inthe form of NICD released from different cellular locations, perhaps affecting signalhalf-life or target gene specificity.

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interactions with partner proteins on vesicular bodies that modu-late Notch trafficking and processing. Recent work in mammaliancell culture has implicated Deltex-1 in recruiting a deubiquitinat-

leavage of Notch at the cell surface to generate a substrate thatan then be constitutively activated by Presenilin. The latter stepay occur at the cell surface or following endocytosis. Without this

igand-induced activation, the transfer of Notch to the late endo-ome/lysosome provides an alternative mechanism to generate aimilar removal of the Notch extracellular domain. Notch traffickedo the lysosomal peripheral membrane would have its cytoplas-

ic domain exposed to the cytoplasm and its extracellular domainxposed to the intra-luminal environment, which may lead to theigand-independent loss of the extracellular domain by an as yetnknown protease. Fig. 1 provides an overview of the endocytosisathway and its relationship to Notch signalling.

Downstream of Presenilin-dependent cleavage, the Deltex andanonical forms of Notch signalling appear to be similar, with botheing dependent on the downstream transcription factor Suppres-or of Hairless [50]. The latter work corrected an earlier report thatuggested Deltex may activate target genes by a different down-

tream mechanism [26].

ental Biology 23 (2012) 437– 442 439

1.3. Alternative trafficking destinations of Notch within theendocytic pathway are associated with signal up or downregulation

Given the extensive role of the late endosomal/lysosomal path-way in down-regulating membrane proteins by degradation, it isunexpected that trafficking to these organelles might be involvedin signal generation. However endosomal sorting of Notch to differ-ent destinations was found to be important in determining whetherthere is a positive or negative outcome of Notch endocytosis on sig-nalling [51]. The relationship between the activities of Deltex andthe Nedd4 family protein Suppressor of deltex (Su(dx)) is illumi-nating as to how endosomal sorting can determine whether Notchis activated or down-regulated. Coexpression of Su(dx) with Del-tex overrides the ability of the latter to stimulate signalling, and thecombined effect of the two proteins is to strongly enhance down-regulation of Notch by Su(dx) [25]. Correlated with the latter, thelocation of Notch is altered from the periphery of Rab7 positivelate endosomal vesicles into the central luminal vesicles, whereNICD is sequestered from access to the cytoplasm. From this it canbe concluded that Deltex has two biological activities: firstly totrigger Notch endocytosis and then subsequently prevent Notchbeing moved from the periphery of the late endosome into themultivesicular bodies that occupy its lumen. The latter function isover-ridden by the activity of Su(dx) which relocalises Notch intothe multivesicular body [25]. Since this depends on the E3 ubiq-uitin ligase HECT domain of Su(dx) then it is possible that directubiquitination of Notch by Su(dx) regulates this sorting decision.Ubiquitination of Notch by Su(dx) has not been detected however,although there are reports that similar Drosophila and mammalianproteins of the Nedd4 family do promote Notch ubiquitination andmay be in part functionally interchangeable with Su(dx) [52,53].Nedd4 family proteins DNedd4 and Itch may also function in partby triggering the degradation of Deltex [52,54], although this doesnot appear to account for the activity of Su(dx) [25 and unpublishedobservations].

The identification of additional proteins that cause Deltex activ-ity to have a negative outcome on signalling, has shed further lighton how the endosomal sorting of Notch in the late endosome isregulated. Proteomics analysis identified the Drosophila �-arrestinprotein Kurtz as a protein binding partner of Deltex [46]. Coexpres-sion of Kurtz with Deltex has similar consequences to that of Su(dx),with the combined expression resulting in strong down-regulationof Notch signalling. In contrast kurtz mutant clones produced anincrease in Notch signalling levels [46]. The strong down-regulationof Notch signalling produced by the combined effect of Deltex andKurtz is suppressed by reducing the activity of the ESCRT III com-plex component Shrub. Mutation or RNAi knock-down of Shrubalso results in a strong-upregulation of the Deltex-induced Notchsignal. In contrast coexpression of Deltex with Shrub results inNotch signal down-regulation. In both circumstances Notch accu-mulates on the Rab7-positive, late endosomal vesicles [28]. Againthe precise location within this vesicle seems to be the key towhether Notch is activated or down-regulated (Fig. 1). Interest-ingly, the difference between up and down-regulation of Notch iscorrelated with its ubiquitination status. Deltex promotes Notchmono-ubiquitination, which is associated with Notch signalling,while the interaction with Kurtz promotes poly-ubiquitination ofNotch associated with down-regulation [28]. It will be interestingto determine whether the activity of Su(dx) is also involved withthis Shrub and Kurtz regulated process, or if it acts by a parallel deci-sion making mechanism. It is possible that Deltex makes multiple

ing protein eIF3f to Notch on vesicle surfaces. The eIF3f protein

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egulates access of Notch-1 to Psn-dependent S3 cleavage andence its activation [55]. However in a different cell context mam-alian Deltex-1 was found to negatively regulate Notch through

romoting its degradation [56]. Other possible activities of Deltex,hould also not be overlooked. Work with mammalian Deltex hasuggested it can localise to the nucleus and negatively regulate thectivity of the soluble Notch intracellular domain [57].

.4. Relationships between different Notch endocytic-dependentctivation mechanisms

The role of endosomal sorting in regulating Notch activations further highlighted by the consequences of mutations in com-onents of the ESCRT (endosomal sorting complex required forransport) I, II and III complexes, which are required to mediatehe transfer of membrane receptors into the multivesicular bodyuring late endosomal maturation [58,59]. Mutations in severalSCRT components result in endosomal Notch accumulation andause ectopic Notch activation, contributing to neoplastic tissuevergrowth [29–31]. Similarly mutation of Lethal (2) giant discslgd) also results in Notch endosomal accumulation and Notchctivation [60–62]. The known role of ESCRT complexes in trans-erring membrane receptors into the multivesicular body andffectively out of contact with the cytoplasm is compatible withhe above-described model of Deltex-induced Notch activationn the endosomal limiting membrane. However Notch accumu-ation in ESCRT complex mutant cells has been shown to occur inrs (Hepatocyte growth factor regulated substrate) positive earlyndosomes [29–31] and progression to the Rab7 positive late endo-ome may not be required for up-regulation of Notch in theseases. It should be noted that the relationships between Notch sig-alling activated by mutations in ESCRT-complexes, mutations ofgd, or by Deltex expression are still to be defined and it shouldot be assumed that the activation-mechanisms are the same,lthough it is likely that Notch activation in all these cases is ligand-ndependent [26,28,31,35]. Intriguing new work suggests that even

ithin the three ESCRT complexes, there is heterogeneity in howoss of function mutations lead to Notch upregulation. As discussedbove, loss of function of the ESCRT-III complex component, Shrub,pecifically enhanced the Deltex-induced Notch signal and causedhe accumulation of Notch in Rab-7 marked late endosomes. In con-rast knock-down of ESCRT I and II components unexpectedly ledo a down-regulation of the Deltex-induced signal [28]. Thereforehere may be a variety of Notch activation mechanisms within thendocytic pathway associated with Notch accumulation in differentocations.

.5. Endocytic-dependent activation of Notch in development

Endocytosis-dependent activation of Notch may be enhanced byoss or gain of function of a variety of endocytosis-related proteins,robably by different molecular mechanisms in different cellu-

ar locations. It is clear that a robust mechanism, depending onransfer of Notch into the multivesicular body, prevents excessiveigand-independent Notch activation within the endocytic path-

ay. However it is important to consider whether endocytosis alsoontributes to signal activation in normal development. If so, howo the different signal activation mechanisms relate to the canon-

cal ligand-dependent signal activation route? Is there a role forSL ligand-independent signalling in normal development? How

s this mechanism regulated and are there other non-DSL ligandshat can input into this pathway? These questions are not straight-

orward to answer. In the Drosophila wing, developmental rolesor components of the late endosomal Notch activation pathwayre revealed in sensitised genetic backgrounds with only one func-ional copy of the Notch gene [25]. Deltex itself has recessive wing

ental Biology 23 (2012) 437– 442

margin notching phenotypes resulting from reduction of Notch sig-nalling at the Dorsal-Ventral (DV) boundary [24]. However it isunlikely that these phenotypes solely reflect loss of late endosomalNotch activation, since loss of function of the AP3 and HOPS down-stream components do not, on their own, result in loss of Notchsignalling at the DV boundary [25 and unpublished observations].Unlike the latter components deltex mutants also lead to a suppres-sion of DSL-ligand induced signalling, reflecting additional roles forDeltex on the ligand-induced pathway [24]. Indeed many of thecomponents of endocytic Notch regulation seem to have roles inboth ligand-dependent and independent activities. For example,Kurtz mutants enhance the phenotype observed in flies devoid ofdeltex [46] implying Kurtz also affects Deltex-independent Notchactivation. Similarly Su(dx) mutants suppress the null deltex phe-notype indicating that at least one function of Su(dx) is to restrictthe activity of Deltex-independent Notch signalling. ParadoxicallySu(dx) also acts indirectly to positively regulate Notch signalling inthe Drosophila ovary germline stem cell niche by antagonising theactivity of the cell-junction scaffold protein ZO-1 [63]. Assigning thedevelopmental requirements of these genes to specific molecularmechanisms is therefore problematic. It is likely however that inmost tissues there is no strict requirement for the late endosomalactivation mechanism, rather this route produces a basal level ofsignalling on which the DSL ligand-induced signal is superimposed.In this way it may contribute to developmental robustness.

In Drosophila embryo midline and nervous system develop-ment the late endosomal Notch activation mechanism may havea more prominent role. This is inferred from the fact that HOPSand AP3 mutants have similar Notch-like phenotypes to deltexmutants, and similarly show increasing penetrance of these phe-notypes at higher temperatures [25]. Indeed HOPS mutants aremore penetrant than deltex mutations suggesting other Deltex-independent routes might lead to late endosomal activation ofNotch, although the presence of additional embryonic phenotypescomplicates the genetic analysis. One location where DSL ligand-independent signalling may be necessary is during Drosophilahaematopoesis, where Notch signalling is required for expansionand maintenance of Crystal cells, at a time when these cells arenot in contact with ligand bearing cells [64]. It is not known how-ever whether this form of ligand-independent signalling reflectsthe late endosomal mechanism, or is more related to that observedin ESCRT complex mutants. A further question is whether othernon-DSL ligands can regulate this activation mechanism. Workin mammalian cell culture on oligodendrocyte differentiation hassuggested that F3/Contactin and NB3 are alternative non-canonicalligands that induce Notch signalling in a Deltex-dependent man-ner [65–68], although whether this reflects a Notch traffickingrole for mammalian Deltex has not been investigated in this sys-tem. Alternatively regulation of endosomal sorting may representa point of cross talk with other signalling pathways. In the aboveexample Notch endosomal sorting in the circulating Crystal cellsis regulated by Sima, the Drosophila homologue of mammalianHypoxia-inducible factor-� (Hif-�). Hypoxic conditions stabiliseHif-� and cause an upregulation of endocytic-dependent Notchsignalling and an expansion of crystal cell number [64].

Seemingly the late endosomal Notch activation mechanism doesnot play a role in DSL ligand-induced Notch signalling [25]. If thisis the case then is there any role, in the signal-receiving cell, forNotch endocytosis in the generation of the canonical signal? Ifso then could this occur in the early endosome by a mechanismmore related to the events that follow the loss of ESCRT complexfunction? This is difficult to demonstrate in vivo because the sepa-

rate requirement of ligand-endocytosis in signal sending cells [69]complicates the genetic analysis. The involvement of endocytosisas a normal requirement for Notch signalling was first proposedthrough analysis of neurogenic mutant phenotypes of shibire, the

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rosophila Dynamin. Using mitotic clones it was found that Shibireas required cell autonomously in both the signal-sending and

ignal-receiving cells [33]. Several subsequent studies producedonflicting results both excluding and favouring a role for endo-ytosis in the S3 cleavage of NEXT. In Drosophila it was foundhat signalling resulting from NEXT expression is not inhibited byemoving Dynamin function [70]. On the other hand studies inammalian cells have indicated that S3 cleavage of NEXT requires

ndocytosis [71], although the interpretation of this work is compli-ated by subsequent analysis showing that a mutation preventingotch endocytosis also results in altered specificity of S3 cleavage.he latter generates an unstable form of NICD [72]. This unstableorm is also generated when the rate of endocytosis of wild typeEXT is increased, suggesting that Notch activation in the endo-

omal pathway may make only a minor contribution to signallingevels. Other studies also indicate that NEXT activation predomi-antly occurs at the cell surface [17,73].

One problem with the above studies is that they examinehe processing and activation of an over-expressed NEXT producthat mimics the consequences of S2 cleavage. This overcomes theomplication of the requirement for ligand-endocytosis in Notchctivation, but leads to a rather non-physiological situation. Recentork has proposed a role for endosomal sorting downstream of

igand-dependent S2 cleavage of full-length Drosophila Notch [74].hyllopod regulates endocytic sorting of membrane-tethered NICD,fter ligand-dependent S2 cleavage, into a down-regulatory latendosomal route. Loss of Phyllopod results in a retention of mem-rane tethered NICD in the early endosome and upregulation of

igand-dependent signalling. This illustrates that, in principle, DSLigand-activated Notch can be S3 cleaved in the endosomal path-

ay. Since Phyllopod expression is under control of Epidermalrowth Factor (EGF) signalling, this represents another point ofross talk between Notch and other signalling pathways at the levelf endosomal sorting.

The Drosophila ovary has also proven useful in identifying normal contribution of receptor endocytosis to generatinghysiological levels of Notch signalling. In the developing egghamber Notch signalling is unidirectional with signalling to Notchxpressed in the follicle cells from Delta expressed in the germline.y generating marked mitotic clones of mutant tissue it is possi-le to specifically remove endosomal trafficking components fromhe signal receiving follicle cells [35]. Mutations that interfere withhe entry of Notch into the early endosome reduce Notch signalling

onitored through the expression of lacZ-reporter constructs andnown target genes, and this appears to be due to reduced access ofotch to Presenilin-dependent S3 cleavage. Mutations that inter-

ere with endosomal acidification also reduce Notch signalling36]. In these studies the endosomal mutants did not completelyemove Notch signalling however. A complication to the analysiss that alternative endocytic routes may be involved [35] making itifficult to examine phenotypes that completely remove endocytic-ependent activation. It is not yet possible therefore to answer theuestion as to whether Notch receptor endocytosis is an obligatorytep in full-length Notch activation.

.6. Conclusions

The emerging picture is that multiple ligand-dependent andndependent activation mechanisms in different cellular compart-

ents may contribute to the overall level of Notch signallingresent in a cell. Depending on genetic and environmental varia-ion, and developmental context, the relative contributions of these

echanisms may vary, and the sum total of these pathways mayrovide for developmental robustness and homeostasis. Further-ore, endosomal sorting represents a node of regulatory cross

alk able to fine tune signalling levels, and may provide routes for

[

ental Biology 23 (2012) 437– 442 441

misactivation or down-regulation of Notch signalling in a varietyof pathogenic conditions.

Acknowledgements

I thank Marian Wilkin, Simon Woodcock and Hideyuki Shimizufor helpful comments and suggestions, Kazuya Hori and SpyrosArtavanis-Tsakonas for discussing data ahead of publication, andacknowledge financial support from the BBSRC and WellcomeTrust.

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