Current Biology, Vol. 14, R129–R138, February 3, 2004, ©2004 Elsevier Science Ltd. All rights reserved. DOI 10.1016/j.cub.2004.01.023

Review

Regulation of Notch Signaling Activity François Schweisguth

Cell-cell signaling mediated by the receptor Notch is used widely across the metazoans to determine cell fate and regulate pattern formation. Notch signals via a conserved regulated intramembrane proteolysis. Recent analyses of the cell biology of the Notch receptor have identified several fundamental mechanisms that contribute to regulate Notch signaling activity in space and time.

Proper development of multicellular organisms requires that cells coordinate their behavior spatially and temporally. Cell-cell communication regulates cell growth, proliferation, survival, fate, differentiation and morphogenesis. Cell-cell signaling mediated by receptors of the Notch family has been involved in all these processes in a wide variety of developmental and physiological contexts in organisms ranging from nematode to man [1]. Not surprisingly, it has also been implicated in various pathologies in humans [2]. The study of Notch is also of particular interest to developmental biologists, as Notch mediates lateral inhibition, a key patterning process that organizes the regular spacing of different cell types within tissues [3] (Box 1). Though Notch is best known for its role in lateral inhibition, it also regulates other important patterning processes, such as the formation of boundaries in both space and time [4,5]. The last few years have witnessed an explosion of new functions for Notch signaling during embryogenesis and adulthood. I will not attempt to review the plethora of the Notch-regulated processes. Instead, I will focus on recent insight into the cell biology of Notch receptor signaling that have emerged from the study of several Notch regulators. I will first describe how Notch signals via the so-called ‘canonical’ pathway, and then discuss some of the regulatory mechanisms that ensure tight spatial-temporal control in the level and/or duration of Notch signaling activity. As many of the genes involved in these regulatory mechanisms have been first identified in Drosophila and Caenorhabditis elegans, this review will primarily describe the role of these genes in the context of fly and nematode development. CSL-Dependent Notch Receptor Signaling Receptors of the Notch family are cell-surface type I transmembrane proteins (see Figure 1 for domain composition). The Notch ligands, Delta (Dl) and Serrate (Ser; known as Jagged in vertebrates), are also type I transmembrane proteins (see [6] for a review). Upon ligand binding, Notch receptors undergo successive proteolytic cleavages that lead to the release of the Notch Intra-Cellular Domain (NICD; Figure 2). NICD is the active form of the receptor. It acts in the nucleus as CNRS UMR 8542, Ecole Normale Supérieure, 46, rue d’Ulm 75230 Paris cedex France. E-mail: [email protected]

a transcriptional regulator. Thus, Notch receptors can also be described as membrane-anchored transcriptional co-activators. Newly synthesized Notch molecules are processed in the trans-Golgi network by proteases of the Furin family [7]. This constitutive processing is required for signaling in mammals [7], but appears to be dispensable in flies [8]. Notch accumulates at the plasma membrane as a heterodimer consisting of an ectodomain called Notch Extra-Cellular Domain (NECD) and a membranetethered intracellular domain called NTM (Figure 2). NECD and NTM interact non-covalently in a Ca2+dependent manner [9]. Ligand-induced activation of Notch renders NTM sensitive to cleavage at the S2 site by extracellular proteases of the ADAM/TACE/Kuzbanian family [10–12]. Ligand-induced activation of the receptor can be mimicked by Ca2+ depletion [9] or by deletion of the Lin12/Notch Repeats (LNR) [13]. S2 cleavage releases the ectodomain of Notch and generates an activated membrane-bound form of Notch called Notch Extracellular Truncation (NEXT). NEXT is further processed at two endomembrane sites, S3 and S4 (Figure 2) [14–16]. These two cleavages are catalyzed by the γ-secretase activity of the PresenilinNicastrin-Aph1-Pen2 protein complex [17,18]. Processing of NEXT releases NICD into the interior of the cell, β peptide extracellularly [15]. The fate and and a small Nβ β are unknown. NICD possible signaling activity of Nβ translocates into the nucleus and assembles into a ternary complex with the CSL (human CBF1, fly Suppressor of Hairless, worm Lag-1) DNA-binding protein

Box 1 Notch signaling mediates both lateral inhibition and induction. A

Lateral inhibition

B

Mutual inhibition

x

Biased inhibition Unidirectional inhibition

Induction y

on

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on

x

z Current Biology

(A) Lateral inhibition: Notch mediates reciprocal inhibitory signaling between cells that have similar developmental potential. Two cells are shown here for simplicity. Reciprocal signaling (top: mutual inhibition) is resolved over time into unidirectional signaling (bottom: unidirectional inhibition). The singling out of the signaling cell (red) results from a self-amplifying feed-back loop in which Notch inhibits the ability of the signal-receiving cell (blue) to produce inhibitory signaling. (B) Induction: Notch mediates unidirectional signaling between two cells, x and y, with distinct developmental potentials. The signal-sending cell (x, in red) activates Notch in the signal-receiving cell (y, in blue). In response to Notch activation, y becomes z.

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Figure 1. Domain composition of Notch. The extracellular domain of Notch consists primarily of EGF repeats (light blue), Kuzbanian ranging from 36 in Drosophila Notch and Numb Numb vertebrate Notch1 to 10 in C. elegans Delta GLP-1 (see [6] for a review). It also Dishevelled Serrate Fringe Deltex Fringe CSL includes three Lin12/Notch repeats • • (LNRs; dark gray). The EGF repeats 11 ** * ** * ** * * * ** and 12 (dark blue) are necessary and sufS1(furin) ficient for binding Dl and Ser [6]. The EGF S2 (TACE/ADAM) S3+S4 (γ-secretase) repeats mutated in split (yellow) and Ax Current Biology (orange) are indicated. The intracellular domain includes six ankyrin repeats (green) and two nuclear localization signals (black dots). The positions of the S1–S4 cleavage sites (arrows), predicted O-fucosylated sites (o; evolutionarily conserved sites are indicated with asterisks), carboxyl terminus of predicted NotchMcd proteins (gray arrowheads) and domains of interaction with selected partners are indicated. Itch/Su(dx)

dissociates the ligand binding unit from the intracellular signaling unit, each receptor molecule can only signal once. Thus, intensity and duration of signaling cannot be regulated by receptor desensitization. Second, signaling is direct and is not relayed by secondary messenger molecules. This property clearly limits the possibilities for signal amplification and cross-talk between different signaling pathways. Third, receptor processing releases extracellular by-products that may either have novel signaling activities or act to downregulate signaling, for instance via titrating the ligands. These properties have obvious implications on how signaling by Notch may be inhibited or buffered in space and time. The dramatic consequences caused by the forced expression of NICD indicate that Notch receptor signaling activity must be tightly regulated; for instance, expression of an activated version of human Notch1 in T-cells causes acute lymphoblastoma [25] and ectopic expression of NICD blocks early neurogenesis in fly and vertebrate embryos [26–28]. Furthermore, whereas the timing and positioning of Notch activation obviously depends on the presence of its activating ligands, the pattern of Notch activity is,

[19] and the Mastermind (Mam)/Lag-3 co-activator [20]. This complex binds specific regulatory DNA sequences and activates the expression of CSL/Notch target genes. In the absence of NICD, CSL can recruit repressor complexes to the cis-regulatory region of the CSL/Notch target genes. Therefore, activation of Notch triggers a switch from repression to activation (Figure 2) [19,21,22]. Notably, endogenous NICD has not been detected in the nucleus of signal-receiving cells. This has led to the suggestion that NICD acts at a very low concentration, below immuno-detection threshold [16]. Properties of Signaling via Regulated Intramembrane Proteolysis Signaling by bi-functional proteins that, like Notch, integrate ligand binding at the cell surface and transcriptional regulation in the nucleus involves regulated intramembrane proteolysis (RIP) [23]. Signaling via RIP is an unusual mode of signal transduction used by a number of unrelated membrane proteins, such as SREBP, APP and N-Cadherin [23,24]. This mode of signaling has several interesting features: First, proteolytic cleavage is irreversible and, as the cleavage physically

Figure 2. A model of CSL-dependent signaling. Dl at the surface of signaling cell (top) binds S1 processed Notch at the surface of the responding cell (bottom). Liganddependent S2 cleavage of Notch generates NEXT, which is further processed at the S3 and S4 sites. This releases NICD (which translocates into the nucleus), β. NICD associates with CSL NECD and Nβ and Mam, thereby triggering a switch from repression, mediated by CSL-corepressor (coR) complexes, to activation.

Signal-sending cell Delta

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however, not coincident with the broad distribution of its ligands, again suggestive of regulatory mechanisms restricting Notch receptor activation. Extracellular Shedding of Notch Ligands Dl and Ser are membrane-bound ligands that bind and activate Notch receptors in trans, i.e., at the surface of neighboring cells. Nevertheless, biochemical studies have indicated that Dl can be cleaved to release a soluble form of Dl, Dl extra-cellular (DlEC), that contains the Notch binding domain [29–35]. The functional significance of DlEC is unclear. In worms, expression of secreted forms of LAG-2 and APX-1, two ligands of the Notch receptors GLP-1 and LIN-12, rescues the lag-2 mutant phenotype [36], demonstrating that soluble ligands can signal. By contrast, secreted forms of Dl and Ser appear to act in a dominant-negative fashion in Drosophila [37] and cleavage of Dl has been suggested to be an important step in switching off the Dl signal [33]. Extracellular shedding of Dl also generates a membrane-bound form that is further processed in a Presenilin-dependent manner [28]. One possible interpretation is that the Presenilin-dependent cleavage of Dl is part of a general clearing mechanism [38]. Alternatively, intracellular forms of Dl may regulate novel signaling events, raising the possibility of bi-directional signaling [31,30]. Determining the functional significance of Dl processing will certainly be an active area of research. Regulation of Dl Signaling Activity by Ubiquitination A critical step for efficient signaling by Dl has recently been uncovered from the analysis of the Drosophila neuralized (neur) and zebrafish mind-bomb (mib) genes, respectively. These two genes, isolated due to their Notch-like mutant phenotypes, regulate Dl ubiquitination [39–42]. Ubiquitin is a 76-amino acid polypeptide, which is covalently linked to protein substrates in a multi-step reaction. Ubiquitin-protein ligases (E3-ligases) recognize specific substrates and catalyze the transfer of ubiquitin to the protein substrate. Ubiquitin was first identified as a tag for proteolytic degradation. More recently, ubiquitin has also been shown to serve as a signal for endocytosis [43]. Neur and Mib are two evolutionarily conserved and structurally distinct E3-ligases. Both Xenopus Neur and zebrafish Mib physically interact with Dl and mediate Dl ubiquitination [39,40]. Moreover, clonal analysis in the fly and transplantation studies in the zebrafish have suggested that Neur and Mib, respectively, act noncell-autonomously to up-regulate Dl signaling activity [40,42,44], suggesting that endocytosis of Dl in the signal-sending cell promotes Notch activation in the signal-receiving cell [45]. How ubiquitination of Dl upregulates Dl signaling activity is not yet clear and various hypotheses have been discussed [46]. Interestingly, Neur-dependent ubiquitination of Dl has also been shown to promote ligand degradation in a Xenopus injection assay [39]. It is thus possible that ubiquitination of Dl promotes both its signaling activity and its degradation. The notion that ubiquitination plays a dual role was first seen in the regulation of VP16 activity. Ubiquitination of the transactivation domain of VP16 up-regulates its activity and promotes

(1) SOP-specific transcription of the neur gene

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Figure 3. Two mechanisms regulate Neur accumulation in signal-sending cells. First, following singling out from groups of equipotent cells, selected sensory organ precursors (SOPs) further inhibit nonselected cells through the SOP-specific expression of the neur gene (red). Each selected SOP then undergoes a series of asymmetric cell divisions to produce a sensory bristle. The first division of the sensory bristle lineage generates the pIIa and pIIb cells. Neur localizes at one pole of the dividing SOP (red crescent) and is unequally segregated into the anterior daughter cell. Neur up-regulates Dl signaling in this anterior cell and thereby promotes the activation of Notch in the posterior cell (signaling ON), which therefore adopts the pIIa fate (left) [40]. In the absence of neur activity, Notch appears to remain inactive in both daughter cells (signaling OFF), which therefore adopt a pIIb fate (right).

its degradation by the proteasome [47]. Obviously, linking molecular activation to the degradation of the activated molecule provides an elegant means of temporal control. The studies on Neur and Mib predict that the regulated accumulation of Neur (or Mib) should contribute to specify when and where Dl signals. Consistent with this prediction, the Drosophila neur gene is specifically expressed in the signal-sending sensory organ precursor cells (SOPs) during adult peripheral neurogenesis (Figure 3). Neur and Notch also regulate the binary pIIa/pIIb fate decision following the asymmetric division of the SOP. Activation of Notch in the posterior cell is required for adoption of the pIIa fate. Conversely, inhibition of Notch in the anterior cell is required for the adoption of the pIIb fate [48]. During mitosis, Neur localizes asymmetrically at one pole of the SOP and is unequally segregated into the signal-sending pIIb cell [44] (Figure 3). Thus, both transcriptional and post-transcriptional mechanisms contribute to restrict Neur accumulation in signal sending cells in Drosophila. Regulation of Receptor-Ligand Interaction via Glycosylation Notch receptors are glycoproteins that are modified by the addition of fucose to specific Serine and Threonine

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Figure 4. A model for Ofut1 function. O-fucose is a modification of serine or threonine residues found on EGF repeats ** * ** * ** * ** ** Ectopic O-fucosylation Low level of containing the amino acid consensus on Notchsplit Ofut1 activity sequence Cxxx(G/A/S)(T/S)C between the second and third cysteine of the EGF repeat [52,58]. According to this consenLow affinity for Delta Increased affinity for Delta High level sus, 23 of the 36 repeats of Notch have of Ofut1 potential O-fucosylation sites (adapted from [50]). It has been proposed that the actual number of O-fucosylated repeats High affinity for Delta Current Biology depends on the level of Ofut1 activity [50]. Notch may be highly modified in cells with high levels of Ofut1 activity, whereas a reduced level of Notch O-fucosylation is predicted in cells with lower levels of Ofut1 activity. A high level of O-fucosylation promotes Dl binding, hence receptor activation. The mutant Notchsplit receptor has one additional predicted O-fucosylation site at EGF repeat 14. This renders Notchsplit receptor more sensitive to Dl [54], which may be due an increased affinity for Delta.

residues in their Epidermal Growth Factor (EGF)-like repeats (Figures 1 and 4). O-fucosylation of Notch is catalyzed by a GDP-fucose protein O-fucosyltransferase encoded by the Ofut1 gene. Loss of Ofut1 function mimicks loss of Notch activity in both flies and mice [49–51]. Dl and Ser/Jagged also appear to be synthesized as glycoproteins modified by O-fucosylation of their EGF-repeats [52]. However, genetic analysis in Drosophila has indicated that Ofut1 functions cell-autonomously [51], suggesting that Notch is the key target of Ofut1. Transfection and RNAi studies in cultured cells have shown that O-fucosylation of Notch promotes receptorligand interaction [53]. The multiple O-fucosyl glycans of modified Notch may be involved in low-affinity binding of lectins, proteins that bind to sugar residues without further modifying them, suggesting an explanation for the large numbers of predicted O-fucosylated EGF repeats (up to 23) [50]. As the expression of Ofut1 appears to be developmentally regulated and overexpression of Ofut1 blocks Notch receptor signaling, Ofucosylation may be used as a regulatory mechanism to control Notch-ligand interaction [50] (Figure 4). Consistent with this hypothesis, a mutation that introduces a new O-fucosylation site at EGF repeat 14 results in the ectopic activation of Notch in the photoreceptor R8 precursor cell, suggesting that the pattern of Notch fucosylation may influence the ability of Notch to respond to Dl in a cell-specific manner [54] (Figure 4). Notch is further glycosylated by the β-1,3-N-acetylglucosaminyl (GlcNac) transferase Fringe [55]. Fringe physically interacts with Notch and modifies O-linked fucose on specific Notch EGF-repeats, including EGFrepeat 12 (EGF12), which is involved in ligand binding [56–59] (Figure 1). Fringe regulates boundary formation in Drosophila and in vertebrates [60]. For example, in the Drosophila wing, expression of fringe in dorsal cells makes these cells more sensitive to Dl, which is expressed in ventral cells, and less sensitive to Ser, which is expressed dorsally [61]. Consistent with these observations, results from binding assays on cultured cells have indicated that modification of Notch by Fringe increases the affinity of Notch for Dl and decreases its affinity for Ser [56,59]. The biological significance of EGF12 glycosylation was further examined by mutating the EGF12 serine residue to which Ofucose is normally attached [59], such that it cannot be

modified neither by Ofut1 nor Fringe. Functional analysis of this mutant form of Notch indicated that O-fucosylation of EGF12 is important for the Fringe-dependent down-regulation of Notch-Ser interactions, implying that EGF12 is a key target of Fringe [59]. By contrast, the Fringe-dependent up-regulation of Notch-Dl interactions appears to involve other EGF repeats [59]. Together, these studies suggest that Dl and Ser have lectin-like properties in that they bind glycosylated Notch with different affinities depending on the extent and nature of the sugar modifications carried by Notch. Another model, which is non-exclusive to the previous one, suggests that Fringe does not modulate the affinity of ligand-receptor interaction. Instead of modulating the ability of Dl and Ser to activate Notch in trans (i.e., Notch receptors on the surface of signal-receiving cells are activated by ligands on the surface of signal-sending cells), Fringe would regulate the ability of Dl and Ser to inhibit Notch in cis (i.e., the ligands act cellautonomously in the signal-receiving cells to block Notch signaling activity). Cis-inhibition has been proposed to result from the formation of receptor-ligand complexes that are retained in the Golgi, thereby preventing the cell from receiving extracellular signals via Notch at its surface [62] (Figure 5). Cis-inhibition contributes to wing margin specification in Drosophila [63,64] and has also been observed in vertebrates [62]. This model proposes that Fringe antagonizes the formation of receptor-ligand complexes in the Golgi and thereby prevents cis-inhibition of Notch from occurring. This model is supported by two sets of observations. First, transfection of increasing levels of mammalian Lunatic Fringe reduces the amounts of receptor–ligand complexes in cultured mammalian cells [62]. Second, many of the O-fucose sites that can be elongated by Fringe map to the EGF-repeats that are mutated in a specific class of Notch mutations called Abruptex (Ax) [58] (Figure 1). Interestingly, Ax mutant receptors behave as hyperactivable forms of Notch [65] that appear to be no longer responsive to cis-inhibition by Dl and Ser [66]. Further in vivo analysis of the trafficking of Notch and of its ligands, which may also be modified by Fringe [52], should help test this model. Regulation of Notch Activity by Endocytosis Notch signaling activity can also be regulated by endocytosis. Internalization of active receptors to the

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Figure 5. A model of cis-inhibition of Notch by Ser. Direct interaction of Ser with newly synthesized Notch blocks export of Notch to the cell surface. Ser, therefore, inhibits the activity of Notch in cis, in the signalreceiving cell (bottom). Elongation of some (or all) O-fucoslyated EGF repeats of Notch by Fringe may reduce the interaction in cis between Ser and Notch and may thereby antagonize cis-inhibition. This effect of Fringe may also be mimicked by Ax mutations.

Delta

Signal-sending cell NECD

S2 Extracellular

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• • • • • •

Release of cis-inhibition by Fringe-mediated glycan elongation Serrate

S1

cis-inhibition of Notch by Serrate

Notch Current Biology

lysosome, where they are degraded, is a general mechanism of desensitization. Down-regulation of the C. elegans Notch family receptor LIN-12 has recently been shown to be mediated by a conserved dileucinebased Down-regulation Targeting Signal (DTS) that may act as a signal for receptor internalization [67]. Endocytosis of Notch is probably a conserved process, as both intracellular and extracellular Notch epitopes are also detected in late endosomal compartments in Drosophila [68]. The functional significance of Notch internalization is, however, not well understood. The identification of ubiquitin E3 ligases targeting Notch for endocytosis should help in dissecting this process. Two possible candidates are Itch and Deltex. The E3 ligase Itch contains a C2-type phospholipid binding motif that possibly targets Itch to the plasma membrane, four WW-motifs involved in the interaction with the intracellular domain of Notch, and a HECT domain with E3 ubiquitin ligase activity. In mammalian cells, Itch interacts with a form of Notch similar to NEXT and promotes its ubiquitination [69]. The fly homolog of Itch, Suppressor of deltex (Su(dx)), was identified genetically as a dominant suppressor of deltex (dx) and behaves as a negative regulator of Notch signaling [70], suggesting that Itch down-regulates Notch. The dx gene encodes a RING finger protein that localizes at the cell cortex, binds to the intracellular domain of Notch and acts as a positive regulator of Notch [71–73]. Whether Itch/Su(dx) and Dx regulate Notch endocytosis, or else regulate the stability and/or activity of processed Notch remains to be established.

Receptor endocytosis may also positively regulate signal transduction. For instance, endocytosis may bring active receptors to a specific intracellular compartment where they associate with their signal transduction machinery. Analysis of the Drosophila shibire (shi) gene, which encodes a GTPase involved in pinching off endocytic vesicles from the plasma membrane, has revealed that endocytosis plays a positive role in Notch receptor signaling. When grown at restrictive temperatures, temperature sensitive shi mutants exhibit a Notch-like phenotype. Results from clonal analysis further suggested that shi activity is required in both the signal-sending and the signal-receiving cells [74]. The basis for the requirement of shi-dependent endocytosis in the signalreceiving cell is not understood. As NICD, but not fulllength Notch, is active in a shi mutant background [74], one hypothesis is that endocytosis may transfer NEXT from the plasma membrane to an intracellular compartment, which contains the γ-secretase activity. Regulation of Notch Signaling by Numb and Sanpodo Further evidence for a regulatory role of endocytosis in Notch signaling has come from the isolation of specific mutant alleles of the α-adaptin gene [75]. αadaptin is one of the four subunits of the endocytic AP-2 complex. Mutations that specifically delete or inactivate the function of the ear-domain of Drosophila α-adaptin result in a bristle phenotype that is associated with hyperactivation of Notch receptor signaling. Both loss of α-adaptin function or ectopic expression of NICD within the sensory bristle lineage results in a

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A Model 1

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Figure 6. Two models for Numb function. (A) In model 1, Numb down-regulates Notch receptor signaling by clearing Notch from the surface of the signal-receiving cell. Numb would act as an adaptor molecule between full-length Notch and/or NEXT and the α-adaptin subunit of the AP-2 complex. Alternatively, Numb may target Spdo for endocytosis (Model 2). Spdo is a positive regulator of Notch that is specifically required for Notch receptor activation in Numb-mediated cell fate decisions. This model predicts that Spdo is active for Notch receptor signaling when localized at the cortex. These two models are not mutually exclusive. (B) The MP2 neuroblast divides asymmetrically to generate two post-mitotic neurons, dMP2 and vMP2, in the developing CNS of wildtype Drosophila embryos (left). Numb (red) is unequally segregated into the future dMP2 neuron and inhibits Notch in this cell. Conversely, Notch is activated in the vMP2 neuron and Spdo is required for Notch activity in this cell. Spdo is detected in both cells. However, it is cortical in vMP2 and cytoplasmic in dMP2. Numb binds to Spdo and appears to regulate the internalization of Spdo, thereby inactivating Spdo. In the absence of Spdo (center), Notch receptor signaling is off in both cells, which therefore adopt the dMP2 fate. Conversely, in the absence of Numb (right), Spdo remains at the cortex and Notch receptor signaling is on in both cells which hence become vMP2.

pIIb-to-pIIa cell fate transformation. Such a transformation is also seen in numb mutant sensory cells, as Numb acts as a cell-fate determinant during the asymmetric SOP division. Numb is unequally segregated into the anterior daughter cell and specifies it as a pIIb cell by inhibiting Notch [76,77]. Numb colocalizes with endocytic vesicles in cultured mammalian cells and was shown to directly bind both the intracellular domain of Notch as well as the ear domain of αadaptin [75,78]. These observations have led to a model according to which Numb acts as an adaptor between α-adaptin and its Notch cargo and thus promotes the down-regulation of Notch by endocytosis [75] (Figure 6A). However, direct evidence for Numbmediated endocytosis of Notch is still missing. A recent analysis of the Drosophila sanpodo (spdo) gene has suggested an alternative model for how Numb may act [79] (Figure 6A). Spdo is a predicted four-pass transmembrane protein required for Notch signaling. Genetic analysis indicates that Spdo acts downstream of Dl and full-length Notch, but upstream of NICD. In the embryo, Spdo accumulates in the MP2 neuroblast, which divides asymmetrically to generate two neurons, vMP2 and dMP2 (Figure 6B). Numb and Notch act in a very similar manner during the vMP2/dMP2 and pIIa/pIIb decisions, with Numb being specifically segregated into dMP2 and pIIb and Notch being activated in vMP2 and pIIa [80,81] (Figure 6B). Strikingly, Spdo localizes to the cortex in vMP and is

required for Notch signaling in this cell, whereas in dMP2, it appears to accumulate in a numb-dependent manner in intracellular vesicles [79]. These observations have suggested a model in which Spdo positively regulates Notch signaling at the plasma membrane (but only in the context of Numb-regulated fate decisions) and Numb inhibits Notch by triggering the endocytosis of Spdo [79] (Figure 6A). Selective Nuclear Response to Notch Activation Signaling by Notch receptors is used in a variety of developmental decisions resulting in different outputs, depending upon the cellular context. Thus, different Notch/CSL targets are expressed in different cells upon ligand stimulation [82]. This indicates that transcriptional responses to Notch activation are highly regulatable and suggests that the selective response of the genome to Notch activation may constitute a major regulatory step in Notch receptor signaling. The molecular basis underlying output specificity is not well understood. Following S3 cleavage, NICD translocates to the nucleus and participates in the formation of DNA-bound transcriptional complexes containing the DNA binding protein CSL (Figure 2). It is not known whether NICD competes with repressor complexes for binding to DNA-bound CSL or whether Mam–NICD–CSL complexes compete with CSL–corepressor complexes for DNA binding. As CSL recruits different co-repressor complexes [19,21,83–85], there

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may be several different mechanisms. Moreover, chromatin structure and/or co-factor availability may also regulate this switch. Rapid progress can be expected in this area of research as the repertoire of Notch/CSL targets will be unraveled by combining in silico identification of CSL target genes [86] and transcriptome analysis. Switching the Signal off by Regulated Degradation Another important aspect in the regulation of NICD activity is its down-regulation via the ubiquitin–proteasome pathway. First insight into this regulatory process came from the identification of sel-10 as a negative regulator of the Notch family receptor LIN-12 in C. elegans [87]. Sel-10 is a Cdc4-related protein containing an F-box and seven WD repeats. By analogy with Cdc4p, Sel-10 has been proposed to recruit a multi-protein SCF complex that has a ubiquitin ligase E3 activity. Functional analysis of mammalian Sel-10 strongly supports this model. Indeed, Sel-10 binds NICD via its WD repeats and, together with three other components of an SCF complex, mediates NICD ubiquitination and degradation by the proteasome [88–90]. Whereas the function of the fly homolog of sel-10 is not yet known, analysis of dominant-negative mutations in two proteasome subunits supports the notion that, also in Drosophila, NICD is subject to proteasomemediated degradation [91]. Rapid degradation of NICD in the nucleus might explain why it has not been possible to detect endogenous NICD in the nucleus [16]. Whether negative regulation by Sel-10 is constitutive and simply required to attenuate the activity of NICD, or is itself subject to developmental regulation remains to be investigated. Signaling Cross-talk A fundamental property of signaling by RIP is that the same molecule integrates the functions of signal reception at the cell surface and of gene regulation in the nucleus. This property is predicted to reduce the possibilities of regulatory cross-talks between the Notch pathway and other signal transduction pathways in the cytoplasm. The various stimuli received by a cell at a given time may, therefore, predominantly be integrated at the level of CSL-regulated genes or at the level of the proteins encoded by these target genes. I will not consider these two levels of signal integration further and will, instead, present a few examples of cross-regulatory interactions that occur upstream of Notch/CSL targets. First, signal integration may take place at the level of Dl expression. A recent analysis of the role of EGFR signaling in photoreceptor differentiation in Drosophila has revealed that activation of the EGFR relieves repression of Dl gene expression by a complex formed by Su(H) and the co-repressor SMRTER [92]. This de-repression requires Strawberry notch (Sno) and Ebi, a F-box /WD40-domain containing protein, both of which physically interact with the Su(H)-SMRTER complex. Thus, activation of the EGFR causes Ebi to target a yet unknown component of the Su(H)-SMRTER repression complex for proteasome-mediated degradation [92]. Second, different signals may be integrated at the level of Notch endocytosis. In C. elegans, the Notch

family receptor LIN-12 is down-regulated in one of the six vulval precursor cells, P6.p, upon Ras signaling [67]. Ras appears to indirectly promote degradation of internalized LIN-12 in the P6.p cell by regulating the expression of yet unknown genes that shift the balance between recycling LIN-12 to the cell surface and targeting LIN-12 toward degradation [67]. Third, the stability of NICD has recently been sugβ has gested to be regulated by phosphorylation. GSK3β been shown to phosphorylate NICD in vitro and inhibiβ decreased the stability of NICD and tion of GSK3β βreduced Notch receptor signaling [93]. Thus, GSK3β mediated phosphorylation appears to stabilize NICD and potentiate its activity. Finally, cross-regulation by other signaling pathways may target Notch regulators. For instance, mouse Numb has been shown to be down-regulated by two E3 ubiquitin ligases, Sina and LNX [94,95], thus raising the possibility that Numb stability may be signal-regulated. Whereas our understanding of signal integration between Notch and other signal transduction pathways is still very preliminary, many of the regulatory steps discussed in this review may be subject to cross-regulatory interactions. Further insights into cross-talk between Notch and other signaling pathways will certainly arise from global proteomic and functional genomic approaches that are currently under way. CSL-Independent Signaling So far, I have only discussed the canonical CSL-dependent pathway in which the key active signaling molecule is NICD and the output is transcription. However, studies in both invertebrate and vertebrate species have also pointed toward CSL-independent signaling activities (see [96] for review). One of the best-documented case of CSL-independent signaling has come from the identification of novel Notch alleles, called NotchMcd [97]. These alleles cause a dominant loss of SOPs. This phenotype is opposite to the Notch or Su(H) loss of function phenotype. However, these mutant alleles are not simple gain-of-function alleles. Indeed, NotchMcd;Su(H) double mutant cells appear to develop as NotchMcd cells, implying that mutant NotchMcd proteins possess a novel activity that does not depend on Su(H). This suggests that NotchMcd receptors signal in a CSL-independent manner. Strikingly, six of the seven NotchMcd alleles encode carboxy-terminally truncated receptors. Notably, this region binds Dishevelled (Dsh), a membrane-associated protein required for Wnt signaling [97] (Figure 1). While the pathway activated by these truncated receptors is not well understood, molecular and genetic interaction data have suggested that mutant NotchMcd proteins escape inhibition by Dsh. Thus, these data raise the possibility that wild-type Notch receptors can signal in a CSL-independent, but Dsh-regulated manner. A possible function for this novel branch of the Notch pathway is the regulation of actin dynamics during dorsal closure in the embryo. Dorsal closure is a complex morphogenetic process that involves signaling by Notch, Dsh and Jun N-terminal Kinase (JNK) [98–100]. Consistent with a CSL-independent function of Notch in this process, expression of truncated

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constructs has indicated that Notch acts in a CSLindependent manner to modulate JNK signaling activity in the dorsal-most cells of the epidermis [99]. Whether NotchMcd proteins influence this process remains to be investigated. Conclusions and Perspectives In this review, I have highlighted the importance of keeping Notch activity under tight control and I have emphasized the diversity of the mechanisms that, despite the apparent simplicity and directness inherent in signaling via RIP, are involved in the regulation of Notch activity. Strikingly, one of the most critical steps in Notch receptor signaling, the S3 cleavage of NEXT, is apparently not subject to regulation. While there is yet no evidence that the γ-secretase activity is subject to developmental regulation, recent studies indicate that the γ-secretase complex is assembled and activated in a stepwise manner, opening the way for possible regulation [101]. Finally, the study of Notch regulation has potential implications for our understanding of other important physiological processes that depend on RIP signaling. This can be illustrated by the binding of the Notch inhibitor Numb to the intracellular domain of APP [102]. Future analysis of the cell biology of Notch receptor signaling may therefore shed light on fundamental cellular regulatory processes. Acknowledgments My apologies go to the authors of many important contributions that could not be discussed and cited due to space limitation. I thank A. Martinez-Arias for enlightening discussions and A. Bardin, A. Israël, E. Lai, M.-A. Michellod, A. Plessis and R. Le Borgne for critical reading.

13.

14.

15.

16.

17. 18. 19. 20.

21.

22.

23.

24.

25.

26.

27.

References 1. Artavanis-Tsakonas, S., Rand, M.D., and Lake, R.J. (1999). Notch signaling: cell fate control and signal integration in development. Science 284, 770-776. 2. Joutel, A., and Tournier-Lasserve, E. (1998). Notch signalling pathway and human diseases. Semin. Cell Dev. Biol. 9, 619-625. 3. Lewis, J. (1998). Notch signalling and the control of cell fate choices in vertebrates. Semin. Cell Dev. Biol. 9, 583-589. 4. Bray, S. (1998). Notch signalling in Drosophila: three ways to use a pathway. Semin. Cell Dev. Biol. 9, 591-597. 5. Pourquie, O. (2003). The segmentation clock: converting embryonic time into spatial pattern. Science 301, 328-330. 6. Fleming, R.J. (1998). Structural conservation of Notch receptors and ligands. Semin. Cell Dev. Biol. 9, 599-607. 7. Logeat, F., Bessia, C., Brou, C., LeBail, O., Jarriault, S., Seidah, N.G., and Israel, A. (1998). The Notch1 receptor is cleaved constitutively by a furin-like convertase. Proc. Natl. Acad. Sci. USA 95, 8108-8112. 8. Kidd, S., and Lieber, T. (2002). Furin cleavage is not a requirement for Drosophila Notch function. Mech. Dev. 115, 41-51. 9. Rand, M.D., Grimm, L.M., Artavanis-Tsakonas, S., Patriub, V., Blacklow, S.C., Sklar, J., and Aster, J.C. (2000). Calcium depletion dissociates and activates heterodimeric notch receptors. Mol. Cell. Biol. 20, 1825-1835. 10. Brou, C., Logeat, F., Gupta, N., Bessia, C., LeBail, O., Doedens, J.R., Cumano, A., Roux, P., Black, R.A., and Israel, A. (2000). A novel proteolytic cleavage involved in Notch signaling: the role of the disintegrin-metalloprotease TACE. Mol. Cell 5, 207-216. 11. Mumm, J.S., Schroeter, E.H., Saxena, M.T., Griesemer, A., Tian, X., Pan, D.J., Ray, W.J., and Kopan, R. (2000). A ligand-induced extracellular cleavage regulates gamma-secretase-like proteolytic activation of Notch1. Mol. Cell 5, 197-206. 12. Lieber, T., Kidd, S., and Young, M.W. (2002). kuzbanian-mediated cleavage of Drosophila Notch. Genes Dev. 16, 209-221.

28.

29.

30.

31.

32.

33.

34.

35.

36.

37.

Lieber, T., Kidd, S., Alcamo, E., Corbin, V., and Young, M.W. (1993). Antineurogenic phenotypes induced by truncated Notch proteins indicate a role in signal transduction and may point to a novel function for Notch in nuclei. Genes Dev. 7, 1949-1965. Jarriault, S., Brou, C., Logeat, F., Schroeter, E.H., Kopan, R., and Israel, A. (1995). Signalling downstream of activated mammalian Notch. Nature 377, 355-358. Okochi, M., Steiner, H., Fukumori, A., Tanii, H., Tomita, T., Tanaka, T., Iwatsubo, T., Kudo, T., Takeda, M., and Haass, C. (2002). Presenilins mediate a dual intramembranous gamma-secretase cleavage of Notch-1. EMBO J. 21, 5408-5416. Schroeter, E.H., Kisslinger, J.A., and Kopan, R. (1998). Notch-1 signalling requires ligand-induced proteolytic release of intracellular domain. Nature 393, 382-386. Fortini, M.E. (2001). Notch and presenilin: a proteolytic mechanism emerges. Curr. Opin. Cell Biol. 13, 627-634. De Strooper, B. (2003). Aph-1, Pen-2, and Nicastrin with Presenilin generate an active gamma-Secretase complex. Neuron 38, 9-12. Bray, S., and Furriols, M. (2001). Notch pathway: making sense of suppressor of hairless. Curr. Biol. 11, R217-R221. Petcherski, A.G., and Kimble, J. (2000). LAG-3 is a putative transcriptional activator in the C. elegans Notch pathway. Nature 405, 364-368. Kao, H.Y., Ordentlich, P., Koyano-Nakagawa, N., Tang, Z., Downes, M., Kintner, C.R., Evans, R.M., and Kadesch, T. (1998). A histone deacetylase corepressor complex regulates the Notch signal transduction pathway. Genes Dev. 12, 2269-2277. Morel, V., and Schweisguth, F. (2000). Repression by suppressor of hairless and activation by Notch are required to define a single row of single-minded expressing cells in the Drosophila embryo. Genes Dev. 14, 377-388. Brown, M.S., Ye, J., Rawson, R.B., and Goldstein, J.L. (2000). Regulated intramembrane proteolysis: a control mechanism conserved from bacteria to humans. Cell 100, 391-398. Marambaud, P., Wen, P.H., Dutt, A., Shioi, J., Takashima, A., Siman, R., and Robakis, N.K. (2003). A CBP binding transcriptional repressor produced by the PS1/epsilon-cleavage of N-cadherin is inhibited by PS1 FAD mutations. Cell 114, 635-645. Ellisen, L.W., Bird, J., West, D.C., Soreng, A.L., Reynolds, T.C., Smith, S.D., and Sklar, J. (1991). AN-1, the human homolog of the Drosophila notch gene, is broken by chromosomal translocations in T lymphoblastic neoplasms. Cell 66, 649-661. Struhl, G., Fitzgerald, K., and Greenwald, I. (1993). Intrinsic activity of the Lin-12 and Notch intracellular domains in vivo. Cell 74, 331345. Lieber, T., Kidd, S., Alcamo, E., Corbin, V., and Young, M.W. (1993). Antineurogenic phenotypes induced by truncated Notch proteins indicate a role in signal transduction and may point to a novel function for Notch in nuclei. Genes Dev. 7, 1949-1965. Rebay, I., Fehon, R.G., and Artavanis-Tsakonas, S. (1993). Specific truncations of Drosophila Notch define dominant activated and dominant negative forms of the receptor. Cell 74, 319-329. Qi, H., Rand, M.D., Wu, X., Sestan, N., Wang, W., Rakic, P., Xu, T., and Artavanis-Tsakonas, S. (1999). Processing of the notch ligand delta by the metalloprotease Kuzbanian. Science 283, 91-94. Klueg, K.M., Parody, T.R., and Muskavitch, M.A. (1998). Complex proteolytic processing acts on Delta, a transmembrane ligand for Notch, during Drosophila development. Mol. Biol. Cell 9, 1709-1723. Bland, C.E., Kimberly, P., and Rand, M.D. (2003). Notch-induced proteolysis and nuclear localization of the Delta ligand. J. Biol. Chem. 278, 13607-13610. Six, E., Ndiaye, D., Laabi, Y., Brou, C., Gupta-Rossi, N., Israel, A., and Logeat, F. (2003). The Notch ligand Delta1 is sequentially cleaved by an ADAM protease and gamma-secretase. Proc. Natl. Acad. Sci. USA 100, 7638-7643. Mishra-Gorur, K., Rand, M.D., Perez-Villamil, B., and ArtavanisTsakonas, S. (2002). Down-regulation of Delta by proteolytic processing. J. Cell Biol. 159, 313-324. Ikeuchi, T., and Sisodia, S.S. (2003). The Notch ligands, Delta1 and Jagged2, are substrates for presenilin-dependent "gamma-secretase" cleavage. J. Biol. Chem. 278, 7751-7754. LaVoie, M.J., and Selkoe, D.J. (2003). The Notch ligands, Jagged and Delta, are sequentially processed by alpha-secretase and presenilin/gamma-secretase and release signaling fragments. J. Biol. Chem. 278, 34427-34437. Fitzgerald, K., and Greenwald, I. (1995). Interchangeability of Caenorhabditis elegans DSL proteins and intrinsic signalling activity of their extracellular domains in vivo. Development 121, 42754282. Sun, X., and Artavanis-Tsakonas, S. (1997). Secreted forms of DELTA and SERRATE define antagonists of Notch signaling in Drosophila. Development 124, 3439-3448.

Current Biology R137

38.

39.

40.

41.

42.

43.

44.

45.

46. 47.

48. 49.

50. 51.

52.

53.

54.

55.

56.

57. 58.

59.

60. 61.

62.

63.

Struhl, G., and Adachi, A. (2000). Requirements for presenilindependent cleavage of notch and other transmembrane proteins. Mol. Cell 6, 625-636. Deblandre, G.A., Lai, E.C., and Kintner, C. (2001). Xenopus neuralized is a ubiquitin ligase that interacts with XDelta1 and regulates Notch signaling. Dev. Cell 1, 795-806. Itoh, M., Kim, C.H., Palardy, G., Oda, T., Jiang, Y.J., Maust, D., Yeo, S.Y., Lorick, K., Wright, G.J., Ariza-McNaughton, L., et al. (2003). Mind bomb is a ubiquitin ligase that is essential for efficient activaiton of Notch signaling by Delta. Dev. Cell 4, 67-82. Lai, E.C., Deblandre, G.A., Kintner, C., and Rubin, G.M. (2001). Drosophila neuralized is a ubiquitin ligase that promotes the internalization and degradation of delta. Dev. Cell 1, 783-794. Pavlopoulos, E., Pitsouli, C., Klueg, K.M., Muskavitch, M.A., Moschonas, N.K., and Delidakis, C. (2001). neuralized Encodes a peripheral membrane protein involved in delta signaling and endocytosis. Dev. Cell 1, 807-816. Bonifacino, J.S., and Weissman, A.M. (1998). Ubiquitin and the control of protein fate in the secretory and endocytic pathways. Annu. Rev. Cell Dev. Biol. 14, 19-57. Le Borgne, R., and Schweisguth, F. (2003). Unequal segregation of Neuralized biases Notch activation during asymmetric cell division. Dev. Cell 5, 139-148. Parks, A.L., Klueg, K.M., Stout, J.R., and Muskavitch, M.A. (2000). Ligand endocytosis drives receptor dissociation and activation in the Notch pathway. Development 127, 1373-1385. Le Borgne, R., and Schweisguth, F. (2003). Notch signaling: endocytosis makes Delta signal better. Curr. Biol. 13, R273-R275. Salghetti, S.E., Caudy, A.A., Chenoweth, J.G., and Tansey, W.P. (2001). Regulation of transcriptional activation domain function by ubiquitin. Science 293, 1651-1653. Posakony, J.W. (1994). Nature versus nurture: asymmetric cell divisions in Drosophila bristle development. Cell 76, 415-418. Shi, S., and Stanley, P. (2003). Protein O-fucosyltransferase 1 is an essential component of Notch signaling pathways. Proc. Natl. Acad. Sci. USA 100, 5234-5239. Okajima, T., and Irvine, K.D. (2002). Regulation of notch signaling by o-linked fucose. Cell 111, 893-904. Sasamura, T., Sasaki, N., Miyashita, F., Nakao, S., Ishikawa, H.O., Ito, M., Kitagawa, M., Harigaya, K., Spana, E., Bilder, D., et al. (2003). neurotic, a novel maternal neurogenic gene, encodes an Ofucosyltransferase that is essential for Notch-Delta interactions. Development 130, 4785-4795. Panin, V.M., Shao, L., Lei, L., Moloney, D.J., Irvine, K.D., and Haltiwanger, R.S. (2002). Notch ligands are substrates for protein Ofucosyltransferase-1 and Fringe. J. Biol. Chem. 277, 29945-29952. Okajima, T., Xu, A., and Irvine, K.D. (2003). Modulation of notchligand binding by protein O-fucosyltransferase 1 and fringe. J. Biol. Chem. 278, 42340-42345. Li, Y., Li, L., Irvine, K.D., and Baker, N.E. (2003). Notch activity in neural cells triggered by a mutant allele with altered glycosylation. Development 130, 2829-2840. Moloney, D.J., Panin, V.M., Johnston, S.H., Chen, J., Shao, L., Wilson, R., Wang, Y., Stanley, P., Irvine, K.D., Haltiwanger, R.S., et al. (2000). Fringe is a glycosyltransferase that modifies Notch. Nature 406, 369-375. Bruckner, K., Perez, L., Clausen, H., and Cohen, S. (2000). Glycosyltransferase activity of Fringe modulates Notch-Delta interactions. Nature 406, 411-415. Ju, B.G., Jeong, S., Bae, E., Hyun, S., Carroll, S.B., Yim, J., and Kim, J. (2000). Fringe forms a complex with Notch. Nature 405, 191-195. Shao, L., Moloney, D.J., and Haltiwanger, R. (2003). Fringe modifies O-fucose on mouse Notch1 at epidermal growth factor-like repeats within the ligand-binding site and the Abruptex region. J. Biol. Chem. 278, 7775-7782. Lei, L., Xu, A., Panin, V.M., and Irvine, K.D. (2003). An O-fucose site in the ligand binding domain inhibits Notch activation. Development 130, 6411-6421. Irvine, K.D. (1999). Fringe, Notch, and making developmental boundaries. Curr. Opin. Genet. Dev. 9, 434-441. Panin, V.M., Papayannopoulos, V., Wilson, R., and Irvine, K.D. (1997). Fringe modulates Notch-ligand interactions. Nature 387, 908-912. Sakamoto, K., Ohara, O., Takagi, M., Takeda, S., and Katsube, K. (2002). Intracellular cell-autonomous association of Notch and its ligands: a novel mechanism of Notch signal modification. Dev. Biol. 241, 313-326. Micchelli, C.A., Rulifson, E.J., and Blair, S.S. (1997). The function and regulation of cut expression on the wing margin of Drosophila: Notch, Wingless and a dominant negative role for Delta and Serrate. Development 124, 1485-1495.

64.

65.

66.

67.

68.

69.

70.

71.

72.

73.

74.

75.

76.

77.

78.

79.

80.

81. 82.

83.

84.

85.

86.

Klein, T., Brennan, K., and Arias, A.M. (1997). An intrinsic dominant negative activity of serrate that is modulated during wing development in Drosophila. Dev. Biol. 189, 123-134. Heitzler, P., and Simpson, P. (1993). Altered epidermal growth factor-like sequences provide evidence for a role of Notch as a receptor in cell fate decisions. Development 117, 1113-1123. de Celis, J.F., and Bray, S.J. (2000). The Abruptex domain of Notch regulates negative interactions between Notch, its ligands and Fringe. Development 127, 1291-1302. Shaye, D.D., and Greenwald, I. (2002). Endocytosis-mediated downregulation of LIN-12/Notch upon Ras activation in Caenorhabditis elegans. Nature 420, 686-690. Kooh, P.J., Fehon, R.G., and Muskavitch, M.A. (1993). Implications of dynamic patterns of Delta and Notch expression for cellular interactions during Drosophila development. Development 117, 493-507. Qiu, L., Joazeiro, C., Fang, N., Wang, H.Y., Elly, C., Altman, Y., Fang, D., Hunter, T., and Liu, Y.C. (2000). Recognition and ubiquitination of Notch by Itch, a hect-type E3 ubiquitin ligase. J. Biol. Chem. 275, 35734-35737. Cornell, M., Evans, D.A., Mann, R., Fostier, M., Flasza, M., Monthatong, M., Artavanis-Tsakonas, S., and Baron, M. (1999). The Drosophila melanogaster Suppressor of deltex gene, a regulator of the Notch receptor signaling pathway, is an E3 class ubiquitin ligase. Genetics 152, 567-576. Diederich, R.J., Matsuno, K., Hing, H., and Artavanis-Tsakonas, S. (1994). Cytosolic interaction between deltex and Notch ankyrin repeats implicates deltex in the Notch signaling pathway. Development 120, 473-481. Matsuno, K., Diederich, R.J., Go, M.J., Blaumueller, C.M., and Artavanis-Tsakonas, S. (1995). Deltex acts as a positive regulator of Notch signaling through interactions with the Notch ankyrin repeats. Development 121, 2633-2644. Matsuno, K., Ito, M., Hori, K., Miyashita, F., Suzuki, S., Kishi, N., Artavanis-Tsakonas, S., and Okano, H. (2002). Involvement of a proline-rich motif and RING-H2 finger of Deltex in the regulation of Notch signaling. Development 129, 1049-1059. Seugnet, L., Simpson, P., and Haenlin, M. (1997). Requirement for dynamin during Notch signaling in Drosophila neurogenesis. Dev. Biol. 192, 585-598. Berdnik, D., Torok, T., Gonzalez-Gaitan, M., and Knoblich, J. (2002). The endocytic protein alpha-adaptin is required for Numb-mediated asymmetric cell division in Drosophila. Dev. Cell 3, 221-231. Guo, M., Jan, L.Y., and Jan, Y.N. (1996). Control of daughter cell fates during asymmetric division: interaction of Numb and Notch. Neuron 17, 27-41. Rhyu, M.S., Jan, L.Y., and Jan, Y.N. (1994). Asymmetric distribution of numb protein during division of the sensory organ precursor cell confers distinct fates to daughter cells. Cell 76, 477-491. Santolini, E., Puri, C., Salcini, A.E., Gagliani, M.C., Pelicci, P.G., Tacchetti, C., and Di Fiore, P.P. (2000). Numb is an endocytic protein. J. Cell Biol. 151, 1345-1352. O'Connor-Giles, K.M., and Skeath, J.B. (2003). Numb inhibits membrane localization of sanpodo, a four-pass transmembrane protein, to promote asymmetric divisions in Drosophila. Dev. Cell 5, 231243. Spana, E.P., Kopczynski, C., Goodman, C.S., and Doe, C.Q. (1995). Asymmetric localization of numb autonomously determines sibling neuron identity in the Drosophila CNS. Development 121, 34893494. Spana, E.P., and Doe, C.Q. (1996). Numb antagonizes Notch signaling to specify sibling neuron cell fates. Neuron 17, 21-26. Cooper, M.T., Tyler, D.M., Furriols, M., Chalkiadaki, A., Delidakis, C., and Bray, S. (2000). Spatially restricted factors cooperate with notch in the regulation of Enhancer of split genes. Dev. Biol. 221, 390-403. Hsieh, J.J., Zhou, S., Chen, L., Young, D.B., and Hayward, S.D. (1999). CIR, a corepressor linking the DNA binding factor CBF1 to the histone deacetylase complex. Proc. Natl. Acad. Sci. USA 96, 2328. Morel, V., Lecourtois, M., Massiani, O., Maier, D., Preiss, A., and Schweisguth, F. (2001). Transcriptional repression by suppressor of hairless involves the binding of a hairless-dCtBP complex in Drosophila. Curr. Biol. 11, 789-792. Barolo, S., Stone, T., Bang, A.G., and Posakony, J.W. (2002). Default repression and Notch signaling: Hairless acts as an adaptor to recruit the corepressors Groucho and dCtBP to Suppressor of Hairless. Genes Dev. 16, 1964-1976. Rebeiz, M., Reeves, N.L., and Posakony, J.W. (2002). SCORE: a computational approach to the identification of cis-regulatory modules and target genes in whole-genome sequence data. Site clustering over random expectation. Proc. Natl. Acad. Sci. USA 99, 9888-9893.

Review R138

87.

Hubbard, E.J., Wu, G., Kitajewski, J., and Greenwald, I. (1997). sel10, a negative regulator of lin-12 activity in Caenorhabditis elegans, encodes a member of the CDC4 family of proteins. Genes Dev. 11, 3182-3193. 88. Oberg, C., Li, J., Pauley, A., Wolf, E., Gurney, M., and Lendahl, U. (2001). The Notch intracellular domain is ubiquitinated and negatively regulated by the mammalian Sel-10 homolog. J. Biol. Chem. 276, 35847-35853. 89. Wu, G., Lyapina, S., Das, I., Li, J., Gurney, M., Pauley, A., Chui, I., Deshaies, R.J., and Kitajewski, J. (2001). SEL-10 is an inhibitor of notch signaling that targets notch for ubiquitin-mediated protein degradation. Mol. Cell. Biol. 21, 7403-7415. 90. Gupta-Rossi, N., Le Bail, O., Gonen, H., Brou, C., Logeat, F., Six, E., Ciechanover, A., and Israel, A. (2001). Functional interaction between SEL-10, an F-box protein, and the nuclear form of activated Notch1 receptor. J. Biol. Chem. 276, 34371-34378. 91. Schweisguth, F. (1999). dominant-negative mutations in the beta2 and beta6 proteasome subunit genes affect alternative cell fate decisions in the Drosophila sense organ lineage. Proc. Natl. Acad. Sci. USA 96, 11382-11386. 92. Tsuda, L., Nagaraj, R., Zipursky, S.L., and Banerjee, U. (2002). An EGFR/Ebi/Sno pathway promotes delta expression by inactivating Su(H)/SMRTER repression during inductive notch signaling. Cell 110, 625-637. 93. Foltz, D.R., Santiago, M.C., Berechid, B.E., and Nye, J.S. (2002). Glycogen synthase kinase-3beta modulates notch signaling and stability. Curr. Biol. 12, 1006-1011. 94. Susini, L., Passer, B.J., Amzallag-Elbaz, N., Juven-Gershon, T., Prieur, S., Privat, N., Tuynder, M., Gendron, M.C., Israel, A., Amson, R., et al. (2001). Siah-1 binds and regulates the function of Numb. Proc. Natl. Acad. Sci. USA 98, 15067-15072. 95. Nie, J., McGill, M.A., Dermer, M., Dho, S.E., Wolting, C.D., and McGlade, C.J. (2002). LNX functions as a RING type E3 ubiquitin ligase that targets the cell fate determinant Numb for ubiquitindependent degradation. EMBO J. 21, 93-102. 96. Martinez Arias, A., Zecchini, V., and Brennan, K. (2002). CSL-independent Notch signalling: a checkpoint in cell fate decisions during development? Curr. Opin. Genet. Dev. 12, 524-533. 97. Ramain, P., Khechumian, K., Seugnet, L., Arbogast, N., Ackermann, C., and Heitzler, P. (2001). Novel Notch alleles reveal a Deltexdependent pathway repressing neural fate. Curr. Biol. 11, 17291738. 98. Noselli, S. (1998). JNK signaling and morphogenesis in Drosophila. Trends Genet. 14, 33-38. 99. Zecchini, V., Brennan, K., and Martinez-Arias, A. (1999). An activity of Notch regulates JNK signalling and affects dorsal closure in Drosophila. Curr. Biol. 9, 460-469. 100. Kaltschmidt, J.A., Lawrence, N., Morel, V., Balayo, T., Fernandez, B.G., Pelissier, A., Jacinto, A., and Martinez Arias, A. (2002). Planar polarity and actin dynamics in the epidermis of Drosophila. Nat. Cell Biol. 4, 937-944. 101. Hu, Y., and Fortini, M.E. (2003). Different cofactor activities in gamma-secretase assembly: evidence for a nicastrin-Aph-1 subcomplex. J. Cell Biol. 161, 685-690. 102. Roncarati, R., Sestan, N., Scheinfeld, M.H., Berechid, B.E., Lopez, P.A., Meucci, O., McGlade, J.C., Rakic, P., and D'Adamio, L. (2002). The gamma-secretase-generated intracellular domain of betaamyloid precursor protein binds Numb and inhibits Notch signaling. Proc. Natl. Acad. Sci. USA 99, 7102-7107.