Protein Interactions

The Notch protein has 36 EGF-like repeats: two of them (numbers 11 and 12) are required for the interaction with the Delta and Serrate ligands. A Notch mutation has been isolated in its Delta- and Serrate-binding domain. It behaves genetically as both a Notch antimorphic and as a loss-of-function mutation. This mutation, NM1, carries a Glu to Val substitution in the Notch EGF repeat 12. The NM1 allele interacts with other Notch alleles such as Abruptex and split and with mutations in the Notch-ligand genes Delta and Serrate (deCelis, 1993).

During wing development in Drosophila, the Notch receptor is activated along the border between dorsal and ventral cells, leading to the specification of specialized cells that express Wingless (Wg) and organize wing growth and patterning. Three genes, fringe(fng), Serrate(Ser) and Delta (Dl), are involved in the cellular interactions leading to Notch activation. The relationship between these genes has been investigated by a combination of expression and coexpression studies in the Drosophila wing. Ser is normally expressed in dorsal cells while Dl is initially expressed by all wing cells. However, their expression soon becomes restricted to the dorso-ventral boundary. In order to study Ser and Dl signaling between dorsal and ventral cells, Dl and Ser were expressed ectopically along the anterior side of the anterior-posterior compartment boundary. These experiments confirm that Ser and Dl induce and maintain each other's expression by a positive feedback loop. Importantly, their ability to induce each others expression is dorsal-ventrally asymmetric, because Ser induces Dl strongly in ventral cells, but only very weakly in dorsal cells, whereas Dl induces Ser expression in dorsal cells, but not in ventral cells. fng is expressed specifically by dorsal cells and functions to position and restrict this feedback loop to the developing dorsal-ventral boundary. This is achieved by fng through a cell-autonomous mechanism that inhibits a cell's ability to respond to Serrate protein and potentiates its ability to respond to Delta protein (Panin, 1997).

To determine the effects of Fringe protein on Ser and Dl activity, margin gene expression and margin bristle formation were asayed while coexpressing these proteins in ventral cells. Ectopic expression of Ser leads to ectopic wing-margin gene expression and adult wing-margin formation in ventral cells along the edges of the ectopic Ser stripe. Misexpression of Fng in ventral cells inhibits these effects of Ser activity, demonstrating that Fng can inhibit Ser signaling. Misexpression of Dl induces ectopic wing-margin formation and Ser expression in dorsal cells but not in ventral cells. However, when Fng is misexpressed in ventral cells, Dl induces Ser expression and margin formation in both dorsal and ventral cells. Thus Fng potentiates Dl signaling, allowing ventral cells to respond to Dl just as dorsal cells normally do. Experiments show that Fng inhibits Ser activity only when it is expressd in receiving cells, and not when expression is restricted to Ser-signaling cells. Activated Notch can induce both Ser and Dl, and activated Notch has similar effects on dorsal and ventral cells, implying that Fng exerts its effects upstream of Notch activation. Because Fng is extracellular, this implies that activity of cell-associated Fng protein differentially modulates the binding and/or activation of Notch by its two ligands (Panin, 1997).

During investigations to further understand the biochemical mechanism by which Delta signaling is regulated, four Delta isoforms have been identified in Drosophila embryonic and larval extracts. The proportions of these four isoforms vary during embryonic development. The L isoform predominates in embryonic extracts during the first half of embryogenesis, whereas three of the four isoforms are present in approximately equal amounts in extracts prepared from animals completing embryogenesis. Immunoprecipitation of Delta from larval extracts reveals that these four isoforms are present in relatively equal amounts. In Drosophila cultured cells programmed to express full-length Delta protein, the Delta L I1, I2, and S isoforms are found in cell extracts, and Delta S is also found in the surrounding medium. The apparent molecular weights of the embryonic and larval Delta S isoforms are substantially less than that of the full-length L isoform; yet these Delta S isoforms react with Delta-specific mAbs 9B and 8A, which are specific for the Delta extracellular domain. Embryonic Delta S has an apparent molecular weight very similar to that of DeltaSEC1, a secreted form of the Delta extracellular domain, truncated at amino acid 573, which has been expressed in Drosophila cultured cells. These findings suggest that Delta S could be a proteolytically processed derivative of Delta L, cleaved within the Delta extracellular domain. One or more proteolytic activities, present during later stages of embryogenesis and in young larvae, is shown to be capable of processing full-length Delta (Klueg, 1998).

Colocalization studies in cellular blastoderm embryos reveal that antibodies to the Delta intracellular domain localize to plasma membranes, as do antibodies to the Delta extracellular domain. However, some subtle differences at this stage in the distributions of Delta extracellular and intracellular domain epitopes are observed. At high magnification, occasional foci of intense staining are observed at or near the plasma membrane with antibodies to the Delta extracellular domain. These foci are not detected with antibodies to the intracellular domain. Before gastrulation, within the mesodermal anlage, antibodies to the intracellular domain localize to vesicular structures as do antibodies to the extracellular domain. Merging these images reveals that these two classes of Delta antibodies colocalize within a majority of these vesicular structures. However, a few vesicles appear to react with only antibodies specific for the extracellular or intracellular domain. Collectively, these localization studies in embryos and cultured cells provide evidence for the existence and distinguishable localization of at least three Delta isoforms in vivo: Delta L, Delta S, and Delta IC (Klueg, 1998).

Delta is demonstrated to be a transmembrane ligand that can be taken up by Notch-expressing Drosophila cultured cells. Cell culture experiments imply that full-length Delta is taken up by Notch-expressing cells. Evidence is presented that suggests this uptake occurs by a nonphagocytic mechanism. If phagocytosis or a similar mechanism mediates clearance of Delta-Notch complexes from the surfaces of Notch+ cells during Delta-Notch interactions in S2 cells, one would predict that large amounts of membrane from Delta+ cells would be taken up by Notch+ cells during this process. Vectors were used supporting expression of either of two full-length Drosophila transmembrane proteins, Boss or Neuroglian, in cotransfections with an expression vector encoding full-length Delta to mark Delta+ cell membranes. In experiments in which Drosophila S2 cells are programmed to express full-length Delta and Boss, and then aggregated with Notch+ cells, Boss is not detectable in Delta+ vesicles within Notch+ cells. However, on occasion, Boss colocalizes with Delta in Delta+ vesicles within Delta+ cells. In similar experiments, using Neuroglian instead of Boss to label plasma membranes, no colocalization of Neuroglian in Delta+ vesicles in Notch+ cells is observed (Klueg, 1998).

The finding of cross talk between two pathways is important news in developmental biology. Dishevelled is a key element in the wingless pathway, and also appears to physically interact with the intracellular domain of Notch. The dishevelled gene interacts antagonistically with Notch and its ligand Delta. A direct physical interaction between Dishevelled and the Notch carboxyl terminus, distal to the cdc10/ankyrin repeats, suggests a mechanism for this interaction. It is proposed that Dishevelled, in addition to transducing the Wingless signal, blocks Notch signaling directly, thus providing a molecular mechanism for the inhibitory cross talk observed between these pathways (Axelrod, 1996).

The Notch signaling pathway plays an important role during the development of the wing primordium, especially of the wing blade and margin. In these processes, the activity of Notch is controlled by the activity of the dorsal specific nuclear protein Apterous, which regulates the expression of the Notch ligand, Serrate, and the Fringe signaling molecule. The other Notch ligand, Delta, also plays a role in the development and patterning of the wing. It has been proposed that Fringe modulates the ability of Serrate and Delta to signal through Notch and thereby restricts Notch signaling to the dorsoventral boundary of the developing wing blade. The results are reported of experiments aimed at establishing the relationships between Fringe, Serrate and Delta during wing development (Klein, 1998).

The initial stages of the development of the wing blade require Notch signaling and lead to the activation of the vestigial boundary enhancer (vgBE) at the interface between dorsal and ventral cells. Ser is not involved in this event and therefore there ought to be another Notch ligand, under the control of ap, that is responsible for the activation of the vgBE. The product of the Delta gene is a good candidate for this function. During the second instar, Dl is expressed throughout the wing disc, but it is slightly upregulated over the ventral region and shortly afterwards, its pattern of expression is identical to that of vgBE, i.e. a 2- to 3- cell-wide stripe that straddles the DV interface. Furthermore, the expression of Delta is similar to that of the vgBE in Ser and ap mutant discs: in ap mutant wing discs, expression of Dl is lost at the time when the wing primordium is induced, whereas in Ser mutants expression is detected until early third instar. This suggests that Delta might be the activating ligand for the Notch-dependent expression of the vgBE, which operates in the absence of Ser. Consistent with this possibility, ectopic expression of Dl can rescue the loss of wing blade tissue and of wing margin characteristic of ap and Ser mutants (Klein, 1998).

Serrate, in turn, along with Delta, refines Notch function at the DV interface. After the establishment and expansion of the wing primordium, there is a new requirement for Notch signalling in the growth and patterning of the wing blade. In this process, both Serrate and Delta act as ligands for Notch and, as in earlier stages, have different patterns of gene expression, which suggests that they might have different functions. However, ectopic expression of either Dl or Ser will rescue the loss of wing tissue and of wing margin characteristic of ap mutants, and this raises the question of why are there two different ligands to achieve the activation of Notch in these early stages of wing development. It is believed that coexpression of both ligands might result in a different degree of Notch activation than would be achieved individually -- this might allow for a finer degree of regulation for Notch activity. In the presence of Serrate, Delta is able to signal although with a reduced activity level, which perhaps reflects a competition between Serrate and Delta for Notch (Klein, 1998).

Cell-cell signaling mediated by the receptor Notch regulates the differentiation of a wide variety of cell types in invertebrate and vertebrate species, but the mechanism for signal transduction following receptor activation is unknown. A recent model proposes that ligand binding induces intracellular processing of Notch; the processed intracellular form of Notch then translocates to the nucleus and interacts with DNA-bound Suppressor of Hairless [Su(H)], a transcription factor required for target gene expression. Intracellular cleaveage has been suggested to occur within either the transmembrane domin or the first 10 amino acids of the cytoplasmic domain. As intracellular processing of endogenous Notch has so far escaped immunodetection, a sensitive nuclear-activity assay was devised to monitor indirectly the processing of an engineered Notch in vivo. First, the non-membrane-tethered intracellular domain of Notch, fused to the DNA-binding domain of Gal4, regulates transcription in a Delta-independent manner. This transcriptional regulation requires Su(H) activity, suggesting that Su(H) may not only target the Notch intracellular domain to the DNA but may also have an additional function. For instance, Su(H) may be required to protect processed Notch from degradation, or participate in transcriptional activation together with processed Notch. Subsequently, full-length Notch, containing the Gal4 DNA-binding domain inserted 27 amino acids carboxy-terminal to the transmembrane domain, activates transcription in a Delta-dependent manner. These results provide indirect evidence for a ligand-dependent intracellular processing event in vivo, supporting the view that Su(H)-dependent Notch signaling involves intracellular cleavage, and transcriptional regulation by processed Notch (Lecourtois, 1998).

Processing of the notch ligand delta by the metalloprotease Kuzbanian

Signaling by the Notch surface receptor controls cell fate determination in a broad spectrum of tissues. This signaling is triggered by the interaction of the Notch protein with what, so far, have been thought to be transmembrane ligands expressed on adjacent cells. Here biochemical and genetic analyses show that the ligand Delta is cleaved on the surface, releasing an extracellular fragment capable of binding to Notch and acting as an agonist of Notch activity. The ADAM disintegrin metalloprotease Kuzbanian is required for this processing event. Given the similar phenotypes produced by loss of Notch signaling and loss-of-function mutations for kuz, it has been suggested that Kuz may be involved in the cleavage of N (Pan, 1996). This hypothesis is not corroborated by recent biochemical studies, indicating that the functionally crucial cleavage of N in the trans-Golgi network is catalyzed by a furinlike convertase (Logeat, 1997). These observations raise the possibility that Notch signaling in vivo is modulated by soluble forms of the Notch ligands (Qi, 1999).

A genetic screen to identify modifiers of the phenotpes associated with the constitutive expression of a dominant negative transgene of kuz (kuzDN) in developing imaginal discs has identified Delta as an interacting gene (X. Wu, W. Wang, and T. Xu, unpublished observation reported by Qi, 1999). Flies expressing this dominant negative kuz construct, despite carrying a wild-type complement of kuz, become semi-lethal when heterozygous for a loss-of-function Delta mutation (X. Wu, W. Wang, and T, Xu, unpublished observation reported by Qi, 1999). In contrast, Delta duplications rescue the phenotypes associated with kuzDN. The kuzDN flies display extra vein material (especially deltas at the ends of the longitudinal veins); wing notching (observed with a low penetrance); extra bristles on the notum, and they also have small rough eyes. When kuzDN flies carry three, as opposed to the normal two, copies of wild-type Notch the bristle and eye phenotypes are not affected, nor are the vein deltas altered. However, the kuzDN phenotypes are effectively suppressed by Delta duplications, indicating that a higher copy number of Dl molecules is capable of overriding the effects of the kuzDN construct (Qi, 1999).

Delta has been shown to be cleaved by Kuz in transfected cultured cells, with the release of the extracellular domain of Delta. Sequencing reveals a putative propeptide processing site that is conserved in all Delta homologs. There is a distinct absence of cleaved Delta in kuz minus embryos but no difference in the processing of Notch. The biological activity of Delta extracellular domain can be detected in culture. Ligand-dependent Notch activation has been demonstrated in cortical neurons, which express endogenous Notch receptors, causing morphological changes as well as retractions of neurites. The same effects are observed when neurons are cultured in the presence of the extracellular domain of Delta. The importance of additional cleavages in Dl, the mode of activity of full-length Dl, and whether the second ligand Serate is also processed are critical questions to resolve. It is now apparent that future analysis of Delta in Notch signaling events must consider its potential as a diffusable ligand (Qi, 1999).

Ligand-receptor interactions and trans-endocytosis of Delta, Serrate and Notch: members of the Notch signalling pathway in Drosophila

Molecular evidence has established that direct heterotypic interactions occur between the Drosophila receptor Notch and the ligands Delta and Serrate, and that homotypic interactions occur between Delta molecules on opposing cell surfaces. Using an aggregation assay developed for Drosophila cultured cells, the affinities of these interactions have been compared. The heterotypic interactions between Notch and the ligands Delta and Serrate have higher affinities than homotypic interactions between Delta molecules. Contrary to previous suggestions, the evidence implies that the interactions between Serrate and Notch are similar in affinity to those between Delta and Notch. Fringe does not detectably affect the ligand-receptor interactions of the Notch pathway in cultured cells. Furthermore, Serrate, like Delta, is a transmembrane ligand that can participate in reciprocal trans-endocytosis of ligand and receptor between expressing cells. These findings imply that qualitative differences between Delta- and Serrate-mediated Notch signaling depend on characteristics other than intrinsic ligand-receptor affinities or the ability to participate in reciprocal ligand and receptor trans-endocytosis (Klueg, 1999).

The Delta extracellular domain and intracellular domain are taken up by adjacent Notch+ Drosophila cultured cells. Using antibodies against the Serrate extracellular domain or a MYC epitope tag in the intracellular domain, it was asked whether the entire Serrate molecule is transferred into neighboring Notch+ cells. In Drosophila Serrate-Notch cell aggregates, the Serrate extracellular domain and intracellular domain are found to be taken up by neighboring Notch+ cells. The fraction of Notch+ cells adjacent to one or more SerrateWTMYC+ cells that contain vesicles positive for the Serrate extracellular domain is 17%. This is similar to the fraction of Notch+ cells that contain vesicles positive for the Serrate intracellular domain (15%). The fraction of Notch+ cells that contain vesicles positive for the Delta extracellular domain (27%) was recorded in parallel as a control (Klueg, 1999).

Delta+ cells adjacent to Notch+ cells contain vesicles positive for the Notch intracellular domain, demonstrating that the entire Notch molecule can be taken up by adjacent Delta+ cells. To determine whether similar trans-endocytosis occurs during Serrate-Notch interactions, trans-endocytosis of Notch into SerrateWTMYC+ cultured cells was examined. Notch is found to be trans-endocytosed, and the percentage of SerrateWTMYC+ cells that contain vesicles positive for the Notch intracellular domain is 38%, similar to the percentage of Delta+ cells that contain vesicles positive for the Notch intracellular domain. Heterotypic aggregates formed after 15 minutes of aggregation were examined, and it was found that trans-endocytosis of Delta and Notch occurs shortly after their interaction is initiated. Similar observations have been repeated for Serrate+-Notch+ heterotypic cell aggregates. Both Serrate and Notch are trans-endocytosed shortly after interaction is initiated. Delta, however, is not taken up by adjacent Delta+ cells in homotypic aggregates (Klueg, 1999).

Ligand endocytosis drives receptor dissociation and activation in the Notch pathway

Receptor and ligand processing have recently come under scrutiny as critical elements in the regulation of Notch signaling. Delta is proteolytically processed to yield at least four isoforms; however, the functional significance of this processing is currently unclear. Processing of Delta may be necessary for signal activation or for downregulation, or may result from protein degradation following clearance of ligand from the cell surface. Notch is processed in a complex manner that is thought to be required for genesis and activation of the receptor. First, Notch is cleaved during transport through the Golgi at a site amino-proximal to the transmembrane domain ('site 1' or 'S1') by a furin-like convertase. Following this cleavage, Notch is 'reassembled' and transported to the cell surface as a heterodimeric receptor. Another cleavage event (termed 'S3') within the intracellular domain has also been shown to occur. The S3 cleavage of Notch is ligand-dependent and produces a Notch intracellular domain fragment that may act, in conjunction with Su(H), in the nucleus as the primary Notch signal transducer. The mechanism that triggers the intracellular domain cleavage is unknown. However, the Notch/lin-12 repeats (LNRs) within the receptor extracellular domain may contribute to regulation of this cleavage. When the LNRs are removed, intracellular domain cleavage occurs in a constitutive manner in the absence of ligand. This has led to the hypothesis that binding of Delta to Notch may result in a cleavage event (termed 'S2') in the Notch extracellular domain. This cleavage would uncouple the LNRs from the remainder of the receptor, allowing the intracellular domain cleavage (S3) to occur constitutively. Recently, Notch S2 cleavage has been demonstrated in mammalian cells (Mumm, 2000 in press, cited in Parks, 2000). S2 cleavage in these cells occurs in response to ligand binding and blocking S2 cleavage results in loss of S3 cleavage, consistent with a proteolytic cascade model of Notch activation (Parks, 2000).

Endocytosis of the ligand Delta (by the signaling cell), possibly by inducing cleavage of the receptor at the S2 site, is required for activation of the receptor Notch during Drosophila development. The Notch extracellular domain (NotchECD) dissociates from the Notch intracellular domain (NotchICD) and is trans-endocytosed into Delta-expressing cells in wild-type imaginal discs. Reduction of dynamin-mediated endocytosis in developing eye and wing imaginal discs reduces Notch dissociation and Notch signaling. Furthermore, dynamin-mediated Delta endocytosis is required for Notch trans-endocytosis in Drosophila cultured cell lines. Endocytosis-defective Delta proteins fail to mediate trans-endocytosis of Notch in cultured cells, and exhibit aberrant subcellular trafficking and reduced signaling capacity in Drosophila. It is suggested that endocytosis into Delta-expressing cells of NotchECD bound to Delta plays a critical role during activation of the Notch receptor and is required to achieve processing and dissociation of the Notch protein (Parks, 2000).

The separation of NotchECD from NotchICD would relieve LNR-mediated repression of the S3 cleavage, which would then occur constitutively to release a non-membrane bound, activated form of NotchICD. Several predictions of this model are borne out. Dl alleles that encode endocytosis-defective ligands are loss-of-function mutations, and these defective ligands fail to support Notch trans-endocytosis in cultured cells and Delta-mediated signaling in vivo. Dynamin function, which is necessary for Notch signaling, is required for Delta endocytosis and for Notch trans-endocytosis in cultured cells, and for dissociation of NotchECD from NotchICD in developing imaginal tissues. In addition, the third epidermal growth factor-like repeat within the Delta extracellular domain is required for Delta endocytosis and Notch trans-endocytosis, and for Delta-dependent signaling during development (Parks, 2000).

Three alternative mechanisms are proposed by which endocytosis may induce receptor activation. (1) After binding of Delta to Notch, molecular strain imparted to Notch by endocytosis in the signaling and receiving cells results in a conformational change that permits access by processing enzyme(s) to the S2 site. (2) The S2 site is masked by proteins that interact with Notch to form a complex. Following Delta binding, endocytosis of Delta and Notch alters intramolecular interactions within the complex, unmasking the S2 site and making it available for cleavage. (3) Endocytosis is not required for Delta-induced S2 cleavage, but is instead required to separate NotchECD and associated proteins from the remainder of the Notch protein, thus relieving inhibition of S3 cleavage by NotchECD. The fact that Serrate-expressing cultured Drosophila cells mediate Notch trans-endocytosis at frequencies similar to those observed for Delta-expressing cells suggests that trans-endocytosis of NotchECD is one aspect of the Notch activation mechanism that is common to Notch ligands (Parks, 2000).

The Abruptex domain of Notch regulates negative interactions between Notch, its ligands and Fringe

The activation of Notch is regulated both by the temporal and spatial distribution of the ligands and by the expression of proteins such as Fringe (Fng) that are able to modulate ligand-receptor interactions. This was first evident in the developing wing, where Notch activity results in the expression of genes such as wingless and cut in a narrow 2- to 4-cell-wide domain at the dorsoventral boundary. In this process, Fng influences the effectiveness of the interactions between Notch and its ligands by preventing Ser-mediated activation and potentiating Notch activation by Dl. The localized activation of Notch initially occurs because Apterous promotes the expression of both Ser and Fng in dorsal cells, while the inhibitory effect of Fng on Ser/Notch restricts Ser signaling primarily to ventral cells. At the same time, the effect of Fng on Dl has the consequence that ventral Dl-expressing cells signal primarily to dorsal cells. A similar process occurs in the eye, where again the compartment-specific expression of fng allows localized activation of Notch at the eye dorsoventral boundary (de Celis, 2000 and references therein).

Conventionally Dl and Ser are considered activating ligands of Notch and, in many instances, their elimination has non-autonomous effects on development that are characteristic of a membrane-associated ligand. However, in the Drosophila wing and eye, both Notch ligands have also been shown to have cell-autonomous inhibitory effects on the activity of the receptor. Thus, the elimination of both ligands in clones of cells in the wing can result in Notch activation within the clone, detected as ectopic ct expression, indicating that a normal function of Dl and Ser is to prevent Notch activation within the cells in which they are expressed. In addition, ectopic expression of Dl or Ser in groups of cells causes Notch activation only in the adjacent cells. Consistent with the suggestion that the inhibitory activity of the ligands relies on interactions occurring between molecules within the same cell, the negative effects of ectopically expressed Ser can be alleviated by co-expression of full-length Notch. The negative effect of the ligands could be instrumental in determining the polarity of Notch signaling: cells expressing higher levels of ligand would have reduced Notch responsiveness compared to adjacent cells with lower ligand levels and hence Notch would be more readily activated in the cells with relatively less ligand. The concept that the relative levels of Notch and Dl are important for signaling is also evident from the phenotypes caused by varying the dosage of these genes. Finally, Dl and Notch have been seen to co-localize on the surface of cultured cells, suggesting that they could interact in the plasma membrane. However, the antagonistic interactions could be occurring anywhere within the cell and the functional domain of Notch involved in this process has not been characterised (de Celis, 2000 and references therein).

The extracellular domain of Notch contains an array of 36 EGF repeats, two of which, repeats 11 and 12, are necessary for direct interactions between Notch with Delta and Serrate. An investigation has been carried out of the function of a region of the Notch extracellular domain where several missense mutations, called Abruptex, are localized. These Notch alleles are characterized by complex complementation patterns and phenotypes that are the opposite of those observed with a loss of Notch function. In Abruptex mutant wing discs, only the negative effects of the ligands and Fringe are affected, resulting in the failure to restrict the expression of cut and wingless to the dorsoventral boundary. It is suggested that Abruptex alleles identify a domain in the Notch protein that mediates the interactions between Notch, its ligands and Fringe that result in suppression of Notch activity (de Celis, 2000).

In wild-type discs, the response of Notch to Dl and Ser is affected by the presence of Fng, which is expressed in dorsal cells. Since the domain of Fng expression corresponds to the region where Dl loses its capacity to antagonize Notch in NAx mutants, an analysis was carried out to see whether NAx mutations have an altered sensitivity to Fng by comparing the consequences of ectopic fng expression in wild-type and NAx discs. As with the ectopic ligand expression, clones of cells expressing fng that cross the dorsoventral boundary inhibit expression of ct except at the clone borders. When the Fng-expressing clones lie in the ventral compartment, ct is induced in the cells at the boundary of the clone, with the result that ct is detected in neighboring fng+ and fng- cells. The ability of Fng to prevent ct expression is reduced when Fng-expressing clones are induced in NAx mutant backgrounds. In a weak NAx allelic combination, the expression of ct is still highest at clone boudaries, but significant expression is detected within the clone. In the more severe mutants, the Fng-expressing cells have little or no inhibitory effect on ct, and there are high levels of Ct throughout the clone. Similar effects are seen when fng misexpression is driven by Gal4-sal. Normally this causes an inhibition of ct expression at the dorsoventral boundary; in NAx mutant discs, however, Ct is detected throughout most of the domain of ectopic Fng-expression. If the NAx domain is significant in the interactions between Notch and Fng, the NAx mutations should modify phenotypes caused by alterations in fng expression. In the allele fngD4, fng is expressed throughout the wing pouch, causing severe scalloping of the wing margin. This correlates with the loss of ct and wg expression at the dorsoventral boundary and the expansion of vvl/drifter expression. In NAx heterozygous flies, the phenotype of fngD4 is reduced both at the level of wing scalloping and the expression of dorsoventral boundary markers. In hemizygous NAx males, both the expression of ct and vvl and the adult phenotype are similar to the expression and phenotype typical of NAx. Taken together, these results suggest that NAx proteins are also deficient in some activity related to the capability of Fng to restrict Notch activity (de Celis, 2000).

The amino-acid sequence of Fng indicates that it could be a glycosyltransferase. Since NAx mutations affect the extracellular domain of Notch, the fact that the NAx alleles have altered behavior with respect to Fng suggests that the mutated domain could be a target for Fng-mediated glycosylation. If the NAx mutations perturb glycosylation of Notch by Fng, this might explain why they only affect the activity of Notch in the imaginal discs and not in the early embryo, since fng is only required at later stages of development. NAx alleles also affect several processes, such as sensory organ development and vein cell differentiation, that do not seem to require fng activity. This indicates that the NAx domain also affects negative interactions between Notch with Dl and Ser independent of fng function (de Celis, 2000).

The results shown here indicate that the NAx domain of Notch is only necessary to mediate the functions of Fng and the ligands that result in the suppression of Notch activity. A comparison between the effects on Fng, Dl and Ser indicates that the interactions between these molecules and Notch are affected to different extents by NAx mutations. For example, although the dominant negative effects of Dl and Fng are dramatically reduced in NAx alleles, these mutations do not appear to compromise the potentiating effect of Fng on Dl activation, since there is still a strong bias towards Dl activity in the dorsal domain where Fng is present. Similarly high levels of ectopic Ser can efficiently suppress Notch activity in NAx backgrounds, even though the phenotype of NAx mutant discs indicates that NAx mutations perturb the dominant negative effects of Ser when it is expressed at normal levels. Each NAx allele has a characteristic strength that is reflected in its phenotype and in the extent of ectopic ct activation. Furthermore, heteroallelic combinations between NAx alleles often result in synergistic phenotypes, a phenomenon called negative complementation. This suggests that the correct conformation of the NAx domain in Notch multimers is critical for the efficiency of the interactions between Notch, its ligands and Fng that determine suppression of Notch activity (de Celis, 2000).

Structural requirements for Notch signaling with Delta and Serrate during the development and patterning of the wing disc of Drosophila

The Delta and Serrate proteins interact with the extracellular domain of the Notch receptor and initiate signaling through the receptor. The two ligands are very similar in structure and have been shown to be interchangeable experimentally; however, loss of function analysis indicates that they have different functions during development and analysis of their signaling during wing development indicates that the Fringe protein can discriminate between the two ligands. This raises the possibility that the signaling of Delta and Serrate through Notch requires different domains of the Notch protein. This possibility has been tested by examining the ability of Delta and Serrate to interact and signal with Notch molecules in which different domains have been deleted. This analysis has shown that EGF-like repeats 11 and 12, the RAM-23 and cdc10/ankyrin repeats and the region C-terminal to the cdc10/ankyrin repeats of Notch are necessary for both Delta and Serrate to signal via Notch. They also indicate, however, that Delta and Serrate utilize EGF-like repeats 24-26 of Notch for signaling, but there are significant differences in the way they utilize these repeats (Lawrence, 2000).

Expression of Delta and Serrate with molecules that lack EGF-like repeats 24-26 produce differing results. Delta can signal through a Notch protein that lacks these repeats, but Serrate cannot. This requirement for EGF-like repeats 24-26 is surprising and indicates that the influence of these repeats on the interactions between Notch and its ligands in cell culture and in vivo might reflect a requirement for these repeats in signaling. Interestingly, there are differences in the way Delta and Serrate require these repeats. In the case of Serrate, the requirement for signaling is absolute, whereas in the case of Delta it is conditional on the presence of EGF-like repeats 17-19, which have been shownto be structurally related to 24-26. These differences are made more clear when comparing the interactions of Delta and Serrate with the dominant negative extracellular Notch constructs (ECNs). Delta cannot signal with full length Notch molecules that lack repeats 10-12 or 17-19 and 24-26, nor interact with ECN molecules that lack these repeats. Although Serrate cannot signal with FLN molecules lacking these repeats, it can, however, interact with the corresponding ECN molecules. This suggests that whereas in the case of Delta a direct interaction with Notch amounts to signaling, this might not be sufficient for Serrate. In the case of Serrate, further modifications of Notch or interactions with other proteins might be required to trigger a signal (Lawrence, 2000).

Notch responds differently to Delta and Wingless in cultured Drosophila cells

Notch, a cell surface receptor, is required for the production of different types of cells during Drosophila development. Notch activates expression of one set of genes in response to ligand Delta and another set of genes in response to the ligand Wingless. Just how Notch initiates these different intracellular activities has been the focus of this study. Cultured cells expressing Notch were treated with Delta or Wingless, and the effect on Notch was examined by Western blotting. Treatment of cells with Delta results in accumulation of ~120-kDa Notch intracellular domain molecules in the cytoplasmic fraction. This form of Notch does not accumulate in cells treated with Wingless; rather, the ~350-kDa full-length Notch molecules accumulate. These results indicate that N responds differently to binding by Delta and by Wingless, and suggest that although the Delta signal is transduced by the Notch intracellular domain released from the plasma membrane, the Wingless signal is transduced by the Notch intracellular domain associated with the plasma membrane. It is proposed that the N receptor is a 'switch' for activation of different signaling pathways during development. Dl binds the EGF-like repeats 11-12 region to shunt the N120-Su(H) complex into the nucleus for turning on the expression of Dl-related genes. Wg binds the EGF-like repeats 19-36 region to send a transcriptional activator to the nucleus for turning on the expression of Wg-related genes (Wesley, 2000).

Schneider cells expressing Notch (S2-N cells) treated with Dl for 1h accumulate ~120-kDa N molecules (N120). Dl binds N in the extracellular region, including EGF-like repeats 11 and 12). S2 cells expressing N molecules lacking this region, NDeltaEGF1-18, do not accumulate N120 molecules in response to treatment with Dl. This indicates that N120 accumulates in response to Dl binding N. N120 is the complete intracellular domain and is similar to the ~120-kDa N intracellular domain molecule shown to accumulate in vivo in response to D (Wesley, 2000).

N120 molecules do not accumulate in S2-N cells treated with Wg for 1, 2, or 5 hours. However, S2-N cells treated with Wg for 5h accumulates ~350-kDa N molecules (N350) but not S2-N cells treated with Dl. N350 is the full-length co-linear N molecule containing both the intracellular and extracellular domains. Wg binds N in the EGF-like repeats 19-36 region. S2 cells expressing N molecules lacking this region, NDeltaEGF19-36, do not accumulate co-linear molecules when treated with Wg for 5h. In contrast, truncated, co-linear NDeltaEGF19-36 molecules containing the Wg binding sites accumulate upon treatment with Wg. These results indicate that accumulation of N350 in S2-N cells is in response to Wg binding N. Accumulation of N350 molecules is also discernible in cells treated with Wg for 2h when the blots are exposed to film for shorter periods. In contrast to Wg-treated cells, Dl-treated cells in the same blots always have lower levels of N350 compared with the levels in untreated cells (Wesley, 2000).

Accumulation of N350 molecules in Wg-treated cells is not due to activity of the endogenous Notch gene, which is rearranged in S2 cells. It is not due to a general increase or stabilization of all proteins in the cells: all N molecules do not accumulate, and the total protein levels in the three lanes are comparable. It is also not due to a Wg effect that is unrelated to N binding but retards N processing for cell surface presentation. Otherwise, co-linear NDeltaEGF19-36 would have also accumulated, but it did not. Thus, whereas Dl binding full-length N results in accumulation of N120, Wg binding results in accumulation of the co-linear N350 (Wesley, 2000).

Treatment of S2-N cells with Dl or Wg for 2 h also results in accumulation of ~55-kDa N molecules (N55). N55 contains only the amino terminus half of the intracellular domain, requires about 2 h to accumulate, and is variably recovered after about 3 h of treatment (Wesley, 2000).

To determine whether the responses observed in S2 cells are general N responses to treatments with Dl and Wg, the experiments were repeated with clone-8 cells that express N endogenously. The results show that N in clone-8 cells responds similarly to N in S2 cells. Treatment with Dl results in accumulation of N120 and not N350, whereas treatment with Wg results in accumulation of N350 and not N120; both Dl and Wg treatments result in accumulation of N55 molecules. The difference in levels of N350 between Dl-treated and Wg-treated cells is obvious after just 2 h of treatment. Clone-8 cells express a higher level of N55 molecules in the absence of any treatment, presumably because they also express Dl endogenously (Wesley, 2000).

When Dl binds N in vivo, the ~120-kDa N intracellular domain is released into the cytoplasm. To determine whether the N120 in these in vitro experiments with Dl also accumulates in the cytoplasm, S2-N cells were fractionated and analyzed following treatments with Dl and Wg. Following treatment with Dl, N120 molecules accumulate in the cytoplasmic fraction. In contrast, N350 molecules accumulate in the membrane fraction following treatment with Wg. N55 molecules are not consistently detected in these experiments as they are very unstable in this fractionation and extraction procedure (Wesley, 2000).

It is not known whether the N120 molecules that accumulate in the cytoplasm in response to Dl are the same as those present in the membranes or whether they are different molecules migrating in the same region of the gel. Membrane-tethered N intracellular domain (Nintra), untethered Nintra, and N120 migrate alongside each other in these gels. N120 molecules associated with the membranes or with the cytoplasm are probably the membrane-tethered or released N intracellular domain, respectively. Accumulation of N350 molecules in response to Wg is likely to be in the intracellular membranes associated with production of the heterodimeric cell surface receptor. N55 is derived from N350 upon activation of Notch signaling by a ligand (Wesley, 2000).

Drosophila Gp150 is required for early ommatidial development through modulation of Notch signaling

Cellular signaling activities must be tightly regulated for proper cell fate control and tissue morphogenesis. The Drosophila leucine-rich repeat transmembrane glycoprotein Gp150 is required for viability, fertility and development of the eye, wing and sensory organs. Gp150 might function in subcellular vesicles to control appropriate intracellular levels of Dl to modulate N signaling. In the eye, Gp150 plays a critical role in regulating early ommatidial formation. Gp150 is highly expressed in cells of the morphogenetic furrow (MF) region, where it accumulates exclusively in intracellular vesicles in an endocytosis-independent manner. Loss of gp150 function causes defects in the refinement of photoreceptor R8 cells and recruitment of other cells, which leads to the formation of aberrant ommatidia. Genetic analyses suggest that Gp150 functions to modulate Notch signaling. Consistent with this notion, Gp150 is co-localized with Delta in intracellular vesicles in cells within the MF region and loss of gp150 function causes accumulation of intracellular Delta protein. Therefore, Gp150 might function in intracellular vesicles to modulate Delta- Notch signaling for cell fate control and tissue morphogenesis (Fetchko, 2002).

Sequence analysis indicates that the gp150 gene consists of six exons and five introns. The gp150 open reading frame (ORF) is restricted to the last four exons, which encode a polypeptide of 1051 amino acids. The extracellular domain of Gp150 contains 18 LRR motifs that might provide a scaffold for mediating protein-protein interactions. In addition, it has several unique structural features that are conserved during evolution. These features include specific potential N-glycosylation sites, cysteine motifs and acidic regions that flank the LRR region. Although the intracellular domain is short, it contains three conserved tyrosine phosphorylation motifs that are potential targets of tyrosine kinases and receptor tyrosine phosphatases. The conserved tyrosine phosphorylation motifs might be used for interaction with SH2 domains of some docking proteins (Fetchko, 2002).

Phenotypic analysis has demonstrated that Gp150 plays a critical role during ommatidial development. In the absence of gp150 function, both the selection and patterning of the R8 cells become aberrant and a small number of ectopic R8 cells appear to be specified. The gp150 mutant eye phenotypes mimic what occurs in loss-of-function mutants of Dl and N, suggesting that Gp150 might facilitate Dl-N signaling. Supporting this hypothesis, the reduction of Dl or N function dominantly enhances the gp150 mutant eye phenotypes (Fetchko, 2002).

Clonal analysis was carried out to reveal in which cells the function of Gp150 is required. By examining phenotypically normal but genetically mosaic ommatidia, it was found that gp150 could be either wild type or mutant in all R cells. Therefore, the function of gp150 does not appear to be required in R cells for normal ommatidial development. Given that R cells are signal-sending cells, which prevent neural differentiation in neighboring precursor cells, this result is consistent with a view that gp150 might be required in cells that function as precursor cells to R cells (Fetchko, 2002).

How might Gp150 modulate Dl-N signaling? So far, Gp150 is the only protein known to co-localize with Dl in intracellular vesicles. In a simple model, Gp150 might facilitate Dl presentation at the cell surface. Gp150 might also promote fusion between Dl-positive MVBs and lysosomes so that the intracellular levels of Dl can be reduced. The latter mechanism might be used to reduce the autonomous inhibitory effect of Dl on N signaling in the signal-receiving cells. In the absence of gp150 function, the levels of intracellular Dl increase, which autonomously blocks the N pathway in N signal-receiving cells and possibly makes these cells more responsive to instructive signals for R cell specification and differentiation, thus resulting in ommatidia with too many R cells. In tissues such as the wing and sensory organs, Dl has been shown to autonomously inhibit N signaling in a signal-receiving cell. In some cases, increased intracellular levels of Dl might allow some Dl to be presented at the cell surface to initiate N signaling on adjacent cells. This would increase lateral inhibition and result in the formation of ommatidia with fewer R cells. The fewer R cell phenotype could also be due to an insufficient number of competent retinal precursor cells that are available for the competing ommatidial clusters during R cell recruitment in gp150 mutants. In any event, the possibility that Gp150 might use other mechanisms to modulate Dl-N signaling cannot be excluded. For instance, the up-regulation of Dl in gp150 mutant eye discs might be due to impaired N signaling, since N signaling can often cause down-regulation of Dl expression in signal-receiving cells. Further work is needed to distinguish among these possibilities (Fetchko, 2002).

N signaling is known to be regulated through mechanisms such as site-specific proteolysis and glycosylation. In addition, N is recognized and ubiquitylated by an E3 ubiquitin ligase Itch, suggesting that N stability could be regulated through the proteasome-mediated pathway. An Itch-related Drosophila protein, Suppressor of deltex, also negatively regulates N signaling. Another negative regulator of N, Numb, has recently been shown to be an endocytic protein, suggesting that Numb regulation of N could occur in the endocytic pathway. The regulation of Dl is also complex. For instance, Dl is proteolytically processed so its extracellular domain can be released. From this work, it is proposed that Gp150 might function in intracellular vesicles, which include endosomes, to adjust appropriate intracellular levels of the Dl protein for modulating N signaling (Fetchko, 2002).

Functional analysis of Gp150 indicates that both the extracellular and intracellular domains of Gp150 are essential. The extracellular domain of Gp150 contains 18 LRR motifs that might provide a scaffold for mediating protein-protein interactions. In addition, it has several unique structural features that are conserved during evolution. These features include specific potential N-glycosylation sites, cysteine motifs and acidic regions that flank the LRR region. Although the intracellular domain is short, it contains three conserved tyrosine phosphorylation motifs that are potential targets of tyrosine kinases and receptor tyrosine phosphatases. The conserved tyrosine phosphorylation motifs might be used for interaction with SH2 domains of some docking proteins. Further studies are required to reveal the functional significance of these conserved structural motifs, which should help reveal mechanisms of Gp150 action in cell fate control and tissue morphogenesis (Fetchko, 2002).

An O-fucosylation site into EGF repeat 14 of Notch confers sensitivity to Dl on N expressed in R8 precursors

The receptor protein Notch is inactive in neural precursor cells despite neighboring cells expressing ligands. Specification of the R8 neural photoreceptor cells, which initiate differentiation of each Drosophila ommatidium, was investigated. The ligand Delta was required in R8 cells themselves, consistent with a lateral inhibitor function for Delta. By contrast, Delta expressed in cells adjacent to R8 could not activate Notch in R8 cells. The split mutation of Notch was found to activate signaling in R8 precursor cells, blocking differentiation and leading to altered development and neural cell death. split does not affect other, inductive functions of Notch. The Ile578-->Thr578 substitution responsible for the split mutation introduced a new site for O-fucosylation on EGF repeat 14 of the Notch extracellular domain. The O-fucose monosaccharide did not require extension by Fringe to confer the phenotype. These results suggest functional differences between Notch in neural and non-neural cells. R8 precursor cells are protected from lateral inhibition by Delta. The protection is affected by modifications of a particular EGF repeat in the Notch extracellular domain. These results suggest that the pattern of neurogenesis is determined by blocking Notch signaling, as well as by activating Notch signaling (Li, 2003).

N signaling in response to Dl is patterned in two distinct ways. In some situations, typified by induction of the wing margin, the expression pattern of Dl contributes to where N will be activated. N remains inactive where Dl is not expressed. In other cases, typified by lateral specification of R8 precursor cells during eye development, N and Dl are expressed homogeneously, and the pattern of N signaling depends on differential activity of the N and Dl proteins. Even though Dl is expressed homogeneously, it is essential in the cells taking R8 precursor fate. The requirement for Dl in the R8 precursor cannot be substituted by Dl expression in the other cells, even though together they contact all of the cells that the R8 precursor contacts. This suggest that the interaction between Dl in cells selected for R8 precursor fate and N in other cells might be qualitatively different from any interaction between Dl on non-R8 cells and N in R8 precursor cells (Li, 2003).

Inactivity of N in R8 precursor cells is not a passive event defined by absence of ligands, because even ubiquitous Dl overexpression fails to activate N in R8 precursor cells. By contrast, a recessive mutation, the split allele of N, now permits N to be activated by Dl in R8 precursor cells but has little or no effect on N signaling in many other contexts. The Dl protein in non-R8 cells is in an active form, because it can activate R8-cell N in the spl mutant (Li, 2003).

The spl mutant affects development of many retinal cell types. There is an R8 cell deficit, many other retinal cells are missing, cell death is elevated and additional cells may take R7 fate. The initiation and maintenance of atonal expression is deficient even before R8 specification begins. Mosaic analysis demonstrates that all these defects depend on the genotype of R8 cells only. Therefore N is activated in spl mutant R8 cells. Other cells must be affected indirectly as a consequence of the abnormal R8 cells. In confirmation of this, activation of the N signal transduction pathway solely in R8 cells recapitulates the spl phenotype, including the effects on other cell types (Li, 2003).

The notion that many cells might be affected indirectly in spl mutants is consistent with the role of R8 cells in founding each ommatidium. R8 cells initiate the cascade of EGF receptor-mediated inductions that recruit most of the retinal cell types, and are required for the survival of unspecified cells. The effectiveness with which R8 cells carry out these roles depends on the level of atonal expression in the R8 precursors. Reduced Atonal expression in the ato2 mutant, which is defective in ato autoregulation, reduces recruitment of other cell fates because EGF receptor is activated in fewer surrounding cells. Elevating Atonal expression by targeted expression in R8 using the G109-68 driver leads to activation of EGF receptor in more cells than normal and recruitment of excess outer photoreceptor cells. Thus, losses of many other cells are an expected consequence of the reduced Atonal expression that occurs in spl mutant R8 cells (Li, 2003).

In addition to producing ligands for the EGF receptor, R8 and other photoreceptor cells also secrete Hh, the primary signal moving the morphogenetic furrow across the eye disc. Reducing Atonal levels in R8 has further phenotypic effects through altered Hh signaling. It is proposed that defective Hh signaling is the likely explanation of non-autonomous effects of spl on the initiation of atonal expression in the morphogenetic furrow (Li, 2003).

The spl mutation also affects differentiation of sensory bristles in the epidermis. As in R8 cells in the eye, sensory organ precursor cells are specified by lateral inhibition but not inhibited by ectopic Dl expression. N signaling is important in cell fate specification within the lineage of cells descended from sensory organ precursors. It is plausible that aberrant N signaling might be responsible for bristle defects in spl mutants, although this has not been examined directly (Li, 2003).

The substitution of Thr for Ile578 in the spl mutation introduces a site for O-fucosylation into EGF repeat 14 of the N extracellular domain. This site is fucosylated in SL2 cells and provides a substrate for the further action of Fringe, an enzyme that functions to extend O-fucose glycans. Comparisons of O-fucosylation sites on clotting factors identified a consensus sequence, C2XXGGS/TC3. Similar sequences are found in eleven EGF repeats of N, although little is known about which EGF repeats are actually modified in vivo. However, site-directed mutagenesis of Factor IX and other proteins indicates that Gly residues at the -1 and -2 positions of the consensus are not essential for fucosylation. This raises the possibility that some of the other EGF repeats that contain C2XXXXS/TC3 sequences might be fucosylated. Indeed EGF repeat 25, which contains C2QNGAS/TC3, is fucosylated by Drosophila SL2 cells and is a substrate for Fringe. SL2 cells fucosylate the sequence C2RNRGTC3 in the spl mutant EGF repeat 14 and the sequence C2LNDGTC3 in wild-type EGF repeat 13. In light of these results, it seems possible that many of the 22 N EGF repeats that contain C2XXXXS/TC3 sequences might be fucosylated. These include the sequence C2QNEGSC3 in EGF repeat 12, required for Dl to bind and activate N. It is important to note that the efficiency of O-fucosylation at all these sites is unknown, as well as the efficiency with which O-fucose is extended by Fringe, so that it is possible that even within the same cell individual N molecules may carry different combinations of O-fucose and of extended O-fucose glycans (Li, 2003).

During eye development, fng mutants have little direct effect on R8 specification. In addition, fng is not required for the spl mutant phenotype. This means that N function during R8 specification is little affected by any extension of O-fucose chains that occurs, unlike N function during wing development. It is possible that O-fucose monosaccharides affect N function during eye development, with or without modification to polysaccharide forms. Consistent with this interpretation, O-fucosylation has been found to be important for many aspects of N function, including others not dependent on Fringe (Li, 2003).

Taken together, these studies suggest that introduction of an O-fucosylation site into EGF repeat 14 confers sensitivity to Dl on N expressed in R8 precursors, but has little effect on N activity in many other cells. One interpretation is that additional O-fucosylation of N increases sensitivity to ligand, so that N activation occurs in R8 precursors. The finding that in the wild type R8 cells are insensitive to Dl also suggests another possibility: that EGF repeat 14 has a normal function inhibiting signaling, and that this function is disrupted by O-fucosylation. These two models cannot be distinguished definitively on the basis of current data. The model that EGF repeat 14 has a normal function blocking N signaling in R8 cells is supported by the recessive genetics of the spl mutation, however, because in heterozygous cells that contain wild-type and O-fucosylated EGF repeat 14, the wild-type protein continues to maintain N inactivity in R8 cells. Since EGF repeat 12, which is essential for many aspects of N signaling, contains a potential O-fucosylation site, one very simplistic hypothesis is that whereas O-fucosylated EGF repeats promote N activity, during lateral inhibition EGF repeats lacking this modification inhibit N activity. It is suggested that during lateral inhibition of neural cells the spatial pattern of N activity is determined by insensitivity of presumptive neural cells to N ligands, and that such insensitivity is regulated by modifications or interactions of EGF repeats on the N extracellular domain (Li, 2003).

Notch modulation by O-fucosyltransferase 1 is essential for Notch interaction with its ligands and for Fringe function

Notch and its ligands are modified by a protein O-fucosyltransferase (O-fut1, also known as Neurotic or Ofut1) that attaches fucose to a serine or threonine within EGF domains. By using RNAi to decrease O-fut1 expression in Drosophila, it has been demonstrated that O-linked fucose is positively required for Notch signaling, including both Fringe-dependent and Fringe-independent processes. The requirement for O-fut1 is cell autonomous, in the signal-receiving cell, and upstream of Notch activation. Therefore, O-fut1 activity is required for the cell's ability to receive ligand signals, and would thus be consistent with the hypothesis that the key substrate of O-fut1 is Notch. The transcription of O-fut1 is developmentally regulated, and surprisingly, overexpression of O-fut1 inhibits Notch signaling. Together, these results indicate that O-fut1 is a core component of the Notch pathway, one that is required for the activation of Notch by its ligands, and whose regulation may contribute to the pattern of Notch activation during development (Okajima, 2002).

A mutation has been isolated in the gene encoding O-fucosyltransferase, and analysis of the mutant phenotype confirms the RNAi studies and reveals an unprecedented example of an absolute requirement of a protein glycosylation event for a ligand-receptor interaction. A novel maternal neurogenic gene, neurotic, is essential for Notch signalling. neurotic functions in a cell-autonomous manner, and genetic epistasis tests reveal that Neurotic is required for the activity of the full-length but not an activated form of Notch. neurotic has been shown to be required for Fringe activity. fringe encodes a fucose-specific ß1, 3 N-acetylglucosaminyltransferase that modulates Notch receptor activity. Neurotic is essential for the physical interaction of Notch with its ligand Delta, and for the ability of Fringe to modulate this interaction in Drosophila cultured cells. These results suggest that O-fucosylation catalysed by Neurotic is also involved in the Fringe-independent activities of Notch and may provide a novel on-off mechanism that regulates ligand-receptor interactions (Sasamura, 2003).

Since O-fucose on Notch has been shown to act as a molecular scaffold for GlcNAc that is elongated by Fng, one would expect that the phenotypes of O-fucosyltransferase mutant might be the same as those of fng. Unexpectedly, however, the nti and fng mutant phenotypes are quite different. Strikingly, the embryonic neurogenic phenotype that is evident in nti mutant, and is an indication of its essential role in Notch signalling, is not evident in fng mutants. Furthermore, it is thought that Fng does not have a significant role in lateral inhibition, while it is involved in the generation of the cell boundary between cells expressing Fng and cells not expressing Fng. Additionally, an in vitro binding assay revealed that Nti is essential for binding between Notch and Delta. Based on the previous findings and the present results, it is proposed that O-glycosylation of Notch EGF repeats has two distinct roles for binding to Delta. (1) O-fucosylation catalysed by Nti is an absolute requirement for binding between Notch and the ligand, and this binding is sufficient to accomplish lateral inhibition. For this function, no additional glycosylation to O-fucose residue is required. This idea is also supported by the observation that in the tissues and organisms that do not express fng, Delta is competent to activate the Notch receptor. In this respect, it is worth noting that in the C. elegans genome there is a highly conserved nti, while a fng homolog is not found. (2) Addition of GlcNAc to the O-fucose residue by Fng enhances the interactions between Notch and Delta, modulating the receptor-ligand interactions. In fact, the expression of fng shows a high degree of regional specificity, and the boundary of the cells expressing and not expressing Fng often defines the border of distinct tissue structures. Thus, the region-specific expression of fng allows modulation of Notch signalling, resulting in generation of complex structure of organs. As expected from the second function of Nti, its function is essential for Fng-dependent modulation of Notch signalling as well as Fng-independent function. In the wing disc, nti is epistatic to fng, and fng requires nti to induce Wg at the dorsal and ventral compartment boundary. Additionally, in the in vitro binding assay, Fng depends on Nti to enhance the binding between Notch and Delta. These lines of evidence indicate that Nti is involved in Fng-dependent modulation of Notch signalling, which is consistent with an O-glycan structure of the Notch EGF repeats (Sasamura, 2003).

Modulation of notch-ligand binding by protein O-fucosyltransferase 1 and fringe

To investigate the molecular basis for the requirement for O-linked fucose on Notch, an assay was carried out of the ability of tagged, soluble forms of the Notch extracellular domain to bind to its ligands, Delta and Serrate. Downregulation of O-fut1 by RNAi in Notch-secreting cells inhibits both Delta-Notch and Serrate-Notch binding, demonstrating a requirement for O-linked fucose for efficient binding of Notch to its ligands. Conversely, over-expression of O-fut1 in cultured cells increases Serrate-Notch binding but inhibits DeltaNotch binding. These effects of O-fut1 are consistent with the consequences of O-fut1 overexpression on Notch signaling in vivo. Intriguingly, they are also the opposite of, and are suppressed by, expression of the glycosyltransferase Fringe, which specifically modifies O-linked fucose. Thus, Notch-ligand interactions are dependent upon both the presence and the type of O-fucose glycans (Okajima, 2003).

The requirement for O-Fut1 in Notch signaling has been demonstrated by RNAi in Drosophila (Okajima, 2002), and by a targeted mutation in the murine Pofut1 gene. One line from a large scale screen for lethal transposable element insertions in Drosophila has an insertion in the 3' end of O-fut1, and is predicted to result in replacement of the seven C-terminal amino acids of O-fut1 with four different amino acids followed by a stop codon. To confirm that this insertion creates an O-fut1 mutation, animals in which patches of cells were made homozygous mutant for this allele were examined by mitotic recombination. These animals exhibit classic Notch mutant phenotypes, such as wing notching, thickened wing veins, and loss of sensory bristles on the notum, consistent with the phenotypes generated by RNAi of O-fut1 (Okajima, 2002). In developing wing imaginal discs, the expression of targets of Notch signaling, such as Wingless, is lost in cells mutant for O-fut1. This mutation (referred to hereafter as O-fut1SH) thus provides an independent demonstration of the requirement for O-fut1 for Notch signaling in Drosophila, and indicates that the seven C-terminal amino acids of O-fut1 are essential for function in vivo. The last four amino acids of O-fut1 conform to a consensus signal for retention in the endoplasmic reticulum, and experiments are in progress to determine whether the loss of function in O-fut1SH is due to loss of enzymatic activity or to mislocalization (Okajima, 2003).

The studies presented here indicate that O-fucosylation is required for the physical binding of Notch to its ligands Dl and Ser. These binding studies are consistent with prior genetic studies, which positioned a requirement for O-fucosylation in signal receiving cells, upstream of the cleavages associated with Notch activation (Okajima, 2002). Although the current results do not exclude the possibility that O-fucose glycans could also act at other steps, and indeed some influence of O-fut1 RNAi on secretion of Notch extracellular domain fusion proteins is detected, the requirement for O-fucose for Notch-ligand binding can in principle account for the requirement for O-fut1 in Notch signaling (Okajima, 2003).

Notably, O-fut1 is required for efficient binding of Notch to both Ser and Dl. This is consistent with the severe Notch phenotypes observed in vivo when O-fut1 is impaired by mutation or RNAi. By contrast, elongation of O-fucose by the GlcNAc transferase Fringe exerts opposing influences on the ability of Notch to bind to Ser and Dl. Fringe has clear and reproducible effects on both Dl-Notch and Ser-Notch binding. Importantly, these effects of Fringe on Notch-ligand binding recapitulate its effects on signaling by these two ligands in Drosophila. The ability of both the O-fucose monosaccharide and elongated forms of O-fucose to influence Notch-ligand binding, the influence of O-fucosylation on binding by both ligands, and the consistent correlations between the effects of O-fucosylation on binding in vitro and its effects on signaling in vivo all argue that O-fucose glycans act at the ligand binding step of Notch signaling (Okajima, 2003).

Beyond their importance to understanding regulation of Notch signaling, these observations thus provide a striking example of glycosylation as a mechanism for modulating protein-protein interactions. With the determination that O-fucosylation affects Notch-ligand binding, attention must now be turned to elucidating the mechanistic basis for this effect (Okajima, 2003). O-fut1 and Fringe always act in Notch-expressing cells to influence Notch signaling and Notch-ligand binding: this implicates Notch itself as the relevant substrate. However, the actual sites of glycosylation on Notch that mediate the effects of these glycosyltransferases remain to be identified. It is also not yet clear whether the importance of O-fucosylation reflects a role for lectin-like recognition of Notch by its ligands or other co-factors, or whether instead O-fucose glycans influence Notch-ligand binding indirectly, by altering the conformation or oligomerization of Notch (Okajima, 2003).

By contrast to the positive requirement for O-fut1 demonstrated by RNAi, over-expression of O-fut1 enhances Ser-Notch binding but inhibits Dl-Notch binding. It is intriguing that elevated O-fut1 expression provides a mechanism for differentially modulating the ability of different Notch ligands to interact with the Notch receptor. Previously, Fringe was the only factor known that could discriminate between the ability of Delta to activate Notch and that of Serrate to activate Notch. Indeed, elevated O-fut1 expression might be a mechanism for increasing the sensitivity of cells to the presence or absence of Fringe. In vivo, Fringe only affects a subset of Notch signaling events, and it remains unclear why certain processes are sensitive to Fringe whilst others are insensitive. Although O-fut1 action is the opposite of Fringe, its effects can be blocked by Fringe; therefore, the relative impact of Fringe on Dl-Notch or Ser-Notch interactions is expected to be greater in tissues where O-fut1 is expressed at higher levels. Indeed, even though expression of Fringe alone has no obvious effect on the patterning of notal bristles, it has a strong effect when O-fut1 is also overexpressed. Overexpression of O-fut1 inhibits Dl-Notch signaling, resulting in the formation of excess sensory bristles, but this effect is partially inhibited by co-expression with Fringe (Okajima, 2002). In addition to increasing the sensitivity of Notch signaling events to the presence or absence of Fringe, elevated O-fut1 expression presents a potential mechanism for modulating Notch signaling independently of Fringe. Although the in vivo relevance of Notch-ligand modulation by increased expression of O-fut1 at endogenous levels of expression remains uncertain, it is noted that certain tissues, such as the lymph gland, express much higher levels of O-fut1 than surrounding cells (Okajima, 2002). Intriguingly then, in most Drosophila tissues Dl is the sole or major Notch ligand. However, in the larval lymph gland, a role for Notch signaling in regulating cell fate decisions during hematopoeisis has recently been described, and Ser, rather than Dl, is the ligand that regulates Notch in this tissue. These observations provide some support for the possibility that transcriptional regulation of O-fut1 might provide a mechanism for Notch pathway regulation (Okajima, 2002), and suggest developmental contexts in which this issue may be investigated further (Okajima, 2003).

Mutation of O-fucose sites in Notch: Effect on Delta activation

Two glycosyltransferases that transfer sugars to EGF domains, OFUT1 and Fringe, regulate Notch signaling. However, sites of O-fucosylation on Notch that influence Notch activation have not been previously identified. Moreover, the influences of OFUT1 and Fringe on Notch activation can be positive or negative, depending on their levels of expression and on whether Delta or Serrate is signaling to Notch. This study describes the consequences of eliminating individual, highly conserved sites of O-fucose attachment to Notch. The results indicate that glycosylation of an EGF domain proposed to be essential for ligand binding, EGF12, is crucial to the inhibition of Serrate-to-Notch signaling by Fringe. Expression of an EGF12 mutant of Notch (N-EGF12f) allows Notch activation by Serrate even in the presence of Fringe. By contrast, elimination of three other highly conserved sites of O-fucosylation does not have detectable effects. Binding assays with a soluble Notch extracellular domain fusion protein and ligand-expressing cells indicates that the NEGF12f mutation can influence Notch activation by preventing Fringe from blocking Notch-Serrate binding. The N-EGF12f mutant can substitute for endogenous Notch during embryonic neurogenesis, but not at the dorsoventral boundary of the wing. Thus, inhibition of Notch-Serrate binding by O-fucosylation of EGF12 might be needed in certain contexts to allow efficient Notch signaling (Lei, 2003).

Although the O-fucose site in EGF12 is essential for Fringe inhibition of Serrate signaling in the wing, Fringe still reduces N-EGF12f:AP-Serrate binding. The decrease in binding is not sufficient to prevent N-EGF12f activation, but there must nonetheless be multiple sites that can contribute to the inhibition of Serrate signaling by Fringe. There must also be distinct sites that mediate the potentiation of Delta-Notch signaling by Fringe, because N-EGF12f:AP-Delta binding is potentiated almost as effectively as N:AP-Delta binding. Importantly then, the effects of Fringe on Delta versus Serrate signaling appear to be mediated, at least to some extent, through distinct sites of O-fucosylation (Lei, 2003).

The importance of additional O-fucose sites is further underscored by the distinct consequences of removal of O-fucose only at EGF12 by the S to A mutation, compared with removal of O-fucose at all sites by Ofut1 mutation or RNAi. Using a cell aggregation assay, EGF11 and EGF12 of Notch have been shown to have a key role in ligand binding. Deletion of EGF11 and EGF12 prevents aggregation between Notch-expressing cells and Delta-expressing cells, and a construct including only EGF11 and EGF12 of Notch is able to confer Delta-binding activity upon cells, albeit with decreased efficiency compared with full-length Notch. Although a role for other EGF repeats in ligand binding has been suggested based on the consequences of expressing fragments of Notch in the wing imaginal disc, and by cell aggregation experiments with mutant Notch proteins, EGF11 and EGF12 have generally been considered to be the key EGF domains for ligand binding. However, because RNAi of Ofut1 in S2 cells indicates that O-fucose is required on Notch for binding to its ligands, yet O-fucosylation of EGF12 is not required for ligand binding, other O-fucosylated EGF domains must also be required for Notch-ligand interactions. Thus, multiple sites are subject to O-fucosylation, but with different phenotypic consequences (Lei, 2003).

Unequal segregation and Degradation/Cleavage of Delta

The Drosophila gene neuralized has long been recognized to be essential for the proper execution of a wide variety of processes mediated by the Notch (N) pathway, but its role in the pathway has been elusive. In this report, genetic and biochemical evidence is presented that Neur is a RING-type, E3 ubiquitin ligase. It has been shown that neur is required for proper internalization of Dl in the developing eye, and it has been demonstrated that ectopic Neur targets Dl for internalization and degradation in a RING finger-dependent manner, and that the two exist in a physical complex. Collectively, these data indicate that Neur is a ubiquitin ligase that positively regulates the N pathway by promoting the endocytosis and degradation of Dl (Lai, 2001).

Previous studies have indicated that Dl not only nonautonomously activates the N pathway in neighboring cells, but can also autonomously inhibit the N pathway. For example, a reduction in Dl autonomously potentiates the ability of a cell to receive an N signal, while misexpression of Dl interferes with the ability of a cell to activate the N pathway and can induce N loss-of-function phenotypes. Neur-mediated destabilization of Dl is thus predicted to increase the ability of a cell to receive the N signal, and is therefore consistent with the observed cell-autonomous function of Neur in promoting N pathway activity. The data also suggest a possible explanation for the dominant-negative effect of Dl lacking the intracellular domain, which is predicted to be immune to regulation by Neur (Lai, 2001).

Endogenous neur does not strongly alter Dl level or subcellular localization in the wing disc as assayed by indirect immunofluorescence microscopy, even though neur mutant cells in the eye disc display a clear defect in their ability to internalize Dl. Notably, neur transcripts are not detected in nonsensory organ precursor cells of proneural clusters, even though their requirement for neur can be demonstrated genetically. This suggests that low levels of endogenous Neur may lead to a modification of Dl levels during adult peripheral neurogenesis that is too subtle to observe in the immunofluorescence assay. Neur-mediated destabilization of Dl in the wing imaginal disc may therefore be easily visualized only in gain-of-function experiments where large amounts of Neur are present. Nevertheless, genetic mosaic experiments have convincingly demonstrated that modest changes in the ligand/receptor ratio have a strong influence on cell fate decisions controlled by the N pathway. Thus, Neur need not greatly modify the level of Dl in order to have a significant effect on the activity of the N pathway and the choice of cell fate (Lai, 2001).

Studies of Drosophila dynamin, encoded by shibire (shi), reveal that the activity of the N pathway is particularly dependent upon endocytosis. Shits1 mutants pulsed at the restrictive temperature phenocopy N mutant phenotypes, including neural hyperplasia and thickening of wing veins. Endocytosis and trafficking of Dl and N are abnormal following reduction of dynamin function, and shi function is required in both N signal-sending and -receiving cells, suggesting that both ligand and receptor are regulated by endocytosis. Curiously, misexpression of not only soluble N IC but also membrane-localized full-length N is completely epistatic to shits, indicating that endocytosis is not essential for signal transduction downstream of the N receptor. The requirement for shi in N signal-receiving cells might be simply explained if it functions in Neur-regulated endocytosis of Dl. In this case, biasing the ligand/receptor ratio by misexpression of full-length N would be sufficient to bypass the requirement for dynamin. Consistent with this, preliminary experiments indicate that the ability of Neur to downregulate Dl is compromised when dynamin function is reduced (Lai, 2001).

It is also emphasized that multiple mechanisms must exist for internalization of Dl, since endocytosis of Dl still occurs in wing and eye disc neur clones, and Dl accumulates in large intracellular apical vesicles in the presence of dominant-negative NeurDeltaRF. In addition, Dl localizes to vesicles in a dynamin-independent fashion in the pupal wing. It is suggested that ubiquitination of Dl by Neur represents a mechanism for regulated endocytosis and subsequent degradation of Dl, but additional means for clearance of Dl from the plasma membrane must exist, possibly including constitutive membrane recycling or pinocytosis (Lai, 2001).

neur is essential for many, but not all, lateral inhibitory and inductive processes mediated by N. For example, neur is absolutely required for multiple steps during PNS development and for lateral inhibition of photoreceptors, but is dispensable for processes such as lateral inhibition of wing veins and induction of wing margin. Nevertheless, ectopic Neur and NeurDeltaRF could interfere with all of these processes, consistent with the proposed function of Neur in regulating Dl, a 'core' N pathway component involved in all of these processes. In light of the findings presented here, attempts were made to identify common features of Neur-dependent Dl-mediated processes (Lai, 2001).

It has been previously observed that Dl and N expression are coincident in some settings and complementary in others. Notably, Dl and N expression are coincident or overlapping in most settings that require Neur, including in proneural clusters of the imaginal discs and the pupal notum, and in the developing eye imaginal disc. Conversely, Dl and N are complementary or highly asymmetric in Neur-independent developmental settings such as disc and pupal wing vein development, and at the wing margin. An attractive hypothesis is that Neur functions to bias the relative levels of N and Dl in settings where both ligand and receptor are coexpressed on a cell-by-cell basis; in other settings where ligand and receptor expression are highly asymmetric or exclusive, Neur may not be required (Lai, 2001).

neuralized encodes a peripheral membrane protein involved in Delta signaling and endocytosis

Activation of the Notch (N) receptor involves an intracellular proteolytic step triggered by shedding of the extracellular N domain (N-EC) upon ligand interaction. The ligand Dl has been proposed to effect this N-EC shedding by transendocytosing the latter into the signal-emitting cell. Dl endocytosis and N signaling are greatly stimulated by expression of neuralized. neur inactivation suppresses Dl endocytosis, while its overexpression enhances Dl endocytosis and Notch-dependent signaling. neur encodes an intracellular peripheral membrane protein. Its C-terminal RING domain is necessary for Dl accumulation in endosomes, but may be dispensable for Dl signaling. The potent modulatory effect of Neur on Dl activity makes Neur a candidate for establishing signaling asymmetries within cellular equivalence groups (Pavlopoulos, 2001).

Static pictures of Dl localization do not allow an unambiguous conclusion of whether intracellular Dl is endocytosed or blocked in its secretory trafficking. The former hypothesis is favored for three reasons: (1) intracellular Dl often colocalizes with endocytosed fluorescent dextran; (2) if Dl were retained in the endoplasmic reticulum or Golgi, it would not be available at the cell surface where signaling is taking place, yet, concomitant with increased endocytosis, Neur is able to stimulate Dl signaling and (3) wt Neur protein is found mostly at the plasma membrane, so it is more likely to affect endocytic events rather than steps in secretory processes (Pavlopoulos, 2001).

The nonautonomous effect of neur- clones on lateral inhibition favors a role for Neur in signal-emitting, rather than signal-receiving, cells. Such a function is consistent with the fact that Neur is an intracellular peripheral membrane protein expressed preferentially in the signal-emitting cells during lateral inhibition, such as the neuroblasts, SOPs, and central provein cells. In agreement with a role for Neur in generating the Dl signal, epistasis analyses have shown that neur is required to express the embryonic neural suppression ('antineurogenic') phenotype associated with ligand-dependent N gain-of-function (gof) mutants. neur is dispensable for the constitutive activity of ligand-independent N variants. Interestingly, some N variants that are Dl independent are also shi (Dynamin) independent. Taken together, these data point to the involvement of Neur and Dynamin in processes upstream of (or parallel to) N activation by Dl. The implication of Neur in endocytic regulation suggests an important role for endocytosis in events leading up to N activation (Pavlopoulos, 2001).

If Dl endocytosis and Dl-N signaling are causally linked, then this analysis of the NeurDeltaRING-GFP mutant poses a paradox: although NeurDeltaRING-GFP does not detectably stimulate Dl endocytic trafficking (or turnover), it retains the ability to enhance Dl signaling. This could mean that the above model is wrong and endocytosis is simply a consequence of Dl-N stimulation, rather than a prerequisite for Dl signaling. Alternatively, the absence of detectable Dl internalization upon coexpression of NeurDeltaRING-GFP does not necessarily preclude the possibility that early endocytic events (e.g., recruitment of Dl into coated pits) that are undetectable by light microscopy are initiated by NeurDeltaRING-GFP. Such events might be sufficient to stimulate ligand-dependent N cleavage and activation. Ultrastructural analysis will be required to distinguish between these alternative models (Pavlopoulos, 2001).

Removal of the Neur RING domain does seem to adversely affect its ability to stimulate N signaling in some contexts: UAS-neurDeltaRING yields phenotypes indicative of a negative effect on N signaling (tufted bristles, thick veins, and notched wings) with most Gal4 driver lines, although in certain cases, positive effects are also observed (shaft to socket transformation). Context-dependent variability with the UAS-neurDeltaRING-GFP construct suggests that these differences do not result from the presence of the GFP moiety but rather from the type of assay employed. In fact, NeurDeltaRING-GFP coexpressed with Dl blocks N signaling within the omb-Gal4 domain, where wt Neur and Dl are able to induce Wg, even though the nonautonomous signaling (at the borders of the omb-Gal4 domain or at the borders of FLP-out clones) appears unaffected by the RING deletion. It is possible then that NeurDeltaRING can exert negative effects on Dl-N signaling in a cell-autonomous fashion and positive effects in a cell-non-autonomous fashion. The cell-autonomous block in N signaling could be due to the block in Dl turnover and its accumulation at the apical membrane, because it has been proposed that high levels of Dl may sequester N receptor molecules in unproductive cis complexes (Pavlopoulos, 2001).

Two major models for Dl signaling have been put forward. In one, the active Dl species is proposed to be the extracellularly cleaved, secreted Dl-EC fragment, because it is produced by the metalloprotease Kuzbanian (Kuz), and the kuz lof phenotype is similar to the N lof phenotype. In the other, binding of cell surface-tethered Dl to N on the apposing cell has a dual impact: activating extracellular cleavage of Notch and mediating the transendocytosis into the signal-sending cell of N-EC complexed with Dl. The observations in this paper suggest that Neur could act intracellularly in the signal-sending cell to promote assembly of a productive Dl-N complex and to trigger its endocytosis. Concomitantly with endocytosis, Neur leads to a drastic reduction in the levels of the Dl-EC fragment, even as Dl-N signaling is increased. It therefore appears unlikely that Dl-EC is the active signal that stimulates N in the wing disk. This leaves unanswered at present the question of why Kuz is needed for N signaling. Perhaps Kuz has pleiotropic activity and acts on some other protein(s) required for N activation, and Kuz-dependent Dl cleavage is a secondary effect. Better characterization of the different Dl isoforms, including their localization and trafficking, will be required to understand the detailed mechanism of Dl-N activation (Pavlopoulos, 2001).

Despite the proposed role of Neur to promote Dl signaling, it is also noted that Dl can signal in the absence of Neur, inasmuch as there are instances of Dl signaling where Neur is not detectably expressed, such as from the germline to ovarian follicle cells. N target gene expression is indeed induced by Dl in the absence of neur. With the caveat that available detection methods may fail to detect low levels of neur expression, it is proposed that two types of Dl signaling may exist: basal signaling that does not require Neur activity and high-intensity signaling that does. During neurogenesis, basal Dl-N signaling probably takes place during early stages among all cells within proneural clusters, where Dl and N are uniformly expressed but Neur is absent. Upon expression of neur by a nascent neural precursor, signaling becomes asymmetric, since the neighboring cells receive more intense signal even though Dl and N levels have not changed. The absolute requirement for neur in neurogenesis suggests that basal 'mutual' inhibition is insufficient to permanently block proneural protein activity. Indeed, the E(spl) bHLH Notch targets, which are the main antagonists of proneural proteins, are not expressed in neur- embryos or clones, suggesting that their expression may be induced only by intense Neur-dependent 'lateral' inhibitory signaling (Pavlopoulos, 2001).

This hypothesis can be extended to propose that Neur may be required more stringently in instances in which a novel asymmetry has to be imposed upon uniform basal N-Dl signaling. neur is not required at the wing DV boundary, where asymmetry is imposed by Fringe or in the egg chamber, where asymmetry is imposed by expression of N and Dl in distinct cells. Similarly, neur is not essential during lateral inhibition within the provein. Despite its expression there and its dramatic effect on Dl localization, neur lof clones yield normal looking veins with only minor thickenings. It is believed that neur is not crucial for this process because wing patterning mechanisms place N and Dl in different cells: Dl expression is most intense within the central proveins and N expression is most intense within the lateral proveins (Pavlopoulos, 2001).

Down-regulation of Delta by proteolytic processing

Notch signaling regulates cell fate decisions during development through local cell interactions. Signaling is triggered by the interaction of the Notch receptor with its transmembrane ligands expressed on adjacent cells. Recent studies suggest that Delta is cleaved to release an extracellular fragment, DlEC, by a mechanism that involves the activity of the metalloprotease Kuzbanian; however, the functional significance of that cleavage remains controversial. Using independent functional assays in vitro and in vivo, the biological activity of purified soluble Delta forms were examined; it is concluded that Delta cleavage is an important down-regulating event in Notch signaling. The data support a model whereby Delta inactivation is essential for providing the critical ligand/receptor expression differential between neighboring cells in order to distinguish the signaling versus the receiving partner (Mishra-Gorur, 2002).

Western analysis of extracts from tissues and cultured Drosophila S2 cells show that the ligand Delta is cleaved at an extracellular site close to the transmembrane domain, shedding a fragment that encompasses most of the extracellular domain (DlEC). Conditioned medium from S2 cells stably expressing Delta (S2-Dl) was used to purify DlEC to homogeneity by affinity chromatography using the 9B monoclonal antibody (C594.9B). Resolution of the highly purified product by SDS-PAGE and silver staining demonstrates two species migrating as a doublet of 63 and 65 kD, with the 63 kD species being the predominant form. NH2-terminal sequence analysis revealed a single sequence consistent with the putative NH2 terminus resulting from the signal peptide cleavage. Direct chemical COOH-terminal sequence analysis determined that the COOH-terminal residue of both isoforms is alanine. These results were corroborated by tryptic digestion followed by mass spectrometry, which revealed the existence of two peptides ending in the sequence LTNA and ... QYGA. It is concluded that Delta is cleaved at two distinct sites: COOH-terminal to Ala581 and Ala593, respectively. Henceforth, these two isoforms are referred to as DlEC581 and DlEC593 (Mishra-Gorur, 2002).

In an attempt to explore the functional significance of the two extracellular cleavages in Delta, truncated soluble molecules mimicking the DlEC581 and DlEC593 were generated. In addition, the Ala581 and Ala593 amino acids were mutagenized to serine (henceforth Ala581Ser and Ala593Ser). The constructs were transfected into Drosophila S2 cells, which endogenously express Kuzbanian but not Delta. Transfection of the DlEC581 and DlEC593 constructs effectively generated soluble secreted products, with DlEC593 exhibiting a slightly different molecular mass, consistent with the 11-amino-acid difference in their COOH-termini. When expressed in S2 cells, both the Ala581Ser and Ala593Ser mutants were cleaved to generate a product of essentially the same size as DlEC. In addition, an Ala581,593Ser double mutant was generated which was also cleaved to generate a product similar to DlEC. The cis or trans requirement of Kuz in Delta cleavage was assessed using the S2 cell-culture system. S2 cells stably expressing either WT Kuz or dominant-negative Kuz, when mixed with S2-Dl cells, do not affect Delta cleavage; Kuz effects are seen only when it is cotransfected with Delta into the same cell (Mishra-Gorur, 2002).

It has been established that cells expressing Notch aggregate with Delta-expressing cells. Although a rigorous, in vivo demonstration that this interaction is direct is still lacking, it is known that specific regions in the extracellular domain of Notch and Delta are necessary and sufficient for aggregation. Attempts were made to examine whether WT DlEC, DlEC581, and DlEC593 interact with Notch and thus inhibit the normal Notch-Delta-mediated cell aggregation (Mishra-Gorur, 2002).

Preincubation of S2-N cells with concentrated conditioned medium from S2-Dl cells causes a >60% inhibition of aggregation rate. In contrast, concentrated conditioned medium from S2 cells stably expressing each of the mutant forms of soluble Delta show essentially no inhibitory effect in the aggregation assay (Mishra-Gorur, 2002).

The medium from S2-Dl-expressing cells was fractionated on an anti-Delta (9B) antibody affinity column, and selected fractions were tested for their inhibitory effect in the aggregation assay. All the inhibitory activity in the flowthrough of the affinity column. Further, the purified DlEC fractions shows essentially no inhibitory activity. A mild inhibition in aggregation (<20%) was seen at concentrations of DlEC >0.5 µM, indicating that DlEC has a very weak affinity for Notch and is not an effective competitive inhibitor of Notch-Delta aggregation. Western blot analysis of the different fractions during purification of DlEC showed that the flow through contains full-length Delta, suggesting that the inhibitory effect could be either attributed to this Delta protein species or to an unknown activity which copurified with it. In any case, it is noted that these experiments reveal the existence of a Notch agonist activity, other than DlEC, in the supernatant of the S2-Dl cells (Mishra-Gorur, 2002).

The S2-Dl-derived inhibitory activity was further examined by size exclusion chromatography in neutral aqueous buffer to avoid the harsh elution conditions of the affinity column (i.e., pH 2.8). When S2-Dl conditioned medium was fractionated on a Sephadex-200 HR FPLC size exclusion column, all of the inhibitory activity was seen to elute in the void volume of the column (Mr>600 kD). Western blot analysis also demonstrated that this fraction was devoid of DlEC, which eluted in subsequent fractions. It is important to note that a band corresponding in size with full-length Delta is seen in the void-volume fractions. These data corroborate the notion that the DlEC fragment does not compete with the Notch-Delta interaction mediating the cell aggregation (Mishra-Gorur, 2002).

This analysis was extended by examining the activity of the soluble Delta molecules using independent in vitro assays of Notch activation. All of the in vitro assays employed consistently indicate that the soluble forms of Delta are not active, and support the notion that cleavage of Delta corresponds to an inactivation of the ligand. However, they also corroborate the existence of a soluble agonist activity in fractions containing small amounts of full length Delta (Mishra-Gorur, 2002).

The in vitro studies were extended by an assessment of the activity of the mutant constructs in transgenic flies. Flies carrying the various Delta mutants were generated and the effects of expression of the different DlEC isoforms and Delta mutants were analyzed in vivo. Expression was driven by the eye specific glass (pGMR) promoter, which is active in all cells posterior to the morphogenetic furrow. Flies expressing DlEC581 and DlEC593 exhibited mild eye phenotypes. The underlying mechanism of the weak effects associated with soluble ligand expression are not understood; however, the severity of phenotypes of the various transgenic lines varies from mild to no phenotype, suggesting a link with the level of over expression. This may correlate with the slight inhibition of aggregation observed with micromolar amounts of purified DlEC in the in vitro aggregation assay (Mishra-Gorur, 2002).

In contrast, a severe glassy eye phenotype is exhibited with the Ala581Ser and Ala593Ser mutants. Significantly, very similar results are seen with pGMR driven overexpression of WT Delta, consistent with the notion that the cleavage site mutations, in addition to being ineffective at preventing cleavage, do not significantly alter the biological activity of Delta. The in vivo activity of the mutant Delta molecules corroborates the results of the cell-based analysis. It is important to note that unlike the strong phenotypes associated with the overexpression of the WT ligand, or the full-length mutant ligands that are normally cleaved in the aforementioned cell based assays, the soluble forms have mild effects. The expression of the soluble Delta isoforms in every context examined could at best elicit only mild phenotypes consistent with the notion that these molecules are inactive (Mishra-Gorur, 2002).

The finding that the proteolytic processing of Delta releases soluble DlEC raised the obvious question of the functional significance of this cleavage. Even though several studies have addressed this question, either directly or indirectly, in flies, nematodes and vertebrates, it is unclear whether this is an antagonistic or agonistic event in Notch signaling. The initial characterization of soluble fractions of Delta suggested an agonistic function for the DlEC. More rigorous biochemical characterization presented here clearly shows Delta proteolysis yields more than one DlEC (DlEC581 and DlEC593), neither of which exhibit significant biological activity. Furthermore, the previously reported soluble activity is most likely attributed to trace levels of full-length Delta in the cell culture media. It is concluded is that the proteolytic processing of Delta is a step that renders this Notch ligand inactive (Mishra-Gorur, 2002).

Previous studies demonstrated a central role for Kuzbanian in Delta processing both in cell-based assays and in vivo by mutant analysis. The existence of two DlEC products (DlEC581/593) indicates more than one cleavage event occurs in the extracellular domain of Delta. It is important to note that the DlEC581 product is far more abundant as compared to DlEC593 and experiments using KuzDN result mainly in the reduction of the 581 form. Whether or not Kuzbanian alone or additional enzyme activity is responsible for these cleavages requires further investigation. Regardless of the mechanism of cleavage, both of the Delta products have proven to be biologically inactive. Therefore, it is reasonable to conclude that processing in general results in ligand inactivation (Mishra-Gorur, 2002).

Based on these results, it is suggested that the agonistic activity, previously reported to be associated with the medium from Delta expressing cells, is not due to the activity of DlEC. However, it is noted that the present study detected the presence of a 'soluble' activity in the medium, raising the possibility that such an activity may after all exist in vivo. Formally at least, this activity can be attributed to the detectable quantities of full length Delta in the medium or to another yet-to-be-determined molecule. It is not inconceivable that soluble, full-length, membrane-associated Delta may in fact be secreted into the medium even if only to act on a neighbor rather than over long distances. For instance, in the case of Wingless, the existence of membrane exovesicles as a vehicle for Wingless delivery has been documented. Whether a soluble, biologically significant Delta activity can be generated by exocytic events remains to be tested, but this is worth considering (Mishra-Gorur, 2002).

Despite the uncertainty of the role of ligand processing, several studies have attempted to use soluble forms of the ligand as an agonist of the receptor with variable success. However, a common element in these studies is that the soluble forms display activity only if they are forced into an oligomeric state either via Fc fusions or by immobilization on a matrix. The biologically inactive DlEC fragment secreted in the medium does not have a natural tendency to oligimerize because it exists in a monomeric state (as judged by gel filtration and centrifugal/sedimentation studies. Furthermore, the inactivity of DlEC expressed in vivo indicates that a biologically relevant mechanism for immobilization of the DlEC so as to make it active is nonexistent. Therefore, it is of utmost importance to consider the physiological relevance of continued attempts to employ soluble ligands as Notch agonists (Mishra-Gorur, 2002).

Irrespective of the potential requirement for oligomerization or immobilization as an essential activation step for the ligand, it has been proposed that endocytosis of the dissociated Notch extracellular domain bound to Delta into the Delta-expressing cells (transendocytosis) is a critical part of the Notch signaling mechanism. If such a mechanism is essential for Notch activation, then the blocking of an endocytic event may result in inhibition of signaling. This notion is also compatible with the in vivo analysis that demonstrates that membrane-tethered forms of either Delta or Serrate lacking the intracellular domain cannot undergo effective endocytosis, and hence behave as antagonists of Notch signaling. However, the present analysis shows that Delta molecules fixed on the cells, similar to a molecule immobilized on a matrix, is still capable of activating the Notch receptor. This observation would then favor the hypothesis that endocytosis of Delta may be a facilitating but not necessarily an essential part of Notch signaling (Mishra-Gorur, 2002).

In assessing the developmental significance of Delta cleavage, the activity of Kuzbanian needs to be examined more closely. Although the initial link between Notch signaling and Kuzbanian was reported to involve Notch processing, genetic data show that multiple copies of Delta can suppress the phenotypes associated with dominant-negative Kuzbanian (KuzDN) expression. This observation is compatible with the notion that Delta cleavage produces an active soluble ligand. However the mechanism of action of KuzDN is not known and it may be equally plausible to consider that KuzDN acts by sequestering Delta, such that the addition of more WT Delta molecules suppress the KuzDN phenotype. It is also worth emphasizing that whereas the dominant-negative forms of Kuzbanian inhibit Delta cleavage, and that Delta cleavage products are not detected in loss of function kuz embryos, it is quite possible that the Kuzbanian-Delta interaction is indirect. The original proposal that Kuzbanian is involved in the proteolytic processing of Notch has been challenged by subsequent experimentation. Indeed, recent reports documenting Kuzbanian cleavage of Notch rely on deletion mutants of the receptor that are susceptible to cleavage, bringing further uncertainty to the physiological relevance of Kuzbanian acting on Notch directly (Mishra-Gorur, 2002).

A model to explain the role of Delta down-regulation by proteolysis must consider the mechanism of action of the Notch ligands. Delta can influence Notch through two modes of action: in trans, where Notch and Delta are presented on adjacent cells and Delta can act as agonist, or in cis, where Notch and Delta are presented on the same cell and Delta (and Serrate) can act as a dominant-negative antagonist. It is well established that cells in tissues undergoing Notch signaling can express Notch and Delta simultaneously. For instance, in the early Drosophila embryo, all cells in the proneural clusters, the group of cells which will eventually segregate into epidermal and neuronal lineages via Notch-Delta signaling, express both Notch and Delta. However, in order for proper signaling to occur, there must be a distinction between a signaling versus a receiving cell. The accumulated studies to date suggest that the critical parameter for a cell to be a receiving or signaling cell is the ratio rather than the absolute expression levels of Delta and Notch. Moreover, feedback loops may be responsible for consolidating and amplifying a given state (ratio). Thus, a mechanism that inactivates Delta in a given cell may contribute to the feedbacks that are necessary to establish a critical expression differential between two neighbors (Mishra-Gorur, 2002).

Mosaic analysis in Drosophila during cell fate acquisition in the neuroectoderm has demonstrated that Kuz is required in cells to receive signals that inhibit the neural fate. These signals are known to be transmitted through the Notch receptor and this cell autonomous effect of Kuz is consistent with studies in nematodes. Similarly, using a cell-culture system, it has been found that dominant-negative or WT forms of Kuzbanian can affect Delta only when cotransfected in the same cell, and have no effect when transfected into adjacent cells. Hence, it is suggested that Kuz acts on Delta in the same cell, although it is not clear whether the cleavage occurs at the cell surface or inside the cell. In either case, it is proposed that this proteolysis renders Delta incapable of interacting with Notch either on an adjacent cell or on the same cell (Mishra-Gorur, 2002).

Therefore, a model is favored whereby proteolytic processing of Delta on a Notch/Delta-expressing cell has the overall effect of rendering that cell the signal receiving cell by (1) alleviating the dominant-negative activity of Delta toward Notch on that cell, and (2) down-regulating the Delta available to signal Notch on adjacent cells. Consistent with this model, a similar role has been proposed for the Neuralized ubiquitin ligase in Delta down-regulation. Interestingly, because Neuralized is not required in all Notch dependent developmental contexts, it has been emphasized that multiple mechanisms must exist to clear Delta from the plasma membrane. The above model is also compatible with the possibility that Kuzbanian may play more than one role in Notch signaling. For example, if Kuz is somehow involved in the activation of the Notch receptor, the existence of an activity such as Kuzbanian in a particular cell, which is able to simultaneously enhance receptor function and inactivate the ligand, is a hypothesis worth testing. One of the predictions of the proposed scheme is that Kuzbanian activity must be differentially regulated between critical neighbors. More experimentation will be necessary to confirm or discount this hypothesis and indeed this model (Mishra-Gorur, 2002).

Unequal segregation of Neuralized biases Notch activation during asymmetric cell division

In Drosophila, Notch signaling regulates binary fate decisions at each asymmetric division in sensory organ lineages. Following division of the sensory organ precursor cell (pI), Notch is activated in one daughter cell (pIIa) and inhibited in the other (pIIb). The E3 ubiquitin ligase Neuralized localizes asymmetrically in the dividing pI cell and unequally segregates into the pIIb cell, like the Notch inhibitor Numb. Furthermore, Neuralized upregulates endocytosis of the Notch ligand Delta in the pIIb cell and acts in the pIIb cell to promote activation of Notch in the pIIa cell. Thus, Neuralized is a conserved regulator of Notch signaling that acts as a cell fate determinant. Polarization of the pI cell directs the unequal segregation of both Neuralized and Numb. It is proposed that coordinated upregulation of ligand activity by Neuralized and inhibition of receptor activity by Numb results in a robust bias in Notch signaling (Le Borgne, 2003).

Recent studies have indicated that endocytosis of Dl is critical for N activation. (1) Dynamin-dependent endocytosis is not only required for signal transduction but is also required in signal-sending cells to promote N activation. (2) Endocytosis-defective Dl proteins have reduced signaling capacity. (3) The E3-ubiquitin ligases Neuralized (Neur) in Drosophila and Mind bomb (Mib) in zebrafish promote endocytosis of Dl and appear to be required for efficient activation of N by Dl. It has been proposed that Dl endocytosis facilitates the S2 cleavage of N at the surface of the signal-receiving cell. Neur is unequally segregated during asymmetric division of the pI cell, upregulates endocytosis of Dl in the pIIb cell, and plays a critical role in generating cell fate diversity. It is proposed that Neur acts as a cell fate determinant during asymmetric cell divisions (Le Borgne, 2003).

To examine whether asymmetry in N ligands distribution may play a role in generating cell fate diversity during asymmetric divisions, the subcellular distribution of Dl and Ser was studied in the sensory organ lineage. In mitotic pI cells, Dl and Ser are uniformly distributed around the cell cortex and are equally partitioned into both daughter cells. In both pI daughter cells, Dl and Ser accumulated at the apical cell cortex as well as in intracellular dots of 0.5 ± 0.2 μm in diameter. These dots are coated by Hrs (hepatocyte growth factor-regulated tyrosine kinase substrate). Hrs binds ubiquitinated proteins via its ubiquitin-interacting motif and sorts endocytic cargos into the lumen of multivesicular bodies (MVBs). Therefore, these Dl-positive vesicles appear to be large endocytic vesicles that probably correspond to MVBs. These Dl-positive vesicles also contain Notch extracellular domain (NECD) and NICD epitopes. Strikingly, a higher number of large Dl-positive vesicles were seen in the anterior signal-sending pIIb cell (5.0 ± 2.2) than in the posterior signal-receiving pIIa cell (2.0 ± 1.5). This asymmetry in Dl endocytosis is established independently of the unequal partitioning of Numb. Indeed, anterior pI daughter cells are shown to accumulate a higher number of Dl-positive vesicles than posterior pI daughter cells in numb2 and numb15 mutant clones. Thus, asymmetry in Dl endocytosis does not depend on Numb (Le Borgne, 2003).

Recent studies have suggested that endocytosis of Dl is promoted by the ubiquitination of Dl by Neur, a RING finger-type E3-ubiquitin ligase required for N signaling. Neur is found in a complex with Dl and is required for Dl ubiquitination. Finally, Neur stimulates the accumulation of Dl into intracellular vesicles in imaginal disc cells. The latter conclusion was, however, based on the analysis of steady-state levels of Dl, making it difficult to unambiguously conclude whether Neur promotes Dl endocytosis or favors direct sorting from the Golgi to intracellular vesicles. To discriminate between these two possibilities and to test whether Neur regulates Dl trafficking in sensory cells, an ex vivo assay was developed for endocytosis. Internalization of Dl was followed in living epithelial cells using antibodies recognizing the extracellular part of Dl. Briefly, the single-layered epithelium corresponding to the pupal notum was dissected and cultured in the presence of anti-Dl antibodies. Following medium changes and fixation, the uptake of anti-Dl antibodies was revealed using secondary antibodies. Anti-Dl antibodies were found to be specifically internalized in the pIIa and pIIb cells. Internalized anti-Dl antibodies colocalize with Dl into large Dl-positive vesicles. Internalization of anti-Dl requires dynamin activity and is not observed at 4°C. Together, these results indicate that anti-Dl interacts with Dl at the cell surface and that Dl-anti-Dl complexes are endocytosed in sensory cells (Le Borgne, 2003).

This assay was used to examine the function of neur. Clones of neur1F65 mutant cells have been shown to exhibit a neurogenic phenotype with too many pI cells being specified. The progeny of these mutant pI cells produce no external sensory structures indicating that pIIa cells have been transformed into pIIb-like cells. These cell fate transformations are associated with defects in Dl trafficking. High levels of anti-Dl remain at the surface of neur1F65 mutant cells and internalization of anti-Dl is drastically reduced. It is concluded that neur is required for the endocytosis of Dl in sensory cells (Le Borgne, 2003).

This defect in Dl endocytosis was quantified on fixed tissues. neur mutant pI cells and pIIb-like progeny cells were found to accumulate high levels of Dl at the cell surface. Accumulation of Dl at the cell surface is consistent with the proposed function of Neur in the internalization and degradation of Dl. Quantification of Dl-positive vesicles in neur mutant clones revealed that mutant pIIb-like cells contain much fewer Dl-positive vesicles than wild-type pIIb cells. Thus, in the absence of neur function, both pI daughter cells have the same reduced number of Dl-positive vesicles. Furthermore, a similar distribution of Dl-containing vesicles is seen in the wild-type pIIa cells, which do not inherit Neur, and in the neur mutant pIIb-like cells. These comparisons indicate that neur is required to upregulate the endocytosis of Dl in the pIIb cell (Le Borgne, 2003).

Upregulation of Dl endocytosis in the pIIb cell may result from higher levels of Neur in this cell. To test this hypothesis, the localization of Neur was examined. The Neur protein is detectable in the pI cell and in its progeny cells, but not in epidermal cells. Neur is perinuclear in prophase and localized asymmetrically at the anterior cortex during prometaphase. At telophase, Neur specifically segregates into the anterior daughter cell. At cytokinesis, Neur uniformly redistributes at the cortex and in the cytoplasm in the pIIb cell. Localization of Neur at mitosis is identical to the one described for Partner of Numb (Pon). Consistently, Neur colocalizes with Pon-GFP throughout mitosis. Asymmetric localization of Neur is also seen in the pIIb and pIIa dividing cells. Specificity of anti-Neur antibodies was demonstrated by absence of staining in neur mutant pI cells. Unequal segregation of Neur does not depend on numb activity. Conversely, unequal segregation of Numb does not depend on neur activity. Thus, the numb-independent unequal segregation of Neur into the pIIb cell provides a simple explanation for the upregulation of Dl endocytosis in the pIIb cell (Le Borgne, 2003).

To test the functional significance of Neur unequal segregation, Neur was overexpressed in pI cells. Overexpression of Neur using neurP72GAL4 fails to affect the unequal partitioning of Neur at pI mitosis and the pIIa/pIIb decision but instead results in a weak double-socket phenotype associated with a shaft-to-socket transformation. This fate transformation is known to result from high levels of Delta-Notch signaling and is opposite that of the socket-to-shaft transformation seen in neur mutant clones. Moreover, this shaft-to-socket transformation may result from the equal partitioning of Neur (but not Numb) in the two pIIa daughter cells which can also be observed at low frequency. Thus, these observations support the notion that unequal segregation of Neur is functionally important (Le Borgne, 2003).

The mechanisms by which Neur localized at the anterior cortex of the dividing pI cell were investigated. The role of the cytoskeleton was studied by applying drugs to cultured nota. Colcemid, a microtubule-depolymerizing agent, was found to have no significant effect. In contrast, both Latrunculin A, an agent that depolymerizes actin microfilaments, and the myosin motor inhibitor butanedione-2-monoxime (BDM) strongly impaired or completely inhibited the asymmetric localization of Neur. Thus, both myosin motor activity and an intact actin cytoskeleton are required for the formation and/or maintenance of the Neur crescent at the anterior cortex of the dividing pI cell. These requirements for Neur localization are similar to the ones seen earlier for Numb and Pon. Neur also behaves in a manner similar to Numb and Pon in that localization of Neur at the anterior cortex of the pI cell depends on planar polarity genes and on the polarity genes discs-large and pins. Moreover, mispartitioning of Neur in dlg and pins mutant cells correlates with a loss in asymmetric internalization of Dl. These data indicate that Neur and Numb share part of the same molecular machinery to localize asymmetrically in the pI cell (Le Borgne, 2003).

Unequal segregation of Neur in the anterior pIIb cell suggests that Neur acts in this cell to promote adoption of the pIIa fate by the posterior cell. To test whether neur activity is indeed required in the pIIb cell, clones within the sensory organ lineage were generated. Mitotic recombination in the pI cell produces one neur mutant cell and one wild-type cell. Importantly, the anterior daughter cell inherits Neur, regardless of its genotype. Thus, when the anterior cell is neur mutant, the posterior cell is predicted to adopt a pIIa fate whatever the requirement for neur activity. However, two different outcomes are predicted when the posterior cell is mutant. If neur activity is required in the signal-receiving cell, the posterior cell is predicted to adopt a pIIb-like fate activity. This should result in a bristle loss phenotype. In contrast, if neur acts in the signal-sending cell, the mutant posterior cell is predicted to become a pIIa cell. This mutant pIIa cell should then produce two mutant cells unable to signal, hence leading to bristle duplication. Mitotic recombination induced at 0-6 hr before puparium formation (PF), when most macrochaete pI cells are specified but have not yet divided, produces flies with double-shaft bristles on the head, thorax and at the wing margin. No macrochaete loss was detectable. This double-shaft phenotype appears to result from wild-type pIIb/mutant pIIa pairs because sensory organs composed of two mutant shaft cells and wild-type pIIb progeny cells were detected at 20 hr after PF. Reciprocally, a sheath-to-neuron transformation was observed in mutant pIIb/wild-type pIIa pairs. These data show that neur is required for the socket/shaft and neuron/sheath fate decisions and further indicate that neur acts in the pIIb cell to specify the pIIa cell (Le Borgne, 2003).

Echinoid facilitates Notch pathway signalling during Drosophila neurogenesis through functional interaction with Delta

The Notch intercellular signalling pathway is important throughout development, and its components are modulated by a variety of cellular and molecular mechanisms. Ligand and receptor trafficking are tightly controlled, although context-specific regulation of this is incompletely understood. During sense organ precursor specification in Drosophila, the cell adhesion molecule Echinoid colocalises extensively with the Notch ligand, Delta, at the cell membrane and in early endosomes. Echinoid facilitates efficient Notch pathway signalling. Cultured cell experiments suggest that Echinoid is associated with the cis-endocytosis of Delta, and is therefore linked to the signalling events that have been shown to require such Delta trafficking. Consistent with this, overexpression of Echinoid protein causes a reduction in Delta level at the membrane and in endosomes. In vivo and cell culture studies suggest that homophilic interaction of Echinoid on adjacent cells is necessary for its function (Rawlins, 2003).

Therefore, both in vivo and in culture Ed protein is strongly associated with Dl at the cell membrane and in the early endosome compartment. Several lines of evidence suggest that Ed self associates in trans. Ed expression promotes the adhesion of cultured cells, while genetic clonal analysis shows that in vivo Ed protein cannot accumulate at the cell membrane if it is absent from the adjacent cell. Moreover, this genetic analysis suggested that such a trans interaction might be important for function (Rawlins, 2003).

Ed is not essential for Notch signalling but has a modulatory effect. The basis of this effect must be relatively subtle, since no strongly visible difference is found in expression pattern, level, or subcellular localization of Dl, N or E(spl) in ed mutant clones. The idea is favored that Ed influences PNC resolution as part of the specific process that drives the singling out of individual SOPs. In other words, it is a part of a 'symmetry breaking' apparatus. There are two lines of evidence to suggest that Ed functions to inhibit the transition from PNC cell to SOP. (1) No more than four SOPs are selected from each PNC even in null ed alleles. (2) ed interacts particularly strongly with ase, which is expressed on the transition from PNC to SOP. It is suggested that the role of ed is analogous to that proposed for sca. Based on analysis in the eye, it is envisaged that singling out causes several cells to begin to become resistant to Dl ('pre-SOPs'), but a specific genetic mechanism involving sca and gp150, encoding a leucine-rich repeat (LRR) protein that is required for viability, fertility and proper development of the eye, wing and sensory organs, causes all but one of these unwanted SOPs to revert and once again become responsive to Dl from the selected precursor. It is hypothesized is that, like sca, ed functions to promote N receptor activation in these pre-SOPs. Despite these similarities between sca and ed function, genetic evidence suggests that they take part in parallel processes. Moreover, Sca and Gp150 are located in late endosomes, whereas Ed is located at the membrane and in early endosomes (Rawlins, 2003).

In vivo and in cultured cells, Ed protein colocalizes very strongly with Dl in cis, both at the membrane and in early endosomes. It is possible that there is a direct molecular interaction between the two proteins, but no evidence has yet been found for this. Such an association may require Ed-Ed homophilic binding. Nevertheless, colocalization suggests a close and specific association with Dl-N signalling. One possibility is that Ed promotes Dl function in the 'true' SOP, leading to more efficient suppression of the emergence of unwanted SOPs. Cis-endocytosis of Dl into the signalling cell is apparently required for activation of the Notch receptor, and one could envisage that ed may enhance this process in the SOP. This is supported by the colocalization of Ed with N and Dl during N activation as observed in this study's cell culture analysis (Rawlins, 2003).

An alternative is that ed may inhibit Dl activity in recipient (non-SOP) cells. There is evidence that such reduction of Dl activity may promote unidirectional signalling in two ways: (1) it would free an SOP from inhibition by surrounding cells; (2) it has been suggested that Dl in recipient cells antagonizes their response to trans signalling, perhaps by cis association of Dl and N. Therefore, Ed inhibition of this antagonistic function of Dl would make non-SOP cells more vulnerable to signalling from the SOP. No difference is seen in Dl distribution and level in ed mutant clones, but it is suspected that this might only be apparent in the pre-SOPs. However, after overexpression of Ed, a striking and specific decrease in Dl is observed both at the membrane and in vesicles. Remarkably, this correlates with SOP loss, which is the opposite phenotype to that normally expected for loss of Dl. Thus, Ed function may be connected to the downregulation of Dl in recipient cells. Proteolysis and endocytosis of Dl have both been implicated as causing its downregulation. It is feasible that Ed promotes one of these processes, for example by helping to present Dl to Kuzbanian for cleavage (Rawlins, 2003).

ed mutants have twinned R8 photoreceptors in the eye and additional es organ SOPs everywhere. A priori one would imagine these phenotypes to have the same genetic and mechanistic basis. They appear, however, to indicate the interaction of ed with two different signalling pathways. Ed negatively regulates Egfr signalling (through direct interaction with pathway components) during R8 specification. This is in contrast to the role of Ed during es organ specification, where it modulates Notch pathway signalling. There are several other reasons for concluding that the R8 and SOP phenotypes of ed mutants, although superficially similar, have different origins. The latter, but not the former, is sensitive to overexpression of Ed protein. For R8, this is explained because Ed is regulated by EGFR post-translationally and so absolute protein levels are unimportant. Sensitivity of SOP singling out to Ed protein levels suggests a different mechanism is at play. Most strikingly, Ed protein is colocalized extensively with N and Dl in the wing disc cells, but not in the eye disc, where interestingly there appears to be very little N and Dl on the cell . Therefore, all this suggests the conclusion that the two phenotypes do indeed have different origins, and moreover that there are significant differences in SOP singling out compared with R8 precursor selection (Rawlins, 2003).

Drosophila Epsin mediates a select endocytic pathway that DSL ligands must enter to activate Notch

Delta/Serrate/Lag2 (DSL) ligands must normally be endocytosed in signal-sending cells via the action of liquid facets (Lqf) to activate Notch on the surface of signal-receiving cells. Surprisingly, however, bulk endocytosis of DSL ligands appears normal in the absence of Lqf. This apparent paradox is resolved by providing evidence that Lqf is unique among adapters that target mono-ubiquitinated cargo proteins for internalization: it allows them to enter a special endocytic pathway that DSL ligands must enter to acquire signaling activity. This requirement can be bypassed by introducing the internalization signal that normally mediates internalization and recycling of the Low Density Lipoprotein (LDL) receptor. On the basis of these results, it is hypothesized that Epsin-mediated endocytosis might be required to allow DSL proteins to be recycled rather than degraded following internalization, possibly to convert them from inactive pro-ligands into active ligands (Wang, 2004).

In screens for mutations affecting wing pattern, six alleles of a single complementation group were obtained that cause phenotypes similar to those caused by the loss of Notch signaling, namely severe wing notching, wing vein thickening and bristle tufts. All six alleles fail to complement existing alleles of liquid facets (lqf), and are associated with nonsense or missense mutations in the lqf-coding sequence (Overstreet, 2003). One new allele, lqf1227, truncates the coding sequence after amino acid 119 in the middle of the ENTH domain, the most N-terminal conserved domain, and abolishes Lqf protein expression in vivo. This allele is referred to as lqf-, and it was used for all experiments described in this study. A transgene containing the intact lqf gene (Cadavid, 2000) rescues the lethality of lqf- homozygotes, as well as all of the mutant phenotypes associated with lqf- clones. Lqf encodes the sole ortholog of vertebrate Epsin1 (Cadavid, 2000); a second Drosophila protein, sometimes referred to as Dm Epsin2 (Overstreet, 2003), lacks several conserved domains found in Lqf and vertebrate Epsin1, and appears instead to be the Drosophila ortholog of vertebrate EpsinR, a functionally distinct Epsin-related protein (Wang, 2004).

In imaginal wing discs, signaling by the DSL ligands Delta (Dl) and Serrate (Ser) specifies the wing margin at the dorsoventral (DV) compartment boundary, and can be assayed by boundary-specific expression of wing margin genes (or their protein products), such as cut, wingless (wg) and vestigial (vg). lqf- clones resemble Dl- Ser- clones or N- clones in that they cause the loss of cut, wg and vg boundary-specific expression when they abut or cross the DV compartment boundary, corroborating the Notch-related phenotypes of lqf- clones observed in the adult wing (Wang, 2004).

The loss of margin gene expression in lqf- clones is not cell autonomous. Instead, wild-type cells can rescue the expression of margin specific genes in adjacent lqf- cells (e.g., cut). Similarly, non-autonomous rescue of lqf- clones was observed in the adult, where the presence of wild-type cells can rescue the ability of neighboring lqf- cells to form single bristles. In both respects, as well as in others, lqf- clones resemble Dl- Ser- clones, but differ from N- clones, which show a strictly cell-autonomous loss of Notch target gene expression (Wang, 2004).

Collectively, these data establish an obligate role for Lqf in Notch signaling, and implicate Lqf in sending, rather than receiving, DSL signals (Wang, 2004).

To determine whether Lqf is required in signal-sending cells, the MARCM technique was used to generate lqf- clones that express either Dl or Ser under Gal4 control. Notch is normally expressed in both the D and V compartments of the wing primordium, but is modified in D cells by the action of the glycosyltransferase Fringe (Fng) so that it responds preferentially to Dl signaling from V cells. Ser is expressed predominantly in D compartment cells, and signals in the opposite direction, activating unmodified Notch in V cells. Clones of cells that express Dl under Gal4 control activate Notch strongly in adjacent wild-type cells only when located in the D compartment, as monitored by the expression of margin-specific genes like cut. Conversely, Ser-expressing clones activate Notch strongly only when located in the V compartment. In both cases, the levels of exogenous Dl and Ser expression are several fold higher than the peak levels of endogenous Dl and Ser generated along the DV boundary, and this overexpression autonomously inhibits the activation of Notch in cells within the clones (Wang, 2004).

Clones of lqf- cells that overexpress either Dl or Ser fail to induce margin gene expression, irrespective of where they are located within the wing primordium. Indeed they behave like simple lqf- clones in blocking normal margin gene expression when they abut, or cross, the DV compartment boundary. Thus, Lqf is required in DSL signal-sending cells to activate Notch in adjacent, signal-receiving cells (Wang, 2004).

Intact Dl and Ser normally accumulate in intracellular puncta, some of which co-localize with the endosomal marker Hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs), as well as at the apical cell surface. By contrast, C-terminally truncated forms of Dl that lack the intracellular domain (DlDeltaC) accumulate predominantly at the cell surface and, like C-terminally truncated forms of Ser (SerDeltaC), cannot activate Notch on the surface of neighboring cells. If such truncated DSL ligands fail to signal because they cannot be endocytosed, replacement of the missing Dl cytosolic domain with heterologous domains that contain other internalization signals should rescue both endocytosis and signaling activity. Moreover, mutations in the internalization signals of these domains should eliminate their rescuing activity. These predictions have been tested and confirmed with two heterologous domains, each containing a different internalization signal (Wang, 2004).

(1) The missing intracellular domain of DlDeltaC was replaced with a 21 amino acid peptide from the Low Density Lipoprotein (LDL) receptor that contains either the wild-type internalization signal FDNPVY, or a mutant signal, ADAAVA. The LDL peptide contains two Lysines; these were replaced by Arginine to avoid their serving as possible acceptors for ubiquitination. Both the wild-type (DlLDL+) and mutant (DlLDLm) chimeric proteins were labeled by the insertion of six copies of the Myc epitope tag in the juxtamembrane portion of the extracellular domain. When expressed in the wing disc, DlLDL+ shows a similar subcellular distribution to wild-type Dl, accumulating on both the apical cell surface and in intracellular puncta. DlLDL+-expressing clones, like wild-type Dl-expressing clones, can induce cut activity in surrounding cells, indicating that the chimeric protein has signaling activity. However, they differ from wild-type Dl-expressing clones in that they only induce cut when located close to the DV boundary. Hence, it is inferred that DlLDL+-expressing clones have reduced signaling activity relative to wild-type Dl-expressing clones, and require the additional boost provided by endogenous signaling from neighboring wild-type cells to activate cut (Wang, 2004).

By contrast, DlLDLm accumulates predominantly on the apical cell surface, but not in intracellular puncta, and lacks signaling activity. Indeed, clones of cells overexpressing DlLDLm that abut the DV boundary block normal Notch signaling across the boundary, as would be expected if DlLDLm can inhibit Notch transduction within the same cell (like wild-type Dl), but is devoid of the capacity to activate Notch in adjacent cells (Wang, 2004).

(2) It was found serendipitously that replacement of the missing cytosolic domain of DlDeltaC with a random peptide, R+, of 50 amino acids (DlR+) also restored normal behavior. DlR+ accumulates in intracellular puncta as well as on the apical cell surface; in addition it activates Notch in neighboring cells. The R+ peptide contains two Lysines that might potentially serve as acceptors for ubiquitination. Replacement of both Lysines with Arginine blocked the rescuing activity of the R+ peptide. The mutant protein, DlRm, accumulated predominantly only on the cell surface and lacked signaling activity; moreover, clones of DlRm that abutted the DV boundary interrupted signaling across the boundary (Wang, 2004).

To assess the possibility that mono-ubiquitination of native Dl, as well as the DlR+ chimera, might suffice to provide an internalization signal, the missing cytosolic domain of DlDeltaC was replaced with Ubiquitin itself, and with a corresponding mutant form of Ubiquitin in which the Isoleucine at position 44 was mutated to Alanine, which functionally inactivates the internalization signal. All seven Lysine residues in each Ubiquitin domain were also replaced by Arginine to avoid additional ubiquitination. Both the resulting proteins, DlUbi+ and DlUbim, accumulated on the cell surface, as well as in intracellular puncta. However, far fewer puncta were found in DlUbim-expressing cells than in DlUbi+-expressing cells, and only DlUbi+ was able to signal to neighboring cells. These data indicate that mono-ubiquitination is sufficient for Dl endocytosis and signalling, and suggest that at least one of the Lysines in the R+ peptide serves as Ubiquitin acceptor, allowing the protein to be internalized and to signal. It is noted that the DlUbi+ protein appears to have only weak signaling activity relative to Dl or DlR+, since induction could only be detected of vg boundary-specific expression, but not cut or wg expression (Wang, 2004).

It is concluded that: (1) the cytosolic domain of Dl is essential for its endocytosis; (2) mono-ubiquitination is sufficient for Dl internalization; and (3) Dl endocytosis is essential for signaling activity (Wang, 2004).

Given that Epsin has been implicated in endocytosis, lqf- cells may fail to send DSL signals because they are generally impaired for endocytosis. However, Dpp, Hh and Wg signaling, Wg internalization, and cell growth and proliferation are not adversely affected by the absence of Lqf, suggesting that endocytosis is not significantly impaired overall (Wang, 2004).

Alternatively, Lqf might be required specifically for the endocytosis of DSL ligands. To assess this possibility, the effects of lqf- clones were first examined in the developing retina, where they have been reported to cause abnormally high levels of Dl on the cell surface, consistent with impaired Dl endocytosis (Overstreet, 2003). Such clones do indeed cause elevated surface expression of Dl, but it was also observed that endogenous Dl transcription (as assayed using a Dl-lacZ reporter gene), is strongly upregulated in the mutant cells, apparently as a consequence of the lack of Notch signaling. Furthermore, Dl staining can be detected in intracellular puncta in such lqf- eye disc clones. Thus, the elevated surface accumulation of Dl observed in lqf- eye clones can be ascribed to elevated Dl expression in the mutant cells, and may not reflect impaired Dl endocytosis (Wang, 2004).

lqf- clones were generated in wing discs expressing uniformly high levels of exogenous Dl under Gal4 control, and Dl staining was compared in lqf- cells and their wild-type neighbors. Under this condition, the level of Dl expression does not vary between wild-type and lqf- cells, simplifying analysis. No difference was detected in the subcellular distribution of Dl between lqf- and adjacent wild-type cells. In both cases, Dl was localized predominantly at the cell surface, as well as in similar numbers of intracellular puncta, many of which co-localize with the endosomal protein Hrs. The same result was obtained in separate experiments in which only the subcellular distribution of endogenous Dl was assayed (i.e., in the absence of overexpressed Dl) (Wang, 2004).

It was reasoned that if the Dl-positive puncta in lqf- clones are indeed endocytic, the appearance of such puncta should change in the absence of hrs activity, which interferes with the maturation of early into late endosomes, and causes the formation of abnormal endosomal structures. To test this, both hrs- and hrs- lqf- clones were generated. Endogenous Dl was found to accumulate in abnormally large puncta in both types of clones, and similar results were obtained when these clones expressed exogenous Dl under Gal4 control. The block in endosomal maturation caused by the removal of Hrs does not interfere with signaling by Dl; nor does it alter the requirement for Lqf. Clones of hrs- cells that express exogenous Dl induce Cut expression in surrounding cells, whereas corresponding hrs- lqf- clones do not (Wang, 2004).

To determine unequivocally whether the abnormal puncta that accumulate Dl in hrs- and hrs- lqf- cells are indeed endosomal, use was made of the finding that Wg secreted from prospective wing margin cells accumulates in similar, abnormally large puncta in hrs- cells positioned at a distance from the secreting cells. The same result was obtained in double mutant hrs- wg- cells, establishing that the accumulation of Wg in these puncta serves as an in vivo marker for endocytosis. Then Wg and Dl staining was examined in triple mutant hrs- wg- lqf- clones that express an HRP-tagged form of Dl under Gal4 control. In this case, as in corresponding hrs- wg- double mutant clones, co-localization of Wg and Dl was observed in large intracellular puncta. Thus, bulk endocytosis of both endogenous and overexpressed Dl appear normal in lqf- cells (Wang, 2004).

Although bulk Dl endocytosis appears unaffected by the absence of Lqf, blockage of a relatively small, but specific, subset of Dl endocytic events might escape detection, and this subset might be crucial for signaling activity. To examine this possibility, Dl was co-expressed together with the E3 Ubiquitin Ligase Neuralized (Neur), under Gal4 control, to drive efficient ubiquitination and internalization of the exogenous Dl. It was reasoned that under these conditions, even modest reductions in the rate of Dl endocytosis might cause an abnormal persistence of Dl at the apical cell surface (Wang, 2004).

Wing discs that express uniformly high levels of Dl under Gal4 control accumulate high levels of Dl on the apical cell surface. However, in discs that co-express high levels of both Dl and Neur, this surface accumulation is strongly reduced and Dl accumulates instead in an abnormally large number of intracellular puncta. Clones of lqf- cells generated in such co-expressing discs do not appear to alter the number or general appearance of these Dl-positive puncta, many of which co-localize with Hrs. However, they do affect the level of Dl staining associated with the apical cell surface (as visualized in discs processed either with, or without, detergent). Such lqf- clones show residual surface staining of Dl, in contrast to neighboring wild-type cells where surface-associated staining is depleted. It is inferred that lqf- cells cannot endocytose Dl as efficiently as their wild-type neighbors, accounting for why a difference was detected under sensitized conditions in which the rate of surface clearance appears to be limiting (Wang, 2004).

Significantly, the residual staining of Dl on the surface of lqf- cells that overexpress Neur and Dl correlates with the failure of these cells to signal. Clones of lqf- cells that overexpress Neur and Dl fail to activate cut in neighboring cells, even though clones of otherwise wild-type cells that overexpress Neur and Dl show enhanced Dl signaling. Hence, it appears that the impairment in Dl endoctyosis detected in lqf- clones in this sensitized background correlates with an absolute block in signaling activity (Wang, 2004).

The cytosolic domain of DSL ligands contains multiple Lysines at least some of which serve as acceptors for Ubiquitin. Lqf contains two Ubiquitin Interacting Motifs (UIMs) (Hofmann, 2001). Hence, mono-ubiquitination of DSL ligands might allow Lqf to target DSL ligands for a special subset of endocytic events that are required for signaling activity. By contrast, bulk endocytosis of DSL ligands mediated by interactions with other Ubiquitin-binding adaptor proteins might not suffice to confer signaling activity. To test this hypothesis, whether the signaling activity of the DlR+ protein depends on Lqf activity, was investigated (Wang, 2004).

Endocytosis and signaling activity of DlR+ depends on the presence of at least one of the two Lysines in the R+ peptide comprising the cytosolic domain. Clones of lqf- cells that express DlR+ fail to induce cut expression in adjacent wing disc cells. However, DlR+ protein in these lqf- clones accumulates both on the apical surface and in intracellular puncta. Moreover, no difference was detected in the punctate, cytosolic accumulation of DlR+ between lqf- and wild-type cells in wing discs that generally overexpress DlR+. Both results indicate that bulk endocytosis of DlR+ is not significantly altered in the absence of Lqf. Because substitution of both Lysines by Arginine blocks internalization and signaling activity of DlRm, it is inferred that DlR+ is targeted for internalization solely by ubiquitination at one or both of these Lysines. Hence, it is suggested that other Ubiquitin-interacting proteins aside from Lqf can target mono-ubiquitinated cargo proteins, such as DlR+ or endogenous Dl, for internalization. However, only Lqf appears able to direct endocytosis of these proteins in a way that allows DSL ligands to signal (Wang, 2004).

Both endocytosis and signaling activity of DlLDL+ depends on the FDNPVY internalization signal. However, unlike either native Dl or DlR+, it was found that clones of lqf- cells expressing DlLDL+ can induce cut expression in adjacent wild-type cells, indicating that the presence of the LDL internalization signal in the chimeric DlLDL+ protein bypasses the requirement for Lqf. As observed for clones of wild-type cells overexpressing DlLDL+, the 'rescued' lqf- clones induced cut only when located close to the DV boundary. Nevertheless, their ability to signal, albeit weakly, contrasts with that of lqf- clones that overexpress native Dl, native Dl plus Neur, or DlR+, all of which are devoid of signaling activity. Hence, it is concluded that the FDNPVY signal directs internalization of DlLDL+ in a manner that permits the protein to acquire signaling activity even in the absence of Lqf activity (Wang, 2004).

Lqf-dependent endocytosis of DSL ligands might be accompanied by modifications of these ligands, either as a pre-requisite for, or a consequence of, signaling activity. To examine this possibility, it was asked whether the size of Dl protein changes as a consequence of Lqf-dependent endocytosis. Initially, clones of wild-type and lqf- cells were generated that express Dl tagged by the insertion of six copies of the Myc epitope in the extracellular juxtamembrane domain, and the profile of Dl peptides that retain the Myc epitope was examined by Western blotting. Under these conditions, similar, complex profiles were observed of Myc-tagged Dl peptides from both wild-type and lqf- cells, corresponding to full-length Myc-Dl protein, as well as several lower molecular weight peptides (Wang, 2004).

This experiment was then repeated using wild-type and lqf- cells that overexpress Neur and Myc-tagged Dl, the sensitized condition under which residual surface expression can be detected of Myc-tagged Dl in lqf-, but not in wild-type, cells. In this case, the profile of Myc-tagged Dl is remarkably simple. Wild-type cells show two bands, one corresponding by size to full-length Myc-tagged Dl (~105 kDa) and the other to a Myc-tagged cleavage product of ~50 kDa. By contrast, lqf- cells show only a single band, corresponding to full-length Myc-tagged Dl. Thus, the failure to clear Dl from the cell surface of lqf- cells is associated with an apparent failure in Dl processing. These results provide evidence for a Lqf-dependent cleavage of Dl that correlates with Lqf-dependent endocytosis and signaling activity (Wang, 2004).

It is noted that the expected size of the Myc-tagged extracellular domain of Dl is ~75 kDa, whereas that of the complementary, Myc-tagged portion of the ligand containing the transmembrane and cytosolic domains is ~40 kDa. Hence, the 50 kDa Myc-tagged cleavage product must be composed of a C-terminal portion of the extracellular domain, and possibly some or all of the transmembrane and cytosolic domains as well. The relationship of this truncated peptide to the active ligand is presently unknown. It could comprise part, or all, of the active ligand, or alternatively, a non-signaling C-terminal fragment cleaved off in the process of generating an N-terminal signaling fragment. Alternatively, it might be a degradation product generated as a consequence of the activation of Notch by Dl (Wang, 2004).

Fat facets and Liquid facets promote Delta endocytosis and Delta signaling in the signaling cells

Endocytosis modulates the Notch signaling pathway in both the signaling and receiving cells. One recent hypothesis is that endocytosis of the ligand Delta by the signaling cells is essential for Notch activation in the receiving cells. Evidence is presented in strong support of this model. In the developing Drosophila eye Fat facets (Faf), a deubiquitinating enzyme, and its substrate Liquid facets (Lqf), an endocytic epsin, promote Delta internalization and Delta signaling in the signaling cells. While Lqf is necessary for three different Notch/Delta signaling events at the morphogenetic furrow, Faf is essential only for one: Delta signaling by photoreceptor precluster cells, which prevents recruitment of ectopic neurons. In addition, the ubiquitin-ligase Neuralized (Neur), which ubiquitinates Delta, is shown to function in the signaling cells with Faf and Lqf. The results presented bolster one model for Neur function in which Neur enhances Delta signaling by stimulating Delta internalization in the signaling cells. It is proposed that Faf plays a role similar to that of Neur in the Delta signaling cells. By deubiquitinating Lqf, which enhances the efficiency of Delta internalization, Faf stimulates Delta signaling (Overstreet, 2004).

Cells with decreased lqf+ activity accumulate Delta on apical membranes and fail to signal to neighboring cells. Three Notch/Delta signaling events were examined in the eye: proneural enhancement, lateral inhibition and R-cell restriction. Loss of lqf+-dependent endocytosis during all three events has identical consequences to loss of Delta function in the signaling cells. It is concluded that lqf+-dependent endocytosis of Delta is required for signaling, supporting the notion that endocytosis in the signaling cells activates Notch in the receiving cells. However, Lqf is not required absolutely for all Delta internalization in the eye. Even in lqf-null cells, which are incapable of Delta signaling, some vesicular Delta is present. Perhaps not all of the vesicular Delta present in wild-type discs results from signaling (Overstreet, 2004).

Genetic studies in Drosophila indicate clearly that deubiquitination of Lqf by Faf activates Lqf activity. Moreover, genetic and biochemical evidence in Drosophila suggests that Faf prevents proteasomal degradation of Lqf. In vertebrates, however, it is thought that epsin is mono-ubiquitinated to modulate its activity rather than poly-ubiquitinated to target it for degradation. If Lqf regulation by ubiquitin also occurs this way in the Drosophila eye, the removal of mono-ubiquitin from Lqf by Faf would activate Lqf activity (Overstreet, 2004).

Whatever the precise mechanism, given that both Faf and Lqf are expressed ubiquitously in the eye, two related questions arise. First, why is Lqf ubiquitinated at all if Faf simply deubiquitinates it everywhere? One possibility is that Faf is one of many deubiquitinating enzymes that regulate Lqf, and expression of the others is restricted spatially. This could also explain why Faf is required only for R-cell restriction. Another possibility is that Faf activity is itself regulated in a spatial-specific manner in the eye disc. Alternatively, Lqf ubiquitination may be so efficient that Faf is needed to provide a pool of non-ubiquitinated, active Lqf. Similarly, Faf could be part of a subtle mechanism for timing Lqf activation. Second, why is Faf essential only for R-cell restriction? One possibility is that there is a graded requirement for Lqf in the eye disc, such that proneural enhancement requires the least Lqf, lateral inhibition somewhat more and neural inhibitory signaling by R2/3/4/5 the most. The mutant phenotype of homozygotes for the weak allele lqfFDD9 supports this idea, as R-cell restriction is most severely affected. Alternatively, Lqf may be expressed or ubiquitinated with dissimilar efficiencies in different regions of the eye disc. More experiments are needed to understand the precise mechanism by which the Faf/Lqf interaction enhances Delta signaling (Overstreet, 2004).

In neur mutants, Delta accumulates on the membranes of signaling cells and Notch activation in neighboring cells is reduced. These results support a role for Neur in endocytosis of Delta in the signaling cells to achieve Notch activation in the neighboring receiving cells, rather than in downregulation of Delta in the receiving cells. Because neur shows strong genetic interactions with lqf and both function in R-cells, Neur and Lqf might work together to stimulate Delta endocytosis. Lqf has ubiquitin interaction motifs (UIMs) that bind ubiquitin. One explanation for how Neur and Faf/Lqf could function together is that Lqf facilitates Delta endocytosis by binding to Delta after its ubiquitination by Neur. This is anattractive model that will stimulate further experiments (Overstreet, 2004).

One exciting observation is that the endocytic adapter Lqf may be essential specifically for Delta internalization. Although, hedgehog, decapentaplegic and wingless signaling pathways have not been examined directly, they appear to be functioning in the absence of Lqf. These three signaling pathways regulate movement of the morphogenetic furrow and are thought to require endocytosis. The furrow moves through lqf-null clones and at the same pace as the surrounding wild-type cells. Moreover, all mutant phenotypes of lqf-null clones can be accounted for by loss of Delta function. Further experiments will clarify whether this apparent specificity means that Lqf functions only in internalization of Delta, or if the process of Delta endocytosis is particularly sensitive to the levels of Lqf (Overstreet, 2004).

Lqf expands the small repertoire of endocytic proteins that are known targets for regulation of cell signaling. In addition to Lqf, the endocytic proteins Numb and Eps15 (EGFR phosphorylated substrate 15) are objects of regulation. In vertebrates, asymmetrical distribution into daughter cells of the alpha-adaptin binding protein Numb may be achieved through ubiquitination of Numb by the ubiquitin-ligase LNX (Ligand of Numb-protein X) and subsequent Numb degradation. In addition, in vertebrate cells, Eps15 is phosphorylated and recruited to the membrane in response to EGFR activation and is required for ligand-induced EGFR internalization. Given that endocytosis is so widely used in cell signaling, endocytic proteins are likely to provide an abundance of targets for its regulation (Overstreet, 2004).

Unidirectional Notch signaling depends on continuous cleavage of Delta

Unidirectional signaling from cells expressing Delta (Dl) to cells expressing Notch is a key feature of many developmental processes. The Drosophila ADAM metalloprotease Kuzbanian-like (Kul) has been shown to play a key role in promoting this asymmetry. Kul cleaves Dl efficiently both in cell culture and in flies, and has previously been shown not to be necessary for Notch processing during signaling. In the absence of Kul in the developing wing, the level of Dl in cells that normally receive the signal is elevated, and subsequent alterations in the directionality of Notch signaling lead to prominent phenotypic defects. Proteolytic cleavage of Dl by Kul represents a general mechanism for refining and maintaining the asymmetric distribution of Dl, in cases where transcriptional repression of Dl expression does not suffice to eliminate Dl protein (Sapir, 2005).

ADAM proteins have a characteristic domain signature, including a signal peptide followed by a pro-domain, a metalloprotease domain possessing a zinc-binding catalytic pocket, and a disintegrin domain. A cysteine-rich region is followed by a transmembrane domain and cytoplasmic tail. Sequence similarity searches have determined that the Drosophila genome harbors five ADAM-family metalloproteases. The full open reading frames of these metalloproteases were obtained from a combination of cDNA clones, reverse-transcription based on gene prediction, and 5' RACE in cases where the cDNAs did not cover the entire coding region (Sapir, 2005).

Among the five Drosophila metalloproteases, a single homolog was identified for TNF-alpha converting enzyme (TACE); two homologs were found for Meltrin-alpha, and two for ADAM10. Analysis of the ADAM family phylogenetic tree identified gene duplication events that took place most probably after the divergence of the ancestors of nematodes and insects. While Kuz shows a high degree of similarity to human ADAM10, another Drosophila protein, which was termed Kuzbanian-like (Kul), exhibits an even higher degree of similarity to ADAM10, especially in the disintegrin domain (which facilitates substrate recognition), as well as in the metalloprotease catalytic domain (Sapir, 2005).

In view of the ability of Kuz to cleave Dl, three other Drosophila ADAM proteins were examined for this activity. Dl was co-expressed with each of the ADAM proteins in Drosophila S2 cells, and the levels of Dl were monitored in the cells and in the medium, by probing with an antibody directed against the Dl extracellular domain. When Dl alone was expressed in the cells, basal cleavage by endogenous proteases was detected by an accumulation of the cleaved form of Dl in the medium. Co-expression of Kuz led to a marked elevation of Dl in the medium, concomitant with a reduction in the levels of the membrane-bound Dl in the cells. Two additional ADAM proteins, Kul and DTACE, exhibited a similar potency of cleaving and releasing Dl to the medium. By contrast, the expression of DMeltrin had no effect (Sapir, 2005).

Serrate (Ser) is a second ligand of Notch in Drosophila and is employed in more restricted biological settings. A similar profile of cleavage was also observed for Ser, which was cleaved by Kuz, Kul and DTACE, but only marginally by DMeltrin. Detection of efficient cleavage in S2 cells, which are grown in suspension, supports the notion of cell-autonomous cleavage. Thus, cleavage of Dl or Ser by ADAM proteins is likely to take place within the same cell, rather than between adjacent cells (Sapir, 2005).

To examine the biological roles of the ADAM proteins that can cleave Dl, it was necessary to compromise their activity in flies. Mutations or P-element insertions in DMeltrin, DTACE and kul are not currently available. Therefore double-stranded RNA (dsRNA) 'knock-down' constructs were generated for each of these genes, directed against a region of minimal similarity with the other family members (Sapir, 2005).

Activation of the Notch pathway is required many times during normal wing development. Especially notable is the role of Notch activation in restricting the width of the wing veins within a pro-vein territory. Utilizing the UAS-GAL4 system, dsRNA constructs of Drosophila ADAM metalloproteases were expressed in the wing. Expression of ds-DMeltrin or ds-DTACE did not lead to any detectable wing phenotypes, even when expressed under the regulation of the potent wing driver MS1096-GAL4. By contrast, induction of ds-kul expression by the same driver gave rise to distorted wings, and loss of the wing margin following induction by sd-GAL4. Expression of ds-kul by the weaker driver, sal-GAL4, resulted in two distinct adult wing phenotypes in the spalt-expression domain, which encompasses the region between veins L2-L4 -- formation of multiple wing hairs, and partial loss of veins. The first phenotype was found to be Notch-independent (Sapir, 2005).

The opposite phenotype with respect to vein loss, i.e. vein thickening, was observed when full-length Kul was overexpressed by sal-GAL4. These phenotypes suggest a functional link between Kul and Notch signaling in patterning the wing veins. Additional defects in the morphology of the wing were observed, and may stem from effects of Kul on other substrates independent of the Notch pathway, in accordance with the additional defects observed following ds-kul misexpression. Compromising the levels of Kuz by a ds-kuz construct resulted in vein thickening, representing a Notch loss-of function. This vein phenotype of ds-kuz is similar to the reported kuz loss-of-function wing phenotype, and is also consistent with observations in cell culture, which have demonstrated that ds-kuz RNA abolished the S2 step of Notch cleavage, while ds-kul RNA had no effect on Notch (Sapir, 2005).

In light of the high sequence similarity between Kul and Kuz, the specificity and activity of ds-kul was verified in cultured cells. When Kul was expressed in S2 cells, both a high molecular weight precursor form, and a cleaved, mature form were detected. Expression of ds-kul eliminated both forms of Kul, but did not affect Kuz protein levels, demonstrating the specificity of ds-kul (Sapir, 2005).

The activity of ds-kul was also examined in vivo. A broad distribution of kul transcripts was detected during all embryonic stages and in the wing imaginal discs. ds-kul expression in the wing reduced endogenous kul mRNA levels, illustrating the potency of ds-kul in vivo (Sapir, 2005).

The structural requirements for Kul function were characterized by examining in S2 cells protein maturation and the role of the different domains in promoting Dl cleavage. Basal cleavage of Dl was enhanced by co-expression of full-length Kul. By contrast, a Kul variant bearing an E-to-A substitution within the metalloprotease catalytic domain (E-A Kul), which abolishes catalytic activity, failed to cleave Dl. This demonstrates the need for an active protease domain in Kul. A similar mutation abolished the catalytic activity of Kuz (Sapir, 2005).

Interestingly, E-A Kul reduces Dl cleavage below the basal level, probably due to formation of a complex between E-A Kul and Dl that is refractive to cleavage by the endogenous proteases. Expression of this construct in the wing gives rise to broadened veins, again probably due to the sequestration of Dl. A similar inhibitory effect in cell culture was detected when expressing a Kul protein lacking the intracellular domain. This domain is important for correct trafficking and sorting of ADAM proteins. Finally, the role of pro-domain removal was analyzed by expressing a form of Kul in which cleavage was blocked by mutating two conserved amino acids at the putative pro-domain cleavage site. This variant full-length form of Kul again failed to carry out Dl cleavage, demonstrating the need of pro-domain removal in order to convert Kul to an active protease (Sapir, 2005).

In the pupal wing, activation of the Notch pathway by Dl contributes to refinement and restriction of the veins. Dl, expressed by the central pro-vein cells, activates Notch in the lateral pro-vein domain, forcing these cells to adopt an inter-vein fate, while the Dl-expressing cells themselves differentiate as veins. Reduction of Notch signaling causes lateral pro-vein cells to adopt a vein cell fate, leading to vein thickening in the adult wing. By contrast, ectopic expression of Dl by the inter-vein cells results in Notch activation in the central pro-vein cells, leading to vein loss (Sapir, 2005).

ds-kul expression gives rise to loss of veins, which is consistent with an effect on Notch signaling. To verify that this is indeed the case, the effects of compromising Kul levels in the pupal wing were monitored by a transcriptional reporter of Notch activation termed Gbe+Su(H)m8, and by following the expression of Dl. In a wild-type pupal wing 30 hours after pupariation, Dl protein is restricted to the future veins, while reporter expression is prominent in the lateral pro-vein cells and excluded from the vein cells. It is interesting to note that reporter expression is detected even several cell rows away from the vein cells, possibly resulting from the capacity of Dl-expressing cells to form far-reaching cellular extensions and protrusions (Sapir, 2005).

Expression of ds-kul by sal-GAL4 had a marked effect on the expression of Dl, as well as on the Notch reporter. Irregular expansion of Dl into the lateral pro-vein territory was observed, while in other parts of the wing Dl expression disappeared from the veins. Notch-reporter expression expanded into the vein cells. Elevation in Dl levels in the lateral pro-vein cells endowed them with the capacity to activate Notch signaling within the vein, demonstrating a role for Kul in maintaining unidirectional signaling by the Notch pathway. These patterns account for the loss of veins seen in the adult wing following expression of ds-kul. The irregular patterns observed in the pupal wing may represent snapshots of a dynamic sequence, which is initiated by expansion of Dl to the lateral pro-vein cells, followed by expansion of Notch activation and loss of Dl expression in the vein cells (Sapir, 2005).

The expansion of Dl-protein distribution in the pupal wing following ds-kul expression implies that Kul normally contributes to the restricted distribution of Dl in this tissue. To examine in more detail the capacity of Kul to cleave Dl in vivo, the larval wing imaginal disc was monitored. Dl protein, detected by an antibody recognizing the extracellular domain, is normally observed as a membrane-associated protein that is elevated in the vein and juxta-margin cells and excluded from the wing margin. Changes in Dl distribution were monitored in discs where Kul was overexpressed by the MS1096 driver. Dl membrane-associated staining in the wing pouch was diminished, and a residual punctate staining appeared, possibly reflecting endocytosis of secreted Dl. Normal Delta distribution was retained in the notum, where Kul was not overexpressed. Similarly, expression of Kul by sal-GAL4 eliminated Dl in the sal domain. To verify that Kul directly affects the cleavage of Dl, rather than Dl expression, clones were generated overexpressing Dl under the regulation of actin-GAL4, in the absence or presence of ectopic Kul. Indeed, the prominent appearance of Dl was completely abolished when Kul was co-expressed in the same clone (Sapir, 2005).

The capacity of Kul to cleave Ser has been demonstrated in S2 cells. To check if Kul can also cleave Ser in vivo, the distribution of Ser was analyzed following overexpression of Kul, using an antibody recognizing the Ser extracellular domain. Ser is normally expressed in the wing disc at this phase in a similar pattern to that of Dl, with a more pronounced appearance in the dorsal side of the pouch. The effects of Kul overexpression on Ser were distinct from the effect on Dl. Ser protein did not disappear, but instead displayed an altered, punctate localization within the cells. Kul overexpression also led to a uniform expansion of Ser expression, especially in the dorsal part of the pouch, where the expression of MS1096-GAL4 is more pronounced. It is not known in which compartment(s) Ser accumulates, nor the mechanism by which Kul overexpression leads to this sequestration (Sapir, 2005).

To test if Kul cleaves Dl cells autonomously, cell clones overexpressing Kul were generated. Dl staining was diminished only in the clone cells, implying a cell autonomous activity of Kul. There was no detectable change in Ser distribution within these small clones. The same cleavage assay carried out with Kuz overexpression revealed a different substrate specificity. Kuz cleaved both Dl and Ser within the overexpression clones. Like Kul, the activity of Kuz is cell autonomous, i.e. restricted to the cells overexpressing the protease (Sapir, 2005).

In the wing disc, Notch signaling plays a key role in defining and maintaining the margin, in two distinct signaling phases. Initially, the asymmetry between the dorsal and ventral compartments defines the margin and induces the expression of Wg by the future margin cells. The process is dictated by expression of Fringe only in the dorsal compartment, facilitating Notch signaling in the two cell rows comprising the border between the two compartments. The wing margin fate is subsequently maintained by complementary unidirectional signals between the margin and juxta-margin cells. In the margin, Notch signaling leads to the expression of Wg and Cut, the latter operating as a transcriptional repressor of Dl. In parallel, Wg activates expression of Dl and Serrate in the juxta-margin cells. High levels of Dl and Serrate prevent Notch activation in these cells. Thus a stable loop of two reinforcing signals is generated (Sapir, 2005).

It was interesting to examine the biological consequences of the response to Kul overexpression. Genetic removal of Dl or Ser alone was not sufficient to alleviate the dominant-negative effect of the remaining ligand on Notch signaling in the juxta-margin cells. However, in the case of Kul overexpression, both ligands were affected, i.e., Dl was efficiently cleaved and Ser was predominantly sequestered within the cells. An expansion was observed in the expression of the Notch-target genes wg and cut. In addition, the expression of Ser was broader. It is assumed that effective removal of Dl in conjunction with sequestration of Ser, give rise to alleviation of their dominant-negative effect on Notch signaling in the juxta-margin cells expressing these ligands. Thus, a residual level of Dl or Ser on the cell surface could trigger Notch signaling within these cells. Activation of Notch subsequently leads to ectopic production of Wg, which in turn spreads to neighboring cells to trigger ectopic expression of Ser (Sapir, 2005).

Does endogenous Kul in the wing disc have a role in maintaining the asymmetric distribution of Dl? Expression of ds-kul by the potent sd-GAL4 driver gives rise to loss of Cut and Wg expression in the margin, and a reduction in the size of the wing pouch. The adult wings that develop are significantly reduced in size and show no indication of veins, and only rudimentary margin bristles in very restricted domains. These results demonstrate that Kul is essential for maintaining the spatial balance of Notch signaling in the wing margin (Sapir, 2005).

Induction of ds-kul by the sal-GAL4 driver does not affect the adult wing margin and results only in a reduction in Wg levels in the wing margin, without a pronounced effect on Cut levels. In view of the role Kul plays in Dl cleavage, it was of interest to test whether the changes in Notch target-gene expression in the wing margin result from elevation in Dl levels within the margin cells. Indeed, higher levels of Dl could be detected in the margin within the sal domain where ds-kul is expressed. By contrast, no effects of ds-kul on the distribution of Ser were observed (Sapir, 2005).

How does Kul activity impinge on the distribution of Dl? Overexpression of Kul in the wing disc results in a dramatic diminution of the levels of Dl. This effect is cell autonomous, i.e., Kul can only eliminate Dl within the cells in which Kul is expressed. It is not known if cleavage takes place once both proteins are localized to the cell surface, or if removal of Dl occurs during trafficking to the cell surface. Since no accumulation of Dl was observed within the cells following Kul overexpression, the first possibility is favored. Kul activity appears to be constitutive (see below), implying that there is no preferential cleavage of Dl by Kul in the receiving cells. Rather, the final outcome is likely to result from the activity of Kul in both cell types. In the receiving cells, where the levels of Dl are low, the proteolytic activity of Kul effectively eliminates the Dl protein. By contrast, in the sending cells expressing high levels of Dl, while Kul may cleave some of the ligand, sufficient levels of Dl remain to allow efficient signaling (Sapir, 2005).

Disruption of Notch unidirectional signaling following removal of Kul highlights the necessity of continuously removing the Dl protein, in order to generate a setting in which it would be hard for the Dl protein to accumulate. Transcriptional repression of Dl expression is not sufficient. For example, in the wing margin, activation of the Notch pathway specifically leads to the induction of E(spl) and Cut, which are transcriptional repressors of Dl expression. Yet, in the absence of Kul, some Dl protein is produced by the margin cells. Similarly, in the pupal wing, activation of Notch in the lateral pro-vein cells induces E(spl) expression. Nevertheless, Dl is produced by these cells when Kul is eliminated. These observations underscore an inherent difficulty in shutting down Dl transcription efficiently. They also imply that even residual levels of Dl have detrimental biological consequences. The constitutive cleavage of Dl by Kul is therefore a crucial safeguard, continuously removing low levels of Dl that have escaped transcriptional repression (Sapir, 2005).

The biological role of Kul was demonstrated in this work in two stages in which Notch signaling refines a pre-existing asymmetry between adjacent cells: the wing margin and the wing veins. In other instances, Notch signaling actually generates the asymmetry between cells. Notch defines the correct number and spacing of differentiated cells within a field of equipotent cells, e.g. in the embryonic neuroectoderm or among pupal sensory organs. In these cases, it is thought that stochastic fluctuations in the levels of Dl, coupled to mechanisms that amplify these changes, lead to differentiation of some cells and concomitant repression of differentiation in the neighboring cells. Kul does not seem to impinge on these process. No effects on the number and organization of neuroblasts were observed following induction of ds-kul by broad maternal and early zygotic drivers. Another avenue of Notch signaling is triggered by asymmetric cell divisions in the sensory neuron precursors. Again, induction of ds-kul by neu-GAL4 does not give rise to any Notch-related phenotypes in the sensory bristles. It is therefore concluded that the activity of Kul appears to be essential for Notch signaling specifically in cases where a pre-existing spatial asymmetry is used to guide the directionality of Notch signaling (Sapir, 2005).

In view of the central role of Kul in Notch signaling, it was important to examine the different junctions in which Kul activity may be regulated. At the transcriptional level, Kul appears to be broadly expressed, in embryos and in imaginal discs. This broad expression is also reflected in the Notch-independent multiple-wing hair phenotype that is observed in all wing cells where ds-kul is induced. It is still possible that the basal level of Kul expression may be elevated in cells where Notch signaling takes place, to reduce the levels of Dl in these cells more efficiently (Sapir, 2005).

At the post-transcriptional level, however, there are several steps in the generation of an active Kul protein, which could be regulated. The protein must be correctly targeted to the plasma membrane, a process that may rely on the cytoplasmic domain of Kul and its interaction with the intracellular trafficking machinery. The precursor form of Kul undergoes processing by Furins, to remove the pro-domain. In the absence of this processing, Kul cannot cleave Dl. Finally, association of Kul with its substrates is mediated by the disintegrin domain, and possibly also by additional proteins that could bias this interaction (Sapir, 2005).

In spite of the sequential processes necessary for the formation of a mature, active Kul protein, there is no evidence that any of these steps is regulated in time or space. The data so far support the notion of a constitutive maturation and processing of Kul. In every cell where Kul was misexpressed, an outcome was observed, as monitored by removal of Dl. In the wing, removal of Kul activity also gives rise to additional phenotypes that are not related to Notch, e.g., the appearance of multiple wing hairs. This phenotype was observed in all cells where ds-kul was expressed, again supporting the notion that Kul is normally expressed and activated uniformly (Sapir, 2005).

In conclusion, while Kul may be broadly active, it enhances and maintains the asymmetrical activation of Notch, by relying on the initial differences in the levels of Dl. Kul effectively removes the ligand from the cells expressing Dl at low levels, while retaining sufficient levels of Dl in the cells that will activate Notch. Thus, a uniform activity of Kul can amplify a bias in the levels of Dl expression, and leads to a strict unidirectional activation of Notch, a process that is central to patterning the organism at multiple stages of development (Sapir, 2005).

Mind-bomb and Neuralized are two distinct E3 ubiquitin ligases that have complementary functions in the regulation of Delta and Serrate signaling in Drosophila

Signaling by the Notch ligands Delta (Dl) and Serrate (Ser) regulates a wide variety of essential cell-fate decisions during animal development. Two distinct E3 ubiquitin ligases, Neuralized (Neur) and Mind-bomb (Mib), have been shown to regulate Dl signaling in Drosophila melanogaster and Danio rerio, respectively. While the neur and mib genes are evolutionarily conserved, their respective roles in the context of a single organism have not yet been examined. Drosophila mind bomb (D-mib) regulates a subset of Notch signaling events, including wing margin specification, leg segmentation, and vein determination, that are distinct from those events requiring neur activity. D-mib also modulates lateral inhibition, a neur- and Dl-dependent signaling event, suggesting that D-mib regulates Dl signaling. During wing development, expression of D-mib in dorsal cells appears to be necessary and sufficient for wing margin specification, indicating that D-mib also regulates Ser signaling. Moreover, the activity of the D-mib gene is required for the endocytosis of Ser in wing imaginal disc cells. Finally, ectopic expression of neur in D-mib mutant larvae rescues the wing D-mib phenotype, indicating that Neur can compensate for the lack of D-mib activity. It is concluded that D-mib and Neur are two structurally distinct proteins that have similar molecular activities but distinct developmental functions in Drosophila (Le Borgne, 2005).

Cell-to-cell signaling mediated by receptors of the Notch (N) family has been implicated in various developmental decisions in organisms ranging from nematodes to mammals. N is well-known for its role in lateral inhibition, a key patterning process that organizes the regular spacing of distinct cell types within groups of equipotent cells. Additionally, N mediates inductive signaling between cells with distinct identities. In both signaling events, N signals via a conserved mechanism that involves the cleavage and release from the membrane of the N intracellular domain that acts as a transcriptional co-activator for DNA-binding proteins of the CBF1/Suppressor of Hairless/Lag-2 (CSL) family (Le Borgne, 2005).

Two transmembrane ligands of N are known in Drosophila, Delta (Dl) and Serrate (Ser). Dl and Ser have distinct functions. For instance, Dl (but not Ser) is essential for lateral inhibition during early neurogenesis in the embryo. Conversely, Ser (but not Dl) is specifically required for segmental patterning. Some developmental decisions, however, require the activity of both genes: Dl and Ser are both required for the specification of wing margin cells during imaginal development. These different requirements for Dl and Ser appear to primarily result from their non-overlapping expression patterns rather than from distinct signaling properties. Consistent with this interpretation, Dl and Ser have been proposed to act redundantly in the sensory bristle lineage where they are co-expressed. Furthermore, Dl and Ser appear to be partially interchangeable because the forced expression of Ser can partially rescue the Dl neurogenic phenotype. Additionally, the ectopic expression of Dl can partially rescue the Ser wing phenotype. The notion that Dl and Ser have similar signaling properties has, however, recently been challenged by the observation that human homologs of Dl and Ser have distinct instructive signaling activity (Le Borgne, 2005).

Endocytosis has recently emerged as a key mechanism regulating the signaling activity of Dl. (1) Clonal analysis in Drosophila has suggested that dynamin-dependent endocytosis is required not only in signal-receiving cells but also in signal-sending cells to promote N activation. (2) Mutant Dl proteins that are endocytosis defective exhibit reduced signaling activity (Parks, 2000). (3) Two distinct E3 ubiquitin ligases, Neuralized (Neur) and Mind-bomb (Mib), have recently been shown to regulate Dl endocytosis and N activation in Drosophila and Danio rerio, respectively. Ubiquitin is a 76-amino-acid polypeptide that is covalently linked to substrates in a multi-step process that involves a ubiquitin-activating enzyme (E1), a ubiquitin-conjugating enzyme (E2), and a ubiquitin-protein ligase (E3). E3s recognize specific substrates and catalyze the transfer of ubiquitin to the protein substrate. Ubiquitin was first identified as a tag for proteins destined for degradation. More recently, ubiquitin has also been shown to serve as a signal for endocytosis. Mib in D. rerio and Neur in Drosophila and Xenopus have been shown to associate with Dl, regulate Dl ubiquitination, and promote its endocytosis. Moreover, genetic and transplantation studies have indicated that both Neur and Mib act in a non-autonomous manner, indicating that endocytosis of Dl is associated with increased Dl signaling activity. Finally, epsin, a regulator of endocytosis that contains a ubiquitin-interacting motif and that is known in Drosophila as Liquid facet, is essential for Dl signaling. In one study, Liquid facets was proposed to target Dl to an endocytic recycling compartment, suggesting that recycling of Dl may be required for signaling. Accordingly, signaling would not be linked directly to endocytosis, but endocytosis would be prerequisite for signaling. How endocytosis of Dl leads to the activation of N remains to be elucidated. Also, whether the signaling activity of Ser is similarly regulated by endocytosis is not known (Le Borgne, 2005 and references therein).

While genetic analysis has revealed that neur in Drosophila and mib in D. rerio are strictly required for N signaling, knockout studies of mouse Neur1 have indicated that NEUR1 is not strictly required for N signaling. One possible explanation is functional redundancy with the mouse Neur2 gene. Conversely, the function of Drosophila mib (D-mib), the homolog of D. rerio mib gene has not previously been characterized (Le Borgne, 2005).

To establish the respective roles of these two distinct E3 ligases in the context of a single model organism, the function of the Drosophila D-mib gene was studied. D-mib, like D. rerio Mib, appears to regulate Dl signaling during leg segmentation, wing vein formation, and lateral inhibition in the adult notum. D-mib is specifically required for Ser endocytosis and signaling during wing development, indicating for the first time that endocytosis regulates Ser signaling. Interestingly, the D-mib activity was found necessary for a subset of N signaling events that are distinct from those requiring the activity of the neur gene. Nevertheless, the ectopic expression of Neur compensates for the loss of D-mib activity in the wing, indicating that Neur and D-mib have overlapping functions. It is concluded that D-mib and Neur are two structurally distinct proteins with similar molecular activities but distinct and complementary functions in Drosophila (Le Borgne, 2005).

This analysis first establishes that D-mib regulates Ser signaling during wing development. (1) Clonal analysis revealed that the activity of the D-mib gene is specifically required in dorsal cells for the expression of Cut at the wing margin. (2) Expression of D-mib in the dorsal Ser-signaling cells is sufficient to rescue the D-mib mutant wing phenotype. (3) Results from an in vivo antibody uptake assay indicate that the endocytosis of Ser (but not of Dl) was strongly inhibited in D-mib mutant cells. This inhibition correlates with the strong accumulation of Ser (but not Dl) at the apical cortex of D-mib mutant cells. Thus, an essential function of D-mib in the wing is to regulate the endocytosis of Ser in dorsal cells to non-autonomously promote the activation of N along the D-V boundary. By analogy, the defective growth of the eye tissue may similarly result from the lack of Ser signaling and of N activation along the D-V boundary. Because (1) D-mib co-localizes with Ser at the apical cortex of wing disc cells, (2) acts in a RING-finger-dependent manner to regulate Ser endocytosis in S2 cells, and (3) physically associates with Ser in co-immunoprecipitation experiments, D-mib may ubiquitinate Ser and directly regulate its endocytosis (Le Borgne, 2005).

This analysis further suggests that endocytosis of Ser is required for Ser signaling. This conclusion is consistent with observations made earlier showing that secreted versions of Ser cannot activate N but instead antagonize Ser signaling. Thus, endocytosis of both N ligands appears to be strictly required for N activation in Drosophila. Different models have been proposed to explain how endocytosis of the ligand, which removes the ligand from the cell surface, results in N receptor activation. Interestingly, the strong requirement for Dl and Ser endocytosis seen in Drosophila is not conserved in Caenorhabditis elegans, in which secreted ligands have been shown to be functional. Noticeably, there is no C. elegans Mib homolog, and the function of C. elegans neur (F10D7.5) is not known. It is speculated that endocytosis of the ligands may have evolved as a means to ensure tight spatial regulation of the activation of Notch (Le Borgne, 2005).

This analysis also establishes that the activity of the D-mib gene is required for a subset of N signaling events that are distinct from those that require the activity of the neur gene. The D-mib gene regulates wing margin formation, leg segmentation, and vein formation, whereas none of these three processes depend on neur gene activity. Conversely, the activity of the neur gene is essential for binary cell-fate decisions in the bristle lineage that do not require the activity of the D-mib gene (no bristle defects were seen in D-mib mutant flies). The activity of the neur gene is also required for lateral inhibition during neurogenesis in embryos and pupae. This process is largely independent of D-mib gene activity since the complete loss of D-mib function resulted only in a mild neurogenic phenotype in the notum. These data thus indicate that the neur and D-mib genes have largely distinct and complementary functions in Drosophila. Whether a similar functional relationship between Neur and D-mib exists in vertebrates awaits the study of the D. rerio neur genes and/or of the murine Mib and Neur genes (Le Borgne, 2005).

The functional differences observed between D-mib and neur cannot be simply explained by obvious differences in molecular activity and/or substrate specificity. Both Neur and D-mib physically interact with Dl and promote the down-regulation of Dl from the apical membrane when overexpressed. Furthermore, Dl signaling appears to require the activity of either Neur or D-mib, depending on the developmental contexts. Specific aspects of the D-mib phenotype in legs and in the notum cannot simply result from loss of Ser signaling and are consistent with reduced Dl signaling, suggesting that D-mib regulates Dl signaling. Consistent with this interpretation, overexpression studies indicate that D-mib up-regulates the signaling activity of Dl, whereas a dominant-negative form of D-mib inhibits it. It is noted, however, that no clear defects in Dl subcellular localization and/or trafficking were observed in D-mib mutant cells. It is conceivable that the contribution of D-mib to the endocytosis of Dl is masked by the activity of D-mib-independent processes that may, or may not, be linked to Dl signaling. It has also been shown that, reciprocally, Neur and D-mib may similarly regulate Ser. Neur and D-mib similarly promote down-regulation of Ser from the cell surface when overexpressed. Moreover, D-mib binds Ser and regulates Ser signaling. Whether endogenous Neur binds and activates Ser remains to be tested. However, the ability of Neur to rescue the D-mib mutant wing phenotype when expressed in dorsal cells strongly indicates that Neur can promote Ser signaling. Together, these data indicate that Neur and D-mib have similar molecular activities (Le Borgne, 2005).

D-mib and Neur may have identical molecular activities but distinct expression patterns, hence distinct functions at the level of the organism. Consistent with this possibility, D-mib is uniformly distributed in imaginal discs, whereas Neur is specifically detected in sensory cells. Importantly, the rescue of the D-mib mutant phenotype by ectopic expression of Neur strongly supports this interpretation. This result further suggests that Neur can regulate Ser signaling. Consistent with this idea, overexpression of Neur in imaginal discs results in a strong reduction of Ser accumulation at the apical cortex. Thus, despite their obvious structural differences, Neur and D-mib appear to act similarly to promote the endocytosis of Dl and Ser. Nevertheless, the observation that D-mib can not compensate for the loss of neur activity in the embryo indicates that D-mib and Neur have overlapping rather than identical molecular activities (Le Borgne, 2005).

In conclusion, Neur and D-mib appear to have similar molecular activities in the regulation of Dl and Ser endocytosis but distinct developmental functions in Drosophila. The conservation from Drosophila to mammals of these two structurally distinct but functionally similar E3 ubiquitin ligases is likely to reflect a combination of evolutionary advantages associated with: (1) specialized expression pattern, as evidenced by the cell-specific expression of the neur gene in sensory organ precursor cells, (2) specialized function, as suggested by the role of murine MIB in TNFα signaling and (3) regulation of protein stability, localization, and/or activity. For instance, Neur, but not D-mib, localizes asymmetrically during asymmetric sensory organ precursor cell divisions (Le Borgne, 2005).

The ubiquitin ligase Drosophila Mind bomb promotes Notch signaling by regulating the localization and activity of Serrate and Delta

Loss- and gain-of-function analyses indicate that the major function of D-mib is to regulate Notch signal transduction. Since Delta is a bona fide substrate of zebrafish Mib, tests were performed for a physical association of D-mib and Delta by co-immunoprecipitation. Cultured cells were co-transfected with Delta and various D-mib expression vectors, and co-immunoprecipitation was performed in both directions. Although Delta did not successfully co-immunoprecipitate full-length D-mib, it did associate with all isoforms that contain the D-mib N terminus and lack the C-terminal RING finger (namely D-mib-N, D-mibDelta3RF and D-mibDeltaRF. Conversely, these same D-mib isoforms efficiently co-immunoprecipitate Delta; full-length D-mib also shows modest association with Delta in this direction. It was consistently observed that the presence of full-length D-mib reduces Delta levels, which might account for why this interaction is poorly detected. Notably, D-mib-N shows the strongest interaction with Delta. In fact, immunoprecipitated D-mib-N brings down both full-length Delta and cleaved DeltaIC, consistent with a direct interaction between the N terminus of D-mib and the intracellular domain of Delta. A truncated D-mib protein lacking the N-terminal domain (D-mib-C) shows no binding to Delta, demonstrating that this region is crucial for association with Delta (Lai, 2005).

Physical association between D-mib proteins and Serrate was tested. D-mib:Serrate interactions appear to be somewhat weaker than D-mib:Delta interactions; however, the overall profile of the different D-mib truncations in association with Serrate and Delta is identical. These findings lead to the conclusion that the N terminus of D-mib mediates physical association with both Drosophila DSL ligands. In addition, full-length D-mib similarly reduces the accumulation of Serrate, indicating that D-mib downregulates both DSL ligands (Lai, 2005).

In vitro data correlate well with in vivo studies, in that all RING-finger-deleted D-mib isoforms that retain the ability to associate with DSL ligands (D-mib-N, D-mibDeltaRF and D-mib3DeltaRF) have at least some ability to inhibit Notch signaling. However, full specificity and activity of D-mib requires inclusion of the ankyrin repeats and the two non-canonical RING fingers. Curiously, there is no significant similarity at the primary amino acid level between the intracellular domains of Delta and Serrate. In this regard, it is relevant to note that Xenopus Neur (X-Neur) robustly regulates Drosophila Delta in vivo, even though there is no significant similarity between the intracellular domains of Delta and X-Delta. D-mib and Neur may therefore recognize a more hidden, possibly structural, feature that is shared by DSL ligands (Lai, 2005).

The interplay between Delta and Serrate proteins and ubiquitin ligases in Notch signaling

Lateral inhibition is a pattern refining process that generates single neural precursors from a field of equipotent cells and is mediated via Notch signaling. Of the two Notch ligands Delta and Serrate, only the former was thought to participate in this process. It is shown in this study that macrochaete lateral inhibition involves both Delta and Serrate. In this context, Serrate interacts with Neuralized, a ubiquitin ligase that was heretofore thought to act only on Delta. Neuralized physically associates with Serrate and stimulates its endocytosis and signaling activity. A mutation was characterized in mib1, a Drosophila homolog of zebrafish mind-bomb, another Delta-targeting ubiquitin ligase. Mib1 affects the signaling activity of Delta and Serrate in both lateral inhibition and wing dorsoventral boundary formation. Simultaneous absence of neuralized and mib1 completely abolishes Notch signaling in both aforementioned contexts, making it likely that ubiquitination is a prerequisite for Delta/Serrate signaling (Pitsouli, 2005).

Until now, it was thought that lateral inhibition in notum SOPs was solely mediated via Dl and that Dl transcriptional upregulation in the nascent neural precursor was crucial for a Dl-N negative feedback loop to establish the neural precursor fate within a group of equivalent cells. These data have refuted both of these models, because endogenous Ser has now been shown to participate in lateral inhibition of macrochaete SOPs and either Dl or Ser uniformly expressed is able to produce a wild-type pattern of macrochaetes. Dl transcriptional upregulation in the absence of Notch signaling in proneural fields does occur, but this modulation does not appear to be a prerequisite for the specification of the wild-type neural precursor, at least in the case of macrochaetes and embryonic neuroblasts. It is possible that the genetically detected N-Dl negative feedback loop may reflect Dl and N activity rather than transcription, although a transcriptional input has been documented. An exciting possibility, given the reliance of DSL activity on ubiquitin ligases, is that this feedback loop targets transcription of neur, rather than Dl. mib1 is an unlikely target as since shows no transcriptional modulation within proneural regions (Pitsouli, 2005).

Although Neur was known to affect Dl localization and function in some instances, ubiquitin ligases were not considered as essential components of Notch signaling. The characterization of Mib1 described here and in recent papers (Lai, 2005; Le Borgne, 2005; Wang, 2005) points to a much more prominent role of these factors. mib1 appears to be required in a large number of Notch-dependent processes where neur is not expressed, e.g., the wing DV boundary. The fact that mib1 neur double mutants appear to lose all ability to perform lateral inhibition strongly supports the hypothesis that Ub ligases may always be required for Dl/Ser signaling. A comprehensive survey of Notch-dependent events with respect to neur and mib1 will test this hypothesis and may uncover additional E3 ligases with this activity; Mib2 represents a potential candidate (Pitsouli, 2005).

The intimate relation between Neur/Mib1 and DSL proteins is generally assayed in three ways: (1) physical association, (2) effects on Dl/Ser endocytosis and (3) effects on Dl/Ser signaling. All of these had been well documented for the Neur-Dl combination and, more recently, for the Mib1-Dl and Mib1-Ser combinations (Lai, 2005; Le Borgne, 2005; Wang, 2005). In the present work the final pair, Neur-Ser, has been added, using all of the above assays. The conclusion, stated simply, is that both Neur and Mib1 associate with and affect the endocytosis and function of both Dl and Ser (Pitsouli, 2005).

Ubiquitination of transmembrane proteins tags them for endocytosis, using a complex of adaptors, including epsin, which carry ubiquitin recognition domains. The simplest scenario for the role of Neur/Mib1 in Dl/Ser signaling would be that they attach ubiquitin to Dl/Ser to trigger endocytosis. Signaling would ensue, either as a consequence of recruiting/clustering ubiquitinated DSL cargo to specialized plasma membrane domains conducive to signaling, or by more elaborate routes involving DSL protein recycling through the endocytic pathway as a prerequisite for their modification/activation (Pitsouli, 2005).

Alternatively, Neur/Mib1 need not ubiquitinate the DSL proteins directly. In the ubiquitin-dependent endocytosis pathway, many of the adaptor proteins are themselves ubiquitinated, possibly favoring the formation of interconnected cargo-adaptor complexes; Neur/Mib1 could have one or more of the adaptors, including themselves, as substrates. DSL protein chimaeras become Mib1 independent if their intracellular domains are substituted with ones bearing alternative internalization motifs (Wang, 2005). Of two such artificial Mib1-independent versions of Dl, one is ubiquitination/epsin-independent (Dl-LDL-receptor fusion), whereas the other (Dl-random-peptide-R fusion) still curiously requires ubiquitination/epsin for activity (Wang, 2004). Nothing is yet known about the native Dl/Ser intracellular domains, other than the puzzling fact that they are neither similar nor evolutionarily conserved, despite apparent conservation of recognition by Neur/Mib (Pitsouli, 2005).

An even more puzzling observation in the light of this model is that some DSL proteins in C. elegans appear to be secreted. Secreted mutants of Drosophila Dl and Ser act as Notch antagonists, consistent with a requirement for endocytosis in DSL signaling. Even C. elegans LAG-2 (a transmembrane DSL) needs EPN-1 (epsin ortholog), in order to signal to GLP-1 (Notch-like) during germline differentiation, which is hard to reconcile with secreted DSL proteins. Apparently, ubiquitination/endocytosis can be bypassed in some contexts, allowing secreted DSL proteins to signal via a yet unknown process (Pitsouli, 2005).

Whatever the molecular details and variations turn out to be, it is becoming clear that ubiquination plays a prominent role in Notch signaling, in both sending and receiving cells. In the latter, Ub ligases downregulate Notch activity either at the membrane or in the nucleus. Besides downregulation, however, Notch ubiquitination is also needed for activation: ubiquitination apparently targets Notch to a compartment where it can be activated by gamma-secretase cleavage. How two ubiquitination/trafficking events, activating DSL proteins in one cell and Notch in another, might be coordinated across the extracellular space is a mystery worth investigating in the future (Pitsouli, 2005).

Sec15, a component of the exocyst, promotes recycling of Delta during the asymmetric division of Drosophila sensory organ precursors

Asymmetric division of sensory organ precursors (SOPs) in Drosophila generates different cell types of the mature sensory organ. In a genetic screen designed to identify novel players in this process, a mutation was isolated in Drosophila sec15, which encodes a component of the exocyst, an evolutionarily conserved complex implicated in intracellular vesicle transport. sec15 sensory organs contain extra neurons at the expense of support cells, a phenotype consistent with loss of Notch signaling. A vesicular compartment containing Notch, Sanpodo, and endocytosed Delta accumulates in basal areas of mutant SOPs. Based on the dynamic traffic of Sec15, its colocalization with the recycling endosomal marker Rab11, and the aberrant distribution of Rab11 in sec15 clones, it is proposed that a defect in Delta recycling causes cell fate transformation in sec15 sensory lineages. The data indicate that Sec15 mediates a specific vesicle trafficking event to ensure proper neuronal fate specification in Drosophila (Jafar-Nejad, 2005).

In a genetic screen designed to identify novel players in Drosophila sensory organ development, a mutation in sec15 was isolated that caused a pIIa to pIIb transformation phenotype. Sec15 is a component of a multiprotein complex called the exocyst or Sec6/8 complex. Mutations in exocyst components were originally isolated in a yeast screen for secretion-defective mutants. Subsequent analysis of the exocyst complex in yeast and mammalian cell culture systems has indicated that it functions in intracellular vesicle transport. In yeast, the exocyst mediates the post-Golgi to membrane targeting of exocytic cargo via an interaction with the Rab GTPase Sec4p. In Madin-Darby canine kidney (MDCK) epithelial cells, the exocyst localizes to areas of cell-cell contact and is involved in basolateral delivery of vesicles. However, none of the studies on the exocyst components have implicated these proteins in cell fate determination. The data suggest that Sec15 mediates highly specific intracellular trafficking events that promote N signaling and thereby ensure proper cell fate specification in Drosophila mechanosensory organs (Jafar-Nejad, 2005).

The various cell types that form an adult sensory organ in Drosophila are generated via asymmetric divisions of a pI and its progeny. Differential activation of the N signaling pathway between the two daughter cells of each division ensures that each sensory organ acquires the proper complement of cell types necessary to function. Sec15, a component of the evolutionarily conserved exocyst complex as reported here, is required for proper cell fate specification of the pI progeny. Studies on sec15 mutations in the eye did not reveal any fate change in the photoreceptors. Loss-of-function mutations in three other exocyst components have been reported previously: sec5 and sec6 in flies and sec8 in mice. sec8 mutant mice die at day E7.5, before the development of specific neuronal populations can be studied. Also, sec5 and sec6 mutations are cell lethal in the Drosophila eye. Therefore, this report is the first to identify a role for an exocyst component in cell fate determination. At this point, it cannot be predicted if sec5 and sec6 also play a role in neuronal cell fate specification. However, given the data obtained from studies of the fly eye, the hypothesis is favored that components of the exocyst may form more than a single functional unit and/or have subunit-specific roles (Jafar-Nejad, 2005).

Live imaging of dividing pI cells indicates that Sec15 is associated with a vesicular compartment that traffics between apical and subapical areas. In sec15 SOPs, an expanded compartment is observed that contains Spdo, N, and Dl. Unlike wt pI and pIIb cells, in which Spdo/N/Dl+ vesicles tend to reside at or above the level of septate junctions, in mutant SOPs these puncta accumulate at the basal side of the cell. Together, these observations suggest that Sec15 is involved in vesicle trafficking to the apical parts of the cell. The defect in the apical trafficking of proteins does not seem to be a general one, since localization of E-Cad and Arm at the adherens junction is not disrupted in mutant tissue. Therefore, the data link a specific vesicle trafficking event to a developmental decision made by sensory precursor cells (Jafar-Nejad, 2005).

Genetic experiments and immunohistochemical stainings strongly suggest that Sec15 and Spdo function in the same pathway in sensory cell fate determination process. It has been proposed, based on studies performed on the asymmetric divisions of Drosophila embryonic neuroblasts, that Spdo promotes N signaling at the membrane of the signal-receiving cell. In contrast, Numb and α-Adaptin in the signal-sending cell might promote endocytosis of Spdo and its removal from the membrane, thereby preventing the reception of signal by this cell. The subcellular distribution of Spdo in pIIa and pIIb cells is similar to its localization in embryonic neuroblast progeny, suggesting that this model might also apply to adult bristle formation. Notably, however, Spdo is observed at or close to the membrane of both pI progeny in sec15 clones. Therefore, while the proposed role for Spdo in promoting N signaling at the membrane of the signal-receiving cell cannot be ruled out, the data suggest a role for Spdo in Dl recycling in the signal-sending cell. It should be noted, though, that these two models are not mutually exclusive. Presence of a significantly higher number of vesicles containing both Dl and N in pIIb compared to the pIIa in wt sensory precursors has been implicated in the ability of the pIIb cell to send the Dl signal. Colocalization of Spdo with Dl in a significant fraction of these vesicles suggests that a defect in Spdo/Dl trafficking in pIIb contributes to the sec15 loss-of-function phenotype (Jafar-Nejad, 2005).

Presence of endocytosed Dl in vesicles that accumulate in sec15 clones implicates these vesicles in the endocytic traffic of Dl. This notion is further supported by the observation that in both wt and sec15 SOPs, the Spdo/Dl/N puncta show a significant colocalization with the endosomal markers Rab5 and HRS. It has recently been proposed that in order to signal, Dl needs to traffic through a specific endocytic compartment, which will lead to recycling of the protein. A defect in Dl recycling is further suggested by the aberrant accumulation of the recycling endosomal marker Rab11 in sec15 clones. The Rab11+ endosomal compartment is thought to be a central trafficking intermediate in both exocytic and endocytic pathways and is shown to control the traffic of cargo from the perinuclear recycling endosomal compartment to the membrane. Interestingly, it has been shown that Sec15 meets the criteria of being an effector for Rab11 in mammalian cell lines: Sec15 physically binds Rab11 in a GTP-dependent manner; Sec15 colocalizes with Rab11 in the perinuclear region of the cells; Sec15 labels structures containing an endocytosed protein in immuno-EM experiments. Similarly, Drosophila Sec15 and Rab11 interact physically and show a high level of colocalization in SOPs. Altogether, these data are compatible with a model in which Sec15 regulates the traffic of a subset of endocytosed Dl to the membrane of the pIIb cell via a Rab11+ recycling endosomal compartment. Sec15 traffics symmetrically in pIIa and pIIb. Therefore, it is proposed that an intrinsic difference between the endocytic traffic of Dl in pIIa and pIIb allows the pIIb cell to employ the Sec15-Rab11 machinery differentially from the pIIa cell and thereby assume the role of signal-sending cell. The most likely mechanisms for the proposed intrinsic difference are unequal segregation of Neur into the pIIb, which promotes Dl endocytosis in this cell, and asymmetric distribution of the Rab11+ recycling endosomes in the pIIb versus pIIa, which is thought to specifically mediate Dl recycling in the pIIb (Jafar-Nejad, 2005).

These data suggest that at least some of the Spdo/N/Dl-containing vesicles that accumulate in the basal areas of sec15 SOPs are of a mixed exo-endocytic nature. This is not unprecedented, since traffic from the TGN to an endosomal compartment has been documented. Accordingly, it has been proposed that some exocytic cargo might pass through the recycling endosome on its way from the TGN to the plasma membrane. Recently, it has been shown that upon exit from the Golgi apparatus, newly synthesized E-Cad fuses with a Rab11+ recycling endosomal compartment before it reaches the plasma membrane. It is interesting to note that members of the exocyst complex have been shown to localize to both the TGN and recycling endosomes in polarizing epithelial cells. Although the recycling endosome has been proposed as an intermediate to transfer the exocytic cargo to the plasma membrane, it is possible that passing through these vesicles somehow enhances the signaling ability of internalized Dl. In other words, presence of Spdo might be part of the specific environment that Dl needs to traffic through. Although Dl endocytosis and recycling are also implicated in N signaling during lateral inhibition, no lateral inhibition defects are observed in sec15 clones. It is proposed that the link to Spdo results in the specificity of the sec15 phenotype to the asymmetric divisions, since loss of spdo similarly does not affect lateral inhibition (Jafar-Nejad, 2005).

In summary, the data indicate that one component of the highly conserved exocyst complex affects the asymmetric division of the sensory precursors in the Drosophila PNS through specific vesicle trafficking events. Components of the exocyst complex are conserved from yeast to human, and several reports have shown parallels between the contribution of asymmetric divisions to Drosophila and vertebrate neurogenesis. Therefore, it is conceivable that Sec15, and perhaps other members of the exocyst complex, are involved in neural cell fate determination in other species (Jafar-Nejad, 2005).

Asymmetric Rab11 endosomes regulate Delta recycling and specify cell fate in the Drosophila nervous system

Drosophila sensory organ precursor (SOP) cells are a well-studied model system for asymmetric cell division. During SOP division, the determinants Numb and Neuralized segregate into the pIIb daughter cell and establish a distinct cell fate by regulating Notch/Delta signaling. This study describes a Numb- and Neuralized-independent mechanism that acts redundantly in cell-fate specification. Trafficking of the Notch ligand Delta is different in the two daughter cells. In pIIb, Delta passes through the recycling endosome which is marked by Rab11. In pIIa, however, the recycling endosome does not form because the centrosome fails to recruit Nuclear fallout, a Rab11 binding partner that is essential for recycling endosome formation. Using a mammalian cell culture system, it was demonstrated that recycling endosomes are essential for Delta activity. These results suggest that cells can regulate signaling pathways and influence their developmental fate by inhibiting the formation of individual endocytic compartments (Emery, 2005).

To test whether Rab11 asymmetry is important for cell-fate specification, Rab11 accumulation in the pIIa cell was induced by nuf expression. Postorbital ES organs, which can easily be scored in fairly high numbers, were used. Cell-fate transformations upon nuf overexpression have been described (Abdelilah-Seyfried, 2000), but surprisingly, they do not occur at high frequency. Such transformations can, however, be observed upon coexpression of constitutively active Rab11. Upon expression of nonphosphorylatable lgl, Numb and Neuralized asymmetry are disrupted, but most ES organs still develop normally. When both pathways are disrupted by coexpression of lgl3A and nuf, however, a large fraction of ES organs shows cell-fate transformations that are consistent with a higher level of Delta activity in pIIa. Lineage analysis shows cell-fate transformations in 44% of postorbital ES organs, and in 18% of these, pIIb cells are transformed into pIIa cells (6% in ES organs expressing lgl3A alone). Twenty-five percent of the cell fate transformations affect the first (SOP) while 75% affect the second (pIIa) division, indicating that Rab11 asymmetry also plays a role in other divisions of the SOP lineage. Taken together, these results suggest that two partially redundant pathways exist to generate asymmetry in the SOP lineage: the Par proteins phosphorylate Lgl to direct Numb and Neuralized into the pIIb cell where they repress Notch or activate Delta, respectively. In the pIIa cell, inhibition of Nuf and Rab11 inhibits Delta by preventing its trafficking through the recycling endosome (Emery, 2005).

These results suggest that cells can also regulate signal transduction pathways by controlling the formation or distribution of whole endocytic compartments. After SOP division, Rab11-positive vesicles accumulate around the centrosome in this cell but not in pIIa. Rab11 plays a well-documented role in controlling vesicular protein transport through recycling endosomes to the plasma membrane (Zerial, 2001). Dominant-negative forms of Rab11 inhibit the recycling of endocytosed Transferrin receptors or recruitment of H+-K+-ATPase to the plasma membrane suggesting that Rab11 regulates trafficking of vesicular cargo through the recycling endosomal compartment. In SOP cells, the asymmetric localization of Rab11 reflects a different ability of pIIa and pIIb cells to recycle the Notch ligand Delta. Rab11 asymmetry is observed 3.5 min after cytokinesis but Delta is in recycling endosomes only 15 min after endocytosis. Thus, the protein is endocytosed before mitosis and recycles back to the plasma membrane in pIIb but not in pIIa. In pIIa, more Delta/Hrs double-positive vesicles are observed, indicating that the protein enters a late-endosomal pathway (Emery, 2005).

Several observations indicate that passage through recycling endosomes is essential for Delta to signal. In a marrow stromal cell line, OP9, inhibition of recycling endosomes dramatically reduces Delta signaling capacity. Similarly, blocking the recycling pathway by overexpression of a dominant-negative form of Rab11 in SOP cells causes relocalization of Delta into enlarged late endosomes. In Drosophila wing discs, Delta has been postulated to pass through a specific endocytic recycling pathway to acquire signaling capacity (Wang, 2004). Finally, Jafar-Nejad (2005) demonstrates that the Rab11 binding partner Sec15 is required both for Delta trafficking and Notch activation in the SOP lineage. Sec15 is a component of the exocyst and is a Rab11 effector (Zhang, 2004). Although Sec15 is not asymmetric itself, it is conceivable that the higher amounts of GTP bound Rab11 in pIIb increase its activity in delivering Delta to the plasma membrane. A difference between Delta trafficking in pIIa and pIIb has been observed previously (Le Borgne, 2003), but both Delta/Hrs vesicles and total number of Delta vesicles were actually higher in pIIb in these previous experiments. While these earlier experiments analyzed the whole two cell stage, this study focusses on the short time interval right after mitosis where Rab11 is asymmetric. This explains the different outcome and might in fact indicate that pIIb cells switch from an initial phase where Delta is recycled to a later phase where trafficking is regulated by neuralized-dependent endocytosis (Emery, 2005).

Although many cell types in different organisms undergo asymmetric cell division, only one mechanism has been identified so far that directs this important biological process in animals. This mechanism involves the Par proteins, which phosphorylate Lgl on one side and direct cell fate determinants to the opposite side of the cell cortex. Several results indicate that other pathways might exist: in dividing progenitor cells of the mammalian brain, Numb segregates into one of the two daughter cells and is required for lineage specification. However, some of these divisions are asymmetric, although their orientation predicts that Numb would be inherited by both daughter cells. In Drosophila SOP cells, lgl3A overexpression affects both Numb and Neuralized localization but has only a minor influence on the asymmetric outcome of the division. The results indicate that the asymmetric distribution of Rab11 is established through a distinct pathway: (1) Rab11 asymmetry is unaffected in SOP cells overexpressing lgl3A; (2) Rab11 is still asymmetric in dlg mutants where Par proteins do not localize and Numb and Neuralized segregate into both daughter cells; (3) Rab11 asymmetry can be uncoupled from Numb and Neuralized localization by the expression of inscuteable; (4) the events responsible for Rab11 asymmetry seem to occur in the pIIa cell, but none of the known determinants is inherited by this daughter cell. Although the observations could also be explained if Numb or Neuralized would relieve a general suppression of recycling endosome formation in the SOP lineage, this is unlikely since Rab11 asymmetry is unaffected in numb or neuralized mutants. More likely, an unknown factor could act on Nuf or the centrosome in the pIIa cell to prevent Rab11 accumulation. Nuf localization is cell cycle regulated, and a key regulatory component could be missing in pIIa. For example, Nuf is highly phosphorylated and differential activity of a kinase or phosphatase could prevent its pericentriolar localization in the pIIa cell. Homologs of Nuf exist and bind to Rab11 in vertebrates. Their expression pattern has not yet been described but it will be interesting to determine whether these homologs regulate Notch signaling in vertebrates and are responsible for asymmetric cell division in the mammalian brain (Emery, 2005).

dEHBP1 controls exocytosis and recycling of Delta during asymmetric divisions

Notch signaling governs binary cell fate determination in asymmetrically dividing cells. A forward genetic screen identified the fly homologue of Eps15 homology domain containing protein-binding protein 1 (dEHBP1) as a novel regulator of Notch signaling in asymmetrically dividing cells. dEHBP1 is enriched basally and at the actin-rich interface of pII cells of the external mechanosensory organs, where Notch signaling occurs. Loss of function of dEHBP1 leads to up-regulation of Sanpodo, a regulator of Notch signaling, and aberrant trafficking of the Notch ligand, Delta. Furthermore, Sec15 and Rab11, which have been previously shown to regulate the localization of Delta, physically interact with dEHBP1. It is proposed that dEHBP1 functions as an adaptor molecule for the exocytosis and recycling of Delta, thereby affecting cell fate decisions in asymmetrically dividing cells (Giagtzoglou, 2012).

This study describes the identification of dEHBP1 as a novel, positive regulator of Notch signaling in asymmetrically dividing cells in the ESO lineage in Drosophila. In the absence of dEHBP1, external cell types, such as socket and shaft cells, are transformed into internal cell types, i.e., neuron and sheath cells, one of the hallmarks of loss of Notch signaling. EHBP1 has been previously studied in mammalian cell culture systems and in vivo in C. elegans. In mammalian adipocytes, EHBP1 affects endocytosis and recycling of the glucose transporter GLUT4 in the context of insulin signaling, depending on its interaction via the NPF motifs present in its N-terminal region with EHD2 or EHD1, respectively. However, the fly and worm EHBP1 lack the NPF motifs, suggesting that the EHD-EHBP1 interaction may have emerged later in evolution. In C. elegans, EHBP1 was shown to impair rab10-mediated endocytic recycling of clathrin-independent endocytosed cargoes, such GLR-1 glutamate receptor. This study shows that dEHBP1 is required in the exocytosis and recycling of Delta, a ligand of the Notch receptor. Notch signaling defects were not reported in C. elegans ehbp1 mutants. Therefore, it would be interesting to investigate whether EHBP1 and its homologues play an evolutionarily conserved role of EHBP1 in Notch signaling (Giagtzoglou, 2012).

dEHBP1 is a ubiquitous protein that is associated with the plasma membrane, enriched at the lateral and basal surface of pII cells, where it colocalizes with F-actin. Live imaging with mCherry-dEHBP1 and immunofluorescent stainings with anti-dEHBP1 antisera also reveal dEHBP1-positive, punctate, intracellular structures within ESO lineages. An extensive analysis with a diverse array of intracellular markers revealed that these punctae colocalize with Rab8, indicating their exocytic nature. Importantly, in C. elegans, EHBP1 physically interacts and colocalizes with Rab8 and Rab10, and controls the recruitment of Rab10 in recycling endosomal structures. However, in the current studies, overexpression of dominant-negative forms of Rab10 or Rab8 in the ESO lineages as well as thoracic clones of a newly identified Rab8 loss-of-function allele do not confer any cell fate phenotypes. Furthermore, no interaction was detected between dEHBP1 and Rab8 or Rab10 in a yeast two-hybrid analysis. Therefore, it is believed that loss of either Rab8 or Rab10 function does not underlie the dEHBP1 mutant phenotypes that are describe (Giagtzoglou, 2012).

Notably, many key players that affect cell polarity or mark subcellular compartments, including Arm, Rab11, Sec15, and F-actin, are not affected by the loss of dEHBP1. In addition, cell fate determinants Numb and Neuralized are correctly segregated upon asymmetric cell division in dEHBP1 mutant cells. However, loss of dEHBP1 specifically affects the abundance and localization of Spdo, a regulator of Notch signaling in asymmetrically dividing ESO cells, and the exocytosis and trafficking of Delta (Giagtzoglou, 2012).

Spdo facilitates reception of Notch signal at the plasma membrane of the signal-receiving cell. Therefore, accumulation of Spdo in dEHBP1−/− ESO clusters and its presence in the plasma membrane should result in a Notch gain of function, instead of the loss-of-function phenotype that was observed. No effects have been observed of Spdo overexpression upon cell fate acquisition in the ESO lineage. Alternatively, the accumulation of Spdo in the absence of dEHBP1 in these cells may reflect defects in its trafficking and membrane localization, which render the activation of Notch signaling more difficult (Giagtzoglou, 2012).

dEHBP1 mutations cannot suppress the gain of function phenotype of overexpressed ligand-independent, activated Notch intracellular domain. In addition, dEHBP1 does not affect the steady-state levels of Notch protein, as well as its endocytosis. Therefore, it is concluded that dEHBP1 functions at a level upstream of presenilin-mediated S3 cleavage of Notch during reception of the signal. Although it cannot be excluded that dEHBP1 functions in the signal-receiving cell, where it may control the trafficking and localization of Spdo, it is concluded that dEHBP1 also functions in the sending of the signal. This conclusion is based on the fact that dEHBP1 mutations are able to suppress the gain of function of Notch phenotype conferred by the overexpression of DaPKCΔN. Overexpressed constitutively active DaPKCΔN places Spdo at the plasma membrane, enabling the activation of Notch signaling. This study found that upon loss of dEHBP1, Spdo is still found at the plasma membrane under conditions of overexpression of DaPKCΔN. Therefore, the suppression of the overexpression phenotype of DaPKCΔN by loss of dEHBP1 may be because of other defects, such as loss of the ability of Delta to signal. Furthermore, loss of dEHBP1 leads to development of additional neurons despite the concomitant ectopic expression of DeltaR+, a variant of Delta, in clones within pupal nota at 36 h APF. Because the steady-state levels of Delta are not affected in dEHBP1−/− ESO lineages, whether dEHBP1 affects Delta trafficking in the signal-sending cell was examined. Upon loss of dEHBP1, the abundance of Delta at the cell surface is significantly reduced, suggesting that exocytosis is defective. Importantly, most of the remaining extracellular Delta protein localizes at the basal side of the signal-sending cell. This suggests that in addition to affecting exocytosis of Delta, dEHBP1 may also play a role in basal-to-apical trafficking of Delta. This leads to a reduced level of Delta at the signaling interface, which interferes with proper Notch signaling in the cell receiving the signal. Although the results do not exclude a possible role of dEHBP1 in other aspects of Delta trafficking, such as endocytosis, reduced exocytosis of Delta should mask an endocytic defect in the assays. The enrichment of dEHBP1 in the basal and lateral area of the plasma membrane, its colocalization with F-actin at the actin-rich structure at the interface of the pIIa and pIIb cells, the reduction of Delta exocytosis in mutant cells, and the absence of Delta at the interface and the apical surface of the ESO cluster in mutant cells indicate a role of dEHBP1 in the Sec15/Rab11 recycling pathway. Indeed, the colocalization of dEHBP1 and Delta in sec15−/− ESO lineages implies that the exocyst component, Sec15, controls exocytosis of Delta, Spdo, and dEHBP1 to the apical plasma membrane through a common compartment. Because loss of dEHBP1 does not affect the localization of either Rab11 or Sec15, it is concluded that sec15 lies more upstream in the trafficking pathway regulating the localization of multiple components, while dEHBP1 functions during the later stages of intracellular trafficking. Furthermore, the physical interaction between dEHBP1 and Sec15 as well as Rab11 suggest a mechanism how dEHBP1 may regulate the membrane localization of Delta via its interaction with Sec15 and Rab11 at the pII cells interface, even though such interaction was detected under transient overexpression conditions. It is proposed (see Model of dEHBP1 function) that dEHBP1 is an adaptor of the Rab11/Sec15-positive, Delta-bearing vesicles required for exocytosis (Giagtzoglou, 2012).

The identification of dEHBP1 provides further compelling evidence that the exocytosis and recycling pathway of Delta during asymmetric divisions is tightly regulated. The recycling pathway of Delta appears to be context dependent, i.e., it is not required in all cells that use Notch signaling. Still, the discovery of dEHBP1 as a novel player in Notch signaling provides the opportunity to test its role in Notch-related neurobiological behaviors, such as sleep and addiction, as well as in Notch-related diseases, as for example in Wiskott-Aldrich syndrome, an immunodeficiency characterized by abnormal differentiation and function of T cell lineages. Furthermore, because the anthrax toxins lethal factor (LF) and edema factor (EF) inhibit the Sec15/Rab11-dependent Delta-recycling pathway in flies and endothelial cells, it would be interesting to hypothesize whether they target dEHBP1 to mediate their toxicity (Giagtzoglou, 2012).

Bearded family members inhibit Neuralized-mediated endocytosis and signaling activity of Delta in Drosophila

Endocytosis of Notch receptor ligands in signaling cells is essential for Notch receptor activation. In Drosophila, the E3 ubiquitin ligase Neuralized (Neur) promotes the endocytosis and signaling activity of the ligand Delta (Dl). This study identifies proteins of the Bearded (Brd) family as interactors of Neur. Tom, a prototypic Brd family member (see Bearded), inhibits Neur-dependent Notch signaling. Overexpression of Tom inhibits the endocytosis of Dl and interferes with the interaction of Dl with Neur. Deletion of the Brd gene complex results in ectopic endocytosis of Dl in dorsal cells of stage 5 embryos. This defect in Dl trafficking is associated with ectopic expression of the single-minded gene, a direct Notch target gene that specifies the mesectoderm. It is proposed that inhibition of Neur by Brd proteins is important for precise spatial regulation of Dl signaling (Bardin, 2006).

In order to identify regulators of Neur, a yeast two-hybrid screen was conducted using as bait the conserved central domain of Neur that comprises the two Neur homology repeats (NHRs). Eighty-four cDNAs were identified, of which 62 encoded members of the Brd gene family: Ocho (39 times [×]), Tom (12×), m4 (7×), Brd (2×), and Bob (2×). Interaction of Neur with m6, mα, and m2 was tested directly. The m6 and mα proteins, but not m2, interacted with Neur in this assay. Tom also interacts with full-length Neur. It is concluded that all Brd family members, with the exception of m2, interact with Neur (Bardin, 2006).

Most Brd proteins share four conserved motifs: (1) a lysine-rich N-terminal region predicted to form an amphipathic α helix; (2) a short NxxNExLE motif, found in all proteins except m2; and (3 and 4) two C-terminal motifs found in only a subset of the Brd family members. Motifs 2 and 3 are the most-conserved motifs among insect Brd homologs. Because Brd and Bob lack motifs 3 and 4, these motifs cannot be strictly required for interaction with Neur. Additionally, clones encoding N-terminally truncated Tom proteins (amino acids 75-58, 58-158, 54-158, and 29-158 of Tom) were recovered in the screen, indicating that the N-terminal region predicted to form an amphipathic helix (amino acids 26-43 of Tom) is not necessary for interaction with Neur. Thus, motif 2 is the only conserved motif present in all the clones that interact with Neur. It is also absent from m2, which is the only family member that does not interact with Neur and fails to inhibit Notch when overexpressed. Deletion and point mutation analysis of Tom further demonstrates that motif 2 is important for Neur binding: (1) truncated versions of Tom lacking motif 2 did not interact with Neur in the two-hybrid assay; (2) internal deletion of this motif strongly impaired interaction in the two-hybrid assay; (3) alanine substitution of either 11 or 4 residues of motif 2 also impair interaction. It is concluded that motif 2 is important for Neur binding (Bardin, 2006).

Activation of DSL signaling by Neur is regulated by multiple mechanisms. A first level of regulation operates at the transcriptional level, both along the DV axis in the early embryo and within proneural clusters during imaginal development. A second level of regulation is seen during asymmetric division of the SOP with the unequal partitioning of Neur at mitosis. This study identifies a third level of regulation based on the inhibition of Neur by Brd family members. All Brd family members (with the exception of m2) interact in the yeast two-hybrid assay with the E3 ubiquitin ligase Neur. The overexpression of Brd genes specifically inhibits Neur-dependent Notch signaling events and leads to a defect in Dl endocytosis. Conversely, loss of the Brd-C that contains six out of the ten Brd genes results in ectopic Dl endocytosis and ectopic expression of the Notch target gene sim in the early embryo. Finally, physical interaction of Tom with Neur appears to inhibit the interaction of Neur with its substrate Dl. A model is proposed whereby proteins of the Brd family antagonize Neur-mediated Dl signaling by inhibiting the interaction of Dl with Neur (Bardin, 2006).

Precise positioning of the mesectoderm results from the integration of different activities that are more broadly distributed along the DV axis. The DV gradient of nuclear Dorsal is interpreted to establish large domains of gene expression. The twist gene is expressed in a large ventral territory that encompasses the mesoderm, whereas the expression of the snail gene becomes restricted to the mesoderm. Twist and Dorsal activate the expression of the sim gene whereas Snail represses it. Neur-dependent Dl signaling in the mesoderm is thought to further restrict sim expression to cells in direct contact with the mesoderm. The signaling activity of Dl is thought to be restricted to the mesoderm because its endocytosis is tightly restricted to the mesoderm in stage 5 embryos. While transcriptional regulation of neur in ventral cells likely contributes to this spatial regulation, it cannot on its own account for the mesoderm-specific regulation of Dl endocytosis. Indeed, high levels of transcripts are detected in ventral cells outside the mesoderm and low levels of transcripts are detected all around the embryo. This suggests that a posttranscriptional inhibitory mechanism exists to ensure that Neur is not active outside the mesoderm. This study shows that Brd proteins inhibit Neur-mediated Dl endocytosis and Notch signaling in nonmesodermal cells. It is also shown that ectopic expression of Tom inhibits the endocytosis of Dl in the mesoderm. This suggests that the repression of the expression of Brd-C genes in the mesoderm is important for Neur to be active in this tissue. Inhibition of Tom expression (and possibly of the other Brd-C genes) in the mesoderm depends on the mesoderm-specific repressor Snail. Accordingly, the ectopic expression of Brd genes in ventral cells of snail mutant embryos may explain the loss of Dl endocytosis and Notch activation that was previously observed in these embryos. It is therefore suggested that the Brd-C genes represent the hypothesized Snail target gene X proposed to act as a negative regulator of Notch signaling and Dl endocytosis (Morel, 2003). Thus, the sharp boundary of Snail expression appears to define the ventral limit of Brd family gene expression, hence the dorsal limit of Neur activity and Dl signaling. In summary, these data support a model whereby the Brd genes prevent ectopic Notch activation in the early embryo and contribute to DV patterning by restricting the mesectoderm territory to a single row of cells (Bardin, 2006).

The function of the Brd genes is probably not restricted to the early embryo. Indeed, several Brd genes are also strongly expressed during early neurogenesis in the embryo as well as in the proneural clusters of the eye, leg, and wing imaginal discs. Proneural cluster expression of the Brd genes may be important to restrict, in space and/or time, the activity of Neur during the process of SOP determination. While Neur appears to be primarily expressed in the presumptive SOP, there is also evidence that Neur may also be expressed at low levels in non-SOP cells. In particular, low-level expression of Neur in non-SOP cells is occasionally seen using neurP72Gal4. It is hypothesized that Brd may act to antagonize this low level of Neur activity in proneural cluster cells. Interestingly, the expression of the neur gene in SOPs is accompanied by the transcriptional repression of the gene by Su(H) in SOPs. The expression of other Brd family genes is excluded from SOPs, suggesting that they may also be repressed by Su(H). Conversely, the positive regulation of Brd gene expression by Notch in non-SOP cells correlates with a loss in Dl signaling activity in these cells. It is therefore speculated that the Brd genes contribute to amplify an initially weak difference in Dl signaling activity between presumptive SOP and non-SOP cells (Bardin, 2006).

The role proposed for the Brd genes in lateral inhibition remains to be investigated. It was found that the deletion of the Brd-C is largely embryonic lethal. However, a few homozygous Brd-C1 escaper flies are observed. These Brd-C1 flies show no detectable defects in bristle density. While this observation indicates that the Brd-C does not play an essential role in the process of SOP selection, the possibility remains that the and m4 genes act redundantly with genes of the Brd-C in this process (Bardin, 2006).

This study shows that all Brd family members (with the exception of m2) interact with Neur in the yeast two-hybrid assay. Interaction of Tom with Neur was further confirmed by coimmunoprecipitation experiments. Importantly, interaction of Tom with Neur correlates with a decrease in the amount of Dl immunoprecipitated by Neur, without affecting the levels of Neur and/or Dl. It is noted, however, that TomΔ2, which interacts weakly with Neur, can still inhibit interaction of Dl with Neur in this assay. The ability of Tom to decrease the Dl-Neur binding in this assay has led to a model whereby Brd family members antagonize Neur-mediated Dl signaling by inhibiting the Neur-Dl interaction. This model is consistent with the observations that overexpression of Tom has no effect on Neur protein levels. It is also consistent with the observation that Tom blocks the activity of NeurC701S. The latter may act in a dominant-negative manner by titrating DSL ligands. Accordingly, Tom could prevent NeurC701S from titrating DSL ligands. Similarly, the failure of Tom to suppress the wing phenotype induced by Mib1C1205S is consistent with the observation that Tom does not bind Mib1 and cannot, therefore, prevent Mib1C1205S from titrating DSL ligands. Whether Brd family members inhibit Neur by competing with Dl for overlapping binding sites remains to be investigated (Bardin, 2006).

These studies have focused on the interaction between Neur and a single Brd family member for the sake of consistency. Tom was chosen because (1) it includes all four conserved motifs present in the various Brd family members; (2) it is the Brd gene that aligns best with the single Anopheles Brd gene; (3) its overexpression gives a strong gain-of-function phenotype; and (4) it is expressed at high levels in stage 5 embryos. Whether all Brd family members similarly act by inhibiting the Neur-Dl interaction remains to be fully investigated. Because all Brd family members (with the exception of m2) have been shown to inhibit Neur-mediated Notch signaling, it is likely that all Brd family members similarly inhibit Neur. This in turn raises the question of the role of the two additional conserved motifs found at the C terminus of Ocho, Tom, mα, m4, and m6 that are also conserved in the single Bombyx and Anopheles Brd homologs (Bardin, 2006).

While Neur has homologs in vertebrates and Xenopus Neur has been suggested to regulate Dl signaling during early neurogenesis, no obvious homologs of the Brd genes are detectable in vertebrate sequenced genomes. This does not, however, exclude the possibility that vertebrate genes encoding Brd-like inhibitors exist. Indeed, motif 2 of Brd may be too short to reliably detect possible Brd homologs in vertebrate genomes by sequence alignments (Bardin, 2006).

This study has shown that Brd family members interact with Neur and block Neur-mediated Dl endocytosis. The activity of the Brd-C is required to spatially restrict Dl signaling along the DV axis in the early embryo (Bardin, 2006).

Dorsal-ventral pattern of Delta trafficking is established by a Snail-Tom-Neuralized pathway

The intracellular trafficking of the Notch ligand Delta plays an important role in the activation of the Notch pathway. This study addresses Snail-dependent regulation of Delta trafficking during the plasma membrane growth of the mesoderm in the Drosophila embryo. Delta is retained in endocytic vesicles in the mesoderm but expressed on the surface of the adjacent ectoderm. This trafficking pattern requires Neuralized. A protocol based on chromosomal deletion and microarray analysis has led to the identification of tom as the target of snail regulating Delta trafficking. Snail represses Tom expression in the mesoderm and thereby activates Delta trafficking. Overexpression of Tom abolishes Delta trafficking and signaling to the adjacent mesoectoderm. Loss of Tom produces mesoderm-type Delta trafficking in the entire blastoderm epithelium and an expansion of mesoectoderm gene expression. It is proposed that Tom antagonizes the activity of Neuralized and thus establishes a sharp mesoderm-mesoectoderm boundary of Notch signaling (De Renzis, 2006).

The Neuralized-dependent trafficking of Delta in the signal-sending cell is required for Notch activation in the receiving cell. Therefore, the activity of Neuralized must be tightly controlled between the signal-sending and signal-receiving cell in order to ensure the correct pattern of Delta trafficking and signal polarity. This study has followed the snail-mediated modulation of Delta trafficking during the cellularization of the Drosophila embryo. The concomitant formation of cell membranes and activation of zygotic transcription has offered some unique advantages for the experiments described in this work. Most importantly, the growth of the plasma membrane is timed with the zygotic expression of snail and neuralized and thus allows a precise staging protocol (De Renzis, 2006).

The experiments demonstrate that snail regulates the mesoderm-specific trafficking of Delta by repressing the expression of tom, and presumably of the other brd genes. In the ectoderm, where the brd class genes are expressed and snail is not, Delta and Notch Extra-Cellular Domain (NECD) have a predominantly cell surface localization. Obvious endocytic vesicles containing these proteins do not accumulate in ectodermal cells. Such vesicles do form in the mesoderm where Snail represses tom expression, and in tom−/− embryos, where the NECD-Delta vesicles extend into the ectoderm. The vesicular trafficking of Delta and NECD characteristic of mesodermal cells requires the ubiquitin ligase Neuralized. Overexpression of Tom recapitulates the loss of function phenotype of neuralized, consistent with the view that Tom may normally function by opposing the role of Neuralized in Delta trafficking. Neuralized expression is dynamic and extends to the lateral region of the embryo, beyond the mesoderm-ectoderm boundary. Thus, on its own it cannot explain the restriction of vesicles to the mesoderm. It is proposed instead that Tom creates a functional boundary for Neuralized by suppressing its activity in the ectoderm. In agreement with this model, in tom−/− embryos the expression of the mesoectoderm gene sim is extended dorsally (De Renzis, 2006).

The expression of sim is regulated by the maternal Dorsal nuclear gradient that directly or indirectly specifies all ventral cell fates. The mechanisms that precisely position sim expression to one row of cells, however, are not completely understood. sim can respond to the Dorsal gradient and can in principle be expressed in the entire ventral region of the embryo. Recent studies suggest that Notch signaling restricts the expression of sim to the mesoectoderm by relieving Suppressor of Hairless [Su(H)]-mediated repression of sim. Su(H) is uniformly distributed throughout the early embryo and represses sim expression in the ectoderm (De Renzis, 2006).

The precision of sim expression and its one cell diameter reflect the bias that zygotic expression of tom introduces on maternal Notch signaling in that region of the embryo. If the mesoderm cells that do not express Tom are the only cells that retain Neuralized activity and can internalize Delta ligand, they might be the only cells capable of sending signal. The snail repression will allow sim expression outside the mesoderm, and thus only those cells could be effective signal recipients. According to this model, the last snail-expressing cell of the mesoderm becomes the signal-sending cell and its immediate dorsal neighbor becomes the signal-receiving cell; i.e., the mesoectoderm. In the absence of Tom, the dynamic expression of Neuralized, which extends dorsally, would make more cells competent to send Notch signal. Indeed, the dorsal expression of sim is more pronounced at the end of mesoderm invagination at the time when the expression of Neuralized has extended to the neuroectoderm (De Renzis, 2006).

The molecular mechanisms by which Tom functions are most likely related to its interaction with Neuralized; this has been demonstrated in a yeast-two hybrid genomic screening (Giot, 2003). Tom may inhibit the ubiquitin ligase activity of Neuralized or it could compete for the interaction between Delta and Neuralized. Future work will be necessary to discriminate between these two possibilities. It will also be important to test the activity of the other Brd family members in the regulation of Neuralized activity. At least eight bearded-like genes (m2, m4, m6, , bob, brd, tom, and ocho) have been identified in the Drosophila genome. An interesting possibility is that different genes in this family may have different effects on Delta trafficking. Any difference may provide important clues into the regulation of the Notch pathway (De Renzis, 2006).

The glycosyltransferase Fringe promotes Delta-Notch signaling between neurons and glia, and is required for subtype-specific glial gene expression

The development, organization and function of central nervous systems depend on interactions between neurons and glial cells. However, the molecular signals that regulate neuron-glial communication remain elusive. In the ventral nerve cord of Drosophila, the close association of the longitudinal glia (LG) with the neuropil provides an excellent opportunity to identify and characterize neuron-glial signals in vivo. This study found that the activity and restricted expression of the glycosyltransferase Fringe (Fng) renders a subset of LG sensitive to activation of signaling through the Notch (N) receptor. This is the first report showing that modulation of N signaling by Fng is important for CNS development in any organism. In each hemisegment of the nerve cord the transcription factor Prospero (Pros) is selectively expressed in the six most anterior LG. Pros expression is specifically reduced in fng mutants, and is blocked by antagonism of the N pathway. The N ligand Delta (Dl), which is expressed by a subset of neurons, cooperates with Fng for N signaling in the anterior LG, leading to subtype-specific expression of Pros. Furthermore, ectopic Pros expression in posterior LG can be triggered by Fng, and by Dl derived from neurons but not glia. This effect can be mimicked by direct activation of the N pathway within glia. These genetic studies suggest that Fng sensitizes N on glia to axon-derived Dl and that enhanced neuron-glial communication through this ligand-receptor pair is required for the proper molecular diversity of glial cell subtypes in the developing nervous system (Thomas, 2007).

This study identified Fng as a means by which a specific subtype of glia, the anterior LG, are made sensitive to N activation, evidence was provided that Dl, expressed on axons, activates N signaling in these glia leading to subtype-specific gene expression. Fng is required for maintenance of Pros expression in the anterior LG, which can also be blocked by antagonism of the N pathway with no effect on their survival or positioning. This is in contrast with studies of pros mutants, which found a role for Pros earlier in CNS development in establishing glial cell number. The role of Pros in mature LG is poorly understood, but it has been proposed to retain mitotic potential in these cells for use in repair or remodeling of the nervous system in subsequent larval or adult stages. It will be important to determine the consequences of lost Pros expression from mature anterior LG, and whether additional features and functions of the anterior LG are controlled by N signaling from axons (Thomas, 2007).

The importance of glycosylation for N function has been demonstrated in vivo. The addition of O-linked fucose to EGF repeats in the N extracellular domain is essential for all N activities and is mediated by O-fucosyltransferase-1 (O-fut1). By contrast, Fng is selectively used in specific developmental contexts, and has been best studied in the formation of borders among cells in developing imaginal tissues. Fng catalyzes the addition of GlcNac to O-linked fucose, to which galactose is then added. The resulting trisaccharide is the minimal O-fucose glycan to support Fng modulation of Notch signaling. Fng activity reduces the sensitivity of N for the ligand Ser but increases its sensitivity for Dl. By contrast with imaginal discs, in which modulation of N sensitivity to both ligands appears to be important, loss of Fng in LG resulted in reduced N activation only, consistent with reduced response to Dl. Expression of Pros in LG can be triggered by Dl derived from neurons but not glia, and this effect can be mimicked by direct activation of the N pathway within glia. Genetic experiments implicate neuron-derived Dl as the relevant N ligand for Pros expression in anterior LG, consistent with the ability of Fng to sensitize N to signaling by Dl. Enriched Fng expression in the anterior LG probably renders them differentially sensitive to sustained N signaling from Dl-expressing axons (Thomas, 2007).

The final divisions of the six LG precursors that give rise to 12 LG are thought to be symmetric, with low levels of Pros first distributed evenly between sibling cells after division. However, Pros is maintained and in fact upregulated in the anterior LG, and downregulated in sibling LG that migrate posteriorly. fng transcripts first appear to be expressed in all LG, then become enriched in the anterior LG and reduced in the posterior LG. It is speculated that refinement of fng expression may involve a positive feedback mechanism to consolidate and enhance N signaling in the anterior LG, since preliminary evidence suggests that N signaling can positively influence fng expression in the LG (Thomas, 2007).

Like Pros, Glutamine synthetase 2 (Gs2) is specifically expressed in the anterior LG but not posterior LG, indicating that these are functionally distinct glial subtypes with respect to their ability to recycle the neurotransmitter glutamate. The specificity of N signaling for Pros but not Gs2 indicates that N signaling is unlikely to influence cell fate decisions in the LG lineage and that Fng is unlikely to be the primary determinant of anterior versus posterior LG identity. Rather, Fng probably serves to consolidate this distinction through sustained N signaling (Thomas, 2007).

NICD is a potent activator of Pros expression in the posterior LG. This leads to a consideration of what factors limit Pros expression to the anterior LG in wild-type animals, since posterior LG are indeed capable of expressing Pros in response to constitutive N activity. (1) Based on analysis of fng mutants and Fng misexpression, it is proposed that Fng is a major determinant. The finding that misexpression of Fng causes ectopic Pros in posterior LG supports the argument that Dl-expressing axons do not contact the anterior LG only. It is likely that they make contact with at least some of the posterior LG. Therefore, in wild-type animals, in which Fng is reduced on posterior LG, contact from the subset of Dl axons is alone not sufficient to drive Pros expression. (2) Misexpression of Dl in all postmitotic neurons led to ectopic expression of Pros in posterior LG, indicating that the restricted expression of Dl on a subset of neurons also limits N activation. (3) N appears to be expressed in most or all LG, though it was also found that overexpression of full-length N caused ectopic expression of Pros. From these data a threshold model is proposed for N activation in LG that invokes a combination of factors, including Fng-regulated N sensitivity, exposure of N to ligand, N expression levels, and perhaps others. Increasing any of these factors can provide sufficient signaling for ectopic Pros induction in posterior LG. In wild-type embryos, these factors are also likely to combine with one another in the anterior LG to achieve supra-threshold N signaling and sustained Pros expression during normal development (Thomas, 2007).

Signaling through N is important for glial cell development in Drosophila, although it is context-dependent. Both an embryonic sensory lineage and the subperineurial CNS glial lineage utilize N activation to promote Gcm expression and glial fate. By contrast, in the sensory organ of adult flies, antagonism of N leads to Gcm expression in the glial precursor cell. In vertebrates, signaling through Notch receptors promotes the differentiation of peripheral glia, Müller glia, radial glia and mature oligodendrocytes. A Fng ortholog, lunatic fringe, is expressed in the developing mouse brain in a pattern consistent with glial progenitors. It will be interesting to determine whether Fng-related proteins in vertebrates have a role in glial cell differentiation, and whether they too can modulate N sensitivity and the context of N signaling between neurons and glia (Thomas, 2007).

The NHR1 domain of Neuralized binds Dl and mediates Dl trafficking and N signaling

Notch signaling, crucial to metazoan development, requires endocytosis of Notch ligands, such as Delta and Serrate. Neuralized is a plasma membrane-associated ubiquitin ligase that is required for neural development and Delta internalization. Neuralized is comprised of three domains that include a C-terminal RING domain and two neuralized homology repeat (NHR) domains. All three domains are conserved between organisms, suggesting that these regions of Neuralized are functionally important. Although the Neuralized RING domain has been shown to be required for Delta ubiquitination, the function of the NHR domains remains elusive. This study shows that neuralized1, a well-characterized neurogenic allele, exhibits a mutation in a conserved residue of the NHR1 domain that results in mislocalization of Neuralized and defects in Delta binding and internalization. Furthermore, a novel isoform of Neuralized is described; it is recruited to the plasma membrane by Delta and this is mediated by the NHR1 domain. Finally, it is shown that the NHR1 domain of Neuralized is both necessary and sufficient to bind Delta. Altogether, these data demonstrate that NHR domains can function in facilitating protein-protein interactions and in the case of Neuralized, mediate binding to its ubiquitination target, Delta (Commisso, 2007; full text of article).

Neuralized contains a phosphoinositide-binding motif required downstream of ubiquitination for Delta endocytosis and Notch signaling

The Notch signaling pathway, which plays a critical role in cell-fate decisions throughout development, is regulated by endocytosis of both the ligand and receptor. Endocytosis of the Drosophila ligands, Delta and Serrate, is required in the signaling cell for signal initiation and requires one of two ubiquitin ligases, Neuralized or Mind bomb. Through in vitro binding assays an interaction has been identified between Neuralized and phosphoinositides, modified membrane lipids that mediate membrane trafficking and signaling. Interactions between phosphoinositides and Neuralized contribute to the membrane localization of Neuralized in the absence of Delta; the phosphoinositide-binding motif is required for Neuralized to endocytose Delta downstream of Delta ubiquitination. Lastly, evidence is provided that this interaction may also be important for vertebrate Neuralized function. These results demonstrate that, through interactions with Neuralized, phosphoinositides may regulate Delta endocytosis and, by extension, Notch signal transduction (Skwarek, 2007).

This study has shown that Neur interacts with PIPs in vitro through a lysine-rich region in the N terminus, and that this PIP-binding motif mediates localization to the cell surface of S2 cells in the absence of Dl. Although the PIP-binding residues are not required for Neur to bind Dl, these residues are required for Neur function during embryonic neurogenesis and, specifically, for Dl internalization. Interestingly, Neur-mediated ubiquitination does not appear to be reduced in the absence of the PIP-binding motif, suggesting that these residues, and possibly PIP binding, are required downstream of Dl ubiquitination. Lastly, evidence is presented to suggest that PIP binding may also be important for vertebrate Neur1 function. Taken together, these data demonstrate that the PIP-binding motif in Neur plays an important role in regulating ligand endocytosis downstream of ligand ubiquitination, and that it is important for the initiation of Notch signaling (Skwarek, 2007).

A role for PIPs in the signal-receiving cell has been demonstrated in previous work. For example, increases in plasma membrane PIP levels due to deletion of the phosphocholine cytidylyltransferase cct1 enhances Notch loss-of-function phenotypes due to increased endocytic activity and, consequently, lower levels of cell-surface receptor (Weber, 2003). In addition, three recent studies have demonstrated an important role of the phospholipid-binding protein Lethal giant discs (Lgd) in the regulation of Notch signaling (Childress, 2006; Gallagher, 2006, Jaekel, 2006). Lgd appears to regulate Notch trafficking at a step downstream of Hrs-dependent sorting, and PIP binding is required for this function. Lastly, overexpression of a kinase-dead version of the Drosophila class II PI-3-kinase, PI3K_68D, phenocopies and enhances Notch loss of function, although it has yet to be determined if this is due to disruption of signal initiation or transduction (MacDougall, 2004). The current data are the first to suggest a specific role for PIPs in the regulation of Notch signaling in the signal-sending cell (Skwarek, 2007).

Neur binds PIPs promiscuously in vitro; however, cell culture studies demonstrate that the PIP-binding motif contributes to constitutive plasma membrane localization, suggesting an important role for the interaction between Neur and PI(4,5)P2. This is supported by the observation that expression of an isolated PH domain from PLC-δ that has been well demonstrated to specifically bind PI(4,5)P2 promotes the redistribution of Neur from the plasma membrane into large intracellular puncta. Although many proteins display promiscuous binding in vitro, including most yeast PH domains, the actual in vivo localization can be mediated through a combination of PIP, protein-protein, and nonspecific electrostatic interactions. Consistent with this, it was shown that, in the absence of the PIP-binding motif, Neur can still interact with Dl, and this interaction is sufficient to recruit Neur to the cell surface. Despite this redundant mechanism for localization, the PIP-binding motif is essential for Neur function during embryonic neurogenesis since the expression of transgenes containing mutations that disrupt PIP binding in vitro cannot rescue neurogenesis in neur−/− embryos. This appears to be due to an inability of Neur to trigger Dl endocytosis in the absence of the PIP-binding motif, suggesting an important role for the interactions mediated by this motif in Neur function in vivo (Skwarek, 2007).

Given that the results demonstrate that in vivo, in the presence of Dl, the PIP-binding mutant is capable of localizing to the plasma membrane and interacting with Dl, it was hypothesized that the PIP-binding residues are not required for the localization of Neur. It remains possible, however, that it was not possible to detect subtle differences in localization. For example, there is evidence for localized differences in plasma membrane microdomains, and the sites of endocytic internalization are probably not of uniform identity. While the specific role the PIP-binding residues may be playing in Neur function is not known, it is possible that they mediate interactions with additional endocytic effector molecules, and they may also be involved in localizing Neur to a specialized membrane subdomain that is important for signaling competent endocytosis (Skwarek, 2007).

The mechanism through which Dl endocytosis activates Notch is unknown. One model proposes that the endocytosis of Notch-bound Dl provides a mechanical force that is required for subsequent cleavage of Notch. A second model suggests that, after endocytosis, Dl is somehow modified to make it more active in a process that depends on Epsin. This modification may involve the recycling of Dl, as the receptor-binding domain of Dl fused to the low-density lipoprotein receptor sorting and recycling motif partially bypasses the requirement for Epsin (though not for ubiquitination) in Notch signal initiation. In support of this model, recycling endosomes have been shown to be required for Dl activity during Drosophila sense-organ development and in mammalian cell culture. The data do not support one model over the other; however, they do highlight the importance of the initial endocytosis step in Notch signaling, which is dependent on the PIP-binding motif in Neur. Due to the fact that Dl trafficking is blocked at endocytosis in Neur PIP-binding motif mutants, it cannot be directly assessed whether this motif, and by extension interactions with PIPs, may play additional roles in Dl trafficking downstream of endocytosis. Given that Neur interacts with multiple PIPs in vitro, and that the requirement for the PIP-binding motif appears to be downstream of Dl ubiquitination, it is possible that an interaction between Neur and phosphoinositides could be involved in additional trafficking steps, as PIPs play diverse and essential roles throughout endosomal trafficking. Consistent with this, colocalization of ectopically expressed Dl and Neur is seen in both Hrs- and Rab11-positive endosomes in Drosophila Kc cells and in vivo, suggesting a possible role for Neur in postendocytic trafficking steps. However, the presence of endogenous Neur in vivo and in Kc and S2 cells has thus far complicated efforts to further address additional requirements for Neur in Dl trafficking. Interestingly, compared to wing discs lacking exogenous Neur, ectopic expression of Neur lacking the PIP-binding motif results in higher levels of Dl both at the cell surface and in internal vesicles, suggesting that expression of this protein may interfere with the normal trafficking of Dl to the lysosome (Skwarek, 2007).

Recently, it has been demonstrated that mouse Neur2 does not appear to play a role in ligand internalization, but is involved in targeting endocytosed Dl to Hrs-positive vesicles (Song, 2006), providing further evidence that Neur is involved in additional trafficking steps. Interestingly, mNeur2 does not contain the N-terminal domain predicted to interact with PIPs in mNeur1, and instead it looks more like the Drosophila Neur isoform, NeurPC, that displays similar localization properties to Neur5Q (Commisso, 2007; Skwarek, 2007).

Mouse Neur1 contains a sequence in its N-terminal region that is similar to a conserved motif present in FYVE domains that has been shown to interact directly with the PI phosphate group of PI3P. It is predicted that this region is required for PIP binding in mNeur1, as it is absolutely conserved in all vertebrate Neur1 protein sequences available, and this study has shown that mutation of 3 residues in this region disrupts PIP binding in vitro. This suggests that interactions between Neur and PIPs may be involved in the function of Neur in both Drosophila and vertebrates, and it provides an interesting example of a protein in which important interactions are conserved despite large sequence changes during evolution (Skwarek, 2007).

To date, this study has assessed the overall functional consequences of loss of the PIP-binding motif during embryonic neurogenesis. Whether the interaction mediated by the PIP-binding residues is required during other Neur-mediated signaling events remains to be determined. For example, it will be interesting to see whether the PIP-binding motif is required for Neur to regulate Notch signaling during the asymmetric divisions of the sense-organ precursor cells, and whether asymmetries in PIP binding may play a role in the asymmetric localization of Neur into a single daughter of the dividing sense-organ precursor (Skwarek, 2007).

Without strict regulation, aberrant Notch signaling leads to cancer and other developmental diseases. Neur and Mib have emerged as crucial regulators of the Notch signaling pathway that are required for receptor endocytosis and signal induction, and they may also play a role in cis inhibition by ligand/receptor interactions within the same cell (Glittenberg, 2006). This study has identified PIPs as interacting with Neur in vitro, and has provided several pieces of evidence that they may also be physiologically relevant ligands in vivo. The PIP-binding motif that was identified is important for Neur function during embryonic neurogenesis, and it was demonstrated that this motif is required for Neur-mediated Dl endocytosis downstream of ligand ubiquitination. It is still unknown what this novel role for Neur may be, and whether PIPs are the critical ligands required for this function. It will be interesting to see whether Mind bomb may also play a role downstream of ligand ubiquitination and to clarify the role of phosphoinositides during ligand trafficking and Notch signaling throughout development (Skwarek, 2007).

Drosophila melanogaster auxilin regulates the internalization of Delta to control activity of the Notch signaling pathway

Mutations were isolated in the Drosophila homologue of auxilin, a J-domain-containing protein known to cooperate with Hsc70 in the disassembly of clathrin coats from clathrin-coated vesicles in vitro. Consistent with this biochemical role, animals with reduced auxilin function exhibit genetic interactions with Hsc70 and clathrin. Interestingly, the auxilin mutations interact specifically with Notch and disrupt several Notch-mediated processes. Genetic evidence places auxilin function in the signal-sending cells, upstream of Notch receptor activation, suggesting that the relevant cargo for this auxilin-mediated endocytosis is the Notch ligand Delta. Indeed, the localization of Delta protein is disrupted in auxilin mutant tissues. Thus, these data suggest that auxilin is an integral component of the Notch signaling pathway, participating in the ubiquitin-dependent endocytosis of Delta. Furthermore, the fact that auxilin is required for Notch signaling suggests that ligand endocytosis in the signal-receiving cells needs to proceed past coat disassembly to activate Notch (Hagedorn, 2006).

To further understand the roles of endocytosis in cell signaling during animal development, loss-of-function mutations were generated in auxilin from an F2 complementation screen in D. melanogaster. From this screen, six loss-of-function mutations in were isolated auxilin. In support of previous biochemical data, it was found that auxilin interacts genetically with Hsc70 and clathrin. In addition, the location of the genetic lesion in one of the alleles suggests that the putative lipid binding tensin domain plays a role in regulating clathrin function. The auxilin mutations also interact specifically with Notch and disrupt several Notch-mediated processes, suggesting that auxilin participates in an endocytic event critical for regulating the Notch cascade. Indeed, this analysis suggests that D. melanogaster auxilin is required for internalization of the Dl proteins that are critical for activating the Notch receptor (Hagedorn, 2006).

This study isolated and characterized mutants in Drosophila auxilin. In support of its well-known biochemical role in Hsc70-mediated disassembly of CCVs, this dAuxI670K mutation was shown to interact genetically with Hsc70-4 and the Clc. The in vivo link between auxilin and Hsc70 is further strengthened by the observation that a nonsense mutation (dAuxW1150X) near the very COOH terminus, where the J-domain is located, can strongly disrupt dAux function. These genetic observations are in agreement with in vivo analyses of auxilin function from other systems, which showed that clathrin function was disrupted in auxilin-deficient cells. In addition, genetic data of dAuxI670K suggest a relevance of the tensin-related domain, a putative lipid binding domain, in clathrin-mediated endocytosis, despite the fact that it does not appear to be required for catalyzing the dissociation of clathrin triskelions from CCVs in vitro (Hagedorn, 2006).

It has been suggested that, in addition to disassembling clathrin coats, auxilin participates in the dynamin-mediated constriction during CCV formation. However, subcellular localization analysis did not reveal dAux proteins colocalizing with clathrin at the cell periphery. Instead, most auxilin proteins appear to be associated with intracellular structures, in regions devoid of clathrin staining. This lack of overlap between dAux and Clc seems more consistent with the notion that auxilin is required for the dissociation of clathrin coats from CCVs under physiological conditions (Hagedorn, 2006).

Analysis of dAux clearly suggests that auxilin plays an important role in the Notch cascade in multiple Notch-dependent processes. Supportive evidence comes from the strong genetic interactions between dAux and Notch and the phenotypic similarities ranging from eye and wing development to neural development during embryogenesis. Moreover, the in vivo function of auxilin in the Notch signaling cascade seems specific, since dAuxI670K has no dominant effect on the phenotype caused by the overexpression of EGFR. Together, these observations argue that dAux acts specifically as a general component in the Notch cascade (Hagedorn, 2006).

Analysis from several groups has suggested that ligand internalization is a key event for Notch activation. The neurogenic phenotypes exhibited by dAuxI670K tissues and other genetic data further support this notion. The distribution of phenotypically mutant clusters in a genotypically mutant clone suggests that dAux acts noncell autonomously. In addition, the epistasis analysis places dAux function upstream of an activated form of Notch. Based on the phenotypic resemblance of dAuxI670K to those reported for neur and lqf, it is suspected that dAux functions along with neur and lqf in the ubiquitin-dependent endocytic pathway in the signal-sending cells (Hagedorn, 2006).

The identification of dAux as a critical factor in Notch ligand endocytosis has strong implications on the mechanism of Notch activation. Unlike Neur and Lqf, which are postulated to tag and sequester cargos into vesicles, auxilin is thought be involved in disassembly of clathrin coats. Thus, the revelation of dAux as another component in this pathway suggests that Dl-containing endocytic vesicles need to proceed past the clathrin uncoating step to activate Notch. One possible mechanism is that recycling of Dl is a prerequisite to form signaling-competent Dl-containing exosomes, although the presence of these structures under physiological conditions remains to be demonstrated. Alternatively, it may be that, as previously proposed, the DSL ligand is not signaling competent before endocytosis but is 'activated' during transit through recycling compartments. Indeed, the transit through Rab11-positive recycling endosomes has been suggested as a critical step for Dl activity. However, although Dl appears to colocalize extensively with coalesced perinuclear Rab11-positive structures in the sensory organ precursor cells, the current analysis found little spatial overlap between Rab11 and Dl in cells near the furrow. One possible explanation for this apparent difference is that the transit of Dl through Rab11-positive structures in the eye disc cells occurs more transiently, therefore evading detection by immunostaining at a steady state (Hagedorn, 2006).

Another explanation for the relevance of ligand endocytosis hypothesizes that Dl internalization causes a mechanical stress on the Notch receptors, which then induces subsequent cleavages. A variation of this model proposes that the objective of Dl internalization is to remove the NECD fragment from the intercellular space so proteolytic processing can occur. If auxilin is solely involved in clathrin-coat disassembly, it will be difficult to reconcile the current data with these two models because the internalization of Dl into CCVs, the presumed force-generating event, should have already been completed in dAux mutants (Hagedorn, 2006).

Auxilin is essential for Delta signaling

Endocytosis regulates Notch signaling in both signaling and receiving cells. A puzzling observation is that endocytosis of transmembrane ligand by the signaling cells is required for Notch activation in adjacent receiving cells. A key to understanding why signaling depends on ligand endocytosis lies in identifying and understanding the functions of crucial endocytic proteins. One such protein is Epsin (Drosophila Liquid facets), an endocytic factor first identified in vertebrate cells. This study shows in Drosophila that Auxilin, an endocytic factor that regulates Clathrin dynamics, is also essential for Notch signaling. Auxilin, a co-factor for the ATPase Hsc70, brings Hsc70 to Clathrin cages. Hsc70/Auxilin functions in vesicle scission and also in uncoating Clathrin-coated vesicles. Like Epsin, Auxilin is required in Notch signaling cells for ligand internalization and signaling. Results of several experiments suggest that the crucial role of Auxilin in signaling is, at least in part, the generation of free Clathrin. These observations in the light of current models for the role of Epsin in ligand endocytosis and the role of ligand endocytosis in Notch signaling (Eun, 2008).

A role for Clathrin in Notch signaling cells was originally inferred from the observation that Chc mutants are strong dominant enhancers of lqf hypomorphs. Since Epsin has both Ubiquitin- and Clathrin-binding motifs, and also binds the plasma membrane, the simplest scenario imaginable for Clathrin and Epsin function in Delta internalization is for Epsin to act as a Clathrin adapter that recognizes ubiquitinated Delta, and brings Clathrin to the membrane for CCV formation. However, in light of evidence that Epsin-dependent endocytosis of ubiquitinated transmembrane proteins such as Delta may not occur through formation of CCVs, it has become unclear how to interpret the Chc/lqf genetic interaction. The results presented in this study point to a crucial role for Clathrin in Notch signaling cells. One intriguing possibility is that Delta internalization depends on Clathrin not because Delta is endocytosed in CCVs, but because Clathrin is a positive regulator of Epsin function. More experiments are required to test this idea (Eun, 2008).

Why do tissues that lack Epsin or Auxilin display Delta-like phenotypes, rather than phenotypes indicating failure of many signaling pathways or even cell death? One possibility is that the apparent specificity of both Epsin and Auxilin might simply reflect the usual redundancy of endocytic protein functions, and an unusual dependence of Notch signaling on efficient endocytosis. Alternatively, a special function of Epsin might be crucial to Notch signaling cells. Two kinds of models have been proposed to explain why Notch signaling requires ligand endocytosis by the signaling cells. One idea (the 'pulling model') is that after receptor binding, ligand endocytosis generates mechanical forces that result in cleavage of the Notch intracellular domain (Notch activation), either by exposing the proteolytic cleavage site on the Notch extracellular domain, or by causing the heterodimeric Notch receptor to dissociate. Alternatively, ligand internalization prior to receptor binding might be required to process the ligand endosomally, and recycle it back to the plasma membrane in an activated form (the 'recycling model'). Epsin might generate an environment particularly conducive to either pulling or recycling, and Auxilin might be required specifically by Notch signaling cells because it activates Epsin, perhaps by providing free Clathrin. Alternatively, Auxilin might be needed to provide free Clathrin because Delta is internalized through CCVs. In this case, if Auxilin is required in Notch signaling solely to provide free Clathrin, the implication would be that efficient CCV uncoating is not important for generating uncoated Delta-containing vesicles per se, which are prerequisite for travel through an endosomal recycling pathway. Further understanding of the role of Auxilin in Notch signaling cells might be key to understanding the role of ligand endocytosis (Eun, 2008).

The clathrin-binding motif and the J-domain of Drosophila Auxilin are essential for facilitating Notch ligand endocytosis

Ligand endocytosis plays a critical role in regulating the activity of the Notch pathway. The Drosophila homolog of auxilin (dAux), a J-domain-containing protein best known for its role in the disassembly of clathrin coats from clathrin-coated vesicles, has recently been implicated in Notch signaling, although its exact mechanism remains poorly understood. To understand the role of auxilin in Notch ligand endocytosis, several point mutations affecting specific domains of dAux were analyzed. In agreement with previous work, analysis using these stronger dAux alleles shows that dAux is required for several Notch-dependent processes, and its function during Notch signaling is required in the signaling cells. In support of the genetic evidences, the level of Delta appears elevated in dAux deficient cells, suggesting that the endocytosis of Notch ligand is disrupted. Deletion analysis shows that the clathrin-binding motif and the J-domain, when over-expressed, are sufficient for rescuing dAux phenotypes, implying that the recruitment of Hsc70 to clathrin is a critical role for dAux. However, surface labeling experiment shows that, in dAux mutant cells, Delta accumulates at the cell surface. In dAux mutant cells, clathrin appears to form large aggregates, although Delta is not enriched in these aberrant clathrin-positive structures. These data suggest that dAux mutations inhibit Notch ligand internalization at an early step during clathrin-mediated endocytosis, before the disassembly of clathrin-coated vesicles. Further, the inhibition of ligand endocytosis in dAux mutant cells possibly occurs due to depletion of cytosolic pools of clathrin via the formation of clathrin aggregates. Together, these observations argue that ligand endocytosis is critical for Notch signaling and auxilin participates in Notch signaling by facilitating ligand internalization (Kandachar, 2008).

From a F2 non-complementation screen, several new dAux alleles were isolated, some of which contain point mutations disrupting specific domains. Consistent with previous analysis of a viable dAux allele, strong dAux mutations affect several Notch-mediated processes, including photoreceptor specification in the eye and DV boundary formation in the wing. These phenotypes are consistent with the genetic interactions exhibited between dAux and Notch and between dAux and lqf . Taken together, these genetic observations strengthen the notion that endocytosis plays a critical role in Notch signaling, and suggest that dAux functions in multiple Notch-dependent events (Kandachar, 2008).

Since the functional importance of endocytosis has been suggested for both the signaling and receiving cells during Notch signaling, it is critical to determine in which cell is dAux function required. Although it has been previously concluded that dAux is needed in the signaling cells, the evidence, obtained from mitotic clones of a weak dAux allele, was less than convincing (Hagedorn, 2006). To adequately address this critical issue, the expression of E(spl), a Notch target gene, was examined in clones mutant for strong dAux alleles. Using these reagents, it is clear that dAux mutant cells at the clone border can still activate Notch (a similar result was seen with Cut and Ato staining), suggesting dAux acts non-cell autonomously. These genetic data imply that the relevant cargo is likely to be the Notch ligand. Indeed, as shown by the surface labeling experiment, Dl internalization is disrupted in dAux mutant cells (Kandachar, 2008).

Inhibition of auxilin function by mutations, RNAi, or injection of inhibitory peptides (Morgan, 2001) is known to interfere with the endocytosis of many molecules. In mammalian cells, inhibition of GAK function causes a decrease in the internalization of EGFR and transferrin. The current observations suggests that, similar to the mammalian cells, dAux participates in the endocytosis of EGFR, although a genetic interaction between DER and dAux was not previously (Hagedorn, 2006). It is possible that this lack of interaction between dAux and DER reflects the low sensitivity of the genetic assay. Alternatively, it may be that a defect in DER internalization does not significantly impact its signaling during eye development. Consistent with this, no drastic increase was observed in the phosphorylation of MAP kinase, a downstream event of DER activation, in dAuxF956* mutant clones. Nevertheless, the data show that, although the developmental defects of dAux resemble those of Notch, Notch ligand is not the sole cargo of auxilin-mediated endocytosis. This apparent specificity of dAux's Notch-like phenotypes suggests that the Notch pathway, compared to other signaling cascades, may be more sensitive to disruptions in the clathrin-mediated endocytosis (Kandachar, 2008).

Sequencing analysis of the dAux alleles revealed that disruptions in the kinase, the PTEN-related region, and the J-domain could all result in abnormal Notch signaling. Noticeably, the screen did not isolate any point mutation in the clathrin-binding motif (CBM), although the deletion analysis suggests that the CBM is critical for dAux function. This apparent discrepancy is likely due to the fact that the CBM domain contains multiple redundant clathrin-binding motifs, thereby obscuring the effect of eliminating one single motif by a point mutation. Interestingly, the removal of the CBM from the yeast auxilin (swa-2) does not completely eliminate its function in vivo. The reason for this difference is unclear but it is possible that swa-2 contains other protein domains capable of substituting for the CBM. Similar to a study of the mammalian GAK , a deletion analysis confirmed the importance of the J-domain, because over-expression of the dAuxdeltaJ construct fails to restore the extra photoreceptor cell defect. The CBM and J domains are thought to facilitate the recruitment of Hsc70 to CCVs, and a fragment consisting of CBM and J domain alone has been shown to support clathrin uncoating in vitro. In support of this notion that the recruitment of Hsc70 to CCVs is likely to be a critical step, over-expression of the CBM and J domain alone could restore the supernumerary Elav-positive cell phenotype (Kandachar, 2008).

Conversely, these observation also implies that the loss of the kinase and PTEN-related region could be compensated by the over-expression of the CBM and J-domain. The PTEN-related region is thought to participate in the membrane recruitment of auxilin during CME. Thus it is imaginable that a defect in the subcellular localization is less deleterious when the fragment consisting of CBM and J-domain is over-expressed. It is unclear how the requirement of kinase domain can be compensated by the over-expression of the CBM and J-domain, as the relevant substrate for dAux kinase domain during Notch signaling is not known. It should be mentioned that elevated expression of dAuxCJ rescued the extra Elav-positive cell phenotype in both dAuxF956* and dAuxL78H (point mutations disrupting the J-domain and the kinase domain respectively), arguing against a scenario in which the kinase domain of endogenous dAuxF956* mutant proteins could complement the over-expressed dAuxCJ in trans. It is possible that some functional redundancy exists between dAux and Numb-associated kinase (NAK, the Drosophila homolog of adaptin-associated kinase) (Chien, 1998), since the kinase domains from both factors are known to phosphorylate adaptor complexes. However, although mutations in subunits of Drosophila AP1 and AP2 complexes have been implicated in other Notch-dependent processes, it is not clear if these adaptor complexes have a role in the Notch processes that were examined. Homozygous α -adaptin mutants do not appear to exhibit a neurogenic phenotype. Furthermore, the removal of one copy of AP2 mu subunit (by a deletion) has no effect on the dAuxI670K rough eye phenotype. In any case, it should be stressed that the kinase and the PTEN-related region do play a role in Notch signaling, since point mutations disrupting these domains cause Notch-like defects, albeit to a weaker extent. Taken together, these results suggest the role of the kinase and the PTEN-related region during Notch ligand endocytosis is less than obligatory (Kandachar, 2008).

What is the role of ligand endocytosis in Notch signaling? It has been suggested that, after receptor-ligand binding, ligand endocytosis may provide a mechanical stress or other types of micro-environment (clustered ligand and receptor, etc.) to facilitate Notch cleavage or NECD shedding. Alternatively, before binding to Notch, the ligands may have to enter a particular recycling pathway to render them active. The linking of dAux to Notch was initially viewed as evidence favoring the latter model because it suggests that ligand endocytosis needs to proceed past clathrin uncoating. However, as an increased level of the Dl appeared to be trapped at the mutant cell surface, not inside CCVs, the linking of dAux to Notch certainly does not exclude the model that ligand internalization per se is critical for Notch signaling. Biochemical analysis has suggested several additional functions for auxilin during the CCV cycle besides uncoating. Although abnormal clathrin distribution was observed in dAux cells, given the resolution of the analysis, it is unclear which particular step(s) were affected. It is possible that mutations in dAux directly inhibit Notch ligand endocytosis by disrupting one or more of these early steps during CCV formation. Alternatively, dAux mutations may indirectly inhibit Notch ligand internalization by causing an excessive formation of non-functional clathrin-dependent structures, thereby decreasing the cytosolic clathrin pool. Indeed, in dAux mutant cells, those large clathrin-positive structures did not appear to contain an elevated level of Dl. Consistent with this, it was recently shown (Eun, 2008) that over-expression of Chc could restore the dAux-associated defects (Kandachar, 2008).

This genetic analysis of strong dAux alleles clearly strengthens the notion that ligand endocytosis plays a critical role in Notch signaling. Furthermore, the deletion analysis suggests that the recruitment of Hsc70 to clathrin is a key event for dAux to facilitate Notch signaling. More importantly, this study showed that Dl accumulates at the cell surface in dAux mutant cells. This suggests that the linking of dAux to the Notch pathway does not exclude the model in which ligand endocytosis activates Notch by physically dissociating the receptor (Kandachar, 2008).

Crumbs is required to achieve proper organ size control during Drosophila head development

Crumbs (Crb) is a conserved apical polarity determinant required for zonula adherens specification and remodelling during Drosophila development. Interestingly, crb function in maintaining apicobasal polarity appears largely dispensable in primary epithelia such as the imaginal discs. This study shows that crb function is not required for maintaining epithelial integrity during the morphogenesis of the Drosophila head and eye. However, although crb mutant heads are properly developed, they are also significantly larger than their wild-type counterparts. In the eye, this is caused by an increase in cell proliferation that can be attributed to an increase in ligand-dependent Notch (N) signalling. Moreover, in crb mutant cells, ectopic N activity correlates with an increase in N and Delta endocytosis. These data indicate a role for Crb in modulating endocytosis at the apical epithelial plasma membrane, which is shown to be independent of Crb function in apicobasal polarity. Overall, this work reveals a novel function for Crb in limiting ligand-dependent transactivation of the N receptor at the epithelial cell membrane (Richardson, 2010).

This demonstrates a novel function for crb in the proper control of head and eye size during Drosophila development. This function is not restricted to the fly head and eye, but also extends to other tissues such as the wing. In the case of the eye, the data indicate that this is linked to a function for Crb in limiting ligand-dependent transactivation of N. In support of this model, a significant increase in endosomes positive for NICD, NECD and Dl was observed in crb mutant eye discs, that correlates with excessive cell proliferation. The eye overgrowth phenotype associated with the loss of crb function is correlated with an increase in NECD/Dl co-endocytosis. The data further indicate that this increase is dependent on the S2 cleavage of N. There is currently no evidence for the requirement of the S2 cleavage to promote endocytosis of N with Dl in cis. Moreover, Dl in cis is thought to inhibit N activation. It is therefore concluded that during eye development, Crb limits ligand-dependent transactivation of the N receptor (Richardson, 2010).

Mutations in the crb gene are associated with a failure to properly polarise the ectoderm along the apicobasal axis in the gastrulating embryo. In human, three Crb orthologues, CRB1, 2 and 3, have been described to date. Interestingly, CRB1 can be differentially spliced to produce the CRB1b isoform, which contains only the extracellular domain, whereas CRB3 lacks the conserved extracellular domain and comprises just the TM and intracellular domains. This suggests independent function for the extracellular and intracellular domains. The function of crb in establishing apicobasal polarity can be rescued in the Drosophila embryo using its intracellular domain anchored to the plasma membrane via the TM domain. Interestingly, crb function in the developing pupal photoreceptor has been linked to stalk membrane endocytosis through the connection of Crb to βHeavy-spectrin. This idea is supported by the finding that overexpression of Crb in either the gastrulating fly ectoderm or fly pupal photoreceptor leads to an increase the length of the apical and stalk membranes, respectively. Interestingly, when examining crb mutant adult photoreceptors, clathrin-coated-like pits were detected in the region of the stalk membrane, which are not normally readily detectable in the WT. Importantly, overexpression of the extracellular domain of Crb linked only to its TM domain is sufficient to cause a striking increase in the length of the stalk membrane in the developing fly photoreceptor. Consistent with this finding is the observation that this transgene can rescue the overgrowth phenotype in crb mutant heads, a phenotype that is correlated with an increased in N/Dl endocytosis. Moreover, the data indicate that Crb function in regulating N activity does not depend on the ability for Crb to interact with βHeavy-spectrin, Moesin, Yurt, Sdt, Patj, or Lin7. This suggests that the extracellular domain of Crb might bind to a component of the extracellular matrix or with itself. It is therefore probable that in the WT, both the extracellular and intracellular domain could synergise to limit endocytosis at the apical membrane (Richardson, 2010).

During Drosophila eye development, N activation at the D/V boundary is thought to promote cell proliferation within the eye primordium via activation of the JAK-STAT pathway. Consistent with this model, overexpression of the NICD in the developing eye discs leads to overgrowth of the eyes. Indeed, flies heterozygous for N, Dl and Ser have smaller eyes than WT flies. However, the width of the corresponding head capsules remains unchanged, arguing that N activity is not required during the head capsule growth. These data suggest that crb function in this epithelium might not be related to N activity, but is instead linked to that of another growth signalling pathway. The data in the eye strongly argue that loss of crb function causes ectopic activation of the N signalling pathway. Moreover, the overgrowth of crb mutant eye tissue can be suppressed by inhibiting the N pathway. Finally, this analysis of crb mutant clones during oogenesis, together with the inhibition of the S2 cleavage, indicates that crb limits ligand-dependent transactivation of N (Richardson, 2010).

Trafficking, and in particular endocytosis, plays a major role in modulating the N signalling pathway. A steady level of N at the cell surface is achieved by a balance between its activation on the way to the plasma membrane and its endocytosis and degradation. Ligand activation also requires the endocytosis and recycling of the ligand back to the cell surface in the signal-sending cell. Upon ligand binding, the transendocytosis of NECD bound to its ligand into the signal-sending cell allows the S2 cleavage of N, and recent work indicates that proteolytic cleavage of N by the γ-secretase complex occurs in the endocytic compartment. In normal conditions, the S2 cleavage of N is required for its subsequent S3/S4 cleavage by the γ-secretase complex. The increase in the number of NECD/Dl endosomes observed in the absence of crb function cannot be explained by Crb's link to the γ-secretase complex and indicates that in the developing eye disc, crb is also required to limit ligand-dependent transactivation of N (Richardson, 2010).

How does Crb act to regulate ligand-dependent N signalling activity? Structural analysis of Crb, N and Dl shows that these proteins all contain extracellular domains that contain multiple EGF-like repeats. This raises the possibility that the extracellular domain of Crb could interact with N and/or Dl via their EGF-like repeats, thus preventing N-Dl binding. Such specificity towards the N pathway is supported by the observation that there is no significant increase in Hrs-positive endosomes in the absence of crb function. Interestingly, other EGF repeat-containing proteins have been shown to inhibit N signalling; Dlk, for example, interacts and inhibits Notch1 in mammalian cells (Baladron, 2005). Alternatively, Crb might limit the rate of endocytosis of N or Dl. One outcome of this could be that Crb limits Dl activation by regulating its endocytosis and subsequent recycling back to the plasma membrane in order for it to become competent for signalling. Another possibility is that Crb might limit the endocytosis of NECD and Dl into the signal-sending cell, which is proposed to be required for the S2 cleavage of N. An increase in either of these endocytic events due to Crb loss-of-function could result in ectopic N activity. It is difficult to differentiate signal-sending versus signal-receiving cells in the context of the early developing eye epithelium, which prevents determination of whether crb function is required in one of these cell types or in both. In addition, it will be interesting to determine how exactly the extracellular domain of Crb modulates endocytosis at the apical membrane, and whether, as suggested by the present study, this might target specific signalling pathways such as the N pathway. Finally, given that crb itself is a transcriptional target of the N pathway, its ability to limit the activity of the γ-secretase complex, together with its function in limiting ligand-dependent transactivation of N, is likely to provide a very robust negative-feedback loop mechanism to regulate N activity during organogenesis (Richardson, 2010).

Drosophila Epsin's role in Notch ligand cells requires three Epsin protein functions: the lipid binding function of the ENTH domain, a single Ubiquitin interaction motif, and a subset of the C-terminal protein binding modules

Epsin is an endocytic protein that binds Clathrin, the plasma membrane, Ubiquitin, and also a variety of other endocytic proteins through well-characterized motifs. Although Epsin is a general endocytic factor, genetic analysis in Drosophila and mice revealed that Epsin is essential specifically for internalization of ubiquitinated transmembrane ligands of the Notch receptor, a process required for Notch activation. Epsin's mechanism of function is complex and context-dependent. Consequently, how Epsin promotes ligand endocytosis and thus Notch signaling is unclear, as is why Notch signaling is uniquely dependent on Epsin. By generating Drosophila lines containing transgenes that express a variety of different Epsin deletion and substitution variants, tests were performed of each of the five protein or lipid interaction modules for a role in Notch activation by each of the two ligands, Serrate and Delta. There are five main results of this work that impact present thinking about the role of Epsin in ligand-expressing cells. First, it was discovered that deletion or mutation of both Ubiquitin interaction motifs (UIM) destroyed Epsin's function in Notch signaling and had a greater negative impact on Epsin activity than removal of any other module type. Second, only one of Epsin's two UIMs was essential. Third, the lipid-binding function of the Epsin-N-terminal homology (ENTH domain) was required only for maximal Epsin activity. Fourth, although the C-terminal Epsin modules that interact with Clathrin, the adapter protein complex AP-2, or endocytic accessory proteins were necessary collectively for Epsin activity, their functions were highly redundant; most unexpected was the finding that Epsin's Clathrin binding motifs were dispensable. Finally, it was found that signaling from either ligand, Serrate or Delta, required the same Epsin modules. All of these observations are consistent with a model where Epsin's essential function in ligand-expressing cells is to link ubiquitinated Notch ligands to Clathrin-coated vesicles through other Clathrin adapter proteins. It is proposed that Epsin's specificity for Notch signaling simply reflects its unique ability to interact with the plasma membrane, Ubiquitin, and proteins that bind Clathrin (Xie, 2012).

Epsin is a complex multi-modular protein that functions differently in different contexts. Each Lqf isoform has two UIMs, two Clathrin binding motifs (CBMs), seven DPW motifs that bind the AP-2 endocytic adapter complex, and two NPF motifs that bind EH-domain-containing endocytic factors such as Eps15. In C. elegans, Drosophila, and mice, Epsin is needed specifically in Notch ligand cells. The structure/function analysis of Epsin performed in this study shows that modules of Epsin associate with the internalization step of endocytosis - the lipid binding function of the ENTH domain and the C-terminal modules that bind proteins present in Clathrin-coated vesicles - are required for Epsin's function in Notch ligand cells. In addition, it was shown that a UIM is necessary (Xie, 2012).

The dispensability of the Cdc42 GAP binding function of the ENTH domain suggests that in ligand cells the primary role of Drosophila Epsin, unlike yeast Ent1, is not regulation of actin dynamics. The other known function of the ENTH domain is the endocytic function, and the results suggest that the ability of the ENTH domain to interact with PIP2 explains why it is needed for maximal Epsin function in Notch ligand cells. These observations are consistent with the lack of typical Notch signaling defects in Drosophila cdc42 mutants. In contrast, flies with mutations in genes for either of two actin regulators, the Arp2/3 complex and WASp, do have notal bristle defects indicative of Notch signaling failure. The notal bristle phenotype described in this study is not due to failure of the Epsin-dependent endocytosis of ligand that activates Notch in all cell types, but instead to failure of ligand transcytosis required in only some cell types to relocalize ligand prior to signaling. The absence of the Arp2/3 complex or WASp in mutants inhibits signaling by blocking traffic of endocytosed Delta to apical microvilli of sensory organ precursors. Whether or not Delta transcytosis in sensory organ precursors also depends on Epsin is unknown. If Epsin is involved, it may be interesting to use the Epsin variant transgenes generated in this study to determine whether or not the Cdc42 GAP interaction function of the ENTH domain is required (Xie, 2012).

There are two types of UIMs: single-sided UIMs that bind one Ubiquitin, and double-sided UIMs that bind two Ubiquitins simultaneously. As the affinity between a UIM and Ubiquitin is low, successful interaction between a mono-ubiquitinated protein and a UIM-containing protein is thought to require either one double-sided UIM, or two single-sided UIMs. Epsins have single-sided UIMs, and so the observation that only one single-sided UIM is required for Drosophila Epsin function in Notch signaling is unexpected. The simplest explanation is that Notch ligands use multiple mono-Ubiquitins or Ubiquitin chains as a signal for Epsin-mediated internalization (Xie, 2012).

Two distinct Lysine residues in the intracellular domains of both Delta and Serrate have been implicated as important for the function of each ligand. In the case of Serrate, simultaneous mutation of both of these Lysines results in a Serrate ligand that can neither activate Notch nor be endocytosed in wing discs. These observations identify two particular Lysines as candidates for the critical Ub attachments, but do not distinguish whether one or both Lysines are required. In the case of Delta, single mutation of either of two specific Lysines results in accumulation of Delta at the cell surface of eye discs and failure to signal. Although Delta is thought to be mono-ubiquitinated, these results suggest the possibility that Delta is multiply mono-ubiquitinated. An alternative explanation for Epsin's ability to promote ligand endocytosis with a single UIM is that mono-ubiquitinated ligands cluster to generate an environment where multiple Ubiquitins attract Epsin to ligand at the plasma membrane (Xie, 2012).

There is compelling evidence that in somatic cells, Notch ligand endocytosis associated with signaling is Clathrin-dependent. First, there are exceedingly strong genetic interactions between the Clathrin heavy chain (Chc) gene and lqf, the gene for Epsin. Flies with only one Chc+ gene copy are wild-type, but this condition is lethal in homozygotes for a normally viable hypomorphic allele of lqf. Second, the Clathrin-coated vesicle uncoating protein Auxilin is, like Epsin, required specifically for Notch signaling in Drosophila and in ligand cells. Given the clear involvement of Clathrin and the lack of strong genetic interaction between α-Adaptin (the gene for an AP-2 subunit) and lqf, the simplest model for Epsin function in Notch signaling was as an adapter protein that links Clathrin and the plasma membrane, independent of AP-2. This model predicted that direct interaction between Epsin and Clathrin would be necessary, and thus the most surprising result of this work is that deletion of the CBMs had no detectable effect on Epsin activity. The dispensability of the CBMs rules out models where Epsin acts as a monomeric Clathrin adapter that links ligand to Clathrin cages (Xie, 2012).

In the Drosophila female germline, Notch signaling requires Epsin but neither Clathrin nor Auxilin. Although this is surprising, Epsin has been shown to function in Clathrin-independent internalization of ubiquitinated transmembrane cargos in vertebrate cell culture. Epsin must therefore function differently in Notch signaling in the female germline than in somatic cells. It is speculated that the ENTH domain and UIMs may be required in germline cells to guide the ubiquitinated proteins into Q6 an endocytic vesicle. However, it is not clear how any of the characterized modules within Epsin's C-terminus might be involved in Clathrin-independent endocytosis. It would be of interest to use the transgenes that were generated in this study to determine which motifs are required in the female germline. Additional experiments could potentially identify unknown C-terminal interaction motifs used in Clathrin-independent endocytosis (Xie, 2012).

Does Epsin function in the same way in the embryo, eye, and wing? The experiments began with the assumption that Epsin functions through the same mechanism in all signaling contexts, and thus it was expected the same Epsin modules would be required for Epsin function in all contexts. Epsin appears to be required in every Notch signaling event and thus could be regarded as a core component of Notch signaling. It therefore seems reasonable to expect that Epsin would function in the same manner in all tissues. The female germline is apparently an exception. Nevertheless, in the three assays used for Epsin activity - rescue of lethality and eye morphology defects due to lqf mutations and rescue of the ability of lqf null cells to activate Cut expression in cells at the D/V boundary in the wing disc - only subtle differences were detected between the eye and the wing in the activity of two Epsin variants, δENTH and δUIM. (The only major difference was with the highly artificial Epsin variant, 4XNPF.) Despite these differences, it is thought that Epsin likely functions the same way in the eye and wing, as well as during embryogenesis. For one, the differences in activity that were observed be explained easily without invoking different mechanisms for Epsin in the eye and wing. Importantly, no even one case was observed where modules were essential in one context (embryogenesis, eye, or wing development) and dispensable in another one. In fact, it is possible to observe all-or-none differences in requirements for Epsin modules. Epsin was found to function outside of Notch ligand cells and modules were found that were dispensable completely in this context yet absolutely essential for Epsin's function in ligand cells (Xie, 2012).

Notch ligands require ubiquitination and (usually) Clathrin-dependent endocytosis, and formation of Clathrin-coated vesicles requires adapter proteins that link the plasma membrane with Clathrin. The absolute necessity of at least one UIM and the observation that the lipid-binding function of the ENTH domain plays a role in ligand cells suggests that Epsin indeed binds ubiquitinated Notch ligands at the plasma membrane.However, as an Epsin derivative lacking CBMs functions as well as wild-type Epsin in ligand cells, the essential role of Epsin in Notch signaling cannot be as a monomeric Clathrin adapter that links Clathrin directly to ligand at the plasma membrane. As any pair of the three types of modules is sufficient for Epsin function (CBMs+DPWs, CBMs+NPFs, or DPWs+NPFs), Epsin must be able to support Notch activation by linking ligand to Clathrin in a variety of different ways. It is speculated that Eps15, the second Drosophila Epsin, is involved because of the three EH-domain proteins in Drosophila (Eps15, Dap160, Past1), none have Clathrin binding motifs, and Eps15 is the only one with motifs for a known Clathrin-binding protein (AP-2). From analysis of mutant phenotypes and genetic interaction studies, there is no evidence for the involvement of Eps15 nor AP-2 in Notch signaling (Xie, 2012). The results presented in this study suggest that Eps15 and AP-2 may play redundant roles in the presence of intact Epsin and this idea could be tested with additional genetic experiments. In light of the evidence indicating a requirement for Clathrin in ligand cells (outside of the germline), the results suggest that Epsin is required absolutely for Notch signaling not because it generates a special endocytic environment, but simply because it is the only UIM-containing endocytic protein with the appropriate complement of interaction modules to target ubiquitinated cargo to Clathrin-coated vesicles (Xie, 2012).

Functional analysis of the NHR2 domain indicates that oligomerization of Neuralized regulates ubiquitination and endocytosis of Delta during Notch signaling

The Notch pathway plays an integral role in development by regulating cell fate in a wide variety of multicellular organisms. A critical step in the activation of Notch signaling is the endocytosis of the Notch ligands Delta and Serrate. Ligand endocytosis is regulated by one of two E3 ubiquitin ligases, Neuralized (Neur) or Mind bomb. Neur is comprised of a C-terminal RING domain, which is required for Delta ubiquitination, and two Neur homology repeat (NHR) domains. Previous studies have shown that the NHR1 domain is required for Delta trafficking. This study shows that the NHR1 domain also affects the binding and internalization of Serrate. Furthermore, it was shown that the NHR2 domain is required for Neur function and that a point mutation in the NHR2 domain (Gly430) abolishes Neur ubiquitination activity and affects ligand internalization. Finally, evidence is provided that Neur can form oligomers in both cultured cells and fly tissues, which regulate Neur activity and, by extension, ligand internalization (Liu, 2012).

Neur is an E3-ubiquitin ligase that plays an essential role in Notch signaling by regulating the endocytosis of Notch ligands. It contains NHR domains, which are rare and conserved between vertebrates and invertebrates but not present in viruses, bacteria, fungi, or plants. In the Drosophila proteome, besides Neuralized, there are two other NHR-containing proteins, CG3894 and Bluestreak. In mammals, proteins containing NHR domains (also known as NEUZ) include the β-catenin regulator OzzE3 and lung-inducible Neuralized-related C3HC4 RING protein (LINCR). Recent studies reported that the human homologue of Bluestreak serves to localize to the centrosome. Although the general role of the NHR domains is unclear, these domains tend to cluster, and most proteins contain two to six NHR domains. The significance of having more than one NHR domain in one protein is to yet be determined (Liu, 2012).

This study has investigated the role of the highly conserved NHR domains in Neur function. The NHR1 domain alone mediates the interaction between Neur and both Delta and Serrate. It was also shown that the NHR2 domain is required for Neur function, and while it is not required for the interaction with Notch ligands, it is involved in Dl internalization, a critical step in Notch activation. Moreover, the NHR domains play a role in Neur oligomerization, which in turn could contribute to Neur ubiquitination activity and ligand endocytosis (Liu, 2012).

The NHR1 domain mediates the interaction between Neur and the Notch ligands Delta and Serrate. Previous studies have shown that the NHR1 domain of Neur is both necessary and sufficient for the interaction with the Notch ligand Dl. Specifically, it was found that a point mutation in a highly conserved glycine residue within the NHR1 domain (G167E) abolishes the ability of Neur to bind Delta. Whether the NHR1 domain was also required to bind to Serrate, however, was unknown. In fact, in vitro studies in vertebrates suggested that the NHR2 domain in mouse Neuralized- like 1 (Neurl1) is sufficient to bind to Jagged1, the mouse orthologue of Serrate. In contrast, the current study found that the NHR2 domain is not required for Neur to bind Serrate in Drosophila and that the interaction is mediated entirely by the NHR1 domain. Other studies reported previously that the motifs on Dl and Ser that mediate the interaction with Neur are conserved. In comparisons of protein sequences, Jagged1 and Serrate share 40.7% similarity overall, while the overall similarity between Jagged1 and Dl is 33.8%. The NHR1 and NHR2 domains from Drosophila Neuralized have the same degree of amino acid similarity with the mouse Neurl1 NHR2 domain (33%). Since there is no clear correlation between protein sequence similarity and the ability of either the NHR1 or the NHR2 domain to interact with Notch ligands, whether NHR1 or NHR2 is important for interacting with ligands is likely to be species dependent. The Neur-Ser interaction was found to be abrogated by the G167E mutation in the NHR1 domain. Given that the NeurG167E mutant still retains ubiquitination activity, it is unlikely to affect overall protein folding. A previously reported structural analysis of the Drosophila NHR1 domain suggested that Gly167 resides in a hydrophobic core and that the Gly167 mutation presumably destabilizes the surrounding microenvironment. Therefore, the Gly167 mutation may result in spatial changes in the neighboring residues of the core, thus abolishing binding to ligands (Liu, 2012).

The G430E mutation reveals a distinct role for the NHR2 domain in the regulation of Neur activity and Delta trafficking. The data demonstrate that the NHR2 domain is required for Neur function in vivo. NeurG430E fails to rescue neur mutant embryos, while NeurNHR2 has some residual activity, which suggests that they affect different aspects of Neur function. The expression of NeurG430 in a heterozygous background, which does not have a neurogenic phenotype on its own, resulted in a significant increase in the percentage of neurogenic embryos, suggesting that NeurG430E has a negative effect on Neur function. In contrast, NeurNHR2 overexpression did not have any effect on heterozygous embryos, suggesting that it behaves as a loss-of-function allele. Despite the fact that the two NHR2 mutant proteins NeurG430E and NeurNHR2 behave differently, they both localize to the plasma membrane in the presence of Delta both in vitro and in vivo, and they are both capable of binding to the Notch ligands Delta and Serrate. However, both mutant proteins affect the extent of Dl internalization to various degrees. NeurG430E exhibits severely compromised ubiquitination activity and is no longer capable of inducing Delta internalization. NeurNHR2, on the other hand, retains ubiquitination activity but is much less efficient than WT Neur at directing Dl internalization in vivo or in Kc cells. The precise mechanism by which NeurNHR2 affects Delta endocytosis is unclear (Liu, 2012).

One possibility is that the NHR2 domain is required for Neur oligomerization. Neur was shown to form NHR domain-mediated oligomers by coimmunoprecipitation experiments. Therefore, the deletion of the NHR2 domain (NeurNHR2) may simply reduce the oligomerization potential of Neur, leading to a decrease in ligand endocytosis. In contrast, the point mutation (NeurG430) might disrupt the overall structure of the NHR2 domain, preventing oligomerization and resulting in a protein that has no ubiquitination activity and therefore can no longer internalize ligands. However, this model cannot fully explain the data, which show that NHR2 does not prevent Neur oligomerization and that Neur oligomerization still occurs in the absence of any NHR domains, suggesting that while the NHR domains may play a role in oligomerization, they are not necessary for this process. The data also show that although the G430E mutant loses ubiquitination activity, the double mutant containing the NHR1 deletion and the G430E mutation retains ubiquitination activity, which argues that G430E does not affect the overall folding of the NHR2 domain (Liu, 2012).

Another possibility is that the NHR1 and NHR2 domains initially form an intramolecular structure that is inactive and must be resolved for ubiquitination to promote ligand internalization. The G430E mutation may lock Neur into an intramolecular conformation through an NHR1-NHR2 interaction, such that this inactive form can still bind to Dl and Ser but cannot form oligomers and has no ubiquitination activity as a consequence of dysfunctional oligomerization. This model, in contrast to the former one, is supported by the data that demonstrate that the G430E mutant no longer forms oligomers and has no ubiquitination activity. Furthermore, when the NHR1 domain was removed from the G430E mutant (NeurNHR1G430E), the ubiquitination activity was restored, suggesting that the intramolecular loop can no longer form, whereas intermolecular interactions between NHR1 and NHR2 domains can occur. It may also explain the negative effect of NeurG430E on NeurWT: although NeurG430E has a reduced ability to bind WT Neur, it is still be able to sequester some portion of NeurWT into a nonfunctional intermolecular oligomer that can no longer ubiquitinate targets. In contrast, the deletion of the NHR2 domain would prevent intramolecular interactions but would be expected to have a reduced ability to form productive oligomers, leading to a defect in ligand internalization. Whether the NHR2 domain has additional roles in recruiting a protein(s) that promotes Notch ligand ubiquitination and endocytosis remains to be determined. Like Neuralized, other RING domain E3 ligases often function as oligomers, and they multimerize in different ways: some form heterodimers, such as Mdm2-MdmX, and some can form homo-oligomers, such as TRAF. The functional significance of RING E3 oligomerization is poorly defined. One previously proposed model is that the oligomerization of E3 ligases may functionally resemble the dimerization of receptor tyrosine kinases in such a way that autoubiquitination yields a mark that serves as a platform to assemble a signaling complex. Consistent with this idea, ubiquitination was seen in anti-Neur IPs when Dl was not present, consistent with the idea that Neur may be autoubiquitinated. Whether this autoubiquitination can initiate a cascade of further downstream ubiquitination events remains to be determined. It is possible that oligomerization is mediated via autoubiquitination and the interaction of ubiquitinated Neur with itself through a ubiquitin-binding motif (UIM). If so, then the NHR1-NHR2 interaction could also function to keep Neur in an inactive state by occluding the putative UIM. Such a model of Ubi-UIM complex formation was previously proposed for other endocytic proteins (Liu, 2012).

In summary, this study has shown that NHR domains are protein-protein interaction modules that are required for many aspects of Neur function. The NHR1 domain mediates the interaction between Neur and its targets Dl and Ser. Both NHR domains appear to regulate Neur activity by affecting its ability to form oligomers and/or interact with proteins required for the endocytosis of Notch ligands. Interestingly, NHR domains have been identified in several other proteins that are conserved between flies and humans. Whether Neur can form heterodimers with these other NHR-containing proteins and whether these heterodimers play a role in Notch signaling and other developmental processes remain to be determined (Liu, 2012).

Cis-interactions between Notch and its ligands block ligand-independent Notch activity

The Notch pathway is integrated into numerous developmental processes and therefore is fine-tuned on many levels, including receptor production, endocytosis, and degradation. Notch is further characterized by a two-fold relationship with its Delta-Serrate (DSL) ligands, as ligands from opposing cells (trans-ligands) activate Notch, whereas ligands expressed in the same cell (cis-ligands) inhibit signaling. This study, carried out in vivo during oogenesis and in cultured Drosophila cells shows that cells without both cis and trans ligands are able to mediate Notch-dependent developmental events during Drosophila oogenesis, indicating ligand-independent Notch activity occurs when the receptor is free of cis and trans ligands. Furthermore, cis-ligands can reduce Notch activity in endogenous and genetically-induced situations of elevated trans-ligand-independent Notch signaling. It is concluded that cis-expressed ligands exert their repressive effect on Notch signaling in cases of trans-ligand independent activation, and a new function of cis-inhibition is proposed which buffers cells against accidental Notch activity (Palmer, 2014: PubMed).

Delta: Biological Overview | Evolutionary Homologs | Regulation | Developmental Biology | Effects of Mutation | References

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