Ligand binding to receptors of the LIN-12/Notch family causes at least two proteolytic cleavages: one between the extracellular and transmembrane domains, and the other within the transmembrane domain. The transmembrane cleavage depends on Presenilin, a protein also required for transmembrane cleavage of beta-APP. The substrate requirements for Presenilin-dependent processing of Notch and other type I transmembrane proteins in vivo has been assayed. Presenilin-dependent cleavage does not depend critically on the recognition of particular sequences in these proteins but rather on the size of the extracellular domain: the smaller the size, the greater the efficiency of cleavage. Hence, Notch, beta-APP, and perhaps other proteins may be targeted for Presenilin-mediated transmembrane cleavage by upstream processing events that sever the extracellular domain from the rest of the protein (Struhl, 2000).

Evidence suggests that Presenilin is a component of a general mechanism that cleaves type I transmembrane proteins in the transmembrane domain, provided that they have a relatively small extracellular domain. Little if any processing occurs when the extracellular domain is greater than 200-300 amino acids. However, as the size of the extracellular domain is reduced incrementally, progressive increases in the efficiency of processing is attained, with proteins having very small extracellular domains (<50 amino acids) exhibiting similar, if not higher, levels of processing to those of full-length Notch in response to ligand. These results support the view that ligand activates LIN-12/Notch proteins by inducing a cleavage of the extracellular domain close to the membrane. Consistent with this proposal, recent biochemical studies indicate that mammalian Notch proteins undergo just such a cleavage event in response to ligand. Although this cleavage could activate the receptor in a number of different ways, these findings indicate that the resulting reduction in the size of the extracellular domain should suffice to convert the remainder of the protein into a substrate for Presenilin-dependent cleavage. Hence, the hypothesis is favored that ligand activates Notch by severing the extracellular domain from the rest of the receptor, a process described as 'ectodomain shedding' for other transmembrane proteins (Struhl, 2000).

Presenilin-dependent processing of betaAPP provides a second example of a possible link between ectodomain shedding and Presenilin-dependent cleavage. betaAPP initially contains a large extracellular domain of approximately 600 amino acids, and the full-length protein is not believed to be a substrate for Presenilin-dependent cleavage. However, full-length betaAPP is a target for cleavage by beta-secretase, a transmembrane aspartyl protease, which cuts at a site around 25 amino acids amino-terminal to the transmembrane domain. This initial cleavage is thought to be responsible for shedding the extracellular domain and for rendering the transmembrane domain susceptible to the Presenilin-associated gamma-secretase activity (Struhl, 2000).

The finding that proteins with diverse transmembrane domains can all be processed in a Presenilin-dependent fashion provided that the extracellular domain is small raises the possibility that such transmembrane cleavages can be viewed as relatively general and indiscriminate scavenging events that allow a cell to clear residual, truncated proteins from the membrane. Such a role could account for the transmembrane cleavages that generate the beta-amyloid peptides, which have no known function in normal cell physiology. However, in the case of LIN-12/Notch receptors, it appears that this cleavage mechanism has been incorporated as a critical step in signal transduction (Struhl, 2000).

Presenilin-dependent cleavage has been implicated in transduction of the unfolded protein response (UPR), which depends on the release and nuclear import of the cytosolic domain of the UPR receptor. Hence, activation of the UPR receptor, like that of Notch, may depend on processing events that cause ectodomain shedding and thereby target the remainder of the receptor for Presenilin-dependent cleavage. It is suggested that LIN-12/Notch proteins and the UPR receptors may belong to a general class of receptors that are activated by ectodomain shedding and which transduce signals by a mechanism involving Presenilin-dependent release of the intracellular domain from the rest of the receptor. It is possible that beta-APP also belongs to this class of receptors, since there is evidence that the intracellular domain of beta-APP interacts via an adaptor protein with a transcription factor (Struhl, 2000).

The mechanism by which ligand might induce dissociation of the Notch extracellular domain from the rest of the protein remains uncertain. Evidence has been found that the Presenilin-dependent cleavage of full-length Notch does not occur in shibire mutant embryos, which are defective in endocytosis due to reduced activity of Dynamin. In contrast, truncated forms of Notch that lack virtually the entire extracellular domain appear to be cleaved in these embryos. These results indicate that the Presenilin-dependent cleavage is not inherently dependent on endocytosis. Instead, endocytosis may be required for upstream events that are necessary to shed the ectodomain and hence to target the rest of the receptor for transmembrane cleavage. For example, endocytosis of the transmembrane ligand Delta in the signaling cell bound to Notch on the receiving cell might expose the Notch extracellular domain to cleavage. Alternatively, Notch may undergo extracellular processing in response to Delta while both proteins are on the cell suface, but endocytosis by the receiving cell might be required to dissociate the cleaved ectodomain from the rest of the receptor (Struhl, 2000).

In mammals, Notch proteins are cleaved at an extracellular Furin site (termed S1) close to the transmembrane domain during their trafficking to the cell membrane. As a consequence, the mature receptor is a heterodimer composed of two components: (1) a large extracellular domain and (2) the remainder of the receptor consisting of a short extracellular stub, the transmembrane domain, and the intracellular domain. In principle, interactions with ligand could activate the receptor by disrupting the association between these two components, causing the ectodomain to be separated from the rest of the protein by displacement rather than by proteolysis. Alternatively, ligand might induce shedding by triggering cleavage at a second site (S2) between the Furin cleavage and the transmembrane domain, a possibility directly supported by biochemical studies of Notch activation in mammalian cell culture. In the case of Drosophila, there is evidence that the mature Notch protein on the cell surface is not normally processed by Furin to form a heterodimer. If the Furin-mediated S1 cleavage does not occur in Drosophila Notch, ectodomain shedding would presumably depend on a ligand-induced S2 cleavage in order to convert the receptor into a substrate for the transmembrane cleavage (referred to as the S3 cleavage), which requires Presenilin (Struhl, 2000).

Recent biochemical evidence in mammals suggests that the metalloprotease TACE can execute the S2 cleavage of Notch in response to ligand. Genetic data in C. elegans and Drosophila suggest that a related metalloprotease, Kuzbanian/SUP-17, is essential for LIN-12/Notch signaling. However, there are conflicting biochemical data concerning whether Kuzbanian cleaves Notch or its ligands, complicating interpretation of whether it plays a direct role in executing the S2 cleavage. The nature of the event that precipitates the S2 cleavage is not known, but genetic evidence in C. elegans raises the possibility that ligand-induced oligomerization is involved (Struhl, 2000).

One determinant of whether a protein is a substrate for Presenilin-dependent cleavage appears to be the size of the extracellular domain. How might the size of the extracellular domain be assayed by the Presenilin-dependent cleavage mechanism? One possibility is that the cleavage mechanism requires the assembly of an active processing complex in close proximity to the transmembrane domain of the substrate. Although Presenilin has been reported to associate with Notch proteins as they move from the endoplasmic reticulum to the cell surface, the presence of a large extracellular domain may interfere sterically with the assembly of the complete complex or with the proteolytic activity of the complex. Another possibility is that the cleavage mechanism recognizes a free amino terminus in close proximity to the transmembrane domain, a condition that may be more likely when the extracellular domain is small. Both of these possible mechanisms are compatible with the finding that there is a progressive decline in cleavage efficiency as the size of the extracellular domain is increased incrementally (Struhl, 2000).

A second factor appears to be the primary sequence of the transmembrane domain. Although all of the transmembane domains tested can be cleaved in a Presenilin-dependent fashion, the amount of cleavage varies. The transmembrane domains of Notch, beta-APP, and Sevenless all appear to be cleaved efficiently, whereas those of Torso, Delta, and GlycophorinA are less efficiently cleaved. Similarly, substitution or deletion of a conserved valine located immediately downstream of the likely S3 cleavage site reduces, but does not eliminate, cleavage in mammalian tissue culture, and evidence has been found in Drosophila. for a reduction in the efficiency of cleavage of such mutated or deleted forms of Notch. These findings suggest that Presenilin-dependent processing may be limited to some extent by the conformational state of the transmembrane domain, a property that is likely to depend on the primary sequence. Nevertheless, it remains striking that many different transmembrane domains, each with a distinct primary sequence, can be cleaved in a Presenilin-dependent fashion. Hence, the protease activity does not appear to require recognition of specific primary sequences (Struhl, 2000).

A third variable that appears to influence substrate specificity is the potential for oligomerization. The transmembrane domain of Glycophorin A, which dimerizes avidly in the membrane, is a relatively poor substrate, whereas a single amino acid substitution, which is expected to severely reduce dimerization of this transmembrane domain, renders it a better substrate for Presenilin-dependent cleavage. Similarly, the presence of an extracellular dimerization domain, a leucine zipper, severely reduces the efficiency of Presenilin-dependent cleavage compared to a control protein that carries a mutated and inactive zipper. Hence, the Presenilin-dependent cleavage reaction appears to work better on isolated monomeric proteins. It is not clear why oligomerization reduces the efficiency of Presenilin-dependent cleavage. One possibility is that the cleavage mechanism depends on the assembly of a protease complex that wraps around a single, isolated transmembrane domain. Another possibility is that oligomerization effectively increases the size of the extracellular domain. The inhibitory effect of oligomerization on Presenilin-dependent cleavage might also be important for stabilizing single-pass transmembrane proteins that normally have short extracellular domains, such as the zeta and eta chains of the T cell receptor CD3 signaling complex. Ligand may intially activate the receptor by inducing oligomerization, but cleavage of the ectodomain may in turn generate truncated proteins that can no longer oligomerize, helping to convert them into substrates for Presenilin-dependent cleavage (Struhl, 2000).

Finally, the amino-to-carboxyl polarity of the transmembrane domain of a protein may also govern whether it is a substrate for Presenilin-mediated cleavage. Notch, betaAPP, and the other proteins assayed are all type I transmembrane domains with amino-to-carboxyl polarity oriented in the extracellular-to-intracellular direction. In contrast, the first transmembrane domains of sterol regulatory element-binding proteins (SREBPs), which are cleaved in response to changes in sterol abundance, have the opposite polarity and do not appear to be Presenilin dependent. There is evidence that Presenilin-dependent cleavage depends on an aspartyl protease activity, perhaps Presenilin itself, and not the S2P metalloprotease, which appears responsible for the transmembrane cleavage of SREBPs. Perhaps these different proteolytic activities reflect distinct mechanisms involved in cleaving type I and type II transmembrane proteins (Struhl, 2000).

Both loss of expression and overexpression of Presenilin suggested a role for this protein in the localization of Armadillo/beta-catenin. In blastoderm stage Presenilin mutants, Arm is aberrantly distributed, often in Ubiquitin-immunoreactive cytoplasmic inclusions predominantly located basally in the cell. These inclusions are not observed in loss of function Notch mutants, suggesting that failure to process Notch is not the only consequence of the loss of Presenilin function. Human presenilin 1 expressed in Drosophila produces embryonic phenotypes resembling those associated with mutations in armadillo; embryos exhibit reduced Armadillo at the plasma membrane; this is likely due to retention of Armadillo in a complex with Presenilin. The interaction between Armadillo/beta-catenin and Presenilin 1 requires a third protein, which may be delta-catenin. These results suggest that Presenilin may regulate the delivery of a multiprotein complex that regulates Armadillo trafficking between the adherens junction and the proteasome (Noll, 2000).

Arm at the cell membrane is associated with E-cadherin, and the continued expression and function of E-cadherin are dependent on the presence of functional Arm. E-cadherin is encoded by the shotgun gene, and shotgun mutant embryos develop poorly formed cuticles due to a loss of cell adhesion. It was reasoned that, if the increased level of cytoplasmic Arm observed in the presence of overexpression of hPS1 correlates with a depletion of the membrane-associated pool of Arm, then overexpression of hPS1 should result in a shotgun-like cuticle phenotype. Indeed most hPS1 embryos that survive long enough to secrete cuticle exhibit phenotypes that resemble the loss of E-cadherin function. In embryos that develop more cuticular elements, additional phenotypes include loss of head structures, variable degrees of segmental fusion, and a 'dorsal open' phenotype. In a small subset of animals (~10%) that survive to secrete cuticle, a weak/moderate wingless-like cuticle phenotype is observed, and this phenotype can be correlated with a failure to maintain Engrailed expression (Noll, 2000).

Because Drosophila PS appears to have a role in the Notch pathway, embryos expressing hPS were labeled with Fas III to assess the integrity of the ventral ectoderm and to look for any indication of hypertrophy of the nervous system. When the ectodermal cells associated with the ventral midline were examined there was evidence of some disruption in patterning as indicated by the meandering of the midline, but the epidermis was found to be intact. Further, when the Fas III-positive cells in the nervous system of these same embryos were visualized the number and location of Fas III-positive cells were found to be normal. This indicates that the nervous system hypertrophy at the expense of ventral ectodermal cell fates that is found as a result of disruption in the Notch signaling pathway does not occur when UAS-hPS1 is expressed. Since Fas III can be found at the cell membrane, the localization of Fas III was examined. Compared to wild-type, there was no obvious mislocalization of Fas III as a result of UAS-hPS1 expression. This suggests that the mislocalization of Arm as a result of UAS-hPS1 expression is not due to some nonspecific effect, but rather reflects a specific interaction been Arm and hPS1 (Noll, 2000).

The resemblance to shotgun mutations of the cuticle phenotypes associated with hPS1 expression strongly implicates dysfunction in adhesion and cytoskeletal organization. To investigate the basis of this phenotype further, phalloidin staining, used to reveal the distribution of actin, was carried out in these embryos at stages before cuticle deposition. UAS-hPS animals that make it through cellularization usually fail to initiate and/or complete dorsal closure, a phenotype resembling that observed in some Arm mutations. At the stage when an accumulation of actin in the peripheral nervous system is clearly apparent, dorsal closure should be approaching completion. However, in the hPS1 animals, the leading edge cells do not undergo the proper change in shape, and there is no accumulation of actin along the dorsal most edge of the cells. Cells all along the dorsoventral axis of the epidermis fail to stretch and take on the proper thin, cuboidal shape. Alterations in the normal distribution of actin due to expression of hPS1 can also be observed in blastoderm stage embryos, concurrent with the mislocalization of Arm to the cytoplasm. In hPS1 animals, the overall amount of actin present at the membrane is greatly reduced, resulting in a thin, spotty phalloidin staining pattern in some regions of the embryo and often in a complete degeneration of the membrane structure in other regions. In areas where hexagonal arrays are not present, large patches of intense phalloidin staining are evident (Noll, 2000).

It is unknown whether the interaction between PS1 and beta-catenin is direct or requires additional factor(s). To address this issue, different mouse beta-catenin deletion constructs were prepared and tested for interaction with PS1. PS1 failed to coimmunoprecipitate with beta-catenin antibodies and beta-catenin did not coimmunoprecipitate with PS1 antibodies. Taken together, these observations suggest that some cofactor or posttranslational modification is required for beta-catenin to bind to PS1, and this cofactor is not present in the yeast two-hybrid or coimmunoprecipitation assays. Because delta-catenin binds directly to PS1, could delta-catenin outcompete beta-catenin from an in vivo complex with PS1 in CHO cells? The minimal delta-catenin interactive fragment requires both the last four Arm repeats and a portion of the carboxy terminal sequence just beyond the Arm repeats. Expression of the delta-catenin DEco fragment (residues 828-1127) almost completely displaces beta-catenin from the PS1 complex. These experiments support the hypothesis that beta-catenin interacts with the hydrophilic loop of PS1 via a third protein, which competes with the DEco fragment of delta-catenin for binding to PS1. The most parsimonious explanation for these observations is that beta-catenin associates with the complex via full-length delta-catenin itself, but is unable to do so in the presence of the DEco fragment alone. If this were the case, then full-length delta-catenin would not be expected to compete beta-catenin from the PS1 complex, as the DEco fragment of delta-catenin did. Furthermore, it should be possible to demonstrate a direct interaction between delta-catenin and beta-catenin. Indeed, in the presence of full-length delta-catenin, beta-catenin is retained in the PS1 complex, suggesting that delta-catenin is capable of mediating beta-catenin association with PS1 (Noll, 2000).

Thus, embryos derived from presenilin germline clone females exhibit mislocalization of Armadillo. These embryos contain cytoplasmic inclusions that are both Arm and Ubiquitin immunoreactive, suggestive of a failure to target Arm to a degradative pathway. A role for PS in regulating the degradation of proteins is suggested by other PS interactions. sel-12, the C. elegans ortholog of PS, interacts with sel-10, a member of the Cdc4p family which targets proteins for Ubiquitin-mediated turnover. Furthermore, the fly ortholog of Cdc41p, Slimb, may target beta-catenin for Ubiquitin/proteasome degradation. Also, the LEF/beta-catenin complex is thought to be affected in its translocation to the nucleus by mutations in PS. A genetic relationship between Drosophila PS and Arm is also suggested by a genetic modifier screen for mutations that can suppress the armadillo mutant phenotype. Together these observations implicate PS in a complex with beta-catenin as a means to target beta-catenin and possibly its cargo for degradation or other functions at remote sites in the cell. Thus the Presenilin/ beta-catenin complex may serve as an endoplasmic reticular staging platform for complex assembly and targeting to a variety of cellular destinations including the proteosome (Noll, 2000).

Before beta-catenin joins alpha-catenin and arrives at the plasma membrane, it forms a 'preadhesion' complex with Cadherin that is required for ER exit and membrane delivery of the complex. The delivery of Arm requires that the cell specify a polar trafficking route to the site of the adherens junctions at the apical part of the cell during blastoderm stages. The fact that many of these inclusions are located basally suggests impaired apical trafficking in the absence of PS. Although apparently reduced, sufficient Arm does reach the adherens junctions in these embryos so they do not develop an early adhesion defect phenotype. Instead, the phenotype includes a neurogenic defect thought to be related to the role of PS in cleaving Notch to generate an active product. The reports of the close resemblance between the Notch and PS phenotypes suggested a highly restricted function for PS: enhancement of Notch function by facilitating Notch cleavage. However, loss of Notch does not produce the Arm inclusions observed with loss of PS. This finding suggests a broader function for PS that extends beyond its role in Notch processing (Noll, 2000).

One site of residence for beta-catenin is in a complex with Axin, APC, and GSK3beta where it mediates regulation of Wnt signaling. Although quantitatively less frequent than the shotgun phenotype among embryos expressing hPS1, phenotypes that resemble the loss of wingless activity were occasionally observed, suggesting that binding to hPS1 also successfully competes beta-catenin away from its signaling pool. In conjunction with the evidence that PS is involved in Notch activation by releasing its cytoplasmic domain these findings suggest another link between the Wingless and Notch pathways. Previous studies reported genetic interactions between wg and N and direct interactions between these pathways via Dishevelled, as well as isolation of wg mutations in screens for genetic modifiers of Notch and vice versa. PS is primarily localized in the ER, but cleavage of Notch occurs either at or close to the cell surface. Transit of PS to the region of the adherens junction could resolve the contradiction between previous views regarding the location of PS in the endoplasmic reticulum and the cleavage of Notch either in or near the plasma membrane. PS is associated with two proteins -- beta-catenin and delta-catenin -- whose destination is the adherens junction. Both Notch and Wingless have also been reported in the region of the adherens junction. A large regulatory complex associated with PS may cleave Notch leaving the released cytoplasmic fragment to translocate to the nucleus and activate transcription or prevent transcription by binding through its carboxy terminus to Dsh. Dishevelled may independently localize to intracellular junctions through its discs large homology (DHR) region or utilize the PS complex to direct the Notch cytoplasmic fragment toward a degradative pathway (Noll, 2000 and references therein).

Alternatively, members of the PS complex such as beta-or delta-catenin may regulate the inhibitory interaction between Notch and Dishevelled. Because both of the putative substrates for PS (Notch and the amyloid precursor protein) transit through the ER, PS-associated proteins may serve in the ER to prevent premature cleavage. If PS cleaves Notch, it is curious that the expression of hPS1 does not induce a Notch activation phenotype. Neither was a Notch activation phenotype reported when the endogenous Drosophila PS was overexpressed. Significant inhibitory controls must be present that either prevent Notch cleavage or prevent the activity of the Notch active fragment. The interactions of beta-catenin with other proteins are complex and numerous. PS1 may delimit the components of the beta-catenin complex at specific cellular locales and allow it to discriminate among potential binding partners. In the case of the beta-catenin/APC complex, GSK3beta can phosphorylate both proteins. Although a direct association of GSK3beta with beta-catenin could not be demonstrated in vitro, it has been observed that Axin simultaneously and directly binds to APC, beta-catenin, and GSK3beta. The binding of all three proteins to axin may coordinate beta-catenin down-regulation by bringing these proteins into proximity (Noll, 2000).

GSK3beta binds PS1 between residues 259 and 298 of the fragment that is N-terminal after endoproteolytic cleavage. This site differs from the delta-catenin binding site on hPS1, which spans residues 319 to 371. Thus PS may coordinate the entry of both beta-catenin and GSK3beta into the complex with APC and Axin. Alternatively, PS may coordinate the trafficking routes of beta-catenin as it assembles and shifts large multicomponent protein complexes to diverse destinations in the cell (Noll, 2000).

The cleavage model for signal transduction by receptors of the LIN-12/Notch family posits that ligand binding leads to cleavage within the transmembrane domain, so that the intracellular domain is released to translocate to the nucleus and activate target gene expression. The familial Alzheimer's disease-associated protein Presenilin is required for LIN-12/Notch signaling, and several lines of evidence suggest that Presenilin mediates the transmembrane cleavage event that releases the LIN-12/Notch intracellular domain. However, doubt was cast on this possibility by a report that Presenilin is not required for the transducing activity of N^ECN, a constitutively active transmembrane form of Notch, in Drosophila. This finding has been reassessed and it has been shown instead that Presenilin is required for activity of N^ECN for all cell fate decisions examined. These results indicate that transmembrane cleavage and signal transduction are strictly correlated, supporting the cleavage model for signal transduction by LIN-12/Notch and a role for Presenilin in mediating the ligand-induced transmembrane cleavage (Struhl, 2001).

The classic Notch-mediated neurogenic interaction occurs during embryonic development, so that some cells in the 'proneural' portion of the ventral ectoderm segregate as neuroblasts, while the others remain in the ectoderm and eventually differentiate into the ventral epidermis. The absence of Notch activity results in neural hyperplasia at the expense of the epidermis, whereas constitutive Notch activity suppresses neuroblast segregation so that all ectodermal cells differentiate as epidermis (Struhl, 2001 and references therein).

Early neuroblast segregation can be readily visualized by the expression of the transcription factor Hb. During wild type development, the initial rounds of neuroblast segregations generate a stereotyped pattern of three anteroposterior columns of Hb-expressing neuroblasts on each side of the ventral midline. Early neural segregations also appear normal in embryos in which N⁺ is ubiquitously expressed from a transgene. In contrast, embryos lacking Notch activity form a broad swath of Hb-expressing neuroblasts in place of the normal pattern of three columns, whereas embryos in which constitutively activated forms of Notch (N^ECN or N^intra) are ubiquitously expressed, are found to lack Hb expression (Struhl, 2001 and references therein).

Embryos lacking maternal and zygotic Presenilin activity, referred to as PS^- embryos, resemble Notch^- embryos. This phenotype results from the absence of Notch signal transducing activity rather than from a marked decrease in Notch protein levels at the plasma membrane. The ability of N⁺, N^ECN, and N^intra to suppress neuroblast formation has been examined in PS^- embryos. Ubiquitous expression of N⁺ or N^ECN fails to suppress neuroblast segregations, so that such embryos appear indistinguishable from PS^- embryos. In contrast, ubiquitous expression of N^intra in PS^- embryos efficiently suppresses neuroblast segregations, as it does in otherwise wild-type embryos (Struhl, 2001).

The intracellular domains of N⁺-GV3 (wild type N) and N^ECN-GV3 do not gain access to the nucleus in PS^- embryos, in contrast to N^intra-GV3, which appears to have ready access. Thus, Notch nuclear access in PS^- embryos appears to correlate with Notch transducing activity: N^intra has access and retains constitutive transducing activity, whereas N^ECN and N⁺ lack access and show no evidence of transducing activity (Struhl, 2001).

Notch activity is required in several distinct processes during the development of the wing imaginal disc. The eponymous Notch phenotype is a notched wing, a consequence of reduced Notch-mediated signaling across the dorsoventral compartment boundary. Notch-mediated signaling also regulates classic neural/ectodermal decisions that control the pattern of mechanosensory bristles on the mesonotum (the dorsal portion of the fuselage of the adult thorax). Finally, Notch signaling is required to resolve thin stripes of wing vein cells from initially broader stripes of 'prevein' tissue, a process essential for normal vein development. The consequences of expressing N^ECN and N^intra in genetically marked clones of PS^- cells for each of these processes was examined. In all cases, in the absence of Presenilin, N^intra retains constitutive transducing activity, whereas N^ECN shows no evidence of transducing activity (Struhl, 2001).

Activation of Notch signaling across the dorsoventral compartment boundary in wing imaginal discs induces a thin stripe of 'edge cells' that straddle the boundary to express the target genes Cut and Wingless (Wg). Cut is a transcription factor that is required for differentiation of the edge cells and Wg is a morphogen that controls growth and patterning of the wing, including specification of the mechanosensory bristles that decorate the wing margin. Clones of cells that lack either Notch or Presenilin activity fail to express either Cut or Wg along the presumptive wing margin. The loss of Cut expression can be visualized in discs by antibody staining; furthermore, in adults, the loss of Wg signaling can be readily assayed morphologically by the presence of large wing notches. Conversely, clones of cells that express constitutively active forms of Notch, such as N^intra or N^ECN, ectopically express both Cut and Wg wherever they arise within the wing blade primordium. Ectopic expression of Wg in turn induces the formation of ectopic sensory mother cells (SMCs) in neighboring wing tissue and also causes ectopic wing outgrowths (Struhl, 2001).

Clones of PS^- cells that express N^ECN or N^intra, as well as a nuclearly localized form of Green fluorescent protein and the Yellow protein (which both allow adult structures to be genetically marked), were generated early during wing disc development by using the MARCM (Mosaic analysis with a repressible cell marker) technique and their effects on Cut expression and growth in the wing blade were assayed. PS^- clones expressing N^ECN that straddle the dorsoventral compartment boundary fail to express Cut. In addition, these clones are associated with severe notching of the adult wing, consistent with loss of Wg signaling. These phenotypes indicate that the constitutive activity of N^ECN in the developing wing depends on Presenilin activity (Struhl, 2001).

In contrast, the constitutive activity of N^intra does not require Presenilin activity. Clones of PS^- cells that express N^intra autonomously express Cut. In addition, they are associated with two phenotypes that indicate that they ectopically express Wg: (1) they induce ectopic wing margin bristles in neighboring wild-type cells; (2) they are associated with bulges in the disc epithelium suggesting excessive wing growth, a possibility confirmed by the behavior of the clones in the adult wing where they are associated with large outgrowths of wing tissue and ectopic rows of margin bristles formed by wild-type cells adjacent to the clone (Struhl, 2001).

During the development of the mesonotum, small 'proneural clusters' of ectodermal cells undergo Notch-mediated interactions so that one cell within the cluster becomes an SMC, whereas the others remain ectodermal. In the absence of Notch or Presenilin function, all cells of the cluster choose the SMC fate, so that a cluster of neurons is produced at the expense of the epidermis. Conversely, the constitutive activity of N^ECN or N^intra prevents any cell from choosing the SMC fate, thereby suppressing bristle formation. All of the SMCs can be marked by the expression of the smc-Z reporter gene, and a subset of these also expresses Cut. Presenilin activity is essential for the constitutive transducing activity of N^ECN during SMC specification. Clones of PS^- cells expressing N^ECN that arise within the mesonotum primordium cause clusters of SMCs to form in place of a single SMC. In contrast, no SMCs appear to segregate within clones of PS^- cells expressing N^intra or clones of PS^- cells expressing N^ECN which also carry the rescuing Tubulinalpha1-PS⁺ transgene. Thus, the constitutive transducing activity of N^ECN in this context also depends on Presenilin (Struhl, 2001).

Cells of initially broad 'provein' regions undergo Notch-mediated cell-cell interactions so that some cells become vein cells whereas the others become intervein cells. In the absence of Notch or Presenilin function, most or all provein cells become vein cells, so that the wing veins are abnormally thick; conversely, constitutive activation of the Notch pathway suppresses vein cell formation. Clones of PS^- cells that express N^ECN can contribute to the adult wing blade, provided that they do not cross the wing margin where Notch signal transduction is essential for activating Wg. Such clones cause a thickened vein phenotype indicating a failure of Notch signal transduction in the provein cells. Because N^intra-expressing PS^- cells as well as Tubulinalpha1-PS⁺ N^ECN-expressing PS^- cells strongly activate Wg expression and cause outgrowths composed primarily of surrounding, wild-type wing cells, whether they have the ability to differentiate as vein cannot readily be assessed. Nevertheless, the finding that N^ECN-expressing PS^- cells form abnormally thickened veins indicates that Presenilin is essential for N^ECN transducing activity in this context as well (Struhl, 2001).