Notch

Table of contents

Notch, Abl and Disabled

Abl is an axonal tyrosine kinase that has yet to be clearly linked to a receptor; Notch is a receptor for which the signaling pathway remains incompletely understood. Genes that interact synergistically with abl are collectively termed HDA loci (haploinsufficient, dependent on Abl). Similar synergistic genetic interactions are often found in genes whose products interact directly, such as the different constituents of multiprotein complexes. While it has not been shown directly that HDA loci encode proteins that associate with Abl, the sequence of Dab makes it a good candidate to bind to the Abl SH2 domain, and indeed the mouse Dab homolog binds to mouse Abl in vitro and to the closely related SRC SH2 domain in vivo. Similarly, the Abl-interacting gene ena is thought to encode a direct substrate of the Abl kinase. Notch and Abl mutations are shown to interact synergistically to produce synthetic lethality and defects in axon extension. These axonal aberrations cannot be accounted for on the basis of changes in cell identity, as the Notch/abl interaction is shown not to cause neurogenic or anti-myogenic phenotypes. Notch is shown to be present in the growth cones of extending axons, and the Abl accessory protein Disabled binds to a signaling domain of Notch in vitro. It is therefore speculated that Disabled and Abl may play a role in Notch signaling in Drosophila axons, perhaps by binding to the Notch intracellular domain (Giniger, 1998).

The gross morphology of the nervous system is typically normal in N/abl embryos, but specific axon tracts fail to develop. Axonal defects are observed in all of the nerve tracts that are known to require Notch, i.e., the CNS longitudinal tracts between neuromeres and the lateral portion of the ISN. In contrast, longitudinal tracts within each neuromere and commissural tracts appear normal, as does the dorsal and ventral portions of the ISN. The penetrance (fraction of embryos affected) and expressivity (number of affected hemisegments per affected animal) of the N/abl axonal phenotype depend on the particular combination of alleles used (Giniger, 1998).

In principle, the axonal defects observe in mature N/abl embryos could reflect a failure either to form axon tracts or to maintain them. Moreover, if the defect is in the initial development of the axon, it could be due to the absence of required substratum cells, the absence or improper identity of the neurons themselves, or else the failure of the actual guidance machinery of the growth cone. To discriminate among these possibilities, the development of pioneer neurons and substratum cells were examined directly for affected axon tracts. The initial extension of pioneer axons were examined in N/abl embryos. Consistent with the terminal phenotype, the combined MP fascicle, the first to form between successive neuromeres, is obviously aberrant from a very early stage (st 13). In contrast, both the anterior and posterior commisures appear to develop normally, as do the longitudinal tracts within the neuromeres. The substratum cells for affected axon tracts were examined. The MP fascicle projects between neuromeres on a specific Fasciclin II-expressing glial cell, LG5, and this is present in affected hemisegments. In the PNS, the direct cellular substratum for ISN extension in the lateral part of the embryo is a cluster of lateral peritracheal cells that lie along the trachea. Examination of stalled motor axons in an N/abl embryo shows that the nerve frays and stalls precisely as it attempts to grow along the trachea. Since substratum cells for affected axon tracts are present in N/abl embryos, the pioneer neurons themselves were examined. The positions and cell body morphologies of the sensory neurons in the PNS provide sensitive assays for the identities of these cells, and these typically appear to be wild type. The neuron aCC that pioneers the ISN and innervates the most dorsal muscle (muscle 1) is readily apparent in N/abl embryos. The neurons that pioneer the MP fascicle within the CNS are MP1, pCC, dMP2, and vMP2, and cells whose positions and axonal morphologies are appropriate for these cells can be seen in affected hemisegments of N/abl embryos (Giniger, 1998).

The observation of morphologically normal pioneer neurons and substratum cells in N/abl embryos is surprising, since perturbation of cell identity seems a priori to be the simplest explanation for the axonal defects in these embryos. Molecular markers for the development of affected pioneer neurons were therefore examined to determine whether their identities were disturbed in some more subtle way. The homeobox proteins Ftz and Eve are expressed in the pioneer neurons aCC and pCC, and changes in the expression of either protein disrupts the guidance of some axons. Eve expression is wild type in these cells in 98% of hemisegments of N^ts/Abl embryos and >85% of hemisegments of DfN⁸/+;abl/abl embryos. Ftz is wild type in these cells in 98% of hemisegments of N^ts/Abl embryos (Giniger, 1998).

Particularly telling tests of neuronal identity in the CNS of N/abl embryos are provided by analysis of Eve expression in the neuron RP2 and Odd protein expression in the pioneers of the MP fascicle. Notch controls the identities and projections of RP2 and of the MP2 progeny cells, and the effect of Notch on the fates of these cells can be assayed by their expression of Eve and Odd, respectively. These are, however, among the neurons whose axons are also affected by the Notch/abl interaction. Thus, if the axonal defects observed in N/abl embryos are due to Notch-dependent alterations of cell identity, one should be able to detect precisely these alterations by assaying the expression of Eve and Odd. In wild-type embryos, Eve is expressed in RP2 but not in its sibling cell (RP2sib). Eve expression is wild type in RP2 and RP2sib in 98% of hemisegments of N^ts/Abl embryos and >88% of hemisegments of DfN⁸/+;abl/abl embryos. At the time that the MP fascicle is pioneered, Odd protein is expressed in the MP1 and dMP2 neurons but not in vMP2 (the sibling cell to dMP2). Notch is responsible for differentiating the fates of dMP2 and vMP2. 97% of st 13/14 N^ts/Abl hemisegments have the proper pattern of Odd-expressing cells. Moreover, upon double staining a N/abl embryo with anti-Odd and anti-FasII, appropriate Odd staining is observed even in a hemisegment in which the MP fascicle has failed to develop. This argues directly against the model that the failure to form the MP fascicle in N/abl embryos arises from a Notch-dependent transformation in the identities of the dMP2 and vMP2 pioneer neurons. Similarly, the notion that PNS axonal defects in N/abl embryos might arise from a Notch-dependent interconversion of identity between sensory neurons and their sibling glia is inconsistent with the observation that the peripheral pattern of 22C10 expression (a marker for PNS neurons) and of Pros expression (a marker for sense organ glia) is generally unaffected by the N/abl interaction (Giniger, 1998).

The experiments above suggest that most of the axonal defects in Notch/abl embryos cannot be accounted for on the basis of observed transformations of pioneer neuron identity. The converse question was therefore asked: whether Notch-dependent transformations of pioneer neuron identity are sufficient to produce axonal defects like those observed in N/abl embryos. Indeed they are not. Embryos were prepared that were N^ts1;elav-GAL4;UAS-Notch and they were shifted to restrictive temperature in mid-embryogenesis. In these embryos, the endogenous Notch is inactivated by the temperature shift after the completion of neuroblast segregation but during the time when neuronal identities are still being specified and prior to axonogenesis. The GAL4 system then restores wild-type Notch to each neuron at about the time it begins to extend its axon, after its identity has been decided. The characteristic pattern of Notch-dependent axonal defects are found in >90% of N^ts1 embryos subjected to a standard temperature shift protocol. In contrast, the axon scaffold of the CNS is rescued to wild type or nearly wild type in >80% of N^ts1;elav-GAL4;UAS-Notch embryos. As assayed by staining with anti-FasII, 49% of embryos show rescue of longitudinal tracts in all hemisegments and 32% of embryos show residual defects in just a single hemisegment. In only 19% of cases do N^ts1;elav-GAL4;UAS-Notch embryos have CNS axonal aberrations that overlap in severity with those observed in the N^ts control. By monitoring the expression of Odd and Eve, it was verified that expression of wild-type Notch via elav-GAL4 does not rescue Notch-dependent defects in cell identity. In temperature-shifted N^ts embryos, 5.7 ± 1.6 Odd-positive neurons are found per neuromere, versus 4 Odd⁺ cells in wild type. By comparison, the number of Odd⁺ cells found in N^ts1;elav-GAL4;UAS-Notch embryos is 5.1 ± 1.2. Analogous results were found for the Notch-dependent transformation of RP2sib to RP2, as assayed with anti-Eve. These data show directly that the Notch-dependent perturbations of cell identity induced in temperature-shifted N^ts embryos are not sufficient to produce the axonal defects observed in these embryos. They therefore provide strong evidence that the requirement for Notch in axon patterning reflects a function of the protein at the time of axon outgrowth, genetically separable from the role of Notch in the establishment of cell identity (Giniger, 1998).

Abl is localized to developing axons: it is thought that Abl works in the axon directly to control cytoskeletal organization and function. Might Notch also act in the axon to control axon extension directly? Notch is known to be present in mature nerves, but its presence in developing nerves, and specifically in growth cones, has not been investigated. Since Notch expression in substratum cells interferes with visualizing growth cones in situ, the localization of Notch protein was examined in primary Drosophila neurons cultured in vitro. Primary fly embryo neurons were differentiated in culture and analyzed either by indirect immunofluorescence with anti-HRP, to characterize neuronal morphologies, or with anti-Notch. All samples were also labeled with anti-Elav, to verify that the cells being examined were neurons. Notch protein is clearly detected on the entire cell surface, including extending axons, and on a variety of bulbous, spiked, and flattened structures at the tips of axons, which have the appearance of growth cones. To test further whether the Notch-containing structures at the ends of axons are bona fide growth cones, cell preparations were double labeled for Notch and for a known growth cone marker, kinesin-ß-galactosidase. Notch protein is present on the growth cones of axons extending in culture (Giniger, 1998).

What might be the physical basis of the N/abl genetic interaction? It is unlikely that the absence of Abl is affecting Notch protein levels, since Western analysis of extracts from homozygous abl^- females detects wild-type amounts of Notch protein. Such a mechanism would be expected to alter Notch-dependent cell identities as well as cell morphologies, and this does not generally occur. Might Abl bind Notch directly? This seems unlikely. While Abl contains a variety of protein interaction domains, Notch does not resemble its known ligands. It has recently been shown that the Drosophila Numb protein includes a PTB domain that binds two sites in the intracellular domain of Notch, even when Notch is not phosphorylated. Recalling that the Abl-interacting gene Disabled includes a PTB domain closely related to the Numb PTB, and which like Numb can bind to nonphosphorylated targets, a test was performed to discover whether Dab can bind the intracellular domain of Notch in vitro (Giniger, 1998).

Three experiments demonstrate that the PTB domain of Drosophila Disabled binds directly to the intracellular domain of Notch in vitro. (1) First, beads bearing a glutathione S-transferase (GST) fusion of the Dab PTB domain were incubated in an extract of total embryo protein. Western analysis of the protein bound by Dab shows that GST-Dab selects Notch protein out of an embryo lysate, whereas GST alone binds only a small amount of Notch nonspecifically. (2) It was next asked what portion of Notch is recognized by Dab. Four protein fragments, each of which represents a distinct functional domain from the intracellular tail of Notch, were expressed. These are the RAM23 region (amino acids 1766-1896), the ankyrin repeats (amino acids 1896-2109), the PEST/OPA region (amino acids 2262-2606), and the notchoid region (amino acids 2612-2703). The four proteins were translated in vitro in reticulocyte lysates and assayed for binding to GST-Dab as above. Of the four Notch domains, only the RAM23 peptide binds to GST-Dab, while none of the four bind to GST alone. This pattern is similar but not identical to the pattern of Notch binding to the Numb PTB: like Dab, the Numb PTB binds to the Notch RAM23 domain but not to the ankyrin repeats or PEST/OPA region. Unlike Dab, Numb does bind to the notchoid domain. (3) Finally, to determine whether the Dab-Notch interaction is direct, a stable and soluble N-terminal fragment of the Notch intracellular domain (amino acids 1767-2235) was purifed from bacteria and its binding to the purified Dab PTB domain was assayed. The Notch intracellular domain is precipitated by GST-Dab beads but not by GST alone, demonstrating that the purified Dab PTB domain can bind directly to purified Notch intracellular domain in vitro (Giniger, 1998).

The data above demonstrate that Notch interacts genetically with Abl and biochemically with Disabled. These results beg the question whether Notch interacts genetically with disabled. Since isolated dab alleles were not available, their genetic interactions with Notch could not be tested directly. It can be asked, however, whether flies that are triply heterozygous for all three mutations, Notch, abl, and dab, display any synthetic phenotypes. Flies were constructed that are both heterozygous for a strong Notch allele (N⁸ or N^55e11) and for one of two unrelated chromosomes that bear strong mutations of both Abl and dab. All pairwise combinations cause defects in eye development, giving rise to flies with rough eyes reminiscent of the defective eyes observed in Abl homozygotes (Giniger, 1998).

Given the genetic and biochemical evidence for Abl-Dab interaction, it is attractive to speculate that Dab may act as an adaptor protein that links Notch to Abl in response to a signal from Delta. Recruitment of Abl by Notch would in turn engage the actin cytoskeleton via mechanisms similar to those that have been studied in vertebrate systems. The notion that Notch may use distinct signaling pathways to control different downstream events is consistent with analysis of other signaling receptors. For example, receptor tyrosine kinases typically bind and activate a complex array of intracellular signaling proteins upon ligand induction, and different downstream signaling pathways are often responsible for different aspects of the induced phenotype. Finally, there is extensive precedent for receptors that control cell fate in some developmental contexts and cell motility or axon extension in others (Giniger, 1998 and references).

Notch and Notchless

notchoid¹ (nd¹) is a viable mutant allele of Notch that causes scalloping of the wing. In a genetic screen for modifiers of Notch activity, searching for mutations that diminish the nd¹ phenotype, mutations in a gene encoding a novel WD40-repeat protein were identified. The gene, called Notchless Nle is conserved, with homologs apparent in Xenopus, mouse and humans. The sel-10 gene of C.elegans encodes a WD40-repeat-containing protein that modifies lin-12 function (lin-12 is a Notch homolog). Although SEL-10 and Notchless both contain WD40 repeats, they are not orthologs. Notchless has nine WD40 repeats rather than the seven repeats found in SEL-10, and does not contain the F-box that characterizes SEL-10 as a CDC4-related protein. SEL-10 does not share a conserved Nle domain in the N-terminus of Notchless. A different C. elegans predicted protein appears to be the ortholog of Notchless. Sequence comparison indicates that the degree of conservation in the N-terminal domain is quite high among the different family members. In the 80 amino acid region corresponding to residues 27-106 of Notchless, sequence identity ranges from 33% (between Drosophila and Saccharomyces cerevisiae) to 61% (between Drosophila and Xenopus proteins). Particular residues are identical in all species examined, suggesting that they may be important for domain structure. It is proposed that this region be called the Nle domain (Royet, 1998).

Notchless loss-of-function mutant alleles dominantly suppress the wing notching caused by nd¹ alleles. Reducing Notchless activity increases Notch activity. Overexpression of Notchless in Xenopus or Drosophila appears to have a dominant-negative effect in that it also increases Notch activity. Deltex is thought to function as a positive regulator of Notch activity. deltex mutant flies show a phenotype resembling a reduction of Notch activity: nicking of the distal region of the wing blade and thickening of the wing veins. Removing one copy of Notchless restores the deltex mutant wing to normal. Thus the effects of reducing deltex activity can be compensated for by simultaneously reducing Notchless activity. Likewise, removing one copy of Notchless enhances the effects of overexpressing Deltex using a heat-shock deltex transgene. These results suggest that Deltex and Notchless act in opposite directions. Biochemical studies show that Notchless binds to the cytoplasmic domain of Notch, suggesting that it serves as a direct regulator of Notch signaling activity (Royet, 1998).

How might Notchless act to reduce Notch activity? Genetic interactions suggest a possible link between Notchless and deltex. deltex mutants resemble weak Notch mutants, suggesting that Deltex helps to increase Notch activity. Deltex protein binds to the CDC10/Ankyrin repeats in the ICN1 domain of Notch, but does not bind to the ICN2 domain. Experiments using the yeast two-hybrid system have shown that Notchless, expressed as an activator fusion protein, binds to the ICN2 domain of Notch, but not to ICN1. This suggests that Notchless is likely to oppose Deltex function indirectly through an opposing activity on Notch, and not by direct competition for binding. Little is known about Deltex function, except that overexpression of Deltex can liberate Su(H) to translocate to the nucleus under conditions where Su(H) is artificially retained in the cytoplasm by binding to overexpressed Notch. It is possible that the balance between Deltex and Notchless activities in some way modulates processing of Notch. The observation that increasing or decreasing Nle has a similar effect on Notch activity raises the possibility that Nle forms a complex with proteins in addition to Notch. If the function of Nle is to bring other components together in a complex and if the level of any component other than Nle is limiting, it is possible that overexpression of Nle could reduce formation of the active complex by sequestering the limiting component(s) into incomplete or inactive complexes. This is easiest to imagine in a complex with several components, but it is also possible in tetramers of two components if a 1:1 stoichiometry is important for activity. Many other explanations could be proposed to account for the dominant-negative behavior of the overexpressed protein. It is worth noting that a similar phenomenon has been reported for Notch itself. Overexpression of wild-type Notch produces a phenotype of thickened veins that resembles that of reducing Notch or Delta activity. This is thought to occur by sequestration of Delta in cells overexpressing Notch, which reduces the ability of these cells to signal productively (Royet, 1998).

Notch, Presenilin and Nicastrin

Evidence has been found of a role for Drosophila Presenilin in Notch processing. Notch acts as a transmembrane cell-surface receptor for intercellular signals during development. It has been proposed that signal transduction involves cleavage and transport of the Notch intracellular domain to the nucleus. Results from Drosophila and mammalian cells indicate that cleavage occurs in or near the transmembrane domain. In mammalian cells, at least one proteolytic event occurs in the extracellular domain during Notch transit to the cell surface, and it has been suggested that ligand-binding might trigger additional extracellular proteolytic processing. Thus Notch proteins undergo proteolytic processing events that resemble the ß- and gamma-secretase cleavages of ß-APP. These parallels, as well as genetic studies of presenilin in C. elegans, indicate that the presenilins may promote proteolytic cleavage during receptor maturation or activation (Levitan, 1998b and references)

To investigate the involvement of presenilin in proteolysis of Notch protein, an in vivo assay was used for ligand-dependent cleavage and nuclear access of the intracellular domain of Drosophila Notch. To identify PS mutants, an examination was made of a collection of recessive-lethal mutations that map to the location identified for PS and that cause a neurogenic phenotype in genetic mosaics (J. Jiang, C.-M. Chen and G. Struhl cited in Struhl, 1999). Two independent mutations, PS^C1 anPS^C2, contain lesions in PS. Both alleles are predicted to cause premature termination and appear to be null alleles. To generate embryos with no PS activity, both maternal and zygotic PS activity were removed by generating PS minus embryos derived from PS minus germ cells. In both PS minus and Notch minus embryos, clusters of neuroblasts segregate at the positions normally occupied by single neuroblasts, as revealed by Hunchback staining. Both PS minus and Notch minus embryos also show extensive neural hyperplasia during subsequent development and die as pharate first-instar larvae lacking both dorsal and ventrical cuticle. In addition, the number of midline cells, as defined by the expression of Single-minded (Sim), is greatly reduced. Notch protein is found predominantly at the plasma membrane and at similar levels in both wild-type and PS minus embryos. Hence, the profound developmental defects in PS minus embryos appears to result from the absence of Notch signal-transducing activity, rather than from a marked decrease in Notch protein at the plasma membrane (Struhl, 1999).

The effect of PS null mutation on nuclear access by the Notch intracellular domain was examined by using three Notch proteins in which the chimaeric transcription factor Gal4-VP16 (GV) was inserted in-frame into Notch just after the transmembrane domain. Nuclear access was assayed by UAS-lacZ expression. N⁺-GV3 functions like the wild-type Notch protein and the intracellular domain gains nuclear access and has signal transducing activity only in the presence of the ligand, Delta. N^ECN-GV3 contains a deletion that removes most of the extracellular domain and causes constitutive signal transducing activity and nuclear access in the absence of Delta. N^intra-GV3 lacks the extracellular and transmembrane domains and also displays ligand-independent nuclear access. The key difference between the two constitutively active forms is that N^ECN-GV3 retains the transmembrane and extracellular juxtamembrane domains, whereas N^intra-GV3 is a cytosolic protein (Struhl, 1999).

In PS minus embryos, neither N⁺-GV3 nor N^ECN-GV3 has access to the nucleus, as indicated by the complete absence of ß-galactosidase (ß-Gal) expression. In contrast, the nuclear access of N^intra-GV3 is unaffected by the absence of presenilin activity. The N⁺-GV3 observation indicates that presenilin activity is normally required for the nuclear access of Notch intracellular domain. Furthermore, the observation that presenilin is needed for nuclear access of N^ECN-GV3, a constitutively active transmembrane form, but not for N^intra-GV3, a constitutively active cytosolic form, suggests that presenilin participates in the release of the intracellular domain from the plasma membrane. Only about 35 amino acids of the Notch extracellular juxtamembrane region remain in the N^ECN-GV3 protein. Thus, if there are specific signals required for presenilin-dependent cleavage, they are likely to be somewhere in this region or within the transmembrane domain (Struhl, 1999). Similar experiments by Y. Ye (1999) confirm these results.

Although these experiments demonstrate that presenilin is necessary for the ligand-dependent nuclear access of the intracellular domain of Notch, it is not known whether presenilin directly mediates proteolytic release of the intracellular domain or if it acts more indirectly, for example by activating a protease or mediating the protease's transit to the plasma membrane. These findings can be incorporated into a model of events involved in Notch signal transduction, in which ligand-binding activates Notch, thereby creating a substrate for presenilin-dependent release of the intracellular domain from the membrane. Although there are other possibilities, release could require the direct participation of presenilin in the proteolytic cleavage of Notch protein in or near the transmembrane domain. Presenilin may play an analogous role in the processing of ß-APP (Struhl, 1999). Experiments with mammalian Notch1 and PS1 show that the two proteins physically interact. The interaction predominantly occurs early in the secretory pathway, prior to Notch cleavage in the Golgi, because PS1 immunoprecipitation preferentially recovers the full-length Notch1 precursor. These results suggest that the genetic relationship between presenilins and the Notch signaling pathway derives from a direct physical association between these proteins in the secretory pathway (Ray, 1999a).

Mutant human presenilins cause early-onset familial Alzheimer's disease and render cells susceptible to apoptosis in cultured cell models. Loss of presenilin function in Drosophila increases levels of apoptosis in developing tissues. Moreover, overexpression of presenilin causes apoptotic and neurogenic phenotypes resembling those of Presenilin loss-of-function mutants, suggesting that presenilin exerts a dominant negative effect when expressed at high levels. In Drosophila S2 cells, Psn overexpression leads to reduced Notch receptor synthesis affecting levels of the intact ~300-kD precursor and its ~120-kD processed COOH-terminal derivatives. Presenilin-induced apoptosis is cell autonomous and can be blocked by constitutive Notch activation, suggesting that the increased cell death is due to a developmental mechanism that eliminates improperly specified cell types. A genetic model is described in which the apoptotic activities of wild-type and mutant presenilins can be assessed, and it is found that Alzheimer's disease-linked mutant presenilins are less effective at inducing apoptosis than wild-type presenilin (Ye, 1999).

The ability of wild-type and, to a lesser extent, mutant forms of Drosophila Psn to induce low levels of apoptosis similar to the apoptotic levels seen in developing imaginal tissues of Psn loss-of-function mutants suggests that the apoptotic effects of Psn may be a secondary consequence of reduced or dominant-negative Psn activity during developmental patterning. In C. elegans and mice, presenilin proteins have been shown to facilitate Notch signaling, and worms or mice lacking presenilin activity display typical Notch or lin-12/glp-1 loss-of-function phenotypes. Similarly, flies lacking functional Psn gene activity exhibit embryonic neurogenic phenotypes and imaginal disc phenotypes that are characteristic of impaired Notch signaling. Moreover, elevated levels of apoptosis have been noted previously in wing imaginal discs of flies having the partial loss-of-function heteroallelic Notch genotype N^ts/N^55e11; neur^A101/+. These observations raise the possibility that the apoptotic effects of PS overexpression may be due to a primary interference with Notch signaling, followed by elimination of cells that have not adopted their proper cell fate by a normal corrective mechanism of developmentally controlled apoptosis (Ye, 1999).

The in vivo genetic model for Psn-mediated apoptosis allowed for an examination of the potential involvement of Notch signaling in the apoptotic response, an important issue that has not been possible to assess in the widely used mammalian cell culture assays for Psn-induced apoptosis. First, it was determined if the UAS-Psn constructs that cause apoptosis in the Drosophila eye when driven by GMR-GAL4 are able to produce Notch pathway phenotypes in other tissues when expressed using suitable GAL4 driver constructs. Several GAL4 driver lines that are active in the wing and cuticle anlagen are indeed capable of producing adult Notch-like phenotypes in the wing blade and thorax, including wing margin notching, vein thickening, ectopic wing margin bristles, ectopic wing vein campaniform sensilla, ectopic thoracic macrochaetae, and missing thoracic microchaetae. These phenotypes are consistent with the notion that Psn overexpression leads to dominant-negative effects, since Psn loss-of-function mutants exhibit similar Notch-like phenotypes. To determine if Psn-induced apoptosis might be an indirect effect of reduced Notch activity, apoptosis was analyzed in imaginal wing discs of the conditional temperature-sensitive Notch mutant N^ts1. Increased levels of programmed cell death are spatiotemporally correlated with progressive loss of Notch activity as visualized by reduced wing-pouch-specific expression of the Notch target gene reporter vg(quadrant enhancer)-lacZ and expansion of proneural cell clusters positive for ac-lacZ expression in the presumptive notum region, suggesting that developmental patterning defects caused by reducing Notch activity directly lead to elimination of affected cells by apoptosis. The next step involved testing whether reduction in the dosage of the wild-type Notch gene or coexpression of constitutively activated Notch is able to suppress or enhance Psn-related apoptotic phenotypes. The rough eye phenotype of GMR-GAL4, 2X UAS-Psn+14 is strongly enhanced in an N^54l9 mutant background bearing only one functional copy of the Notch gene. Apoptosis caused by either Psn overexpression or removal of Psn gene function is also dramatically suppressed by coexpression of constitutively activated Notch in the retina. These studies show that genetic removal or overexpression of Psn in developing Drosophila tissues is able to induce Notch-like phenotypes, as well as apoptosis, and that when genetic methods are used to compensate for effects on Notch signaling, the levels of apoptosis are dramatically reduced. These results, together with the observed correlation between impaired Notch signaling and high levels of developmental apoptosis, offer a potential explanation of Psn-mediated apoptosis as a developmental response to a primary failure in cellular patterning events requiring Psn activity for proper Notch synthesis or signaling (Ye, 1999).

To further elucidate the molecular mechanism underlying the Psn overexpression phenotypes, Notch processing and trafficking was examined in S2 cells cotransfected with Psn and Notch. When expressed before Notch induction, wild-type Psn and the various Psn mutants lead to reduced Notch protein levels, affecting both the full-length and the processed COOH-terminal fragments of Notch. In agreement with genetic results, the biochemical effect appears to be more pronounced for wild-type Psn than for the mutant forms. The expression of a nonmembrane-bound control protein, Suppressor of Hairless, is not affected by either the wild-type or the mutant Psn proteins. The effect on Notch synthesis is unlikely to be due to increased protein degradation, because ectopic expression of the same Psn construct in S2 cells after Notch induction has no detectable effect on Notch protein levels. In addition, live cell surface immunostainings reveal that Notch protein is trafficked and inserted into the cell membrane normally in spite of the reduced protein levels caused by Psn overexpression (Ye, 1999).

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).

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).

Nicastrin is involved Notch signaling and along with Presenilin forms part of a large multiprotein complex. Drosophila nicastrin (nic) mutants display characteristic Notch-like phenotypes. Genetic and inhibitor studies indicate a function for Nicastrin in the gamma-secretase step of Notch processing, similar to Presenilin. Further, Nicastrin is genetically required for signaling from membrane-anchored activated Notch. In the absence of Nicastrin, Presenilin is destabilized and mature C-terminal subunits are absent. Partially processed Notch accumulates apically in nicastrin and presenilin mutant follicle cells. nicastrin and presenilin mutations disrupt the spectrin cytoskeleton, suggesting that the gamma-secretase complex has another function in Drosophila in addition to its role in processing Notch and the second target, ß amyloid protein precursor (APP). Nicastrin might recruit gamma-secretase substrates into the proteolytic complex as a prerequisite for Presenilin maturation and active complex assembly (Hu, 2002; López-Schier, 2002).

By isolating and characterizing nicastrin loss-of-function mutants in Drosophila, it has been demonstrated that Nicastrin is required both genetically and biochemically for proteolysis of membrane-tethered forms of Notch and associated Notch signaling activity. The Drosophila nicastrin mutants exhibit defects in Notch signaling and intramembranous cleavage that are indistinguishable from those seen in presenilin mutants. Notch-like phenotypes are observed in various tissues at all stages of development, and may be attributed to a specific failure in the gamma-secretase-like cleavage of Notch that normally generates an ~120 kDa intracellular signaling fragment from the activated receptor. Identical biochemical effects are observed for Drosophila Notch with the gamma-secretase inhibitor DFK-167, supporting the role of Nicastrin in this proteolytic event and suggesting that Drosophila Notch processing might be a useful model for investigating pharmacological inhibitors of gamma-secretase. Analysis of Notch immunoreactivity in genetic mosaics reveals that homozygous loss of nicastrin activity results in a subtle overaccumulation of Notch proteins at the apical intracellular surface. Similar observations have been reported for Drosophila Psn mutants, and are consistent with the idea that nic and Psn are necessary for cleavage and release of membrane-anchored Notch C-terminal fragments from the cell surface (Hu, 2002).

The proteolysis of Notch was examined in nic mutant tissues, since Presenilin is required for proteolytic activation of the Notch receptor during signaling. Notch is synthesized as an ~300 kDa full-length protein, the majority of which is cleaved at a lumenal site by furin proteases within the trans-Golgi network. As a result, most mature Notch receptor at the cell surface is a heterodimer consisting of a large ~200 kDa N-terminal extracellular EGF-homologous region joined noncovalently to a smaller C-terminal Notch subunit containing a short extracellular stalk, the transmembrane domain, and the intracellular domain. Activation of this heterodimeric receptor is accompanied by removal of the extracellular domain by TNFalpha-converting enzyme (TACE)-mediated proteolysis at an extracellular site located just beyond the transmembrane domain or by subunit dissociation under some experimental conditions. In Drosophila, the TACE-related metalloprotease Kuzbanian is thought to execute this postfurin second cleavage. This event generates a membrane-anchored C-terminal segment of Notch, containing an intact intracellular domain and a short extracellular stub. This product is then efficiently cleaved within the membrane by a Presenilin-associated gamma-secretase-like activity, thereby liberating a soluble intracellular Notch fragment that translocates to the nucleus and activates transcription of Notch target genes (Hu, 2002 and references therein).

Notch proteins from nic mutants, Psn mutants, and wild-type animals were subjected to Western immunoblot analysis following extraction under gentle hypotonic shock conditions, which allows the selective resolution of the mature processed forms of Notch that comigrate as an ~120 kDa smear under harsher SDS-urea extraction conditions. This protocol also results in efficient ligand-independent activation of Notch, presumably due to dissociation of the extracellular Notch subunit at low ionic concentrations. As is the case with numerous studies involving ligand-independent truncated forms of Notch, the increased Notch activation achieved in this manner facilitates the analysis of Notch processing by generating relatively high levels of gamma-secretase-derived Notch polypeptides. All four nic loss-of-function mutations result in the complete absence of a faster migrating ~120 kDa major C-terminal fragment of Notch (DNIC-2) and overaccumulation of a more slowly migrating C-terminal ~120 kDa fragment (DNIC-1). A third ~120 kDa C-terminal fragment (DNIC-3) that migrates slightly faster than DNIC-2 is unaltered in the nic mutants, relative to wild-type. These effects are identical to those observed with homozygous loss-of-function mutations in the Drosophila Psn gene. Equivalent results were obtained using a cultured Drosophila Schneider-2 (S2) cell assay in which Psn and nic function were selectively eliminated by RNA-mediated interference (Hu, 2002).

To determine which of the three ~120 kDa C-terminal fragments corresponds to the gamma-secretase-generated signaling product of Notch, the inhibition profile of each band was characterized in S2 cells using a variety of pharmacological compounds and expression constructs. Mutagenesis of the putative furin cleavage site in the Notch expression construct, coexpression of a dominant-negative Kuzbanian construct, or treatment of the cells with Brefeldin A all blocked production of DNIC-1 and -2 with no observable effects on DNIC-3. Notch immunoreactivity was undetectable at the extracellular surface of the Brefeldin A-treated cells. Conversely, DNIC-3 alone is blocked by treatment of the cells with a broad spectrum of protease inhibitors that have no discernible effect on DNIC-1 and -2. These results suggest that DNIC-1 most likely corresponds to the C-terminal fragment of Notch produced by furin-mediated cleavage and/or the slightly smaller fragment thought to be generated following subsequent Kuzbanian cleavage. The data do not allow for the unambiguous identification of DNIC-1, because the dominant-negative Kuzbanian protein might interact with the extracellular region of Notch containing the nearby furin and Kuzbanian cleavage sites, interfering with both processing events. Since DNIC-2 shows a similar inhibition profile as DNIC-1, but is specifically blocked by loss of Psn or nic function with a corresponding overaccumulation of DNIC-1, DNIC-2 appears to be derived from DNIC-1. The inference that DNIC-2 is generated by gamma-secretase-like cleavage of DNIC-1 was confirmed by the finding that the appearance of DNIC-2 is specifically blocked by treatment of S2 cells with the pharmacological gamma-secretase inhibitor DFK-167. Unlike DNIC-1 and -2, DNIC-3 appears to be derived from a different Notch processing pathway that is independent of Brefeldin A-sensitive anterograde protein transport through the ER and Golgi compartments (Hu, 2002).

To confirm these results using a genetic assay, the ability of constitutively activated forms of Notch to signal in the absence of nic gene function was examined. A soluble intracellular C-terminal fragment of Notch lacking all extracellular and transmembrane sequences [N(intra)] possesses intrinsic signaling activity and suppresses neural precursor cell specification independent of both Notch ligand and Presenilin. In contrast, a membrane-anchored C-terminal fragment of Notch lacking only the extracellular domain (DeltaECN) also signals constitutively in the absence of ligand, but requires Presenilin activity for intramembranous cleavage and liberation of the signal-transducing intracellular Notch domain. The effects of N(intra) and DeltaECN on SOP formation in larval imaginal wing discs of wild-type flies and nicastrin mutant flies was assessed using immunostaining for the proneural antigen Scabrous to monitor SOP differentiation. N(intra) displays nearly complete SOP inhibition in both wild-type and nic mutant flies, whereas DeltaECN blocks SOP formation only in wild-type animals and has no detectable inhibitory effect on SOP formation in the nic mutants. Identical results were confirmed for DeltaECN and N(intra) in Psn mutants. The similar genetic requirements for Presenilin and nicastrin during signaling by membrane-anchored DeltaECN, but not by the soluble intracellular domain N(intra), implies that Nicastrin is needed specifically for the Presenilin-associated intramembranous cleavage of Notch during signaling, consistent with biochemical studies with nic mutants and RNAi-treated S2 cells (Hu, 2002).

The presenilins and nicastrin, a type 1 transmembrane glycoprotein, form high molecular weight complexes that are involved in cleaving the beta-amyloid precursor protein (betaAPP) and Notch in their transmembrane domains. The former process (termed gamma-secretase cleavage) generates amyloid beta-peptide (Abeta), which is involved in the pathogenesis of Alzheimer's disease. The latter process (termed S3-site cleavage) generates Notch intracellular domain (NICD), which is involved in intercellular signalling. Nicastrin binds both full-length betaAPP and the substrates of gamma-secretase (C99- and C83-betaAPP fragments), and modulates the activity of gamma-secretase. Nicastrin is shown in this study to bind to membrane-tethered forms of Notch (substrates for S3-site cleavage of Notch), and, although mutations in the conserved 312-369 domain of nicastrin strongly modulate gamma-secretase, they only weakly modulate the S3-site cleavage of Notch. Thus, nicastrin has a similar role in processing Notch and betaAPP, but the 312-369 domain may have differential effects on these activities. In addition, the Notch and betaAPP pathways do not significantly compete with each other (Chen, 2001).

To determine where Nicastrin (Nct) acts in the Notch signal transduction pathway, advantage was taken of transgenic Notch constructs that bypass some of the processing steps required for signaling. (1) hs-N^FL was used as a control; it expresses full-length Notch protein under the control of a heat shock promoter. Like endogenous Notch, the protein from this construct requires both the ligand-induced S2 cleavage [this involves cleavage of the extracellular domain of Notch to produce a transient form of the receptor called NEXT (Notch extracellular truncation)] and the Psn-dependent S3 cleavage (an intramembranous cleavage to release the intracellular domain of Notch, which translocates to the nucleus, where it acts as a transcriptional activator in association with Suppressor of Hairless protein. (2) hs-N^ECN is a deletion of the extracellular domain of Notch beyond the S2 cleavage site, and therefore mimics NEXT. This protein signals independently of ligand, but still requires the S3 cleavage to release the intracellular domain of Notch from the membrane. (3) hs-N^ICD expresses the intracellular domain of the receptor and requires neither ligand, the S2 nor S3 cleavages, for signaling. The expression of hs-N^FL has no effect on the development of wild-type embryos, whereas hs-N^ECN and hs-N^ICD disrupt germ band retraction because their ligand-independent signaling overactivates the Notch pathway. To test which of these constructs requires Nct for signaling, each was expressed in embryos that lack both maternal and zygotic Nct activity. hs-N^ECN and hs-N^FL have no effect on the neurogenic phenotype of nct null embryos. In contrast, hs-N^ICD expression strongly rescues the nct phenotype: most embryos form patches of normal cuticle, and in some cases, the wild-type cuticular pattern is almost completely restored. Thus, the nct null mutation blocks Notch signaling after the S2 cleavage but before the release of the intracellular domain from the membrane, indicating that Nct is required for the S3 cleavage, as is Psn (López-Schier, 2002).

Presenilins and the human Nct associate with Notch during its passage through the secretory pathway, raising the possibility that they also function in earlier steps in Notch processing. Since the experiments above only reveal the last step at which these proteins are required, the behavior of Notch protein was followed in the follicle cells, where the downregulation of Notch in response to Delta binding can be visualized. Notch accumulates on the apical side of these cells until stage 7 of oogenesis, when Delta signals to trigger its proteolysis. Thus, Notch disappears from the apical membrane of cells that contact wild-type germline cells, whereas high levels of apical Notch persist in follicle cells that contact Delta germline clones, and this can be detected with antibodies against both the extracellular (N^ECD) and intracellular domains (N^ICD) of the protein. This reduction of apical Notch staining in response to Delta still occurs in Su(H) mutant follicle cell clones, in which Notch signaling in the nucleus is blocked. Therefore the disappearance of most Notch from the apical membrane appears to be a direct consequence of ligand-induced processing, and is not due to downregulation of the receptor in response to the activation of the signaling pathway. In nct and psn mutant follicle cells, apical N^ECD staining disappears at stage 7 as it does in wild-type, indicating that the ligand-dependent S2 cleavage and subsequent degradation of N^ECD occur normally. Unlike wild-type cells, however, mutant cells accumulate a processed form of Notch that can be stained with the anti-N^ICD antibody, and this is concentrated at the apical side of the cells and in intracellular clusters that may be endocytic vesicles. Thus, nct or psn mutant cells can transport Notch to the plasma membrane and process it to form a functional receptor that binds Delta and undergoes the S2 cleavage, indicating that both proteins are specifically required for the S3 cleavage, but not for any earlier steps in the pathway (López-Schier, 2002).

The processed form of Notch that accumulates on the apical side of nct and psn mutant follicle cells presumably corresponds to NEXT, which is the membrane-tethered product of the S2 cleavage and the substrate for the S3 cleavage. To test this possibility, Western blots of extracts from wild-type and embryos lacking maternal and zygotic Nct or Psn were probed with the anti-N^ICD antibody. In addition to the 300 kDa band that corresponds to the uncleaved form of Notch, nct and psn null embryos accumulate a 120 kDa species that is barely detectable in the wild-type extracts. This band migrates at the expected size of NEXT and N^ICD, which cannot be distinguished on these gels as they differ by only 3 kDa. This band is unlikely to be N^ICD, however, because the results above demonstrate that nct and psn block the S3 cleavage (López-Schier, 2002).

To investigate whether Nct is required for the localization or stability of Psn, nct^agro clones of varying sizes were generated in the follicular epithelium. In wild-type follicle cells, Psn protein shows a punctate distribution in the cytoplasm that may correspond to the endoplasmic reticulum, and it localizes at the cell cortex. When nct clones are analyzed early in oogenesis, the mutant cells show a normal distribution of Psn, and this is also the case for small clones in late-stage egg chambers. In contrast, very large clones in late-stage egg chambers show a strong reduction in the levels of Psn. Thus, Nct seems to be required for the long-term stability of Psn in these cells. Since small nct clones show a completely penetrant Notch loss-of-function phenotype but have normal levels of Psn, this effect of Nct on Psn stability is unlikely to cause the defect in Notch signaling in these cells (López-Schier, 2002).

During an analysis of follicle cell clones, an additional function of Nct and Psn in the organization of the submembranous spectrin cytoskeleton was discovered. In wild-type cells, ß_Heavy-spectrin (ß_H-spectrin) associates with alpha-spectrin to form tetramers that localize to the apical membrane, while the basolateral spectrin cytoskeleton is composed of ß-spectrin/alpha-spectrin complexes. In psn and nct follicle cell clones, ß_H-spectrin does not localize to the apical membrane. In contrast, ß-spectrin localization to the basolateral membrane is unaffected in mutant clones. The apical localization of ß_H-spectrin requires its association with alpha-spectrin and vice versa, and the distribution of alpha-spectrin was therefore examined in follicle cell clones. Surprisingly, both psn and nct mutant cells show an increase in the amount of cortical alpha-spectrin, with the highest levels on the apical side. alpha-spectrin therefore appears to be recruited to the apical membrane of mutant cells by a novel mechanism that does not require its association with either ß subunit, neither of which localizes apically in these cells (López-Schier, 2002).

To test whether the mislocalization of alpha- and ß_H-spectrin in nct and psn mutants is a consequence of the defect in Notch signaling, the localization of both proteins was examined in follicle cell clones of a null allele of Notch (N^55e11) and in wild-type cells that abut Delta mutant germline clones. ß_H-spectrin is recruited normally to the apical membrane in both cases, while alpha-spectrin localizes uniformly around the cell cortex at the same level as in wild-type cells. These results indicate that Psn and Nct have a novel function independent of their role in Notch signaling that somehow affects the organization of the spectrin cytoskeleton (López-Schier, 2002).

The absence of apical ß_H-spectrin in nct and psn mutant cells suggested that their apical-basal polarity might be disrupted, and the localization of a variety of other polarity markers was therefore examined. The overall polarity of the cells is unaffected in either nct or psn mutant clones, since Coracle, Neurotactin, DE-Cadherin, Armadillo (ß-catenin), alpha-catenin, and Notch itself are localized to the proper membrane domains. However, DE-Cadherin, Armadillo, and alpha-catenin accumulate to much higher levels in mutant cells than in wild-type. These three proteins are components of the Cadherin adhesion complex, and are enriched at the sites of adherens junction formation at the apical margins of the cell. Their overaccumulation in mutant cells may therefore be linked to the loss of apical ß_H-spectrin and the apical enrichment of alpha-spectrin, although the nature of this link remains unclear (López-Schier, 2002).

Previous studies have shown that almost all Psn is associated with the ER and intermediate compartment and that there is little or no protein at the plasma membrane, where APP and Notch reside. This discrepancy, which has been called the 'spatial paradox,' raises the question of where in the cell the S3 cleavage occurs. One possible solution to this paradox is suggested by the observation that small amounts of Psn can be coimmunoprecipitated with Notch at the cell surface. Both Nct and Psn have been shown to interact with Notch in the secretory pathway, and a minor fraction of the S3 protease complex could therefore be transported to the plasma membrane through binding to its future substrate. A second possibility is that the active protease resides in an intracellular compartment, and that the products of S2 cleavage of Notch are internalized and transported to this site. This analysis provides strong evidence for the first model. Since Nct and Psn are required for the S3 cleavage of Notch, the substrate for this cleavage, NEXT, accumulates in mutant cells. Most NEXT remains closely associated with the apical membrane, arguing against the existence of a major transport pathway to an intracellular compartment. Although a small amount of NEXT is found in intracellular clusters, these do not correspond to the major sites of Psn localization, and may be endocytic vesicles. Furthermore, recent data indicate that endocytosis is not required for the S3 cleavage of NEXT, because a membrane-tethered Notch derivative lacking the extracellular domain can signal normally in shibire mutant embryos, in which Dynamin-dependent endocytosis is blocked. Taken together, these results strongly suggest that S3 cleavage occurs predominantly at the plasma membrane (López-Schier, 2002).

Modeling clinically heterogeneous Presenilin mutations with transgenic Drosophila

To assess the potential of Drosophila to analyze clinically graded aspects of human disease, a transgenic fly model was developed to characterize Presenilin (PS) gene mutations that cause early-onset familial Alzheimer's disease (FAD). FAD exhibits a wide range in severity defined by ages of onset from 24 to 65 years. PS FAD mutants have been analyzed in mammalian cell culture, but conflicting data emerged concerning correlations between age of onset and PS biochemical activity. Choosing from over 130 FAD mutations in Presenilin-1, 14 corresponding mutations at conserved residues were introduced in Drosophila Presenilin (Psn) and their biological activity in transgenic flies was assessed by using genetic, molecular, and statistical methods. Psn FAD mutant activities were tightly linked to their age-of-onset values, providing evidence that disease severity in humans primarily reflects differences in PS mutant lesions rather than contributions from unlinked genetic or environmental modifiers. This study establishes a precedent for using transgenic Drosophila to study clinical heterogeneity in human disease (Seidner, 2006).

Presenilin is an evolutionarily conserved polytopic membrane protein that is part of the multisubunit γ-secretase complex responsible for intramembranous cleavage of several transmembrane proteins, including APP, Notch, Delta, DCC, ErbB4, N-Cadherin, and E-Cadherin. Familial Alzheimer's disease (FAD) mutations in Presenilin-1 (PS1) and PS2 alter proteolytic processing of APP to generate more toxic Aβ42 peptides that accelerate amyloid plaque formation in brain tissues. PS mutations also contribute to neurodegeneration and cognitive decline through amyloid-independent mechanisms, involving altered regulation of receptor signaling and intracellular kinase pathways (Seidner, 2006).

The Drosophila APP ortholog, APPL, lacks homology to APP within the Aβ peptide region, and loss of APPL produces subtle behavioral deficits that are difficult to measure quantitatively. A more suitable substrate to monitor Presenilin (Psn) FAD mutant activity in Drosophila is the Notch receptor, the most extensively characterized fly γ-secretase substrate. Psn is required throughout Drosophila development for Notch signaling, and a wide variety of Notch-related phenotypes exist that range from severe embryonic lethality to specific defects in adult tissues. This phenotypic range is amenable to characterizing variable degrees of function among different Psn FAD mutants. In addition, genetic and molecular reagents are available to characterize Notch biochemical cleavage and subsequent target-gene activation in Drosophila (Seidner, 2006).

Previous studies have successfully used transgenic mice and C. elegans to assess the genetic properties of human PS FAD mutants, and one study suggested a potential difference between two FAD variants. However, quantitative comparison between FAD mutants was not practical because of heterologous expression methods and difficulties controlling transgene copy numbers. Two studies in mammalian cell culture showed little or no relationship between average ages of onset and Aβ42 secretion levels, contrary to one study reporting a strong correlation (Seidner, 2006).

This study assessed PS FAD mutant function in Drosophila by expressing 14 FAD-linked mutant psn transgenes in animals lacking endogenous psn function. The mutations were selected on the basis of the following criteria: (1) they span the entire protein, (2) relatively large numbers of families and affected individuals have been identified for most mutations, and (3) their ages of onset range from 24 to 65 years, or are possibly asymptomatic (E318G and F175S). Thirteen of the mutations affect residues that are conserved in fly Psn, and the remaining one alters a conservatively substituted residue. To achieve physiological expression of the transgenes, a 1.5 kb promoter of the endogenous psn gene was defined, termed PEPC (Presenilin endogenous promoter cassette), . At the larval-pupal transition, loss of psn function causes highly uniform lethality that is fully rescued by a wild-type PEPC-psn transgene but not by wild-type human PS1 or PS2 transgenes driven by PEPC. To circumvent this technical problem with human PS transgenes, each FAD mutation was engineered into the fly psn gene (Seidner, 2006).

Examining transgenic lines expressing PEPC-psn FAD mutants in a psn null genetic background, eight distinct phenotypic categories were defined that represent a graded series in order of increasing rescue ability, as follows: (1) prepupal lethal, (2) late prepupal lethal, (3) pupal lethal, (4) pharate lethal, (5) severe neurogenic/adult lethal, (6) moderate neurogenic/adult viable, (7) weak neurogenic/adult viable, and (8) morphologically normal. Quantitative analysis of survival rates, incidence of dorsoscutellar bristle duplications, wing notching, wing vein defects, and other morphological features was performed to assign each transgenic line to the appropriate rescue category (Seidner, 2006).

The phenotypic categories are consistent with an ordered series attributable to progressive increases in Psn-dependent Notch activation, but it was important to verify that they do not instead represent the effects of Psn on other substrates. In the canonical Notch pathway, ligand binding to the Notch receptor leads to ectodomain removal and subsequent γ-secretase-mediated intramembranous cleavage of Notch. The liberated intracellular domain, termed NICD, translocates to the nucleus and participates directly in transcriptional regulation of target genes, including the Enhancer of split m7 (E(spl)m7) gene in Drosophila. Activation of Notch signaling within proneural cell clusters destined to give rise to adult sensory organs results in restricted expression of the proneural marker scabrous (sca). Visualization of sca expression in the larval imaginal wing disc with a sca-lacZ transgene provides evidence for a progressive range of Notch signaling with different PEPC-psn transgenes (Seidner, 2006).

Biochemical evidence for progressively increasing Psn function was obtained by immunoblot analysis of Notch cleavage products across the spectrum of PEPC-psn FAD phenotypes. Representative PEPC-FAD transgenic lines of the different categories exhibit partial-to-complete failure in γ-secretase-mediated NICD production. Interestingly, although transgenic lines belonging to categories 2–4 exhibit significant levels of biological rescue activity, they do not produce levels of NICD detectable by immunoblot analysis. These results demonstrate that genetic assays such as these are valuable for studying low-level or tissue-specific aspects of γ-secretase substrate cleavage that might not be amenable to biochemical analysis (Seidner, 2006).

To determine whether the PEPC-psn FAD phenotypic series is similarly correlated with Notch target-gene activation, semiquantitative RT-PCR was used to monitor activation of the E(spl)m7 gene. Normalizing transcriptional activation to two control genes, a progressive increase in E(spl)m7 transcript levels was observed across categories 1-8, confirming that they represent graded increases in γ-secretase-dependent Notch activation. To verify this relationship, a temperature-sensitive Notch allele termed N^ts1 was used. Incremental levels of Notch activity obtained by raising N^ts1 flies at different temperatures produced phenotypes matching those seen in the Psn FAD mutant series, but were independent of manipulations involving endogenous or transgenic Psn. Levels of E(spl)m7 transcriptional activation seen in these N^ts1 phenotypic classes resembled closely those observed in the corresponding PEPC-psn FAD classes, confirming the progressive increase in Notch target-gene activation across this phenotypic spectrum. Taken together, these results validate the use of the phenotypic criteria to characterize varying degrees of PEPC-psn FAD transgene activity toward the endogenous γ-secretase substrate Notch (Seidner, 2006).

A limitation of transgenic analysis in Drosophila is that the transgenes are inserted at essentially random genomic locations, leading to variations in expression as a result of local position effects at different sites. These effects are a confounding factor in comparisons of relative degrees of rescue function, which reflects properties of the primary lesion in each Psn mutant protein as well as the expression level of each transgenic insertion. Therefore, from four to 16 independent insertions were generated for each transgene, and each insertion was scored with multiple quantitative morphological criteria and assigned to the appropriate phenotypic category. Visual inspection of the results suggests a positive overall trend between increasing age of onset in human FAD pedigrees and increasing biological activity for these FAD mutants in transgenic Drosophila (Seidner, 2006).

Statistical tests demonstrate an overall correlation across the dataset as a whole, but the correlation is not smooth. Most FAD mutants appear to cluster in groups separated by discontinuities, with a few mutants that are noticeably different from nearby mutants. Mutants having similar ages of onset are, in general, not statistically different from one another. Nevertheless, the mutants can be statistically grouped into three distinct classes: strong (L166P, L173W, P436Q, V272A, and L235P), intermediate (M146L, M139V, H163R, E280A, A246E, and G206A), and weak (A79V, F175S, and E318G). Strong mutants differ most clearly from the weak ones in terms of function, whereas the intermediate group shows more modest differences compared to either of these two flanking groups. Pairwise comparisons of aggregated mutant groups confirmed these classes. The strong mutant group is significantly different from both the intermediate and weak groups. Despite its age of onset of ~60, the A79V mutant is more similar to the weak mutants than the intermediate ones, as confirmed by pairwise comparisons of aggregated intermediate and weak groups with A79V assigned to either the intermediate or the weak group (Seidner, 2006).

Similarly, two mutants, L235P and P436Q, are less functional in transgenic flies than might be predicted on the basis of their ages of onset, whereas another mutant, M139V, is somewhat more functional than expected. Whether these disparities reflect inadequate age-of-onset data, modifier effects in the corresponding human pedigrees, or an unknown feature of the fly assay system is not clear. Finally, the E318G and F175S mutants, proposed to be either very weak pathogenic mutants or functionally normal polymorphisms, are statistically indistinguishable from the wild-type transgene in the genetic assay, consistent with the idea that they are nonpathogenic polymorphisms. Overall, the results support the assertion that disease severity in early-onset Alzheimer's disease is primarily determined by PS mutant lesion type as opposed to unlinked genetic or environmental modifiers, as was also deduced from a study of Aβ secretion levels in PS FAD mutant transfected cells (Seidner, 2006).

A few other interesting patterns emerge from these comparisons. Mutants for which relatively few independent lines were obtained, most notably H163R, fail to show significant differences when compared to other mutant groups. On the basis of the complete dataset, it is estimated that ~10 independent lines of a given mutant are required to obtain statistically useful data. This problem might be circumvented by employing a 'knock in' strategy to precisely replace the endogenous psn gene with FAD variants, an approach that is not yet reliable in Drosophila. Additionally, one source of experimental noise is that transgenes occasionally insert into locations where they are poorly expressed or damaged during insertion, as is evident from a few instances involving the 'asymptomatic' F175S and E318G mutant and wild-type transgenes. Normalization of functional read-outs for each transgene insertion relative to its mRNA or protein expression level in the appropriate transcript null or protein null psn mutant background should reduce this noise and lead to further refinement of the statistical data (Seidner, 2006).

These findings establish the validity of using transgenic Drosophila or other heterologous organisms to evaluate clinically heterogeneous aspects of human diseases with a clearly defined genetic etiology. Transgenic Drosophila offer several advantages to augment more traditional clinical assessments as well as transgenic mouse models. Transgenic flies are relatively inexpensive and rapid to produce, large numbers of independent lines can be easily generated, and limitless numbers of progeny for each line can be examined under controlled genetic conditions. These features of the assay might make it useful for obtaining a rapid estimate of approximate disease severity for new PS1 and PS2 mutations, especially for those with limited pedigree data, small numbers of affected individuals, or suspected environmental or genetic confounding factors. Although the primary goal of this study was to assess an array of genetically diverse FAD mutant variants in a more standardized genetic background, the transgenic flies characterized could be used to study the effects of suspected modifiers or search for new modifiers of PS function. The transgenic lines could also be combined with APP-expressing transgenes to investigate more directly the role of PS FAD mutants in APP cleavage, amyloid-peptide accumulation, and neurotoxicity in a fly model. The correlation observed between the effects of different FAD mutations on Drosophila Notch signaling and human disease onset underscores recent proposals that in addition to APP processing, more global perturbations in pathways involving other γ-secretase substrates should be considered in early-onset Alzheimer's disease. Finally, the results offer encouragement that additional transgenic Drosophila models might be developed to investigate clinical heterogeneity in other human diseases (Seidner, 2006).

Notch cleavage by Furin

Notch (N) is a large transmembrane protein that acts as a receptor in an evolutionarily conserved intercellular signaling pathway. Because of this conservation, it has been assumed that biochemical events mediating N function are identical in all species. For instance, intracellular maturation by furin protease and subunit assembly leading to the formation of a heterodimeric cell surface N receptor are thought to be central to N function in both mammals and flies. However, in Drosophila the majority of N appears to be full-length. It has not been determined whether this full-length N protein is on the cell surface. Experimental evidence indicates that unlike mammalian N, the majority of Drosophila N on the cell surface is full-length and that in Drosophila, in vivo, furin cleavage is not required for biological activity. The behavior of fly and mouse N can be interchanged simply by swapping the regions in which the mammalian furin-like cleavage site is located (Kidd, 2002).

In spite of the fact that Drosophila N and mammalian N1 proteins show extensive sequence homology, most vertebrate N1 exists on the cell surface as a heterodimer, while most of Drosophila N on the cell surface is full-length. Further, a Drosophila N protein (N^BCLexA) which cannot be cleaved by furin is functional in the canonical N pathway in vivo. Because the only detectable form of N on the Drosophila cell surface is the full-length form, this suggests that in contrast to mammals, full-length N is the functional form of N in Drosophila (Kidd, 2002).

A caveat to these conclusions might be that the expression of N^BCLexA is being driven by a heterologous promoter, and that at physiological levels of expression furin cleavage is required for normal N function. If furin cleavage of N is required for transport to the cell surface, the ability of N^BCLexA to suppress the neurogenic phenotype of N^- embryos might be due to overexpression somehow bypassing a need for furin cleavage for transport. However it is thought that the impaired trafficking of N^BCLexA to the cell surface is due to incorrect folding rather than lack of furin cleavage. Furthermore, when expressed in embryos under control of the same heterologous promoter, Nmf^LexA is cleaved, presumably by furin. This suggests that expression of N under control of a heterologous promoter does not saturate endogenous furin. [Nmf is a fly/mouse N hybrid in which the region between the LNG repeats and the transmembrane domain (the furin-dependent cleavage region) of fly N (amino acids 1593-1744) was replaced with the corresponding region of mouse N1 (mN1, amino acids 1562-1725)]. Additionally, full-length endogenous N is found on the cell surface, clearly indicating that furin cleavage is not required for transport. It might, however, remain possible that although furin cleavage is not required for the transport of N, it is required for normal levels of N activity on the cell surface. Perhaps furin cleaved N is more biologically active than full-length N, and therefore upon binding its ligand Dl is rapidly processed leaving only full-length N on the cell surface. This is unlikely because N^BCLexA, which cannot be cleaved by furin, is found on the cell surface at levels considerably lower than wild-type protein expressed from the same promotor and has almost wild type activity. This observation argues against a strict requirement for furin cleavage for N function, and the less than wild type activity seen with N^BCLexA is in accord with the reduced amount of this protein on the cell surface (Kidd, 2002).

The difference in the structures of fly and mammalian cell surface N does not appear to be due to a difference between Drosophila and mammalian cells; both types appear to have equivalent abilities to process proteins with the mammalian furin-dependent cleavage region (FDCR) and it has been demonstrated that Drosophila and mammalian furins have similar specificities. In mammals the N heterodimer results from cleavage by a furin-like convertase during export of N through the trans-golgi. Inhibition of furin by alpha1-PDX results in the absence of the N heterodimer and the commensurate appearance of full-length N on the cell surface. Similarly in Drosophila cells, cell surface mN1 is heterodimeric, and cleavage of N proteins containing the mammalian FDCR (Nmf) can be inhibited by alpha1-PDX with the commensurate appearance of full-length N on the cell surface (Kidd, 2002).

In mammalian cells when furin cleavage is blocked by mutating the N furin cleavage sites no N heterodimer is found on the cell surface and there is no increase in full-length N on the cell surface. A similar result is seen in Drosophila cells when the same mutations are introduced into Nmf and also with N^BCLexA in vivo. However, because even in mammalian cells furin cleavage is not obligatory for N to appear on the cell surface, the absence of cleavage in the mutants most likely result from disruptions to N secondary structure. It has been established that the endoplasmic reticulum (ER) of eukaryotes has mechanisms that prevent the export of improperly folded, potentially non-functional proteins. There exists in mammals a family of ER storage diseases that result from retention in the ER of proteins with mutations that cause only minor changes in primary structure. For example, a majority of cystic fibrosis cases result from a single amino acid deletion in the cystic fibrosis transmembrane conductance regulator. Although it is thought that this protein has near wild type function as a chloride conducting channel, it never reaches the membrane. Support for the notion that N is subject to similar quality control is provided by the finding that mutations in sel-9, the Caenorhabditis elegans p24 homolog, increase the activity of Lin-12 and Glp-1 proteins that are mutated in their extracellular domains. A mutation in sel-9 also allows for the trafficking to the plasma membrane of a mutant Glp-1 protein with a mutation in its extracellular domain that in a sel-9⁺ background is retained within the cell. Since the only form of N that can be detected on the Drosophila cell surface is the full-length form, the weaker suppression of the N neurogenic phenotype conferred by N^BCLexA compared to N^LexA is most likely accounted for by the impaired trafficking of N^BCLexA to the plasma membrane rather than to its inability to be cleaved by furin. A similar trafficking defect is observed in N molecules deleted for regions of the extracellular domain that do not encompass the FDCR. In fact the cleavage of one of these mutant N proteins to produce the activated soluble cytoplasmic domain as defined by association with Su(H) occurs to a lesser degree than does the corresponding cleavage of N^BCLexA (Kidd, 2002).

Despite the apparently equal abilities of Drosophila and mammalian cells to process N proteins with the mammalian FDCR, fly N and mouse N behave differently when expressed in either mammalian cells or Drosophila. In contrast to mammalian N, the most abundant form of N on the cell surface of Drosophila is the full-length protein. There is in addition a small amount of a 250 kDa protein, which by virtue of its relative size, immunoreactivity and susceptibility to biotinylation probably comprises the entire extracellular domain of N. However, the Drosophila 250 kDa protein is not associated with the intracellular domain indicating that it does not correspond to the 240 kDa protein produced by the ligand independent cleavage of mammalian N by furin. Preliminary data suggest that it may be due to the previously described ligand dependent cleavage of N. In addition in Drosophila S2 cells, where under certain conditions cleavage of N can be observed, generation of the N 250 kDa protein unlike the 240 kDa protein of Nmf, is not inhibited by alpha1-PDX. The data presented suggest there is a qualitative difference between fly and mouse N. Swapping the domain cleaved by furin in the mouse and a corresponding region of fly N results in mouse N appearing on the human 293T cell surface as the full-length protein and fly N as a heterodimer comparable in structure to native mouse N in either Drosophila embryos or third instar larval tissues. Thus, with respect to proteolysis and heterodimer formation, important differences between mouse and fly N are localized to the interval between the transmembrane domain and the LNG repeats where the mammalian furin cleavage site is located (Kidd, 2002).

Three sites within the FDCR of mN1 have been identified that had to be mutated in order to abolish furin cleavage. The major cleavage site is between R1654 and E1655. Mutation of the preceding four amino acids RQRR, which match the consensus furin cleavage sequence RxxR, partially abolishes furin cleavage, while mutating these amino acids along with changes in two additional dibasic sequences completely prevents cleavage. Comparison of mouse and fly N indicates that none of these sequences are conserved between the two species. The closest match in flies in the region corresponding to the mouse furin cleavage site are residues 1667_¯1670, RKNK followed by I. RKNKI is also found in Scalloped, the N homolog of the fellow Dipteran, Lucilia cuprina (Blowfly) but it is not known if Scalloped is processed. Glp-1 of C. elegans (an Ecdysozoa as is Drosophila) has the sequence RRYR, matching the minimal furin cleavage site. However, in contrast to Drosophila N, Glp-1 has been shown to be processed, and in this case the intracellular domain is known to be associated with a glycoprotein, presumably the Glp-1 extracellular domain. A furin consensus sequence has been found in N proteins of all the deuterostomes that have been examined. The sequence RxRR is conserved in Xenopus, zebra fish and the ascidian Halocyntheia, but there are no data indicating whether the proteins are cleaved. Sea urchin N contains the sequence RAVR and most is processed into extra- and intra-cellular halves, but it is not known if the two fragments associate to form a heterodimeric protein. Because N proteins of both deuterostomes and C. elegans are processed, while Drosophila N is not, these comparisons suggest that furin cleavage of N is the ancestral form and that its loss in Drosophila is a derived characteristic (Kidd, 2002).

Two mutants of the Drosophila FDCR have been generated. In one, K1670 of Drosophila N was changed to R creating the sequence (RKNR) that matches the minimal furin cleavage site (RxxR). In the second mutant, the Drosophila sequence RKNK (1667_¯1670) was changed to RQRR, the sequence of mouse N that is cleaved by Drosophila furin. Preliminary experiments indicate that neither of these changes results in heterodimeric fly N appearing on the cell surface. Possibly other sequence differences between mouse and fly N are responsible for their different susceptibility to furin cleavage (Kidd, 2002).

Regulation of somatic myosin activity by protein phosphatase 1β controls Drosophila oocyte polarization

The Drosophila body axes are established in the oocyte during oogenesis. Oocyte polarization is initiated by Gurken, which signals from the germline through the epidermal growth factor receptor (Egfr) to the posterior follicle cells (PFCs). In response the PFCs generate an unidentified polarizing signal that regulates oocyte polarity. A loss-of-function mutation of flapwing, which encodes the catalytic subunit of protein phosphatase 1β (PP1β) was identified that disrupts oocyte polarization. PP1β, by regulating myosin activity, controls the generation of the polarizing signal. Excessive myosin activity in the PFCs causes oocyte mispolarization and defective Notch signaling and endocytosis in the PFCs. The integrated activation of JAK/STAT and Egfr signaling results in the sensitivity of PFCs to defective Notch. Interestingly, the results also demonstrate a role of PP1β in generating the polarizing signal independently of Notch, indicating a direct involvement of somatic myosin activity in axis formation (Sun, 2011).

The AP body axis of Drosophila is established during oogenesis through intracellular communication between the oocyte and the somatic follicle cells. Correct oocyte polarity requires a polarizing signal generated by the PFCs, in response to an earlier signal (Gurken) that is secreted from the oocyte and received by the PFCs via Egfr. Previous studies have shown that genes regulating PFC proliferation, differentiation and epithelial polarity must function normally to render the PFC competent to signal back to the oocyte; however, the nature of this polarizing signal is still unknown, neither is it clear how the signal is produced or transmitted from the PFCs to the germline. This study reports a direct role of Drosophila PP1β in the production of the polarizing signal. Loss of PP1β in the PFCs due to the flw^FP41 mutation causes a disruption of the oocyte MT polarity and the mislocalization of determinants of embryonic AP polarity indicative of a defect in the polarizing signal. This oocyte polarity defect was not observed with anterior or lateral follicle cell clones mutant for flw^FP41, demonstrating that the activity of PP1β is required in the PFCs to repolarize the oocyte. It was also shown that heterozygous mutants of positive regulators of myosin activity suppress the oocyte polarity defect, whereas constitutive activation of Rok or expression of a mutant myosin targeting subunit in the PFCs induces a similar oocyte polarity phenotype. This supports the conclusion that myosin activity controls the polarizing signal in the PFCs (Sun, 2011).

The fact that elevated myosin activity in the PFCs interferes with the production of the polarizing signal raises the question of the specific function of myosin in this process. There are two separable effects of elevated myosin activity in the PFCs: an effect on Notch signaling and a Notch-independent effect. Loss of Notch signaling in the follicle cells inhibits the developmental progress of the PFCs and results in the disruption of the formation of the AP polarity in the oocyte. In flw^FP41 PFC clones, the cells are still responsive to the patterning signals of Egfr and the JAK/STAT pathway and the mutant PFCs are able to adopt the posterior fate as indicated by the expression of pnt-lacZ. Therefore, the major problem in the generation of the polarizing event by loss of PP1β is not cell specification or cell survival. Instead, it is proposed that loss of Notch signaling directly affects the production of the polarizing signal, and that myosin activity is further required for the proper generation of this signal independently of its effects on Notch signaling, as discussed below (Sun, 2011).

It was shown that defective Notch signaling in flw^FP41 mutant PFCs can be rescued by expression of NICD, but not by full-length Notch or Notch extracellular truncation (NEXT). This indicates that myosin hyperactivation through loss of PP1β disrupts Notch signaling probably at the level of the final Notch cleavage. This cleavage, which is γ-secretase dependent and generates the functional NICD, is subject to regulation at the level of endosomal trafficking. In mutants that disrupt entry of the receptor into early endosomes, Notch accumulates at the cell surface or below the plasma membrane with significantly reduced signaling activity. In mutants affecting the function of the Vacuolar ATPase, Notch signaling is also blocked at the step of the third cleavage, indicating that this cleavage requires an endosomal environment. An elevated level of Notch protein at the cell surface and in early and late endosomal compartments in the subapical cell cortex is observed in the flw^FP41 mutant PFCs. It is therefore likely that the defective Notch activity in flw^FP41 is caused by a failure of the receptor to efficiently enter early endosomes and subsequent sorting compartments. Such a defect in endosomal trafficking might be a direct consequence of abnormal myosin activity. The regulation of the actin cytoskeleton and of actin motor proteins plays an important role in the endocytic pathway in yeast and mammalian cells. In Drosophila embryos, cortical actin regulates endocytic dynamics at early cellularization. In addition, studies in mammalian cell culture have shown that Rho, Rok and myosin II directly regulate phagocytosis, revealing important roles of myosin II in the process of endocytosis. However, loss of PP1β does not cause a significant block in endocytosis in all cell types. It was found that flw^FP41 clones in the eye discs allow apparently normal Notch signaling to occur and do not show ectopic Notch accumulation. Also no an overt endocytic defect in mutant eye disc cells was detected by performing a trafficking assay. In addition, mutant clones in anterior and lateral follicle cells did not show a defect in Notch signaling. This indicates a particular sensitivity of the PFCs to problems in Notch endocytosis and Notch activation, which is due to the coordinated activities of JAK/STAT and Egfr signaling (Sun, 2011).

The data strongly suggest that PP1β has an independent role in axis formation apart from its effects on regulating Notch cleavage and activation. Excessive myosin activity resulting from constitutive Rok activity, or from expression of a mutant myosin targeting subunit in the PFCs, disrupts Stau localization without inducing a measurable Notch phenotype. Additionally, expression of NICD only marginally suppresses Stau mislocalization caused in the flw^FP41 mutant cells, whereas it strongly rescues the Notch signaling defect. Therefore, oocyte polarity defects were observed by myosin misregulation even in the presence of normal Notch signaling (Sun, 2011).

The effects of excessive myosin activity are also different from those of the Hippo pathway, which is also specifically required in the PFCs for axis formation. Similar to flw^FP41, hippo mutant PFCs are defective in Notch signaling and result in oocyte mispolarization, and these defects are restricted to PFCs. However, previous studies demonstrate that the effects of the Hippo pathway are mediated solely by its effects on Notch. Hippo signaling itself appears to occur normally in the flw^FP41 mutant follicle cells (Sun, 2011).

The abnormal accumulation of membrane proteins suggests a general membrane trafficking problem associated with myosin hyperactivation. It raises the possibility that PP1β regulates the polarizing signal, which might be a membrane associated protein, by controlling its intracellular trafficking as it is trafficked to the cell surface. However, hyperactive myosin caused by loss of PP1β function might also directly impede the interaction between the PFCs and the oocyte, possibly by affecting the function of cellular structures, such as microvilli, required for the presentation of the polarizing signal on the apical surface of the PFCs to the oocyte. Higher levels of components of apical membrane complexes as well as of the adherens junction proteins were observed on the apical surface, which might result from changes in the underlying actin cytoskeleton caused by excessive myosin activity. Consequently, changes in the membrane properties, especially on the apical side that contacts the germline, might also change cell surface protein interactions between the PFCs and the oocyte, which might then affect the transmission of the polarizing signal. A very local effect on oocyte polarity is observed when a subset of PFCs are mutant for flw^FP41, where Staufen protein is still localized correctly in the oocyte underneath the wild-type cells, but is absent from the region underneath the mutant cells. This strongly suggests that the polarizing signal is not freely diffusing over longer distances, and points to local interactions between the PFCs and the oocyte (Sun, 2011).

One very puzzling aspect of the flw^FP41 phenotype is the fact that the phenotypes of defective Notch signaling and cell overproliferation are restricted to the PFCs. Position-dependent phenotypes have been observed in mutants disrupting the epithelial integrity of the follicle cells, such as dlg1 and crb mutants. There, defects of the epithelial architecture, such as multilayering, are mostly observed at the poles of the egg chamber. In mutants of the Hippo pathway, dramatic Notch defects are observed in PFC clones but only modest ones in clones at other sites of the epithelium. Such position-dependent responses might be due to the special terminal positions of the cells at the poles where they could experience more mechanical stress than the lateral cells. Excessive myosin activity caused by loss of PP1β function might exacerbate the mechanical forces experienced by the PFCs, leading to posterior-restricted phenotypes. Alternatively, signaling events specific to subpopulations of follicle cells might cause the cells to react differentially to the loss of common gene products. Strikingly, it was found that the hyperactive myosin can lead to loss of Notch signaling and overproliferation when the Egfr pathway is activated in anterior follicle cells where JAK/STAT activity is normally present. Even the lateral cells produced these phenotypes when subject to the combined activity of JAK/STAT and Egfr signaling. Therefore, whereas loss of PP1β function elevates myosin activity in all the mutant cells independent of cell position, the coordinated activation of JAK/STAT and Egfr signaling creates a sensitized intracellular environment in the PFCs and renders them particularly susceptible to phenotypes such as defects in protein trafficking due to myosin misregulation. It is likely that particular targets of the combined activity of Egfr and JAK/STAT enhance the defects generated by the elevated myosin activity; however, it is presently unknown what these target proteins might be (Sun, 2011).

Overall this study has shown that the regulation of myosin activity by PP1β is crucial in the posterior follicle cells where overactive myosin interferes with intracellular trafficking and with the generation of the posterior polarizing signal. This demonstrates the importance of the general cellular physiology in both signal transduction as well as signal generation, and adds a layer of complexity to the analysis of developmental signals important for cell specification (Sun, 2011).

Notch and Mastermind

During signaling by the Notch receptor, Notch's intracellular domain is cleaved, moves to the nucleus and associates with a DNA-binding protein of the CSL class (CSL for CBF1, Suppressor of Hairless [Su(H)], LAG-1); as a result, target genes are transcriptionally activated. In C. elegans, a glutamine-rich protein called LAG-3 forms a ternary complex with the Notch intracellular domain and LAG-1 and appears to serve as a transcriptional activator that is critical for signaling. Although database searches have failed to identify a LAG-3-related protein, it has been surmised that Notch signaling in other organisms might involve an analogous activity. To search for a LAG-3-like activity in mice, a modified yeast two-hybrid screen was used similar to that used to identify LAG-3. Briefly, a complex bait was used to screen a library of mouse cDNAs fused to the Gal4 activation domain (Clontech). That bait included mouse CBF1 fused to the Gal4 DNA-binding domain (GD) as well as the intracellular domain of mouse Notch1. The bait proteins were co-expressed from a pBridge vector. Out of 6 million transformants, one positive with similarity to Drosophila Mastermind and human KIAA0200 was recovered. A focus was placed on this clone because Drosophila Mastermind is known to be critical for Notch signaling. The murine ortholog of Mastermind is called mMam1, and the human one hMam1. The mMam1 fragment recovered in the two-hybrid screen consists of 62 amino acids and included a conserved region present in both fly and human Mastermind proteins (Petcherski, 2000).

To explore the idea that Mastermind might have a role similar to LAG-3 in Notch signaling, a series of two-hybrid assays was conducted. mMam1 binds mCBF1-GD in the presence of either Notch1 or Notch3, but not in their absence. It was next asked whether Drosophila Mastermind might participate in a similar complex in flies. A fusion protein was used carrying the Gal4 activation domain and the amino-terminal 198 amino acids of fly Mastermind (dMam [1-198]; henceforth called dMam), which includes the conserved region of Mastermind that is critical for complex formation among mouse components. dMam was found to bind Su(H) strongly in the presence of the fly Notch intracellular domain, but not in its absence (Petcherski, 2000).

The interchangeability of proteins from different species was examined. Remarkably, the fly protein, dMam, interacts with murine Notch1 or Notch3 and murine CBF1, and mMam1 interacts with fly Notch and Su(H). In contrast, C. elegans LAG-3 does not form a complex with either murine or fly components, and mMam and dMam do not complex with worm components. It is concluded that both fly and murine Mastermind proteins form a ternary complex with either fly or murine receptors and CSL proteins. This interchangeability underscores the similarity between the fly and murine Notch pathways. Although murine Mastermind is not described, a full-length cDNA sequence for human Mastermind is available. Comparison of human and fly Mastermind sequences reveals only one short region of significant similarity that is limited to 60 amino acids at the amino terminus. Therefore, despite a low overall sequence similarity between mouse and Drosophila Mastermind proteins, the region crucial for complex formation is conserved (Petcherski, 2000).

The importance of Notch's ankyrin repeats for complex formation was examined. In C. elegans, formation of the ternary complex is dependent on the ankyrin repeats of the Notch-related receptor GLP-1. To ask whether the same situation holds for the murine complex, two missense mutants, M1 and M2, were used, each of which bears amino-acid substitutions in the fourth ankyrin repeat of mNotch1. Consistent with results in C. elegans, both M1 and M2 compromise interactions among Notch1, CBF1 and either mMam1 or dMam (Petcherski, 2000).

What is the role of Mastermind in Notch signaling? Previous studies have suggested a role in transcriptional control. In Drosophila, Mastermind is a nuclear protein and is bound to chromatin. Furthermore, in Drosophila, Mastermind acts downstream of Notch in signaling. The amino-acid sequences of both human and fly Mastermind proteins are rich in glutamine and proline, a common feature in transcriptional activators. In the work reported here, a physical link between Mastermind and the major CSL transcription factor of the Notch pathway is described. The interaction of both mMam and dMam with the Notch intracellular domain and CBF1 relies on the receptor's ankyrin repeats. These repeats are essential for Notch signaling and the transcriptional response. In C. elegans, point mutations in the ankyrin repeats severely compromise signaling by the Notch-related receptor GLP-1. In tissue culture cells, the M1 and M2 point mutations abolish receptor function and compromise the activation of transcription by Notch signaling. The simplest explanation for all these findings is that Mastermind functions as a transcriptional activator for Notch signaling (Petcherski, 2000).

Important parallels exist between LAG-3 in C. elegans and Mastermind in Drosophila and mammals. (1) All of these proteins form a ternary complex with an intracellular fragment of Notch and a CSL DNA-binding protein. (2) Mutations in the fourth ankyrin repeat of the receptor compromise ternary complex formation for C. elegans and mouse proteins, as is reported here. (3) All three proteins are rich in glutamine and proline: 27.6% in LAG-3, 29.4% in dMam and 22% in hMam1. (4) LAG-3 and Mastermind function downstream of Notch in C. elegans and Drosophila, respectively. It is proposed that LAG-3 and Mastermind perform analogous functions as activators for Notch (Petcherski, 2000).

What is the evolutionary relationship between LAG-3 and Mastermind? An intriguing idea is that LAG-3 and Mastermind share a common ancestor. The conservation in amino-acid sequence between Mastermind orthologs is much lower than is found for other components of the pathway: whereas hMam1 and dMam share similarity only in a stretch of 60 amino acids within a much larger protein, Notch and CSL proteins show high similarity (44.8% and 74.5% identity for hNotch1/dNotch and hCBF1/Su[H], respectively) over most of their length between these same species. It therefore seems plausible that the absence of similarity between LAG-3 and Mastermind may reflect a high rate of amino-acid substitution in these proteins rather than a distinct evolutionary origin (Petcherski, 2000).

Nipped-A and Notch signaling

The Notch receptor controls development by activating transcription of specific target genes in response to extracellular signals. The factors that control assembly of the Notch activator complex on target genes and its ability to activate transcription are not fully known. This study shows, through genetic and molecular analysis, that the Drosophila Nipped-A protein is required for activity of Notch and its coactivator protein, Mastermind, during wing development. Nipped-A and Mastermind also colocalize extensively on salivary gland polytene chromosomes, and reducing Nipped-A activity decreases mastermind binding. Nipped-A is the fly homologue of the yeast Tra1 and human TRRAP proteins and is a key component of both the SAGA and Tip60 (NuA4) chromatin-modifying complexes. Like Nipped-A, the Ada2b component of SAGA and the Domino subunit of Tip60 are also required for Mastermind function during wing development. Based on these results, it is proposed that Nipped-A, through the action of the SAGA and Tip60 complexes, facilitates assembly of the Notch activator complex and target gene transcription (Gause, 2006).

Nipped-A mutations were isolated in a genetic screen for factors that regulate activation of cut by the wing margin enhancer, and it was found that they reduce Notch activity both at the wing margin and in the developing wing veins. Heterozygous Nipped-A mutations increase the severity of the mutant wing margin and blade reduction phenotype caused by the weak loss-of-function Notch (N^nd-1) mutation and decrease the severity of the vein-shortening phenotype caused by a gain-of-function Notch mutation (N^Ax-E2)(Gause, 2006).

Other genetic data also indicate that Nipped-A is important for Notch signaling. Mastermind is a coactivator protein required for transcriptional activation by Notch, and heterozygous Nipped-A mutations dramatically increase the weak wing-nicking phenotype caused by heterozygous mastermind mutations. The vestigial gene is directly activated by Notch, and flies heterozygous for both Nipped-A and vestigial mutations display wing margin defects. The Notch intracellular fragment binds to the Suppressor of Hairless [Su(H)] protein on target genes, and a Nipped-A Su(H) double mutant displays a dominant wing-nicking phenotype. Together, the effects that the Nipped-A dosage has on the mutant phenotypes displayed by Notch, mastermind, and vestigial mutants indicate that Nipped-A encodes a factor critical for Notch activity in the developing wing (Gause, 2006).

Two Nipped-A mutants have point mutations in the gene encoding the Drosophila homologue of the yeast Tra1 and mammalian TRRAP proteins. Tra1/TRRAP is a key component of the SAGA and Tip60 (NuA4) chromatin-remodeling complexes in yeast, flies, and humans (Gause, 2006).

Tra1/TRRAP is a direct target of transcriptional activators and helps them recruit the SAGA and Tip60 chromatin modification complexes to aid in gene activation. Mammalian Tra1/TRRAP was first identified as a coactivator that interacts directly with the Myc and E2F activators. Tra1/TRRAP is also a target of several other activators in yeast and mammalian cells, including Gal4, E1A, VP16, nuclear receptors, and p53. Tra1/TRRAP contains an ATM-phosphatidylinositol-3 (PI-3) kinase-like domain near the C terminus that is important for recruitment of histone acetyltransferase (HAT) activity in mammalian cells. The C terminus is also critical for interaction of yeast Tra1 with acidic activators (Gause, 2006 and references therein).

There is evidence that SAGA, which contains Tra1/TRRAP and the Gcn5/PCAF HAT, may be involved in transcriptional activation by the Notch complex. Several components of the Notch activator complex are known and functionally identical in worms, flies, and mammals. Upon binding of ligands such as Serrate or Delta to the extracellular EGF repeats of Notch, an intracellular fragment of Notch (NICD) is proteolytically released, allowing it to enter the nucleus, where it interacts with a DNA-bound CSL [CBF1/Su(H)/Lag-1] protein. NICD helps recruit the Mastermind coactivator. An N-terminal region of Mastermind interacts with both the CSL protein and an ankyrin repeat domain of NICD. The p300/CBP (CREB-binding protein) HAT coactivator is recruited by interactions with both the NICD ankyrin repeats and a specific region in the N-terminal half of Mastermind. The Gcn5/PCAF HAT is also recruited by the Notch activator complex in cultured mouse cells; this requires the ankyrin repeat region of NICD. The NICD ankyrin repeats bind other proteins, such as Mastermind and CBP, and thus it is possible that these proteins are also required to recruit Gcn5/PCAF. Because Tra1/TRRAP is the SAGA subunit targeted by several transcriptional activators, it is a distinct possibility that it is required for recruitment of Gcn5/PCAF by the Notch activator complex (Gause, 2006).

This study presents a molecular genetic analysis of several Nipped-A mutations that provides new insights into the roles of the Tra1/TRRAP protein and its complexes in Notch signaling. Reducing the Nipped-A gene dosage by half reduces both Mastermind and Notch activities during wing development and, surprisingly, certain mutant alleles can replace one copy of wild-type Nipped-A. These data also show that other subunits of the SAGA and Tip60 complexes that contain Nipped-A are required for Mastermind and Notch function in wing development and that Nipped-A is required for binding of Mastermind to chromosomes. Taken together, the results indicate that Nipped-A plays multiple roles in Notch signaling (Gause, 2006).

The evidence provided here, combined with the finding that two Nipped-A mutants have point mutations in the Tra1/TRRAP gene, demonstrates conclusively that Nipped-A encodes Tra1/TRRAP. All EMS-induced Nipped-A alleles sequenced to date have point mutations in the Tra1/TRRAP gene that affect the protein coding sequence or, in one case, the 3' UTR. A seventh allele generated by gamma rays, Nipped-A³²³, does not produce Tra1/TRRAP mRNA. Additional Nipped-A mutant alleles have been sequenced, and all contain point mutations that alter the protein coding sequence (Gause, 2006).

The results show that the major Nipped-A transcript differs from a previously reported splicing pattern, which appears to be a rare variant. Antibodies against a polypeptide encoded largely by the rare exons detect a weak Tra1/TRRAP signal in Western blot assays of concentrated nuclear extracts or purified complexes, confirming that the variant produces Tra1/TRRAP protein in vivo. The rare transcript does not, however, support at least one essential function of Nipped-A and Notch signaling in the wing margin because mutation of a splice site in Nipped-A^NC106 for an exon that is not included in the rare variant is lethal and causes defects in Notch signaling. Nipped-A^NC106, however, had little effect on the N^Ax-E2 wing vein phenotype, raising the possibility that the alternatively spliced product can support Notch function in developing wing veins (Gause, 2006).

An unexpected finding is that the Nipped-A^NC105 allele, which encodes the N-terminal 2,048 residues of Tra1/TRRAP, suffices to replace one wild-type copy of Nipped-A to support Notch and Mastermind function in vivo. This was unexpected because the protein encoded by Nipped-A^NC105 lacks the ATM-PI3 kinase motif which, in mammalian cell culture experiments, is required for Tra1/TRRAP to associate with Gcn5 and Tip60. One possible explanation is that the C terminus of the Nipped-A protein is not required for Notch and Mastermind function and that the truncated protein can replace the full-length protein. Because the effects of the Nipped-A mutations on Notch functions in wing development could only be studied in the presence of a wild-type allele, it is also possible that a truncated protein somehow increases the activity of the remaining full-length Nipped-A protein. The truncated protein could not be detected in Western blot assays of extracts or by immunostaining, suggesting that if this is the case, only a small amount of the mutant protein is sufficient. It is considered improbable that linked second-site mutations are masking effects of Nipped-A^NC105 on both Notch mutant phenotypes and the mastermind phenotype. Many mutations have effects similar to Nipped-A, and few have opposing effects, and it would likely require multiple mutations to counteract the effects of Nipped-A^NC105 on all three phenotypes. It is also unlikely that there is a linked second-site mutation that counteracts the effects of Nipped-A^NC105 by increasing the expression of wild-type Nipped-A, because mutant embryos and larvae show the expected decrease in full-length Nipped-A protein (Gause, 2006).

The Nipped-A^NC194 allele, which encodes residues 1 to 1500, had a significant effect on both of the Notch mutant phenotypes but did not increase the severity of the wing-nicking phenotype displayed by mam^g2. Again, this differs from null alleles of Nipped-A, which affect all three phenotypes, suggesting that Nipped-A^NC194 retains sufficient activity to replace one copy of the wild type in support of Mastermind activity. Again, one possible explanation is that Nipped-A residues 1 to 1500 are sufficient to support Mastermind function, although it is conceivable that the truncated protein somehow increases the activity of the remaining wild-type Nipped-A protein. It was not possible to detect this truncated protein, suggesting that if a truncated protein is responsible, only low levels are required. Despite extensive screens with a deficiency collection and candidate genes, no mutations that suppress mastermind mutant phenotypes have been mapped to chromosome 2. Thus, it is unlikely that a linked second-site mutation masks an effect of Nipped-A^NC194 on the mastermind phenotype. Similar to Nipped-A^NC105, heterozygous Nipped-A^NC194 mutants display the expected reduced levels of full-length protein, although the possibility cannot be excluded of a subtle increase in the expression of the wild-type Nipped-A allele that is sufficient to rescue the mastermind phenotype but not the Notch mutant phenotypes (Gause, 2006).

Isolation and analysis of additional Nipped-A truncation alleles and development of more sensitive biochemical assays will lead to a fuller understanding of how Nipped-A alleles encoding truncated proteins support Notch signaling (Gause, 2006).

The experiments presented in this study indicate that the roles of Nipped-A in supporting Mastermind function likely involve both the SAGA and Tip60 complexes. The Ada2b protein is specific to SAGA, and Ada2b mutations affect the mastermind phenotype but not the two Notch mutant phenotypes. It is thought unlikely that the effect of the Ada2b mutations is more specific than Nipped-A mutations because the mastermind phenotype is more sensitive. As shown by the Nipped-A^NC96 hypomorph, the N^nd-1 phenotype is more sensitive to the Nipped-A dosage than is mastermind. Moreover, the Nipped-A^NC194 allele has a specificity opposite that of the Ada2b mutations and affects the Notch mutant phenotypes but not the mastermind phenotype. Combined, the contrasts in the effects of Ada2b and various Nipped-A mutations show that Nipped-A and its complexes play multiple roles in Notch signaling. They suggest that the SAGA complex, or at least the Ada2b subunit, is more specific for Mastermind function and that Nipped-A has additional functions (Gause, 2006).

Another possibility raised by the specificity of the effects of Ada2b mutations for effects on Mastermind activity in wing margin development is that Mastermind may have functions in margin development independent of Notch. For example, Mastermind could conceivably function as a coactivator for other activator proteins in addition to Notch. This possibility is consistent with the binding of Mastermind to several sites in polytene chromosomes, including the ecdysone-dependent puffs (Gause, 2006).

The Domino protein, a putative ATPase remodeling enzyme, is a subunit of the Tip60 complex. The N^nd-1 and N^Ax-E2 phenotypes and the Mastermind phenotype are modified by domino mutations, although the effect on N^Ax-E2 is modest. These effects are similar to those of the Nipped-A^NC106 allele and thus suggest that the Tip60 complex also supports Mastermind function and Notch signaling during wing development. It is possible, however, that Domino functions independently of Tip60 and Nipped-A because the human Domino homologue SRCAP interacts directly with the CBP HAT enzyme that interacts with Mastermind. Nevertheless, the likely involvement of the Tip60 complex raises the possibility that histone exchange could facilitate transcriptional activation by Notch because, in addition to acetylating histone H4, Tip60 exchanges histone H2 variants during DNA repair (Gause, 2006).

As revealed by immunostaining of salivary gland polytene chromosomes, at least one function of Nipped-A is to regulate the binding of Mastermind to chromosomes. The reduction in binding of Mastermind to polytene chromosomes caused by the hypomorphic Nipped-A^NC96 and Nipped-A^NC186 alleles is dramatic. Supporting the idea that Nipped-A directly regulates Mastermind binding, virtually all sites on polytene chromosomes that bind Mastermind also bind Nipped-A. A few possible explanations for these results are envisioned. The SAGA and Tip60 complexes that contain Nipped-A could acetylate Mastermind, proteins in the Notch activator complex, and/or possibly histones to facilitate binding of the Notch activator complex to chromatin. These modifications could be made by Gcn5 and/or Tip60, which acetylate histones H3 and H4, respectively. Alternatively, Nipped-A or its complexes could bind to chromosomes cooperatively with Mastermind. This would be consistent with the published observation that the ankyrin repeats of the NICD fragment of Notch, which help recruit Mastermind to the Notch activator complex, are also required to recruit Gcn5/PCAF SAGA subunit in transfected mouse cells. Both the Ada2b component of SAGA and the Domino subunit of Tip60 affect Mastermind function, so it is likely that Nipped-A supports Mastermind function in more than one way (Gause, 2006).

Because the evidence suggests that Nipped-A supports Mastermind function through both the SAGA and Tip60 chromatin-modifying complexes, it is theorized that, in addition to controlling the binding of Mastermind to chromosomes, Nipped-A could also cooperate with Mastermind to recruit these complexes to facilitate transcriptional activation through chromatin modification (Gause, 2006).

The data indicate that the SAGA complex, or at least its Ada2b subunit, is not required for some functions of Nipped-A in Notch signaling. Unlike Nipped-A and domino mutations, Ada2b mutations did not affect Notch mutant phenotypes, while they did enhance the phenotype caused by a mastermind mutation. It is postulated, therefore, that the Tip60 complex is also required for functions of Nipped-A beyond controlling the binding of Mastermind to chromosomes. The Tip60 complex could affect the expression of Notch activator complex components, or it could modify proteins in the Notch activator complex. It is also possible that Tip60 modifies chromatin to either aid binding of the Su(H) protein to the Notch target genes or, as mentioned above, to aid transcriptional activation by the Notch activator complex. In any case, the evidence indicates that two subunits of Tip60, Nipped-A and Domino, play more than one role in Notch signaling during wing development (Gause, 2006).

Enhancer of Split Gene Complex: architecture and coordinate regulation by Notch, Cohesin and Polycomb group proteins

The cohesin protein complex functionally interacts with Polycomb group (PcG) silencing proteins to control expression of several key developmental genes, such as the Drosophila Enhancer of split gene complex [E(spl)-C]. The E(spl)-C contains twelve genes that inhibit neural development. In a cell line derived from central nervous system, cohesin and the PRC1 PcG protein complex bind and repress E(spl)-C transcription, but the repression mechanisms are unknown. The genes in the E(spl)-C are directly activated by the Notch receptor. This study shows that depletion of cohesin or PRC1 increases binding of the Notch intracellular fragment (NICD) to genes in the E(spl)-C, correlating with increased transcription. The increased transcription likely reflects both direct effects of cohesin and PRC1 on RNA polymerase activity at the E(spl)-C, and increased expression of Notch ligands. By chromosome conformation capture this study found that the E(spl) C is organized into a self-interactive architectural domain that is co-extensive with the region that binds cohesin and PcG complexes. The self-interactive architecture is formed independently of cohesin or PcG proteins. It is posited that the E(spl)-C architecture dictates where cohesin and PcG complexes bind and act when they are recruited by as yet unidentified factors, thereby controlling the E(spl)-C as a coordinated domain (Schaaf, 2013).

These studies investigated the regulation of the E(spl)-C complex by cohesin, PRC1, and the Table of contents

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