Effects of Mutation or Deletion

Table of contents

Notch genetic interaction with Presenilin

Recent studies have suggested a role for presenilins in the Notch-signaling pathway, but their specific function within this pathway remains unclear. The Drosophila Presenilin gene and protein have been characterized and their interaction with Notch has been studied in both mutants and transgenics. The Drosophila PS protein is proteolytically cleaved and broadly expressed during development with the highest levels in neurons within the larval CNS. Mutations in Drosophila PS genetically interact with Notch and result in an early pupal-lethal phenotype characterized by defects in eye and wing development and incomplete neuronal differentiation within the larval CNS. Moreover, processing of Notch in the Golgi by the furin protease is unaffected in PS mutants and Notch is present and may even accumulate on the plasma membrane of neuroblasts in the larval CNS of PS mutants. In contrast, overexpression of PS in transgenics causes Notch to accumulate in the cytoplasm. Taken together, these results indicate that Drosophila PS is required for proper neuronal differentiation and may regulate the subcellular localization of Notch proteins within cells, necessary for Notch accumulation and subsequent signaling capabilities (Guo, 1999).

To examine the function of PS, a series of overlapping deletions was generated by imprecise P-element excision of P4, a homozygous viable P-element insertion located ~1 kb 3' from the PS gene. Six deletions were generated, three of which remove portions of the PS coding region (PS^W20, Dps^W6, and PS^W11). All of the deletions fail to complement one another, and none produce any detectable PS protein as determined by Western blot analysis on extracts from mutant larvae. The largest deletion (PS^W20) deletes the PS gene as well as portions of the lipoic acid synthase and the 50 S ribosomal protein L15 genes and is embryonic lethal. PS^W11 and PS^W6 that delete PS and the 50S ribosomal protein L15 genes are lethal during the second larval instar, and these larvae are small and grow at a much slower rate (Guo, 1999).

In addition to the deletion mutants in PS two EMS alleles, PS³⁰ and PS⁴⁶ have been characterized. These EMS alleles were originally identified in a lethal screen over the deficiency Df(3L)ri-79c/TM3 that uncovers the PS locus and has break points at 77B-C;77F-78A. Both EMS lines fail to complement each other as well as the two PS deletions tested, PS^W6 and PS^W11. To determine the molecular basis of these mutations, both PS³⁰ and PS⁴⁶ were sequenced. To date, no mutation within the coding region of PS³⁰ could be detected and alterations in surrounding regulatory sequences are being sought. A single missense mutation was found in PS⁴⁶, consisting of a proline to leucine substitution at amino acid 507 within the highly conserved C-terminal domain of PS (Guo, 1999).

To address the function of PS during development, a test was made of the phenotype in flies of the genotype PS⁴⁶/PS^W6 or PS⁴⁶/PS^W11, that is, flies that are mutant for PS but wild-type for lipoic acid synthase and hemizygous for the 50 S ribosomal protein L15 gene. These mutants are early pupal lethal; the third instar larvae form a pupal case, but no adult structures develop. When the phenotype of the mutants is examined during the late third larval instar stage, the mutant larvae have underdeveloped eye and wing imaginal discs. Although the eye imaginal disc forms, it fails to undergo proper neuronal differentiation. The wing imaginal disc is also smaller with the most severe defects observed in the region that will give rise to the wing blade. Furthermore, Wingless expression, which is normally detected in two domains within the developing disc, is disrupted in PS mutant discs. In contrast, both the antennae and leg imaginal discs appeared normal. Both the lethality as well as the wing and eye phenotypes could be rescued using a PS transgene driven by a heatshock promoter. To determine whether there was any evidence of neuronal differentiation within the eyes and optic lobes of these pupal lethal mutants, the expression of two neuronal markers, Elav and Cut, that are broadly expressed in neurons during postembryonic development, were examined. In wild-type eye-antennae imaginal discs of third larval instars, Elav is expressed in photoreceptor neurons. Cut is absent from the eye disc at this stage but is broadly expressed in the antennae disc. At later stages, Cut can be detected in cone cells and sensory organ precursors that give rise to interommatidial bristles. Wild-type discs stain positively for Elav (eye) and Cut (antennae), whereas mutant eye imaginal discs appear small and underdeveloped and fail to express Elav. Furthermore, although both Cut and Elav could be detected in the developing optic lobes, the highly organized, laminar pattern of expression that is normally found in wild-type optic lobes fails to develop in PS mutants. This suggests that PS is required for eye and wing imaginal disc development, and although it is not required for the development of the optic lobes, they fail to differentiate properly without it (Guo, 1999).

To examine the link between PS and Notch, the ability of PS mutants to interact genetically with Notch was examined. Specifically, each PS mutant was crossed to several alleles of Notch and dosage-sensitive interactions were sought in transheterozygotes. An examination was made of the ability of the point mutant PS⁴⁶ and the deletions PS^W6, PS^W11, and PS^W20 to interact with the deficiency Df(1)N-8/In(1)dl-49, y1 Hw1 m2 g4. These interactions give rise to dominant notching of the wing blade. Transheterozygotes containing one copy of the Notch deficiency and one copy of the PS⁴⁶ mutation or a PS deletion all exhibit an enhanced wing phenotype. In addition, both PS alleles enhance the phenotype of N^nd-3, a loss-of-function allele of Notch that gives rise to a thickened wing vein phenotype at 29°C. Specifically, transheterozygotes between N^nd-3 and PS alleles have enhanced thickening of wing veins and notches at the tips of the wing similar to what is observed in stronger Notch alleles. This suggests that PS mutations reduce Notch signaling within cells. In agreement with this, PS alleles suppress the interrupted wing vein phenotype observed in the gain-of-function N^Ax-1 allele. Finally, PS mutants also enhance the phenotype of the Delta allele Dl[7] that is characterized by thickened wing veins and small deltas located near cross-veins. Specifically, transheterozygotes between Dl[7] and PS mutants exhibit a greater extent of thickening and deltas at the wing veins. Taken together, these results indicate that PS mutations reduce Notch signaling and support a role for PS in the Notch-signaling pathway (Guo, 1999).

To gain insight into the mechanism underlying PS and Notch interactions, immunocytochemical studies were performed to examine the subcellular distribution of Notch within the larval CNS of PS mutants. Using an antibody that recognizes the Notch intracellular domain, it has been found that Notch is expressed at high levels within neuroblasts throughout the proliferative centers in the developing optic lobes and at somewhat lower levels in neuroblasts within the thoracic ganglia of third larval instar CNS. Within wild-type neuroblasts, Notch is distributed throughout the cytoplasm and on the plasma membrane. In contrast, the overall distribution of Notch within the optic lobes is disrupted in PS mutants, and the protein levels are reduced in the cytoplasm and appear to be preferentially retained on the plasma membrane of specific neuroblasts. To distinguish between primary effects of PS on Notch localization versus secondary defects caused by altered differentiation of PS mutants, the distribution of Notch protein within neuroblasts of second instar larvae was examined. At this stage, Notch is expressed in small groups of cells that consist of a large neuroblast surrounded by smaller ganglia mother cells. Within these groups of cells Notch appears throughout the cytoplasm and at high levels at the plasma membrane in regions of contact between the neuroblast and its progeny. There is little, if any, accumulation of Notch at the plasma membrane in regions that contact the remaining surrounding cells. In contrast, Notch distribution is altered with less in the cytoplasm and more staining seen over the entire plasma membrane of neuroblasts in PS mutants. In agreement with this observation, processing of Notch, found to give rise to a functional heterodimeric receptor on the cell surface, is unaffected in PS mutants (Guo, 1999).

The reciprocal experiment was performed to determine whether overexpression of PS also affects the subcellular distribution of Notch. Specifically, the GAL4/UAS system was used to target expression of PS to specific subsets of cells within a developing tissue. Specifically, a pannier-GAL4 line was used to drive expression of UAS-Dps in regions of the wing imaginal disc that will give rise to the adult notum and subsets of neurons within the eye imaginal disc or a cut-GAL4 line that drives expression in the presumptive wing margin. Notch protein is found to specifically accumulate within PS-expressing cells and appears to be primarily localized within the cytoplasm. In contrast, neighboring cells that do not overexpress PS show no change in Notch expression or distribution. Notch specifically accumulates in PS-overexpressing cells but not in the adjacent cells (internal control) that only express basal levels of PS. Taken together, these results show PS does not affect the ability of Notch to undergo furin-dependent cleavage within the Golgi and to accumulate on the plasma membrane. Rather, PS may be affecting the subcellular distribution of Notch and later cleavage events leading to defects in cell signaling (Guo, 1999).

In the absence of ligand interaction, proteolytic processing of Notch does not occur. Alternatively, PS could be required for internalization of the receptor-ligand complex and subsequent proteolytic processing because it is not known where within the cell these cleavage events occur. Although presenilin in mammals is thought to reside predominantly in the endoplasmic reticulum (ER) and Golgi, consistent with possible roles in early stages of protein processing, some evidence suggests that it may transiently reach the plasma membrane where it could participate in endocytic processes. In fact, in Drosophila, PS has been shown to accumulate within vesicular structures throughout the cytoplasm, including the ER and Golgi, and is also found at the apical regions of cells in some tissues. A role for presenilin in protein processing or trafficking has been suggested previously on the basis of its subcellular localization and its ability to affect the processing of APP in vertebrates. APP, like Notch, encodes a single-pass transmembrane protein found at the plasma membrane that is known to undergo several cleavage steps giving rise to two secreted peptides, Abeta(40) and Abeta(42-43). Recent studies in transgenic mice have shown that presenilins can affect the processing of both Notch and APP by mechanisms that are unknown. Current models suggest that presenilins may directly cause cleavage of Notch and APP or, alternatively, affect the ability of secretases to cleave. Alternatively, presenilins may play a more general role in regulating the subcellular distribution of transmembrane proteins like APP and Notch within the cell, thereby physically altering their ability to interact with proteases necessary for their proper signaling functions (Guo, 1999 and references therein).

Antioxidant proteins TSA and PAG interact synergistically with Presenilin to modulate Notch signaling in Drosophila

Alzheimer's disease (AD) pathogenesis is characterized by senile plaques in the brain and evidence of oxidative damage. Oxidative stress may precede plaque formation in AD; however, the link between oxidative damage and plaque formation remains unknown. Presenilins are transmembrane proteins in which mutations lead to accelerated plaque formation and early-onset familial Alzheimer's disease. Presenilins physically interact with two antioxidant enzymes thiol-specific antioxidant (TSA) and proliferation-associated gene (PAG) of the peroxiredoxin family. The functional consequences of these interactions are unclear. In the current study a presenilin transgene was expressed in wing and sensory organ precursors of the fly. This caused phenotypes typical of Notch signaling loss-of-function mutations. While expression of TSA or PAG alone produced no phenotype, co-expression of TSA and PAG with presenilin led to an enhanced Notch loss-of-function phenotype. This phenotype was more severe and more penetrant than that caused by the expression of Psn alone. In order to determine whether these phenotypes were indeed affecting Notch signaling, this experiment was performed in a genetic background carrying an activated Notch (Abruptex) allele. The phenotypes were almost completely rescued by this activated Notch allele. These results link peroxiredoxins with the in vivo function of Presenilin, which ultimately connects two key pathogenetic mechanisms in AD, namely, antioxidant activity and plaque formation, and raises the possibility of a role for peroxiredoxin family members in Alzheimer's pathogenesis (Wangler, 2011).

The underlying genetic heterogeneity of Alzheimer's disease has made understanding the pathogenesis of the disease difficult without the aid of model genetic organisms. It is clear that plaque formation and oxidative damage are key pathogenetic mechanisms in AD. The presence of early oxidative stress in mice with Presenilin mutations lends further support for the idea that plaque formation and oxidative damage are connected in AD. However, how these two pathogenetic processes are linked is not known. This study used Drosophila as a model system to explore a protein-protein interaction between presenilin and two peroxiredoxin family members TSA and PAG, which may provide a link between oxidative stress and plaque formation in AD (Wangler, 2011).

There is genetic and biochemical evidence that peroxiredoxin family members play a role in the pathogenesis of AD. There are six peroxiredoxin family members in humans. Of these genes PRDX1 (PAG) was implicated in physical interaction with human Presenilin-1 and is primarily expressed in glia. PRDX2 (TSA) is globally expressed in the brain and has increased expression in the frontal cortex of patients with AD and is implicated in Parkinson’s disease and amyotrophic lateral sclerosis . Another family member PRDX6 has been more directly implicated in AD by linkage studies. The PRDX6 protein displays markedly increased expression in glia surrounding amyloid plaques in AD brain. Overall these studies support a role for some of these family members in AD pathogenesis. Furthermore, they demonstrate an increase in the expression of PRDX family members in AD brain, an effect which the data suggest could impact the function of Presenilin. These observations suggest the use of a study of models of the clinically relevant pathogenetic mechanism (Wangler, 2011).

One advantage of the Drosophila wing as an assay system is the ability to make use of a wealth of knowledge and an array of genetic tools for analyzing wing development. For example, this study observed phenotypes with overexpression of Psn which recapitulated Notch loss-of-function, and a substantial increase was observed in the penetrance and severity of these phenotypes when human TSA and PAG were co-expressed with Psn. This led to the hypothesis that TSA and PAG enhance the dominant negative effect of overexpressed Psn. Whether this enhancement was mediated by the Notch pathway was tested by suppressing this effect with an activated allele of Notch. Since Psn acts in conjunction with three other components that comprise γ- secretase, it is likely that overexpression of Psn acts in a dominant negative fashion to inhibit Notch signaling by altering the normal stoichiometry of γ-secretase subunits. The results suggest that association of Psn with TSA or PAG aggravates this effect of Psn overexpression. Interestingly, this seemed to occur with the human peroxiredoxin isoforms in the presence of the Drosophila Psn transgene. While the portion of the PSEN1 protein which interacts with TSA and PAG is only partially conserved with Psn, these observations suggest a functionally significant effect across species. Furthermore, an exacerbation of phenotype was seen in response to overexpression of peroxiredoxin antioxidant enzymes, conditions that one might expect to provide oxidative protection. One possible explanation for these findings is that by associating with Psn, TSA and PAG further reduce the complete intact γ-secretase complex. Alternatively, Notch inactivation could be a result of either degradation or abnormal processing of the endogenous presenilin in response to the expression of Psn along with the mammalian transgenes. Either possibility could have obvious disease relevance, as it seems clear that increased expression of these proteins occurs in AD brain, and the current data suggest that such an increase in expression could have an impact on Presenilin function and therefore indirectly could influence plaque formation (Wangler, 2011).

While these results support a role for peroxiredoxins PAG and TSA in modifying the Notch inactivation phenotype caused by Psn overexpression, it a direct role for these proteins in the processing of amyloid precursor protein, or in the pathogenesis of AD cannot be infered. Loss-of-function studies are needed to determine the mechanism of peroxiredoxin function and whether there is a clear role for endogenous peroxiredoxin in Notch signaling or amyloid cleavage. Further experiments in mammalian cells will be needed to determine whether peroxiredoxin family members are indeed involved in amyloid processing. Nonetheless, this study has demonstrated that Drosophila provides an effective system for testing the in vivo relevance of AD related protein-protein interactions. As the wing assay for Psn activity has proven robust in validating a suspected protein-protein interaction with Presenilin, it should be amenable to screening for new unknown functionally interacting partners of Psn (Wangler, 2011).

Fringe regulation of Notch signaling

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

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

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

In wild-type discs, the response of Notch to Dl and Ser is affected by the presence of Fng, which is expressed in dorsal cells. Since the domain of Fng expression corresponds to the region where Dl loses its capacity to antagonize Notch in N^Ax mutants, an analysis was carried out to see whether N^Ax mutations have an altered sensitivity to Fng by comparing the consequences of ectopic fng expression in wild-type and N^Ax discs. As with the ectopic ligand expression, clones of cells expressing fng that cross the dorsoventral boundary inhibit expression of ct except at the clone borders. When the Fng-expressing clones lie in the ventral compartment, ct is induced in the cells at the boundary of the clone, with the result that ct is detected in neighboring fng⁺ and fng^- cells. The ability of Fng to prevent ct expression is reduced when Fng-expressing clones are induced in N^Ax mutant backgrounds. In a weak N^Ax allelic combination, the expression of ct is still highest at clone boudaries, but significant expression is detected within the clone. In the more severe mutants, the Fng-expressing cells have little or no inhibitory effect on ct, and there are high levels of Ct throughout the clone. Similar effects are seen when fng misexpression is driven by Gal4-sal. Normally this causes an inhibition of ct expression at the dorsoventral boundary; in N^Ax mutant discs, however, Ct is detected throughout most of the domain of ectopic Fng-expression. If the N^Ax domain is significant in the interactions between Notch and Fng, the N^Ax mutations should modify phenotypes caused by alterations in fng expression. In the allele fng^D4, fng is expressed throughout the wing pouch, causing severe scalloping of the wing margin. This correlates with the loss of ct and wg expression at the dorsoventral boundary and the expansion of vvl/drifter expression. In N^Ax heterozygous flies, the phenotype of fng^D4 is reduced both at the level of wing scalloping and the expression of dorsoventral boundary markers. In hemizygous N^Ax males, both the expression of ct and vvl and the adult phenotype are similar to the expression and phenotype typical of N^Ax. Taken together, these results suggest that N^Ax proteins are also deficient in some activity related to the capability of Fng to restrict Notch activity (de Celis, 2000).

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

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

Notch interaction with Suppressor of deltex

In Drosophila, Suppressor of deltex [Su(dx)] mutations display a wing vein gap phenotype resembling that of Notch gain of function alleles. The Su(dx) protein may therefore act as a negative regulator of Notch but its activity on actual Notch signalling levels has not been previously demonstrated. Su(dx) is shown to regulate the level of Notch signalling in vivo, upstream of Notch target genes and in different developmental contexts, including a previously unknown role in leg joint formation. Overexpression of Su(dx) is capable of blocking both the endogenous activity of Notch and the ectopic Notch signalling induced by the overexpression of Deltex, an intracellular Notch binding protein. In addition, using the conditional phenotype of the Su(dx)^sp allele, it has been shown that loss of Su(dx) activity is rapidly followed by an up-regulation of E(spl)mß expression, the immediate target of Notch signal activation during wing vein development. While Su(dx) adult wing vein phenotypes are quite mild, only affecting the distal tips of the veins, the initial consequence of loss of Su(dx) activity is more severe than previously thought. Using a time-course experiment it has been shown that the phenotype is buffered by feedback regulation illustrating how signalling networks can make development robust to perturbation (Mazaleyrat, 2003).

To begin to unravel the mechanism of action of Su(dx), it is an important prerequisite to establish whether Su(dx) acts on the Notch pathway itself, or whether the genetic interactions observed reflect an indirect, parallel, or downstream activity. The data argue that Su(dx) can indeed negatively regulate Notch signalling, upstream of the immediate Notch target genes. (1) It has been shown, using the temperature sensitivity of the Su(dx)^sp wing vein gap phenotype, that Su(dx) loss of function is rapidly followed by the up-regulation of E(spl)mß expression in the pupal wing. (2) In third instar wing imaginal discs, it has been shown that in two enhancing genetic backgrounds, Su(dx) loss of function results in the up-regulation of wingless, another Notch target gene at the D-V boundary. (3) Su(dx) overexpression in the wing imaginal disc is capable of down-regulating the Notch-dependent expression of three genes, wingless and cut at the D-V boundary, and the vg^BE-LacZ element at both the D-V and the A-P boundaries. These data show that Su(dx) is capable of downregulating Notch in different developmental contexts and that its activity on Notch is not limited to the particular situation of wing vein development (Mazaleyrat, 2003).

Su(dx) is capable of blocking the stimulation of Notch signalling, which is induced by the overexpression of Deltex, a regulatory protein which binds to the Notch intracellular domain. Thus these data suggest that the activity of Su(dx) lies upstream of the regulation of Notch target gene expression but downstream of, or at the level of, Deltex. This, together with the rapidity of the response of increased Notch signalling that is observed following Su(dx) loss of function, supports the hypothesis that Su(dx) acts directly on the Notch pathway. In vivo data are thus consistent with the in vitro observation that a related mammalian Nedd4 family protein, Itch, can promote the ubiquitination of the Notch1 intracellular domain (Mazaleyrat, 2003).

The phenotype of Su(dx)^sp was examined in two different enhancing genetic backgrounds and different consequences on the spatial distribution of ectopic Notch activation were obtained at the wing disc D-V boundary, as monitored by wingless expression. Su(dx) mutations alone have no wing margin phenotype. wingless expression was investigated in the background of Notch alleles that enhance the Su(dx) wing vein phenotype, i.e., notchoid¹ (nd¹) and Abruptex^E2 (Ax^E2). Ectopic wingless expression in nd¹;Su(dx)^sp discs is restricted to the ventral side of the D-V boundary, but is found on both sides of this boundary in Ax^E2;Su(dx)^sp discs. A similar ventral compartment-specific Notch activation is observed when Serrate is expressed along the anterior-posterior axis, while expression of constitutively active Notch intracellular domain does not show such a restriction (Mazaleyrat, 2003).

This spatial restriction of the ectopic Serrate-induced response has been explained by the inhibitory effect of dorsally expressed Fringe, which represses Serrate-dependent but not Delta-dependent Notch activation through the glycosylation of the Notch extracellular domain. Normally, Serrate, which is expressed in the dorsal compartment, is only able to signal to Notch in adjacent ventral cells where Notch is not modified by Fringe. A possible interpretation of these data therefore is that the ectopic Notch activation in the nd¹;Su(dx)^sp combination is similarly blocked in the dorsal compartment by Fringe. If this is the case then it is unlikely that this interaction results in a constitutive activation of the Notch receptor, since the latter would be independent of Fringe. The Abruptex class of mutations have been shown to make the Notch receptor less sensitive to the down-regulatory effect of Fringe and this may explain why the Ax^E2;Su(dx)^sp combination, unlike the nd¹;Su(dx)^sp combination, allows ectopic Notch activity on both sides of the D-V boundary (Mazaleyrat, 2003).

Frequently in studies of the effects of mutations on gene expression, the patterns detected represent only the end point of the aberrant developmental history. Here it was possible to exploit the temperature-sensitive nature of the Su(dx)^sp wing vein gap phenotype to investigate the evolving expression pattern of two Notch regulated genes, E(spl)mß and rhomboid. The shift to the nonpermissive temperature was closely followed by increased expression of the Notch target gene E(spl)mß and a concomitant decrease in rhomboid expression in the wing vein precursor cells. The latter is expected because rhomboid expression is repressed by E(spl)mß. The initial elevation in E(spl)mß expression level was found to be transient, peaking around 45 min. Subsequently E(spl)mß expression levels are progressively reduced and are lost altogether from regions of the vein precursor territories that corresponded to positions of vein gaps found in the adult wings. rhomboid expression has been proposed to be necessary to activate EGF receptor signalling, which in turn is required to maintain Notch signalling levels. A loss of rhomboid expression due to elevated Notch activation would therefore be predicted to cause a subsequent reduction in Notch activity via decreased EGF receptor signalling. In turn this would be predicted to lead to the derepression of rhomboid expression. In a time-course experiment the operation of this predicted feedback loop was followed for the first time, and the oscillation in Notch signalling activity observed is in broad agreement with this model. However the data are not completely in agreement. Up-regulation of E(spl)mß expression was seen followed by moderation of the raised levels in intervein territories where rhomboid is not detectably expressed and presumably is therefore not involved in the feedback regulation in these cells. This implies that an additional uncharacterized means of feedback control might be in operation (Mazaleyrat, 2003).

The implementation of the feedback loop makes Drosophila wing vein development relatively robust to perturbations of Notch activity. The final adult phenotype may depend on the kinetics of the feedback loop leading to the restoration of EGF receptor signal required to drive cells into the vein cell fate, compared to the kinetics of the process of commitment itself. The variable sensitivity of different parts of the wing veins could therefore be due to different times at which cells in different regions pass through a critical point at which they irreversibly commit to a vein cell or intervein cell fate. These data illustrate an important point that in a mutant background a cell can ultimately adopt a wild-type fate even though its developmental history is altered, providing the interacting signals produce a network which is robust enough to withstand and adjust for the perturbation. Such interacting networks could be important buffers for development against genetic variation in a population. It is speculated that this robustness together with other forms of redundancy may help to mask wider activities of Su(dx) that are uncovered in different genetic backgrounds (Mazaleyrat, 2003).

In the light of the above discussion it is interesting that the data have uncovered, in an enhancing nd¹ genetic background, a previously unknown function of Su(dx) in leg development. The resulting extra joint phenotype is consistent with a role for wild-type Su(dx) in down-regulating the Notch pathway. It is interesting that the extra joints observed in the tarsus of nd;Su(dx)^sp are of reversed polarity. In the third instar and pupal leg, Serrate, Delta, and fringe are expressed in largely overlapping domains proximal to the site of joint formation. In the wild-type leg, the joints always form distal to the stripes of high Serrate and Delta expression and not proximal. However, in polarity mutants such as dsh¹, extra joints of reversed polarity are formed just proximal to the high levels of Delta and Serrate expression and are coincident with ectopic Notch activation. It has been proposed that repressor elements possibly involving planar polarity signalling, together with Fringe, repress Notch activation proximal to the stripe of high Serrate and Delta expression, and the nd¹; Su(dx)^sp combination may thus be able to overcome this inhibition. Overexpression of Su(dx) using the PtcGAL4 driver, which drives along the A-P boundary of the leg disc, causes fusion of the tarsal joints and a shortening of the leg. This is again consistent with the role of Su(dx) being to inhibit Notch signalling, because loss of function of Notch, Serrate, and Delta results in fusion between leg segments and reduced leg growth (Mazaleyrat, 2003).

Interestingly while Deltex expression does not block the Notch down-regulatory activity of Su(dx), it does inhibit the latter’s wing overgrowth phenotype. This uncoupling of phenotypes suggests that Su(dx) has multiple activities. One activity down-regulates the Notch signal and thus blocks the ectopic wing margin and wing growth phenotype induced by Deltex overexpression. The overexpression of Deltex may in turn titrate Su(dx) away from a second activity responsible for a distinct wing overgrowth phenotype. This could explain how the coexpression of these two proteins fails to produce a wing overgrowth when the expression of each singly does result in an overgrowth phenotype (Mazaleyrat, 2003).

Additional Su(dx) activities may also explain an unexpected interaction of Su(dx) with daughterless (Smith, 2002). Loss of function of Su(dx) enhances the daughterless phenotypes during ovary development, similar to the enhancement of daughterless shown by loss of function mutations of Notch. This is in contrast to what would be predicted if the activity of Su(dx) in the ovary was a negative regulator of Notch and therefore supports the hypothesis that Su(dx) may have more than one role (Mazaleyrat, 2003).

In conclusion, data provide support for a direct role, in vivo, for Su(dx) in the regulation of the Notch pathway in different developmental contexts. The phenotype of Su(dx) may be moderated by the feedback activity of interlocking networks of signals and other means of developmental redundancy. Further analysis of interacting genetic backgrounds should reveal the full scope of Su(dx)-dependent functions, which may also include additional activities beyond Notch down-regulation (Mazaleyrat, 2003).

Notch activity in neural cells triggered by a mutant allele with altered glycosylation

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

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

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

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

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

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

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

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

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

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

The nature, extent, and consequences of genetic variation in the opa repeats of Notch in Drosophila

Polyglutamine (pQ) tracts are abundant in proteins co-interacting on DNA. The lengths of these pQ tracts can modulate their interaction strengths. However, pQ tracts > 40 residues are pathologically prone to amyloidogenic self-assembly. This study assesses the extent and consequences of variation in the pQ-encoding opa repeats of Notch in Drosophila melanogaster. Sanger sequencing was used to genotype opa sequences (5'-CAX repeats), which have resisted assembly using short sequence reads. While most sampled lines carry the major allele opa31 encoding Q13HQ17 or the opa32 allele encoding Q13HQ18, many lines carry rare alleles encoding pQ tracts > 32 residues: opa33a (Q14HQ18), opa33b (Q15HQ17), opa34 (Q16HQ17), opa35a1/opa35a2 (Q13HQ21), opa36 (Q13HQ22), and opa37 (Q13HQ23). Only one rare allele encodes a tract < 31 residues: opa23 (Q13-Q10). This opa23 allele shortens the pQ tract while simultaneously eliminating the interrupting histidine. The study introgressed these opa variant alleles into common backgrounds, and measured the frequency of Notch-type phenotypes. Homozygotes for the short and long opa alleles have defects in embryonic survival and sensory bristle organ patterning, and sometimes show wing notching. Consistent with functional differences between Notch opa variants, it was found that a scute inversion carrying the rare opa33b allele suppresses the bristle patterning defect caused by achaete/scute insufficiency, while an equivalent scute inversion carrying opa31 manifests the patterning defect. These results demonstrate the existence of potent pQ variants of Notch, and the need for long read genotyping of key repeat variables underlying gene regulatory networks (Rice, 2015).

Mutational alteration of sites of O-fucosylation of Notch: Influence on Notch signaling

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

To begin to identify EGF domains whose O-fucosylation influences Notch activation, S was substituted at the O-fucose attachment site for A, and T for V. A or V can be found at this position in other EGF repeats of Notch or its ligands, and hence are unlikely to cause disruptions of EGF structure. Focus was placed on four EGF repeats of Notch: 12, 24, 26 and 31. EGF24, EGF26 and EGF31 were chosen because they lie in or near the region of Notch to which the N^Ax alleles map, and because they contain highly conserved O-fucose sites that conform to the original consensus sequence. EGF12 was chosen because it corresponds to one of two EGF repeats identified as necessary and sufficient for Notch-ligand binding in a cell aggregation assay, and because it contains a potential O-fucose site in all cloned Notch receptors with 36 EGF repeats. Although this site does not conform to the original consensus for O-fucosylation, EGF12 of Notch1 has been shown to be glycosylated by O-FucT-1 and Fringe in CHO cells. O-fucosylation of Drosophila Notch EGF12 in Drosophila cells was confirmed by assessing the ability of a fragment of Notch isolated from S2 cells to serve as an in vitro substrate for Fringe (Lei, 2003).

Therefore, EGF12 is a biologically relevant site of O-fucosylation. O-fucose is attached to an S or T. Consequently, when that amino acid is changed to one that lacks a terminal hydroxyl group, O-fucosylation of the EGF domain cannot occur. Consistent with this, the S to A mutation eliminates the ability of a Notch fragment including EGF12 to serve as a substrate for Fringe. For several reasons, the observed differences between N-EGF12f and wild-type Notch can be attributed to this absence of glycosylation, rather than to the amino acid change per se. Substitution of an S with an A is a conservative change, and the two amino acids differ only by an oxygen atom. A is found at this location in other EGF repeats (e.g., EGF36 of Drosophila Notch, and EGF7 and EGF19 of mammalian Notch1), and hence is unlikely to disrupt the EGF structure. Indeed, this same mutation in EGF26 does not result in a detectable phenotype. A distinct amino acid change in EGF12, the E491V mutation in N^M1, results in a strong loss-of-function phenotype, as would be predicted for a gross structural change in the ligand-binding domain (Lei, 2003).

By contrast, the phenotype of N-EGF12f is consistent with that which would be expected of a Notch receptor that had lost a functional site of glycosylation by Fringe. Expression of N-EGF12f results in an ectopic activation of Notch in dorsal wing cells that is insensitive to Fringe, yet dependent upon endogenous ligand expression. Binding studies further show that Serrate is able to bind to this mutant form of Notch even in the presence of Fringe, which contrasts with the lack of detectable Serrate binding to wild-type Notch expressed in the presence of Fringe. Based on these observations, it is concluded that EGF12 is an essential site for inhibition of Serrate-to-Notch signaling by the Fringe glycosyltransferase (Lei, 2003).

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

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

Among the 15 Notch receptors with 36 EGF repeats in sequence databases, an average of 20 of the 36 EGF repeats contain potential sites for O-fucosylation. However, only three EGF repeats contain O-fucose sites in all of these 15 Notch receptors: EGF12, EGF26 and EGF27. Thirteen other EGF domains contain sites that are somewhat conserved (i.e., an O-fucose site is found in that repeat in 11 or more of the 15 Notch protein sequences), including EGF24 (13/15 Notch receptors) and EGF31 (14/15 Notch receptors). These conserved sites for O-fucosylation cluster in an N-terminal region, and in a more C-terminal region centered around the N^Ax mutations. This general pattern of conservation (most Notch receptors have many sites, but only a few sites are absolutely conserved) suggests that at least some aspects of OFUT1 and Fringe regulation might be achieved through glycosylation of regions of Notch, rather than through glycosylation of specific EGF repeats. The lack of effect of mutation of individual, highly conserved EGF repeats in the N^Ax region is consistent with this suggestion, and experiments to analyze the consequences of mutation of arrays of O-fucose sites are in progress (Lei, 2003).

Notch ligands activate Notch receptors expressed by neighboring cells, but inhibit Notch receptors expressed by the same cell. Elevated expression of the Notch extracellular domain can also inhibit the ability of ligands to signal to neighboring cells. Thus, one apparent consequence of the transmembrane nature of Notch ligands is that Notch activation depends not simply on the ability of ligand to bind receptor, but also on a competition between intracellular and intercellular interactions. Previously, most attention has focused on the impact of different levels of expression on this competition. But the balance in this competition can also be shifted by adjusting the affinity between Notch and its ligands. Indeed, even though most studies have focused on the ability of Fringe to inhibit the response of a cell to Serrate, the ability of cells to send a Serrate signal appears to be enhanced by co-expression with Fringe, which is consistent with the idea that decreasing intracellular Serrate-Notch interactions increases the amount of Serrate available to signal to neighboring cells (Lei, 2003).

Cell-based binding assays indicate that the O-fucose site in EGF12 is not just important for Fringe-dependent inhibition: even the presence of the O-fucose monosaccharide at this site inhibits Serrate binding. The presence of an inhibitory site of O-fucosylation in EGF12 was unexpected given the general positive requirement for O-fucose in Notch signaling. However, the presence of an inhibitory site can be rationalized in terms of a competition between intracellular and intercellular Notch-ligand interactions. The competition model implies that it is important, at least in certain contexts, for Notch not to bind too strongly to its ligands. One such context is probably the DV boundary of the Drosophila wing, because Notch ligands are expressed on both sides of the compartment boundary, and Notch is activated on both sides of the compartment boundary. Thus, it is suggested that N-EGF12f is unable to rescue normal Notch activation at the DV boundary because its increased affinity for ligands enhances intracellular binding to a degree that interferes with the ability of a cell to send and receive Notch signals. Notably, EGF12 is apparently essential for both intercellular and intracellular Notch-ligand interactions (Lei, 2003).

The highly conserved presence of an O-fucose site in EGF12 suggests that inhibition of ligand binding by the O-fucosylation of EGF12 might be of widespread importance. However, if O-fucosylation of EGF12 was constitutive, it would simply counteract the positive influence of O-fucosylation at other sites. If, by contrast, O-fucosylation of EGF12 was regulated, then differential O-fucosylation of EGF12 could occur, and could serve as a mechanism of Notch regulation. Intriguingly then, EGF12 is distinguished from other potential O-fucose sites by the presence of an acidic amino acid (E or D) at the -2 position relative to the O-fucose attachment site. None of the other EGF repeats in Notch contain an acidic amino acid at this position, yet 13/15 Notch receptor proteins contain an acidic amino acid at this position in EGF12. It is not yet known what fraction of Notch receptors in a cell are modified at any of the potential sites of O-fucosylation, but the presence of this conserved sequence difference suggests that EGF12 might be O-fucosylated under different conditions, or with a different efficiency, than other EGF domains, and hence that differential fucosylation of this site might serve as a regulatory mechanism (Lei, 2003).

Notch and Scabrous

Notch and Delta are required for lateral inhibition during eye development. They prevent a tenfold excess in R8 photoreceptor cell specification. Mutations in two other genes, Scabrous and Gp150, result in more modestly increased R8 specification. Their roles in Notch signaling have been unclear. Both sca and gp150 are required for ectopic Notch activity that occurs in the split mutant. Similar phenotypes show that sca and gp150 genes act in a common pathway. Gp150 was required for all activities of Sca, including inhibition of Notch activity and association with Notch-expressing cells that occur when Sca is ectopically expressed. Mosaic analysis found that the gp150 and sca genes were required in different cells. Gp150 concentrates Sca protein in late endosomes. A model is proposed in which endosomal Sca and Gp150 promote Notch activation in response to Delta, by regulating acquisition of insensitivity to Delta in a subset of cells (Li, 2003).

A Notch mutation, split, specifically elevates Notch activity in the neural cells. The split mutation alters glycosylation of the N extracellular domain and leads to inappropriate N activity within R8 precursor cells in the developing eye. Specifically, a Ile578-->Thr578 substitution responsible for the split mutation introduces a new site for O-fucosylation on EGF repeat 14 of the Notch extracellular domain. This suggests functional differences between Notch in neural and non-neural cells. Thus factors specifically regulate the inactivity of N in neural cells and contribute to the spatial pattern of neurogenesis (Li, 2003).

Genetic studies have identified several genes whose mutations interact with the split allele. One gene has been reported where deletion of a single allele is sufficient to suppress the spl phenotype. This gene encodes the secreted protein Scabrous. In addition, in the homozygous absence of sca, the spl mutation has no detectable effect, i.e. spl mutant and wild-type N behave indistinguishably. Conversely duplications of sca enhance the spl phenotype. These results indicate that activity of N in neural cells depends critically on sca. By contrast, none of the well-known components of N signaling behave as such dose-sensitive genetic modifiers of spl. Special alleles of Dl were also recovered as dominant spl suppressors, consistent with the finding that in spl the N activity in neural cells is ligand dependent (Li, 2003).

The molecular role of Scabrous in the Notch pathway is not yet clear. Mutations of sca cause defects in the spacing and number of sensory mother cells in the epidermis and of R8 precursor cells in the retina, two founder cell types for adult peripheral nervous system. The sca mutations act cell nonautonomously. Because N acts cell autonomously in the specification of these same cell types it was suggested that sca encodes an extracellular ligand for the receptor protein N. This hypothesis proved difficult to confirm, however, since sca mutations affect only a subset of Notch functions, have weaker effects than N null mutations, and since no direct interaction between the Sca and N proteins has been demonstrated. Other ideas have been proposed: that Sca acts to scaffold N to the extracellular matrix to downregulate N activity; that it acts to preserve epithelial structure within proneural regions and so enhance function of other N ligands, or acts independently of N to arrest ommatidial rotation (Li, 2003).

Other findings strongly suggest that Sca and N proteins are closely associated in vivo. When Sca is overexpressed in the developing wing, N activity and specification of the wing margin are prevented, even though wing margin specification is independent of Sca function in the wild type. Sca protein appears to prevent Dl from activating of N in this ectopic expression assay. The results strongly suggest that Sca protein targets N signaling, although not defining the exact role of Sca in normal development. When ectopically expressed in pupal retina, Sca protein is preferentially stabilized in cells expressing N and such stability depends on EGF repeats 19-26 of the N extracellular domain. Dl and Ser signal through EGF repeats 10-12. The association with Sca occurs independently of N signaling activity. Chemical crosslinking of Drosophila embryos detects Sca protein in a complex with N, consistent with a close association between the proteins in vivo. Sca protein also appears to stabilize N protein on the surface of tissue culture cells. It remains uncertain, however, whether the interaction is direct or mediated by other proteins, or where in the cell it occurs (Li, 2003 and references therein).

Another gene required for proper eye and bristle patterning has recently been described. Mutations at the Gp150 locus cause defects in ommatidial development and cuticular bristle development that are similar to those seen in sca homozygotes (Fetchko, 2002). Gp150 protein was originally isolated biochemically as a phosphoprotein target of the receptor tyrosine phosphatase DPTP10D. Recent work shows that Gp150 is located in endosomes and interacts with the Notch pathway (Li, 2003).

This study explores the relationship of Sca and Gp150. Gp150 is required for neural Notch activity in the spl mutant, and it is concluded that the Sca and Gp150 proteins must act in a common pathway, with Gp150 acting downstream in cells that respond to secreted Sca protein. Gp150 is required for all Sca activities yet identified, including those of ectopic expression and association with Notch in vivo. Sca is localized to endosomes along with Gp150. It is proposed that an endosomal pathway downregulates N activity in neural cells, and that Sca and Gp150 oppose this pathway to permit N activity in a subset of non-neural cells. Accordingly, Sca and Gp150 activate N indirectly, via effects on N downregulation (Li, 2003).

One piece of evidence that Sca interacts with N comes from colocalization studies in the pupal retina. Notch protein distribution is unusually asymmetric in pupal retina, being excluded from the differentiating ommatidia but expressed in the surrounding pigment cell lattice. Sca expressed transiently and uniformly from the hsp70 promoter accumulates in N-expressing cells, implying an interaction of some kind between the proteins (Li, 2003).

To test whether Gp150 was required for Sca to associate with N, Sca was expressed in pupal retinas from gp150 mutants. Prior to heat shock, pupal retinas from HS-sca transgenic flies lack Sca protein until specification of interommatidial bristle precursors begins. Within 20 minutes of mild heat shock newly synthesized Sca protein was cytoplasmic and uniformly distributed. As Sca protein was secreted and decayed, protein transiently accumulates in the Notch-expressing pigment cell lattice, usually between 40-80 minutes after heat shock. Notch protein is still expressed in the pigment cell lattice of gp150 mutants, but heat-shock induced Sca protein shows no accumulation in these cells. Thus, gp150 is required for Sca to accumulate in N-expressing cells in this assay (Li, 2003).

Sca deletion proteins were used to investigate further how Sca associates with N. Sca comprises an N-terminal coiled-coil, previously found to be sufficient for sca function, and the C-terminal fibrinogen related domain (FReD) that increases the activity of the protein. Flies transgenic for truncated Sca proteins under control of the heat shock promoter were prepared. Neither the ScaDelta41-514 protein encoding the FReD nor the N-terminal sequences encoded by ScaDelta513-773 accumulates in N-expressing cells to the same degree as does full-length Sca. There seems to be more accumulation with the ScaDelta41-514 protein, as if the FReD makes more contribution to Sca accumulating in N-expressing cells (Li, 2003).

It is suggested that neural cells in the spl mutant mimic a subset of non-neural cells that approach neural fate in wild-type development, and that Sca and Gp150 chiefly contribute to N signaling in such cells. It is proposed that during lateral inhibition to select neural precursor cells, activation of N signaling is only one part of the story. Inactivation of N signaling in cells taking the neural fate is also required. It is suggested that neural cells in which N is inactive have passed through a transient stage in which a low level of incipient N signaling is a normal occurrence prior to neural determination. In this model, Sca and Gp150 normally function to sustain N activity in potential neural cells (or to block or delay N inactivation in potential neural cells). Accordingly, Sca and Gp150 increase N signaling by the same mechanism both in wild-type cells on the verge of neural specification and in spl mutant cells struggling to maintain N inactivity. This model predicts that absence of Sca or Gp150 could lead to N inactivity in too many cells and specification of extra neural precursor cells. This is consistent with the sca and gp150 mutant phenotypes. This model is consistent with the presence of Sca and Gp150 in endosomes, as it posits that they regulate inactive N molecules, not the process of N activation that occurs at the cell surface (Li, 2003).

The model suggests two slightly different routes for the inhibition of neural fate by N. In some cells, activation of N by Dl is sufficient. As a by-product of the protection of future neural cells from Dl, there appear to be other cells that are also at risk for protection from Dl. By antagonizing protection, Sca and Gp150 promote N activity in such cells and prevent too many cells taking neural fate (Li, 2003).

The pathway of N activation in which ligands trigger proteolytic cleavages to release the intracellular domain is thought to occur at the cell surface, and none of these reactions is thought to involve endosomes. N activation by trans-endocytosis of the N extracellular domain has been proposed, but this involves endosomes in the signal sending cell, which is not where mosaic analysis finds Gp150 to be required. Endocytosis has been proposed both to downregulate N activity and to promote N activity by removing inactive and inhibitory forms of both N and its ligands from the cell surface. Although the current data are probably consistent with previous models for Sca function in increasing the sensitivity or range of N signaling, both the idea that sca and gp150 are most important in cells where N signaling would otherwise be downregulated, and the location of their products away from the cell surface supports the view that these proteins specifically affect a downregulatory mechanism, rather than acting directly on N activation. Since the ectopic N activity in the spl mutant depends on Dl, it is inferred that sca and gp150 promote ligand-dependent N activation (Li, 2003).

Several new models can be proposed. One model is that either before or after Dl binding, endocytosis reduces the amount of surface N available for activation. Sca and Gp150 might antagonize such endocytosis, or permit endocytosed N to be activated, either by permitting gamma-secretase to act on endocytosed intermediates or by their return to the cell surface. A second model incorporates the observation that in addition to activating N signaling on neighboring cells, N ligands can `cis-inactivate' N signaling in the same cell. Protection of neural cells from N activation by Dl might reflect an increased cis-inactivation in neural cells. In this model, Sca and Gp150 would antagonize cis-inactivation, e.g. by removing Dl or N from cis-inactivatory interactions at the cell surface or in endosomes. Interestingly, Dl is also present in Gp150-positive vesicles. Elevated intracellular Dl levels have been observed in gp150 mutants, suggesting that intracellular Dl may antagonize N signaling (Li, 2003).

One problem for these models is that changes in the cell surface levels of N or Dl have not been detected during the selection of neural cells. It remains possible that there are changes in subsets of the detectable N or Dl proteins that are somehow particularly important for signaling. It is interesting to note that endocytosis is also implicated in N regulation within neural stem cell lineages. Asymmetric divisions during sensory organ lineages deliver Numb protein to particular daughter cells, where Numb then inhibits N signaling through binding to N and to alpha-adaptin, an adaptor for endocytosis via clathrin-coated pits. Although presumed to promote N endocytosis, numb and alpha-adaptin result in no detectable reduction in N protein levels despite blocking N activity. In nematodes, endocytosis has been proposed to permit downregulation of the N-homolog lin-12 by Ras. Perhaps endosomes provide an environment where N signaling components are neither degraded nor removed permanently from the cell surface, but rerouted or modified to change their signaling properties (Li, 2003).

Notch and Friend of echinoid

echinoid (ed) encodes a cell-adhesion molecule (CAM) that contains immunoglobulin domains and regulates the Egfr signaling pathway during Drosophila eye development. Genetic mosaic and epistatic analysis, has suggested that Ed, via homotypic interactions, activates a novel, as yet unknown pathway that antagonizes Egfr signaling. Alternatively, later studies indicate that Ed inhibits Egfr through direct interactions. Another body of work suggests that Ed functions as a homophilic adhesion molecule, and also engages in a heterophilic trans-interaction with Drosophila Neuroglian (Nrg), an L1-type CAM. Co-expression of ed and nrg in the eye exhibits a strong genetic synergy in inhibiting Egfr signaling. This synergistic effect requires the intracellular domain of Ed, but not that of Nrg. A model for this interaction suggest that Nrg acts as a heterophilic ligand and activator of Ed, which in turn antagonizes Egfr signaling (Spencer, 2003 and references therein; Islam, 2003 and references therein).

Complicating the picture even further is an analysis of a paralogue of Ed termed friend of echinoid (fred). ed and fred transcription units are adjacent to one another, approximately 100 kilobases apart on chromosome arm 2L, but they are divergently transcribed in opposite directions. Fred acts in close concert with the Notch signaling pathway. Suppression of fred function results in specification of ectopic SOPs in the wing disc and a rough eye phenotype. Overexpression of N, Su(H), and E(spl)m7 suppresses the fred RNAi phenotypes. Accordingly, decreasing Su(H) or overexpression of Hairless enhances the fred RNAi phenotypes. Thus fred, a paralogue of ed, shows close genetic interaction with the Notch signaling pathway. The weak genetic interaction observed between fred and components of the Egfr pathway also links fred to the Egfr pathway; however, analysis of additional components of the Egfr pathway are necessary to determine Fred's role in the Egfr signaling (Chandra, 2003).

In order to study the function of fred, the heritable and inducible double-stranded RNA-mediated interference (RNAi) method was used. For this study, transcript sequence of fred was cloned as a dyad symmetric molecule in the pUAST vector and transgenic lines established. Expression of the construct was induced by crossing the transgenic lines to tissue- and/or stage-specific GAL4 driver lines. Transcription of a dyad symmetric molecule results in a RNA that snaps back to give rise to a dsRNA with a hairpin loop; this mediates the degradation of the corresponding endogenous mRNA. A 638-bp region of fred was selected for this analysis based on minimal similarity to ed sequence (Chandra, 2003).

The Notch signaling pathway is involved in limiting the SOP fate to a single cell within each proneural cluster. Since degradation of fred mRNA leads to formation of ectopic SOPs, it was of interest to see if the Notch signaling pathway genes functionally interact with fred in this process and, thus, may modulate the fred RNAi phenotype. To this end, four Notch pathway genes, Notch (N), Suppressor of Hairless [Su(H)], Hairless (H), and E (spl) m7 were tested for genetic interactions with fred (Chandra, 2003).

Overexpression of Notch leads to a loss of sensory organs and hair to socket transformation. Expression of a UAS-Notch (UAS-N) construct with pnr-GAL4 results in flies that show loss of most of the bristles from the dorsal-most region of the thorax. In addition, occasionally, bristle to socket transformation is observed. When fred dsRNA and Notch are expressed simultaneously by using the pnr-GAL4 driver, the flies show a phenotype that is intermediate between that of the two individual phenotypes. Although overexpression of Notch could suppress the cuticular holes and ectopic microchaeta formation, the thoraces of these flies still had some of the phenotypes associated with RNAi-mediated suppression of fred, such as a pinched notum and a smaller scutellum (Chandra, 2003).

The observations that changes in the activity of four genes of the Notch signaling pathway can either suppress or enhance the phenotypes associated with the suppression of fred function suggest that fred is functioning in close concert with the Notch signaling pathway. Reduction in the activity of a Notch signaling pathway gene, Su(H) results in an enhancement of the fred RNAi phenotype. In contrast, ectopic expression of Notch signaling pathway genes, Notch, Su(H), and E(spl)m7 suppresses, to different degrees, different aspects of the fred RNAi phenotype in the developing wing, thorax, and eye. In contrast, overexpression of Hairless (a negative regulator of the Notch pathway) enhances the phenotypes induced by Fred suppression. It is presently not clear whether Fred defines a separate pathway for SOP determination or if it shares downstream components of the Notch signaling pathway. The remarkable degree to which ectopic expression of an E(spl) complex bHLH transcription factor results in a nearly complete suppression of phenotypes associated with fred degradation strongly supports the idea of very close functional interactions. These observations, furthermore, raise the possibility that E(spl) complex genes and/or other genes of the Notch signaling pathway act downstream of fred function (Chandra, 2003).

Notch and Tantalus, a potential link between Notch signalling and chromatin-remodelling complexes

The tantalus (tan) gene encodes a protein that interacts specifically with the Polycomb/trithorax group protein Additional sex combs (ASX). Both loss-of-function and gain-of-function mutations in tan cause tissue-specific defects in the eyes, wing veins and bristles of adult flies (Dietrich, 2001). Since these defects are also typical for components of the Notch signalling pathway, it was of interest to determine if Tan interacts with this pathway. Through examination of ectopic tan phenotypes, it was found that Tan specifically disrupts all three major processes associated with the N signalling pathway (boundary formation, lateral inhibition, and lineage decisions). Furthermore, ectopic tan expression abolishes expression of two N target genes, wingless and cut, at the dorsal-ventral boundary of the wing. An interaction between tan and N was also observed using a genetic assay that detects interactions between tan and Asx. The observed ability of Tan to move between the cytoplasm and nucleus, and to associate with DNA, provides a potential mechanism for Tan to respond to N signalling (Dietrich, 2005).

The tantalus (tan) gene was identified in a yeast two-hybrid screen designed to uncover interactors for the Polycomb and trithorax group (Pc-G and trx-G) protein Additional sex combs (Asx; Dietrich, 2001). The Pc-G and trx-G protein complexes act to maintain states of transcription during development in a wide range of organisms. Asx is ubiquitously expressed, but has tissue-specific functions, which led to the postulation that it most likely requires the cooperation of tissue-specific cofactors. Asx and Tan interact directly in vitro as well as in yeast and co-localize to approximately 35 of the 66 Tan polytene chromosome binding sites. Mutants of Asx and tan interact genetically during bristle formation. Although tan is expressed throughout embryogenesis and larval development, higher levels of expression are observed in some tissues (e.g., the morphogenetic furrow of the eye disc; Dietrich, 2001). Flies homozygous for tan null mutations exhibit a variety of sensory organ defects including roughened eyes, bristle loss and duplication and minor defects in distal wing vein formation, but are otherwise viable and fertile. These results led to the proposal that Tan acts as a tissue-specific cofactor for Asx function during sensory organ development. However, its relatively ubiquitous expression left it unclear as to why Tan function appears to be limited to this relatively small subset of tissues (Dietrich, 2005).

The limited loss-of-function tan phenotype, and the stronger, more widespread phenotypes caused by ectopic expression, are similar to those found with other components of the N signalling pathway. A loss-of-function Suppressor of deltex allele, for instance, displays only a weak wing vein gap phenotype that is similar to gain-of-function mutations in Notch. However, ectopic expression of Suppressor of deltex led to more severe phenotypes such as fusions of the leg tarsals, as seen with ectopically expressed tan (Dietrich, 2005).

Unlike most other N pathway genes, it appears unlikely that tan is a downstream transcriptional target of N signalling since tan expression is more or less ubiquitous. This probability is supported by the lack of high affinity binding sites for the Suppressor of Hairless protein, the effector of transcriptional regulation by the N signalling pathway, in the vicinity of the tan gene. However, to unequivocally verify that tan is not a transcriptional target of N signalling, it will be important to analyse tan gene expression in greater detail. Although the possibility that Tan is acting in a parallel pathway cannot be ruled out, the ability of Tan to specifically interfere with the three N-related processes described in this study (boundary formation, lateral inhibition, and lineage decisions), to negatively regulate two N target genes and to genetically interact with N suggests that Tan is more directly involved in N signalling (Dietrich, 2005).

While the data are consistent in their implication of a N and Tan interaction, they differ in the types of relationship implied. The genetic enhancement experiments tended to indicate a positive interaction, while most of the ectopic expression assays implied a negative interaction. There are several possible explanations for these seemingly conflicting results. One possibility is that ectopic expression of Tan, superimposed upon ubiquitous expression of the endogenous gene, may result in the sequestering of N signalling components into incomplete or non-productive complexes (i.e. a dominant-negative effect). A second possible explanation is the complexity of most N-regulated processes, which typically involve a number of sequential events. Loss-of-function and gain-of-function scenarios could well affect these processes at different steps, at different times and in different ways. A third explanation, also stemming from the complexity of the N signalling process, is that compensating effects within the pathway may respond differently to gain or loss of Tan function. For example, it has previously been shown that E(spl)-C genes, which are normally up-regulated by N signalling, can be up-regulated in Su(H) mutant backgrounds. This is because E(spl)-C gene expression can be activated by both the Su(H) and bHLH proteins, and bHLH expression is up-regulated in the absence of Su(H) expression. Clearly, a full understanding of the role(s) of Tan during these steps will require further analyses at the temporal and cellular level (Dietrich, 2005).

The most interesting aspect of the analyses of tan and N signalling is the potential link between Pc-G/trx-G gene regulation and the N signalling pathway. Although the effects of the Pc-G and trx-G complexes have been most extensively analysed for the homeotic genes, it is clear from mutational analyses and the number of sites on polytene chromosomes bound by different family members, that Pc-G and trx-G complexes must regulate other genes as well. These results suggest that, at some level, Tan in conjunction with Asx could interact with N signalling components to modulate Pc-G/trx-G function. Interestingly, ectopically expressed Tan is capable of cycling between the cytoplasm and nucleus, raising the possibility that Tan entry into the nucleus could be associated, either directly or indirectly, with N signalling. The coupling of Tan entry into the nucleus with N signalling could help explain the paradox of a ubiquitous expression pattern but specific sensory functions for tan (Dietrich, 2005).

Tan possesses inherent DNA binding ability (Dietrich, 2001), and once localised to the nucleus it is possible that Tan could be recruited to specific targets of N signalling. A subsequent interaction between Tan and Asx would make a link to the Pc-G and trx-G proteins. There is currently no evidence to suggest that Tan and N signalling are always linked. Tan may cooperate with N only during specific signalling events. It will be important to assess whether Tan nuclear localisation is altered in cells where N signalling has been activated to determine if these events are functionally coupled (Dietrich, 2005).

Bre1 is required for Notch signaling and histone modification

Notch signaling controls numerous cell fate decisions during animal development. These typically involve a Notch-mediated switch in transcription of target genes, although the details of this molecular mechanism are poorly understood. dBre1 has been identified as a nuclear component required cell autonomously for the expression of Notch target genes in Drosophila development. dBre1 affects the levels of Su(H) in imaginal disc cells, and it stimulates the Su(H)-mediated transcription of a Notch-specific reporter in transfected Drosophila cells. Strikingly, dBre1 mutant clones show much reduced levels of methylated lysine 4 on histone 3 (H3K4m), a chromatin mark that has been implicated in transcriptional activation. Thus, dBre1 is the functional homolog of yeast Bre1p, an E3 ubiquitin ligase required for the monoubiquitination of histone H2B and, indirectly, for H3K4 methylation. These results indicate that histone modification is critical for the transcription of Notch target genes (Bray, 2005).

The lethal allele E132 was fortuitously identified among a collection of mutants that modify the wing notching phenotype caused by Armadillo depletion. Genetic mapping of the lethality associated with E132 placed this at 64E8, and it was found to be allelic to an existing mutation, l(3)01640, caused by the P element insertion P1541. Using plasmid rescue of the P element, the site of insertion was localized to the first intron of the open reading frame CG10542, which encodes a predicted protein of 1044 amino acids. The insertion site is 48 nucleotides upstream of the translation initiation codon. Precise excision of P1541 restores viability, confirming that the P element insertion and, by inference, E132 are lethal alleles of CG10542. In support of this, ubiquitous overexpression of the full-length protein encoded by CG10542 rescues the lethality of E132 or P1541 mutant embryos and sustains development to give essentially normal adult flies (with a few minor defects including slightly reduced bristles). CG10542 encodes a conserved protein with close relatives in mammals, C. elegans, plants, and fungi. The Drosophila protein has been named dBre1, after its relative Bre1p in the yeast S. cerevisiae (Bray, 2005).

The hallmarks of the Bre1 proteins are a C-terminal RING finger domain linked to an extensive N-terminal coiled-coil region. The 39 amino acid C3HC4 RING domain is flanked on both sides by ~15 conserved amino acids, suggesting that the fly and mammalian proteins are true orthologs of yeast Bre1p. RING domains are typically found in E3 ubiquitin ligases and frequently mediate the interaction with the E2 ubiquitin-activating enzyme while the other parts of the protein are involved in substrate recognition. The RING domains are therefore critical to catalyze the transfer of ubiquitin from the E2 to the substrate. To confirm the functional importance of the RING domain in dBre1, tests were performed to see whether an N-terminal fragment of dBre1 that lacks the RING domain (ΔRING) could rescue dBre1 mutants. No rescue was observed with any of the 4 transgenic lines (from a total of 814 flies scored), confirming that the RING domain is essential for the function of dBre1 as it is for yeast Bre1p (Bray, 2005).

To examine the subcellular location of full-length dBre1 and the derivative that lacks the RING domain, both forms of the protein were tagged with GFP at the N terminus. Both GFP-dBre1 and GFP-ΔRING are predominantly nuclear in embryonic and imaginal disc cells, although a low level of protein is also detectable in the cytoplasm. This nuclear-cytoplasmic distribution is similar to that of a ΔRING derivative of human Bre1-B when it is overexpressed in mammalian cells. Thus dBre1 appears to be a nuclear protein, like its mammalian counterpart, and deletion of the RING domain does not alter its subcellular distribution even though it abolishes its ability to rescue the mutants (Bray, 2005).

To investigate the role of dBre1 in the fly, homozygous mutant clones were generated in the imaginal disc precursors of the adult structures. Surprisingly, it was found that the majority of defects were similar to those caused by defects in Notch signaling. Thus, adult flies bearing E132 or P1541 mutant clones show notches in the wing margin and aberrant spacing of wing margin bristles, wing blistering and vein defects, fusions of leg segments, and loss of notal bristles and rough eyes. Most of these phenotypes are characteristic of reduced Notch signaling and are distinct from those produced by loss-of-function of other signaling pathways, such as Wingless, Dpp, or Hedgehog signaling that also operate during imaginal disc development. The phenotypic data suggest therefore that dBre1 has a role in promoting Notch signaling (Bray, 2005).

To confirm this, the expression of Notch target genes was examined in dBre1 mutant clones. Since dBre1 mutant clones are considerably smaller than their matched wild-type twin clones, the Minute technique was used to compensate for the growth defect of the mutant clones. In wing imaginal discs, cut and Enhancer of split [E(spl)] are expressed along the prospective wing margin, and their expression depends directly on Notch signaling. Cut expression is absent in large E132 mutant clones, and is lost (3/11) or reduced (6/11) in most P1541 mutant clones. Likewise, E(spl) expression is lost cell autonomously from all E132 mutant clones in the wing. Conversely, expression of spalt, a target of Dpp signaling in the wing, is not reduced in P1541 and E132 mutant cells, indicating that the effects of dBre1 mutation are relatively specific. Similar results are obtained in the eye, where E(spl) expression is also disrupted in E132 clones. Expression in the neurogenic region at the furrow is lost, and elsewhere it is absent or severely reduced, except in the basal layer of undifferentiated cells where expression is independent of Notch. In addition, a derepression of the neuronal cell marker Elav was observed in eye disc clones. The latter indicates excessive neuronal recruitment due to diminished Notch-mediated lateral inhibition (note, however, that the phenotypes are not identical to those produced by complete absence of Notch, which in the eye results in loss of neuronal markers because Notch is needed to promote neural development by alleviating Su(H)-mediated repression. These results demonstrate that dBre1 functions in multiple developmental contexts and, specifically, that it is required for the subset of Notch functions that involve Su(H)-dependent activation of Notch target genes (Bray, 2005).

To further confirm the importance of dBre1 during Notch signaling, it was asked whether any genetic interactions could be detected between overexpressed dBre1 or ΔRING and mutations in Notch (N) or its ligand Delta (Dl). Indeed, overexpression of either protein in the wing disc results in adult phenotypes. In each of 5 ΔRING-expressing lines, mild if consistent mutant phenotypes were observed in both males and females, namely upward-curved wings (due to stronger expression in the dorsal wing compartment), tiny vein deltas, and a significant decrease in wing size. These defects are more severe after overexpression of ΔRING in dBre1 heterozygotes, indicating that ΔRING acts as a weak dominant-negative. Consistent with this, excess ΔRING significantly enhances the phenotypes of N/+ and Dl/+ heterozygotes, resulting in increased vein thickening and additional vein material and, in the case of N/+, also in more frequent wing notching. These genetic interactions support the link between dBre1 and Notch signaling (Bray, 2005).

Excess full-length dBre1 in wing discs causes vein defects whose strength, however, varies considerably between different dBre1-expressing lines, and between males and females (probably because the ms1096.GAL4 driver produces higher expression levels in males). In most lines (4/6), vein thickening and additional vein material were observe only in males, while female wings appear normal. These vein defects in male wings are suppressed to almost normal in dBre1 heterozygotes, suggesting that they are due to increased levels of functional dBre1 protein. The remaining 2 lines produce similar vein defects also in females. Unexpectedly, these defects are enhanced in N/+ and Dl/+ heterozygotes, suggesting that the overexpressed dBre1 interferes with Notch signaling, rather than enhancing it as might have been expected. This anomalous result could be explained if dBre1 is part of a multiprotein complex, in which case its overexpression might interfere with the function of this complex by titrating one of its components. Nevertheless, the genetic interactions between overexpressed dBre1 and Notch and Delta further underscore the link between dBre1 and Notch signaling (Bray, 2005).

To test whether dBre1 directly influences Notch-dependent transcription, Drosophila S2 cells were transfected with Flag-tagged or untagged dBre1, and the activity of a Notch-specific reporter containing 4 Su(H) binding sites (NRE, a luciferase derivative of Gbe+Su(H)_m8) was measured in the presence or absence of low levels of N^ICD. As a control, a reporter was used with mutant Su(H) binding sites [NME, or Gbe+Su(H)_mut]. These experiments reveal a significant stimulation of the NRE reporter by dBre1, especially in the presence of N^ICD. The degree of stimulation is similar to that observed when the ubiquitin ligase Hdm2 is added to transcription assays of Tat activity. dBre1 also elicits a slight stimulation of NME. The fact that overexpressed dBre1 has stimulatory effects on Notch in the transfection assays but not in imaginal discs presumably reflects differences either in the levels of dBre1 or in the amounts of other limiting factors in the two cell contexts. Nevertheless, the transfection assays reveal an intrinsic potential of dBre1 in stimulating the transcription mediated by Su(H) and its coactivator N^ICD (Bray, 2005).

All these results point to a role of dBre1 in promoting Notch signaling. Since other ubiquitin ligases have been shown to influence the levels of specific protein components of the Notch pathway, whether there were any alterations to Notch, Delta, or Su(H) levels in dBre1 mutant clones was investigated. While there are no detectable changes in Notch or Delta staining in dBre1 mutant cells, the levels of Su(H) staining are enhanced slightly but consistently, and cell autonomously, in mutant clones of both dBre1 alleles, regardless of the location of these clones within the disc. This is also obvious in clones induced early in larval development in a non-Minute background in which the mutant dBre1 clones remain small. As an aside, these clones reveal that individual dBre1 mutant cells are enlarged, reminiscent of the yeast bre1p mutant which also shows a 'large cell'phenotype. This phenotype has not been observed in cells lacking Notch signaling, so this aspect of dBre1 function appears distinct from its role in the Notch pathway, and suggests that there are additional molecular targets. Nevertheless, the elevated levels of Su(H) in the dBre1 mutant clones identify Su(H) as one molecular target of dBre1 and suggest that, in the wild-type, dBre1 may expose Su(H) to ubiquitin-mediated degradation. The effects on Su(H) are consistent with the cell-autonomous action of dBre1 on Notch target gene expression, but the fact that removal of dBre1 has a stabilizing effect on Su(H) appears to contradict its stimulating effect on Notch-dependent transcription. Since Su(H) functions as both a repressor and an activator, this may be explained if loss of dBre1 specifically stabilizes the repressor complex. Alternatively, the effect of dBre1 mutations on Su(H) may reflect an indirect bystander activity of dBre1 (Bray, 2005).

Finally, it was asked whether dBre1 has a similar molecular function as its relative yeast Bre1p. The latter is required for the monoubiquitination of histone H2B, which is a prerequisite for the subsequent methylation of histone H3 at K4 by SET1-containing complexes. H3K4 methylation appears to be a chromatin mark for transcriptionally active genes, and yeast bre1p mutants show defects in the transcription of inducible genes that have been ascribed to the lack of H2B ubiquitination and H3K4 methylation at the promoters of these genes. Since there are no in vitro assays for H2B ubiquitination and no antibodies that detect this modified form of H2B, effects of dBre1 mutations on the linked H3K4 methylation were investigated. Wing discs bearing dBre1 mutant clones were stained with an antibody specific for trimethylated H3K4 (H3K4m). This revealed a significant reduction of H3K4m in P1541 mutant clones. More strikingly, in clones of the stronger E132 allele, H3K4m is barely detectable. In contrast, staining of these clones with an antibody against H3K9m does not show any changes in the mutant territory, indicating that the effect in dBre1 mutant clones on the methylation of H3K4 is relatively specific. It is noted that, in wild-type wing discs, there is slight modulation of trimethylated H3K4, with higher levels at the dorsoventral boundary where Notch is activated. However, Notch mutant cells retain robust H3K4m staining, although occasionally show slightly lowered levels compared to adjacent wild-type cells. Thus, the reduced H3K4m staining in dBre1 mutant cells is primarily due to an activity loss of dBre1 rather than due to loss of Notch signaling. Based on its effects on tri-methylated H3K4, it is concluded that dBre1 is indeed the functional homolog of yeast Bre1p. Furthermore, it appears that the activity of dBre1 is essential for the bulk of trimethylated H3K4 in imaginal disc cells (Bray, 2005).

In yeast, H2B ubiquitination and H3K4 methylation are associated with sites of active transcription, but the only identified natural target gene is GAL1. In Drosophila, the target genes of dBre1 evidently include genes regulated by Notch, given the requirement of dBre1 for their transcription. It is therefore conceivable that Su(H) may have a role in targeting dBre1 to their promoters (although it was not possible to detect direct binding or coimmunoprecipitation between dBre1 and Su(H). It is puzzling that dBre1 has a slight destabilizing effect on Su(H), despite being an activating component of Notch signaling. It is believed that this could be a bystander effect of dBre1: evidence suggests that the Bre1p-mediated monoubiquitination of H2B leads to a transient recruitment of proteasome subunits to chromatin, and that the subsequent methylation of H3K4 depends on the activity of these proteasome subunits. Their transient presence at specific target genes may have a destabilizing effect on nearby DNA binding proteins, and the mildly increased levels of Su(H) in dBre1 mutant cells could therefore reflect a failure of proteasome recruitment due to loss of H2B monoubiquitination (Bray, 2005).

Perhaps the most interesting implication of the results is that the dBre1-mediated monoubiquitination of H2B and methylation of H3K4 may be critical steps in the transcription of Notch target genes. Indeed, it appears that the Notch target genes belong to a group of genes whose transcription is particularly susceptible to the much reduced levels of H3K4m in dBre1 mutant cells. Based on the dBre1 mutant phenotypes, there are likely to be other genes in this group, including for example genes controlling cell survival and cell size. Nevertheless, it would appear that the transcription of Notch target genes is particularly reliant on the activity of dBre1. Other examples are emerging where the transcriptional activity of a subset of signal responsive genes is particularly sensitive to the function of a particular chromatin modifying and/or remodelling factor. This sensitivity presumably reflects the molecular mechanisms used by signaling pathways to activate transcription at their responsive enhancers. Understanding why Notch-induced transcription is particularly susceptible to loss of dBre1 function will require knowledge of these underlying molecular mechanisms (Bray, 2005).

Drosophila Rtf1 functions in histone methylation, gene expression, and Notch signaling

The Rtf1 subunit of the Paf1 complex is required for proper monoubiquitination of histone H2B and methylation of histone H3 on lysines 4 (H3K4) and 79 in yeast Saccharomyces cerevisiae. Using RNAi, the role of Rtf1 in histone methylation and gene expression was examined in Drosophila. Drosophila Rtf1 (dRtf1) is required for proper gene expression and development. Furthermore, RNAi-mediated reduction of dRtf1 results in a reduction in histone H3K4 trimethylation levels on bulk histones and chromosomes in vivo, indicating that the histone modification pathway via Rtf1 is conserved among yeast, Drosophila, and human. Recently, it was demonstrated that histone H3K4 methylation mediated via the E3 ligase Bre1 is critical for transcription of Notch target genes in Drosophila. This study demonstrates that the dRtf1 component of the Paf1 complex functions in Notch signaling (Tenney, 2006).

The Notch signaling pathway is required for regulation of cell fate decisions throughout metazoan development. Although Notch signaling is known to activate transcription of numerous target genes, little is known regarding the molecular details of this process. Recently, Bray (2005) reported that the Drosophila homologue of yeast Bre1 is required for Notch target gene expression in Drosophila. Because Rtf1 is also required for regulation of Rad6/Bre1 in yeast, whether dRtf1 is required for proper Notch signaling was tested (Tenney, 2006).

The hypomorphic Notch allele N^nd-1 causes limited nicking in the margin of the adult wing. Mutations in factors required for Notch signaling enhance this wing nicking; strong enhancement results in a reduced wing width. Mild, nonlethal activation of two independent dRtf1 RNAi knockdown transgenes, dRtf1–10A and dRtf1–17C, by an Hsp70–Gal4 driver at 27°C decreased the width of N^nd-1 wings relative to a no-RNAi control (y w) and relative to RNAi directed against dEloA (dEloA–18D). Both dRtf1 RNAi insertions gave significant reductions in wing width relative to the dEloA and y w controls. There was no significant difference in wing width between y w and dEloA RNAi, demonstrating that the effect on N^nd-1 was not due to ectopic Gal4 expression (Tenney, 2006).

Notch and Metabolism

Notch stimulates growth by direct regulation of genes involved in the control of glycolysis and the tricarboxylic acid cycle

Glycolytic shift is a characteristic feature of rapidly proliferating cells, such as cells during development and during immune response or cancer cells, as well as of stem cells. It results in increased glycolysis uncoupled from mitochondrial respiration, also known as the Warburg effect. Notch signalling is active in contexts where cells undergo glycolytic shift. This study tested whether metabolic genes are direct transcriptional targets of Notch signalling and whether upregulation of metabolic genes can help Notch to induce tissue growth under physiological conditions and in conditions of Notch-induced hyperplasia. Genes mediating cellular metabolic changes towards the Warburg effect were shown to be direct transcriptional targets of Notch signalling. They include genes encoding proteins involved in glucose uptake, glycolysis, lactate to pyruvate conversion and repression of the tricarboxylic acid cycle. Even a short pulse of Notch activity is able to elicit long-lasting metabolic changes resembling the Warburg effect. Loss of Notch signalling in Drosophila wing discs as well as in human microvascular cells leads to downregulation of glycolytic genes. Notch-driven tissue overgrowth can be rescued by downregulation of genes for glucose metabolism. Notch activity is able to support growth of wing during nutrient-deprivation conditions, independent of the growth of the rest of the body. Notch is active in situations that involve metabolic reprogramming, and the direct regulation of metabolic genes may be a common mechanism that helps Notch to exert its effects in target tissues (Slaninova, 2016).

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