Table of contents

General considerations

The Notch extracellular domain has 36 EGF-like repeats. Mutational studies show that two of these, numbers 11 and 12, are required for the interaction of Notch with Serrate and Delta ligands (de Celis, 1993). Additionally, Abruptex mutations of the Notch locus alter EGF repeats numbers 24-29, reducing the ability of Notch to bind to Notch ligands (de Celis, 1994). Cytoplasmic retention of Suppressor of Hairless requires the intracellular cdc10/ankyrin repeats of Notch physically bind Su(H) (Rebay, 1993). Deltex regulates the subcellular localization of Su(H) via antagonistic interactions with Notch ankyrin repeats (Fortini, 1994 and Matsuno, 1995).

Notch interactions with its ligands

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

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

In the developing imaginal wing disc of Drosophila, cells at the dorsoventral boundary require localized Notch activity for specification of the wing margin. The Notch ligands Serrate and Delta are required on opposite sides of the presumptive wing margin and, even though activated forms of Notch generate responses on both sides of the dorsoventral boundary, each ligand generates a compartment-specific response. Serrate, which is expressed in the dorsal compartment, does not signal in the dorsal regions due to the action of the fringe gene product. Using ectopic expression, it is shown that regulation of Serrate by Fringe occurs at the level of protein and not Serrate transcription. Furthermore, replacement of the N-terminal region of Serrate with the corresponding region of Delta abolishes the ability of Fringe to regulate Serrate without altering Serrate-specific signaling (Fleming, 1997).

Specification of the dorsal-ventral compartment boundary in the developing Drosophila wing disc requires activation of Notch from its dorsal ligand Serrate and its ventral ligand Delta. Both Notch ligands are required in this process: one cannot be substituted for the other. In the wing disc, expression of BD(G), a dominant-negative, truncated form of Serrate, is capable of inhibiting Notch activation in the ventral but not the dorsal compartments. BD(G) can act as a general antagonist of both Serrate and Delta mediated Notch interactions. However, BD(G) retains the Serrate protein domain targeted by Fringe, hence BD(G)'s antagonistic effects are restricted in the dorsal wing disc. Implicit in these results is the suggestion that binding of a ligand to Notch is not sufficient for Notch activation. The specificity of the Notch signal generated by interactions with Serrate and Delta originates from regions residing outside of the Notch binding domains of these molecules; other properties attributable to Notch ligands are required for Notch activation. Thus, ligand binding to Notch is a necessary but insufficient step toward Notch activation (Hukriede, 1997).

The functions of artificially constructed secreted forms of the two known Drosophila Notch ligands, Delta and Serrate, were examined by expressing them under various promoters in the Drosophila developing eye and wing. The phenotypes associated with the expression of secreted Delta (DlS) or secreted Serrate (SerS) forms mimic loss-of-function mutations in the Notch pathway. Both genetic interactions between DlS or SerS transgenics and duplications or loss-of-function mutations of Delta or Serrate indicate that DlS and SerS behave as dominant negative mutations. Expression of DlS and SerS in the eye results in a rough eye phenotype. This phenotype is enhanced by loss-of-function Delta and gain-of function Suppressor of hairless. These observations were extended to the molecular level by demonstrating that the expression of Enhancer of split mdelta, a target of Notch signaling, is down-regulated by SerS. The antagonistic nature of the two mutant secreted ligand forms in the eye is consistent with their behavior in the wing, where they are capable of down-regulating wing margin specific genes in an opposite manner to the effects of the endogenous ligands. For example, wingless expression is down-regulated where a SerS expressing stripe crosses the dorsal/ventral boundary. The secreted ligands also interfere with wing vein specification. This analysis uncovers secreted molecular antagonists of Notch signaling and provides evidence of qualitative differences in the actions of the two ligands DlS and SerS (Sun, 1997).

The activities of Serrate protein were analyzed in wing development. An important outcome of Serrate activity is the induction, through Notch, of the wing margin at the dorsal-ventral interface of the wing imaginal disc. Analysis of the function of Ser indicates that excess Ser can titrate out Notch function in the developing wing, an effect that is suppressed by an increase in the dosage of Notch. Since Serrate has been shown to bind Notch, this effect can be interpreted as an induction of a dominant negative activity of Serrate on Notch. Ubiquitous expression of Ser in the wing pouch throughout the development of the wing curtails wing development and produces wings that lack most of the margin and adjacent tissue (Klein, 1997).

Serrate can activate or inactivate Notch in a concentration-dependent manner as revealed by the expression of two targets of Notch activity: wingless and Enhancer of split. E(spl) is expressed in a stripe that straddles the DV interface at the beginning of the third larval instar. Ectopic expression of Ser product reduces the normal margin and produces two new margins on the ventral side of the developing wing. Ectopic expression of Ser in a Su(H) mutant has no effect on disc development or patterning and results in discs that are identical to those of Su(H) mutants. This demonstrates that the activity of Serrate during wing development requires Su(H). While the inactivation produced by ectopic Ser is likely to be mediated by a dominant negative effect over Notch, the activation is similar to that elicited by Delta and requires the product of the suppressor of Hairless gene (Klein, 1997).

Expression of Ser leads to smaller wings with thick veins. When wingless and Serrate are coexpressed, the resulting flies bear large wings that are covered with bristles. These wings have a different shape from those in wild-type: they lack a defined margin and are more round rather than elongated. This experiment shows that increased functional Wingless not only can suppress the dominant negative effect of Serrate expression, but can cooperate with Serrate to promote wing development. These wings are very similar to those that result from the expression of Delta and indicate that wingless enables Serrate to activate Notch. Coexpression of Ser and Notch generates very large wings, bigger than those that result from expression of Delta or coexpression of Serrate and wingless. These large wings have a clearly define margin with an antineurogenic phenotype. These results indicate that regulation of the concentration of Serrate during development must be an important way of regulating its activity (Klein, 1997).

Two different models are proposed. In one view the role of Serrate is to bind Notch at the DV interface to free Notch-bound Delta, which then would trigger events at the DV boundary that lead to wing outgrowth. This view is consistent with the observation of a dominant negative activity associated with Serrate and with the ability of Serrate to mimic Delta by activating Notch, leading to signaling through Su(H) and the consequent outgrowth of the wing. A different view sees Serrate as the active component of the signaling system, either alone or in combination with Wingless (Klein, 1997).

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

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

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

A feedback mechanism exists in which Fringe functions to inhibit Serrate by targeting Notch. In contrast to Delta, the effects of ectopic expression of Ser on wild-type discs are restricted to ventral cells. This has led to the suggestion that there is an inhibitor of Serrate activity in dorsal cells and that this inhibitor is under the control of ap. Consistent with this proposal, ectopic expression of Ser in ap mutants is found to be able to induce the expression of downstream targets of Notch in 'dorsal' cells. A variety of arguments have led to the proposal that the dorsal inhibitor of Serrate function is encoded by the fng gene. For example, ectopic expression of Ser with ptcGAL4 results in the activation of Notch targets in two parallel stripes in ventral cells of the developing wing blade, and this can be observed as early as the beginning of the third instar. When Ser is coexpressed with fng, the anterior stripe, but not the posterior one, is lost completely in late third instar discs. Correspondingly the ectopically induced margin structures are reduced to a posterior stripe with characteristics of the posterior compartment. While Fringe suppresses the function of Serrate cell autonomously, it enhances its signaling ability in a nonautonomous manner. Fringe is thought to dampen Serrate signaling by affecting its interaction with Notch, but no evidence has been presented to support this suggestion. Increasing the concentration of Notch appears to be able to titrate the effects of fng. Furthermore, the effects of ectopic expression of fng are partially suppressed by the expression of Notch with fng and are exaggerated by expressing dominant negative Notch molecules with fng. Altogether, these results strongly suggest that a target of Fringe activity is the Notch molecule itself (Klein, 1998).

Fringe functions to inhibit Serrate signaling via Notch. The activity of Fringe can inhibit Serrate signaling by enhancing the intrinsic dominant negative activity of Serrate over Notch. Expression of Ser throughout the late wing disc leads to a strong broadening of the wing veins and a moderate increase in the number of bristles in the notum. Both of these neurogenic phenotypes can be suppressed by coexpressing Notch with Ser, indicating that they are due to a dominant negative effect of Serrate. Ectopic expression of fng alone in the same pattern results in nicked wings with normal veins and a reduction of bristles in the notum, which is associated with the loss of sensory organ precursors. Coexpression of fng with Ser suppresses the extra vein phenotype caused by misexpression of Ser and, therefore, supports the notion that Fringe reduces the ability of Serrate to bind Notch. Fringe is shown to impinge on Notch signaling by the observation that the action of Fringe requires the activity of Su(H). Fringe is not able to rescue the defects caused by Su(H) mutants (Klein, 1998).

It has been suggested that the secreted protein Scabrous (Sca) might be a Notch ligand acting in the peripheral nervous system. The Sca protein was purified and a cell line expressing 18,000 Notch molecules per cell surface was used to test Sca binding by coimmunoprecipitation, cell adhesion assays, and binding with labeled Sca. No interaction was detected between gp300sca and Notch or the related protein Delta, suggesting that Sca acts through a distinct mechanism (Lee, 1996).

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

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

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

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

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

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

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

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

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

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

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

Signaling by Notch lacking a C-terminus

The cell surface receptor Notch is required during development of Drosophila for differentiation of numerous tissues. Notch is often required for specification of precursor cells by lateral inhibition and subsequently for differentiation of tissues from these precursor cells. Certain embryonic cells and tissues that develop after lateral inhibition, like the connectives and commissures of the central nervous system, are enriched for a form of Notch not recognized by antibodies made against the intracellular region carboxy-terminal of the CDC10/Ankyrin repeats. Western blotting and immunoprecipitation analyses show that Notch molecules lacking this region are produced during embryogenesis and form protein complexes with the ligand Delta. Experiments with cultured cells indicate that Delta promotes accumulation of a Notch intracellular fragment lacking the carboxyl terminus. Furthermore, Notch that lacks the carboxyl terminus functions as a receptor for Delta. These results suggest that Notch activities during development include generation and activity of a truncated receptor designated NdeltaCterm (Wesley, 2000b).

This analysis of N molecules in embryos and S2 cells shows the following: (1) whereas the cells undergoing lateral inhibition in the developing embryo are enriched for N molecules recognized by both the amino and carboxyl terminus antibodies, the cells and tissues produced subsequent to lateral inhibition are enriched for N molecules not recognized by the carboxyl terminus antibody; (2) correspondingly, Dl forms complexes with the full-length N during lateral inhibition period, and with the N molecule lacking the carboxyl terminus in the period after lateral inhibition; (3) N molecules lacking the carboxyl terminus (NdeltaCterm, NdeltaCtermTMintra, and NdeltaCtermintra) are produced during embryogenesis; (4) S2 cells expressing N receptors containing the carboxyl terminus (NFull) treated with S2-Dl cells accumulate an intracellular N molecule lacking the carboxyl terminus, NdeltaCtermTMintra; (5) NdeltaCterm is the most likely substrate for production of NdeltaCtermTMintra; (6) NdeltaCterm functions as a receptor for Dl, with the NdeltaCtermintra (comprised mostly of the CDC10/Ankyrin repeats) as its activated signaling molecule, and the da gene is responsive to its signals (Wesley, 2000b).

Based on the results summarized above, the following hypothetical model for N functions during embryogenesis is presented. Lateral inhibition starts with NFull receptor containing the full signaling potential. The back and forth lateral inhibition signaling between interacting cells leads to carboxyl terminus processing of the full-length N molecules present inside the cells (i.e., those not involved in Dl binding) and production of the NdeltaCterm receptors. Cells expressing higher levels of NdeltaCterm become the neuronal precursor cells and cells expressing higher levels of NFull become the epidermal precursor cells. NFull disappears in neuronal precursor cells and NdeltaCterm, a secondary receptor with restricted signaling potential, functions during differentiation of the nervous system. Epidermal precursor cells expressing only NFull, or appreciable levels of both NFull and NdeltaCterm, continue the same process during differentiation of the epidermis. Advance from signaling by NFull to signaling by NdeltaCterm would mean that those cells have attained a degree of irreversibility in their differentiation process. For example, once NdeltaCterm becomes the sole N receptor in the neuronal precursor cells, these cells can only proceed along the neuronal differentiation path. N would continuously function in this manner to both specify and restrict cell fates during differentiation of a cell lineage (Wesley, 2000b).

NdeltaCterm lacks the Dishevelled-binding region, one of the Numb-binding regions, the OPA sequence, and the PEST sequence. Therefore, it is likely that loss of one or more of these features is involved in restricting the differentiation possibilities for a cell. Dishevelled and Numb are known to antagonize Su(H) activities. Proteolytic removal of their binding sites is likely to eliminate antagonisms to Su(H) activities and promote activities of facilitators like Deltex. This might contribute to the lateral inhibition process and selection of precursor cells for neuronal fates. In contrast, production of NdeltaCtermintra lacking the Su(H)-binding sites from NdeltaCterm receptor might promote neuronal fates by promoting activities of Hairless or Numb or Achaete (through Daughterless). It is also possible that Disabled, which functions with N during differentiation of the CNS after lateral inhibition, can signal from NdeltaCterm and not NFull. Thus, production and functions of NFull and NdeltaCterm might provide directionality to N functions at successive stages of differentiation. All these properties of NFull, NdeltaCterm, and the proteins interacting or not interacting with these two receptors, may be involved during differentiation of the adult sensory organ (bristle) wherein Su(H) activity is required for determination of some fates and not others (Wesley, 2000b).

There is no evidence, one way or the other, about involvement of Su(H) in transducing signals from NdeltaCterm. Regulation of expression of E(spl)C genes by NdeltaCterm seems to indicate that the canonical Su(H)-mediated lateral inhibition pathway is involved. However, E(spl)C gene expression could be regulated by an alternate pathway. NdeltaCterm regulates da, not NFull. Daughterless protein, an activator of proneural proteins and proneural genes, also activates expression of E(spl)C. Two differences in the activities of the intracellular domains of NFull and NdeltaCterm seem very likely. (1) The RAM23 region in the intracellular domain of N is important for Su(H) activities related to NFull, Nintra and lateral inhibition. It appears that NdeltaCtermintra lacks most of this region, if not all. (2) The sequence carboxy-terminal of the CDC10/Ankyrin repeats is required for transcriptional activation upon binding DNA. Since NdeltaCterm lacks this sequence, it might activate genes indirectly through inactivation of a constitutive repressor or stabilization of RNA. NFull containing the carboxyl terminus would activate genes directly from DNA. Thus, it is possible that NFull and NdeltaCterm might signal through different pathways with some shared outcomes at certain stages of development, like expression of E(spl)C genes. Su(H) might be functioning with both pathways, albeit in different ways (Wesley, 2000b).

Production of N receptors with restricted signaling potential may be important for another reason. NFull binds different ligands and regulates different genes in response to them. Removal of the carboxyl terminus after initiation of NFull signaling by one ligand might set the cell on a differentiation path specific to that ligand. For example, removal of the carboxyl terminus in neuronal precursor cells after Delta-specific lateral inhibition signaling might make NdeltaCterm in these cells either unresponsive to Wingless functioning in the epidermis differentiation pathway, or responsive to Wingless in the manner specific to neuronal differentiation pathway. Treatment of full-length N with Wingless results in accumulation of a N molecule lacking the Dl-binding region. This secondary N receptor may be produced during epidermogenesis to eliminate the antagonism to Wingless functions presented by the Dl-binding site. Non-response or pathway-specific response to a second ligand may be necessary for development given the broad overlap in distributions of different N ligands. Thus, expression of a particular secondary N receptor might indicate both the differentiation path taken by a cell and the degree to which this cell has differentiated from cells in the parent population (Wesley, 2000b).

The molecular phenotypes of the nd3 allele suggest that EGF-like repeat 2 might be an important component in the regulation of NdeltaCterm production during embryogenesis. It seems possible that the EGF-like repeat array of N might include two classes of repeats, one containing repeats that bind ligands outside the cells and the other containing repeats that target Notch for different kinds of processing inside the cell. Such a function for EGF-like repeats might explain why Nintra does not produce NdeltaCtermTMintra. These molecules might lack the appropriate EGF-like repeats to target them to the right place for carboxyl terminus processing. An interesting extension of this possibility is that there are different targeting EGF-like repeats responsive to different ligands (Wesley, 2000b).

The regulation of da expression by NdeltaCterm may be significant for embryogenesis. da genetically interacts with Notch; it is required for development of the nervous system from neuroblasts but not for lateral inhibition, and the Daughterless protein promotes DNA-binding activities of the proneural Achaete-Scute Complex proteins. Both NdeltaCterm and Daughterless protein accumulate in segregating neuroblasts raising the possibility that NdeltaCterm is involved in this upregulation of da expression. Accordingly, nd3 embryos that overproduce NdeltaCterm also overproduce da RNA in the neuroblasts (Wesley, 2000b).

In the embryo, da is expressed at low levels in almost all cells but is upregulated in certain cells including the segregating neuroblasts. S2 cells expressing NFull and NdeltaCterm receptors have lower levels of DA RNA than S2 cells without N. In response to Dl, only S2-NdeltaCterm cells increase expression of DA RNA, but only to the level observed in cells without N. Therefore, it appears possible that with the expression of different forms of N, developing cells acquire an ability to differentially regulate the otherwise constitutive da expression. Such differential regulation might be important for suppressing the activities of Achaete-Scute Complex proteins in the developing epidermis where NFull is expected to function, but not in the developing nervous system where NdeltaCterm is expected to function. Since both N receptors have the ability to activate E(spl)C, the timing and sequence of expression of NFull and NdeltaCterm may also be important for development (Wesley, 2000b).

Notch nuclear repressor complex

Inducible RNA interference uncovers the Drosophila protein Bx42 as an essential nuclear cofactor involved in Notch signal transduction

The UAS/GAL4 two component system was used to induce mRNA interference (mRNAi) during Drosophila development. In the adult eye the expression from white transgenes or the resident white locus is significantly repressed by the induction of UAS-wRNAi using different GAL4 expressing strains. By induced RNAi it was demonstrated that the conserved nuclear protein Bx42 is essential for the development of many tissues. Phenotypically the effects of Bx42 RNAi resemble those obtained for certain classes of Notch mutants, pointing to an involvement of Bx42 in the Notch signal transduction pathway. The wing phenotype following overexpression of Suppressor of Hairless is strongly enhanced by simultaneous Bx42 RNAi induction in the same tissue. Target genes of Notch signaling like cut and Enhancer of split m8 were suppressed by induction of Bx42 RNAi (Negeri, 2002).

Phenotypically, the consequences of Bx42RNAi often resemble effects obtained by interference with components of the Notch pathway. Studies of protein interaction in vitro suggest an involvement of Bx42 and its human homolog Skip in the Notch signal transduction. Both Skip and Bx42 were found to interact with Notch-IC, CBF1 and components of the CBF1 corepressor complex like SMRT, N-CoR, CIR, Sin3a and HDAC2 proteins (Zhou, 2000a; Zhou, 2000b; Zhang, 2001). By yeast two hybrid interaction and coimmunoprecipitation it was found that Bx42 physically interacts with the Drosophila CBF1-homolog Su(H) and with its antagonist Hairless, for which so far no vertebrate counterpart is known. The current study presents evidence that these interactions are biologically meaningful. Ubiquitous early induction of Bx42 RNAi results in embryos with dorsal cuticle only, a phenotype similar to Notch mutations. Induction of Bx42 RNAi in the eye disc results in an eye to antenna transformation as is observed following overexpression of dominant negative forms of Notch in the same tissue. Both effects could be interpreted that Bx42 normally functions as a coactivator of the Notch pathway. However, Other studies have demonstrated that overexpression of Notch-IC in the eye-antennal discs results in the formation of ectopic antennae too, but only if the eyeless function is reduced by a hypomorphic mutation. eyeless is one of the master regulators for eye development and functions in a cross-regulatory circuitry together with six other master regulatory genes. One of them is dachshund, whose human homolog Ski is a known interactor for the Bx42 human homolog Skip (Dahl, 1998). Thus, the observed eye antennal transformation by Bx42 RNAi could also be interpreted as a downregulation of eye master regulatory genes via diminished dachshund activity and a simultaneous derepression of the Notch pathway. Negative interference of Bx42 with Notch signaling is consistent with the results of ptc-GAL4 driven induction of Bx42 RNAi. A similar loss of scutellar bristles is observed on ptc-GAL4-driven overexpression of the Notch-ankyrin repeats, a part of Notch-IC involved in active signal transductio. However, this is not fully understood, since overexpression of Notch extracellular domains missing certain EGF repeats results in a similar antineurogenic phenotype. Moreover, Notch mutant clones result in a loss of bristles as well. A negative role of Bx42 in Notch signaling is suggested by the wing phenotype of dpp-GAL4/Bx42RNAi. Following reduction of Bx42 the veins are thinner or missing consistent with a Notch gain of function (Negeri, 2002).

Ectopic expression of Su(H) prevents sensory organ development in a similar manner to activated Notch. This may reflect an excess of lateral inhibition or it may be due to interference with the establishment of the correct fates in the progeny of the sensory organ precursor cells. The failure of sensory organ formation in the scutellum and the wing following local Bx42 RNAi may be related to a gain of Su(H) function. The enhancement of the Su(H) overexpression phenotype by Bx42 RNAi and the similar effects of Bx42 RNAi and Su(H) overexpression on Notch target genes strongly support this argument and suggest a functional relationship between both proteins (Negeri, 2002).

Although the data suggest a negative role, it is not believed that Bx42 protein acts as a repressor within the Notch pathway. Earlier work, which suggested an activation function for Su(H) was at odds with data demonstrating a repressive role for its mammalian homolog CBF1. More recent work provides evidence for Su(H) acting as a switch between repression and activation of Notch target genes. It is proposed that Bx42 contributes to the switch provided by the Su(H) protein. How this is accomplished can only be speculated at the moment. By its direct interaction with Su(H) Bx42 may stabilize a switching complex. By its direct interaction Bx42 could recruit Hairless into the complex contributing to its repressive function. Modification or removal of Bx42 protein (as by RNAi) would result in a destabilization of this repressive complex allowing to switch to the active state. Similarly, due to squelching by a large excess of overexpressed Su(H), only a reduced amount of Bx42 protein would be available to stabilize the repression complex. It is important to emphasize that Bx42 is able to physically interact with Notch-IC (Zhou, 2000a) and may act as a switching protein in the recruitment of activators as well (Negeri, 2002).

An important role in Notch signaling and functional relation between Su(H) and Bx42 were also suggested when the effects of reducing Bx42 on Notch target gene expression were studied. Both cut and E(spl)m8 were suppressed by Bx42 RNAi as was the vestigial quadrant element (vgQE) enhancer, indicating a Bx42 activating function for these genes in wild type. wingless expression was not affected under these conditions, excluding Bx42 RNAi induced cell death as an explanation for the observed effects. The effects on Notch target gene expression are consistent with the proposed role for Bx42 as part of a switch. Following Su(H) overexpression repression of cut, E(spl)m8 and the vgQE enhancer was demonstrated. wingless, on the other hand, was not affected. The similar effects of Su(H) overexpression and Bx42 RNAi on Notch target gene expression underscores the close relationship in the function of both proteins (Negeri, 2002).

Besides its involvement in Notch signaling the observed phenotypic effects of Bx42 RNAi suggest that the Bx42 protein is involved in other signaling pathways as well. Data from its vertebrate homolog, which indicate that Bx42 takes part in nuclear receptor pathways, are supported by observations on chromosomal binding to sites occupied by the ecdysone receptor complex. A possible interaction with Dachshund, one of the master regulators in eye development, which is widely expressed in the nervous system, has already been mentioned. It remains to be established how Bx42 is involved in these other pathways (Negeri, 2002).

Structural and functional analysis of the repressor complex in the Notch signaling pathway of Drosophila melanogaster

CSL is the nuclear effector of the Notch signaling pathway and is required for both repression and activation of transcription from Notch target genes. In the absence of a signal, CSL functions as a transcriptional repressor by interacting with corepressor proteins, such as SHARP, SMRT/NCoR, KyoT2, and CIR. CSL-corepressor interactions function to localize histone deacetylase and histone demethylase activity at Notch target genes, which converts the local chromatin into a condensed, transcriptionally silent state. On pathway activation, the ICN binds CSL, and together with Mam, forms a transcriptionally active ternary complex that ultimately displaces corepressors from CSL and upregulates transcription from Notch target genes. In mammals, a number of corepressors have been shown to interact with the BTD of CSL, similar to ICN, which provides a model in which ICN displaces or outcompetes corepressors for binding to CSL. Thus, there are potentially two modes of repression mediated by corepressors: 1) at the transcription or chromatin level, in which the recruitment of HDAC/HDM-containing complexes by corepressors silences gene expression -- this mode of transcriptional repression is independent of Notch; and 2) at the protein level, by which corepressors and ICN compete for binding to CSL (Maier, 2011).

Although several of the mammalian corepressors have fly orthologues, these molecules do not seem to be generally involved in repressing transcription from Notch target genes in flies. A complex involving the SMRT homologue SMRTER negatively regulates Notch signaling during the specification of a subset of nonneuronal cell types in the developing Drosophila retina. However, mammalian SMRT is believed to contact CBF1 directly, whereas SMRTER does not bind Su(H) on its own. The Drosophila orthologue of SHARP/MINT, termed Spen, which genetically inhibits Notch signaling in the context of eye development, is presumably not a transcriptional repressor of Notch target genes in this process. Recently it was shown that Spen is required for the activation rather than the repression of Notch target genes during the development of hemocytes. Moreover, the region of SHARP/MINT that has been defined to interact with CSL is not conserved in Spen. Although formally SHARP/MINT might act as a functional Hairless analogue in mammals, the role of the structurally related Spen proteins seems largely diverse in different organisms (Maier, 2011).

In flies, the major antagonist of Notch signaling is the transcriptional corepressor Hairless, which is ubiquitously expressed in all tissues. Hairless binds the transcription factor Su(H), as well as the corepressors Groucho and CtBP, which serves to localize the transcriptional repression machinery in the nucleus to Notch target genes, thereby repressing gene expression. Removal of the Groucho and CtBP-binding sites from Hairless does not completely eliminate its activity as a repressor, suggesting that, similar to other corepressors, Hairless might compet.e with ICN for binding Su(H). However, the molecular mechanism by which Su(H) is converted from a repressor to an activator complex is unclear (Maier, 2011).

This work investigated the molecular details of the Notch repressor complex in Drosophila. The analysis was multidisciplinary in nature, using biophysical, biochemical, cellular, and in vivo assays to characterize the protein-protein interface between Hairless and Su(H). Hairless was shown to forms a high-affinity 1:1 complex with Su(H) (~1 nM Kd) but only interacts with the CTD, which is in stark contrast to mammalian CSL-corepressor interactions, which are largely mediated through BTD contacts. Previous ITC binding studies of the mammalian Notch components Notch1 (ICN) and RBP-J showed that the Kd of the ICN/RBP-J complex is ~10 nM, suggesting that Su(H)-Hairless and Su(H)-Notch interactions are likely of comparable affinity (Maier, 2011).

Given the similar affinities of ICN and Hairless for Su(H), the question arises whether ICN and Hairless compete for binding to CSL. On one hand, gel-shift assays with purified protein components showed that ICN can displace Hairless from Su(H) independent of Mastermind. On the other hand, it was shown that residues on Su(H) that are important for Notch ANK and Mam binding to CTD do not affect interactions with Hairless. These data suggest that the ICN- and Hairless-binding sites on Su(H) do not overlap. If the ANK domain of ICN and Hairless are competing for binding to the CTD of Su(H), then there is an additional factor to consider: based on binding studies of the mammalian proteins, the vast majority of the binding energy for the Su(H)-ICN complex comes from the RAM domain interaction with the BTD of Su(H), whereas the isolated CTD-ANK interaction is of very low affinity. This represents at least a 10,000-fold difference in the affinities of ANK and Hairless for CTD, which suggests that the ANK domain of ICN would seem to be a very poor competitor for removing Hairless from Su(H) (Maier, 2011).

How then is ICN able to supplant Hairless from Su(H) in order to activate transcription from Notch target genes? Certainly additional experiments will be required to fully address this question; however, at present the following hypothesis is favored: the binding of ICN to Su(H), that is, the RAM-BTD interaction, results in allosteric changes in Su(H) that decreases its overall affinity for Hairless, thereby making ANK a more effective competitor for CTD. Consistent with this notion, gel-shift experiments showed that Hairless was far less effective at removing ICN from Su(H), even when Hairless was present in vast excess (Maier, 2011).

The studies also analyzed two absolutely conserved residues in Hairless (L235 and F243) for their contribution to binding Su(H). Whereas F243 was dispensable for binding, L235 was absolutely required for binding Su(H) in vitro. Mutation of this site to aspartate abrogated binding but did not change the secondary structure content of Hairless, which suggests that L235 lies at the Su(H)-Hairless interface. Given the conservation of F243 but its dispensability for Su(H) binding, this perhaps suggests that this residue is important for interacting with other nuclear factors. Consistent with in vitro binding results, the Hairless mutant L235D failed to assemble a repressor complex with Su(H) in cellular and in vivo assays in the fly. In fact, the L235D mutant was as deficient in repression as the Hairless deletion mutant ΔNT, which removes residues 232-270, emphasizing the importance of this contact in forming the Su(H)-Hairless complex (Maier, 2011).

In conclusion, the fly Notch repressor complex shows similarities and differences compared with the mammalian complex. Despite the high degree of sequence and likely structural conservation, Su(H) in Drosophila differs from mammalian CBF1/RBP-J in that it has no repressor activity on its own; overexpression of Su(H) in cell culture and in vivo results in a Notch gain-of-function phenotype. It is not until the binding of Hairless that Su(H) is transformed into a repressor. Of interest, this study showed that Hairless bound the mammalian CSL orthologue CBF1 nearly as avidly as Su(H), which suggests that the Hairless-binding site on the CTD has been conserved in mammals. In accordance, a potent repression of Notch transcriptional activity was found in cultured mammalian cells by the Hairless NT construct. This raises the possibility of identifying Hairless homologues in other organisms or potentially other transcriptional coregulators that use the Hairless-binding site on CTD, which may be indicative of an as-yet-unidentified mode in mammals to repress Notch signaling. Nonetheless, detailed knowledge of Su(H)-Hairless interactions can now be used to develop molecules that target Notch transcription complexes and either enforce or disrupt their activity, thereby opening new therapeutic avenues (Maier, 2011).

Table of contents

Notch continued: Biological Overview | Evolutionary Homologs | Regulation | Post-transcriptional regulation of Notch mRNA | Developmental Biology | Effects of Mutation | References

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