Two Distinct E3 Ubiquitin Ligases Have Complementary Functions in the Regulation of Delta and Serrate Signaling in Drosophila

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

Notch ligand activity is modulated by glycosphingolipid membrane composition in Drosophila melanogaster

Endocytosis of the transmembrane ligands Delta (Dl) and Serrate (Ser) is required for the proper activation of Notch receptors. The E3 ubiquitin ligases Mindbomb1 (Mib1) and Neuralized (Neur) regulate the ubiquitination of Dl and Ser and thereby promote both ligand endocytosis and Notch receptor activation. In this study, we identify the α1,4-N-acetylgalactosaminyltransferase-1 (α4GT1) gene as a gain of function suppressor of Mib1 inhibition. Expression of α4GT1 suppressed the signaling and endocytosis defects of Dl and Ser resulting from the inhibition of mib1 and/or neur activity. Genetic and biochemical evidence indicate that α4GT1 plays a regulatory but nonessential function in Notch signaling via the synthesis of a specific glycosphingolipid (GSL), N5, produced by α4GT1. Furthermore, we show that the extracellular domain of Ser interacts with GSLs in vitro via a conserved GSL-binding motif, raising the possibility that direct GSL–protein interactions modulate the endocytosis of Notch ligands. Together, our data indicate that specific GSLs modulate the signaling activity of Notch ligands.     

Frizzled/PCP-dependent asymmetric neuralized expression determines R3/R4 fates in the Drosophila eye

Planar cell polarity (PCP) is a common feature in many epithelia, reflected in cellular organization within the plane of an epithelium. In the Drosophila eye, Frizzled (Fz)/PCP signaling induces cell-fate specification of the R3/R4 photoreceptors through regulation of Notch activation in R4. Except for Dl upregulation in R3, the mechanism of how Fz/PCP signaling regulates Notch in this context is not understood. We demonstrate that the E3-ubiquitin ligase Neuralized (Neur), required for Dl-N signaling, is asymmetrically expressed within the R3/R4 pair. It is required in R3, where it is also upregulated in a Fz/PCP-dependent manner. As is the case for Dl, N activity in R4 further represses neur expression, thus, reinforcing the asymmetry. We demonstrate that Neur asymmetry is instructive in correct R3/R4 specification. Our data indicate that Fz/PCP-dependent Neur expression in R3 ensures the proper directionality of Dl-N signaling during R3/R4 specification.     

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

The receptor Notch and its ligands of the Delta/Serrate/LAG2 (DSL) family are the central components in the Notch pathway, a fundamental cell signaling system that regulates pattern formation during animal development. Delta is directly ubiquitinated by Drosophila and Xenopus Neuralized, and by zebrafish Mind bomb, two unrelated RING-type E3 ubiquitin ligases with common abilities to promote Delta endocytosis and signaling activity. Although orthologs of both Neuralized and Mind bomb are found in most metazoan organisms, their relative contributions to Notch signaling in any single organism have not yet been assessed. We show here that a Drosophila ortholog of Mind bomb (D-mib) is a positive component of Notch signaling that is required for multiple Neuralized-independent, Notch-dependent developmental processes. Furthermore, we show that D-mib associates physically and functionally with both Serrate and Delta. We find that D-mib uses its ubiquitin ligase activity to promote DSL ligand activity, an activity that is correlated with its ability to induce the endocytosis and degradation of both Delta and Serrate (see also Le Borgne et al., 2005). We further demonstrate that D-mib can functionally replace Neuralized in multiple cell fate decisions that absolutely require endogenous Neuralized, a testament to the highly similar activities of these two unrelated ubiquitin ligases in regulating Notch signaling. We conclude that ubiquitination of Delta and Serrate by Neuralized and D-mib is an obligate feature of DSL ligand activation throughout Drosophila development.     

Distinct intracellular motifs of Delta mediate its ubiquitylation and activation by Mindbomb1 and Neuralized

DSL proteins are transmembrane ligands of the Notch receptor. They associate with a RING (really interesting new gene) family E3 ubiquitin ligase, either Neuralized (Neur) or Mindbomb 1 (Mib1), as a prerequisite to signaling. Although Neur and Mib1 stimulate internalization of DSL ligands, it is not known how ubiquitylation contributes to signaling. We present a molecular dissection of the intracellular domain (ICD) of Drosophila melanogaster Delta (Dl), a prototype DSL protein. Using a cell-based assay, we detected ubiquitylation of Dl by both Neur and Mib1. The two enzymes use distinct docking sites and displayed different acceptor lysine preferences on the Dl ICD. We generated Dl variants that selectively perturb its interactions with Neur or Mib1 and analyzed their signaling activity in two in vivo contexts. We found an excellent correlation between the ability to undergo ubiquitylation and signaling. Therefore, ubiquitylation of the DSL ICD seems to be a necessary step in the activation of Notch.     

An obligatory role of mind bomb-1 in notch signaling of mammalian development

Background

The Notch signaling pathway is an evolutionarily conserved intercellular signaling module essential for cell fate specification that requires endocytosis of Notch ligands. Structurally distinct E3 ubiquitin ligases, Neuralized (Neur) and Mind bomb (Mib), cooperatively regulate the endocytosis of Notch ligands in Drosophila. However, the respective roles of the mammalian E3 ubiquitin ligases, Neur1, Neur2, Mib1, and Mib2, in mammalian development are poorly understood.

Methodology/Principal Findings

Through extensive use of mammalian genetics, here we show that Neur1 and Neur2 double mutants and Mib2^−/− mice were viable and grossly normal. In contrast, conditional inactivation of Mib1 in various tissues revealed the representative Notch phenotypes: defects of arterial specification as deltalike4 mutants, abnormal cerebellum and skin development as jagged1 conditional mutants, and syndactylism as jagged2 mutants.

Conclusions/Significance

Our data provide the first evidence that Mib1 is essential for Jagged as well as Deltalike ligand-mediated Notch signaling in mammalian development, while Neur1, Neur2, and Mib2 are dispensable.     

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

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

The atypical cadherin Flamingo links Frizzled and Notch signaling in planar polarity establishment in the Drosophila eye

Planar cell polarity is established in the Drosophila eye through distinct fate specification of photoreceptors R3 and R4 by a two-tiered mechanism employing Fz and Notch signaling: Fz signaling specifies R3 and induces Dl to activate Notch in R4. We show that the atypical cadherin Flamingo (Fmi) plays critical, but distinct, roles in both R3 and R4. Fmi is first enriched at equatorial cell borders of R3/R4, positively interacting with Fz/Dsh. Subsequently, Fmi is upregulated in R4 by Notch and functions to downregulate Dl expression by antagonizing Fz signaling. This in turn amplifies and enforces the initial Fz-signaling bias in the R3/R4 pair. Our results reveal differences in the planar cell polarity genetic circuitry between the eye and the wing.     

Both inhibition and activation of Notch signaling rely on a conserved Neuralized-binding motif in Bearded proteins and the Notch ligand Delta

Lateral inhibition is one of the key functions of Notch signaling during animal development. In the proneural clusters that give rise to Drosophila mechanosensory bristles, Delta (Dl) ligand in the sensory organ precursor (SOP) cell is targeted for ubiquitination by the E3 ligase Neuralized (Neur), resulting in activation of Dl's capacity to signal to the Notch receptor on neighboring cells. The cells that receive this signal activate a genetic program that suppresses their SOP fate potential, insuring that only a single SOP develops within each cluster. Using multiple lines of investigation, we provide evidence that members of the Bearded family of proteins (BFMs) inhibit Dl activation in non-SOP cells by binding to Neur and preventing it from interacting with Dl. We show that this activity of BFMs is dependent on the conserved NXXN motif, and report the unexpected finding that several BFMs include multiple functional copies of this motif. We find that a conserved NXXN motif in the intracellular domain of Dl is responsible for its interaction with Neur, indicating direct competition between Dl and BFMs for binding to Neur, and we show that Neur-dependent endocytosis of Dl requires the integrity of its NXXN motif. Our results illuminate the mechanism of an important regulatory event in Notch signaling that appears to be conserved between insects and crustaceans.     

The loss of Drosophila APG4/AUT2 function modifies the phenotypes of cut and Notch signaling pathway mutants

A P-element line ( P0997) of Drosophila melanogaster in which the P element disrupts the Drosophila homolog of the Saccharomyces cerevisiae gene APG4/AUT2 was identified during the course of screening for cut ( ct) modifiers. The yeast gene APG4/AUT2 encodes a cysteine endoprotease directed against Apg8/Aut7 and is necessary for autophagy. The P0997 mutation enhances the wing margin loss associated with ct mutations, and also modifies the wing and eye phenotypes of Notch (N), Serrate (Ser), Delta (Dl), Hairless (H), deltex (dx), vestigial (vg) and strawberry notch (sno) mutants. These results therefore suggest an unexpected link between autophagy and the Notch signaling pathway.     

Drosophila neuralized is a ubiquitin ligase that promotes the internalization and degradation of delta.

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

Sequential Notch Signalling at the Boundary of Fringe Expressing and Non-Expressing Cells

Wing development in Drosophila requires the activation of Wingless (Wg) in a small stripe along the boundary of Fringe (Fng) expressing and non-expressing cells (FB), which coincides with the dorso-ventral (D/V) boundary of the wing imaginal disc. The expression of Wg is induced by interactions between dorsal and ventral cells mediated by the Notch signalling pathway. It appears that mutual signalling from dorsal to ventral and ventral to dorsal cells by the Notch ligands Serrate (Ser) and Delta (Dl) respectively establishes a symmetric domain of Wg that straddles the D/V boundary. The directional signalling of these ligands requires the modification of Notch in dorsal cells by the glycosyltransferase Fng and is based on the restricted expression of the ligands with Ser expression to the dorsal and that of Dl to the ventral side of the wing anlage. In order to further investigate the mechanism of Notch signalling at the FB, we analysed the function of Fng, Ser and Dl during wing development at an ectopic FB and at the D/V boundary. We find that Notch signalling is initiated in an asymmetric fashion on only one side of the FB. During this initial asymmetric phase, only one ligand is required, with Ser initiating Notch-signalling at the D/V and Dl at the ectopic FB. Furthermore, our analysis suggests that Fng has also a positive effect on Ser signalling. Because of these additional properties, differential expression of the ligands, which has been a prerequisite to restrict Notch activation to the FB in the current model, is not required to restrict Notch signalling to the FB.     

Regulation of EGFR and Notch signaling by distinct isoforms of D-cbl during Drosophila development

Cells receive and interpret extracellular signals to regulate cellular responses such as proliferation, cell survival and differentiation. However, proper inactivation of these signals is critical for appropriate homeostasis. Cbl proteins are E3-ubiquitin ligases that restrict receptor tyrosine kinase (RTK) signaling, most notably EGFR (Epidermal Growth Factor Receptor), via the endocytic pathway. Consistently, many mutant phenotypes of Drosophila cbl (D-cbl) are due to inappropriate activation of EGFR signaling. However, not all D-cbl phenotypes can be explained by increased EGFR activity. Here, we report that D-Cbl also negatively regulates Notch activity during eye and wing development. D-cbl produces two isoforms by alternative splicing. The long isoform, D-CblL, regulates the EGFR. We found that the short isoform, D-CblS, preferentially restricts Notch signaling. Specifically, our data imply that D-CblS controls the activity of the Notch ligand Delta. Taken together, these data suggest that D-Cbl controls the EGFR and Notch/Delta signaling pathways through production of two alternatively spliced isoforms during development in Drosophila.     

The neuralized homology repeat 1 domain of Drosophila neuralized mediates nuclear envelope association and delta-dependent inhibition of nuclear import

Signaling by the Notch (N) pathway is critical for many developmental processes and requires complex trafficking of both the N receptor and its transmembrane ligands, Delta (Dl) and Serrate. neuralized encodes an E3 ubiquitin ligase required for N ligand internalization. Neuralized (Neur) is conserved from worms to humans and comprises two Neur homology repeat (NHR) domains, NHR1 and NHR2, and a carboxyl-terminal RING domain. We have previously shown that the RING domain is required for ubiquitin ligase activity and that NHR1 mediates binding to Dl, a ubiquitination target. In Drosophila, Neur associates with the plasma membrane and hepatocyte responsive serum phosphoprotein-positive endosomes. Here we demonstrate that Neur also exhibits nuclear envelope localization. We have determined that Neur subcellular localization is regulated by nuclear trafficking and that inhibition of chromosome region maintenance 1, a nuclear export receptor, interferes with Neur nuclear export, trapping Neur in the nucleus. Moreover, we demonstrate that nuclear envelope localization is mediated by the Neur NHR1 domain. Interestingly, Dl expression in Schneider cells is sufficient to inhibit Neur nuclear import and inhibition occurs in an NHR1-dependent manner, suggesting that Neur nuclear localization occurs in contexts where Dl expression is either low or absent. Consistent with this, we found that Neur exhibits nuclear trafficking and associates with the nuclear envelope in the secretory cells of the larval salivary gland and that overexpression of Dl can reduce Neur localization to the nucleus. Altogether, our data demonstrate that Neur localizes to the nuclear envelope and that this localization can be negatively regulated by Dl, suggesting a possible nuclear function for Neur in Drosophila.     

Distinct roles for Mind bomb, Neuralized and Epsin in mediating DSL endocytosis and signaling in Drosophila

Ligands of the Delta/Serrate/Lag2 (DSL) family must normally be endocytosed in signal-sending cells to activate Notch in signal-receiving cells. DSL internalization and signaling are promoted in zebrafish and Drosophila, respectively, by the ubiquitin ligases Mind bomb (Mib) and Neuralized (Neur). DSL signaling activity also depends on Epsin, a conserved endocytic adaptor thought to target mono-ubiquitinated membrane proteins for internalization. Here, we present evidence that the Drosophila ortholog of Mib (Dmib) is required for ubiquitination and signaling activity of DSL ligands in cells that normally do not express Neur, and can be functionally replaced by ectopically expressed Neur. Furthermore, we show that both Dmib and Epsin are required in these cells for some of the endocytic events that internalize DSL ligands, and that the two Drosophila DSL ligands Delta and Serrate differ in their utilization of these Dmib- and Epsin-dependent pathways: most Serrate is endocytosed via the actions of Dmib and Epsin, whereas most Delta enters by other pathways. Nevertheless, only those Serrate and Delta proteins that are internalized via the action of Dmib and Epsin can signal. These results support and extend our previous proposal that mono-ubiquitination of DSL ligands allows them to gain access to a select, Epsin-dependent, endocytic pathway that they must normally enter to activate Notch.     

Echinoid mutants exhibit neurogenic phenotypes and show synergistic interactions with the Notch signaling pathway

During neurogenesis in Drosophila, groups of ectodermal cells are endowed with the capacity to become neuronal precursors. The Notch signaling pathway is required to limit the neuronal potential to a single cell within each group. Loss of genes of the Notch signaling pathway results in a neurogenic phenotype: hyperplasia of the nervous system accompanied by a parallel loss of epidermis. Echinoid (Ed), a cell membrane associated Immunoglobulin C2-type protein, has previously been shown to be a negative regulator of the EGFR pathway during eye and wing vein development. Using in situ hybridization and antibody staining of whole-mount embryos, we show that Ed has a dynamic expression pattern during embryogenesis. Embryonic lethal alleles of ed reveal a role of Ed in restricting neurogenic potential during embryonic neurogenesis, and result in a phenotype similar to that of loss-of-function mutations of Notch signaling pathway genes. In this process Ed interacts closely with the Notch signaling pathway. Loss of ed suppresses the loss of neuronal elements caused by ectopic activation of the Notch signaling pathway. Using a temperature-sensitive allele of ed we show, furthermore, that Ed is required to suppress sensory bristles and for proper wing vein specification during adult development. In these processes also, ed acts in close concert with genes of the Notch signaling pathway. Thus the extra wing vein phenotype of ed is enhanced upon reduction of Delta (Dl) or Enhancer of split [E(spl)] proteins. Overexpression of the membrane-tethered extracellular region of Ed results in a dominant-negative phenotype. This phenotype is suppressed by overexpression of E(spl)m7 and enhanced by overexpression of Dl. Our work establishes a role of Ed during embryonic nervous system development, as well as adult sensory bristle specification and shows that Ed interacts synergistically with the Notch signaling pathway.