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. Drosophila mind bomb (D-mib) 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. It is concluded that D-mib and Neur are two structurally distinct proteins that have similar molecular activities but distinct developmental functions in Drosophila (Le Borgne, 2005).

Cell-to-cell signaling mediated by receptors of the Notch (N) family has been implicated in various developmental decisions in organisms ranging from nematodes to mammals. N is well-known for its role in lateral inhibition, a key patterning process that organizes the regular spacing of distinct cell types within groups of equipotent cells. Additionally, N mediates inductive signaling between cells with distinct identities. In both signaling events, N signals via a conserved mechanism that involves the cleavage and release from the membrane of the N intracellular domain that acts as a transcriptional co-activator for DNA-binding proteins of the CBF1/Suppressor of Hairless/Lag-2 (CSL) family (Le Borgne, 2005).

Two transmembrane ligands of N are known in Drosophila, Delta (Dl) and Serrate (Ser). Dl and Ser have distinct functions. For instance, Dl (but not Ser) is essential for lateral inhibition during early neurogenesis in the embryo. Conversely, Ser (but not Dl) is specifically required for segmental patterning. Some developmental decisions, however, require the activity of both genes: Dl and Ser are both required for the specification of wing margin cells during imaginal development. These different requirements for Dl and Ser appear to primarily result from their non-overlapping expression patterns rather than from distinct signaling properties. Consistent with this interpretation, Dl and Ser have been proposed to act redundantly in the sensory bristle lineage where they are co-expressed. Furthermore, Dl and Ser appear to be partially interchangeable because the forced expression of Ser can partially rescue the Dl neurogenic phenotype. Additionally, the ectopic expression of Dl can partially rescue the Ser wing phenotype. The notion that Dl and Ser have similar signaling properties has, however, recently been challenged by the observation that human homologs of Dl and Ser have distinct instructive signaling activity (Le Borgne, 2005).

Endocytosis has recently emerged as a key mechanism regulating the signaling activity of Dl. (1) Clonal analysis in Drosophila has suggested that dynamin-dependent endocytosis is required not only in signal-receiving cells but also in signal-sending cells to promote N activation (Seugnet, 1997). (2) Mutant Dl proteins that are endocytosis defective exhibit reduced signaling activity (Parks, 2000). (3) Two distinct E3 ubiquitin ligases, Neuralized (Neur) and Mind-bomb (Mib), have recently been shown to regulate Dl endocytosis and N activation in Drosophila and Danio rerio, respectively. Ubiquitin is a 76-amino-acid polypeptide that is covalently linked to substrates in a multi-step process that involves a ubiquitin-activating enzyme (E1), a ubiquitin-conjugating enzyme (E2), and a ubiquitin-protein ligase (E3). E3s recognize specific substrates and catalyze the transfer of ubiquitin to the protein substrate. Ubiquitin was first identified as a tag for proteins destined for degradation. More recently, ubiquitin has also been shown to serve as a signal for endocytosis. Mib in D. rerio and Neur in Drosophila and Xenopus have been shown to associate with Dl, regulate Dl ubiquitination, and promote its endocytosis. Moreover, genetic and transplantation studies have indicated that both Neur and Mib act in a non-autonomous manner, indicating that endocytosis of Dl is associated with increased Dl signaling activity. Finally, epsin, a regulator of endocytosis that contains a ubiquitin-interacting motif and that is known in Drosophila as Liquid facet, is essential for Dl signaling. In one study, Liquid facets was proposed to target Dl to an endocytic recycling compartment, suggesting that recycling of Dl may be required for signaling. Accordingly, signaling would not be linked directly to endocytosis, but endocytosis would be prerequisite for signaling. How endocytosis of Dl leads to the activation of N remains to be elucidated. Also, whether the signaling activity of Ser is similarly regulated by endocytosis is not known (Le Borgne, 2005 and references therein).

While genetic analysis has revealed that neur in Drosophila and mib in D. rerio are strictly required for N signaling, knockout studies of mouse Neur1 have indicated that NEUR1 is not strictly required for N signaling. One possible explanation is functional redundancy with the mouse Neur2 gene. Conversely, the function of Drosophila mib (D-mib), the homolog of D. rerio mib gene has not previously been characterized (Le Borgne, 2005).

To establish the respective roles of these two distinct E3 ligases in the context of a single model organism, the function of the Drosophila D-mib gene was studied. D-mib, like D. rerio Mib, appears to regulate Dl signaling during leg segmentation, wing vein formation, and lateral inhibition in the adult notum. D-mib is specifically required for Ser endocytosis and signaling during wing development, indicating for the first time that endocytosis regulates Ser signaling. Interestingly, the D-mib activity was found necessary for a subset of N signaling events that are distinct from those requiring the activity of the neur gene. Nevertheless, the ectopic expression of Neur compensates for the loss of D-mib activity in the wing, indicating that Neur and D-mib have overlapping functions. It is concluded that D-mib and Neur are two structurally distinct proteins with similar molecular activities but distinct and complementary functions in Drosophila (Le Borgne, 2005).

This analysis first establishes that D-mib regulates Ser signaling during wing development. (1) Clonal analysis revealed that the activity of the D-mib gene is specifically required in dorsal cells for the expression of Cut at the wing margin. (2) Expression of D-mib in the dorsal Ser-signaling cells is sufficient to rescue the D-mib mutant wing phenotype. (3) Results from an in vivo antibody uptake assay indicate that the endocytosis of Ser (but not of Dl) was strongly inhibited in D-mib mutant cells. This inhibition correlates with the strong accumulation of Ser (but not Dl) at the apical cortex of D-mib mutant cells. Thus, an essential function of D-mib in the wing is to regulate the endocytosis of Ser in dorsal cells to non-autonomously promote the activation of N along the D-V boundary. By analogy, the defective growth of the eye tissue may similarly result from the lack of Ser signaling and of N activation along the D-V boundary. Because (1) D-mib co-localizes with Ser at the apical cortex of wing disc cells, (2) acts in a RING-finger-dependent manner to regulate Ser endocytosis in S2 cells, and (3) physically associates with Ser in co-immunoprecipitation experiments, D-mib may ubiquitinate Ser and directly regulate its endocytosis (Le Borgne, 2005).

This analysis further suggests that endocytosis of Ser is required for Ser signaling. This conclusion is consistent with observations made earlier showing that secreted versions of Ser cannot activate N but instead antagonize Ser signaling. Thus, endocytosis of both N ligands appears to be strictly required for N activation in Drosophila. Different models have been proposed to explain how endocytosis of the ligand, which removes the ligand from the cell surface, results in N receptor activation. Interestingly, the strong requirement for Dl and Ser endocytosis seen in Drosophila is not conserved in Caenorhabditis elegans, in which secreted ligands have been shown to be functional. Noticeably, there is no C. elegans Mib homolog, and the function of C. elegans neur (F10D7.5) is not known. It is speculated that endocytosis of the ligands may have evolved as a means to ensure tight spatial regulation of the activation of Notch (Le Borgne, 2005).

This analysis also establishes that the activity of the D-mib gene is required for a subset of N signaling events that are distinct from those that require the activity of the neur gene. The D-mib gene regulates wing margin formation, leg segmentation, and vein formation, whereas none of these three processes depend on neur gene activity. Conversely, the activity of the neur gene is essential for binary cell-fate decisions in the bristle lineage that do not require the activity of the D-mib gene (no bristle defects were seen in D-mib mutant flies). The activity of the neur gene is also required for lateral inhibition during neurogenesis in embryos and pupae. This process is largely independent of D-mib gene activity since the complete loss of D-mib function resulted only in a mild neurogenic phenotype in the notum. These data thus indicate that the neur and D-mib genes have largely distinct and complementary functions in Drosophila. Whether a similar functional relationship between Neur and D-mib exists in vertebrates awaits the study of the D. rerio neur genes and/or of the murine Mib and Neur genes (Le Borgne, 2005).

The functional differences observed between D-mib and neur cannot be simply explained by obvious differences in molecular activity and/or substrate specificity. Both Neur and D-mib physically interact with Dl and promote the down-regulation of Dl from the apical membrane when overexpressed. Furthermore, Dl signaling appears to require the activity of either Neur or D-mib, depending on the developmental contexts. Specific aspects of the D-mib phenotype in legs and in the notum cannot simply result from loss of Ser signaling and are consistent with reduced Dl signaling, suggesting that D-mib regulates Dl signaling. Consistent with this interpretation, overexpression studies indicate that D-mib up-regulates the signaling activity of Dl, whereas a dominant-negative form of D-mib inhibits it. It is noted, however, that no clear defects in Dl subcellular localization and/or trafficking were observed in D-mib mutant cells. It is conceivable that the contribution of D-mib to the endocytosis of Dl is masked by the activity of D-mib-independent processes that may, or may not, be linked to Dl signaling. It has also been shown that, reciprocally, Neur and D-mib may similarly regulate Ser. Neur and D-mib similarly promote down-regulation of Ser from the cell surface when overexpressed. Moreover, D-mib binds Ser and regulates Ser signaling. Whether endogenous Neur binds and activates Ser remains to be tested. However, the ability of Neur to rescue the D-mib mutant wing phenotype when expressed in dorsal cells strongly indicates that Neur can promote Ser signaling. Together, these data indicate that Neur and D-mib have similar molecular activities (Le Borgne, 2005).

D-mib and Neur may have identical molecular activities but distinct expression patterns, hence distinct functions at the level of the organism. Consistent with this possibility, D-mib is uniformly distributed in imaginal discs, whereas Neur is specifically detected in sensory cells. Importantly, the rescue of the D-mib mutant phenotype by ectopic expression of Neur strongly supports this interpretation. This result further suggests that Neur can regulate Ser signaling. Consistent with this idea, overexpression of Neur in imaginal discs results in a strong reduction of Ser accumulation at the apical cortex. Thus, despite their obvious structural differences, Neur and D-mib appear to act similarly to promote the endocytosis of Dl and Ser. Nevertheless, the observation that D-mib can not compensate for the loss of neur activity in the embryo indicates that D-mib and Neur have overlapping rather than identical molecular activities (Le Borgne, 2005).

In conclusion, Neur and D-mib appear to have similar molecular activities in the regulation of Dl and Ser endocytosis but distinct developmental functions in Drosophila. The conservation from Drosophila to mammals of these two structurally distinct but functionally similar E3 ubiquitin ligases is likely to reflect a combination of evolutionary advantages associated with: (1) specialized expression pattern, as evidenced by the cell-specific expression of the neur gene in sensory organ precursor cells, (2) specialized function, as suggested by the role of murine MIB in TNFα signaling (Jin, 2002) and (3) regulation of protein stability, localization, and/or activity. For instance, Neur, but not D-mib, localizes asymmetrically during asymmetric sensory organ precursor cell divisions (Le Borgne, 2005).

GENE STRUCTURE

cDNA clone length - 4073

Bases in 5' UTR - 164

Exons - 4

Bases in 3' UTR - 228

PROTEIN STRUCTURE

Amino Acids - 1226

Structural Domains

Neur and Mib proteins completely differ in primary structure. Drosophila Neur is a 754-amino-acid protein that contains two conserved Neur homology repeats of unknown function and one C-terminal catalytic really interesting new gene (RING) domain. D. rerio Mib (also known as DIP-1 in the mouse is a 1,030-amino-acid protein with one ZZ zinc finger domain surrounded by two Mib/HERC2 domains, two Mib repeats, eight ankyrin repeats, two atypical RING domains, and one C-terminal catalytic RING domain. Both genes have been conserved from flies to mammals (Le Borgne, 2005).

date revised: 1 October 2005

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