InteractiveFly: GeneBrief

mind bomb 1 : Biological Overview | Regulation | Developmental Biology | Effects of Mutation | Evolutionary Homologs | References

BIOLOGICAL OVERVIEW

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).

REGULATION

Transcriptional Regulation

Temporal ChIP-on-chip reveals Biniou as a universal regulator of the visceral muscle transcriptional network

Smooth muscle plays a prominent role in many fundamental processes and diseases, yet understanding of the transcriptional network regulating its development is very limited. The FoxF transcription factors are essential for visceral smooth muscle development in diverse species, although their direct regulatory role remains elusive. A transcriptional map of Biniou (a FoxF transcription factor) and Bagpipe (an Nkx factor) activity is presented as a first step to deciphering the developmental program regulating Drosophila visceral muscle development. A time course of chromatin immunoprecipitatation followed by microarray analysis (ChIP-on-chip) experiments and expression profiling of mutant embryos reveal a dynamic map of in vivo bound enhancers and direct target genes. While Biniou is broadly expressed, it regulates enhancers driving temporally and spatially restricted expression. In vivo reporter assays indicate that the timing of Biniou binding is a key trigger for the time span of enhancer activity. Although bagpipe and biniou mutants phenocopy each other, their regulatory potential is quite different. This network architecture was not apparent from genetic studies, and highlights Biniou as a universal regulator in all visceral muscle, regardless of its developmental origin or subsequent function. The regulatory connection of a number of Biniou target genes is conserved in mice, suggesting an ancient wiring of this developmental program (Jakobsen, 2007; full text of article).

The dynamic enhancer binding of Biniou suggested that the timing of Biniou occupancy is important for the timing of enhancer activity. To assess this in vivo, a number of regions from each of the three temporal clusters were linked to a GFP reporter. The timing of enhancer activity was assayed in vivo by in situ hybridization in transgenic embryos, to avoid time delays due to GFP protein folding and protein perdurance. All regions examined drive expression in a subset of Biniou-expressing cells and recapitulate all or part of the target genes' expression. This study focused on their temporal activity (Jakobsen, 2007).

The initiation of enhancer activity closely matches the first time point of Biniou binding for >90% of enhancers examined (10 of 11 CRMs). The early-bound enhancers (ttk, fd64a-e, lame duck (lmd), bap3) drive expression at stages 10-11, reflecting the binding of Biniou at these stages of development. Similarly, all four continuous-bound enhancers (HLH54F, otk, mib2, bap-FH) initiate expression at the first time period when Biniou binds. The two late-bound enhancers, in contrast, do not initiate expression at stages 10 or 11 of development, matching the lack of Biniou binding during these stages. Instead, the expression of the fd64a late enhancer initiates at stage 13, while the ken enhancer initiates VM expression at stage 14. This shift in the initiation of activity mirrors Biniou binding to these enhancers at stages 12-13 and 13-14, respectively. The only exception is the CG2330 enhancer, which initiates expression at stage 11, while Biniou enhancer binding was first detected at stage 13-14). As the expression of endogenous CG2330 does not initiate until stage 13, the apparent discrepancy in enhancer activity may simply reflect the exclusion of some regulatory motifs within the limits of the cloned region (Jakobsen, 2007).

Remarkably, the duration of enhancer activity is also tightly correlated with the time span of Biniou binding in 10 out of 11 CRMs examined. This is particularly striking in the early-bound enhancers: When Biniou ceases to bind to these CRMs (lmd, ttk, fd64a early, and bap3), their ability to regulate expression is lost. The converse is also true. Continuous Biniou binding correlates with continuous enhancer activity, specifically for bap-FH, HLH54F, and otk. The exception is the mib2 enhancer. In the context of this module Biniou binding it is not sufficient to maintain enhancer activity in the VM at late developmental time points (Jakobsen, 2007).

Taken together, these data indicate that the timing of Biniou enhancer binding is predictive for temporal enhancer activity in the large majority of cases (Jakobsen, 2007).

Mind-bomb regulates the cell-surface level of Ser and regulates Ser signaling

The subcellular localization of D-mib. Anti-D-mib antibodies were generated that specifically detected D-mib on Western blots and on fixed tissues. Using these antibodies, D-mib was detected in all imaginal disc cells. D-mib subcellular distribution was examined in epithelial cells located along the edge of the wing discs because cross-sectional imaging affords better resolution along the apical-basal axis. D-mib co-localizes with Ser, Dl, and N at the apical cortex. Dl and Ser are also detected in large intracellular vesicles that probably correspond to multivesicular bodies in that they also stained for hepatocyte growth factor-regulated tyrosine kinase substrate. The intracellular dots seen with the anti-D-mib antibodies are distinct from the Dl- and Ser-positive dots and appear to result from background staining. The reduced cytoplasmic staining seen in D-mib mutant cells suggests that D-mib is also present in the cytoplasm. A similar localization at the apical cortex and in the cytoplasm is seen for a functional yellow fluorescent protein (YFP)::D-mib fusion protein. These localization data suggest that D-mib may act at the apical cortex to regulate the activity of Dl and/or Ser (Le Borgne, 2005).

To test whether this specific increase in the level of Ser at the apical cortex results from reduced Ser endocytosis in D-mib mutant cells, the endocytosis of Ser was followed in living imaginal discs using an antibody uptake assay. Briefly, dissected wing discs were cultured for 15 min in the presence of antibodies that recognize the extracellular part of Ser or Dl, then washed, cultured for another 45 min in medium without antibodies, and then fixed. The uptake of anti-Ser and anti-Dl antibodies was then assessed using secondary antibodies. Using this assay, it was found that anti-Ser-and anti-Dl antibodies are internalized in wild-type epithelial cells. The complete loss of D-mib activity in D-mib¹ wing discs does not significantly change the internalization of anti-Dl antibodies, indicating that D-mib is not required for Dl endocytosis in this tissue. However, the loss of D-mib activity strongly inhibits the endocytosis of anti-Ser. Moreover, high levels of anti-Ser antibodies were seen at the apical surface, confirming that D-mib mutant cells accumulate high levels of Ser at their surface. It is therefore concluded that D-mib is specifically required for the endocytosis of Ser in wing discs (Le Borgne, 2005).

Ubiquitin-mediated endocytosis is thought to depend on monoubiquitination. Thus, by analogy with the function of Mib in D. rerio (Chen, 2004), it is suggested that D-mib may directly monoubiquitinate Ser. Consistent with this hypothesis, it was shown that D-mib binds Ser. Moreover, a mutation in the C-terminal catalytic RING domain of D-mib abolished its ability to internalize Ser in transfected S2 cells, implying that the E3 ubiquitin ligase activity of D-mib is required for Ser internalization. Biochemical analysis of the ubiquitination events regulated by D-mib will be needed to further define the mechanism by which D-mib regulates the endocytosis of Ser in vivo (Le Borgne, 2005).

The regulation of Ser endocytosis by D-mib suggests that D-mib may regulate Ser signaling. Ser expression is restricted to dorsal cells in second instar wing imaginal discs. Ser in dorsal cells signals across the D-V boundary to activate N in ventral cells. If D-mib is required for Ser signaling during wing development, then loss of D-mib activity in dorsal cells should affect the specification of the wing margin in a non-autonomous manner. Loss of D-mib activity in large dorsal clones of D-mib² mutant cells results in a loss of Cut expression at the D-V interface. The lack of Cut expression in wild-type ventral cells abutting the D-V boundary indicates that D-mib is required for Ser signaling by dorsal cells and acts in a non-autonomous manner to activate N in ventral cells. Conversely, loss of D-mib activity in large ventral clones does not disrupt margin specification, indicating that D-mib is not strictly required for Dl signaling by ventral cells. However, a narrowing of the Cut-positive margin is observed, suggesting that D-mib contributes to regulating the level of Dl signaling. Of note, ventral D-mib mutant cells express Cut, implying that D-mib is not required for N signal transduction (Le Borgne, 2005).

Next, whether expression of D-mib in dorsal cells is sufficient to rescue the D-mib wing phenotype was tested. D-mib was expressed in dorsal cells of D-mib²/D-mib³ mutant discs using Ser-GAL4. Similarly to the expression of the Ser gene, Ser-GAL4 expression is restricted to dorsal cells in second/early third instar larvae and is weakly expressed in ventral cells in mid/late third instar larvae, i.e., after margin cell specification. Expression of D-mib in dorsal cells is sufficient to rescue growth of the wing pouch and of the expression of Cut in margin cells in D-mib mutant discs. This result confirms that D-mib regulates Ser signaling by dorsal cells (Le Borgne, 2005).

A similar rescue was observed with a YFP::D-mib protein, indicating that YFP::D-mib is functional. YFP::D-mib localizes at the apical cortex and in the cytoplasm, as seen for endogenous D-mib. YFP::D-mib co-localizes with Dl and Ser at the apical cortex of cells expressing low levels of YFP::D-mib. However, cells expressing high levels of YFP::D-mib showed a strong reduction in the level of both Dl and Ser at the cortex, further indicating that D-mib down-regulates the levels of both Ser and Dl at the apical cortex (Le Borgne, 2005).

Protein Interactions

Loss- and gain-of-function analyses indicate that the major function of D-mib is to regulate Notch signal transduction. Since Delta is a bona fide substrate of zebrafish Mib, tests were performed for a physical association of D-mib and Delta by co-immunoprecipitation. Cultured cells were co-transfected with Delta and various D-mib expression vectors, and co-immunoprecipitation was performed in both directions. Although Delta did not successfully co-immunoprecipitate full-length D-mib, it did associate with all isoforms that contain the D-mib N terminus and lack the C-terminal RING finger (namely D-mib-N, D-mibDelta3RF and D-mibDeltaRF. Conversely, these same D-mib isoforms efficiently co-immunoprecipitate Delta; full-length D-mib also shows modest association with Delta in this direction. It was consistently observed that the presence of full-length D-mib reduces Delta levels, which might account for why this interaction is poorly detected. Notably, D-mib-N shows the strongest interaction with Delta. In fact, immunoprecipitated D-mib-N brings down both full-length Delta and cleaved Delta^IC, consistent with a direct interaction between the N terminus of D-mib and the intracellular domain of Delta. A truncated D-mib protein lacking the N-terminal domain (D-mib-C) shows no binding to Delta, demonstrating that this region is crucial for association with Delta (Lai, 2005).

Physical association between D-mib proteins and Serrate was tested. D-mib:Serrate interactions appear to be somewhat weaker than D-mib:Delta interactions; however, the overall profile of the different D-mib truncations in association with Serrate and Delta is identical. These findings lead to the conclusion that the N terminus of D-mib mediates physical association with both Drosophila DSL ligands. In addition, full-length D-mib similarly reduces the accumulation of Serrate, indicating that D-mib downregulates both DSL ligands (Lai, 2005).

In vitro data correlate well with in vivo studies, in that all RING-finger-deleted D-mib isoforms that retain the ability to associate with DSL ligands (D-mib-N, D-mibDeltaRF and D-mib3DeltaRF) have at least some ability to inhibit Notch signaling. However, full specificity and activity of D-mib requires inclusion of the ankyrin repeats and the two non-canonical RING fingers. Curiously, there is no significant similarity at the primary amino acid level between the intracellular domains of Delta and Serrate. In this regard, it is relevant to note that Xenopus Neur (X-Neur) robustly regulates Drosophila Delta in vivo, even though there is no significant similarity between the intracellular domains of Delta and X-Delta. D-mib and Neur may therefore recognize a more hidden, possibly structural, feature that is shared by DSL ligands (Lai, 2005).

Role of conserved intracellular motifs in Ser signalling, cis-inhibition and endocytosis

Notch is the receptor in a signalling pathway that operates in a diverse spectrum of developmental processes. Its ligands (e.g. Serrate) are transmembrane proteins whose signalling competence is regulated by the endocytosis-promoting E3 ubiquitin ligases, Mindbomb1 and Neuralized. The ligands also inhibit Notch present in the same cell (cis-inhibition). This study identifies two conserved motifs in the intracellular domain of Serrate that are required for efficient endocytosis. The first, a dileucine motif, is dispensable for trans-activation and cis-inhibition despite the endocytic defect, demonstrating that signalling can be separated from bulk endocytosis. The second, a novel motif, is necessary for interactions with Mindbomb1/Neuralized and is strictly required for Serrate to trans-activate and internalise efficiently but not for it to inhibit Notch signalling. Cis-inhibition is compromised when an ER retention signal is added to Serrate, or when the levels of Neuralized are increased, and together these data indicate that cis-inhibitory interactions occur at the cell surface. The balance of ubiquitinated/unubiquitinated ligand will thus affect the signalling capacity of the cell at several levels (Glittenberg, 2006; full text of article).

DEVELOPMENTAL BIOLOGY

Effects of Mutation or Deletion

The closest Drosophila homolog of the vertebrate mib gene is the predicted gene CG5841, D-mib. A P-element inserted into the 5' untranslated region of the D-mib gene was recently isolated. Insertion of this P-element confers late pupal lethality. Lethality was reverted by precise excision of the P-element, suggesting that insertion of this P-element is a D-mib mutation, referred to as D-mib¹. A 13.6-kb deletion that removes the entire D-mib coding region was selected by imprecise excision of this P-element. This deletion represents a null allele of D-mib and was named D-mib². This deletion also deletes the 3' flanking RpS31 gene. The D-mib¹ and D-mib² mutant alleles do not complement the l(3)72Cda^J12 and l(3)72Cda^I5 lethal mutations that have been mapped to the same cytological interval as the D-mib gene. This indicates that these two lethal mutations are D-mib mutant alleles, and they were therefore renamed D-mib³ and D-mib⁴, respectively. The D-mib¹ and D-mib³ mutations behave as genetic null alleles. In contrast, D-mib⁴ is a partial loss-of-function allele because flies trans-heterozygous for D-mib⁴ and any other D-mib null alleles are viable (Le Borgne, 2005).

These four mutations identify the CG5841 gene as D-mib by the following evidence: (1) lethality of homozygous D-mib¹ pupae is associated with the insertion of a P-element into the 5' UTR of the D-mib gene; (2) genomic sequencing of the D-mib³ allele revealed the presence of a stop codon at position 258. This allele is therefore predicted to produce a truncated protein devoid of the catalytic RING domain, consistent with D-mib³ being a null allele. Genomic sequencing of the D-mib⁴ allele showed that this mutation is associated with a valine-to-methionine substitution at a conserved position in the second Mib repeat. (3) Western blot analysis showed that the D-mib protein was not detectable in imaginal disc and brain complex extracts prepared from homozygous D-mib¹ and D-mib¹/D-mib² larvae. (4) The leaky, GAL4-independent expression of a UAS-D-mib transgene fully rescues the lethality of D-mib¹/D-mib² flies. Thus, this analysis identified both complete and partial D-mib loss-of-function alleles (Le Borgne, 2005).

Complete loss of zygotic D-mib activity in homozygous D-mib¹ and trans-heterozygous D-mib²/D-mib³, D-mib¹/D-mib³ and D-mib¹/D-mib² individuals leads to late pupal lethality. Mutant pupae die as pharate adults showing ectopic macrochaetes, increased microchaete density on the dorsal thorax, short legs lacking tarsal segmentation, and nearly complete loss of eye and wing tissues. Tissue losses are associated with a dramatic reduction in size of the eye field and of the wing pouch in mutant discs of third instar larvae. Hypomorphic D-mib²/D-mib⁴ mutant flies showed ectopic sensory organs, rough eyes, small wings, and thickened veins(Le Borgne, 2005).

All these phenotypes may result from reduced N signaling. More specifically, the bristle and leg phenotypes are likely to result from reduced signaling by Dl (and not by Ser). Indeed, a reduction in Dl-mediated lateral inhibition can result in ectopic sensory organs and increased bristle density on the body surface. In contrast, a complete loss of Ser signaling had no effect on bristle density. Likewise, loss of Dl signaling has been shown to result in short unsegmented legs, similar to the ones seen in the absence of D-mib activity, whereas a complete loss of Ser activity led to the formation of elongated unsegmented legs. Finally, the vein phenotype seen in D-mib hypomorphic flies is similar to the one seen in Dl^ts mutant flies. Together, these observations suggest that D-mib regulates Dl signaling in several developmental contexts. Consistent with this conclusion, D-mib has been shown to bind Dl and promote Dl signaling, and overexpression of D-mib down-regulates the accumulation of Dl at the cell surface (Le Borgne, 2005).

The function of D-mib during wing development was studied in more detail. Growth of the wing pouch depends on the activity of an organizing center located at the dorsal-ventral (D-V) boundary. This boundary is established in first instar larvae and is defined by the apterous expression boundary. Apterous activates the expression of the Ser and fringe genes in dorsal cells. High levels of Ser in dorsal cells activate N in trans in ventral cells and suppress N activation in cis in dorsal cells, whereas Fringe modifies N in dorsal cells such that dorsal cells located at the D-V boundary respond to Dl. Thus, composite signaling by Ser and Dl leads to symmetric N activation in margin cells located along the D-V boundary. N then regulates the expression of the vestigial and wingless (wg) genes that cooperate to promote growth of the wing pouch. N also regulates expression of the cut gene in margin cells. Thus, loss of N signaling results in a reduction in size of the wing pouch accompanied by the loss of cut and wg expression along the D-V boundary (Le Borgne, 2005).

A complete loss of Cut and Wg accumulation and wg-lacZ expression was observed in the central region of third instar D-mib mutant wing discs. Thus, the D-mib wing phenotype may result from defective N inductive signaling at the D-V boundary. It is concluded that the activity of the D-mib gene is required for the specification of the wing margin and, hence, growth of the wing pouch. Interestingly, wing margin formation and expression of Cut are not affected by the complete loss of neur activity. Similarly, loss of neur activity has no detectable effect on leg segmentation and vein determination, two processes shown here to depend on D-mib gene activity. It is therefore concluded that D-mib and neur have distinct and complementary functions in Drosophila (Le Borgne, 2005).

A functional assay was then used to genetically position the requirement for the D-mib gene activity relative to Ser and N. Expression of an activated version of N, Ncdc10, led to the activation of Cut and promoted growth in dorsal cells of D-mib²/D-mib³ mutant discs. This indicates that D-mib acts at a step upstream of N activation. By contrast, elevated levels of Ser expression failed to restore Cut expression and growth of the wing pouch in D-mib²/D-mib³ mutant larvae. This confirms that Ser signaling requires the activity of the D-mib gene, i.e., that D-mib acts downstream of Ser (Le Borgne, 2005).

The different requirements for neur and D-mib gene activity may suggest that Neur and D-mib have distinct molecular activities. Alternatively, this difference may reflect a difference in gene expression. Consistent with the latter hypothesis, the neur gene is not expressed in wing pouch and wing margin cells, where it is not required, and appears to be expressed only in sensory cells, where it is required. By contrast, D-mib appears to be uniformly expressed in imaginal discs. To test this hypothesis, whether the forced ubiquitous expression of the neur gene can suppress the D-mib loss-of-function phenotype was examined. Expression of Neur, using actin-GAL4, restores growth of the wing pouch and formation of the wing margin. Moreover, expression of Neur in dorsal cells, using Ser-GAL4, is sufficient to rescue growth of the wing pouch as well as the expression of Cut in margin cells in D-mib mutant discs. It is concluded that ectopic expression of Neur compensates for the loss of D-mib activity (Le Borgne, 2005).

In a converse experiment, it was found that the neur-driven expression of D-mib, using neur^PGAL4, does not rescue the cuticular neurogenic phenotype of neur^PGAL4/neur^1F65 embryos. Three UAS-D-mib transgenic lines were tested, and none showed detectable rescue whereas the two UAS-neur lines used as positive controls either fully or partially rescued the cuticular neurogenic phenotype of neur^PGAL4/neur^1F65 embryos. This indicates that a key function of Neur in the embryo cannot be provided by D-mib. It is therefore suggested that Neur and D-mib functions overlap but are not strictly identical (Le Borgne, 2005).

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. A Drosophila ortholog of Mind-bomb (D-mib) has been shown in this study to be a positive component of Notch signaling that is required for multiple Neuralized-independent, Notch-dependent developmental processes. Furthermore, D-mib associates physically and functionally with both Serrate and Delta. 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. 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. It is concluded that ubiquitination of Delta and Serrate by Neuralized and D-mib is an obligate feature of DSL ligand activation throughout Drosophila development (Lai, 2005).

Mind-bomb was originally characterized in zebrafish through forward genetic studies of a novel locus that was absolutely required for Notch-mediated lateral inhibition of neural precursors. The presence of a clear Drosophila ortholog of Mind-bomb was somewhat of a surprise then, given that: (1) almost without exception, different species display similar functional requirements for evolutionarily conserved components of the Notch pathway; (2) this locus never emerged from any of the extensive Drosophila genetic screens for neurogenic genes and components of the Notch pathway; and (3) it is a large locus that might be expected to have been relatively easily hit, as has proven to be the case in zebrafish (Lai, 2005).

Although D-mib mutants have in fact been isolated, they are only very weakly neurogenic (Le Borgne, 2005; Melendez, 1995). This might partially explain how it was missed in earlier screens for components of the Notch pathway. By contrast, D-mib is absolutely required for the execution of several other Notch-regulated development events, including wing margin specification, eye growth and leg joint specification. Misexpression of full-length and dominant-negative truncations of D-mib affects Notch-mediated pattern formation even more broadly, including many settings that do not normally require D-mib. Biochemical and genetic experiments demonstrate that D-mib associates with both Drosophila DSL ligands, and promotes their internalization and signaling activity. However, dominant-negative D-mibDeltaRF binds Delta and Serrate but interferes with their normal trafficking and inhibits their signaling capacity. It is inferred that D-mibDeltaRF binding to both Delta and Serrate occludes endogenous Neur and D-mib from ubiquitinating and activating DSL ligands. This binding likely underlies the broad capacity of D-mibDeltaRF to inhibit Notch activation in virtually all settings of Notch signaling (Lai, 2005).

Curiously, NeurDeltaRF potentiates Delta signaling during wing margin induction just as full-length Neur does, even though ectopic NeurDeltaRF otherwise strongly inhibits Notch signaling. An explanation is lacking for this difference between NeurDeltaRF and D-mibDeltaRF, but it might hint at a functional difference between these DSL-regulating ubiquitin ligases. In almost every other regard, however, the activities and functions of D-mib/D-mibDeltaRF are highly reminiscent of Neur/NeurDeltaRF. In fact, D-mib can functionally replace Neur in a series of developmental decisions in vivo. Conversely, contemporaneous studies show that Neur can functionally replace D-mib during wing margin specification (Le Borgne, 2005). Nevertheless, the essential endogenous requirements for neur and D-mib are quite distinct, in that they are genetically required for different developmental processes and the respective mutants have differential effects on DSL ligands. Despite potent effects of ectopic D-mib on Delta localization and activity, Delta is mislocalized primarily only in neur mutant tissue, whereas Serrate is mislocalized primarily only in D-mib mutant tissue (Le Borgne, 2005; Lai, 2005).

This apparent specificity is unexpected, since D-mib is expressed ubiquitously, and is therefore present in all Delta-expressing cells. Does endogenous D-mib normally regulate Delta as implied by its ability to associate with Delta, induce Delta endocytosis, and potentiate Delta signaling activity? A close examination of D-mib mutants reveals certain phenotypes that are either stronger than those of Serrate mutants (i.e., leg truncation) or are more suggestive of Delta loss of function (i.e., wing vein deltas and a mildly neurogenic phenotype in the adult thorax) (Le Borgne, 2005). These observations collectively imply that another ubiquitin ligase may co-regulate Delta and thereby partially compensate for loss of D-mib. Neur is a possible, but relatively poor, candidate to supply this function. Although it has a demonstrated role in regulating Delta, neur expression in imaginal tissue is restricted mostly to neural precursors and photoreceptors. A more tantalizing candidate is D-mibl (CG17492), which may also prove to regulate DSL ligands. In support of this, systematic yeast two-hybrid screening has identified a specific interaction between D-mibl and Delta. Therefore, the in vivo function of D-mibl with regard to the regulation of DSL ligands deserves future investigation (Lai, 2005).

Both Drosophila DSL ligands are regulated by ubiquitin ligases that promote ligand endocytosis. Still, the mechanism by which endocytosis promotes DSL ligand activity is still unclear. An earlier proposal was that Delta endocytosis might facilitate Notch proteolytic processing by helping to unmask the S2 Notch cleavage site. Other models suggested that ligand endocytosis might promote ligand clustering or clearance of extracellular N^ECD. Most recently, genetic studies of the epsin Liquid facets (Lqf), an apparently DSL ligand-specific endocytic component, have led to further insight into this mechanism. In particular, a provocative model was put forth suggesting that Lqf directs Delta into an endocytic recycling compartment, and that Delta recycling back to the plasma membrane is a prerequisite for ligand activation. The finding that Serrate is similarly regulated by endocytosis via D-mib suggests further avenues for testing this model. For example, it will be informative to ask whether lqf shows defects in Serrate trafficking, or if the requirement of Serrate for D-mib can be bypassed by shunting it through an endocytic recycling pathway (Lai, 2005).

Even though Neur and D-mib promote DSL ligand activity by stimulating ligand endocytosis, they also efficiently induce ligand degradation. This might conceptually be at odds with the proposition that ligand recycling back to the plasma membrane underlies DSL ligand activation. These activities might be reconciled if ubiquitination permits a portion of the DSL ligand pool to enter the select Lqf-mediated recycling pathway, but directs the bulk of DSL ligands for degradation. Consistent with this, Lqf is strictly required for DSL ligand activation, but is not required for bulk endocytosis of DSL ligands . If ubiquitination is prerequisite for DSL ligand activation but also makes DSL ligands prone to degradation, this would prevent endless recycling of activated ligands and thereby limit the temporal extent of Notch pathway activation. The strategy of coupling activation with downregulation is seen with Notch itself. Ligand-induced Notch cleavage liberates activated Notch^intra, which is a potent regulator of gene expression as a nuclear co-activator for Su(H). However, nuclear Notch^intra also becomes a substrate for ubiquitination by the ubiquitin ligase Sel-10, and is rapidly degraded. Coupled activation and downregulation allows for precise temporal control of signaling by limiting the lifetime of activated signaling components (Lai, 2005).

The presence of Neur and Mib homologs in both fly and vertebrate genomes suggests that both proteins were present and regulated DSL ligands in the ancient common ancestor of these species. There is a surprising functional overlap between these structurally unrelated ubiquitin ligases in regulating DSL ligand activity. What, then, was the rationale of evolving such different proteins to perform the same function? The genetic implication that Neur and D-mib preferentially regulate Delta and Serrate, respectively, belies the ability of these enzymes to interact with and regulate the localization and signaling activity both DSL ligands. While it remains to be seen whether Neur regulates Serrate in addition to its documented substrate Delta, D-mib directly and efficiently regulates both Delta and Serrate. Therefore, these ubiquitin ligases did not obviously co-evolve with different classes of DSL ligands (Lai, 2005).

Another possible explanation lies in the curious observation that Neur is genetically required mostly in settings that involve 'lateral inhibitory' Notch signaling, wherein Notch restricts a cell fate among equipotent cells. By contrast, D-mib is required largely in settings that involve 'inductive' Notch signaling, which occurs between non-equivalent cell populations. This apparent division of labor raises the possibility that different ubiquitin ligases could help to specify the appropriate response to Notch activation in each developmental setting (Lai, 2005).

This hypothesis, however, is not particularly supported by the observations that D-mib and Neur can functionally replace each other in a variety of processes. Neither does this correlation hold up in other species, because Neur mediates lateral inhibition of neural precursors in flies, whereas Mib mediates lateral inhibition of neural precursors in fish. This latter finding highlights the plasticity in how these ubiquitin ligases have been deployed during evolution, and is consistent with a model in which fish Mib has subsumed the function of fly Neur during neurogenesis (or vice versa). This may have occurred by appropriate changes in the transcriptional regulation of these genes. Given this likely scenario, one wonders whether it might not have been more evolutionarily expedient to have diversified the function of duplicated, paralogous genes. There are indeed multiple neur and mib genes in vertebrates, and two mib genes in flies. Of course, it might be argued that a similar conundrum concerns the co-existence of the HECT domain and the RING finger/U box as unrelated protein domains that both catalyze E3 ubiquitin ligation (Lai, 2005).

How general is the requirement for DSL ligand endocytosis across evolution? The neurogenic mutant phenotypes of Drosophila neur and zebrafish mib, along with the involvement of Xenopus neur in lateral inhibition, show that DSL ligand ubiquitination is required in both invertebrates and vertebrates. However, thorough loss-of-function genetic studies are incomplete in any organism and are complicated by the duplication of neur and/or mib genes. For example, knockout of murine Neur1 does not affect Notch signaling, possibly due to functional overlap with Neur2 (Lai, 2005).

The present work clearly demonstrates that the vast majority of Notch-regulated settings during Drosophila development are strictly dependent on either Neur or D-mib. Thus, DSL ligand ubiquitination and endocytosis appears to be obligate in Drosophila. In light of this, the situation in nematodes provides an interesting possible counter-example. C. elegans lacks a recognizable Mind-bomb ortholog, but does possess a single Neur gene. However, in contrast to what has been found in Drosophila, where intracellular deletions of the DSL ligands have a dominant-negative activity, the extracellular domains of the DSL ligands LAG-2 and APX-2 can fully rescue the lag-2 mutant and can activate Notch signaling ectopically. A more recent analysis actually revealed a large family of putative secreted DSL ligands in the worm, at least one of which (DSL-1) is a bona fide DSL ligand. This suggests that nematodes may have dispensed with ubiquitination and endocytosis of DSL ligands in at least some settings of Notch signaling. Nevertheless, a nematode ortholog of epsin/Lqf (Ce-epn-1) participates in Notch signaling during germline development. The functional relationships amongst Ce-epn-1, nematode DSL ligands and any potential DSL-regulating E3s in nematodes remain to be determined (Lai, 2005).

Distinct roles for Mind-bomb, Neuralized and Epsin in mediating DSL endocytos 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 (Liquid facets), a conserved endocytic adaptor thought to target mono-ubiquitinated membrane proteins for internalization. Evidence is presented 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, both Dmib and Epsin are required in these cells for some of the endocytic events that internalize DSL ligands, and 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 the 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 (Wang, 2005).

To date, two E3 ubiquitin ligases have been implicated in DSL signaling: zebrafish Mind-bomb (Mib) and Drosophila Neuralized (Neur). Both proteins have been shown to promote DSL ubiquitination, endocytosis and signaling. Moreover, loss-of-function mutations in zebrafish mib and Drosophila neur cause essentially the same hallmark phenotype exemplifying a failure of DSL-signaling in their respective organisms; namely, a dramatic hyperplasia of the embryonic nervous system at the expense of the epidermis. These observations have led to the suggestion that zebrafish Mib and Drosophila Neur are functional homologs. Yet, the two proteins show only limited sequence homology; moreover they appear to be members of distinct Mib and Neur ubiquitin ligase families, each having true orthologs in both vertebrate and invertebrate genomes. As a consequence, the relative roles of Mib and Neur are not known in any animal system and this uncertainty complicates the use of mutations in these genes to assay the role of ubiquitination in DSL endocytosis and signaling (Wang, 2005).

Using newly isolated mutations in dmib, evidence has been found that Dmib and Neur constitute functionally related ubiquitin ligases that are normally required for DSL signaling in different developmental contexts. In the developing Drosophila wing disc, Dmib is required for inductive signaling across the D-V compartment boundary, as well as for the refinement of wing vein primordia, both contexts in which Neur is normally not required or expressed. However, Dmib plays only a modest role in specifying sensory organ precursor (SOP) cells, and little or no role in the subsequent segregation of distinct cell types that form each sensory organ. Instead, Neur appears to provide the essential ubiquitin ligase activity required for DSL signaling in these latter two contexts. Similarly, in the embryo, where Neur is required for most DSL signaling events, Dmib has little or no apparent role. Indeed, embryos devoid of Dmib activity hatch as viable first instar larvae; moreover they develop into pharate adults that show only a limited subset of Notch-related mutant phenotypes, each of which appears to reflect the failure of a particular DSL signaling event that does not, normally, depend on Neur. Thus, it is inferred that Dmib and Neur share a common ubiquitin ligase activity that is essential for DSL ligands to signal (Wang, 2005).

Le Borne (2005) has published similar findings indicating a role for Dmib in DSL signaling, and the capacity of ectopic Neur to substitute for Dmib during wing development. The results differ, however, in that this analysis of clones of dmib^- cells that express Ser, Dl, or the Dl^R+ chimera appears to indicate an absolute requirement for Dmib in sending both Drosophila DSL signals, Dl and Ser. By contrast, Le Borgne interprets his data as evidence for a regulatory rather than an obligatory role of Dmib in sending DSL signals, as well as for a lesser role of Dmib in Dl signaling compared with Ser signaling. Differences in experimental design, particularly in the means used to define or infer the identity of DSL signaling and Dmib-deficient cells, could account for the different conclusions reached (Wang, 2005).

It is noted that if Mib and Neur ligases have overlapping molecular functions in all animal systems, as they have in Drosophila, there is no compelling reason why the ligases would need to be deployed in the same way in different animal species. Instead, any given DSL-signaling process might depend on Mib in one animal system but on Neur in another, as appears to be the case for neurogenesis in zebrafish and Drosophila (Wang, 2005).

In contrast to the selective requirement for Dmib and Neur in overlapping subsets of DSL signaling contexts, Epsin is required for most or all DSL signaling events. This difference is expected if ubiquitination of DSL ligands by either Dmib or Neur is normally a prerequisite for Epsin-mediated endocytosis, and hence for signaling activity (Wang, 2005).

Do Dmib and Neur directly bind and ubiquitinate DSL ligands and thereby confer signaling activity by targeting them for Epsin-mediated endocytosis? Although ectopic Dmib and Neur activity are both associated with enhanced DSL ubiquitination, endocytosis and signaling, there is still no compelling evidence that either ligase directly binds and ubiquitinates DSL proteins, or that Dmib/Neur-dependent ubiquitination of DSL ligands confers signalng activity. However, this study shows that the obligate requirement for Dmib for Dl-signaling by wing cells can be bypassed by replacing the cytosolic domain of Dl with a random peptide, R⁺, that may serve as the substrate for ubiquitination by an unrelated ubiquitin ligase. This result provides in vivo evidence that Dmib/Neur-dependent ubiquitination of DSL ligands is normally essential to confer signaling activity. Moreover, the failure of the chimeric Dl^R+ ligand to bypass the requirment for Epsin, supports the interpretation that ubiquitination of DSL ligands confers signaling activity because it targets them for Epsin-mediated endocytosis (Wang, 2005).

During wing development, Ser and Dl both serve as unidirectional signals that specify the 'border' cell fate in cells across the D-V compartment boundary, and both are found to be equally dependent on Dmib and Epsin function for signaling activity. However, the two ligands differ in the extent to which they accumulate on the cell surface, and to which they are cleared from the surface as a consequence of Dmib and Epsin activity. Specifically, most Ser accumulates in cytosolic puncta rather than on the cell surface, whereas the reverse is the case for Dl. Furthermore, removing either Dmib or Epsin activity results in a dramatic and abnormal retention of Ser on the cell surface, whereas it has no detectable effect on the surface accumulation of Dl. Similar results for Dmib were also obtained by Le Borgne (2005). Thus, it appears that most Ser is efficiently cleared from the cell surface by the actions of Dmib and Epsin, whereas most Dl remains on the cell surface, irrespective of Dmib and Epsin activity. This unexpected difference provides two insights (Wang, 2005).

(1) In a previous analysis of the role of Epsin, focus was placed almost exclusively on Dl endocytosis and signaling and failed to obtain direct evidence that Epsin is required for normal DSL endocytosis, despite the obligate role for Epsin in sending both Dl and Ser signals. Instead, such a requirement could be detected only in experiments in which surface clearance of over-expressed Dl was abnormally enhanced by ectopically co-expressing Neur, or could only be inferred from experiments in which the requirement for Epsin was bypassed by replacing the cytosolic domain of Dl with the well-characterized endocytic recycling signal from the mammalian low density lipoprotein (LDL) receptor. By contrast, the different endocytic behavior of Ser has now allowed direct evidence to be obtained that Dmib and Epsin are both required for normal DSL endocytosis (Wang, 2005).

(2) It was found that even though bulk endocytosis of Ser depends on both Dmib and Epsin activity, neither requirement appears absolute. Instead, the accumulation of Ser can still be detected in cytosolic puncta in both Dmib- and Epsin-deficient cells. Moreover, a difference is detected in the abnormal cell surface accumulation of Ser in Dmib-deficient versus Epsin-deficient cells; significantly more Ser appears to accumulate in the absence of Dmib than in the absence of Epsin. It is inferred that both Dl and Ser are normally internalized by multiple endocytic pathways, only some of which depend on ubiquitination of the ligand, and only a subset of these that depends on Epsin. However, the two ligands normally utilize these pathways to different extents, most Ser being internalized by ubiquitin- and Epsin-dependent pathways, and most Dl being internalized by alternative pathways. It is presumed that this difference reflects the presence of different constellations of internalization signals in the two ligands, especially the presence of signals in Delta, but not Ser, that target the great majority of the protein for internalization pathways that do not depend on ubiquitination or Epsin. Nevertheless, only those molecules of Ser and Dl that are targeted by ubiquitination to enter the Epsin-dependent pathway have the capacity to activate Notch; all other routes of entry that are normally available appear to be non-productive in terms of signaling. These results reinforce previous evidence that endocytosis of DSL ligands, per se, is not sufficient to confer signaling activity; instead, DSL ligands must normally be internalized via the action of Epsin to signal (Wang, 2005).

Why must DSL ligands normally be internalized by an Epsin-dependent endocytic mechanism to activate Notch? Two general classes of explanation are considered. In the first, Epsin confers signaling activity by regulating an early event in DSL endocytosis that occurs before internalization. For example, Epsin might cluster DSL ligands in a particular way or recruit them to a select subset of coated pits or other endocytic specializations. Alternatively, Epsin-mediated invagination of these structures might control the physical tension across the ligand/receptor bridge linking the sending and receiving cell, creating a sufficiently strong or special mechanical stress necessary to induce Notch cleavage or ectodomain shedding. In the second class of models, Epsin acts by regulating a later event in DSL endocytosis that occurs after internalization. For example, Epsin might direct, or accompany, DSL proteins into a particular recycling pathway that is essential to convert or repackage them into ligands that can activate Notch upon return to the cell surface. In both cases, internalization of DSL ligands via the other endocytic routes normally available to them would not provide the necessary conditions, even in the extreme case of Dl, which appears to be internalized primarily by these other pathways (Wang, 2005).

The present results do not distinguish between these models. However, recent studies of Epsin-dependent endocytosis in mammalian tissue culture cells suggest that Epsin may direct cargo proteins to different endocytic specializations or pathways, depending on their state of ubiquitination. They also suggest that interactions between Epsin and components of the core Clathrin endocytic machinery normally regulate where and how Epsin internalizes target proteins. Both properties might govern how DSL proteins are internalized, allowing the ligands to gain access to the select endocytic pathway they need to enter to activate Notch (Wang, 2005).

The interplay between Delta and Serrate proteins and ubiquitin ligases in Notch signaling

Lateral inhibition is a pattern refining process that generates single neural precursors from a field of equipotent cells and is mediated via Notch signaling. Of the two Notch ligands Delta and Serrate, only the former was thought to participate in this process. It is shown in this study that macrochaete lateral inhibition involves both Delta and Serrate. In this context, Serrate interacts with Neuralized, a ubiquitin ligase that was heretofore thought to act only on Delta. Neuralized physically associates with Serrate and stimulates its endocytosis and signaling activity. A mutation was characterized in mib1, a Drosophila homolog of zebrafish mind-bomb, another Delta-targeting ubiquitin ligase. Mib1 affects the signaling activity of Delta and Serrate in both lateral inhibition and wing dorsoventral boundary formation. Simultaneous absence of neuralized and mib1 completely abolishes Notch signaling in both aforementioned contexts, making it likely that ubiquitination is a prerequisite for Delta/Serrate signaling (Pitsouli, 2005).

Until now, it was thought that lateral inhibition in notum SOPs was solely mediated via Dl and that Dl transcriptional upregulation in the nascent neural precursor was crucial for a Dl-N negative feedback loop to establish the neural precursor fate within a group of equivalent cells. These data have refuted both of these models, because endogenous Ser has now been shown to participate in lateral inhibition of macrochaete SOPs and either Dl or Ser uniformly expressed is able to produce a wild-type pattern of macrochaetes. Dl transcriptional upregulation in the absence of Notch signaling in proneural fields does occur, but this modulation does not appear to be a prerequisite for the specification of the wild-type neural precursor, at least in the case of macrochaetes and embryonic neuroblasts. It is possible that the genetically detected N-Dl negative feedback loop may reflect Dl and N activity rather than transcription, although a transcriptional input has been documented. An exciting possibility, given the reliance of DSL activity on ubiquitin ligases, is that this feedback loop targets transcription of neur, rather than Dl. mib1 is an unlikely target as since shows no transcriptional modulation within proneural regions (Pitsouli, 2005).

Although Neur was known to affect Dl localization and function in some instances, ubiquitin ligases were not considered as essential components of Notch signaling. The characterization of Mib1 described here and in recent papers (Lai, 2005; Le Borgne, 2005; Wang, 2005) points to a much more prominent role of these factors. mib1 appears to be required in a large number of Notch-dependent processes where neur is not expressed, e.g., the wing DV boundary. The fact that mib1 neur double mutants appear to lose all ability to perform lateral inhibition strongly supports the hypothesis that Ub ligases may always be required for Dl/Ser signaling. A comprehensive survey of Notch-dependent events with respect to neur and mib1 will test this hypothesis and may uncover additional E3 ligases with this activity; Mib2 represents a potential candidate (Pitsouli, 2005).

The intimate relation between Neur/Mib1 and DSL proteins is generally assayed in three ways: (1) physical association, (2) effects on Dl/Ser endocytosis and (3) effects on Dl/Ser signaling. All of these had been well documented for the Neur-Dl combination and, more recently, for the Mib1-Dl and Mib1-Ser combinations (Lai, 2005; Le Borgne, 2005; Wang, 2005). In the present work the final pair, Neur-Ser, has been added, using all of the above assays. The conclusion, stated simply, is that both Neur and Mib1 associate with and affect the endocytosis and function of both Dl and Ser (Pitsouli, 2005).

Ubiquitination of transmembrane proteins tags them for endocytosis, using a complex of adaptors, including epsin, which carry ubiquitin recognition domains. The simplest scenario for the role of Neur/Mib1 in Dl/Ser signaling would be that they attach ubiquitin to Dl/Ser to trigger endocytosis. Signaling would ensue, either as a consequence of recruiting/clustering ubiquitinated DSL cargo to specialized plasma membrane domains conducive to signaling, or by more elaborate routes involving DSL protein recycling through the endocytic pathway as a prerequisite for their modification/activation (Pitsouli, 2005).

Alternatively, Neur/Mib1 need not ubiquitinate the DSL proteins directly. In the ubiquitin-dependent endocytosis pathway, many of the adaptor proteins are themselves ubiquitinated, possibly favoring the formation of interconnected cargo-adaptor complexes; Neur/Mib1 could have one or more of the adaptors, including themselves, as substrates. DSL protein chimaeras become Mib1 independent if their intracellular domains are substituted with ones bearing alternative internalization motifs (Wang, 2005). Of two such artificial Mib1-independent versions of Dl, one is ubiquitination/epsin-independent (Dl-LDL-receptor fusion), whereas the other (Dl-random-peptide-R fusion) still curiously requires ubiquitination/epsin for activity (Wang, 2004). Nothing is yet known about the native Dl/Ser intracellular domains, other than the puzzling fact that they are neither similar nor evolutionarily conserved, despite apparent conservation of recognition by Neur/Mib (Pitsouli, 2005).

An even more puzzling observation in the light of this model is that some DSL proteins in C. elegans appear to be secreted. Secreted mutants of Drosophila Dl and Ser act as Notch antagonists, consistent with a requirement for endocytosis in DSL signaling. Even C. elegans LAG-2 (a transmembrane DSL) needs EPN-1 (epsin ortholog), in order to signal to GLP-1 (Notch-like) during germline differentiation, which is hard to reconcile with secreted DSL proteins. Apparently, ubiquitination/endocytosis can be bypassed in some contexts, allowing secreted DSL proteins to signal via a yet unknown process (Pitsouli, 2005).

Whatever the molecular details and variations turn out to be, it is becoming clear that ubiquination plays a prominent role in Notch signaling, in both sending and receiving cells. In the latter, Ub ligases downregulate Notch activity either at the membrane or in the nucleus. Besides downregulation, however, Notch ubiquitination is also needed for activation: ubiquitination apparently targets Notch to a compartment where it can be activated by gamma-secretase cleavage. How two ubiquitination/trafficking events, activating DSL proteins in one cell and Notch in another, might be coordinated across the extracellular space is a mystery worth investigating in the future (Pitsouli, 2005).

Neuronal morphogenesis is regulated by the interplay between cyclin-dependent kinase 5 and the ubiquitin ligase mind bomb 1

Neuronal communication requires the coordinated assembly of polarized structures including axons, dendrites, and synapses. This study reports the identification of a ubiquitin ligase mind bomb 1 (Mib1) in the postsynaptic density and the characterization of its role in neuronal morphogenesis. Expression of Rat Mib1 inhibits neurite outgrowth in cell culture and its gene deletion enhances synaptic growth at the neuromuscular junction in Drosophila. The analysis of Rat Mib1 interactome by mass spectrometry revealed that Mib1 primarily interacts with membrane trafficking proteins [e.g., EEA1 (early endosomal antigen 1), Rab11-interacting proteins, and SNAP25 (synaptosomal-associated protein of 25 kDa)-like protein] and cell adhesion components (e.g., catenin, coronin, dystrobrevin, and syndecan), consistent with its previously reported function in protein sorting. More interestingly, Mib1 is associates with deubiquitinating enzymes, BRCC36 and the mammalian ortholog of fat facets, and a number of kinases, such as casein kinase II, MARK (microtubule affinity regulating kinase)/PAR1, and cyclin-dependent kinase 5 (CDK5). Further characterization of the Mib1-CDK5 interaction indicated that the N-terminal domain of Mib1 directly binds to the regulatory subunit p35 of the CDK5 complex. In cell culture, Mib1 induces the relocalization of p35/CDK5 without affecting its degradation. Surprisingly, p35/CDK5 downregulates the protein level of Mib1 by its kinase activity, and completely rescues the Mib1-induced inhibitory effect on neurite morphology. p35/CDK5 also genetically interacts with Mib1 in the fly according to the rough-eye phenotype. The data strongly support that the negative interplay between Mib1 and p35/CDK5 may integrate the activities of multiple pathways during neuronal development (Choe, 2007; full text of article).

A Drosophila model with Mib1 loss-of-function was used to examine its physiological role and interaction with p35/CDK5. A P-element insertion was identified in the fly gene of CG5841, the Drosophila ortholog of Mib1. The homozygote was affirmed to be a mib1-null allele, because it showed pupal lethality that was rescued by precise removal of the transposon in the 5' untranslated region of the gene, or by the expression of transgenic mib1. The lack of full-length mib1 expression in the allele was verified by Western blotting. In Drosophila, the synaptic structure at the larval neuromuscular junction (NMJ) is a well defined system with which to study synaptic structure and neurotransmission, and the number of synaptic boutons is an established index for synaptic growth. By counting synaptic boutons in the wild-type and the mutant larvae, it was found that the loss of mib1 causes the synaptic overgrowth, and the number of synaptic boutons increased 85%. This finding suggests that Mib1 plays a conserved negative role in the formation of synaptic structure (Choe, 2007).

The genetic interaction between p35/CDK5 and mib1 was tested by monitoring adult eye phenotype. Overexpression of p35/CDK5 causes a rough eye phenotype, whereas the heterozygotic line of the mib1 mutant had a smooth eye phenotype. Crossing the p35/CDK5 transgenic line with the mib1 mutant line showed that the partial loss of mib1 in the heterozygote enhances the rough eye phenotype induced by p35/CDK5. This result also supports the negative regulation between p35/CDK5 and Mib1 in neuronal development (Choe, 2007).

EVOLUTIONARY HOMOLOGS

Mind bomb is a ubiquitin ligase that is essential for efficient activation of Notch signaling by Delta

Lateral inhibition, mediated by Notch signaling, leads to the selection of cells that are permitted to become neurons within domains defined by proneural gene expression. Reduced lateral inhibition in zebrafish mib mutant embryos permits too many neural progenitors to differentiate as neurons. Positional cloning of mib revealed that it is a gene in the Notch pathway that encodes a RING ubiquitin ligase. Mib interacts with the intracellular domain of Delta to promote its ubiquitylation and internalization. Cell transplantation studies suggest that mib function is essential in the signaling cell for efficient activation of Notch in neighboring cells. These observations support a model for Notch activation where the Delta-Notch interaction is followed by endocytosis of Delta and transendocytosis of the Notch extracellular domain by the signaling cell. This facilitates intramembranous cleavage of the remaining Notch receptor, release of the Notch intracellular fragment, and activation of target genes in neighboring cells (Itoh, 2003).

Analysis of Mind bomb in zebrafish: Three modules of zebrafish Mind bomb work cooperatively to promote Delta ubiquitination and endocytosis

Precise regulation of Notch signaling activity is critical for development of many different tissues. The zebrafish insertional mutation Hi904 attenuates Notch signaling, and is allelic to mind bomb. Mind bomb protein displays E3 ubiquitin ligase activity in vitro, and it is associated with Delta and enhances its ubiquitination and internalization in transfected cells. By functional analysis of three conserved regions of Mind bomb, it is shown that the N-terminal half is required for Delta association, the ankyrin repeats are important for Delta internalization, and the ring fingers are required for Delta ubiquitination. Thus, the three functionally distinct modules of Mind bomb work cooperatively to regulate Notch signaling by associating with, ubiquitinating, and internalizing Delta (Chen, 2004).

The role of the SPT6 chromatin remodeling factor in zebrafish embryogenesis

Somitogenesis is a highly controlled process that results in segmentation of the paraxial mesoderm. Notch pathway activity in the presomitic mesoderm is fundamental for management of synchronized gene expression which is necessary for regulation of somitogenesis. An embryonic lethal mutation, SBU2, was isolated that causes somite formation defects very similar to Notch pathway mutants. SBU2 mutants generate only 6-7 asymmetrically arranged somites. However, in contrast to Notch pathway mutants, these mutants do not maintain previously formed somite boundaries and by 24 hpf, almost no somite boundaries remain. Other developmental processes disrupted in SBU2 mutants include tail morphogenesis, muscle fiber elongation, pigmentation, circulatory system development and neural differentiation. These defects are the result of a nonsense mutation within the spt6 gene (see Drosophila Spt6). spt6 encodes a transcription elongation factor that genetically interacts with the Paf-1 chromatin remodeling complex. SBU2 mutant phenotypes could be rescued by microinjection of spt6 mRNA and microinjection of spt6 morpholinos phenocopied the mutation. Real-time PCR analysis revealed that Spt6 is essential for the transcriptional response to activation of the Notch pathway. Analysis of sbu2;mib double mutants indicates that Spt6 deficiency suppresses the neurogenic effects of the mib. Altogether, these results demonstrate that Spt6 is critical for somite formation in zebrafish and suggest that some defects observed in spt6 mutants result from alterations in Notch signaling. However, additional Spt6 mutant phenotypes are likely caused by vital functions of Spt6 in other pathways (Kok, 2007).

Delta-Notch signalling and the patterning of sensory cell differentiation in the zebrafish ear: evidence from the mind bomb mutant

Mechanosensory hair cells in the sensory patches of the vertebrate ear are interspersed among supporting cells, forming a fine-grained pattern of alternating cell types. Analogies with Drosophila mechanosensory bristle development suggest that this pattern could be generated through lateral inhibition mediated by Notch signalling. In the zebrafish ear rudiment, homologues of Notch are widely expressed, while the Delta homologues deltaA, deltaB and deltaD, coding for Notch ligands, are expressed in small numbers of cells in regions where hair cells are soon to differentiate. This suggests that the delta-expressing cells are nascent hair cells, in agreement with findings for Delta1 in the chick. According to the lateral inhibition hypothesis, the nascent hair cells, by expressing Delta protein, would inhibit their neighbours from becoming hair cells, forcing them to be supporting cells instead. The zebrafish mind bomb mutant has abnormalities in the central nervous system, somites, and elsewhere, diagnostic of a failure of Delta-Notch signalling: in the CNS, it shows a neurogenic phenotype accompanied by misregulated delta gene expression. Similar misregulation of delta genes is seen in the ear, along with misregulation of a Serrate homologue, serrateB, coding for an alternative Notch ligand. Most dramatically, the sensory patches in the mind bomb ear consist solely of hair cells, which are produced in great excess and prematurely; at 36 hours post fertilization, there are more than ten times as many as normal, while supporting cells are absent. A twofold increase is seen in the number of otic neurons also. The findings are strong evidence that lateral inhibition mediated by Delta-Notch signalling controls the pattern of sensory cell differentiation in the ear (Haddon, 1998).

Analysis of Mind bomb in zebrafish: Delta-Notch signaling regulates oligodendrocyte specification

Oligodendrocytes, the myelinating cell type of the central nervous system, arise from a ventral population of precursors that also produces motoneurons. Although the mechanisms that specify motoneuron development are well described, the mechanisms that generate oligodendrocytes from the same precursor population are largely unknown. By analyzing mutant zebrafish embryos, it has been found that Delta-Notch signaling is required for spinal cord oligodendrocyte specification. Using a transgenic, conditional expression system, it was also learned that constitutive Notch activity promotes formation of excess oligodendrocyte progenitor cells (OPCs). However, excess OPCs are induced only in ventral spinal cord at the time that OPCs normally develop. These data provide evidence that Notch signaling maintains subsets of ventral spinal cord precursors during neuronal birth and, acting with other temporally and spatially restricted factors, specifies them for oligodendrocyte fate (Park, 2003).

Because mouse embryos that are homozygous for null mutations of Delta or Notch genes die at early stages ofeural development, there is little information that addresses the requirement of Notch signaling for vertebrate CNS glial specification. This limitation can be circumvented through analysis of mice in which Notch1 is conditionally inactivated in the cerebellum. These mice prematurely express neuronal markers and have reduced number of mutant cerebellar cells that express the glial marker GFAP. In an alternative approach, neurospheres can derived from Delta-like 1 mutant mice. After culturing, mutant neurospheres produce excess neurons and a deficit of oligodendrocytes and astrocytes compared with controls. Additionally, retinas of mice that are homozygous for a mutation of Hes5, which encodes a downstream effector of Notch signaling, have fewer Müller glia than the wild type. These observations are consistent with the idea that Delta-Notch signaling regulates neuronal-glial fate decisions (Park, 2003).

Several lines of evidence point toward a role for Delta-Notch signaling in regulating specification of motoneuron and oligodendrocyte fates in zebrafish. (1) Prospective primary motoneurons are usually replaced when they are removed at the 11-somite stage. This is similar to observations that ablated neuroblasts were replaced by neighboring cells in grasshoppers and raises the possibility that primary motoneurons, like grasshopper neuroblasts, inhibit neighboring precursors from adopting the same fate. (2) Prospective primary motoneurons expressed higher levels of the two Delta-related genes dla and dld than neighboring cells, indicating that Notch ligands are present at the right time and place to regulate specification of cells that arise in close proximity to primary motoneurons. (3) Mutant zebrafish that had reduced levels of Notch signaling had excess primary motoneurons and a concomitant deficit of later-born secondary motoneurons, showing that Delta-Notch signaling regulates specification of neural precursors for different neuronal fates. Finally, medial neural plate cells, which occupy ventral spinal cord upon completion of neurulation, give rise to primary motoneurons and oligodendrocytes. Thus, Delta proteins expressed by primary motoneurons can regulate specification of nearby cells for oligodendrocyte fate (Park, 2003).

dla^-/-;dld^-/- and mib^-/- embryos [mind bomb (mib)] encodes a ubiquitin ligase necessary for efficient Notch signaling] do not produce OPCs or premyelinating oligodendrocytes. Additionally, neural precursors prematurely exit the cell cycle and differentiated as neurons in these embryos. Since secondary motoneurons and oligodendrocytes arise after primary motoneurons, one interpretation of the data is that Notch signaling prevents a subset of ventral spinal cord precursors from developing as primary motoneurons, enabling them to take later neuronal or oligodendrocyte fates. In this view, downregulation of delta gene expression during primary motoneuron differentiation would result in a decrease of Notch activity in neighboring precursors. A release from Notch-mediated inhibition soon after primary motoneuron specification might allow a cell to develop as a secondary motoneuron, whereas a later release might result in oligodendrocyte development. Thus, temporal regulation of Notch signaling might underlie the temporal switch in production of primary motoneurons to secondary motoneurons to oligodendrocytes (Park, 2003).

A switch between production of neurons and glial cells has been proposed to be regulated by bHLH proteins. In the ventral spinal cord, motoneuron and oligodendrocyte precursors expressed Olig bHLH proteins, which are structurally similar to proneural Ngns. During the period of motoneuron production, a subset of Olig⁺ cells expressed Ngns. Later, Ngn expression subsides, coincident with the time at which oligodendrocytes are thought to be specified. These observations, coupled with various functional tests, led to the proposal that Ngn and Olig proteins create a simple bHLH protein code in which Ngn and Olig expression together specify motoneuron development and Olig alone, upon Ngn downregulation, specifies oligodendrocyte development (Park, 2003).

The data provide evidence supporting the importance of a bHLH protein code to motoneuron and oligodendrocyte specification and show that Delta-Notch signaling is required to establish the code. The failure to restrict ngn1 expression to a subset of medial neural plate cells in Notch signaling deficient zebrafish embryos correlates with formation of excess neurons, consistent with observations that Notch signaling inhibits proneural genes expression and neuronal development in vertebrate and invertebrate embryos. Furthermore, dla^-/-;dld^-/- and mib^-/- embryos fail to maintain a proliferative population of olig2⁺ cells. This is interpreted to mean that, in the absence of Delta-Notch mediated inhibition, uniformly high levels of Ngns cause all olig2⁺ neural precursors to stop dividing and differentiate as neurons at the expense of oligodendrocytes. Thus, in normal embryos, high levels of Notch activity prevents ngn gene expression in a subset of olig2⁺ neural precursors, reserving them to produce other cell types, such as oligodendrocytes, at a later time. In this view, Delta-Notch signaling might play a purely permissive role in neural cell fate diversification, by regulating the ability of neural precursors to respond to other instructive signals (Park, 2003).

Analysis of Mind bomb in zebrafish: Neurogenic phenotype of mind bomb mutants leads to severe patterning defects in the zebrafish hindbrain

Failure of Notch signaling in zebrafish mind bomb mutants results in a neurogenic phenotype where an overproduction of early differentiating neurons is accompanied by the loss of later-differentiating cell types. The hindbrain phenotype of mib mutants has been characterized in detail. Hindbrain branchiomotor neurons (BMNs) are reduced in number but not missing in mib mutants. In addition, BMN clusters are frequently fused across the midline in mutants. Mosaic analysis indicates that the BMN patterning and fusion defects in the mib hindbrain arise non-cell autonomously. Ventral midline signaling is defective in the mutant hindbrain, in part due to the differentiation of some midline cells into neural cells. Interestingly, while early hindbrain patterning appears normal in mib mutants, subsequent rhombomere-specific gene expression is completely lost. The defects in ventral midline signaling and rhombomere patterning are accompanied by an apparent loss of neuroepithelial cells in the mutant hindbrain. These observations suggest that, by regulating the differentiation of neuroepithelial cells into neurons, Notch signaling preserves a population of non-neuronal cells that are essential for maintaining patterning mechanisms in the developing neural tube (Bingham, 2003).

Analysis of Mind bomb in zebrafish: Notch activation regulates the segregation and differentiation of rhombomere boundary cells in the zebrafish hindbrain

During segmentation of the vertebrate hindbrain, a distinct population of boundary cells forms at the interface between each segment. Little is known regarding mechanisms that regulate the formation or functions of these cells. A potential role of Notch signaling has been investigated; in the zebrafish hindbrain, radical fringe is expressed in boundary cells and delta genes are expressed adjacent to boundaries, consistent with a sustained activation of Notch in boundary cells. Mosaic expression experiments reveal that activation of the Notch/Su(H) pathway regulates cell affinity properties that segregate cells to boundaries. In addition, Notch signaling correlates with a delayed neurogenesis at hindbrain boundaries and is required to inhibit premature neuronal differentiation of boundary cells. These findings reveal that Notch activation couples the regulation of location and differentiation in hindbrain boundary cells. Such coupling may be important for these cells to act as a stable signaling center (Cheng, 2004).

Studies of neurogenesis in the zebrafish hindbrain have shown that differentiation first occurs at rhombomere centers, and only at late stages are neurons formed at the boundaries between rhombomeres. The spatial and temporal pattern of neurogenesis is reflected by the expression of delta genes that mark early neuroblasts: expression is excluded from rhombomere boundaries, and by 24 hr occurs in stripes adjacent to the boundaries. These observations are consistent with Delta mediating a lateral inhibition in a manner analogous to its widely utilized role in the neural epithelium, in which Delta expression by early neuroblasts activates Notch and suppresses neurogenesis and delta expression in adjacent cells. Indeed, ectopic expression of dominant-active Su(H) suppresses delta expression throughout the hindbrain. An important role of the lateral inhibition of neurogenesis is to maintain the progenitor pool of neural epithelial cells that is required for the continued generation of neurons. mind bomb (mib) mutant embryos have a strong Notch pathway deficiency due to mutation of a ubiquitin ligase required for Delta ligand activity. Boundary markers are severely depleted in mib mutant embryos -- this suggests that lateral inhibition maintains the neural epithelium not only in nonboundary regions but also at hindbrain boundaries. Consistent with a role for Notch activation in maintaining boundary cells, following mosaic expression of dominant-active Su(H) in mib mutants, the expressing cells sort to boundaries and boundary marker gene expression is rescued (Cheng, 2004).

These findings reveal that two responses to the activation of Notch are coupled at rhombomere boundaries in the zebrafish hindbrain: the regulation of cell affinity properties of boundary cells and the suppression of neurogenesis. This begs the question of why neurogenesis is delayed at rhombomere boundaries. An attractive possibility is suggested by the observation that signaling centers in the neural epithelium such as the floor plate and roof plate do not undergo neurogenesis and have a low rate of cell proliferation. By enabling the maintenance of a relatively stable number of signaling cells, the suppression of differentiation and proliferation is a simple way to maintain a constant amount of signal. By analogy, the suppression of neurogenesis and proliferation at rhombomere boundaries may reflect that the radical fringe-dependent expression of wnt1 by rhombomere boundary cells is involved in patterning of the zebrafish hindbrain. The regulation by Notch of both cell affinity and the suppression of differentiation at rhombomere boundaries would thus provide a coupling between maintenance of the location and number of signaling cells (Cheng, 2004).

Analysis of Mind bomb in zebrafish: Delta-Notch signalling and retinal development in zebrafish

Notch-Delta signaling has been implicated in several alternative modes of function in the vertebrate retina. To further investigate these functions, retinas from zebrafish embryos were examined in which bidirectional Notch-Delta signaling was inactivated either by the mind bomb (mib) mutation, which disrupts E3 ubiquitin ligase activity, or by treatment with gamma-secretase inhibitors, which prevent intramembrane proteolysis of Notch and Delta. Inactivating Notch-Delta signaling does not prevent differentiation of retinal neurons, but it disrupts spatial patterning in both the apical-basal and planar dimensions of the retinal epithelium. Retinal neurons differentiate, but their laminar arrangement is disrupted. Photoreceptor differentiation is initiated normally, but its progression is slowed. Although confined to the apical retinal surface as in normal retinas, the planar organization of cone photoreceptors is disrupted: cones of the same spectral subtype are clumped rather than regularly spaced. In contrast to neurons, Muller glia fail to differentiate, suggesting an instructive role for Notch-Delta signaling in gliogenesis (Bernardos, 2005).

Delta-Notch signalling controls commitment to a secretory fate in the zebrafish intestine

The transparency of the juvenile zebrafish and its genetic advantages make it an attractive model for study of cell turnover in the gut. BrdU labelling shows that the gut epithelium is renewed in essentially the same way as in mammals: the villi are lined with non-dividing differentiated cells, while cell division is confined to the intervillus pockets. New cells produced in the pockets take about 4 days to migrate out to the tips of the villi, where they die. Monoclonal antibodies have been generated to identify the absorptive and secretory cells in the epithelium, and these antibodies were used to examine the role that Delta-Notch signalling plays in producing the diversity of intestinal cell types. Several Notch receptors and ligands are expressed in the gut. In particular, the Notch ligand DeltaD (Delta1 in the mouse) is expressed in cells of the secretory lineage. In an after eight (aei) mutant, where DeltaD is defective, secretory cells are overproduced. In mind bomb (mib), where all Delta-Notch signalling is believed to be blocked, almost all the cells in the 3-day gut epithelium adopt a secretory character. Thus, secretory differentiation appears to be the default in the absence of Notch activation, and lateral inhibition mediated by Delta-Notch signalling is required to generate a balanced mixture of absorptive and secretory cells. These findings demonstrate the central role of Notch signalling in the gut stem-cell system and establish the zebrafish as a model for study of the mechanisms controlling renewal of gut epithelium (Crosnier, 2005).

Mib-Jag1-Notch signalling regulates patterning and structural roles of the notochord by controlling cell-fate decisions

In the developing embryo, cell-cell signalling is necessary for tissue patterning and structural organization. During midline development, the notochord plays roles in the patterning of its surrounding tissues while forming the axial structure; however, how these patterning and structural roles are coordinated remains elusive. This study identified a mechanism by which Notch signalling regulates the patterning activities and structural integrity of the notochord. Mind bomb (Mib) was found to ubiquitylate Jagged 1 (Jag1) and is essential in the signal-emitting cells for Jag1 to activate Notch signalling. In zebrafish, loss- and gain-of-function analyses showed that Mib-Jag1-Notch signalling favours the development of non-vacuolated cells at the expense of vacuolated cells in the notochord. This leads to changes in the peri-notochordal basement membrane formation and patterning surrounding the muscle pioneer cells. These data reveal a previously unrecognized mechanism regulating the patterning and structural roles of the notochord by Mib-Jag1-Notch signalling-mediated cell-fate determination (Yamamoto, 2010).

PAR-1 phosphorylates Mind bomb to promote vertebrate neurogenesis

Generation of neurons in the vertebrate central nervous system requires a complex transcriptional regulatory network and signaling processes in polarized neuroepithelial progenitor cells. This study demonstrates that neurogenesis in the Xenopus neural plate in vivo and mammalian neural progenitors in vitro involves intrinsic antagonistic activities of the polarity proteins PAR-1 and aPKC. Furthermore, Mind bomb (Mib), a ubiquitin ligase that promotes Notch ligand trafficking and activity, is a crucial molecular substrate for PAR-1. The phosphorylation of Mib by PAR-1 results in Mib degradation, repression of Notch signaling, and stimulation of neuronal differentiation. These observations suggest a conserved mechanism for neuronal fate determination that might operate during asymmetric divisions of polarized neural progenitor cells (Ossipova, 2009).

Mammalian Mind bomb homologs

The zebrafish gene mind bomb encodes a protein that positively regulates the Delta-mediated Notch signaling. It interacts with the intracellular domain of Delta to promote its ubiquitination and endocytosis. In a search for the mouse homologue of zebrafish mind bomb, two homologues in the mouse genome were cloned: a mouse orthologue (mouse mib1) and a paralogue, named mind bomb-2 (mib2), which is evolutionarily conserved from Drosophila to human. Both Mib1 and Mib2 have an E3 ubiquitin ligase activity in their C-terminal RING domain and interact with Xenopus Delta (XD) via their N-terminal region. Mib2 is also able to ligate ubiquitin to XD and shift the membrane localization of Delta to intracellular vesicles. Importantly, Mib2 rescues both the neuronal and vascular defects in the zebrafish mib^ta52b mutants. In contrast to the functional similarities between Mib1 and Mib2, mib2 is highly expressed in adult tissues, but almost not at all in embryos, whereas mib1 is abundantly expressed in both embryos and adult tissues. These data suggest that Mib2 has functional similarities to Mib1, but might have distinct roles in Notch signaling as an E3 ubiquitin ligase (Koo, 2005a).

Mind bomb 1 (Mib1) has been identified as a ubiquitin ligase that promotes the endocytosis of Delta. Mice lacking Mib1 die prior to embryonic day 11.5, with pan-Notch defects in somitogenesis, neurogenesis, vasculogenesis and cardiogenesis. The Mib1-/- embryos exhibit reduced expression of Notch target genes Hes5, Hey1, Hey2 and Heyl, with the loss of N1icd generation. Interestingly, in the Mib1-/- mutants, Dll1 accumulates in the plasma membrane, while it is localized in the cytoplasm near the nucleus in the wild types, indicating that Mib1 is essential for the endocytosis of Notch ligand. In accordance with the pan-Notch defects in Mib1-/- embryos, Mib1 interacts with and regulates all of the Notch ligands, jagged 1 and jagged 2, as well as Dll1, Dll3 and Dll4. These results show that Mib1 is an essential regulator, but not a potentiator, for generating functional Notch ligands to activate Notch signaling (Koo, 2005b).

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date revised: 1 November 2010

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