Neuralized is in the same developmental family as Delta and Notch. These neurogenic genes prevent the overproduction of neurons at the expense of epidermal tissues. Without neuralized function neural hyperplasia (too many nerve cells) occurs, followed by cell death.

To define the requirement of neuralized in regulating cell-cell interactions required for Drosophila sense organ development, two independent neu alleles were used to generate mutant clones. neu is found to be required for determination of cell fates within the proneural cluster; cells mutant for neu autonomously adopt neural fates when adjacent to wild-type cells. Furthermore, neu is required within the sense organ lineage to determine the fates of daughter cells and accessory cells. To gain insight into the mechanism by which neu functions, the GAL4/UAS system was used to express wild-type and epitope-tagged neu constructs. Neu protein is localized primarily at the plasma membrane. It is proposed that the function of neu in sense organ development is to affect the ability of cells to receive Notch-Delta signals and thus modulate neurogenic activity that allows for the specification of non-neuronal cell fates in the sense organ (Yeh, 2000).

To assay the effects of loss of neu during SO development, mutant clones were generated using FLP/FRT-mediated somatic recombination. For these purposes, two alleles of neu were recombined onto third chromosome arms containing FRT sequences at 82B. neu^A101 is a hypomorphic allele resulting from the insertion of a lacZ enhancer trap into the upstream regulatory region of the neu locus, while neu^1F65 is an amorphic ethylmethane sulfonate-induced allele. Both alleles produce severe hyperplasia of the embryonic nervous system leading to lethality and both fail to complement any other known neu allele. Flies heterozygous for neu^A101 and carrying a source of FLP display a bristle tufting phenotype affecting both macrochaetae and microchaetae. The severity of the phenotype ranges from duplicated bristles to tufts containing several bristles. Supernumerary macrochaetae and microchaeate are always found in characteristically normal positions. With the exception of extreme cases of microchaetae tufting observed only at the anterior-most part of the notum, regions between bristles and bristle tufts do not appear to be affected. This suggests that neu functions to prevent cells from adopting SOP fates within the proneural cluster (Yeh, 2000).

The effects of neu^A101 clones are not limited to bristles on the notum. Tufting is also observed with adult head sensilla surrounding the eye and ocelli. In addition, bristle sensilla throughout the body, including the dorsal and ventral abdomen, also appear to form tufts. As was observed for macrochaetae, these tufts always occur in the location where normal bristles are formed. neu^A101 clones also give rise to defects in the adult eye. The severity of the phenotype ranges from ectopic interommatidial bristles and aberrant ommatidial size to scarring and defective photoreceptor development. In addition, defects in wing development, including irregular wing margin sensory bristle formation and ectopic wing vein formation, are observed (Yeh, 2000).

To determine whether the supernumerary bristles (i.e. tufting) that are observed in mutant clones are due to commitment to the SOP fate, advantage was taken of the fact that the neu mutant allele neu^A101 is a lacZ enhancer trap line in the neu locus that can be used as a marker of SOP determination. neu^A101 expression can be detected within third larval instar wing imaginal discs in primary SOPs that give rise to macrochaetae on the notum and sensory bristles along the wing margin. As development proceeds, expression of neu^A101 can also be detected in secondary SOPs as well as the accessory cells that are associated with each primary SOP. neu^A101 is similarly expressed in SOPs on the pupal notum that will give rise to microchaetae. Since the appearance and differentiation of each macrochaete SOP is well documented, it is possible to examine the fate of each SOP at particular developmental time points. For example, the primary SOPs that will give rise to bristles along the adult wing margin are determined during late third larval instar, but do not divide until 5-10 h after puparium formation (APF). Therefore, any supernumerary ß-gal positive cells along the wing margin that are observed during third larval instar development are most likely primary SOPs rather than secondary SOPs. Using the piMyc marker to identify neu^A101 clones, it was found that supernumerary SOPs arise from neu^A101 cells. Since supernumerary SOPs are not observed in ectopic locations in the wing disc this suggests that neu functions normally in the proneural cluster to determine epidermal cell fates (Yeh, 2000).

neu mutant clones were also generated using neu^1F65. In this case, it was found that mutant clones give rise to a balding phenotype characterized by the absence of chaetae on the adult notum. This is consistent with a role for neu in the determination of both pIIa/pIIb and accessory cell fates. In N and Dl mutant clones, loss-of-function during secondary SOP and accessory cell fate determination causes cells to assume a neuronal cell fate. To determine whether neu^1F65 clones give rise to similar alterations in secondary SOP and accessory cell fates, pupal nota (24 h APF) were dissected and stained with the neuronal marker 22C10. In wild-type notum at this stage, 22C10 expression is detected in a single neuron comprising each individual sense organ (as identified by the double axon processes. In contrast, 22C10 expression in pupal nota from neu^1F65 clones reveals clusters of 22C10-positive cells that all display the double axon processes associated with neuronal differentiation. The presence of more than four 22C10 positive cells in some clusters further demonstrates that supernumerary primary SOPs are determined in neu mutant clones and that the descendants of these mutant SOPs differentiate to assume neuronal cell fates. Taken together, these data demonstrate that neu affects multiple cell fate decisions required for the proper development of sense organs. Like N and Dl, neu is required for proper determination of the pIIa cell fate, and is also required for determination of the thecogen cell fate in the pIIIb lineage (Yeh, 2000).

Using mosaic analysis, a role for neu in sense organ development has been demonstrated. To determine whether neu is required autonomously in this process, neu mutant clones were generated that were genetically marked with y in a y⁺ background. Somatic recombination was induced by heat-shocking flies during late embryogenesis. The supernumerary bristles arise from neu^A101 cells; mixtures of wild-type and mutant bristles are never observed (Yeh, 2000).

To ascertain the ability of mutant cells to send or receive the signal that prevents neural determination, and thus delineate autonomous versus non-autonomous neu function, adult clones were examined using the epidermal hair marker pawn (pwn), which can be used to identify clonal boundaries on the adult cuticle. Since pwn affects bristle morphology (producing truncated bristles), mutant cells can be identified as well. If mutant bristles can be found at the clonal boundary, and these are unaffected by neighboring wild-type cells, then neu must be required autonomously to inhibit neuronal cell fates. In contrast, if wild-type bristles are found at the boundary next to neu mutant cells, then neu must act non-autonomously within cells since they fail to suppress neighboring cells from becoming SOs. Mutant bristles are found to exist at clonal boundaries next to wild-type cells more frequently than wild-type bristles next to mutant cells (81% versus 19%, respectively). Furthermore, mutant bristles at the clonal boundary are observed as either single bristles or tufts. Thus, neu mutant cells are affected in their ability to receive the signal that prevents neural determination and they form SOs at clonal borders despite the presence of wild-type cells. The ability of mutant cells to send a signal does not appear to be affected since mixtures of wild-type and mutant bristles are not observed. Taken together, these results clearly demonstrate that neu functions cell autonomously during SOP determination to specify epidermal fates in Drosophila (Yeh, 2000).

To understand how neu could function in the signaling process that allows for epidermal cell fate determination, the expression pattern of neu during SOP determination was examined using in situ hybridization techniques on staged third larval instar wing imaginal discs. neu is undetectable in proneural clusters prior to SOP determination. The first detectable neu expression occurs within SOPs in wing discs of late third larval instars. neu expression was also examined within the notum at 24 h APF where its expression was found to be associated with the neuron of each SO cluster. At this stage, all the accessory cells of each SO have been determined and the neuron can be identified based on its shape (Yeh, 2000).

To determine where Neu protein functions within the cell, transgenic lines were generated that express wild-type or myc epitope-tagged neu constructs in the vector pUAST. To ensure that the myc tag did not disrupt Neu function, the ability to rescue the neu^1F65 mutant allele with both the wild-type and myc-tagged construct was compared. The neu^1F65 allele produces a severe neurogenic phenotype characterized by hyperplasia of the central and peripheral nervous system, and complete lack of ventral cuticle. Using a ptc-GAL4 line to drive expression, it was found that both constructs are equally able to partially rescue the neurogenic phenotype. This indicates that the myc epitope does not disrupt the Neu protein and that the fusion protein is functionally wild-type. In addition to being able to rescue neu^IF65 embryonic phenotypes, it was found that ectopic expression of either tagged or untagged neu constructs yield identical adult phenotypes characterized by missing macrochaetae and incomplete wing vein formation (Yeh, 2000).

The myc-tagged UAS-neu lines were then crossed to a sca-GAL4 line that drives expression of the transgene in proneural clusters in third larval instar wing imaginal discs. It was found that myc-tagged Neu is primarily localized at the plasma membrane. Double labelling with an antibody to alpha-spectrin, a structural protein found associated with the plasma membrane confirms this localization. Myc-tagged Neu protein expressed in third larval instar salivary glands is also localized at the plasma membrane. Since the N signaling pathway is not active during this stage of salivary gland development (whereas N signaling is active in the proneural cluster) Neu localization does not appear to be affected by N signaling. This suggests that Neu functions at the plasma membrane to affect neurogenic signaling (Yeh, 2000).

The amino acid sequence of Neu predicts a protein containing a C-terminal RING finger domain that is often found in DNA binding proteins. However, there has been no evidence to demonstrate that Neu functions in the nucleus. Also, it has been demonstrated that some RING fingers have functions outside the nucleus. Myc-tagged Neu has been found to be closely associated with the plasma membrane. While this does not exclude the possibility that endogenous Neu, like Notch, may exist at low levels within the nucleus or elsewhere in the cell, it suggests that neurogenic signaling does not require nuclear Neu. The finding that Neu protein associates with the plasma membrane suggests a possible role in promoting or modulating neurogenic signaling at the receptor/ligand level. One possible model is that neu affects the ability of the cell to receive or propagate signals by affecting N, and that the function of neu in the proneural cluster is to promote differences in the level of N-Dl signaling activity required for mutual inhibition. According to this model, initial low levels of neu expression within the proneural cluster would be required to promote differences in neurogenic activity. Through mutual inhibition (mechanisms that involve feedback between the proneural and neurogenic genes) these differences would then be amplified leading to selection of a single SOP. In the absence of neu function, the formation of multiple SOPs would be the result of loss in the ability to receive a N-Dl signal. Expression of neu would then be upregulated in the SOP, and neu would function during the SO lineage in a similar manner to allow cells to respond to N-Dl signaling. Ectopic expression of neu allows all cells within the field to signal equally, effectively causing gain-of-function N phenotypes. Interestingly, the RING finger in Neu shares high homology to the RING finger found in the oncogene c-cbl. c-cbl has been shown to have ubiquitin ligase activity and affects the strength of receptor tyrosine kinase (RTK) signaling activity by targeting RTKs for degradation. The ubiquitin ligase activity has been shown to be conferred by a domain encompassing the RING finger domain. Whether the RING finger in Neu regulates N-Dl signaling by targeting either N or Dl for ubiquitylation, remains to be determined (Yeh, 2000).

cDNA clone length - 3.2 kb

Bases in 5' UTR - 259

Exons - three

Bases in 3' UTR - 1447 -- The protein appears to use different polyadenylation sites to generate cRNA of different lengths (Price, 1993).

PROTEIN STRUCTURE

Amino Acids - 754

Structural Domains

Neuralized is a zinc finger protein with a C3-H-C4 zinc finger DNA binding motif (Price, 1993).

Evolutionary Homologs

Unlike other zinc finger C3-H-C4 proteins, including two Drosophila Polycomb group genes (Posterior sex combs and suppressor two of zeste), the NEUR zinc finger motif is found near the carboxy terminus of the protein (Price, 1993).

The loss of chromosome 10 is the most frequent genetic alteration found in malignant astrocytomas. In particular, the long arm of chromosome 10 has two or more common deletion regions where tumor suppressor genes may be located. In this study, deletion mapping of 44 malignant astrocytomas was performed using 12 microsatellite markers on chromosome 10q: the minimal common region of loss of heterozygosity (LOH) is present between D10S192 and D10S566 localized at 10q25.1. Subsequently, a novel gene, termed h-neu, was identified within the region frequently deleted and it was found that h-neu encodes a protein with strong homology to the Drosophila Neuralized (Neu) protein. h-neu mRNA is expressed at very low levels in human malignant astrocytoma tissues and the majority of glioma cell lines examined, while normal brains express h-neu transcript. Furthermore, DNA sequencing analysis of the h-neu transcript reveals that one of the glioma cell lines, U251MG, has a single nucleotide substitution that results in an amino acid change from glycine (GGC) to serine (AGC) at codon 253. It is hypothesized that h-neu plays a role in determination of cell fate in the human central nervous system and may act as a tumor suppressor whose inactivation could be associated with malignant progression of astrocytic tumors (Nakamura, 1998).

A human homolog of the Drosophila neuralized gene has been described as a potential tumor suppressor gene in malignant astrocytomas. A murine homolog of the Drosophila and human Neuralized genes has been isolated and, in an effort to understand its physiological function, mice have been derived with a targeted deletion of this gene. Surprisingly, mice homozygous for the introduced mutation do not show aberrant cell fate specifications in the central nervous system or in the developing mesoderm. This is in contrast to mice with targeted deletions in other vertebrate homologs of neurogenic genes such as Notch, Delta, and Cbf-1. Male Neuralized null mice, however, are sterile due to a defect in axoneme organization in the spermatozoa that leads to highly compromised tail movement and sperm immotility. In addition, female Neuralized null animals are defective in the final stages of mammary gland maturation during pregnancy (Vollrath, 2001).

Neurogenic genes in the Notch receptor-mediated signaling pathway play important roles in neuronal cell fate specification as well as neuronal differentiation. The Drosophila gene neuralized is one of the neurogenic genes. A mouse homolog of Drosophila neuralized, m-neu1, has been cloned: the m-neu1 transcript is expressed in differentiated neurons. Mice deficient for m-neu1 are viable and morphologically normal, but exhibit specific defects in olfactory discrimination and hypersensitivity to ethanol. These findings reveal an essential role of m-neu1 in ensuring proper processing of certain information in the adult brain (Ruan, 2001).

To isolate mouse homolog(s) of Drosophila neuralized (d-neu), a cloning strategy of low-stringency hybridization screening was used, using d-neu cDNA to isolate a full-length m-neu1 from a mouse embryonic cDNA library. Sequence analysis of m-neu1 reveals an ORF encoding a predicted protein of 574 aa. M-Neu1 and D-Neu display extensive similarity throughout the protein, including a C-terminal C3HC4 RING zinc finger domain (amino acids 521-560, 55% identity). The RING zinc finger domain represents a small protein module that uses zinc ions for stability. It is found in a wide variety of functionally distinct proteins and may act as a novel protein-interaction module. Evolutionary conservation of the C-terminal C3HC4 RING zinc finger domain is underscored by the sequences of one Caenorhabditis elegans neuralized homolog (C-Neu) in the database, which bears a similar extent of homology to M-Neu1 as does D-Neu, and a human homolog of Drosophila neuralized, which is 94% identical to M-Neu1 at the amino acid level. These data suggest that neuralized is an evolutionarily conserved molecule (Ruan, 2001).

The embryonic and adult expression of m-neu1 was examined by Northern blot and RT-PCR. A 4.3-kb m-neu1 transcript was detected. The m-neu1 transcript is expressed from embryo to adult, similar to d-neu. Whereas low levels of m-neu1 mRNA can be detected at embryonic day 7.5 (E7.5), higher levels of m-neu1 mRNA are detected later in embryogenesis. In the adult, the expression of m-neu1 appears to be highly restricted to the brain and was undetectable in other tissues including heart, liver, kidney, intestine, lung, spleen, and skeletal muscle (Ruan, 2001).

The spatial distribution of m-neu1 transcript was analyzed by in situ hybridization. At E8.5 (embryos with 11-13 pairs of somites), m-neu1 is mainly expressed in the nervous system, although it is also expressed in somites and the first branchial arch. The m-neu1 transcript is detected throughout the neural tube along the rostral-caudal neural axis. In the brain, m-neu1 is highly expressed in the forebrain neural fold and the hindbrain neural fold. Later during embryogenesis, the m-neu1 transcript is detected in differentiated neurons in the brain and the spinal cord, as well as in sensory neurons of the olfactory epithelium and the vomeronasal organ. At postnatal stage, the m-neu1 transcript is also detected in the nervous system. In the adult brain, the m-neu1 transcript is expressed in several regions with high expression in the cerebral cortex, cerebellum, striatum, hippocampus, and dentate gyrus. When expressed in cultured mouse neuroblastoma Neuro2a cells, a fusion protein of the m-neu1 gene product and the green fluorescence protein (GFP) is primarily in the cytoplasm (Ruan, 2001).

m-neu1 is widely expressed in differentiated neurons in the central nervous system. m-neu1 is also expressed in sensory neurons in the olfactory epithelium and in the vomeronasal organ. It was speculated that m-neu1 may play a role in neuronal function in adult mice. Therefore, the behaviors of adult m-neu1^-/- mice were evaluated by a battery of behavioral tests, including a rotarod assay for motor coordination, open field exploration, seizure induction by pentylenetetrazol or kainic acid, olfactory discrimination, pain sensation by hot-plate assay, aggression by resident-intruder assay, and maternal behavior by pup retrieval test. From these many tests, only the following behavioral abnormalities in m-neu1^-/- mice were observed: m-neu1^-/- mice exhibit an olfactory discrimination defect and m-neu1^-/- mice are hypersensitive to ethanol effects on motor coordination (Ruan, 2001).

Olfactory discrimination is assessed by using a conditioned avoidance procedure in which exposure to an odorant in a drinking tube is associated with an aversive stimulus: LiCl injection. Animals subsequently avoid the test odorant when reexposed to the odorant at a later time. A two-bottle preference procedure was used to assess avoidance of odorant. Measurement of preference ratio (volume of odorant solution consumed/total solution consumed) as determined in this two-bottle preference assay has been shown to be an assessment of olfactory function rather than taste. In this behavioral assay, no significant differences were observed in total fluid intake between wild-type mice and m-neu1^-/- mice. However, significant differences were observed in preference ratio between wild-type mice and m-neu1^-/- mice. The defect most likely reflects a reduced ability of the mutant mice to detect the odorant rather than a deficiency in associative learning (Ruan, 2001).

Ethanol effects on motor coordination were examined by using the rotarod assay. Performance of this task involves both the cerebellum and striatum, where the m-neu1 gene is highly expressed. Before ethanol injection, no significant difference was observed in the duration that wild-type mice and m-neu1^-/- mice remained on rotarod (the latency to falling off). However, after ethanol injection, a significant difference was observed in such latencies on rotarod between wild-type mice and m-neu1^-/- mice. Thus, m-neu1^-/- mice are hypersensitive to ethanol effects on motor coordination. Analysis of the blood ethanol concentration showed no difference between wild-type mice and m-neu1^-/- mice. Because ethanol triggers widespread apoptotic neurodegeneration in developing brain, ethanol-induced apoptotic neurodegeneration was examined in m-neu1^-/- mice and wild-type littermates and no differences were found between these two genotypes (Ruan, 2001).

Notch signaling in Drosophila requires a RING finger (RF) protein encoded by neuralized. The Xenopus homolog of neuralized (Xneur) is expressed where Notch signaling controls cell fate choices in early embryos. Overexpressing XNeur or putative dominant-negative forms in embryos inhibits Notch signaling. As expected for a RF protein, XNeur fulfills the biochemical requirements of ubiquitin ligases. Wild-type XNeur decreases the cell surface level of the Notch ligand, XDelta1, while putative inhibitory forms of XNeur increase it. Evidence is provided that XNeur acts as a ubiquitin ligase for XDelta1 in vitro. It is proposed that XNeur plays a conserved role in Notch activation by regulating the cell surface levels of the Delta ligands, perhaps directly, via ubiquitination (Deblandre, 2001).

Transcriptional Regulation

single-minded is activated in a single band of mesectoderm through the actions of Notch and neuralized. Neuralized acts in all processes involving the Notch pathway (Hartenstein, 1992).

Targets of Activity

Neurogenic genes neuralized and Notch are required for the specification of mesectoderm. They control at the transcriptional level the repression of proneural genes and the activation of single-minded in the anlage of the mesectoderm (Martin-Bermudo, 1995).

Wingless targets neuralized at the wing disc margin. wingless-expressing cells induce only their immediate neighbors to express neur whereas wg-expressing cells exert a long range influence on Distal-less and vestigial expression. Thus WG appears to have the capacity to define multiple distinct outputs regulating the expression of target genes at different threshold concentrations. neur is induced in isolated cells close to the D/V boundary rather than in a swath of cells, all of which appear to be responding in a uniform fashion to WG. neur expressing cells are neuroblasts that arise from a population of proneural cells by a process of lateral specification. In this case, WG appears to define the population of proneural cells, and these cells then send signals other than WG, which are transduced by the Notch receptor, leading to the segregation of the neur expressing cells (Zecca, 1996).

Neuralized involvement in Delta signaling and endocytosis

Activation of the Notch (N) receptor involves an intracellular proteolytic step triggered by shedding of the extracellular N domain (N-EC) upon ligand interaction. The ligand Dl has been proposed to effect this N-EC shedding by transendocytosing the latter into the signal-emitting cell. Dl endocytosis and N signaling are greatly stimulated by expression of neuralized. neur inactivation suppresses Dl endocytosis, while its overexpression enhances Dl endocytosis and Notch-dependent signaling. neur encodes an intracellular peripheral membrane protein. Its C-terminal RING domain is necessary for Dl accumulation in endosomes, but may be dispensable for Dl signaling. The potent modulatory effect of Neur on Dl activity makes Neur a candidate for establishing signaling asymmetries within cellular equivalence groups (Pavlopoulos, 2001).

Static pictures of Dl localization do not allow an unambiguous conclusion of whether intracellular Dl is endocytosed or blocked in its secretory trafficking. The former hypothesis is favored for three reasons: (1) intracellular Dl often colocalizes with endocytosed fluorescent dextran; (2) if Dl were retained in the endoplasmic reticulum or Golgi, it would not be available at the cell surface where signaling is taking place, yet, concomitant with increased endocytosis, Neur is able to stimulate Dl signaling and (3) wt Neur protein is found mostly at the plasma membrane, so it is more likely to affect endocytic events rather than steps in secretory processes (Pavlopoulos, 2001).

The nonautonomous effect of neur^- clones on lateral inhibition favors a role for Neur in signal-emitting, rather than signal-receiving, cells. Such a function is consistent with the fact that Neur is an intracellular peripheral membrane protein expressed preferentially in the signal-emitting cells during lateral inhibition, such as the neuroblasts, SOPs, and central provein cells. In agreement with a role for Neur in generating the Dl signal, epistasis analyses have shown that neur is required to express the embryonic neural suppression ('antineurogenic') phenotype associated with ligand-dependent N gain-of-function (gof) mutants. neur is dispensable for the constitutive activity of ligand-independent N variants. Interestingly, some N variants that are Dl independent are also shi (Dynamin) independent. Taken together, these data point to the involvement of Neur and Dynamin in processes upstream of (or parallel to) N activation by Dl. The implication of Neur in endocytic regulation suggests an important role for endocytosis in events leading up to N activation (Pavlopoulos, 2001).

If Dl endocytosis and Dl-N signaling are causally linked, then this analysis of the NeurDeltaRING-GFP mutant poses a paradox: although NeurDeltaRING-GFP does not detectably stimulate Dl endocytic trafficking (or turnover), it retains the ability to enhance Dl signaling. This could mean that the above model is wrong and endocytosis is simply a consequence of Dl-N stimulation, rather than a prerequisite for Dl signaling. Alternatively, the absence of detectable Dl internalization upon coexpression of NeurDeltaRING-GFP does not necessarily preclude the possibility that early endocytic events (e.g., recruitment of Dl into coated pits) that are undetectable by light microscopy are initiated by NeurDeltaRING-GFP. Such events might be sufficient to stimulate ligand-dependent N cleavage and activation. Ultrastructural analysis will be required to distinguish between these alternative models (Pavlopoulos, 2001).

Removal of the Neur RING domain does seem to adversely affect its ability to stimulate N signaling in some contexts: UAS-neurDeltaRING yields phenotypes indicative of a negative effect on N signaling (tufted bristles, thick veins, and notched wings) with most Gal4 driver lines, although in certain cases, positive effects are also observed (shaft to socket transformation). Context-dependent variability with the UAS-neurDeltaRING-GFP construct suggests that these differences do not result from the presence of the GFP moiety but rather from the type of assay employed. In fact, NeurDeltaRING-GFP coexpressed with Dl blocks N signaling within the omb-Gal4 domain, where wt Neur and Dl are able to induce Wg, even though the nonautonomous signaling (at the borders of the omb-Gal4 domain or at the borders of FLP-out clones) appears unaffected by the RING deletion. It is possible then that NeurDeltaRING can exert negative effects on Dl-N signaling in a cell-autonomous fashion and positive effects in a cell-non-autonomous fashion. The cell-autonomous block in N signaling could be due to the block in Dl turnover and its accumulation at the apical membrane, because it has been proposed that high levels of Dl may sequester N receptor molecules in unproductive cis complexes (Pavlopoulos, 2001).

Two major models for Dl signaling have been put forward. In one, the active Dl species is proposed to be the extracellularly cleaved, secreted Dl-EC fragment, because it is produced by the metalloprotease Kuzbanian (Kuz), and the kuz lof phenotype is similar to the N lof phenotype. In the other, binding of cell surface-tethered Dl to N on the apposing cell has a dual impact: activating extracellular cleavage of Notch and mediating the transendocytosis into the signal-sending cell of N-EC complexed with Dl. The observations in this paper suggest that Neur could act intracellularly in the signal-sending cell to promote assembly of a productive Dl-N complex and to trigger its endocytosis. Concomitantly with endocytosis, Neur leads to a drastic reduction in the levels of the Dl-EC fragment, even as Dl-N signaling is increased. It therefore appears unlikely that Dl-EC is the active signal that stimulates N in the wing disk. This leaves unanswered at present the question of why Kuz is needed for N signaling. Perhaps Kuz has pleiotropic activity and acts on some other protein(s) required for N activation, and Kuz-dependent Dl cleavage is a secondary effect. Better characterization of the different Dl isoforms, including their localization and trafficking, will be required to understand the detailed mechanism of Dl-N activation (Pavlopoulos, 2001).

Despite the proposed role of Neur to promote Dl signaling, it is also noted that Dl can signal in the absence of Neur, inasmuch as there are instances of Dl signaling where Neur is not detectably expressed, such as from the germline to ovarian follicle cells. N target gene expression is indeed induced by Dl in the absence of neur. With the caveat that available detection methods may fail to detect low levels of neur expression, it is proposed that two types of Dl signaling may exist: basal signaling that does not require Neur activity and high-intensity signaling that does. During neurogenesis, basal Dl-N signaling probably takes place during early stages among all cells within proneural clusters, where Dl and N are uniformly expressed but Neur is absent. Upon expression of neur by a nascent neural precursor, signaling becomes asymmetric, since the neighboring cells receive more intense signal even though Dl and N levels have not changed. The absolute requirement for neur in neurogenesis suggests that basal 'mutual' inhibition is insufficient to permanently block proneural protein activity. Indeed, the E(spl) bHLH Notch targets, which are the main antagonists of proneural proteins, are not expressed in neur^- embryos or clones, suggesting that their expression may be induced only by intense Neur-dependent 'lateral' inhibitory signaling (Pavlopoulos, 2001).

This hypothesis can be extended to propose that Neur may be required more stringently in instances in which a novel asymmetry has to be imposed upon uniform basal N-Dl signaling. neur is not required at the wing DV boundary, where asymmetry is imposed by Fringe or in the egg chamber, where asymmetry is imposed by expression of N and Dl in distinct cells. Similarly, neur is not essential during lateral inhibition within the provein. Despite its expression there and its dramatic effect on Dl localization, neur lof clones yield normal looking veins with only minor thickenings. It is believed that neur is not crucial for this process because wing patterning mechanisms place N and Dl in different cells: Dl expression is most intense within the central proveins and N expression is most intense within the lateral proveins (Pavlopoulos, 2001).

In Drosophila, Notch signaling regulates binary fate decisions at each asymmetric division in sensory organ lineages. Following division of the sensory organ precursor cell (pI), Notch is activated in one daughter cell (pIIa) and inhibited in the other (pIIb). The E3 ubiquitin ligase Neuralized localizes asymmetrically in the dividing pI cell and unequally segregates into the pIIb cell, like the Notch inhibitor Numb. Furthermore, Neuralized upregulates endocytosis of the Notch ligand Delta in the pIIb cell and acts in the pIIb cell to promote activation of Notch in the pIIa cell. Thus, Neuralized is a conserved regulator of Notch signaling that acts as a cell fate determinant. Polarization of the pI cell directs the unequal segregation of both Neuralized and Numb. It is proposed that coordinated upregulation of ligand activity by Neuralized and inhibition of receptor activity by Numb results in a robust bias in Notch signaling (Le Borgne, 2003).

Recent studies have indicated that endocytosis of Dl is critical for N activation. (1) Dynamin-dependent endocytosis is not only required for signal transduction but is also required in signal-sending cells to promote N activation. (2) Endocytosis-defective Dl proteins have reduced signaling capacity. (3) The E3-ubiquitin ligases Neuralized (Neur) in Drosophila and Mind bomb (Mib) in zebrafish promote endocytosis of Dl and appear to be required for efficient activation of N by Dl. It has been proposed that Dl endocytosis facilitates the S2 cleavage of N at the surface of the signal-receiving cell. Neur is unequally segregated during asymmetric division of the pI cell, upregulates endocytosis of Dl in the pIIb cell, and plays a critical role in generating cell fate diversity. It is proposed that Neur acts as a cell fate determinant during asymmetric cell divisions (Le Borgne, 2003).

To examine whether asymmetry in N ligands distribution may play a role in generating cell fate diversity during asymmetric divisions, the subcellular distribution of Dl and Ser was studied in the sensory organ lineage. In mitotic pI cells, Dl and Ser are uniformly distributed around the cell cortex and are equally partitioned into both daughter cells. In both pI daughter cells, Dl and Ser accumulated at the apical cell cortex as well as in intracellular dots of 0.5 ± 0.2 μm in diameter. These dots are coated by Hrs (hepatocyte growth factor-regulated tyrosine kinase substrate). Hrs binds ubiquitinated proteins via its ubiquitin-interacting motif and sorts endocytic cargos into the lumen of multivesicular bodies (MVBs). Therefore, these Dl-positive vesicles appear to be large endocytic vesicles that probably correspond to MVBs. These Dl-positive vesicles also contain Notch extracellular domain (NECD) and NICD epitopes. Strikingly, a higher number of large Dl-positive vesicles were seen in the anterior signal-sending pIIb cell (5.0 ± 2.2) than in the posterior signal-receiving pIIa cell (2.0 ± 1.5). This asymmetry in Dl endocytosis is established independently of the unequal partitioning of Numb. Indeed, anterior pI daughter cells are shown to accumulate a higher number of Dl-positive vesicles than posterior pI daughter cells in numb² and numb¹⁵ mutant clones. Thus, asymmetry in Dl endocytosis does not depend on Numb (Le Borgne, 2003).

Recent studies have suggested that endocytosis of Dl is promoted by the ubiquitination of Dl by Neur, a RING finger-type E3-ubiquitin ligase required for N signaling. Neur is found in a complex with Dl and is required for Dl ubiquitination. Finally, Neur stimulates the accumulation of Dl into intracellular vesicles in imaginal disc cells. The latter conclusion was, however, based on the analysis of steady-state levels of Dl, making it difficult to unambiguously conclude whether Neur promotes Dl endocytosis or favors direct sorting from the Golgi to intracellular vesicles. To discriminate between these two possibilities and to test whether Neur regulates Dl trafficking in sensory cells, an ex vivo assay was developed for endocytosis. Internalization of Dl was followed in living epithelial cells using antibodies recognizing the extracellular part of Dl. Briefly, the single-layered epithelium corresponding to the pupal notum was dissected and cultured in the presence of anti-Dl antibodies. Following medium changes and fixation, the uptake of anti-Dl antibodies was revealed using secondary antibodies. Anti-Dl antibodies were found to be specifically internalized in the pIIa and pIIb cells. Internalized anti-Dl antibodies colocalize with Dl into large Dl-positive vesicles. Internalization of anti-Dl requires dynamin activity and is not observed at 4°C. Together, these results indicate that anti-Dl interacts with Dl at the cell surface and that Dl-anti-Dl complexes are endocytosed in sensory cells (Le Borgne, 2003).

This assay was used to examine the function of neur. Clones of neur^1F65 mutant cells have been shown to exhibit a neurogenic phenotype with too many pI cells being specified. The progeny of these mutant pI cells produce no external sensory structures indicating that pIIa cells have been transformed into pIIb-like cells. These cell fate transformations are associated with defects in Dl trafficking. High levels of anti-Dl remain at the surface of neur^1F65 mutant cells and internalization of anti-Dl is drastically reduced. It is concluded that neur is required for the endocytosis of Dl in sensory cells (Le Borgne, 2003).

This defect in Dl endocytosis was quantified on fixed tissues. neur mutant pI cells and pIIb-like progeny cells were found to accumulate high levels of Dl at the cell surface. Accumulation of Dl at the cell surface is consistent with the proposed function of Neur in the internalization and degradation of Dl. Quantification of Dl-positive vesicles in neur mutant clones revealed that mutant pIIb-like cells contain much fewer Dl-positive vesicles than wild-type pIIb cells. Thus, in the absence of neur function, both pI daughter cells have the same reduced number of Dl-positive vesicles. Furthermore, a similar distribution of Dl-containing vesicles is seen in the wild-type pIIa cells, which do not inherit Neur, and in the neur mutant pIIb-like cells. These comparisons indicate that neur is required to upregulate the endocytosis of Dl in the pIIb cell (Le Borgne, 2003).

Upregulation of Dl endocytosis in the pIIb cell may result from higher levels of Neur in this cell. To test this hypothesis, the localization of Neur was examined. The Neur protein is detectable in the pI cell and in its progeny cells, but not in epidermal cells. Neur is perinuclear in prophase and localized asymmetrically at the anterior cortex during prometaphase. At telophase, Neur specifically segregates into the anterior daughter cell. At cytokinesis, Neur uniformly redistributes at the cortex and in the cytoplasm in the pIIb cell. Localization of Neur at mitosis is identical to the one described for Partner of Numb (Pon). Consistently, Neur colocalizes with Pon-GFP throughout mitosis. Asymmetric localization of Neur is also seen in the pIIb and pIIa dividing cells. Specificity of anti-Neur antibodies was demonstrated by absence of staining in neur mutant pI cells. Unequal segregation of Neur does not depend on numb activity. Conversely, unequal segregation of Numb does not depend on neur activity. Thus, the numb-independent unequal segregation of Neur into the pIIb cell provides a simple explanation for the upregulation of Dl endocytosis in the pIIb cell (Le Borgne, 2003).

To test the functional significance of Neur unequal segregation, Neur was overexpressed in pI cells. Overexpression of Neur using neur^P72GAL4 fails to affect the unequal partitioning of Neur at pI mitosis and the pIIa/pIIb decision but instead results in a weak double-socket phenotype associated with a shaft-to-socket transformation. This fate transformation is known to result from high levels of Delta-Notch signaling and is opposite that of the socket-to-shaft transformation seen in neur mutant clones. Moreover, this shaft-to-socket transformation may result from the equal partitioning of Neur (but not Numb) in the two pIIa daughter cells which can also be observed at low frequency. Thus, these observations support the notion that unequal segregation of Neur is functionally important (Le Borgne, 2003).

The mechanisms by which Neur localized at the anterior cortex of the dividing pI cell were investigated. The role of the cytoskeleton was studied by applying drugs to cultured nota. Colcemid, a microtubule-depolymerizing agent, was found to have no significant effect. In contrast, both Latrunculin A, an agent that depolymerizes actin microfilaments, and the myosin motor inhibitor butanedione-2-monoxime (BDM) strongly impaired or completely inhibited the asymmetric localization of Neur. Thus, both myosin motor activity and an intact actin cytoskeleton are required for the formation and/or maintenance of the Neur crescent at the anterior cortex of the dividing pI cell. These requirements for Neur localization are similar to the ones seen earlier for Numb and Pon. Neur also behaves in a manner similar to Numb and Pon in that localization of Neur at the anterior cortex of the pI cell depends on planar polarity genes and on the polarity genes discs-large and pins. Moreover, mispartitioning of Neur in dlg and pins mutant cells correlates with a loss in asymmetric internalization of Dl. These data indicate that Neur and Numb share part of the same molecular machinery to localize asymmetrically in the pI cell (Le Borgne, 2003).

Unequal segregation of Neur in the anterior pIIb cell suggests that Neur acts in this cell to promote adoption of the pIIa fate by the posterior cell. To test whether neur activity is indeed required in the pIIb cell, clones within the sensory organ lineage were generated. Mitotic recombination in the pI cell produces one neur mutant cell and one wild-type cell. Importantly, the anterior daughter cell inherits Neur, regardless of its genotype. Thus, when the anterior cell is neur mutant, the posterior cell is predicted to adopt a pIIa fate whatever the requirement for neur activity. However, two different outcomes are predicted when the posterior cell is mutant. If neur activity is required in the signal-receiving cell, the posterior cell is predicted to adopt a pIIb-like fate activity. This should result in a bristle loss phenotype. In contrast, if neur acts in the signal-sending cell, the mutant posterior cell is predicted to become a pIIa cell. This mutant pIIa cell should then produce two mutant cells unable to signal, hence leading to bristle duplication. Mitotic recombination induced at 0-6 hr before puparium formation (PF), when most macrochaete pI cells are specified but have not yet divided, produces flies with double-shaft bristles on the head, thorax and at the wing margin. No macrochaete loss was detectable. This double-shaft phenotype appears to result from wild-type pIIb/mutant pIIa pairs because sensory organs composed of two mutant shaft cells and wild-type pIIb progeny cells were detected at 20 hr after PF. Reciprocally, a sheath-to-neuron transformation was observed in mutant pIIb/wild-type pIIa pairs. These data show that neur is required for the socket/shaft and neuron/sheath fate decisions and further indicate that neur acts in the pIIb cell to specify the pIIa cell (Le Borgne, 2003).

Neuralized function as a ubiquitin ligase

Genetic and phenotypic studies suggest that Neuralized (Neu) plays a role within the N-Dl pathway. Neu is required at the plasma membrane for functional activity and its RING finger domain acts as an E3 ubiquitin ligase. These data suggest that the role of Neu is to target components of the N-Dl pathway for ubiquitination, allowing for propagation and/or regulation of the signal (Yeh, 2001).

The recent finding that RING fingers may confer E3 ubiquitin ligase activity suggests that Neu may also function in this manner. To directly test this possibility, the following GST fusion proteins were made: a full-length GST-Neu protein, GST-NeuDeltaRING (Neu N-terminal region from amino acids 1-423 that lacks the RING finger domain), and GST-NeuRING (Neu protein from amino acids 631-754 containing the RING finger). A fourth fusion protein consisting of the RING finger domain with a cysteine to serine mutation in the absolutely conserved cysteine residue at position 701 was also made (GST-NeuRINGC701S). These fusion proteins were then tested in an in vitro assay that measures the ability of a protein to catalyze the formation of multiubiquitin chains in a reaction containing E1 and E2 enzymes, ubiquitin, and ATP. The addition of a protein with E3 ubiquitin ligase activity (as a GST fusion protein) leads to polyubiquitination of the GST-E3 fusion protein that can be detected by probing Western blots with anti-ubiquitin. In this assay, both the full-length GST-Neu and the RING finger domain GST-NeuRING had E3 ligase activity, as revealed by the presence of polyubiquitinated products. Reactions lacking the essential E2 subunit did not contain polyubiquitinated proteins, nor did those containing GST alone, demonstrating the specificity of the activity conferred by the GST-Neu fusion proteins. Neither Neu protein lacking the RING finger domain, GST-NeuRING, nor a mutant Neu RING finger, GST-NeuRINGC701S, had E3 ligase activity. Taken together, these results show that Neu can catalyze the formation of multiubiquitin chains in an E2-dependent manner, demonstrating that, in vitro, Neu functions as an E3 ubiquitin ligase or as part of an E3 complex and that this activity requires the RING finger domain (Yeh, 2001).

Since genetic data suggest that neu positively propagates N signals, it is thought that Neu does not function to target N protein for ubiquitin-mediated degradation. Rather, Neu may play a role in N receptor activation (perhaps through a proteolytic event) or may relieve inhibition of the N pathway by targeting an inhibitor for degradation. Several studies have recently shown that both ubiquitination and endocytosis are important in regulating the N signaling pathway. Furthermore, endocytosis of both the N receptor or its ligand Dl has also been shown to be an important mechanism by which signaling is controlled during development. Clearly, ubiquitination can be used as a signal for many cellular events, and identifying which components of the N signaling pathway are targeted by Neu will aid in the understanding of how N signals propagate within the cell (Yeh, 2001).

Mind-bomb and Neuralized are two distinct E3 ubiquitin ligases that have complementary functions in the regulation of Delta and Serrate signaling in Drosophila

Signaling by the Notch ligands Delta (Dl) and Serrate (Ser) regulates a wide variety of essential cell-fate decisions during animal development. Two distinct E3 ubiquitin ligases, Neuralized (Neur) and Mind-bomb (Mib), have been shown to regulate Dl signaling in Drosophila melanogaster and Danio rerio, respectively. While the neur and mib genes are evolutionarily conserved, their respective roles in the context of a single organism have not yet been examined. 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. (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 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).

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).

Regulation of membrane localization of Sanpodo by lethal giant larvae and neuralized in asymmetrically dividing cells of Drosophila sensory organs

In Drosophila, asymmetric division occurs during proliferation of neural precursors of the central and peripheral nervous system (PNS), where a membrane-associated protein, Numb, is asymmetrically localized during cell division and is segregated to one of the two daughter cells (the pIIb cell) following mitosis. numb has been shown genetically to function as an antagonist of Notch signaling, and also as a negative regulator of the membrane localization of Sanpodo, a four-pass transmembrane protein required for Notch signaling during asymmetric cell division in the central nervous system (CNS). lethal giant larvae (lgl) is required for Numb-mediated inhibition of Notch in the adult PNS. Sanpodo is expressed in asymmetrically dividing precursor cells of the PNS and Sanpodo internalization in the pIIb cell is dependent on cytoskeletally-associated Lgl. Lgl specifically regulates internalization of Sanpodo, likely through endocytosis, but is not required for the endocytosis Delta, which is a required step in the Notch-mediated cell fate decision during asymmetric cell division. Conversely, the E3 ubiquitin ligase Neuralized is required for both Delta endocytosis and the internalization of Sanpodo. This study identifies a hitherto unreported role for Lgl as a regulator of Sanpodo during asymmetric cell division in the adult PNS (Roegiers, 2005).

This analysis of Sanpodo function in the adult PNS suggests that, as in the embryo, Sanpodo is expressed only in asymmetrically dividing precursor cells and is required for cell fates dependant on high levels of Notch signaling, perhaps through the direct interaction between Sanpodo and the full length Notch receptor. Sanpodo also interacts directly with Numb in vivo, and in both the embryonic CNS and the adult PNS, numb inhibits plasma membrane association of Sanpodo. Therefore, it appears that Sanpodo plays a similar role in asymmetrically dividing precursor cells in both the CNS and PNS in Drosophila (Roegiers, 2005).

Although there are many similarities between the mechanisms of asymmetric cell divisions in embryonic neuroblasts and adult sensory organ precursor cells, one difference involves the role of lgl. In neuroblasts, lgl is required along with another cortical tumor suppressor, dlg, to target Numb to a basal crescent during mitosis, whereas in pI cells, only dlg is required for Numb crescent formation. While lgl is dispensable for segregation of Numb to the pIIb cell following pI cell mitosis, lgl is required for the inhibition of Notch signaling in the pIIb cell. Based on the current study, it is proposed that Lgl functions with Numb to remove Sanpodo from the membrane, leading to down regulation of the Notch signaling pathway in the pIIb cell. Through what mechanism might Lgl regulate Sanpodo localization? Studies in Drosophila, yeast, and vertebrate cells have implicated Lgl as both a regulator of exocytosis, through its interaction with t-SNARES, and as cytoskeletal effector. In this study, no phenotypes suggesting gross defects in exocytosis were detected; in fact, increased accumulation of the membrane protein Sanpodo at the plasma membrane is seen in lgl mutants. Accumulation of Sanpodo at the plasma membrane in lgl mutants resembles the phenotype of three endocytic proteins Numb, alpha-Adaptin, and Shibire, suggested that lgl may have a broader role in vesicle traffic. Although a potential role for Lgl in endocytosis is observed, this role appears to be specific to Sanpodo, since endocytosis of Delta occurs normally in lgl mutants, suggesting that Lgl is not required for bulk endocytosis. Increasingly, selective endocytosis is being implicated as an important regulator of signaling pathways. Two recent studies demonstrate that Liquid facets, an endocytic epsin participates in the Neuralized-mediated Delta endocytosis, apparently by targeting mono-ubiquitinated Delta to a specific, activating, endocytic compartment. The Notch receptor is also subjected to an ubiquitin-mediated endocytic step required for activation via the E3 ubiquitin ligase Deltex, which targets Notch to the late endosome. However, the roles of Liquid facets and Deltex have not been explored in asymmetrically dividing neural precursors. One possible function for Lgl could be to direct Sanpodo toward a specific endocytic compartment. Alternatively, Lgl may be involved indirectly, by targeting molecules required for Sanpodo endocytosis to the membrane region. This scenario would be more consistent with Lgl's role as an exocytic regulator. An alternative hypothesis may be that Lgl regulates Sanpodo localization through its interaction with the cytoskeleton. Lgl functions as an inhibitor of non-muscle myosin II function in both Drosophila and yeast. The data suggests that cytoskeletal association of Lgl is required for regulating Sanpodo localization, because phosphorylation of Lgl by aPKC, which causes an autoinhibitory conformational change in Lgl that disrupts the association with the cytoskeleton, causes membrane accumulation of Sanpodo. It remains to be determined if Sanpodo endocytosis requires inhibition of myosin II activity (Roegiers, 2005).

Previously, Numb and Neuralized had been implicated in two complementary, and possibly independent, mechanisms to determine cell fate in PNS precursor cells. Numb functions to inhibit Notch autonomously by internalizing Sanpodo in the pIIb cell: while Neuralized acts on Delta in the pIIb cell to induce Notch signaling non-autonomously in the pIIa cell. Both neuralized-dependant uptake of Delta and Sanpodo internalization require dynamin function, suggesting that these steps rely on endocytosis. Unexpectedly, it was found that loss of neuralized function affects both Delta internalization and Sanpodo internalization. Failure to internalize Delta into the pIIb cell causes a cell fate transformation of the pIIa cell into a pIIb cell in neuralized mutants, and this transformation occurs despite the accumulation of Sanpodo at the membrane, suggesting that accumulation of Sanpodo at the membrane is not sufficient to induce Notch signaling in the pIIb cell in the absence of neuralized. It is unclear whether membrane accumulation of Sanpodo in neuralized mutants is due to a direct interaction between Neuralized and Sanpodo, perhaps through ubiquitination of Sanpodo, or through an indirect mechanism. Regardless, the data show that regulation of Sanpodo membrane localization is not completely independent of neuralized function. In summary, this study suggests that Sanpodo is regulated by both neuralized and lgl, while Delta is regulated by neuralized independently of lgl. In addition, this study shows that lgl appears to contribute to the endocytosis of Sanpodo, which suggests a broader role for lgl in vesicle trafficking, which may have important implications for its role as a tumor suppressor. Could the regulation of Notch signaling by Sanpodo, Lgl and Numb be conserved across species? Sequence analysis did not reveal any homologues of Sanpodo beyond other insect species. However, loss of function studies of the mouse homologues of Drosophila numb and lgl in the developing brain show strikingly similar phenotypes. Targeted numb/numblike knockouts in dorsal forebrain and Lgl1 knockouts cause profound disorganization of the layered regions of the cortex and striatum and formation of rosettelike accumulations of neurons. These phenotypes may indicate that Numb and Lgl function together to regulate Notch signaling in mouse neurogenesis as well as in Drosophila PNS development, but a functional homologue sanpodo has yet to be identified in the mouse (Roegiers, 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).

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

Endocytosis of Notch receptor ligands in signaling cells is essential for Notch receptor activation. In Drosophila, the E3 ubiquitin ligase Neuralized (Neur) promotes the endocytosis and signaling activity of the ligand Delta (Dl). This study identifies proteins of the Bearded (Brd) family as interactors of Neur. Tom, a prototypic Brd family member, inhibits Neur-dependent Notch signaling. Overexpression of Tom inhibits the endocytosis of Dl and interferes with the interaction of Dl with Neur. Deletion of the Brd gene complex results in ectopic endocytosis of Dl in dorsal cells of stage 5 embryos. This defect in Dl trafficking is associated with ectopic expression of the single-minded gene, a direct Notch target gene that specifies the mesectoderm. It is proposed that inhibition of Neur by Brd proteins is important for precise spatial regulation of Dl signaling (Bardin, 2006).

In order to identify regulators of Neur, a yeast two-hybrid screen was conducted using as bait the conserved central domain of Neur that comprises the two Neur homology repeats (NHRs). Eighty-four cDNAs were identified, of which 62 encoded members of the Brd gene family: Ocho (39 times [×]), Tom (12×), m4 (7×), Brd (2×), and Bob (2×). Interaction of Neur with m6, mα, and m2 was tested directly. The m6 and mα proteins, but not m2, interacted with Neur in this assay. Tom also interacts with full-length Neur. It is concluded that all Brd family members, with the exception of m2, interact with Neur (Bardin, 2006).

Most Brd proteins share four conserved motifs: (1) a lysine-rich N-terminal region predicted to form an amphipathic α helix; (2) a short NxxNExLE motif, found in all proteins except m2; and (3 and 4) two C-terminal motifs found in only a subset of the Brd family members. Motifs 2 and 3 are the most-conserved motifs among insect Brd homologs. Because Brd and Bob lack motifs 3 and 4, these motifs cannot be strictly required for interaction with Neur. Additionally, clones encoding N-terminally truncated Tom proteins (amino acids 75-58, 58-158, 54-158, and 29-158 of Tom) were recovered in the screen, indicating that the N-terminal region predicted to form an amphipathic helix (amino acids 26-43 of Tom) is not necessary for interaction with Neur. Thus, motif 2 is the only conserved motif present in all the clones that interact with Neur. It is also absent from m2, which is the only family member that does not interact with Neur and fails to inhibit Notch when overexpressed. Deletion and point mutation analysis of Tom further demonstrates that motif 2 is important for Neur binding: (1) truncated versions of Tom lacking motif 2 did not interact with Neur in the two-hybrid assay; (2) internal deletion of this motif strongly impaired interaction in the two-hybrid assay; (3) alanine substitution of either 11 or 4 residues of motif 2 also impair interaction. It is concluded that motif 2 is important for Neur binding (Bardin, 2006).

Activation of DSL signaling by Neur is regulated by multiple mechanisms. A first level of regulation operates at the transcriptional level, both along the DV axis in the early embryo and within proneural clusters during imaginal development. A second level of regulation is seen during asymmetric division of the SOP with the unequal partitioning of Neur at mitosis. This study identifies a third level of regulation based on the inhibition of Neur by Brd family members. All Brd family members (with the exception of m2) interact in the yeast two-hybrid assay with the E3 ubiquitin ligase Neur. The overexpression of Brd genes specifically inhibits Neur-dependent Notch signaling events and leads to a defect in Dl endocytosis. Conversely, loss of the Brd-C that contains six out of the ten Brd genes results in ectopic Dl endocytosis and ectopic expression of the Notch target gene sim in the early embryo. Finally, physical interaction of Tom with Neur appears to inhibit the interaction of Neur with its substrate Dl. A model is proposed whereby proteins of the Brd family antagonize Neur-mediated Dl signaling by inhibiting the interaction of Dl with Neur (Bardin, 2006).

Precise positioning of the mesectoderm results from the integration of different activities that are more broadly distributed along the DV axis. The DV gradient of nuclear Dorsal is interpreted to establish large domains of gene expression. The twist gene is expressed in a large ventral territory that encompasses the mesoderm, whereas the expression of the snail gene becomes restricted to the mesoderm. Twist and Dorsal activate the expression of the sim gene whereas Snail represses it. Neur-dependent Dl signaling in the mesoderm is thought to further restrict sim expression to cells in direct contact with the mesoderm. The signaling activity of Dl is thought to be restricted to the mesoderm because its endocytosis is tightly restricted to the mesoderm in stage 5 embryos. While transcriptional regulation of neur in ventral cells likely contributes to this spatial regulation, it cannot on its own account for the mesoderm-specific regulation of Dl endocytosis. Indeed, high levels of transcripts are detected in ventral cells outside the mesoderm and low levels of transcripts are detected all around the embryo. This suggests that a posttranscriptional inhibitory mechanism exists to ensure that Neur is not active outside the mesoderm. This study shows that Brd proteins inhibit Neur-mediated Dl endocytosis and Notch signaling in nonmesodermal cells. It is also shown that ectopic expression of Tom inhibits the endocytosis of Dl in the mesoderm. This suggests that the repression of the expression of Brd-C genes in the mesoderm is important for Neur to be active in this tissue. Inhibition of Tom expression (and possibly of the other Brd-C genes) in the mesoderm depends on the mesoderm-specific repressor Snail. Accordingly, the ectopic expression of Brd genes in ventral cells of snail mutant embryos may explain the loss of Dl endocytosis and Notch activation that was previously observed in these embryos. It is therefore suggested that the Brd-C genes represent the hypothesized Snail target gene X proposed to act as a negative regulator of Notch signaling and Dl endocytosis (Morel, 2003). Thus, the sharp boundary of Snail expression appears to define the ventral limit of Brd family gene expression, hence the dorsal limit of Neur activity and Dl signaling. In summary, these data support a model whereby the Brd genes prevent ectopic Notch activation in the early embryo and contribute to DV patterning by restricting the mesectoderm territory to a single row of cells (Bardin, 2006).

The function of the Brd genes is probably not restricted to the early embryo. Indeed, several Brd genes are also strongly expressed during early neurogenesis in the embryo as well as in the proneural clusters of the eye, leg, and wing imaginal discs. Proneural cluster expression of the Brd genes may be important to restrict, in space and/or time, the activity of Neur during the process of SOP determination. While Neur appears to be primarily expressed in the presumptive SOP, there is also evidence that Neur may also be expressed at low levels in non-SOP cells. In particular, low-level expression of Neur in non-SOP cells is occasionally seen using neur^P72Gal4. It is hypothesized that Brd may act to antagonize this low level of Neur activity in proneural cluster cells. Interestingly, the expression of the neur gene in SOPs is accompanied by the transcriptional repression of the mα gene by Su(H) in SOPs. The expression of other Brd family genes is excluded from SOPs, suggesting that they may also be repressed by Su(H). Conversely, the positive regulation of Brd gene expression by Notch in non-SOP cells correlates with a loss in Dl signaling activity in these cells. It is therefore speculated that the Brd genes contribute to amplify an initially weak difference in Dl signaling activity between presumptive SOP and non-SOP cells (Bardin, 2006).

The role proposed for the Brd genes in lateral inhibition remains to be investigated. It was found that the deletion of the Brd-C is largely embryonic lethal. However, a few homozygous Brd-C1 escaper flies are observed. These Brd-C1 flies show no detectable defects in bristle density. While this observation indicates that the Brd-C does not play an essential role in the process of SOP selection, the possibility remains that the mα and m4 genes act redundantly with genes of the Brd-C in this process (Bardin, 2006).

This study shows that all Brd family members (with the exception of m2) interact with Neur in the yeast two-hybrid assay. Interaction of Tom with Neur was further confirmed by coimmunoprecipitation experiments. Importantly, interaction of Tom with Neur correlates with a decrease in the amount of Dl immunoprecipitated by Neur, without affecting the levels of Neur and/or Dl. It is noted, however, that TomΔ2, which interacts weakly with Neur, can still inhibit interaction of Dl with Neur in this assay. The ability of Tom to decrease the Dl-Neur binding in this assay has led to a model whereby Brd family members antagonize Neur-mediated Dl signaling by inhibiting the Neur-Dl interaction. This model is consistent with the observations that overexpression of Tom has no effect on Neur protein levels. It is also consistent with the observation that Tom blocks the activity of NeurC701S. The latter may act in a dominant-negative manner by titrating DSL ligands. Accordingly, Tom could prevent NeurC701S from titrating DSL ligands. Similarly, the failure of Tom to suppress the wing phenotype induced by Mib1C1205S is consistent with the observation that Tom does not bind Mib1 and cannot, therefore, prevent Mib1C1205S from titrating DSL ligands. Whether Brd family members inhibit Neur by competing with Dl for overlapping binding sites remains to be investigated (Bardin, 2006).

These studies have focused on the interaction between Neur and a single Brd family member for the sake of consistency. Tom was chosen because (1) it includes all four conserved motifs present in the various Brd family members; (2) it is the Brd gene that aligns best with the single Anopheles Brd gene; (3) its overexpression gives a strong gain-of-function phenotype; and (4) it is expressed at high levels in stage 5 embryos. Whether all Brd family members similarly act by inhibiting the Neur-Dl interaction remains to be fully investigated. Because all Brd family members (with the exception of m2) have been shown to inhibit Neur-mediated Notch signaling, it is likely that all Brd family members similarly inhibit Neur. This in turn raises the question of the role of the two additional conserved motifs found at the C terminus of Ocho, Tom, mα, m4, and m6 that are also conserved in the single Bombyx and Anopheles Brd homologs (Bardin, 2006).

While Neur has homologs in vertebrates and Xenopus Neur has been suggested to regulate Dl signaling during early neurogenesis, no obvious homologs of the Brd genes are detectable in vertebrate sequenced genomes. This does not, however, exclude the possibility that vertebrate genes encoding Brd-like inhibitors exist. Indeed, motif 2 of Brd may be too short to reliably detect possible Brd homologs in vertebrate genomes by sequence alignments (Bardin, 2006).

This study has shown that Brd family members interact with Neur and block Neur-mediated Dl endocytosis. The activity of the Brd-C is required to spatially restrict Dl signaling along the DV axis in the early embryo (Bardin, 2006).

Dorsal-ventral pattern of Delta trafficking is established by a Snail-Tom-Neuralized pathway

The intracellular trafficking of the Notch ligand Delta plays an important role in the activation of the Notch pathway. This study addresses Snail-dependent regulation of Delta trafficking during the plasma membrane growth of the mesoderm in the Drosophila embryo. Delta is retained in endocytic vesicles in the mesoderm but expressed on the surface of the adjacent ectoderm. This trafficking pattern requires Neuralized. A protocol based on chromosomal deletion and microarray analysis has led to the identification of tom (see Bearded) as the target of snail regulating Delta trafficking. Snail represses Tom expression in the mesoderm and thereby activates Delta trafficking. Overexpression of Tom abolishes Delta trafficking and signaling to the adjacent mesoectoderm. Loss of Tom produces mesoderm-type Delta trafficking in the entire blastoderm epithelium and an expansion of mesoectoderm gene expression. It is proposed that Tom antagonizes the activity of Neuralized and thus establishes a sharp mesoderm-mesoectoderm boundary of Notch signaling (De Renzis, 2006).

The Neuralized-dependent trafficking of Delta in the signal-sending cell is required for Notch activation in the receiving cell. Therefore, the activity of Neuralized must be tightly controlled between the signal-sending and signal-receiving cell in order to ensure the correct pattern of Delta trafficking and signal polarity. This study has followed the snail-mediated modulation of Delta trafficking during the cellularization of the Drosophila embryo. The concomitant formation of cell membranes and activation of zygotic transcription has offered some unique advantages for the experiments described in this work. Most importantly, the growth of the plasma membrane is timed with the zygotic expression of snail and neuralized and thus allows a precise staging protocol (De Renzis, 2006).

The experiments demonstrate that snail regulates the mesoderm-specific trafficking of Delta by repressing the expression of tom, and presumably of the other brd genes. In the ectoderm, where the brd class genes are expressed and snail is not, Delta and Notch Extra-Cellular Domain (NECD) have a predominantly cell surface localization. Obvious endocytic vesicles containing these proteins do not accumulate in ectodermal cells. Such vesicles do form in the mesoderm where Snail represses tom expression, and in tom^−/− embryos, where the NECD-Delta vesicles extend into the ectoderm. The vesicular trafficking of Delta and NECD characteristic of mesodermal cells requires the ubiquitin ligase Neuralized. Overexpression of Tom recapitulates the loss of function phenotype of neuralized, consistent with the view that Tom may normally function by opposing the role of Neuralized in Delta trafficking. Neuralized expression is dynamic and extends to the lateral region of the embryo, beyond the mesoderm-ectoderm boundary. Thus, on its own it cannot explain the restriction of vesicles to the mesoderm. It is proposed instead that Tom creates a functional boundary for Neuralized by suppressing its activity in the ectoderm. In agreement with this model, in tom^−/− embryos the expression of the mesoectoderm gene sim is extended dorsally (De Renzis, 2006).

The expression of sim is regulated by the maternal Dorsal nuclear gradient that directly or indirectly specifies all ventral cell fates. The mechanisms that precisely position sim expression to one row of cells, however, are not completely understood. sim can respond to the Dorsal gradient and can in principle be expressed in the entire ventral region of the embryo. Recent studies suggest that Notch signaling restricts the expression of sim to the mesoectoderm by relieving Suppressor of Hairless [Su(H)]-mediated repression of sim. Su(H) is uniformly distributed throughout the early embryo and represses sim expression in the ectoderm (De Renzis, 2006).

The precision of sim expression and its one cell diameter reflect the bias that zygotic expression of tom introduces on maternal Notch signaling in that region of the embryo. If the mesoderm cells that do not express Tom are the only cells that retain Neuralized activity and can internalize Delta ligand, they might be the only cells capable of sending signal. The snail repression will allow sim expression outside the mesoderm, and thus only those cells could be effective signal recipients. According to this model, the last snail-expressing cell of the mesoderm becomes the signal-sending cell and its immediate dorsal neighbor becomes the signal-receiving cell; i.e., the mesoectoderm. In the absence of Tom, the dynamic expression of Neuralized, which extends dorsally, would make more cells competent to send Notch signal. Indeed, the dorsal expression of sim is more pronounced at the end of mesoderm invagination at the time when the expression of Neuralized has extended to the neuroectoderm (De Renzis, 2006).

The molecular mechanisms by which Tom functions are most likely related to its interaction with Neuralized; this has been demonstrated in a yeast-two hybrid genomic screening (Giot, 2003). Tom may inhibit the ubiquitin ligase activity of Neuralized or it could compete for the interaction between Delta and Neuralized. Future work will be necessary to discriminate between these two possibilities. It will also be important to test the activity of the other Brd family members in the regulation of Neuralized activity. At least eight bearded-like genes (m2, m4, m6, mα, bob, brd, tom, and ocho) have been identified in the Drosophila genome. An interesting possibility is that different genes in this family may have different effects on Delta trafficking. Any difference may provide important clues into the regulation of the Notch pathway (De Renzis, 2006).

DEVELOPMENTAL BIOLOGY

Embryonic

See the embryonic expression pattern of neur at the Berkeley Drosophila Genome Project Patterns of Gene Expression Site.

During cellularization, neur is expressed in the ventral region in the anlagen for the entire mesoderm, mesectoderm, and possibly the medial most row of ectodermal cells. Another neurogenic locus, mastermind, behaves similarly.

During germ band elongation, NEUR transcripts are found throughout the ectoderm. When neuroblasts begin to segregate, NEUR becomes restricted to a subset of cells in a pattern of segmentally repeated stripes. There are also neuroblasts that express neur in the anlage of the brain and in the stomatogastric nervous system (Boulianne, 1991).

Larval

Neuralized is required for the development of the adult nervous system. It is expressed in sensory organ precursors in the wing discs, leg discs, and haltere disc In the eye disc, neur is expressed strongly in cells of the posterior side of the morphogenetic furrow, and weakly on the anterior side. Neur is expressed in regions of cell proliferation including the brain and ventral ganglia. neur is also expressed in follicular cells in oogenesis (Boulianne, 1991).

The origin of a set of precisely located sense organs in the notum and wing of Drosophila has been studied in transformant flies where lacZ is expressed in the progenitor cells of the sense organs (the sensory mother cells) and in their progeny. neuralized mutant allele neu^A101 is a lacZ enhancer trap line in the neu locus that can be used as a marker of SOP determination. The temporal pattern of appearance and divisions is described for the sensory mother cells that will form the eleven macrochaetes and the two trichoid sensilla of the notum, and five campaniform sensilla on the wing blade. The complete pattern of sensory mother cells develops in a strict sequence that extends over most of the third larval instar and the first 10 h after puparium formation. The delay between the onset of lacZ expression and the first differentiative division ranges from 30 h, in the case of the earliest mother cells, to 2 h for the latest mother cells. The first division shows a preferential orientation that is also specific for each sensory mother cell. Up to this stage, there is no marked difference between the three types of mechanosensory organs (Huang, 1991).

Frizzled/PCP-dependent asymmetric Neuralized expression determines R3/R4 Fates in the Drosophila eye

Planar cell polarity (PCP) is a common feature in many epithelia, reflected in cellular organization within the plane of an epithelium. In the Drosophila eye, Frizzled (Fz)/PCP signaling induces cell-fate specification of the R3/R4 photoreceptors through regulation of Notch activation in R4. Except for Dl upregulation in R3, the mechanism of how Fz/PCP signaling regulates Notch in this context is not understood. The E3-ubiquitin ligase Neuralized (Neur), required for Dl-N signaling, is asymmetrically expressed within the R3/R4 pair. It is required in R3, where it is also upregulated in a Fz/PCP-dependent manner. As is the case for Dl, N activity in R4 further represses neur expression, thus, reinforcing the asymmetry. Neur asymmetry is show to be instructive in correct R3/R4 specification. These data indicate that Fz/PCP-dependent Neur expression in R3 ensures the proper directionality of Dl-N signaling during R3/R4 specification (del Alamo, 2006).

PCP establishment in the eye depends on the specification of photoreceptors R3 and R4 in two steps. First, Fz signaling occurs at higher levels in R3, and second, as a consequence, Dl signaling is directed from R3 to the R4 precursor, where N specifies R4 fate. This study shows that neur is required for proper Dl-N signaling directionality in the R3/R4 pair. In the absence of neur, defects occur in R3/R4 cell-fate specification and PCP. Importantly, neur expression is upregulated in R3 in a Fz/PCP-dependent manner. Finally, this study shows that the asymmetry in neur expression is required for PCP specification (del Alamo, 2006).

Neur is an E3-ubiquitin ligase known to enhance Dl signaling in a variety of Dl-N mediated processes, including lateral inhibition or lateral specification events (e.g., pIIb to pIIa specification in sensory organ development). This study shows that neur is required for lateral specification in R3 for Dl to signal to R4. Analysis of R3/R4 Dl mosaics revealed that the Dl mutant cell always acquires R4 fate, while the wt cell acquires R3 fate. This is consistent with neur analysis showing that in 94.2% of the cases, ommatidia mutant only in R3 showed a PCP defect, indicating that neur is required only in R3, the signal-sending cell (del Alamo, 2006).

There is, nevertheless, a difference between the PCP phenotypes of Dl and neur mosaic ommatidia: Dl mosaics show reversed polarity (chirality flips) when R3 is mutant, while the equivalent neur^IF65 mosaics show mostly a symmetric phenotype (89.5% of ommatidia displaying chirality defects). It is likely that the cold-sensitive neur^IF65 allele is not null and it is not clear if remaining Dl activity is present in the absence of Neur, accounting for the difference (del Alamo, 2006).

Mib1, another E3-ubiquitin ligase-regulating signaling by Dl and Serrate (Ser, the other N ligand in flies), has no effect on PCP specification. These results are in agreement with data showing that Neur and Mib1 have complementary functions. Taken together, the data indicate that neur but not mib1 is required for R3/R4 specification (del Alamo, 2006).

Previous studies suggested that neur has a permissive role in Dl-N signaling. In lateral inhibition processes, neur is expressed in proneural clusters, whereas in asymmetric cell division, Neur is selectively inherited by one of the daughter cells. In either case, Neur makes the cell in which it is expressed competent for Dl signaling. In the eye, Dl is enriched in R3 as a result of Fz signaling, and this study provides evidence that Neur is enriched in R3 and that this enrichment is also regulated by Fz/PCP signaling. While neur is initially expressed in both cells, the data indicate that Fz/PCP-dependent R3 upregulation of neur is necessary and sufficient for Dl signaling directionality. Since Neur affects Dl activity posttranslationally, Dl is still upregulated in R3 when Neur is misexpressed. This implies that the elimination of the difference in Neur levels between the R3/R4 precursors affects the direction of Dl-N signaling. These data indicate that the Neur expression asymmetry, mediated by Fz/PCP signaling, is instructive for R3/R4 specification (del Alamo, 2006).

The phenotypes resulting from Neur misexpression are relatively mild. Only when both Dl and Neur are coexpressed, chirality defects are induced, suggesting that differential expression of both factors in the R3/R4 cell pair is instructive for cell fate. Furthermore, other factors could also be present in R3/R4 precursors to ensure robustness of the cell-fate decision. These observations suggest a complex network of molecular interactions between Fz/PCP and Notch signaling (del Alamo, 2006).

Effects of mutation or deletion

neuralized mutants display no ventral cuticle and undergo hypertrophy of the central nervous system (Jurgens, 1984). The neural hyperplasia caused by mutations in neur can be suppressed by the presence of another neurogenic mutation (Brand, 1988). Mutant alleles of neur cause hypertrophy in nautilus expressing mesodermal cells (Corbin, 1991).

Loss of any one of several neurogenic genes of Drosophila results in overproduction of embryonic neuroblasts at the expense of epidermoblasts. The activities of the dominant, gain-of-function proteins indicate that Notch functions as a signal transducing receptor during ectoderm development. Production of antineurogenic Notch proteins in embryos deficient for the other neurogenic genes allowed functional dependencies to be established. Delta, mastermind, bigbrain, and neuralized appear to function in elaboration of a signal upstream of Notch. Genes of the Enhancer of split complex act after Notch. The cytoplasmic domain of Notch contains nuclear localization sequences that function in cultured cells, and one of the Notch antineurogenic proteins, the cytoplasmic domain, accumulates in nuclei in vivo (Lieber, 1993).

The Notch pathway plays a key role in the formation of many tissues and cell types in Metazoans. Notch acts in two pathways to determine muscle precursor fates. The first is the 'standard' Notch pathway, in which Delta activates the Notch receptor, which then translocates into the nucleus in conjunction with Su(H) to reprogram transcription patterns and bring about changes in cell fates. The second pathway is poorly defined, but known to be independent of the ligands and downstream effectors of the standard pathway. The standard pathway is required in many different developmental contexts; it was of interest to determine if there is a general requirement for the novel pathway. The novel Notch pathway is required for the development of each of five examined cell types. Holonull Notch mutants (mutants null for maternal and zygotic Notch) have a more extreme phenotype than null mutants for Su(H), Delta, neuralized or mastermind. In Notch holonull embryos, clusters of 10 or 15 eve expressing RP2-like cells are found in place of a normal single RP2. The phenotype for the other neurogenic genes is far less severe. Notch and other neurogenic genes are involved in the determination of the mesectoderm and the visceral mesoderm. The Notch holonull phenotype is more severe in both cases than that of other holonull embryos. These results indicate that the novel pathway is a widespread and fundamental component of Notch function. Both Notch pathways operate in the differentiation of the same cell types. In such cases, the novel pathway acts first and appears to set up or limit the size of equivalence groups. The standard pathway then acts within the equivalence groups to limit individual cell fates (Rusconi, 1999).

Proneural enhancement by Notch overcomes Suppressor-of-Hairless repressor function in the developing Drosophila eye: Effect of neuralized mutation

The receptor protein Notch plays a conserved role in restricting neural-fate specification during lateral inhibition. Lateral inhibition requires the Notch intracellular domain to coactivate Su(H)-mediated transcription of the Enhancer-of-split Complex. During Drosophila eye development, Notch plays an additional role in promoting neural fate independent of Su(H) and E(spl)-C, and this finding suggests an alternative mechanism of Notch signal transduction. Genetic mosaics were used to analyze the proneural enhancement pathway. Proneural enhancement involves upregulation of proneural gene expression in single cells that will become neurons. In Drosophila eye development, Notch (N) is required for proneural enhancement in addition to lateral inhibition. The molecular mechanism of proneural enhancement has not been determined. As in lateral inhibition, the metalloprotease Kuzbanian, the EGF repeat 12 region of the Notch extracellular domain, Presenilin, and the Notch intracellular domain are required. By contrast, proneural enhancement becomes constitutive in the absence of Su(H), and this leads to premature differentiation and upregulation of the Atonal and Senseless proteins. Ectopic Notch signaling by Delta expression ahead of the morphogenetic furrow also causes premature differentiation. It is concluded that proneural enhancement and lateral inhibition use similar ligand binding and receptor processing but differ in the nuclear role of Su(H). Prior to Notch signaling, Su(H) represses neural development directly, not indirectly through E(spl)-C. During proneural enhancement, the Notch intracellular domain overcomes the repression of neural differentiation. Later, lateral inhibition restores the repression of neural development by a different mechanism, requiring E(spl)-C transcription. Thus, Notch restricts neurogenesis temporally to a narrow time interval between two modes of repression (Li, 2001).

In the developing eye, lateral inhibition restricts the proneural gene atonal (ato) to individual R8 photoreceptor cells, which found each ommatidium. Earlier, ato must first have reached levels of activity sufficient to sustain expression by autoregulation, in conjunction with its bHLH heterodimer partner encoded by daughterless (da) and with a zinc-finger protein encoded by senseless (sens). Such 'proneural enhancement' depends on N and Dl but not on Su(H) or E(spl)-C. Clones of cells mutant for the E(spl)-C or for Su(H) lead to neural hyperplasia because they lack lateral inhibition, but clones of cells mutant for N or Dl show reduced neural differentiation because they lack proneural enhancement. These divergent phenotypes show that proneural enhancement occurs by a mechanism distinct from that of lateral inhibition (Li, 2001).

Mosaic analysis with Notch pathway mutations have been used to elucidate the mechanism of proneural enhancement. Requirements similar to those of canonical N signaling for processed forms of Dl, Notch EGF repeats 10-12, and proteolytic processing of the N intracellular domain have been found. Proneural enhancement is independent of any Su(H)-mediated gene activation but is mimicked by the complete absence of Su(H) protein, and this indicates that proneural enhancement depends on the disruption of Su(H)-mediated gene repression (Li, 2001).

The phenotypes of other mutations can be compared to the E(spl) or N phenotypes. A neurogeni