BIOLOGICAL OVERVIEW

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).

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).

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).

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 neurogenic mutant phenotype indicates a role in lateral inhibition, not in proneural enhancement. A hyponeural phenotype indicates a requirement in proneural enhancement (Li, 2001).

Different forms or complexes of N intracellular domain might be required to antagonize Su(H)-mediated repression during proneural enhancement from those that coactivate Su(H)-mediated gene transcription. The possible role of bib, mam, and neur in proneural enhancement has not been assessed. The bib gene encodes a transmembrane protein required for lateral inhibition in embryonic neurogenesis. Ommatidia that are mutant for bib contain occasional extra photoreceptor cells, and some ommatidia have multiple R8 cells. Ato expression begins and progresses normally, but posterior to the morphogenetic furrow small clusters of two or three cells, instead of single cells as in the wild type, often retain Ato expression. Sections through the adult retinas often reveal ommatidia with extra photoreceptor cell rhabdomeres, both of the R8/R7 small rhabdomere class and of the larger R1-R6 outer photoreceptor class. Since bib affects lateral inhibition only slightly, it is possible that an equally subtle requirement for bib in proneural enhancement might be undetected in these experiments (Li, 2001).

These findings suggest a model for proneural enhancement. The release of N intracellular domain in response to Dl derepresses genes that are repressed by Su(H). The relevant targets do not require Su(H)-mediated transcriptional activation, so deletion of Su(H) mimics N signaling. The mechanism contrasts with lateral inhibition. N signaling provides N intracellular domain as a coactivator for Su(H), which is essential for the transcription of E(spl)-C. Lateral inhibition cannot proceed in the absence of Su(H) because blocking repression by Su(H) is not sufficient for E(spl)-C transcription (Li, 2001).

neuralized represents one of the strong neurogenic mutants in Drosophila. Mutants of this class display, among other phenotypes, a strong overcommitment to neural fates at the expense of epidermal fates. The role of neu during adult development was analyzed by using mutant clonal analysis, misexpression of wild-type and truncated forms of Neu, and examination of genetic interactions with N-pathway mutations. neu has been found to be required cell-autonomously for lateral inhibition during peripheral neurogenesis and for multiple asymmetric cell divisions in the sensory lineage. In contrast, neu is apparently dispensable for other N-mediated processes, including lateral inhibition during wing vein development and wing margin induction. Misexpression of wild-type Neu causes defects in both peripheral neurogenesis and wing vein development, while a truncated form lacking the RING finger is further capable of inhibiting formation of the wing margin. In addition, the phenotypes produced by misexpression of wild-type and truncated Neu proteins are sensitive to the dosage of several N-pathway components. Finally, using epitope-tagged Neu proteins, Neu was localized to the plasma membrane and a novel morphology to the sensory organ precursor cells of wing imaginal discs was revealed. Collectively, these data indicate a key role for neu in the reception of the lateral inhibitory signal during peripheral neurogenesis (Lai, 2001a).

During the preparatory stages of this study, similar results concerning the phenotypes of adult neu clones were reported by Yeh, 2000, including the tufting phenotype of neu^A101 clones and a balding phenotype of neu^IF65 clones. These results extend the observations of Yey, 2000, in demonstrating a requirement for neu in both socket-shaft and in sheath-neuron cell fate choices. In fact, neu is required for all steps during PNS development that depend on N activity, including lateral inhibition in proneural clusters and three asymmetric cell divisions in the sensory lineage. A strict requirement for neu during lateral inhibition of the R8 photoreceptor fate has also been demonstrated. Since the neu enhancer trap A101 is active in vein cells, analogous to its expression in SOPs, one might have expected neu to function in both settings. However, neu is not required for certain other N-dependent processes in larval development, including formation of the wing margin and lateral inhibition during vein development. There is precedent for such a discrepancy in the expression and apparent function of neurogenic genes. For example, deletions of the E(spl)-C fail to induce defects in wing margin integrity, despite the observations that multiple E(spl) genes are active along the wing margin by reporter or in situ analysis, and mutant clones of the E(spl)-C fail to activate Cut at the wing margin. It may be that there is an overlapping or redundant function of neu that operates in the restriction of vein fates. Although there is a single Neu ortholog in Drosophila, two other Drosophila genes encoding NHR-domain proteins have been identified. To date all Neuralized orthologs identified, from multiple invertebrate and vertebrate species, share the following structure: two copies of a novel domain termed the neuralized homology repeat (NHR) followed by a C-terminal RING finger (Nakamura, 1998). The functional relationship of these proteins to Neu, if any, remains to be determined (Lai, 2001a).

An important conclusion of this work concerns the autonomy of neu function, at least with respect to lateral inhibition within proneural clusters. Yeh (2000) similarly concludes that neu functions autonomously; however, that study was based primarily on characterization of the hypomorphic allele neu^A101. The null allele neu^IF65 similarly behaves autonomously both in adult phenotype as well as with respect to cell fate choices assayed during imaginal disc and pupal development. The autonomy of neu appears to be contradictory to the reported localization of neu transcript and enhancer trap activity to the SOP, a cell fate inhibited by N signaling. A possible reconciliation is that very low levels of neu, below those found by conventional means of detection, may be sufficient for lateral inhibition. A parallel situation may exist for N itself, since subdetectable levels of nuclear N^IC are sufficient for target gene activation. Upregulation of neu in the SOP might then be a consequence of its particular transcriptional regulation that might not actually reflect a function with respect to the SOP-epidermal fate decision. Alternatively, upregulation of neu in the SOP might be required for successive alternative cell fate decisions in the sensory lineage, which also depends on neu (Lai, 2001a).

The first point to consider in constructing a model for Neu function is the role of the only previously identified protein domain in Neu, the RING finger. Diverse functions have been ascribed to various RING fingers, including functioning as DNA-, RNA-, and protein-interaction domains. However, recent reports have concluded that RING fingers may function generally as E3 ubiquitin ligases. The effect of deleting the Neu RING finger can be interpreted as leading to a strong increase in its activity, since much higher levels of wild-type Neu are required to generate antimorphic phenotypes seen with low levels of NeuDeltaRING. A similar effect is observed when the C-terminal RING finger is deleted from the Drosophila inhibitor of apoptosis-1 (DIAP-1); the ability of truncated DIAP-1 to block cell death is strongly increased relative to the full-length protein. It is hypothesized that the C-terminal RING finger of Neu may possess ubiquitin ligase activity that negatively regulates Neu by recruitment of the ubiquitination machinery (Lai, 2001a).

The unusual behavior of the full-length Neu protein in overexpression assays must be considered. In contrast to the report of Yeh, 2000, it was found that Neu overexpression produces both gain- and loss-of-function phenotypes. Lower levels of ectopic Neu result in loss of sensory organs and truncation of wing veins, while higher levels of Neu expression result in tufted sensory organs and wing vein thickening. The former phenotypes phenocopy over-activation of the Notch pathway and are the opposite of the phenotype of neu clones in PNS development, while the latter phenotypes resemble a failure of Notch-pathway activity and are similar to the phenotype of neu clones in PNS development (Lai, 2001a).

The ability of Neu to induce both gain- and loss-of-function phenotypes when overexpressed is most consistent with a model in which Neu functions as part of a multiprotein complex. Under conditions of elevated expression, the formation of the active complex may be encouraged, resulting in a gain-of-function phenotype. However, under conditions of highly elevated expression, components of the complex are titrated into inactive minicomplexes, causing a loss-of-function phenotype. This progression of causing gain-of-function phenotypes at lower levels and loss-of-function phenotypes at higher levels is indeed what is observed with Neu misexpression (Lai, 2001a).

Although a model in which the Neu RING finger may have ubiquitin ligase activity is favored, there is ample precedent for RING fingers to function as protein-interaction domains; indeed, the two functions need not necessarily be exclusive. In addition, both the complexity of the NHR domain and its rarity in the Drosophila proteome also make it an excellent candidate to mediate specific protein-protein interactions. Thus, the domain structure of Neu provides further reason to hypothesize that, in accord with its behavior in overexpression assays in vivo, Neu may function as part of a multiprotein complex. Indeed, deletion of the RING finger domain results in a protein (NeuDeltaRING) with potent dominant-negative activity. Misexpression of the single-NHR derivatives Neu-NHR1, Neu-NHR2, and NeuDeltaNHR1, causes either a mild or no phenotype with respect to N-regulated cell fate decisions, though this model might predict they should have dominant negative activity similar to that of NeuDeltaRING. It is possible that two NHR domains are required to interact with the appropriate target in the N pathway, although it is also possible that these single-NHR proteins are either unstable or inappropriately localized in vivo (Lai, 2001a).

The final points to consider in models of Neu function are its apparent localization to the plasma membrane and its cell-autonomous function, at least with respect to the adoption of the SOP fate; these conclusions are in general agreement with the report by Yeh (2000). Both of these are characteristics of the N receptor as well. N is epistatic to neu; a duplication of the N locus alleviates the neu null phenotype and overexpression of constitutively activated N bypasses the requirement of neu. Placement of Neu at the plasma membrane is based on misexpression of tagged proteins, and thus awaits verification with antibodies specific to Neu. However, a reasonable model that incorporates these observations is that Neu functions in a multiprotein complex that is somehow involved in the activation of the N receptor at the cell membrane. Recent observations that cis-interactions between Dl and N may be important in regulating the ability of a cell to send and respond to Dl signals suggest a further possibility that Neu may modulate Dl-N interactions within the same cell. Current efforts are aimed at identifying Neu-interacting proteins, which may make evident the molecular function of Neu in the N pathway (Lai, 2001a).

The role of neuralized during eye development is examined in this paper. Neur is required in a cell-autonomous fashion to restrict R8 and other photoreceptor fates and is involved in lateral inhibition of interommatidial bristles but is not required for induction of the cone cell fate. The latter contrasts with the absolute requirement for Suppressor of Hairless and the Enhancer of split-Complex for cone cell induction. Using gain-of-function experiments, it is further demonstrated that ectopic wild-type and truncated Neur proteins can interfere with multiple N-controlled aspects of eye development, including both neur-dependent and neur-independent processes (Lai, 2001b).

Thus neur is required only for a subset of N-dependent cell fate choices. Notably, neur is essential for lateral inhibition of the R8 photoreceptor fate. Thus, neur is essential for lateral inhibitory processes involving two distinct populations of imaginal disc cells, R8 cells and sensory organ precursors. In light of these findings, it is curious that neur is dispensable for lateral inhibition during wing vein determination. N also mediates a variety of inductive events, and neur is required for some of these (determination of the mesectoderm) but not for others (determination of the wing margin, induction of cone cells). Overall, there does yet not appear to be an obvious way to categorize all Neur-dependent N-mediated processes (Lai, 2001b).

Although N is known to be involved in induction of the cone cell fate, the precise role of the N pathway in this process in unclear. N signaling via Su(H) activates expression of D-Pax2 in cone cells; however, cone cell development in D-pax2 mutants is abnormal but not eliminated. E(spl)bHLH proteins are also expressed in cone cells, and this expression [as well as other aspects of retinal E(spl)bHLH expression] is Su(H)-dependent. In addition, cone cells fail to differentiate in eyes mutant for either Su(H) or E(spl)-C. These results suggest that the full canonical N pathway is required for cone cell induction. Because the requirement for E(spl)-C in cone cell induction is cell-autonomous, one possibility is that E(spl)bHLH proteins may repress the activity of another repressor of the cone cell fate. The ETS-domain repressor Yan has recently been shown to be capable of directly repressing at least two genes that are expressed in cone cells (D-pax2 and prospero) and may thus be a target of E(spl)bHLH repression during cone cell induction (Lai, 2001b and references therein).

The RING finger domains from several otherwise unrelated proteins have recently been shown to have ubiquitin ligase activity, suggesting a model in which Neur may directly ubiquitinate a target protein whose degradation is required for N-pathway activity. The dominant-negative activity of NeurRF might then be reasonably interpreted as an isoform that can bind its cognate target but is unable to mediate its degradation, resulting in a failure of N signaling. Although we have shown that endogenous Neur is required for only a subset of N-controlled processes, we find that ectopic Neur and NeurRF proteins are able to affect a wide variety of N-pathway-dependent processes, including those that require, and others that are independent of, endogenous neur. Examples of the latter class include the ability of Neur and NeurRF to interfere with lateral inhibition of wing veins and the ability of NeurRF to compromise formation of the wing margin and growth of the retinal portion of the eye disc. These observations suggest that Neur affects the function of a 'core' component of the N pathway. Finally, it has been shown that in two different settings, during lateral inhibition of sensory organ precursors and of R8 cells, neur acts cell-autonomously. An attractive candidate target of Neur ubiquitin ligase activity that is consistent with all of these observations is Delta. Although activation of the N pathway by Delta is nonautonomous, it has been shown that Delta also autonomously interferes with the ability of a cell to activate the N pathway. Degradation of Delta by Neur might then autonomously potentiate the ability of a cell to receive a signal and activate the N pathway. Tests of this hypothesis are currently underway (Lai, 2001b).

The Drosophila gene neuralized has long been recognized to be essential for the proper execution of a wide variety of processes mediated by the Notch (N) pathway, but a deeper understanding of its role in the pathway has been elusive. In this report, genetic and biochemical evidence is presented that Neur is a RING-type, E3 ubiquitin ligase. It has been shown that neur is required for proper internalization of Dl in the developing eye, and it has been demonstrated that ectopic Neur targets Dl for internalization and degradation in a RING finger-dependent manner, and that the two exist in a physical complex. Collectively, these data indicate that Neur is a ubiquitin ligase that positively regulates the N pathway by promoting the endocytosis and degradation of Dl (Lai, 2001c).

Previous studies have indicated that Dl not only nonautonomously activates the N pathway in neighboring cells, but can also autonomously inhibit the N pathway. For example, a reduction in Dl autonomously potentiates the ability of a cell to receive an N signal, while misexpression of Dl interferes with the ability of a cell to activate the N pathway and can induce N loss-of-function phenotypes. Neur-mediated destabilization of Dl is thus predicted to increase the ability of a cell to receive the N signal, and is therefore consistent with the observed cell-autonomous function of Neur in promoting N pathway activity. The data also suggest a possible explanation for the dominant-negative effect of Dl lacking the intracellular domain, which is predicted to be immune to regulation by Neur (Lai, 2001c).

Endogenous neur does not strongly alter Dl level or subcellular localization in the wing disc as assayed by indirect immunofluorescence microscopy, even though neur mutant cells in the eye disc display a clear defect in their ability to internalize Dl. Notably, neur transcripts are not detected in nonsensory organ precursor cells of proneural clusters, even though their requirement for neur can be demonstrated genetically. This suggests that low levels of endogenous Neur may lead to a modification of Dl levels during adult peripheral neurogenesis that is too subtle to observe in the immunofluorescence assay. Neur-mediated destabilization of Dl in the wing imaginal disc may therefore be easily visualized only in gain-of-function experiments where large amounts of Neur are present. Nevertheless, genetic mosaic experiments have convincingly demonstrated that modest changes in the ligand/receptor ratio have a strong influence on cell fate decisions controlled by the N pathway. Thus, Neur need not greatly modify the level of Dl in order to have a significant effect on the activity of the N pathway and the choice of cell fate (Lai, 2001c).

Studies of Drosophila dynamin, encoded by shibire (shi), reveal that the activity of the N pathway is particularly dependent upon endocytosis. Shi^ts1 mutants pulsed at the restrictive temperature phenocopy N mutant phenotypes, including neural hyperplasia and thickening of wing veins. Endocytosis and trafficking of Dl and N are abnormal following reduction of dynamin function, and shi function is required in both N signal-sending and -receiving cells, suggesting that both ligand and receptor are regulated by endocytosis. Curiously, misexpression of not only soluble N ^IC but also membrane-localized full-length N is completely epistatic to shi^ts, indicating that endocytosis is not essential for signal transduction downstream of the N receptor. The requirement for shi in N signal-receiving cells might be simply explained if it functions in Neur-regulated endocytosis of Dl. In this case, biasing the ligand/receptor ratio by misexpression of full-length N would be sufficient to bypass the requirement for dynamin. Consistent with this, preliminary experiments indicate that the ability of Neur to downregulate Dl is compromised when dynamin function is reduced (Lai, 2001c).

It is also emphasized that multiple mechanisms must exist for internalization of Dl, since endocytosis of Dl still occurs in wing and eye disc neur clones, and Dl accumulates in large intracellular apical vesicles in the presence of dominant-negative NeurDeltaRF. In addition, Dl localizes to vesicles in a dynamin-independent fashion in the pupal wing. It is suggested that ubiquitination of Dl by Neur represents a mechanism for regulated endocytosis and subsequent degradation of Dl, but additional means for clearance of Dl from the plasma membrane must exist, possibly including constitutive membrane recycling or pinocytosis (Lai, 2001c).

neur is essential for many, but not all, lateral inhibitory and inductive processes mediated by N. For example, neur is absolutely required for multiple steps during PNS development and for lateral inhibition of photoreceptors, but is dispensable for processes such as lateral inhibition of wing veins and induction of wing margin. Nevertheless, ectopic Neur and NeurDeltaRF could interfere with all of these processes, consistent with the proposed function of Neur in regulating Dl, a 'core' N pathway component involved in all of these processes. In light of the findings presented here, attempts were made to identify common features of Neur-dependent Dl-mediated processes (Lai, 2001c).

It has been previously observed that Dl and N expression are coincident in some settings and complementary in others. Notably, Dl and N expression are coincident or overlapping in most settings that require Neur, including in proneural clusters of the imaginal discs and the pupal notum, and in the developing eye imaginal disc. Conversely, Dl and N are complementary or highly asymmetric in Neur-independent developmental settings such as disc and pupal wing vein development, and at the wing margin. An attractive hypothesis is that Neur functions to bias the relative levels of N and Dl in settings where both ligand and receptor are coexpressed on a cell-by-cell basis; in other settings where ligand and receptor expression are highly asymmetric or exclusive, Neur may not be required (Lai, 2001c).

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date revised: 10 February 2004  

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