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).

Neuralized mediates asymmetric division of neural precursors by two distinct and sequential events: promoting asymmetric localization of Numb and enhancing activation of Notch-signaling

In the CNS, the evolutionarily conserved Notch pathway regulates asymmetric cell fate specification to daughters of ganglion mother cells (GMCs). The E3 Ubiquitin ligase protein Neuralized (Neur) is thought to activate Notch-signaling by the endocytosis of Delta and the Delta-bound extracellular domain of Notch. The intracellular Notch then initiates Notch-signaling. Numb blocks N-signaling in one of the two daughters of a GMC, allowing that cell to adopt a different identity. Numb is asymmetrically localized in a GMC and is segregated to only one of the two daughter cells. In the typical GMC-1 --> RP2/sib lineage, it was found that loss of Neur activity causes symmetric division of GMC-1 into two RP2s. It was further found that Neur asymmetrically localizes in a late GMC-1 to the Numb domain and Neur mediates asymmetric division via two distinct, sequential mechanisms: by promoting the asymmetric localization of Numb in a GMC-1 via down-regulation of the transcription factor Pdm1, followed by enhancing the Notch-signaling via trans-potentiation of Notch in a cell committed to become a sib. In neur mutants the GMC-1 identity is not altered but Numb is non-asymmetrically localized due to an up-regulation of Pdm1. Thus, both its daughters inherit Numb, which prevents Notch from specifying a sib identity. Neur also enhances Notch since in neur; numb double mutants, both sibling cells often adopt a mixed fate as opposed to an RP2 fate observed in Notch; numb double mutants. Furthermore, over-expression of Neur can induce both cells to adopt a sib fate similar to gain of function Notch. These results tie Numb and Notch-signaling through a single player, Neur, thus giving a more complete picture of the events surrounding asymmetric division of precursor cells. It was also shown that Neur and Numb are interdependent for their asymmetric-localizations (Bhat, 2011).

The results in this paper tie the localization of Numb and the signaling-processing of Notch through a single upstream player, Neur. This gives a more complete picture of the events that surround asymmetric division of neural precursor cells. The E3 Ubiquitin ligase protein Neur regulates asymmetric division of Numb and Notch-sensitive neural precursor cells in the CNS via two distinct, sequential mechanisms: first, by promoting the asymmetric localization of Insc and Numb in GMCs and second, via non-cell autonomously potentiating or enhancing the activation of Notch signaling in the Numb-negative daughter cell. While Neur is known to activate Notch-signaling by the endocytosis of Delta and the Delta-bound extracellular domain of Notch, an earlier role for it in asymmetric division via Insc and Numb localization has not been discovered. In fact, these results show that this is the primary role for Neur in generating asymmetry in the CNS. That Neur plays a secondary role or a role which is not absolute in the potentiation or enhancement of Notch signaling is indicated by the finding that in neur; numb double mutants, both sibling cells often but not always adopt a mixed fate as opposed to an RP2 fate seen in Notch; numb double mutants. If the role of Neur in Notch potentiation in this lineage is an absolute one, the same result would have been seen in neur; numb as N; numb mutants. Furthermore, over-expression of Neur can induce both cells to adopt a sib fate similar to gain of function Notch, however, the penetrance of this effect is weak (Bhat, 2011).

Previous studies had shown that the RP2-sib binary fate decision is regulated by unequal segregation of the Notch regulator Numb. The simplest interpretation of the current results would suggest that Neur is required for sib fate specification via Notch. However, the results indicate that the requirement of Neur for sib-specification to a daughter cell of a GMC-1 via regulating Notch is preceded by its requirement in GMC-1 for Numb localization, where Neur itself is expressed and becomes asymmetrically localized to the basal Numb-domain. Thus, the loss of sib identity in neur mutants appears to be mainly due to the non-asymmetric localization of Insc and Numb in GMC-1. Moreover, the levels of Pdm1 are responsive to both loss of function neur (Pdm1 level is up-regulated) and gain of function neur (the Pdm1 level is down-regulated), which are more likely a consequence of Neur function within GMC-1. This regulation of Insc and Numb localization appears to be via regulation of Pdm1 levels inside GMC-1, whereas regulating Notch processing is later and the source of Neur is from outside. By regulating asymmetric localization of Numb, Neur ensures that one of the two daughters is free of Numb, thus, later on the activation of Notch-signaling in that cell can occur. The source of Neur for this Notch processing appears to be from outside of the lineage since a division-arrested GMC-1 in numb; cyc A double mutant can still adopt a sib fate. Thus, the two roles of Neur in this lineage are distinct and separable. But then is it possible Notch has a role in the asymmetric localization of Numb and this activity of Notch is regulated by Neur? It certainly is possible but then one would have to disregard the presence of asymmetrically localized Neur in a GMC-1 as anything but of no consequence to the asymmetric division of GMC-1. It should also be pointed out that the identity of GMC-1 per se in neur is not altered, if it did, two neurons of some other identities would have been seen, not RP2s (or sibs) (Bhat, 2011).

A previous study in the sensory system of the PNS indicated that Neur protein localizes asymmetrically in the pI cell of SOP. It then segregates to pIIb, where it is thought to enhance the endocytosis of Dl to promote N activation in the pIIa cell. This represents a trans-differentiation mechanism to specify different cell fates. The results confirm the findings in SOP lineage but at the same time extends the data on SOP lineage in that this trans-determination process is a potentiation step to mediate a more efficient Notch-signaling-processing, but it is not necessarily a deterministic one. What is new and different from the SOP lineage is that Neur controls not only the asymmetric localization of Numb during mitosis, but also controls the localization of Insc, an apical cue that controls spindle orientation and participates in Numb basal localization. In neur mutant cells, Insc is no longer asymmetric indicating that Neur is somehow needed to localize Insc. The fact that Neur is somehow needed for Insc localization is also consistent with the finding that genetically insc is epistatic to neur, therefore that it is downstream of neur (Bhat, 2011).

Finally, while insc is epistatic to neur in the RP2 lineage defect in insc; neur double mutants, as for the neurogenic phenotype, neur is epistatic. This is not surprising since epistasis relationships are lineage/cell-type/tissue specific, depending upon whether or not the two genes in question are expressed in the same lineage and if the two single mutants give the same (or opposing) phenotype. Insc has no role during the neural versus ectodermal fate decisions and loss of function for insc does not cause a neurogenic phenotype, hence, the neurogenic phenotype of neur mutants is not expected to be present (epistatic) in the double mutant (Bhat, 2011).

It is clear from the results that Neur regulates asymmetric division of GMCs in the CNS. This was examined in at least two different GMCs, the GMC of the RP2/sib lineage (GMC-1 or GMC4-2a of NB4-2) and the GMC of the aCC/pCC lineage (GMC-1 or GMC1-1a of NB1-1). In neur, these GMCs symmetrically divide to generate two of the same cells, RP2 neurons in the case of GMC-1 and aCC neurons in the case of GMC1-1a. It is thought that many more GMC lineages are affected by loss of function for neur. Being a neurogenic protein, Neur is also involved in selecting neural versus ectodermal fates for the neuroectodermal cells. Due to its neurogenic property, the mutant will generate extra copies of many of the NBs in the nerve cord, which in turn, will generate more of the GMCs and neurons. Several lines of evidence indicate that symmetric division of a GMC indeed occurs at a high frequency in the CNS in neur mutants. For example, GMC-1 normally generates an RP2 and a sib, RP2 is larger than the sib and the two have distinct gene expression profiles and patterns. This is also the case for aCC/pCC pairs—they also have distinct gene expression profiles. These specific criteria were used to separate the ones that are generated by the symmetric division from those generated due to a neurogenic effect of neur mutation (Bhat, 2011).

Several additional pieces of evidence indicate a role for Neur in generating asymmetry. These include the asymmetric localization of Neur in GMCs, non-asymmetric localization of Numb in GMC-1 in neur mutants, non-asymmetric localization of Neur in numb mutants, genetic interaction results and effect on downstream players such as Pdm and Numb. All these results point to a specific role for Neur in regulating asymmetric mitosis of precursor cells (Bhat, 2011).

The results show that Neur itself is asymmetrically localized in GMC-1 to the Numb-domain and opposite to that of the Insc-domain (Neur is also localized to the basal end of several NBs, the significance of which is not known). In neur mutants, both Insc and Numb are not localized but found uniformly distributed along the cell cortex. This suggests that Neur is upstream of Insc and Numb localization but not their expression per se. The levels of Numb or Insc are also not affected in neur mutants indicating that Neur does not participate in Numb degradation (via ubiquitination, or otherwise). There is no evidence that Neur has a direct role in the localization of Numb. Do these results therefore mean Neur basically regulates the identity or the fate (i.e. gene expression program) of the GMC-1 prior to its division and therefore that Neur has only one function, which is potentiating Notch signaling? The GMC-1 was examined in neur mutants with several different GMC-1 markers (Eve, Pdm1, Zfh-1, Spectrin, etc.) and with the exception of a higher than normal Pdm1 in a late GMC-1, none of these markers were affected. A higher than normal levels of Pdm1 does not change the identity of a GMC-1. Indeed, several studies have shown that high levels of Pdm1 or its sister protein Pdm2 will induce a GMC-1 to undergo symmetric division to produce two GMC-1s and then two RP2s and two sibs. In order for a GMC-1 to change its identity, many of its genes should be turned off and a new set of genes has to be initiated. Such a drastic change does not occur in GMC-1 of neur mutants. Similarly, an identity change should result in this GMC-1 in neur mutants to produce different sets of neurons, which it does not. Instead, it produces two RP2s. Given these results and that Neur is necessary for the normal localization of Numb, whether this is directly mediated or indirectly mediated, the conclusion that Neur regulates asymmetric division at two different levels during the lineage development is based on firm grounds (Bhat, 2011).

The main question is how might Neur regulate Insc and Numb localization. A clue to this question comes from previous studies. It was shown that over-expression of Pdm POU transcription factors (Pdm1 or Pdm2) in GMC-1 causes non-localization of Insc and Numb and their segregation to both daughter cells of GMC-1; these cells then adopt an RP2 fate, with Numb blocking the N-signaling from specifying a sib fate. Pdm1 was up-regulated in GMC-1 in neur mutants and down-regulated with over-expression of Neur. This shows that the localization of Insc and Numb is altered in neur mutants indirectly via the up-regulation of Pdm protein. At the moment, it is not clear how an up-regulation of Pdm alters Insc or Numb localization. A most likely possibility is that Pdm proteins, being transcription factors, their over-expression may cause changes in the expression of genes that are needed for the proper localization of Insc and Numb but without altering the cell-identity itself (since this cell still produces RP2 neurons and not some other neurons). These conclusions are all consistent with the overall expression pattern and mutant effects of pdm genes: Pdm proteins are down-regulated in GMC-1 prior to its division, loss of function for Pdm causes loss of GMC-1 identity (Bhat, 2011).

The gain of function for these pdm genes indicates that the GMC-1 division is quite sensitive to varying levels and timings of expression of these POU proteins. For example, a high level of pdm gene expression in a GMC-1 from pdm transgenes causes a symmetric division of GMC-1 into two GMC-1s and then each of these GMC-1s generates an RP2 and a sib. In contrast, a symmetric division of GMC-1 into two RP2s can also be observed in these embryos. In this case, the cells from the GMC-1 express Zfh1; a GMC-1 does not continually express (a late GMC-1 about to divide does express Zfh1 at a very low level), a sib transiently expresses Zhf-1, and an RP2 stably expresses Zfh-1. Moreover, both these cells inherit Insc and Numb. No more cells are produced from these two cells, and each of these cells generates a projection as that of an RP2. When these genes are over-expressed for a prolonged period of time, a GMC-1 divides multiple times producing a GMC-1 and a differentiated progeny: First two unequal sized cells are observed. Only one of the two (the smaller cell) expresses markers such as Zfh1. Later on, three cells, and then five cells, etc., are sequentially seen, all in a tight cluster; from these clusters, as many as 5 RP2s are formed. Indeed, with this prolonged over-expression of pdm genes for 90 min from a heat shock promoter causes hemisegments with all the above types of divisions depending upon the time of over-expression. In contrast, it is not clear what the sensitivity range of GMC-1 is to varying concentrations in terms of the kind of division pattern generated. One clue to this comes from an earlier study, that GMC-1 in embryos carrying a duplication chromosome for the chromosomal region containing the two POU genes undergo a single self-renewing asymmetric division of GMC-1. This suggests that when the copy numbers for these genes are doubled, this presumably results in producing twice the amount of these proteins (from their own promoters), and causes a single self-renewing division. Having said that, it was also found that in neur mutants a GMC-1 rarely divides symmetrically into two GMC-1s and then each produces a sib and an RP2, or a GMC-1 dividing more than once with self-renewing asymmetric division as in pdm-GOF situations (Bhat, 2011).

Based on these results with gain of function for pdm genes, a loss of function for pdm genes should suppress the neur defects. However, this experiment is not possible to do since loss of function for the pdm genes causes loss of GMC-1 identity (GMC-1 becomes some other GMC) and therefore GMC-1 is undetectable with GMC-1 markers (Bhat, 2011).

While the exact mechanism as to how the level of Pdm1 is up-regulated in GMC-1 of neur mutants or down-regulated when Neur is over-expressed in GMC-1, is not known, one possibility is that Neur is involved in the degradation of Pdm1 in GMC-1. This scenario is most likely since Neur has the RING domain, one of the signature domains for E3 Ubiquitin-ligase proteins involved in protein degradation. Neur has also been shown to ubiquitinate proteins in vitro. One indication that Neur might be involved in the degradation of Pdm1 is the result that while ectopic or over-expression of full length neur from a transgene down-regulated Pdm1 and resulted in the same phenotype as loss of function for pdm genes, a similar ectopic or over-expression of a neur transgene missing the RING domain (Hs-neur^ΔRF) did not result in a down-regulation of Pdm1 or resulted in any phenotypes. Pdm1 appears to be specifically affected in GMC-1 of the RP2/sib lineage and not in other cells where Pdm proteins are present. Even if the up-regulation of Pdm proteins in neur mutants is via an indirect mechanism, say via factor X or Y, the results define a major role for Neur in regulating asymmetric division prior to the Notch-potentiation role of Neur: regulating Numb localization via down-regulating (directly or indirectly) Pdm proteins (Bhat, 2011).

Results from the analysis of neur, neur; numb double mutant embryos and neur gain-of-function embryos show that Neur functions to increase the efficiency of Notch-signaling but not essential for it. None of the previous studies have made this important distinction. Previous results have indicated that Neur activates Notch-signaling via endocytosis of Delta and the Delta-bound extracellular domain of Notch. However, in neur null mutants (embryos homozygous for a deficiency that removes neur completely), sib specification still occurs in ~ 10% of the hemisegments. While this may arguably be due to a partial redundancy for neur, there is another line of evidence that suggests a role for Neur in enhancing the efficiency of Notch signaling. That is, while in Notch; numb double mutants both daughter cells of a GMC-1 adopt an RP2 fate (note that for the specification of an RP2 fate Numb is needed only when there is an intact Notch-signaling), in neur; numb double mutants the daughters often adopt a mixed identity. This result indicates that Notch is still able to specify some features of a sib identity (i.e., reduced levels of Eve expression) in the absence of Neur activity. If Neur is absolutely needed for Notch signaling, the double mutant results would have been exactly the same as Notch; numb double mutants where both daughters adopt an unambiguous RP2 fate (Bhat, 2011).

In contrast, the results from Neur over-expression experiments indicate that when present at high levels Neur is able to overcome the Numb block and induce both the progeny of GMC-1 to adopt a sib fate. This phenotype is strikingly similar to the phenotype observed with the over-expression of the intracellular domain of Notch or the phenotype in numb mutants. These results suggest that over-expression of Neur leads to processing of Notch in the cell that has Numb. It is also pointed out that the source of Neur for the trans-effect on Notch-signaling need not be only from the “RP2” cell, but may also be from the neighboring cells. This is indicated by the previous result that while the GMC-1 in embryos mutant for cyclin A adopts an RP2 fate, the same GMC-1 in cyclin A; numb double mutants adopts a sib fate (Bhat, 2011).

These results show that the asymmetric basal localization of Numb in neur mutants and Neur in numb mutants is affected. This shows the interdependence of localization of these two proteins. Whether there is any initial localization of Numb or Neur in the two mutants was examined to determine if the localization of the one protein falls apart in the absence of localization of the other. However, no such initial localization was observed for either of the two proteins. It is possible that both Neur and Numb control the same pathway(s) that directly or indirectly mediates localization of the other. Perhaps Neur and Numb interact physically with each other in the cytoplasm prior to any localization and it is this Neur-Numb complex that gets localized to the basal pole; in the absence of either of the two proteins, no such complex is formed, and no localization occurs. This model has not been tested due to lack of appropriate reagents. In contrast, loss of Numb-localization in neur could be due to loss of Insc localization; loss of Neur localization in numb mutants could be more direct where Neur is downstream of Numb and Numb mediates directly or indirectly the localization of Neur. The function of Neur in GMC-1, however, appears to be required for the down-regulation of Pdm and allow localization of such proteins as Insc. Thus, Neur is both upstream and downstream of Numb in GMC-1. Another important distinction between Neur and Numb is that while non-asymmetric localization of Numb in GMC-1 will lead to both daughters of GMC-1 inheriting Numb and adopting RP2 fates, a non-asymmetric localization of Neur and inheritance of Neur by both daughters will not make them adopt an RP2 fate, but a sib fate (Bhat, 2011).

In numb mutants, the localization of Neur is affected in such a way that both daughters inherit Neur. Does this have a consequence? The results argue that unlike Numb there is no consequence to the non-asymmetric localization and segregation of Neur to both daughters. For instance, in wild type the sib cell does not inherit Neur, thus, the potentiation of Notch in this cell by Neur occurs in a cell non-autonomous mechanism (removing the extracellular domain of Notch bound by Delta) and there is no role for Neur in the sib itself. Thus, in numb mutants although both daughters inherit Neur, they still adopt a sib fate (Bhat, 2011).

Crumbs complex-directed apical membrane dynamics in epithelial cell ingression

Epithelial cells often leave their tissue context and ingress to form new cell types or acquire migratory ability to move to distant sites during development and tumor progression. Cells lose their apical membrane and epithelial adherens junctions during ingression. However, how factors that organize apical-basal polarity contribute to ingression is unknown. This study shows that the dynamic regulation of the apical Crumbs polarity complex is crucial for normal neural stem cell ingression. Crumbs endocytosis and recycling allow ingression to occur in a normal timeframe. During early ingression, Crumbs and its complex partner the RhoGEF Cysts support myosin and apical constriction to ensure robust ingression dynamics. During late ingression, the E3-ubiquitin ligase Neuralized facilitates the disassembly of the Crumbs complex and the rapid endocytic removal of the apical cell domain. These findings reveal a mechanism integrating cell fate, apical polarity, endocytosis, vesicle trafficking, and actomyosin contractility to promote cell ingression, a fundamental morphogenetic process observed in animal development and cancer (Simoes, 2022).

Drosophila NBs are an outstanding model for scrutinizing the cellular machineries underpinning an EMT-like process with high temporal and spatial resolution. While ingressing, a single NB sequentially loses AJs responding to tensile forces exerted by two pools of actomyosin: a planar polarized pool enriched at anterior-posterior junctions, which disassembles first, and a pulsatile pool at the free apical cortex, which further tugs on shrinking junctions in a ratchet-like manner. However, while actomyosin forces reduce the apical perimeter, it remained unclear how cells lose their apical domain and how polarity regulators contribute to the dynamics of apical domain loss. This study demonstrates that regulation of the Crb complex plays a key role in orchestrating apical domain loss during ingression. During early ingression, cells shrink their apical domain while retaining total levels of Crb, which is crucial for maintaining normal actomyosin in NBs and NICs (neighboring non-ingressing cells) to generate the tension balance required for normal ingression dynamics. During late ingression, Crb is rapidly lost from the apical domain, a process initiated by an interaction between Neur and Sdt, which causes the disassembly of the Crb complex. The loss of the Crb complex then precipitates the concurrent loss of the apical membrane and AJs. A similar regulatory interplay between Crb, Sdt, and Neur was also observed during early neurogenesis in the Drosophila optic lobes. Here, neural stem cells emerge from a wave front in the optic lobes rather than ingress as individual cells. Despite these topological differences, the Crb/Sdt/Neur module appears to be a common cell biological regulator of EMT during early neurogenesis (Simoes, 2022).

Endocytosis and endocytic degradation and recycling are requirements for normal NB ingression dynamics. The apical membrane of neuroepithelial cells is much more active endocytically than the basolateral membrane. Notably, Crb is endocytosed during apical contractions and re-secreted during expansions, suggesting that myosin-driven cell contact contraction promotes endocytosis, consistent with recent data from mammalian cells, whereas expansions allow for enhanced secretion. Blocking endocytosis increases surface levels of Crb and Ecad as expected, and prevents NB ingression, whereas enhancing endocytosis accelerates ingression. Moreover, endocytic trafficking plays a key role in determining ingression speed. Loss of ESCRT complex-mediated degradation appears to enhance apical Crb and slow ingression, whereas loss of Retromer-mediated recycling dramatically reduced surface Crb and accelerated ingression. In fact, Retromer-compromised embryos showed the fastest ingression speed of any condition examined, suggesting that the Retromer not only recycles Crb but also other factors that counteract apical domain loss in NBs. Crb turnover during early ingression maintains a steady Crb surface abundance. During late ingression, Crb endocytosis is enhanced during both apical contraction and expansion as a result of the disruption of the Crb-Sdt interaction by Neur, which likely makes the Crb cytoplasmic tail accessible to the Clathrin adapter AP2. AP2 binds to Crb competitively with Sdt, facilitating the rapid endocytic removal of Crb and the apical membrane (Simoes, 2022).

The relationship between actomyosin contraction, endocytosis, Crb protein levels, and their function in NB ingression illuminates the complexity and robustness of morphogenesis. Disrupting several individual molecular processes, while changing the dynamics of ingression, rarely abrogates ingression entirely. First, although endocytosis is essential for ingression, strong depletion of endocytotic regulator delayed ingression but did not prevent delamination in most cases. Similarly, a dramatic depletion of myosin, which is essential for apical constriction of NBs, extended the ingression period but did not block delamination. Evidence of the mutual dependency of endocytosis and myosin-driven contractions in NBs. Contractions foster endocytosis, whereas endocytosis promotes contractions, a co-dependency that likely finetunes ingression dynamics. Second, maintaining Crb in the apical membrane (through overexpression or expression of a non-endocytosable form of Crb) delayed ingression but did not prevent it in most cases, suggesting that Crb membrane persistence is overcome by other mechanisms such as the Neur-dependent disassembly of the Crb complex. Moreover, failure to resolve the Crb complex in late ingression caused part of the apical membrane to persist as apical plugs. Nevertheless, NB delaminate, detach from apical plugs and animals are viable, suggesting that nervous system development proceeds rather normally. Crb surface abundance is dependent on endocytosis and versicle trafficking, and Crb regulates junctional myosin which contributes to apical contraction. Together, these findings highlight the multilayered regulation of ingression through the co-dependent interactions between apical polarity, vesicle trafficking, and actomyosin contractions (Simoes, 2022).

EMT is thought to be initiated by the expression of EMT transcription factors (EMT-TFs) of the Snail, Zeb, or bHLH families that downregulate key adhesion or polarity proteins such as Ecad and Crb. NBs are specified through the combined action of proneural genes that include bHLH proteins of the Achaete-Scute complex, the Snail family protein Worniu, and the SoxB family protein SoxNeuro. However, although genes that encode Ecad and Crb are transcriptionally downregulated in NBs, this repression does not appear relevant for NB ingression. Replacing endogenous Ecad with a transgene expressing Ecad under the control of a ubiquitous promoter had no impact on NB ingression dynamics. This study shows that surface levels of Crb remained high in NBs during early ingression before Crb is rapidly removed by endocytosis during late ingression. This raises the question of how the upregulation of proneural genes in presumptive NBs elicits enhanced actomyosin contractility and endocytic removal of apical membrane and junctions (Simoes, 2022).

One proneural gene target is neur. Neur is found throughout the neuroepithelium participating in Delta-Notch-mediated lateral inhibition to select the NB from an equivalence group of 5-7 cells. Neur upregulation in ingressing NBs is thought to be part of a positive feedback that stabilizes NB fate through persistent asymmetric Delta-Notch signaling. The increase in Neur may also be important for the effective disruption of the Crb complex to destabilize the apical domain. Neur can disrupt the Crb complex across the epithelium but is normally prevented from doing so by Bearded proteins that act as inhibitors of Neur. Increasing Neur concentration may overcome this inhibition in NBs. This raises the possibility that the proneural gene-dependent upregulation of neur contributes to the timing of ingression, consistent with the observation that in Neur-depleted embryos ingression is prolonged. Furthermore, Neur may enhance actomyosin contractility in NBs seen in late ingression as was reported for Neur in the Drosophila mesoderm. It is hypothesized therefore that Neur could be a central regulator of NB selection and ingression, stabilizing NB fate, driving apical membrane constriction through actomyosin contraction, and disrupting the Crb complex to remove apical membrane and junctions. Interestingly, it was also noted that during ingression, the number of alternative isoforms of Sdt is limited to Sdt3, the isoform susceptible to Neur. Hence, NBs appear to develop the molecular competence for apical membrane removal at least in part through rebalancing Sdt splice forms (Simoes, 2022).

The loss of apical-basal polarity is an early event during EMT marked by the loss of epithelial AJs that can trigger expression of EMT-TFs and the disassembly of cell junctions. However, the findings of this study indicate that the loss of apical-basal polarity in NBs is preceded by a period (~20 min; early ingression) of ratcheted apical contractions that reduce the apical area of delaminating cells. The maintenance of normal Crb levels during early ingression is crucial for normal ingression dynamics. Crb stabilizes junctional myosin through its effector, Cyst, which is recruited to the junctional domain by the Crb complex (Silver, 2019). Although the loss of Crb or loss of Cyst causes similar reductions of junctional myosin in the neuroepithelium (Silver, 2019), NB ingression was consistently faster in Cyst-compromised embryos than in controls. In contrast, NBs in Crb-compromised embryos showed much larger variability of ingression speeds, with a small fraction of NBs ingressing rapidly while the majority was slower than controls. Thus, it is likely that Crb makes other contributions to regulating NB ingression in addition to its Cyst-mediated function in supporting junctional actomyosin. Interestingly, the mouse Crb homolog Crb2 is required for myosin organization and ingression during gastrulation. The predominant defect in Crb2-compromised mice appears to be a failure of ingression, which may be similar to the fraction of NBs showing slower than normal ingression seen with the loss of Drosophila Crb. To what extent the differences in cell behavior caused by the loss of Crb and Crb2 depend on the biomechanical specifics of the tissue context or result from differences in molecular pathways in which Crb and Crb2 operate remains to be explored (Simoes, 2022).

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).

Neur1 and Neur2 are required for hippocampus-dependent spatial memory and synaptic plasticity

Neur1 and Neur2, mouse homologs of the Drosophila neur gene, consist of two neuralized homology repeat domains and a RING domain. Both Neur1 and Neur2 are expressed in the whole adult brain and encode E3 ubiquitin ligases, which play a crucial role in the Notch signaling pathways. A previous study reported that overexpression of Neur1 enhances hippocampus-dependent memory, whereas the role of Neur2 remains largely unknown. This study aimed to elucidate the respective roles of Neur1 and Neur2 in hippocampus-dependent memory using three lines of genetically modified mice: Neur1 knock-out, Neur2 knock-out, and Neur1 and Neur2 double knock-out (D-KO). The results showed that spatial memory was impaired when both Neur1 and Neur2 were deleted, but not in the individual knock-out of either Neur1 or Neur2. In addition, basal synaptic properties estimated by input-output relationships and paired-pulse facilitation did not change, but a form of long-term potentiation that requires protein synthesis was specifically impaired in the D-KO mice. These results collectively suggest that Neur1 and Neur2 are crucially involved in hippocampus-dependent spatial memory and synaptic plasticity (Jang, 2020).

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).

Neural precursor-specific expression of multiple Drosophila genes is driven by dual enhancer modules with overlapping function

Transcriptional cis-regulatory modules (CRMs), or enhancers, are responsible for directing gene expression in specific territories and cell types during development. In some instances, the same gene may be served by two or more enhancers with similar specificities. This study shows that the utilization of dual, or 'shadow', enhancers is a common feature of genes that are active specifically in neural precursor (NP) cells in Drosophila. By genome-wide computational discovery of statistically significant clusters of binding motifs for both proneural activator (P; Scute, for example) proteins and basic helix-loop-helix (bHLH) repressor (R; Hairy/Enhancer of split (Hes) class) factors (a 'P+R' regulatory code), NP-specific enhancer modules were identified associated with multiple genes expressed in this cell type. These CRMs are distinct from those previously identified for the corresponding gene, establishing the existence of a dual-enhancer arrangement in which both modules reside close to the gene they serve. Using wild-type and mutant reporter gene constructs in vivo, P sites in these modules were shown to mediate activation by proneural factors in 'proneural cluster' territories, whereas R sites mediate repression by bHLH repressors, which serves to restrict expression specifically to NP cells. These results identify the first direct targets of these bHLH repressors. Finally, using genomic rescue constructs for neuralized (neur), it was demonstrated that each of the gene's two NP-specific enhancers is sufficient to rescue neur function in the lateral inhibition process by which adult sensory organ precursor (SOP) cells are specified, but that deletion of both enhancers results in failure of this event (Miller, 2014).

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).

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

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

The NHR1 domain of Neuralized binds Dl and mediates Dl trafficking and N signaling

Notch signaling, crucial to metazoan development, requires endocytosis of Notch ligands, such as Delta and Serrate. Neuralized is a plasma membrane-associated ubiquitin ligase that is required for neural development and Delta internalization. Neuralized is comprised of three domains that include a C-terminal RING domain and two neuralized homology repeat (NHR) domains. All three domains are conserved between organisms, suggesting that these regions of Neuralized are functionally important. Although the Neuralized RING domain has been shown to be required for Delta ubiquitination, the function of the NHR domains remains elusive. This study shows that neuralized¹, a well-characterized neurogenic allele, exhibits a mutation in a conserved residue of the NHR1 domain that results in mislocalization of Neuralized and defects in Delta binding and internalization. Furthermore, a novel isoform of Neuralized is described; it is recruited to the plasma membrane by Delta and this is mediated by the NHR1 domain. Finally, it is shown that the NHR1 domain of Neuralized is both necessary and sufficient to bind Delta. Altogether, these data demonstrate that NHR domains can function in facilitating protein-protein interactions and in the case of Neuralized, mediate binding to its ubiquitination target, Delta (Commisso, 2007; full text of article).

Neuralized contains a phosphoinositide-binding motif required downstream of ubiquitination for Delta endocytosis and Notch signaling

The Notch signaling pathway, which plays a critical role in cell-fate decisions throughout development, is regulated by endocytosis of both the ligand and receptor. Endocytosis of the Drosophila ligands, Delta and Serrate, is required in the signaling cell for signal initiation and requires one of two ubiquitin ligases, Neuralized or Mind bomb. Through in vitro binding assays an interaction has been identified between Neuralized and phosphoinositides, modified membrane lipids that mediate membrane trafficking and signaling. Interactions between phosphoinositides and Neuralized contribute to the membrane localization of Neuralized in the absence of Delta; the phosphoinositide-binding motif is required for Neuralized to endocytose Delta downstream of Delta ubiquitination. Lastly, evidence is provided that this interaction may also be important for vertebrate Neuralized function. These results demonstrate that, through interactions with Neuralized, phosphoinositides may regulate Delta endocytosis and, by extension, Notch signal transduction (Skwarek, 2007).

This study has shown that Neur interacts with PIPs in vitro through a lysine-rich region in the N terminus, and that this PIP-binding motif mediates localization to the cell surface of S2 cells in the absence of Dl. Although the PIP-binding residues are not required for Neur to bind Dl, these residues are required for Neur function during embryonic neurogenesis and, specifically, for Dl internalization. Interestingly, Neur-mediated ubiquitination does not appear to be reduced in the absence of the PIP-binding motif, suggesting that these residues, and possibly PIP binding, are required downstream of Dl ubiquitination. Lastly, evidence is presented to suggest that PIP binding may also be important for vertebrate Neur1 function. Taken together, these data demonstrate that the PIP-binding motif in Neur plays an important role in regulating ligand endocytosis downstream of ligand ubiquitination, and that it is important for the initiation of Notch signaling (Skwarek, 2007).

A role for PIPs in the signal-receiving cell has been demonstrated in previous work. For example, increases in plasma membrane PIP levels due to deletion of the phosphocholine cytidylyltransferase cct1 enhances Notch loss-of-function phenotypes due to increased endocytic activity and, consequently, lower levels of cell-surface receptor (Weber, 2003). In addition, three recent studies have demonstrated an important role of the phospholipid-binding protein Lethal giant discs (Lgd) in the regulation of Notch signaling (Childress, 2006; Gallagher, 2006, Jaekel, 2006). Lgd appears to regulate Notch trafficking at a step downstream of Hrs-dependent sorting, and PIP binding is required for this function. Lastly, overexpression of a kinase-dead version of the Drosophila class II PI-3-kinase, PI3K_68D, phenocopies and enhances Notch loss of function, although it has yet to be determined if this is due to disruption of signal initiation or transduction (MacDougall, 2004). The current data are the first to suggest a specific role for PIPs in the regulation of Notch signaling in the signal-sending cell (Skwarek, 2007).

Neur binds PIPs promiscuously in vitro; however, cell culture studies demonstrate that the PIP-binding motif contributes to constitutive plasma membrane localization, suggesting an important role for the interaction between Neur and PI(4,5)P₂. This is supported by the observation that expression of an isolated PH domain from PLC-δ that has been well demonstrated to specifically bind PI(4,5)P₂ promotes the redistribution of Neur from the plasma membrane into large intracellular puncta. Although many proteins display promiscuous binding in vitro, including most yeast PH domains, the actual in vivo localization can be mediated through a combination of PIP, protein-protein, and nonspecific electrostatic interactions. Consistent with this, it was shown that, in the absence of the PIP-binding motif, Neur can still interact with Dl, and this interaction is sufficient to recruit Neur to the cell surface. Despite this redundant mechanism for localization, the PIP-binding motif is essential for Neur function during embryonic neurogenesis since the expression of transgenes containing mutations that disrupt PIP binding in vitro cannot rescue neurogenesis in neur^−/− embryos. This appears to be due to an inability of Neur to trigger Dl endocytosis in the absence of the PIP-binding motif, suggesting an important role for the interactions mediated by this motif in Neur function in vivo (Skwarek, 2007).

Given that the results demonstrate that in vivo, in the presence of Dl, the PIP-binding mutant is capable of localizing to the plasma membrane and interacting with Dl, it was hypothesized that the PIP-binding residues are not required for the localization of Neur. It remains possible, however, that it was not possible to detect subtle differences in localization. For example, there is evidence for localized differences in plasma membrane microdomains, and the sites of endocytic internalization are probably not of uniform identity. While the specific role the PIP-binding residues may be playing in Neur function is not known, it is possible that they mediate interactions with additional endocytic effector molecules, and they may also be involved in localizing Neur to a specialized membrane subdomain that is important for signaling competent endocytosis (Skwarek, 2007).

The mechanism through which Dl endocytosis activates Notch is unknown. One model proposes that the endocytosis of Notch-bound Dl provides a mechanical force that is required for subsequent cleavage of Notch. A second model suggests that, after endocytosis, Dl is somehow modified to make it more active in a process that depends on Epsin. This modification may involve the recycling of Dl, as the receptor-binding domain of Dl fused to the low-density lipoprotein receptor sorting and recycling motif partially bypasses the requirement for Epsin (though not for ubiquitination) in Notch signal initiation. In support of this model, recycling endosomes have been shown to be required for Dl activity during Drosophila sense-organ development and in mammalian cell culture. The data do not support one model over the other; however, they do highlight the importance of the initial endocytosis step in Notch signaling, which is dependent on the PIP-binding motif in Neur. Due to the fact that Dl trafficking is blocked at endocytosis in Neur PIP-binding motif mutants, it cannot be directly assessed whether this motif, and by extension interactions with PIPs, may play additional roles in Dl trafficking downstream of endocytosis. Given that Neur interacts with multiple PIPs in vitro, and that the requirement for the PIP-binding motif appears to be downstream of Dl ubiquitination, it is possible that an interaction between Neur and phosphoinositides could be involved in additional trafficking steps, as PIPs play diverse and essential roles throughout endosomal trafficking. Consistent with this, colocalization of ectopically expressed Dl and Neur is seen in both Hrs- and Rab11-positive endosomes in Drosophila Kc cells and in vivo, suggesting a possible role for Neur in postendocytic trafficking steps. However, the presence of endogenous Neur in vivo and in Kc and S2 cells has thus far complicated efforts to further address additional requirements for Neur in Dl trafficking. Interestingly, compared to wing discs lacking exogenous Neur, ectopic expression of Neur lacking the PIP-binding motif results in higher levels of Dl both at the cell surface and in internal vesicles, suggesting that expression of this protein may interfere with the normal trafficking of Dl to the lysosome (Skwarek, 2007).

Recently, it has been demonstrated that mouse Neur2 does not appear to play a role in ligand internalization, but is involved in targeting endocytosed Dl to Hrs-positive vesicles (Song, 2006), providing further evidence that Neur is involved in additional trafficking steps. Interestingly, mNeur2 does not contain the N-terminal domain predicted to interact with PIPs in mNeur1, and instead it looks more like the Drosophila Neur isoform, Neur^PC, that displays similar localization properties to Neur^5Q (Commisso, 2007; Skwarek, 2007).

Mouse Neur1 contains a sequence in its N-terminal region that is similar to a conserved motif present in FYVE domains that has been shown to interact directly with the PI phosphate group of PI3P. It is predicted that this region is required for PIP binding in mNeur1, as it is absolutely conserved in all vertebrate Neur1 protein sequences available, and this study has shown that mutation of 3 residues in this region disrupts PIP binding in vitro. This suggests that interactions between Neur and PIPs may be involved in the function of Neur in both Drosophila and vertebrates, and it provides an interesting example of a protein in which important interactions are conserved despite large sequence changes during evolution (Skwarek, 2007).

To date, this study has assessed the overall functional consequences of loss of the PIP-binding motif during embryonic neurogenesis. Whether the interaction mediated by the PIP-binding residues is required during other Neur-mediated signaling events remains to be determined. For example, it will be interesting to see whether the PIP-binding motif is required for Neur to regulate Notch signaling during the asymmetric divisions of the sense-organ precursor cells, and whether asymmetries in PIP binding may play a role in the asymmetric localization of Neur into a single daughter of the dividing sense-organ precursor (Skwarek, 2007).

Without strict regulation, aberrant Notch signaling leads to cancer and other developmental diseases. Neur and Mib have emerged as crucial regulators of the Notch signaling pathway that are required for receptor endocytosis and signal induction, and they may also play a role in cis inhibition by ligand/receptor interactions within the same cell (Glittenberg, 2006). This study has identified PIPs as interacting with Neur in vitro, and has provided several pieces of evidence that they may also be physiologically relevant ligands in vivo. The PIP-binding motif that was identified is important for Neur function during embryonic neurogenesis, and it was demonstrated that this motif is required for Neur-mediated Dl endocytosis downstream of ligand ubiquitination. It is still unknown what this novel role for Neur may be, and whether PIPs are the critical ligands required for this function. It will be interesting to see whether Mind bomb may also play a role downstream of ligand ubiquitination and to clarify the role of phosphoinositides during ligand trafficking and Notch signaling throughout development (Skwarek, 2007).

Functional analysis of the NHR2 domain indicates that oligomerization of Neuralized regulates ubiquitination and endocytosis of Delta during Notch signaling

The Notch pathway plays an integral role in development by regulating cell fate in a wide variety of multicellular organisms. A critical step in the activation of Notch signaling is the endocytosis of the Notch ligands Delta and Serrate. Ligand endocytosis is regulated by one of two E3 ubiquitin ligases, Neuralized (Neur) or Mind bomb. Neur is comprised of a C-terminal RING domain, which is required for Delta ubiquitination, and two Neur homology repeat (NHR) domains. Previous studies have shown that the NHR1 domain is required for Delta trafficking. This study shows that the NHR1 domain also affects the binding and internalization of Serrate. Furthermore, it was shown that the NHR2 domain is required for Neur function and that a point mutation in the NHR2 domain (Gly430) abolishes Neur ubiquitination activity and affects ligand internalization. Finally, evidence is provided that Neur can form oligomers in both cultured cells and fly tissues, which regulate Neur activity and, by extension, ligand internalization (Liu, 2012).

Neur is an E3-ubiquitin ligase that plays an essential role in Notch signaling by regulating the endocytosis of Notch ligands. It contains NHR domains, which are rare and conserved between vertebrates and invertebrates but not present in viruses, bacteria, fungi, or plants. In the Drosophila proteome, besides Neuralized, there are two other NHR-containing proteins, CG3894 and Bluestreak. In mammals, proteins containing NHR domains (also known as NEUZ) include the β-catenin regulator OzzE3 and lung-inducible Neuralized-related C3HC4 RING protein (LINCR). Recent studies reported that the human homologue of Bluestreak serves to localize to the centrosome. Although the general role of the NHR domains is unclear, these domains tend to cluster, and most proteins contain two to six NHR domains. The significance of having more than one NHR domain in one protein is to yet be determined (Liu, 2012).

This study has investigated the role of the highly conserved NHR domains in Neur function. The NHR1 domain alone mediates the interaction between Neur and both Delta and Serrate. It was also shown that the NHR2 domain is required for Neur function, and while it is not required for the interaction with Notch ligands, it is involved in Dl internalization, a critical step in Notch activation. Moreover, the NHR domains play a role in Neur oligomerization, which in turn could contribute to Neur ubiquitination activity and ligand endocytosis (Liu, 2012).

The NHR1 domain mediates the interaction between Neur and the Notch ligands Delta and Serrate. Previous studies have shown that the NHR1 domain of Neur is both necessary and sufficient for the interaction with the Notch ligand Dl. Specifically, it was found that a point mutation in a highly conserved glycine residue within the NHR1 domain (G167E) abolishes the ability of Neur to bind Delta. Whether the NHR1 domain was also required to bind to Serrate, however, was unknown. In fact, in vitro studies in vertebrates suggested that the NHR2 domain in mouse Neuralized- like 1 (Neurl1) is sufficient to bind to Jagged1, the mouse orthologue of Serrate. In contrast, the current study found that the NHR2 domain is not required for Neur to bind Serrate in Drosophila and that the interaction is mediated entirely by the NHR1 domain. Other studies reported previously that the motifs on Dl and Ser that mediate the interaction with Neur are conserved. In comparisons of protein sequences, Jagged1 and Serrate share 40.7% similarity overall, while the overall similarity between Jagged1 and Dl is 33.8%. The NHR1 and NHR2 domains from Drosophila Neuralized have the same degree of amino acid similarity with the mouse Neurl1 NHR2 domain (33%). Since there is no clear correlation between protein sequence similarity and the ability of either the NHR1 or the NHR2 domain to interact with Notch ligands, whether NHR1 or NHR2 is important for interacting with ligands is likely to be species dependent. The Neur-Ser interaction was found to be abrogated by the G167E mutation in the NHR1 domain. Given that the NeurG167E mutant still retains ubiquitination activity, it is unlikely to affect overall protein folding. A previously reported structural analysis of the Drosophila NHR1 domain suggested that Gly167 resides in a hydrophobic core and that the Gly167 mutation presumably destabilizes the surrounding microenvironment. Therefore, the Gly167 mutation may result in spatial changes in the neighboring residues of the core, thus abolishing binding to ligands (Liu, 2012).

The G430E mutation reveals a distinct role for the NHR2 domain in the regulation of Neur activity and Delta trafficking. The data demonstrate that the NHR2 domain is required for Neur function in vivo. NeurG430E fails to rescue neur mutant embryos, while NeurNHR2 has some residual activity, which suggests that they affect different aspects of Neur function. The expression of NeurG430 in a heterozygous background, which does not have a neurogenic phenotype on its own, resulted in a significant increase in the percentage of neurogenic embryos, suggesting that NeurG430E has a negative effect on Neur function. In contrast, NeurNHR2 overexpression did not have any effect on heterozygous embryos, suggesting that it behaves as a loss-of-function allele. Despite the fact that the two NHR2 mutant proteins NeurG430E and NeurNHR2 behave differently, they both localize to the plasma membrane in the presence of Delta both in vitro and in vivo, and they are both capable of binding to the Notch ligands Delta and Serrate. However, both mutant proteins affect the extent of Dl internalization to various degrees. NeurG430E exhibits severely compromised ubiquitination activity and is no longer capable of inducing Delta internalization. NeurNHR2, on the other hand, retains ubiquitination activity but is much less efficient than WT Neur at directing Dl internalization in vivo or in Kc cells. The precise mechanism by which NeurNHR2 affects Delta endocytosis is unclear (Liu, 2012).

One possibility is that the NHR2 domain is required for Neur oligomerization. Neur was shown to form NHR domain-mediated oligomers by coimmunoprecipitation experiments. Therefore, the deletion of the NHR2 domain (NeurNHR2) may simply reduce the oligomerization potential of Neur, leading to a decrease in ligand endocytosis. In contrast, the point mutation (NeurG430) might disrupt the overall structure of the NHR2 domain, preventing oligomerization and resulting in a protein that has no ubiquitination activity and therefore can no longer internalize ligands. However, this model cannot fully explain the data, which show that NHR2 does not prevent Neur oligomerization and that Neur oligomerization still occurs in the absence of any NHR domains, suggesting that while the NHR domains may play a role in oligomerization, they are not necessary for this process. The data also show that although the G430E mutant loses ubiquitination activity, the double mutant containing the NHR1 deletion and the G430E mutation retains ubiquitination activity, which argues that G430E does not affect the overall folding of the NHR2 domain (Liu, 2012).

Another possibility is that the NHR1 and NHR2 domains initially form an intramolecular structure that is inactive and must be resolved for ubiquitination to promote ligand internalization. The G430E mutation may lock Neur into an intramolecular conformation through an NHR1-NHR2 interaction, such that this inactive form can still bind to Dl and Ser but cannot form oligomers and has no ubiquitination activity as a consequence of dysfunctional oligomerization. This model, in contrast to the former one, is supported by the data that demonstrate that the G430E mutant no longer forms oligomers and has no ubiquitination activity. Furthermore, when the NHR1 domain was removed from the G430E mutant (NeurNHR1G430E), the ubiquitination activity was restored, suggesting that the intramolecular loop can no longer form, whereas intermolecular interactions between NHR1 and NHR2 domains can occur. It may also explain the negative effect of NeurG430E on NeurWT: although NeurG430E has a reduced ability to bind WT Neur, it is still be able to sequester some portion of NeurWT into a nonfunctional intermolecular oligomer that can no longer ubiquitinate targets. In contrast, the deletion of the NHR2 domain would prevent intramolecular interactions but would be expected to have a reduced ability to form productive oligomers, leading to a defect in ligand internalization. Whether the NHR2 domain has additional roles in recruiting a protein(s) that promotes Notch ligand ubiquitination and endocytosis remains to be determined. Like Neuralized, other RING domain E3 ligases often function as oligomers, and they multimerize in different ways: some form heterodimers, such as Mdm2-MdmX, and some can form homo-oligomers, such as TRAF. The functional significance of RING E3 oligomerization is poorly defined. One previously proposed model is that the oligomerization of E3 ligases may functionally resemble the dimerization of receptor tyrosine kinases in such a way that autoubiquitination yields a mark that serves as a platform to assemble a signaling complex. Consistent with this idea, ubiquitination was seen in anti-Neur IPs when Dl was not present, consistent with the idea that Neur may be autoubiquitinated. Whether this autoubiquitination can initiate a cascade of further downstream ubiquitination events remains to be determined. It is possible that oligomerization is mediated via autoubiquitination and the interaction of ubiquitinated Neur with itself through a ubiquitin-binding motif (UIM). If so, then the NHR1-NHR2 interaction could also function to keep Neur in an inactive state by occluding the putative UIM. Such a model of Ubi-UIM complex formation was previously proposed for other endocytic proteins (Liu, 2012).

In summary, this study has shown that NHR domains are protein-protein interaction modules that are required for many aspects of Neur function. The NHR1 domain mediates the interaction between Neur and its targets Dl and Ser. Both NHR domains appear to regulate Neur activity by affecting its ability to form oligomers and/or interact with proteins required for the endocytosis of Notch ligands. Interestingly, NHR domains have been identified in several other proteins that are conserved between flies and humans. Whether Neur can form heterodimers with these other NHR-containing proteins and whether these heterodimers play a role in Notch signaling and other developmental processes remain to be determined (Liu, 2012).

DEVELOPMENTAL BIOLOGY

Embryonic

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).

Apoptosis ensures spacing pattern formation of Drosophila sensory organs

In both vertebrates and invertebrates, developing organs and tissues must be precisely patterned. One patterning mechanism is Notch/Delta-mediated lateral inhibition. Through the process of lateral inhibition, Drosophila sensory organ precursors (SOPs) are selected and sensory bristles form into a regular pattern. SOP cell fate is determined by high Delta expression and following expression of neurogenic genes like neuralized. SOP selection is spatially and temporally regulated; however, the dynamic process of precise pattern formation is not clearly understood. In this study, using live-imaging analysis, it was shown that the appearance of neuralized-positive cells is random in both timing and position. Excess neuralized-positive cells are produced by developmental errors at several steps preceding and accompanying lateral inhibition. About 20% of the neuralized-positive cells show aberrant cell characteristics and high Notch activation, which not only suppress neural differentiation but also induce caspase-dependent cell death. These cells never develop into sensory organs, nor do they disturb bristle patterning. This study reveals the incidence of developmental errors that produce excess neuralized-positive cells during sensory organ development. Notch activation in neuralized-positive cells determines aberrant cell fate and typically induces caspase-dependent cell death, as detected using SCAT3, a fluorescence resonance energy transfer (FRET) indicator for effector caspase activity. Apoptosis is utilized as a mechanism to remove cells that start neural differentiation at aberrant positions and timing and to ensure robust spacing pattern formation (Koto, 2011).

The Drosophila sensory organ is a typical model for the study of Notch/Delta-mediated lateral inhibition. Tracking the process of cell fate determination in each cell lineage is presumed to be effective in revealing the mechanisms behind precise pattern formation (Koto, 2011).

The first finding in this study is that bristle patterning starts in a random fashion: about 20% of neuralized-positive cells are fated to become aberrant SOP-like cells, and Notch signaling is involved in determining the fate of SOP-like cells. However, the mechanisms proposed for the production of SOP-like cells in previous reports do not coincide perfectly with the current observations. Previous studies showed that the conventional model of Notch/Delta-mediated lateral inhibition is not sufficient to produce the precise bristle pattern but that cell-autonomous interaction or filopodia-mediated intermittent Notch/Delta signaling makes lateral inhibition robust enough to suppress the neural differentiation of surrounding cells. In contrast, the current results suggest that lateral inhibition from adjacent SOPs is not the sole source of Notch activation in SOP-like cells, because a portion of SOP-like cells preceded the nearby SOPs. Also, SOP-like cells showed ongoing Notch activity even in the absence of adjacent SOPs. One possible explanation for these SOP-like cells failing to develop into sensory organs may be that they appear too early in the developmental time course and cannot complete the developmental program to become sensory organs in a cell-autonomous manner. In any case, the decrease in SOP-like cells in the N^55e11 heterozygous mutant reliably suggests that Notch activation in cells that start neural differentiation contributes to the determination of their cell fate as aberrant SOP-like cells (Koto, 2011).

Dynamic oscillation of the Notch effector gene Hes1 has been observed in neural progenitors of the developing mouse brain with the aid of a short-half-life indicator using ubiquitinated firefly luciferase. In Drosophila sensory organ development, the technical limitations of the GFP reporter make it difficult to confirm this type of oscillation pattern in Notch signaling. However, given that Notch oscillation occurs in cells in the proneural stripe regions at the beginning of SOP selection, it is conceivable that SOP-like cells might be the product of fluctuating Notch signaling at inappropriate times, developmentally speaking (Koto, 2011).

The second finding in this study is that a program of caspase-dependent cell death specifically eliminates SOP-like cells. Ablating an incipient SOP removes Notch/Delta-mediated lateral inhibition and allows a nearby epithelial cell to become the SOP, as has also been observed in the embryonic central nervous system of grasshoppers. However, the adjacent SOP-like cells never develop into sensory organs, suggesting that the fate of SOP-like cells is irreversible. By observing the nuclear morphology along with an indicator for caspase activation, it was noted that in the process of sensory organ development, only SOP-like cells showed the typical features of programmed cell death. These results indicate that programmed cell death ensures robust pattern formation by eliminating aberrantly differentiated cells (Koto, 2011).

The significance of programmed cell death in pattern formation has been well studied, especially in the development of the fly eye. Each ommatidium is composed of eight photoreceptor neurons and six support cells, consisting of four cone cells and two primary pigment cells. Between each ommatidium, remaining cells form the interommatidial lattice. Excess pigment cells are eliminated through programmed cell death. Notch functions within the interommatidial lattice to induce cell death, and the primary pigment cells send a survival signal to adjacent cells. The life-and-death fate of interommatidial cells is decided by their position and the cells to which they are attached. In the case of sensory organ formation, Notch signaling is crucial in determining the aberrant cell fate of SOP-like cells. However, Notch activation alone seems insufficient to induce programmed cell death, because the surrounding epithelial cells do not disappear, even though they exhibit high levels of Notch activation during sensory organ development. Therefore, some factor that marks neural differentiation in SOP-like cells may be required to induce cell suicide. This study found that ectopic neuralized expression did not induce the aberrant cell fate or cell death in epithelial cells, suggesting that neuralized itself is not essential in determining the aberrant cell fate of SOP-like cells. Therefore, to determine how apoptosis is induced in SOP-like cells, the effect of Notch activation in neuralized-positive cells was examined at the one-cell stage using the temporal and regional gene expression targeting (TARGET) system with tub-GAL80^ts. As reported previously, activated Notch induced the multiple-sockets phenotype. At the same time, about 50% of neuralized-positive cell lineages died, accompanied by nuclear fragmentation, causing a dramatic bald phenotype that was observed in the adult flies. These findings suggest that the combination of neural differentiation in the SOP lineage and Notch activation switches on cell death signaling. One possible future approach to searching for the killing factor expressed in SOP-like cells would be gene profiling using laser microdissection (Koto, 2011).

When the apoptotic pathway is blocked, the inhibition of cell death results in cell fate transformation. In C. elegans, cell death survivors in ced-3 mutants exhibit an ambiguous cell fate. The most disruptive alternative cell fate occurs when the remaining cells differentiate into tumor-like proliferating cells, as shown in the development of the Drosophila serotonin lineage. Under apoptosis-deficient conditions, other types of cell death occur, such as necrosis or autophagic cell death. These alternate reactions could mask the incidence of programmed cell death; therefore, it is possible that the role of the apoptotic pathway has been missed in the case of sensory organ development. This study has shown that SOP-like cells differentiate into epithelial cells when the cell death pathway is blocked. Time-lapse imaging made it possible to trace the transient fate of dying SOP-like cells, revealing the contribution of programmed cell death in the SOP selection process. Although the function of apoptosis has been emphasized in various developmental processes, the principle message is that several pathways exist to overcome the appearance of excess or aberrant cells and to make the developmental process more robust. This study reveals that programmed cell death plays an important role in overcoming innately induced developmental errors and contributing to robust neural cell selection (Koto, 2011).

Regulation of epithelial polarity by the E3 ubiquitin ligase Neuralized and the Bearded inhibitors in Drosophila

Understanding how epithelial polarity is established and regulated during tissue morphogenesis is a major issue. This study identified a regulatory mechanism important for mesoderm invagination, germ-band extension and transepithelial migration in the Drosophila embryo. This mechanism involves the inhibition of the conserved E3 ubiquitin ligase Neuralized by proteins of the Bearded family. First, Bearded mutant embryos exhibited a loss of epithelial polarity associated with an early loss of the apical domain. Bearded regulated epithelial polarity by antagonizing neuralized. Second, repression of Bearded gene expression by Snail was required for the Snail-dependent disassembly of adherens junctions in the mesoderm. Third, neuralized was strictly required to promote the downregulation of the apical domain in the midgut epithelium and to facilitate the transepithelial migration of primordial germ cells across this epithelium. This function of Neuralized was independent of its known role in Notch signalling. Thus, Neuralized has two distinct functions in epithelial cell polarity and Notch signalling (Chanet, 2012).

In this study, the Brd genes were identified as Sna molecular targets acting as key regulators of gastrulation. Second, it was shown that the Brd genes are required for the stabilization of the apical domain in the ectoderm at stages 6–8. Third, it was shown that Neur is the main target of Brd for the maintenance of epithelial polarity and that neur is required for the remodelling of the midgut epithelium at stage 10. Fourth, this function of Neur in the regulation of epithelial polarity is independent of its known activity in Notch signalling. Fifth, a model is proposed for the regulation of PGC transepithelial migration based on the regulation in space and time of the activity of Neur by its Brd inhibitors (Chanet, 2012).

The genetic analysis of gastrulation in Drosophila has indicated that Sna regulates both apical constriction and SAJ disassembly. How Sna regulates these two processes has remained unknown and the existence of a zygotic gene 'x' corresponding to a Sna target repressed in the mesoderm with the ability to block SAJ disassembly has been postulated. This study shows that the Brd genes have the proposed properties of this 'x'gene. Indeed, it was shown that Brd proteins inhibited junction disassembly in sna mutant embryos. Moreover, the early expression of the Tom and mαgenes was sufficient to inhibit apical constriction and delay mesoderm invagination. One possible interpretation is that Brd proteins regulate gastrulation by inhibiting Neur. As loss of zygotic neur did not perturb junction disassembly nor mesoderm invagination, it is suggested that maternal Neur may be sufficient for these processes. Alternatively, Brd proteins might regulate gastrulation through an unknown Neur-independent mechanism (Chanet, 2012).

One of the main findings of this study is the identification of the E3 ubiquitin ligase Neur as a regulator of epithelial polarity in the Drosophila embryo. This activity of Neur did not involve Dl–Notch signalling. How Neur stabilizes both the apical domain and adherens junctions is at present unclear; the loss of the apical domain may possibly result from increased apical endocytosis, as seen during apical constriction in Xenopus. Interestingly, increased apical endocytosis in Cdc42-compromised embryos correlated with adherens junction destabilization. Similarly, inhibition of Rab11 led to a loss of Crb preceding adherens junction. Whether increased Neur activity disrupts the balance between apical endocytosis and recycling remains to be investigated. Importantly, cross-regulatory interactions between apical polarity complexes, Rho/Rok signalling, actomyosin and adherens junctions make it difficult to predict the molecular targets of Neur on the sole basis of phenotypic analysis. Nevertheless, components and/or regulators of the Crb complex are possible candidates. Indeed, similarly to Brd proteins, Crb seems to stabilize epithelial polarity during cell–cell intercalation at germ-band extension. Moreover, high Neur levels correlate with low Crb in wild-type neuroblasts and neuroblast polarity did not depend on Brd activity. Finally, loss of Brd led to the premature apical relocalization of adherens junctions in the ectoderm, an effect that is consistent with the proposed role of Crb in excluding Baz and adherens junctions from the apical domain. Whether and how Neur counteracts the function of Crb will require the molecular identification of the Neur targets (Chanet, 2012).

Another important finding is the identification of Neur and Brd as developmental regulators of the transepithelial migration of primordial germ cells (PGCs). Importantly, the migration of PGCs may be used as a model system to investigate the molecular basis of the process of transepithelial migration that is important for innate immune response in humans, as neutrophils migrate across the endothelium to reach sites of inflammation or infection. PGC migration was shown to involve a developmentally regulated remodelling of the midgut epithelium as suggested earlier and that Neur and Brd are essential regulators of this remodelling. Further analysis of the role of Neur in this morphogenetic regulation may shed light on conserved mechanisms involved in transepithelial migration (Chanet, 2012).

Finally, the finding that Neur functions both in Notch signalling and cell polarity raises the exciting possibility that these two processes are coupled. Indeed, Neur-dependent endocytosis of Dl occurs along the basolateral membrane of signal-sending cells. Thus, epithelial remodelling by Neur might facilitate Dl activity along the basolateral membrane. It is speculated that Neur may mechanistically link Dl signalling and epithelial polarity remodelling to ensure efficient signalling. As Neur plays an essential role in Notch signalling in Drosophila but not in mammals, it is possible that the evolutionarily conserved function of Neur is not the regulation of Notch activity but rather the remodelling of epithelial cells. Accordingly, Neur would have been co-opted for the regulation of Dl signalling in insects. It will thus be of interest to examine the potential role of Neur in epithelial polarity in mammals (Chanet, 2012).

A re-examination of the selection of the sensory organ precursor of the bristle sensilla of Drosophila melanogaster

The bristle sensillum of the imago of Drosophila is made of four cells that arise from a sensory organ precursor cell (SOP). This SOP is selected within proneural clusters (PNC) through a mechanism that involves Notch signalling. PNCs are defined through the expression domains of the proneural genes, whose activities enables cells to become SOPs. They encode tissue specific bHLH proteins that form functional heterodimers with the bHLH protein Daughterless (Da). In the prevailing lateral inhibition model for SOP selection, a transcriptional feedback loop that involves the Notch pathway amplifies small differences of proneural activity between cells of the PNC. As a result only one or two cells accumulate sufficient proneural activity to adopt the SOP fate. Most of the experiments that sustained the prevailing lateral inhibition model were performed a decade ago. This study re-examined the selection process using recently available reagents. The data suggest a different picture of SOP selection. They indicate that a band-like region of proneural activity exists. In this proneural band the activity of the Notch pathway is required in combination with Emc to define the PNCs. A sub-group in the PNCs was found from which a pre-selected SOP arises. The data indicate that most imaginal disc cells are able to adopt a proneural state from which they can progress to become SOPs. They further show that bristle formation can occur in the absence of the proneural genes if the function of emc is abolished. These results suggest that the tissue specific proneural proteins of Drosophila have a similar function as in the vertebrates, which is to determine the time of emergence and position of the SOP and to stabilise the proneural state (Troost, 2015).

This study has re-examined the development of the SOP of the MC (macrochaetae) using recently available reagents. Evidence was found that strongly suggests that the range of the Notch signal is restricted to the next cell: The elevated expression of Notch activity reporter Gbe+Su(H) around the SOP is observed only in adjacent cells. In addition, cells of PNCs that are not able to receive the Notch signal, but can send a strong signal to adjacent wildtype cells, cannot prevent a wildtype cell from adopting the SOP fate at a distance of two cell diameters away. Likewise, cells that are not able to send a signal cannot be prevented by wildtype SOPs from adopting the SOP fate more than one cell diameter away. These results suggest that the discovered filopodia of the SOP, which contact more remotely located cells do not extend the range of the inhibitory signal to these cells (Troost, 2015).

This study reveals the existence of a band of proneural activity. The PNCs are regions of elevated proneural activity in this band, rather than discrete clusters. In the band, the Notch pathway exerts an additional novel function, which defines the extent of the PNCs. In the absence of Notch function, most cells in the proneural band accumulate high levels of proneural activity that allows them to become SOPs. Thus, the pathway suppresses the proneural activity and the SOP fate in cells located between the PNCs in the proneural band. The short range of the Notch signal indicates that it is probably local mutual signalling among direct neighbours that generates the necessary Notch activity (mutual inhibition). The expression of Dl and Ser and the overall activity of Gbe+Su(H) (with exception of the halos) is unchanged in the absence of Ac and Sc. This suggests that the widespread activity of Notch in the notum that prevents most cells in the proneural band to become SOPs is not influenced by the proneural factors. It provides a baseline activity of Notch that suppresses the proneural activity in the band to prevent the formation of ectopic SOPs (Troost, 2015).

The presented results indicate that a subgroup within the PNCs exists, which is operationally defined via the requirement of the activity of Neur. The existence of a subgroup has previously been suggested on basis of experiments with a temperature sensitive allele of Notch. These data and the ones presented here, suggest that the cells of the subgroup require Notch activity that is stronger than the baseline activity to be inhibited from adopting the SOP fate. This increase in activity is generated by the nascent SOP through a Neur enhanced Dl signal: This study found that if only one cell in the subgroup is neur positive, it can prevent all other neur mutant members to adopt the SOP fate. Thus, initiating the expression of Neur first, is a critical step for a cell to adopt the SOP fate, since it allows a cell to strongly inhibit its neighbours. The inhibitory signal prevents the accumulation of sufficient proneural activity to also activate Neur in the neighbours. This inhibition is probably reflected in the observed halo of Gbe+Su(H) expression around SOPs. The findings are in good agreement with a previous study that showed that the level of Neur in a cell is a critical factor for the formation of the SOP of the microchaetae (mc) (Troost, 2015).

Loss of Notch activity results in expression of Neur and a dramatic increase in proneural activity in all cells of the PNC. Moreover, the nascent SOP, which contains the highest proneural activity, is the only cell that initiates Neur expression during normal development and expression of neur is abolished in ac sc mutant discs. These data indicate, that high proneural activity is required for the expression of Neur. Thus, the cell in the subgroup with the highest proneural activity is the cell that will express Neur first. The expression of Neur enables it to inhibit its neighbours from adopting the SOP fate by suppressing their proneural activity (Troost, 2015).

One generated through mutual signalling, which is not regulated by Ac and Sc and is sufficient to inhibit all cells in the proneural band outside the neur subgroup to become SOPs. This signalling requires the ubiquitously expressed Mib1 and antagonises the activity of Ac, Sc and Da. However, there is residual activity of Notch in mib1 mutants sufficient to prevent most cells from adopting the SOP fate. This residual activity is generated either independently of E3 ligases or by another unknown E3-ligase. In any case this component contributes to the baseline activity of the Notch pathway in addition to Mib1. The second activity on top of the baseline activity in the neur subgroup is generated by a Neur mediated strong signal from the nascent SOP. This signal suppresses the proneural activity of the other members of the neur subgroup. It is dependent on proneural activity, which initiates the expression of Neur. Thus, lateral inhibition is probably operating after the emerging SOP reaches a threshold of proneural activity. It serves to prevent the formation of supernumerary SOPs in the neur group and assures that other cells can generate the necessary SOP in case the selected one is lost (Troost, 2015).

How is the neur subgroup defined? It was found that the PNCs are small in their beginning, comprising the number of cells typical for the subgroup. These cells probably also constitute the small groups of SOPs observed in early third instar discs mutant for Psn. It is likely that E(spl)m8-SM expression defines this subgroup since this study shows that it is expressed in a small group of cells from which the SOP arises. This construct contains only one E box, the binding sites for Ac and Sc, and response to high proneural activity. It is therefore believed that the cells of the early PNC are the neur group and possess the highest proneural activity (Troost, 2015).

During normal development, a cell with more proneural activity is already recognisable at the early phase of the PNCs. This suggests the existence of a pre-selection mechanism that assures that one cell in the neur-subgroup is advanced in its development. Evidence for such a mechanism has been also previously found during rescue experiments studying the function of the proneural genes Ac and Sc. This study has obtained additional experimental evidence for this pre-selecting mechanism: In neur clones one of the cells is advanced in its development towards the SOP fate. Moreover, clonal analysis of kuz and Psn mutants revealed that wildtype cells at positions in the PNC where the SOP arises cannot be prevented from adopting the SOP fate, even if a mutant SOP that cannot be inhibited (e.g., kuz mutant), is its neighbour. The mutant cells can generate a strong inhibitory Notch signal. This indicates that the pre-selecting mechanism renders the wildtype SOP immune to the signal. The nature of this mechanism is not clear, nor whether it is always the same cell in a cluster that is selected (Troost, 2015).

Recent work demonstrated that in the eye disc a regulatory loop between Da and Emc assures correct expression of both factors and results in their complementary expression. Consequently, loss of emc function results in an increase of expression of Da. The consequences of this up-regulation for the proneural state of the mutant cells have not been investigated in detail. A published study focused on the eye imaginal disc and revealed that a few of the mutant cells in clones could adopt the neural fate. The neural cells do not express Runt, a marker expressed in the normal neural cells. Thus, the loss of emc does not result in the complete determination of the neural fate. The state of the vast majority of the cells in clones remained unknown. This study observed up-regulation of proneural activity in emc clones already in early third instar wing imaginal discs, indicating that it is an immediate reaction to the loss of emc function. Some of these cells progress to become SOPs. The increase in proneural activity was also observed in emc clones of the leg disc. Thus, the cells of imaginal discs must be permanently inhibited from adopting a proneural state through the activity of Emc. It has to be pointed out that this situation is remarkably similar to that in the early vertebrate embryo, where all cells of the blastula adopt the proneural state unless they are inhibited through BMP signalling. The cells of the neural plate maintain the proneural state due to the presence of BMP antagonists (Troost, 2015).

In the eye disc and during oogenesis expression of Emc is regulated by the Notch pathway. This study failed to find evidence that supports a regulatory relationship between Emc and the pathway in the notum during SOP development, since the loss of Psn function did not affect the expression of EMC. However, it has been previously shown that the expression of Emc along the dorso-ventral boundary in the wing primordium depends on the activity of the Notch pathway. This correlates well with the finding that this domain is independent of the activity of Da. However, the genetic network of the wing is significantly different from that in the notum. For example Notch signalling induces the expression of Wg along the D/V boundary. However, its expression in the proximal wing and in the notum is independent of the activity of the Notch pathway. This appears to be true also for the different domains of expression of Emc (Troost, 2015).

This study found that the function of ac and sc is dispensable for bristle development in the absence of emc function. How is the SOP fate initiated in these emc ac sc triple mutant cells? It is believed that the activity of Da is sufficient for SOP development in this situation for the following reasons: 1) Da is expressed ubiquitously and is required for the formation of all external sense organs. 2) Strong over-expression of Da induces bristle formation in cells that lack the whole AS-C. In contrast, over-expression of Sc fails to induce SOP formation in the absence of Da. 3) Da can form homodimers that bind to the same DNA target sequences as Ac/Da and Sc/Da heterodimers in bend-shift assays. 4) Loss of emc activity increases the activity of Da. This study show that this increase is independent of the activity of Ac and Sc. 5) The results show that Da regulates the expression of sca independently of Ac and Sc. 6) It has been shown that the mammalian homologue of Da, E2A, acts without its class II partners during B-cell development. Thus, it is likely that in the absence of function of emc, ac and sc, Da forms active homo-dimers that initiate the required neural program (Troost, 2015).

While it is clear that the activity of Ac and Sc is required during normal development, the formation of normal bristles in their absence after concomitant loss of emc function raises the question about their function. The data suggest that an important function is the neutralisation of Emc through formation of heterodimers with it or with Da. This releases Da from inactive heterodimers with Emc. The neutralisation of Emc by Ac and Sc, which are expressed in precise spatial and temporal regulated patterns, allows the differentiation of neural precursors at the correct position and time. The recent finding that a Sc variant without its transactivation domain is fully active fits well to this view of the function of Ac and Sc. Thus, through their intricate and dynamic expression, Ac and Sc and other tissue specific proneural factors determine when and where a neural precursor cell develops. In this view the function of the tissue-specific proneural genes of Drosophila, is similar to that in mammals where their orthologs also promote differentiation of neural precursors in a proneural field, the neural plate, at correct positions and time (Troost, 2015).

Based on the current results, a working model is suggested for the selection of the SOP of the MC: The differential expression of Emc defines a proneural band in the notum with changing proneural activity. The PNCs in this band are determined and positioned through the cluster-like expression of Ac and Sc, which increases the proneural activity at these positions. A baseline of activity of the Notch pathway generated by mutual inhibition prevents cells between the PNCs to accumulate high levels of proneural activity. In addition, it prevents cells located in the PNC, but outside the neur group, to accumulate high proneural activity required for adopting the SOP fate (Troost, 2015).

In the PNCs, expression of Ac and Sc neutralise Emc. Consequently, the proneural activity increases dramatically, since the released Da can form homodimers and/or heterodimers with Ac or Sc. The cells of the initial small PNCs later constitute the neur subgroup. The cells of this subgroup have the highest level of proneural activity and experience this activity also for the longest time. Within this subgroup a cell is pre-selected to become the SOP by a so far unidentified mechanism. Hence, it is the first to reach the threshold level of proneural activity required to initiate the expression of Neur. The expression of Neur enables it to efficiently inhibit the other cells of the subgroup through lateral inhibition. As a consequence these cells never accumulate sufficient proneural activity to activate Neur expression and to become a SOP. The strong signal also further activates the expression of Brd proteins that inhibit the activation of Neur, which might be accidentally activated weakly in one of the neighbours. This activation contributes to the precision of determination process. Thus, a combination of mutual and lateral inhibition mediated by the Notch pathway operates in the PNC during the determination of the SOP. Only the lateral inhibition component depends on proneural activity through transcriptional activation of expression of Neur (Troost, 2015).

The model differs from the lateral inhibition model in the following points: No feedback loop between expression of Dl and proneural activity and, hence, no differential Dl expression is required. Instead the future SOP is pre-selected and advanced in its development. Subgroups within a proneural band defined through its requirement of Neur exist. In this subgroup the activation of the expression of Neur is critical for SOP development since it enables a cell to potently inhibit its neighbours. The pre-selection mechanism favours a cell at the right position to initiate the expression of Neur before the others of the Neur group and therefore secures its development as SOP. Moreover, the existence of mutual signalling explains the inhibition of cells in the proneural band outside the subgroup without the necessity of signalling of Dl over longer distances (Troost, 2015).

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 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 functions cell-autonomously to regulate a subset of Notch-dependent processes during adult Drosophila development

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).

neuralized is essential for a subset of Notch pathway-dependent cell fate decisions during Drosophila eye development

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 it has been shown that endogenous Neur is required for only a subset of N-controlled processes, it is found 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).

Drosophila Neuralized is a ubiquitin ligase that promotes the internalization and degradation of Delta

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).

Fat facets and Liquid facets promote Delta endocytosis and Delta signaling in the signaling cells: A proposed role for Neur in endocytosis of Delta

Endocytosis modulates the Notch signaling pathway in both the signaling and receiving cells. One recent hypothesis is that endocytosis of the ligand Delta by the signaling cells is essential for Notch activation in the receiving cells. Evidence is presented in strong support of this model. In the developing Drosophila eye Fat facets (Faf), a deubiquitinating enzyme, and its substrate Liquid facets (Lqf), an endocytic epsin, promote Delta internalization and Delta signaling in the signaling cells. While Lqf is necessary for three different Notch/Delta signaling events at the morphogenetic furrow, Faf is essential only for one: Delta signaling by photoreceptor precluster cells, which prevents recruitment of ectopic neurons. In addition, the ubiquitin-ligase Neuralized (Neur), which ubiquitinates Delta, is shown to function in the signaling cells with Faf and Lqf. The results presented bolster one model for Neur function in which Neur enhances Delta signaling by stimulating Delta internalization in the signaling cells. It is proposed that Faf plays a role similar to that of Neur in the Delta signaling cells. By deubiquitinating Lqf, which enhances the efficiency of Delta internalization, Faf stimulates Delta signaling (Overstreet, 2004).

In neur mutants, Delta accumulates on the membranes of signaling cells and Notch activation in neighboring cells is reduced. These results support a role for Neur in endocytosis of Delta in the signaling cells to achieve Notch activation in the neighboring receiving cells, rather than in downregulation of Delta in the receiving cells. Because neur shows strong genetic interactions with lqf and both function in R-cells, Neur and Lqf might work together to stimulate Delta endocytosis. Lqf has ubiquitin interaction motifs (UIMs) that bind ubiquitin. One explanation for how Neur and Faf/Lqf could function together is that Lqf facilitates Delta endocytosis by binding to Delta after its ubiquitination by Neur. This is anattractive model that will stimulate further experiments (Overstreet, 2004).

The roles of cis-inactivation by Notch ligands and of neuralized during eye and bristle patterning in Drosophila

The receptor protein Notch and its ligand Delta are expressed throughout proneural regions yet non-neural precursor cells are defined by Notch activity and neural precursor cells by Notch inactivity. Not even Delta overexpression activates Notch in neural precursor cells. It is possible that future neural cells are protected by cis-inactivation, in which ligands block activation of Notch within the same cell. The Delta-ubiquitin ligase Neuralized has been proposed to antagonize cis-inactivation, favoring Notch activation. Cis-inactivation and the role of Neuralized has not yet been studied in tissues where neural precursor cells are resistant to nearby Delta, however, such as the R8 cells of the eye or the bristle precursor cells of the epidermis. Overexpressed ligands block Notch signal transduction cell-autonomously in non-neural cells of the epidermis and retina, but do not activate Notch nonautonomously in neural cells. High ligand expression levels are required for cis-inactivation, and Serrate is more effective than Delta, although Delta is the ligand normally regulating neural specification. Differences between Serrate and Delta depend on the extracellular domains of the respective proteins. Neuralized acts cell nonautonomously in signal-sending cells during eye development, inconsistent with the view that Neuralized antagonizes cis-inactivation in non-neural cells. It is concluded that Delta and Neuralized contribute cell nonautonomously to Notch signaling in neurogenesis, and the model that Neuralized antagonizes cis-inactivation to permit Notch activity and specification of non-neural cells is refuted. The molecular mechanism rendering Notch insensitive to paracrine activation in neural precursor cells remains uncertain (Li, 2004).

One difference between neural and non-neural cells may be neur, which has been proposed to relieve cis-inactivation cell autonomously by endocytosing Dl, or to promote paracrine signaling in experiments where neur appears nonautonomous. Neur might make non-neural cells less sensitive to cis-inactivation, so that only high Delta levels would be effective (Li, 2004).

Cell autonomy of neur function in the eye was investigated using FLP-mediated mitotic recombination in neur heterozygous larve to induce cell clones homozygous for neur¹, a loss of function neur allele. Mitotic recombination was induced late in the third larval instar to generate small neur mutant clones. Mosaic adult eyes were sectioned and the cellular contribution of neur mutant cells recorded. In many cases presence of neur mutant cells was associated with changes in the number of photoreceptor cells. Ommatidia with too many or too few photoreceptor cells were both observed, as for other neurogenic mutations. Less often, ommatidia containing one or more neur mutant cells differentiated 8 photoreceptor cells in the normal arrangement. Forty such mosaic ommatidia were examined in more detail to identify any cells where neur function might be dispensable (Li, 2004).

Ommatidia almost never developed normally with neur mutant R8 cells. Only a single example was found. If neur activates N signaling by antagonizing cis-inactivation, then one would expect that neur would be required in cells where N is active, but dispensable where N is inactive. On this basis neur should not be required in R8 cells. By contrast the data suggested that R8 is where neur is most important. It has been found that Dl is also required in R8 cells. The possibility is excluded that either neur or Dl is required directly in the execution of the R8 differentiation pathway because many ectopic R8 cells differentiate in large neur or Dl mutant clones, or when the whole eye is mutant. Instead the data suggest that ommatidia with neur or Dl mutant R8 cells could not develop normally because neur acts in R8 to promote Dl-mediated activation of N in neighboring cells (Li, 2004).

To explore further when neur acts autonomously or nonautonomously, other aspects of retinal N signaling were also examined. During ommatidial development, Notch signaling breaks the symmetry of the R3/R4 pair. Dl from R3 activates N in R4. neur mutant cells were five times as likely to take R4 fate as R3 fate. Thus neur is important for the nonautonomous signaling activity of Dl from the R3 cell but not required autonomously for activity of N in the R4 cell. Only rarely can a neur mutant R3 cell activate N in a neighboring R4 cell, but neur R4 cells can be activated in response to wild type R3 cells (Li, 2004).

Further data from abnormally constructed ommatidia support the importance of neur in R3. These were ommatidia where the R3/R4 pair remained symmetrical. 17 symmetrical ommatidia were found with two R3 cells in place of R3/R4. In 3 such ommatidia both R3-like cells were mutant for neur. In 13 of the other 14 cases the cell in the location that should normally have become R3 was neur mutant; in a single case the cell positioned to become R4 was neur mutant. These symmetrical ommatidia indicate that when R3 cells lack neur function, the neighboring cell receives insufficient Dl signaling to take R4 fate and instead is transformed into a second R3 (Li, 2004).

N signaling is further required for R7 specification. N is activated in R7 precursors by Dl from neighboring R1 and R6 cells. R1 and R6 act redundantly but if both R1 and R6 are Dl mutant then the R7 precursor adopts R1/6-like morphology. R7 was frequently neur mutant in normally-constructed ommatidia, so neur is not essential in the R7 precursor cell. R1 and R6 were never both mutant for neur in normal ommatidia. One ommatidium was found in which both R1 and R6 were neur mutant. In this ommatidium the cell in the R7 position was wild type for neur but had R1/6-like morphology. These results indicate that neur, like Dl, is not required for N activity in the R7 cell itself. neur may be required nonautonomously in R1 and R6 for proper R7 specification (Li, 2004).

Pleiotropic fitness effects of the Tre1-Gr5a region in Drosophila melanogaster

Understanding how genotypic variation influences variation in brain structures and behavioral phenotypes represents a central challenge in behavioral genetics. In Drosophila, the neuralized (neur) gene plays a key role in development of the nervous system. Different P-element insertional mutations of neur allow the development of viable and fertile adults with profoundly altered behavioral phenotypes that depend on the exact location of the inserted P element. The neur mutants exhibit reduced responsiveness to noxious olfactory and mechanosensory stimulation and increased aggression when limited food is presented after a period of food deprivation. These behavioral phenotypes are correlated with distinct structural changes in integrative centers in the brain, the mushroom bodies, and the ellipsoid body of the central complex. Transcriptional profiling of neur mutants revealed considerable overlap among ensembles of coregulated genes in the different mutants, but also distinct allele-specific differences. The diverse phenotypic effects arising from nearby P-element insertions in neur provide a new appreciation of the concept of allelic effects on phenotype, in which the wild type and null mutant are at the extreme ends of a continuum of pleiotropic allelic effects (Rollmann, 2008).

Distinct P-element insertions at nearby locations in the neur gene give rise to profoundly different effects on adult behaviors, and aberrant startle-induced locomotor responses, olfactory responses, and aggression correlate with different structural alterations in integrative brain centers, the mushroom bodies, and the ellipsoid body of the central complex. Similar pleiotropic allelic effects with differential effects on life span, resistance to heat stress and starvation, and preference for trehalose intake were observed previously for P-element insertions in the Tre1-Gr5a region (Rollmann, 2006). In addition, different naturally occurring polymorphisms in Catsup, which encodes a negative regulator of tyrosine hydroxylase, are associated with phenotypic variation in sternopleural bristle number, environmental plasticity of abdominal bristle number, and starvation resistance (Carbone, 2006). The diverse phenotypic effects arising from nearby P-element insertions in neur contribute to an emerging new appreciation of the concept of allelic effects on phenotype, in which the wild type and null mutant are at the extreme ends of a continuum of pleiotropic allelic effects. Subtle alterations in transcript abundance for splice variants may contribute to these pleiotropic effects, which would be in line with the subtle regulatory variations that have been associated with phenotypic effects on human and rodent behaviors (Rollmann, 2008).

Previous studies have shown that the introduction of a single P-element in the genome gives rise to widespread altered transcriptional regulation and that about two-thirds of genes with altered transcriptional regulation in a P-element-disrupted background are candidate genes affecting the trait. Moreover, such altered transcriptional profiles can define a functional context for the disrupted gene. The results from an expression microarray analysis show that insertions of P elements in neur result in a genomewide cascade of transcripts with altered expression. Proteolytic and degradative enzymes feature prominently among coregulated genes. One protease-encoding transcript that features notably in these transcriptional profiles is Tequila, which has been implicated in synaptic plasticity in the mushroom bodies during memory formation (Didelot, 2006). One could speculate that the different, yet overlapping patterns of transcriptional profiles with altered expression of proteolytic enzymes and peptidoglycan recognition precursor proteins may reflect alterations in neural connectivity, which could contribute to the different behavioral effects. In addition, changes in structure of the mushroom bodies and ellipsoid body could also arise from developmental effects of neuralized (Sambandan, 2006; Rollmann, 2008 and references therein).

The behavioral phenotypes of neur mutant flies are reminiscent of those encountered in patients suffering from neuropsychiatric and neurodegenerative disorders, including reduced responsiveness to environmental stimuli and increased aggressive behavior. Neurodegenerative and neuropsychiatric disorders ranging from bipolar disorder, schizophrenia, and antisocial personality disorder to Alzheimer's and Parkinson's disease are often accompanied by behavioral alterations, such as indifference to stimuli, hypokinesia, hyperactivity, and aggression. Apathy and aggressive behavior have a catastrophic impact on the social functioning of neuropsychiatric patients. In addition, these behaviors represent some of the most difficult to treat symptoms. Whereas it is likely that underlying genetic architectures that may predispose to such behavioral syndromes are heterogeneous and complex, this study has demonstrated that reduced responsiveness to environmental stimuli together with increased aggressive behavior can arise from a single hypomorphic mutation at the neur locus in Drosophila and that these behavioral defects are associated with distinct subtle alterations in neuroanatomy (Rollmann, 2008).

Neuralized is expressed in the alpha/beta lobes of adult Drosophila mushroom bodies and facilitates olfactory long-term memory formation

Memory formation involves multiple molecular mechanisms, the nature and components of which are essential to understand these processes. Drosophila is a powerful model to identify genes important for the formation and storage of consolidated memories because the molecular mechanisms and dependence of these processes on particular brain regions appear to be generally conserved. Evidence that the highly conserved ubiquitin ligase Neuralized (Neur) is expressed in the adult Drosophila mushroom body (MB) α/β lobe peripheral neurons and is a limiting factor for the formation of long-term memory (LTM). Loss of one copy of neur gene results in significant LTM impairment, whereas overexpression of Neur in the peripheral neurons of the α/β lobes of the adult MBs results in a dosage-dependent enhancement of LTM. In contrast, learning, early memories, or anesthesia-resistant memory are not affected. The role of Neuralized in LTM formation is restricted within the neurons of the periphery of the α/β lobes; it is suggested that this structural subdivision of the MBs participates in the formation of LTM (Pavlopoulos, 2008).

The results demonstrate that Neur plays an essential and limiting role for LTM formation in differentiated neurons. This is supported by complementary experiments that demonstrate dramatic reduction of memory in neur mutant heterozygotes and significant enhancement of LTM upon elevation of Neur levels only within MB neurons that normally express the gene. Consistent with this, the memory deficit in neur mutant heterozygotes is reversed upon UAS-neur transgene expression specifically in the adult MB α/β lobe peripheral neurons. The enhancement of LTM is proportional to Neur dosage and does not have a developmental etiology. These observations strongly suggest that Neur acts to enhance LTM by modulating mechanisms within neurons of the α/β lobe periphery (Pavlopoulos, 2008).

Recent evidence indicates functional specialization of the MB axonal projections, with an essential role for the α/β lobes in LTM. In agreement, these data strongly suggest that Neur is required for LTM formation within c772- and MB247-expressing neurons in the periphery of the α/β lobes. In support of this, c772 and MB247 neurons were recently shown to be important for appetitive LTM. However, the c739 neurons in the center of the α lobes were shown to harbor a LTM cellular trace, evident as enhanced Ca²⁺ influx, 9 or 24 h after spaced training. In contrast, elevation of Neur within these neurons did not enhance LTM. Because the cellular memory trace within c739 neurons appeared 9 h after training, it may characterize a late phase of LTM consolidation or maintenance. This is distinct from the requirement for elevated Neur within the initial 3 h after training, likely representing involvement in LTM formation. Then, at least two different LTM generating systems may exist, one Neur-dependent in neurons at the periphery of the α/β lobes and another Neur-independent in the central c739 neurons of the α lobe. Alternatively, considering the spatiotemporal differences in the requirement for Neur function and cellular trace appearance, LTM formation may occur within c772 neurons and be later transferred to the central c739 neurons for storage and recall. Consistent with the later notion, neurotransmission from the α/β lobes and specifically from c739 neurons was reported to be essential for memory retrieval, and particular neuronal populations of the MBs seem to be used sequentially in distinct phases of aversive and appetitive memory processing. A recent report implicated the γ lobes in long-term courtship conditioning memory, and it is likely that, in contrast to olfactory LTM, these lobes are specifically required for this type of consolidated memory. It is noteworthy, however, that full rescue of the long-term courtship memory deficit in the orb2 mutants in that study was achieved with 201Y and c772 drivers, which, in addition to γ lobes, also express in the α and α/β lobes, respectively. Therefore, in agreement with previous reports, at least the α lobe in addition to the γ appears to be required for efficient long-term courtship memory. Thus, these data support the proposed structural and functional division of the MBs, provide functional validation of the suggested model, and refine the role of neuronal subpopulations of the α/β lobes in LTM (Pavlopoulos, 2008).

Interestingly, Neur overexpression yielded significant LTM with a single 12-pairing cycle in a dosage-dependent manner, whereas multiple spaced cycles are necessary for equivalent LTM in control animals. Therefore, Neur appears to facilitate LTM formation and to be limiting for this process. The limiting role of Neur in memory is in agreement with the low abundance of the protein in the MBs and indicates that its levels are tightly regulated. In fact, the RING domain of Neur has been shown to be critical for Neur proteosomal degradation. Consistently, deletion of the RING domain resulted in increased Neur levels, suggesting that the ubiquitin-proteasome system likely regulates the abundance of the endogenous Neur protein in the MBs (Pavlopoulos, 2008).

Neur has an established role as an E3 ubiquitin ligase involved in the regulation of N signaling in developmental contexts, and this could explain its action in LTM. This is consistent with increasing evidence for the critical role of ubiquitination in the regulation of synaptic strength and long-term changes in plasticity and in agreement with the potential role of N in LTM. In particular, conditional overexpression of N enhances LTM with a single 12-pairing cycle, whereas reduction of N within MBs results in impaired LTM. Surprisingly, expression of a truncated form of Neur (NeurDR) yielded pronounced elevation of memory. The role of the NeurDR remains unclear because its function in N signaling in development is controversial. Studies report a dominant negative effect of NeurDR, whereas others suggest a gain-of-function phenotype. An explanation for this puzzling question comes from evidence for redundant function of Neur and the E3 ubiquitin ligase Mind bomb (Mib), which also regulates the endocytosis of N ligands and N activation. Although the endogenous requirements for Neur and Mib are quite distinct and the two ligases are mostly expressed in different cell types, they also coexist and act synergistically or redundantly for N activation in developmental contexts. For example, the establishment of the wing DV boundary requires Mib but not Neur. In that context, absence of Mib is rescued by ectopic expression of Neur. Importantly, ectopic expression of NeurDR in the wing pouch results in overactivation of N signaling that could be due to potentiation by Mib, as the effect of NeurDR in mib^−/− clones, in the same developmental context, is abolished. It is possible that NeurDR besides ubiquitination has additional, yet unknown, stimulatory activity, but this activity can be manifested only when Mib is present and the ubiquitination of N ligands is feasible. Potentiation of NeurDR by Mib in the adult MBs and its increased stability indicated by the current data could explain the pronounced increase of memory, similar to what has been observed for N signaling activation in the wing pouch. Elucidation of the yet unknown expression patterns of Mib, N, and its ligands in the adult Drosophila brain is intriguing and will help validate the above model (Pavlopoulos, 2008).

Neur function in LTM through an N-independent mechanism is an alternative possibility. One aspect of such Neur function is supported by detection of Neur in the nucleus in contexts where N and its ligands are absent. Nuclear localization of Neur does not depend on the RING but the Neuz1 domain, also essential for its interaction with N ligands, and it is abolished upon ectopic expression of Dl. Moreover, earlier studies have shown that the mouse and rat Neurl1 shuttle between cytoplasm and nucleus in Neuro2a cells, their nuclear localization increases in response to neuronal differentiation, and they are potent inhibitors of transcription. Interestingly, the activity of Neurl1 in transcription was found to be dependent on the Neuz domains but not the RING. Consistent with these observations and given that LTM depends on transcription regulation, a novel function of Neur in the nucleus that does not involve its ubiquitin ligase activity and operates in LTM formation is challenging, and it is worth further investigation (Pavlopoulos, 2008).

Although the exact role of Neur in LTM remains to be resolved, these findings emphasize the importance of Neur for this process. It seems that alternative mechanisms of Neur function may be operative in different contexts depending on the regulated compartmentalization of the protein and the function of its domains. The questions of what mechanisms operate in memory formation will be resolved by future work (Pavlopoulos, 2008).

An unexpected link between Notch signaling and ROS in restricting the differentiation of hematopoietic progenitors in Drosophila

A fundamental question in hematopoietic development is how multipotent progenitors achieve precise identities, while the progenitors themselves maintain quiescence. In Drosophila larvae, multipotent hematopoietic progenitors support the production of three lineages, exhibit quiescence in response to cues from a niche, and from their differentiated progeny. Infection by parasitic wasps alters the course of hematopoiesis. This study addresses the role of Notch (N) signaling in lamellocyte differentiation in response to wasp infection. Notch activity is moderately high and ubiquitous in all cells of the lymph gland lobes, with crystal cells exhibiting the highest levels. Wasp infection reduces Notch activity, which results in fewer crystal cells and more lamellocytes. Robust lamellocyte differentiation is induced even in N mutants. Using RNA interference-knockdown of N, Serrate, and Neuralized, and twin clone analysis of a N null allele, this study shows that all three genes inhibit lamellocyte differentiation. However, unlike its cell-autonomous function in crystal cell development, Notch's inhibitory influence on lamellocyte differentiation is not cell-autonomous. High levels of reactive oxygen species in the lymph gland lobes, but not in the niche, accompany NRNAi-induced lamellocyte differentiation and lobe dispersal. These results define a novel dual role for Notch signaling in maintaining competence for basal hematopoiesis: while crystal cell development is encouraged, lamellocytic fate remains repressed. Repression of Notch signaling in fly hematopoiesis is important for host defense against natural parasitic wasp infections. These findings can serve as a model to understand how reactive oxygen species and Notch signals are integrated and interpreted in vivo (Small, 2013).

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date revised: 18 February 2024
  

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