org Interactive Fly, Drosophila



Transcriptional Regulation

Engrailed is expressed in subsets of interneurons that do not express Connectin or appreciable Neuroglian, whereas other neurons that are Engrailed negative strongly express these adhesion molecules. Connectin and Neuroglian expression are virtually eliminated in interneurons when engrailed expression is driven ubiquitously in neurons, and greatly increased when engrailed genes are lacking in mutant embryos. The data suggest that Engrailed is normally a negative regulator of Connectin and neuroglian. These are the first two effector genes identified in the nervous system of Drosophila as regulatory targets for Engrailed. It is argued that differential Engrailed expression is crucial in determining the pattern of expression of cell adhesion molecules and thus constitutes an important determinant of neuronal shape and perhaps connectivity. In wild-type embryos, all neurons that express engrailed also express invected. The converse is not true, however. Neurons, which lie anterior to the predominant Engrailed/Invected stripe in the CNS, express invected but not engrailed. This is the best example to date of differing expression of engrailed and invected in an identified cell type. Connectin is also expressed in SNa and SNc motor neurons, which are Engrailed negative. When Engrailed is expressed in all neurons, Connectin is not downregulated but slightly upregulated in these motor neurons, in contrast to the effect on interneurons. Since Engrailed can act either as a repressor or as an activator, it is possible that ectopic Engrailed directly activates Connectin in the motor neurons (Siegler, 1999).

MicroRNA-8 promotes robust motor axon targeting by coordinate regulation of cell adhesion molecules during synapse development

Neuronal connectivity and specificity rely upon precise coordinated deployment of multiple cell-surface and secreted molecules. MicroRNAs have tremendous potential for shaping neural circuitry by fine-tuning the spatio-temporal expression of key synaptic effector molecules. The highly conserved microRNA miR-8 is required during late stages of neuromuscular synapse development in Drosophila. However, its role in initial synapse formation was previously unknown. Detailed analysis of synaptogenesis in this system now reveals that miR-8 is required at the earliest stages of muscle target contact by RP3 motor axons. The localization of multiple synaptic cell adhesion molecules (CAMs) is dependent on the expression of miR-8, suggesting that miR-8 regulates the initial assembly of synaptic sites. Using stable isotope labelling in vivo and comparative mass spectrometry, this study found that miR-8 is required for normal expression of multiple proteins, including the CAMs Fasciclin III (FasIII) and Neuroglian (Nrg). Genetic analysis suggests that Nrg and FasIII collaborate downstream of miR-8 to promote accurate target recognition. Unlike the function of miR-8 at mature larval neuromuscular junctions, at the embryonic stage it was found that miR-8 controls key effectors on both sides of the synapse. MiR-8 controls multiple stages of synapse formation through the coordinate regulation of both pre- and postsynaptic cell adhesion proteins (Lu, 2014).

Protein Interactions

A family of ankyrin-binding glycoproteins including alternatively spliced products has been identified in rat brain. The sequence of ankyrin-binding glycoprotein (ABGP) shares 72% homology with chicken neurofascin, a neural cell adhesion molecule. These proteins are closely related to Neuroglian. It is hypothesized that ankyrin-binding activity is shared by all of these Ig superfamily proteins (Davis, 1993).

Neuroglian can transmit positional information directly to Ankyrin and thereby polarize its distribution in Drosophila tissue culture cells. Ankyrin is not normally associated with the plasma membrane of S2 tissue culture cells. Upon expression of an inducible neuroglian minigene, however, cells aggregate into large clusters and Ankyrin becomes concentrated at sites of cell-cell contact. Spectrin is also recruited to sites of cell contact in response to neuroglian expression. The accumulation of Ankyrin at cell contacts requires the presence of the cytoplasmic domain of Neuroglian. Whereas Ankyrin is strictly associated with sites of cell-cell contact, Neuroglian is more broadly distributed over the cell surface. A direct interaction between Neuroglian and Ankyrin can be demonstrated using yeast two-hybrid analysis. Thus, Neuroglian appears to be activated by extracellular adhesion so that Ankyrin and the membrane skeleton selectively associate with sites of contact and not with other regions of the plasma membrane (Dubreuil, 1996).

The L1-family of cell adhesion molecules is involved in many important aspects of nervous system development. Mutations in the human L1-CAM gene cause a complicated array of neurological phenotypes; however, the molecular basis of these effects cannot be explained by a simple loss of adhesive function. Human L1-CAM and its Drosophila homolog Neuroglian are rather divergent in sequence, with the highest degree of amino acid sequence conservation between segments of their cytoplasmic domains. In an attempt to elucidate the fundamental functions shared between these distantly related members of the L1-family, it is demonstrated that the extracellular domains of mammalian L1-CAMs and Drosophila Neuroglian are both able to induce the aggregation of transfected Drosophila S2 cells in vitro. To a limited degree they even interact with each other in cell adhesion and neurite outgrowth assays. The cytoplasmic domains of human L1-CAM and neuroglian are both able to interact with the Drosophila homolog of the cytoskeletal linker protein ankyrin. Moreover the recruitment of ankyrin to cell-cell contacts is completely dependent on L1-mediated cell adhesion. These findings support a model of L1 function in which the phenotypes of human L1-CAM mutations result from a disruption of the link between the extracellular environment and the neuronal cytoskeleton (Hortsch, 1998a).

Expression of the Drosophila cell adhesion molecule Neuroglian in S2 cells leads to cell aggregation and the intracellular recruitment of ankyrin to cell contact sites. The region of neuroglian that interacts with ankyrin was localized and the mechanism that limits this interaction to cell contact sites was investigated. Yeast two-hybrid analysis and expression of neuroglian deletion constructs in S2 cells have identified a conserved 36-amino acid sequence that is required for ankyrin binding. Mutation of a conserved tyrosine residue within this region reduces ankyrin binding and extracellular adhesion. However, residual recruitment of ankyrin by this mutant Neuroglian molecule is still limited to cell contacts, indicating that the lack of ankyrin binding at noncontact sites is not caused by tyrosine phosphorylation. A chimeric molecule, in which the extracellular domain of Neuroglian was replaced with the corresponding domain from the adhesion molecule Fasciclin II, also selectively recruits ankyrin to cell contacts. Thus, outside-in signaling by neuroglian in S2 cells depends on extracellular adhesion, but does not depend on any unique property of its extracellular domain. It is proposed that the recruitment of ankyrin to cell contact sites depends on a physical rearrangement of Neuroglian in response to cell adhesion, and that ankyrin binding plays a reciprocal role in stabilizing the adhesive interaction (Hortsch, 1998b).

The gene spalt is expressed in the embryonic central nervous system of Drosophila but its function in this tissue is still unknown. To investigate this question, a combination of techniques was used to analyse spalt mutant embryos. Electron microscopy shows that in the absence of Spalt, the central nervous system cells are separated by enlarged extracellular spaces populated by membranous material at 60% of embryonic development. Surprisingly, the central nervous system from slightly older embryos (80% of development) exhibited almost wild-type morphology. An extensive survey by laser confocal microscopy has revealed that the spalt mutant central nervous system has abnormal levels of particular cell adhesion and cytoskeletal proteins. Time-lapse analysis of neuronal differentiation in vitro, lineage analysis and transplantation experiments have each confirmed that the mutation causes cytoskeletal and adhesion defects. The data indicate that in the central nervous system, spalt operates within a regulatory pathway which influences the expression of the ß-catenin Armadillo, its binding partner N-Cadherin, Notch, and the cell adhesion molecules Neuroglian, Fasciclin 2 and Fasciclin 3. Effects on the expression of these genes are persistent but many morphological aspects of the phenotype are transient, leading to the concept of sequential redundancy for stable organization of the central nervous system (Cantera, 2002).

A possible interpretation of sal phenotype would be that components of cell adhesion are seriously compromised in the CNS of sal embryos during early stage 16. To test this hypothesis specific antibodies and laser confocal microscopy were used to survey the expression of molecules known to be important for cell adhesion in embryonic CNS at early stage 16. All the markers are detectably expressed in Df(2L)32FP-5;sal445 mutant embryos at both stages, and their spatial patterns of expression in the CNS are normal, showing that sal is not essential for any of these proteins to be expressed. However, the quantification of fluorescence intensity revealed that most markers were present in abnormally high or low levels. In transheterozygous Df(2L)32FP-5;sal445 mutants at early stage 16, when the strong transmission electron microscopy TEM phenotype is manifest, lower fluorescence levels were measured for Armadillo, N-Cadherin, Neuroglian, Fasciclin 2 and Fasciclin 3; higher fluorescence levels were measured for Notch; and levels similar to wild type for Neurotactin, Neurexin IV and Faint Sausage. Comparison between wild-type, heterozygous and null sal mutant embryos revealed a stepwise decrease in the fluorescence levels for Armadillo and N-Cadherin, indicating that the effect of the mutation is dominant (Cantera, 2002).

Fluorescence levels were measured at the stage when the TEM phenotype is reverted (stage 17). The wild-type fluorescence for the three markers studied in this regard (Armadillo, Fasciclin 2, Neuroglian) changes between early stage 16 and stage 17, indicating that during this short developmental interval the levels of cell adhesion proteins are regulated. Relative to these new wild-type levels, the three proteins that are not affected during the expression of the TEM phenotype (Neurotactin, Neurexin IV and Faint Sausage) remain normal in the mutant. The levels of Notch switch from abnormally high to slightly lower than normal. All other markers still exhibit lower-than-normal fluorescence levels, with the exception of N-Cadherin, which exhibits a partial recovery. Taken together, these data led to the conclusions that the expression of sal is necessary to maintain correct dynamic levels of several adhesion molecules in the CNS and that sal exerts this function in a persistent and dominant fashion (Cantera, 2002).

The rapid recovery of sal CNS during the course of stage 16 could be explained by the robustness inherent to a system in which adhesion is mediated by a combination of proteins and the possible compensatory effect mediated by upregulation of other members of the system. However, an alternative view is proposed. The ultrastructural recovery may as well reflect the normal dynamics of combinations of adhesion proteins expressed successively along embryonic development. From this point of view, the rapid recovery from the adhesion phenotype will reflect the normal transition between two particular combinations of adhesion proteins expressed at early or late stage 16. For this to be valid, the expression levels of several adhesion proteins must change along this interval during normal development. Interestingly, the data do support this possibility, since the fluorescence levels for Armadillo, Fasciclin 2 and Neuroglian change between stages 16 and 17 in wild-type CNS. Whether sal is required for the regulation of a combination of cell adhesion and cytoskeletal proteins at a particular developmental stage could be tested by deleting the expression of Sal exclusively in CNS tissue within short developmental intervals. This approach could now be possible using techniques based on combinations of the GAL4-UAS system and RNA interference (Cantera, 2002).

FIGQY phosphorylation defines discrete populations of L1 cell adhesion molecules at sites of cell-cell contact and in migrating neurons

Phosphorylation of neurofascin, a member of the L1 family of cell adhesion molecules (L1 CAMs), at the conserved FIGQY-tyrosine abolishes the ankyrin-neurofascin interaction. This study provides the first evidence, in Drosophila melanogaster and vertebrates, for the physiological occurrence of FIGQY phosphorylation in L1 family members. FIGQY tyrosine phosphorylation is localized at specialized cell junctions, including paranodes of sciatic nerve, neuromuscular junctions of adult rats and Drosophila embryos, epidermal muscle attachment sites of Drosophila, and adherens junctions of developing epithelial cells of rat and Drosophila. In addition, FIGQY-phosphorylated L1 CAMs are abundantly expressed in regions of neuronal migration and axon extension, including the embryonic cortex, the neonatal cerebellum and the rostral migratory stream, a region of continued neurogenesis and migration throughout adulthood in the rat. Based on these results, physiological FIGQY-tyrosine phosphorylation of the L1 family likely regulates adhesion molecule-ankyrin interactions establishing ankyrin-free and ankyrin-containing microdomains and participates in an ankyrin-independent intracellular signaling pathway at specialized sites of intercellular contact in epithelial and nervous tissue (Jenkins, 2001).

A phospho-FIGQY polyclonal antibody recognizes neuroglian on immunoblots. Reactivity of the phospho-FIGQY antibody was examined in Drosophila embyros between developmental stages 12 and 15 that were either heterozygous or homozygous for the neuroglian null mutation. Neuroglian nulls can be distinguished from heterozygous embryos by the absence of Neuroglian labeling. In triple labeled embryos, FIGQY-phosphotyrosine immunoreactivity was present in the nervous system and epidermal epithelium of heterozygous embryos but was conspicuously absent in neuroglian null embryos. The lack of immunoreactivity in neuroglian null fly embryos provides striking confirmation of the specificity of the phospho-FIGQY polyclonal antibody for neuroglian. Pre-incubation of the phospho-FIGQY polyclonal antibody with tyrosine phosphorylated nFIGQY peptide eliminated the signal, while a comparable amount of the non-phosphorylated peptide did not. The fact that signal is displaced with phospho- but not dephospho-FIGQY peptide provides further evidence that the antibody is specific for FIGQY-phosphorylated neuroglian (Jenkins, 2001).

FIGQY-phosphorylated Neuroglian localizes to the developing neuromuscular junction of Drosophila embryos at stage 16/17, as indicated by overlap with the junctional marker Fasciclin II. It was not possible to distinguish presynaptic from postsynaptic sites in Drosophila embryos. The presence of neuroglian at the neuromuscular junction was confirmed by double labeling with the anti-neuroglian antibody 1B7 and the phospho-FIGQY polyclonal antibody. At this stage, the presynaptic terminal has contacted the muscle but has not yet formed the varicosities that develop into the characteristic synaptic boutons of the larval neuromuscular junction. FIGQY phosphorylation was no longer evident in the larval neuromuscular junction, indicating the developmental nature of this phosphorylation event in Drosophila and suggesting that the specific function of FIGQY phosphorylation at the neuromuscular junction is different in Drosophila and vertebrates (Jenkins, 2001).

In addition to this junctional staining, some phospho-FIGQY labeling did not overlap with Fas II. These structures correspond to sites of muscle-tendon cell attachment, which are epidermal muscle attachment sites, as indicated by their overlap with the position-specific integrin PSalpha2. While neuroglian is present at these attachment sites, it is not much enriched suggesting that the neuroglian that is present is highly FIGQY-phosphorylated. The phospho-FIGQY signal was displaced by pre-incubation of the antibody with phosphorylated nFIGQY peptide (Jenkins, 2001).

Neuroglian expression occurs in multiple epithelial tissues of Drosophila. Epithelial tissues of wild-type embryos were examined between stages 12 and 15 for FIGQY-phosphorylated neuroglian. Neuroglian (phosphorylated plus dephorylated forms) is localized along the basolateral domains of epithelial cells of the hindgut while FIGQY-phosphotyrosine label is highly enriched in an apicolateral domain identified as adherens junctions by co-localization with the adherens junction marker Armadillo/ß-catenin. The merged image demonstrates the striking overlap between FIGQY-phosphorylated Neuroglian and Armadillo at adherens junctions. Some FIGQY-phosphorylated neuroglian is also present at the interface between gut epithelium basolateral membranes and visceral muscle, and probably corresponds to hemi-adherens junctions based on location (Jenkins, 2001).

This study provides the first physiological insight, in both Drosophila and vertebrates, into the cellular localization of a functionally important phosphorylation event in a major class of adhesion molecules. L1-type cell adhesion molecules phosphorylated at the FIGQY tyrosine are localized to specialized sites of cell-cell contact, including paranodes of adult sciatic nerve, the neuromuscular junction of adult rats and Drosophila embyros, muscle-tendon junctions of Drosophila, and adherens junctions of developing epithelial cells of rat and Drosophila. In C. elegans, FIGQY-phosphorylated LAD-1 also is localized to adherens junctions of epithelial tissues and sites of body wall-muscle attachment. In addition, FIGQY-phosphorylated neurofascin and L1 are abundantly expressed in regions of neuronal migration and axon extension in the vertebrate central nervous system, including the embryonic cortex, the neonatal cerebellum and the adult rostral migratory stream. These findings establish that FIGQY-tyrosine phosphorylation of L1-type family members first characterized in cultured neuroblastoma cells, also occurs under physiological conditions and is evolutionarily conserved between flies, nematodes and mammals (Jenkins, 2001).

Neuroglian activates Echinoid to antagonize the Drosophila EGF receptor signaling pathway

echinoid (ed) encodes a cell-adhesion molecule (CAM) that contains immunoglobulin domains and regulates the Egfr signaling pathway during Drosophila eye development (Bai, 2001). Genetic mosaic and epistatic analysis, has suggested that Ed, via homotypic interactions, activates a novel, as yet unknown pathway that antagonizes Egfr signaling (Bai, 2001). Alternatively, later studies indicate that Ed inhibits Egfr through direct interactions (Rawlins, 2003; Spencer, 2003). Another body of work suggests that Ed functions as a homophilic adhesion molecule, and also engages in a heterophilic trans-interaction with Drosophila Neuroglian (Nrg), an L1-type CAM. Co-expression of ed and nrg in the eye exhibits a strong genetic synergy in inhibiting Egfr signaling. This synergistic effect requires the intracellular domain of Ed, but not that of Nrg (Islam, 2003). A model for this interaction suggest that Nrg acts as a heterophilic ligand and activator of Ed, which in turn antagonizes Egfr signaling (Islam, 2003).

Since Egfr activity is required for the differentiation of both photoreceptor (except R8) and cone cells, the numbers of these cell types per ommatidum was used as a readout for Egfr activity in the eye disc. Flies with a mutation in the ed gene produce extra photoreceptor and cone cells. By contrast, overexpression of ed in the eye leads to a reduction of the number of photoreceptor cells per ommatidium. These findings together with additional genetic evidence indicates that Ed uses an independent pathway to antagonize Egfr signaling, and it is postulated that this inhibition might be initiated by a homophilic binding activity of Ed (Bai, 2001). To explore the possibility that Ed could be involved in heterophilic interactions with other Ig domain CAMs, a genetic overexpression screen was constructed. It was reasoned that if ed acts as a heterophilic receptor, overexpression of both the Ed receptor and its potential ligand(s) should have a synergistic effect on the inhibition of Egfr signaling, which results in a reduced number of cone and photoreceptor cells. In addition, both adhesion molecules must normally be co-expressed and colocalized in the developing eye disc in order to engage in a functional heterophilic adhesive interaction (Islam, 2003).

The GMR-GAL4 driver line was used to co-express UAS-ed with several available UAS and EP lines that drive overexpression of various Ig domain-containing adhesion molecules. Ectopic expression of Ed in the eye results in a rough eye phenotype and a loss of photoreceptor and cone cells (Bai, 2001). On average, 10%-15% of ommatidia were missing photoreceptor or cone cells. By contrast, overexpression of either the neuronal nrg180 or the non-neuronal nrg167 isoform alone has no effect on the number of photoreceptor or cone cells. However, co-expression of both ed and nrg180 (or nrg167) results in a more severe rough eye phenotype with a reduction of the number of ommatidia, a varying size of ommatidia and a decrease in the number of bristles. In addition, a significantly higher percentage of ommatidia contained fewer photoreceptor and cone cells. No synergistic effects were detected when ed was overexpressed together with other CAMs, such as Drosophila Fasciclin 2 or human L1CAM (Islam, 2003).

To document the interaction between Ed and Nrg further, the effect of overexpression of ed was examined in female flies, that had only one copy of the nrg gene. nrg1 is a nrg null allele. A reduction in half of the nrg gene dosage significantly suppresses the cone cell loss phenotype, but not the loss of photoreceptor cells; both these effects were caused by GMR-GAL4-driven UAS-ed expression. Together, these results demonstrate a specific genetic interaction between ed and both protein isoforms of nrg (Islam, 2003).

Both a genetic interaction between ed and nrg and their direct heterophilic trans-binding have been demonstrated. The synergistic effect of ed and nrg could be caused by a unidirectional signaling mechanism with either Ed as the receptor (and Nrg as the ligand) or Nrg as the receptor (and Ed as the ligand). Another possibility is that both Ed and Nrg act as receptor molecules (with Nrg and Ed as ligands, respectively) in mediating a bi-directional signaling process. To distinguish between these three possibilities, the UAS-Gal4 system was used to co-express in the developing Drosophila eye disc ed and nrgGPI, an artificial isoform of Nrg that lacks the intracellular Nrg domain. Overexpression of nrgGPI alone causes no phenotype. However, the synergistic effect between Ed and Nrg on the percentage of ommatidia lacking photoreceptor and cone cell was fully retained for this genetic combination. By contrast, co-expression of native nrg180 and a truncated artificial isoform of Ed, which lacks the intracellular Ed domain (ed intra), does not exhibit a genetic synergy in the eye disc. Similar results were obtained when ed intra and either nrg167 or nrgGPI were co-expressed. This indicates that the intracellular domain of Ed is essential for repressing Egfr signaling (Islam, 2003).

In summary, these results suggest that in this context Nrg primarily functions as a heterophilic ligand of Ed and thereby activates Ed in the signal-receiving cell. As a result of its interaction with Ed, Nrg antagonizes Egfr signaling non-autonomously. By contrast, there is no evidence from the experimental assay system for suggesting any signaling from Ed to Nrg. Consistent with this model, it was found that the ectopic expression of edC50, which contains only the transmembrane domain and the last 50 amino acids of the Ed intracellular domain, but lacks the extracellular Ed domain, also causes a reduced number of photoreceptor and cone cells (Islam, 2003).

Taken together, these results support a model whereby Nrg functions as a heterophilic ligand of Ed and activates Ed in the signal-receiving cells to antagonize Egfr signaling. Ed is the first identified heterophilic, extracellular partner of Nrg. In this context, Nrg functions as a ligand to activate Ed in the signal-receiving cells. This unidirectional signaling mechanism from Nrg to Ed is further supported by the observation that overexpression of edC50 alone can reduce Egfr signaling. By contrast, co-expression of nrg180 and ed intra does not exhibit any genetic synergy in influencing Egfr signaling. Thus, the results fail to support a bi-directional signaling mechanism from Ed to Nrg. Because it is not known whether the intracellular domain of Ed may also be required for signaling out and for activating Nrg in neighboring cells, a signaling process from Ed to Nrg still remains a possibility. The overexpression effect of edC50 on the Egfr signaling varies between different lines and tends to be weaker than that observed for ed and nrg co-expression. It is not clear whether this simply reflects differential expression levels for EdC50 or whether it lacks the full activity of a wild-type Ed (Islam, 2003).

The non-neuronal isoform of Nrg (Nrg167) is expressed in the non-neuronal, epithelial cells of eye imaginal discs. It exhibits a similar effect on Ed (and thereby the Egfr signaling pathway) as does the neuronal Nrg isoform (Nrg180), which is expressed by the photoreceptor cells. Therefore, Nrg167 is probably the major Nrg isoform that inhibits the intrinsic Egfr signaling for basally located, undifferentiated cells. Although cell mixing experiments clearly show that Ed and Nrg protein interact with each other in a trans-type modus, the results neither prove nor disprove that they might also interact in a cis-type modus. In fact, some Ig-domain CAMs, such as axonin 1/TAG1, interact with L1-type proteins exclusively in a functional cis-type interaction (Islam, 2003).

Genetic evidence indicates that Nrg is a cell-autonomous, positive regulator of Egfr signaling in neuronal cells that express both Nrg and Egfr. However, in the developing Drosophila eye disc Nrg functions non-autonomously as a ligand of Ed and activates Ed in the neighboring cells to repress downstream Egfr signaling. Thus, depending on the cellular context, Nrg can act both as an autonomous activator, as well as a non-autonomous inhibitor of the Egfr signaling pathway (Islam, 2003).

Genetic mosaic analysis indicates that ed acts in a cell non-autonomous manner (Bai, 2001). Since the intracellular domain of Ed is required for Egfr signal repression, it is proposed that through its homophilic interaction Ed transmits a negative signal in the receiving cell and antagonizes the Egfr pathway. In this study, a homophilic adhesive activity of Ed has been demonstrated, and it is further shown that ed also acts autonomously as a heterophilic receptor of Nrg. Thus, Ed appears to influence Egfr signaling through both homophilic (non-autonomous) and heterophilic (autonomous) interactions, but the relative contribution derived from either interaction is unknown. Flies that are mutant for ed have extra photoreceptor and cone cells. By contrast, when shifting temperature-sensitive nrg3 larvae to the restrictive temperature during the third instar larval stage, a wild-type number of Elav- and Cut-positive cells was observed. Therefore, the Nrg-mediated heterophilic activity of Ed in repressing Egfr signaling appears to be redundant with the homophilic activity of Ed (Islam, 2003).

Further studies are required to reveal the molecular mechanism by which ed inhibits the Egfr signaling pathway. Equally, with both ed and nrg widely expressed in the developing Drosophila eye disc, it remains to be revealed how the two opposing effects of nrg on Egfr activity might contribute to a differential cellular segregation and the development of different ommatidial cell types (Islam, 2003).

Control of axonal sprouting and dendrite branching by the Nrg-Ank complex at the neuron-glia interface

Neurons are highly polarized cells with distinct subcellular compartments, including dendritic arbors and an axon. The proper function of the nervous system relies not only on correct targeting of axons, but also on development of neuronal-class-specific geometry of dendritic arbors. To study the intercellular control of the shaping of dendritic trees in vivo, cell-surface proteins expressed by Drosophila dendritic arborization (da) neurons were sought. One of them was Neuroglian (Nrg), a member of the Ig superfamily; Nrg and vertebrate L1-family molecules have been implicated in various aspects of neuronal wiring, such as axon guidance, axonal myelination, and synapse. A subset of the da neurons in nrg mutant embryos exhibited deformed dendritic arbors and abnormal axonal sprouting. Functional analysis in a cell-type-selective manner strongly suggested that those da neurons employed Nrg to interact with the peripheral glia for suppressing axonal sprouting and for forming second-order dendritic branches. At least for the former role, Nrg functioned in concert with the intracellular adaptor protein Ankyrin (Ank). Thus, the neuron-glia interaction that is mediated by Nrg, together with Ank under some situations, contributes to axonal and dendritic morphogenesis (Yamamoto, 2006).

Phenotypes in a number of genetic backgrounds, cell-type-selective rescue, and knockdown experiments indicated the requirement of the Nrg-Ank complex at the neuron-peripheral glia interface. One prominent phenotype was axonal sprouting at the ddaE root that had not been associated with a glial process; this ectopic branch displayed both morphological and molecular characteristics of dendrites. Nrg-mediated cell interactions may restrict distributions of molecules such as Nod-βgal and Tau-βgal. These findings present an interesting similarity to roles of glial membranes or AnkryinG and L1 family molecules at the node of Ranvier. In the rat spinal cord, oligodendrocyte myelin glycoprotein (OMgp) is localized in oligodendroglia-like cells whose processes ensheath the node. OMgp null mice exhibit a defect in node-paranode domain segregation and show collateral sprouting at the node where the glial process was barely detected. AnkryinG and L1 family molecules are also known to play pivotal roles in demarcating subdomains at the node and the axon initial segment (AIS) in Purkinje cells. In this study, penetrance of the sprouting was relatively low. This could be due to functional overlap between Nrg and other adhesive molecules, as shown in sensory axon guidance. In addition to suppressing axonal sprouting, Nrg expression on both sides of the neuron-glia interface recovered formation of second-order dendritic branches. On the basis of all these results, it is speculated that the axon-glia communication may contribute to suppression of ectopic sprouting from axons and to segregation of axon versus soma domains, which might be necessary to properly transport molecules that are necessary for higher-order branching in pre-existing dendritic arbors. It would be intriguing to visualize how the Nrg-mediated neuron-glia interaction controls distributions of candidate components of branching machineries. Therefore, the ddaE-glia interaction may provide a general model whereby mechanisms of formation of neuronal subcellular compartments could be investigated (Yamamoto, 2006).

Post-transcriptional Regulation

Tissue-specific alternative pre-mRNA splicing is a widely used mechanism for gene regulation and the generation of different protein isoforms, but relatively little is known about the factors and mechanisms that mediate this process. Tissue-specific RNA-binding proteins might mediate alternative pre-mRNA splicing. In Drosophila melanogaster, the RNA-binding protein encoded by the elav (embryonic lethal abnormal visual system) gene is a candidate for such a role. The ELAV protein is expressed exclusively in neurons, and is important for the formation and maintenance of the nervous system. In this study, photoreceptor neurons genetically depleted of ELAV, and elav-null central nervous system neurons, were analyzed immunocytochemically for the expression of neural proteins. In both situations, the lack of ELAV corresponds with a decrease in the immunohistochemical signal of the neural-specific isoform of Neuroglian, which is generated by alternative splicing. Furthermore, when ELAV is expressed ectopically in cells that normally express only the non-neural isoform of Neuroglian, the generation of the neural isoform of Neuroglian is observed. It is concluded that Drosophila ELAV promotes the generation of the neuron-specific isoform of Neuroglian by the regulation of pre-mRNA splicing. The findings reported in this paper demonstrate that ELAV is necessary, and the ectopic expression of ELAV in imaginal disc cells is sufficient, to mediate neuron-specific alternative splicing. Although performed in vivo, these experiments do not exclude an indirect effect of ELAV, as for example, on the stability of a neuroglian-specific splicing factor (Koushika, 1996).

Drosophila neural-specific protein, ELAV, has been shown to regulate the neural-specific splicing of three genes: neuroglian (nrg), erect wing, and armadillo. Alternative splicing of the nrg transcript involves alternative inclusion of a 3'-terminal exon. Using a minigene reporter, it has been shown that the nrg alternatively spliced intron (nASI) has all the determinants required to recreate proper neural-specific RNA processing seen with the endogenous nrg transcript, including regulation by ELAV. An in vitro UV cross-linking assay revealed that ELAV from nuclear extracts cross-links to four distinct sites along the 3200 nucleotide long nASI; one EXS is positioned at the polypyrimidine tract of the default 3' splice site. ELAV cross-linking sites (EXSs) have in common long tracts of (U)-rich sequence rather than a precise consensus; moreover, each tract has at least two 8/10U elements; their importance is validated by mutant transgene reporter analysis. Further, criteria are proposed for ELAV target sequence recognition based on the four EXSs, sites within the nASI that are (U) rich but do not cross-link with ELAV, and predicted EXSs from a phylogenetic comparison with Drosophila virilis nASI. These results suggest that ELAV regulates nrg alternative splicing by direct interaction with the nASI (Lisbin, 2001).

A model is invisioned in which, in the absence of ELAV, the default 3' splice site is solely used, despite lacking a consensus branch-point sequence and a proximal polypyrimidine tract. It is hypothesized that the conserved intron elements, along with the default exon and polyadenylation site(s) and perhaps other as yet unrecognized sequences, conspire to positively maintain splicing exclusively to an otherwise weak default 3' splice site. In the presence of ELAV, however, this positive maintenance is disrupted or partially disrupted, either by directly competing with poly(U)-binding proteins, or by countering the effects of the conserved sequences, leading to 3' splice site recognition of the more consensus-like but distal neural-specific 3' splice site. This model has predictive value. For example, the exclusive use of the default 3' splice site could be compromised by deletion of one or both conserved intron elements. Further studies will be needed to address these questions (Lisbin, 2001).

Neuroglian, Gliotactin, and the Na+/K+ ATPase are essential for septate junction function

One essential function of epithelia is to form a barrier between the apical and basolateral surfaces of the epithelium. In vertebrate epithelia, the tight junction is the primary barrier to paracellular flow across epithelia, whereas in invertebrate epithelia, the septate junction (SJ) provides this function. New proteins have been identified that are required for a functional paracellular barrier in Drosophila. In addition to the previously known components Coracle (Cora) and Neurexin (Nrx), four other proteins [Gliotactin, Neuroglian (Nrg), and both the alpha and ß subunits of the Na+/K+ ATPase (see Na pump α subunit) and Nervana 1 and Nervana 2)] are required for formation of the paracellular barrier. In contrast to previous reports, it is demonstrated that the Na pump is not localized basolaterally in epithelial cells, but instead is concentrated at the SJ. Data from immunoprecipitation and somatic mosaic studies suggest that Cora, Nrx, Nrg, and the Na+/K+ ATPase form an interdependent complex. Furthermore, the observation that Nrg, a Drosophila homolog of vertebrate neurofascin, is an SJ component and is consistent with the notion that the invertebrate SJ is homologous to the vertebrate paranodal SJ. These findings have implications not only for invertebrate epithelia and barrier functions, but also for understanding of neuron-glial interactions in the mammalian nervous system (Genova, 2003).

The SJ has historically been thought of as an invertebrate-specific junction; however, recent studies of the vertebrate nervous system have identified a junction that is both molecularly and structurally homologous, the paranodal SJ (PSJ) (Einheber, 1997; Tepass, 2001). The PSJ occurs between neurons and the glial cells that myelinate them, the oligodendrocytes and Schwann cells. Each glial cell wraps around and contacts the neuron multiple times in a spiral pattern to form the paranodal loops. The PSJ forms between the paranodal loops and the neuron and keeps the node of Ranvier distinct from the internodal region by providing a seal between the neuron and glial cell. This seal provides a barrier within the neuronal membrane that separates Na+ channels at the node of Ranvier from K+ channels under the glial cells, and a paracellular diffusion barrier between the neuron and the ensheathing glial cell. Consistent with these structural and functional similarities, the invertebrate epithelial SJ and the vertebrate PSJ also display similarities at the molecular level. Caspr (contactin-associated protein; also known as paranodin), a mammalian homolog of Nrx, is located on the neuronal face of the PSJ (Einheber, 1997), where it interacts with protein 4.1 (Menegoz, 1997), which is homologous to Drosophila Cor (Genova, 2003).

To identify additional components of the Drosophila SJ, a collection of P element insertion mutations was screened for a phenotype attributable to a loss of the paracellular barrier. Two genes, Na pump alpha subunit (Atpalpha) and Nervana 2 (Nrv2), which encodes the ß subunit of the Na+/K+ ATPase) were identified as essential for the barrier function of the SJ. In addition, Neuroglian (Nrg), which is homologous to known components of the PSJ, and Gli, which is necessary for the blood-brain barrier, were tested and found to be necessary for the paracellular barrier. Direct immunostaining, epitope-tagged expression constructs, and GFP-tagged proteins indicate that Nrv2, ATPalpha, and Nrg localize to the SJ, and that they are interdependent for this localization. In keeping with this finding, the existence of a protein complex containing Cora, Nrx, Nrg, and Nrv is demonstrated. Taken together, these results suggest a novel complex involving the Na+/K+ ATPase that is necessary for establishing and maintaining the primary paracellular barrier in invertebrate epithelia, the SJs. Thus these studies provide new insights into the structure and function of SJs in both invertebrate epithelial cells and in the homologous PSJ of the vertebrate nervous system (Genova, 2003).

A novel approach has been used to identify components of the pleated SJ, which provides the barrier to paracellular diffusion in Drosophila epithelial cells. Three independent lines of evidence indicate that the proteins encoded by these genes are essential to the structure and function of epithelial SJs. (1) Mutations in all four identified loci, Nrg, Gli, Nrv2, and Atpalpha, disrupt the paracellular barrier of the salivary gland epithelium and alter the ultrastructure of epithelial SJs. (2) The proteins encoded by three of these genes localize to the region of the SJ as judged by antibody staining of fixed tissues and observation of GFP-tagged proteins expressed in living epithelial cells (reagents were unavailable for observations of the fourth protein, Gli). (3) Somatic mosaic studies and IP experiments indicate that these proteins form an interdependent complex at the SJ. This complex also includes two previously identified SJ components, Nrx, a transmembrane protein, and Cora, a membrane-associated cytoplasmic protein with a FERM domain (Genova, 2003).

One of the most intriguing results of this study is the identification of the Na+/K+ ATPase as a functional member of the SJ. Mutations in either the alpha subunit (ATPalpha) or ß subunit (NRV2) disrupt the paracellular barrier of the embryonic salivary gland and this functional loss corresponds to the structural loss of septae in the junction. Although the SJ is localized just basal to the adherens junction near the apical end of the cell, previous characterizations of the Na+ pump have described it as having a basolateral localization. The localization of the Na+/K+ ATPase was examined using immunofluorescence; both subunits are found highly concentrated at the SJ in imaginal epithelia. In embryonic epithelia, the results differed depending upon the fixation and staining method; methanol treatment resulted in staining that appeared basolateral whereas staining of embryos fixed without methanol was localized to the SJ. Observations of GFP-exon trap lines enabled the confirmation that both ATPase subunits localize to the SJ in live embryos and imaginal epithelia. These results are limited to the examination of ectodermally derived epithelia such as the embryonic epidermis, foregut, hindgut, and salivary glands. Interestingly, the midgut does not contain pleated SJs but rather smooth SJs, and so observed differences in subcellular localization of the ATPase may be cell type dependent (Genova, 2003).

The Nrv2 and Nrv1 genes encode ß subunits of the Na+/K+ ATPase that differ in their cytoplasmic tails. The P-element insertion (l(2)k13315) disrupts the Nrv2 gene product but appears to have no affect on the Nrv1 protein. In addition, both NRV2.1 and NRV2.2 are able to rescue the dye diffusion phenotype of l(2)k13315 whereas NRV1 is not. Together these results indicate that l(2)k13315 is a mutation in the Nrv2 locus, and that NRV2 normally functions in the SJ. Although both NRV2 and NRV1 were previously described as being nervous system specific, evidence from immunostaining and from a GFP gene trap inserted within the Nrv2 locus indicates that Nrv2 is highly expressed in epithelial cells. Because NRV1 expression is not affected by the l(2)k13315 mutation and l(2)k13315 homozygous mutant cells in the wing imaginal disc lack NRV staining, it is proposed that Nrv1 is nervous system specific and epidermal cells express only NRV2 (Genova, 2003).

The observation that an Nrv1 transgene cannot rescue the Nrv2 dye diffusion phenotype, even though it localizes to the SJ when ectopically expressed in epithelial cells, suggests that the proteins encoded by these genes, although quite similar in structure, are functionally distinct. Given the sequence diversity within the cytoplasmic tail, the observation that when expressed ectopically all three proteins localize to the SJ strongly suggests that this localization is mediated by the extracellular or transmembrane domain, rather than by the intracellular domain. This complex pattern of ß subunit expression and functional interactions suggests a surprising degree of functional regulation of the Na+/K+ ATPase in epithelial and neuronal cells (Genova, 2003).

The question still remains, What is the function of localizing the Na pump to such a specialized membrane domain, one of whose functions is to create a paracellular diffusion barrier? Several characteristics of the Na pump might be important in SJ function. Previous studies suggest that the Na+/K+ ATPase functions in cell adhesion, though whether its role is structural or regulatory is unclear. Other studies suggest that the Na pump could function as a scaffold on which proteins essential for the paracellular barrier are organized. For example, both subunits bind to a variety of proteins, from those involved in signal transduction to cytoskeletal elements. In addition, it is possible that the ion pumping activity of the Na pump actively participates in the formation or maintenance of the diffusion barrier. Studies in mammalian cells have demonstrated a requirement for the ATPase, and specifically the Na+ gradient it produces, in cell polarity, adhesion, and the formation of tight junctions. Because the tight junction is responsible for creating the paracellular barrier in vertebrate epithelial cells, the ATPase might perform a similar function in the paracellular barrier of the Drosophila SJ. Further experiments, using point mutations that specifically affect the pump function of the ATPase, could address these questions (Genova, 2003 and references therein).

Cora has been shown to bind to the cytoplasmic tail of Nrx in the SJ. Studies of the PSJ have shown that the mammalian homologs of Nrx and Nrg interact via their extracellular domains. Together, these observations suggest the existence of a multiprotein complex at the SJ in which Cora binds to Nrx, which in turn binds to Nrg. The finding that Nrx and Nrg coimmunoprecipitate when either anti-Cora or anti-Nrg antibodies are used to immunoprecipitate is consistent with this model. Because Drosophila epithelial cells express all three proteins, it is not possible to rigorously distinguish whether this interaction occurs within the same cell or between adjacent cells. However, the observation that wild-type cells are unable to efficiently assemble Cora and Nrx at the boundary with cor- cells suggests that intercellular interaction with the same complex on adjacent cells is required for SJ assembly. In addition, Nrv is found to coimmunoprecipitates with both Cora and Nrx. Nrg has not been detected in this complex, suggesting that the interaction between NRV2 and the Cora-Nrx complex occurs independently of Nrg, perhaps on the cytoplasmic side of the membrane. Although these results imply the possibility of an interaction between Cora and the cytoplasmic tail of NRV2, this seems unlikely in light of observations that NRV1, 2.1, and 2.2 all localize to the SJ, despite having different cytoplasmic tails. Thus, it is more likely that the interaction between Cora and the ATPase occurs either through Nrx or the alpha subunit (Genova, 2003).

Somatic mosaic analysis has demonstrated that this complex of Cora, Nrx, Nrv, ATPalpha, and Nrg can be disrupted without affecting overall polarity, or other components of the SJ. No component essential for the paracellular barrier has been identified that is unaffected in mutant cells, suggesting that the substrate upon which this complex assembles has yet to be found. Previous studies have demonstrated that Ankyrin binds both the cytoplasmic domain of Nrg and, as has been described in mammalian cells, the alpha subunit of NA+/K+ ATPase. In addition, Ankyrin colocalizes with Nrg at points of Nrg-induced S2 cell adhesion complexes. Thus, one candidate for a substrate upon which this complex assembles is Ankyrin, a well-known member of the membrane skeleton (Genova, 2003).

Other candidate proteins for this scaffold are Scribble and Dlg. Both of these proteins are required early in Drosophila development for the establishment of epithelial cell polarity and growth control. If either is absent from epithelial cells, then the apical junctional complexes do not properly form and epithelial integrity is lost. Thus, Scribble and Dlg may be among the first constituents of the SJ upon which the subsequently expressed SJ proteins assemble (Genova, 2003).

Previous studies have suggested that the SJ may function in intercellular signaling, particularly in the regulation of cell proliferation. For example, dlg, which encodes a PDZ repeat-containing, membrane-associated guanylate kinase protein, has tumor suppressor functions. Loss of function dlg mutations are characterized by disruption of apical-basal polarity and an overproliferation of the larval imaginal discs. However, it is not known whether this overproliferation is due to a direct involvement of Dlg in a signal transduction cascade or to the disruption of apical-basal polarity within epithelial cells that could result in a disruption of apical signaling complexes. In addition to dlg, cor mutations were first isolated as dominant suppressors of a gain of function allele of the EGF receptor, EgfrElp (also known as EgfrE3), suggesting that Cora may function to positively regulate EGFR pathway function. Interestingly, a recent study of Nrg function in the developing Drosophila nervous system has proposed that it positively regulates EGF receptor function during axon guidance. The role of Nrg in regulating EGFR function in epithelial cells has not been investigated, but preliminary results indicate that Nrg mutations also dominantly suppress the rough eye caused by EgfrElp. This result may suggest that Nrg (or the entire complex) must be localized to the SJ in epithelial cells to regulate Egfr function. Alternatively, it is possible that the SJ complex is necessary to maintain polarized localization of the Egfr to the apical membrane, though no effect of cor mutations on Egfr localization has been observed (Genova, 2003).

The recent discovery of molecular, structural, and functional similarities between the invertebrate epithelial SJ and the vertebrate PSJ in the nervous system gives added significance to the identification of new SJ components in Drosophila. In addition to Cora/protein 4.1 and Nrx/paranodin, the SJ and PSJ have been shown to share neurofascin-155 and a Drosophila homolog, Nrg. This level of molecular homology strongly suggests that these two SJs are structurally and functionally homologous as well. It is therefore somewhat surprising that published reports indicate that the Na pump is uniformly distributed along the axonal membrane rather than being restricted to the PSJ (Genova, 2003 and references therein). One possible explanation is that only a subset of the several Na+/K+ ATPase isoforms found in the mammalian genome is localized to the PSJ, and that these isoforms have not yet been studied. Similarly, it is not known if the mammalian homologs of Drosophila Gli, the neuroligins, might localize to the PSJ, or if the Drosophila homolog of contactin, a protein that interacts with Nrx/paranodin, localizes to the SJ. Although it is possible that the invertebrate epithelial SJ and vertebrate PSJ are fundamentally different in some respects, this is unlikely given the remarkable degree of similarity between these two junctions. In any case, it is clear that the genetic and genomic tools available in Drosophila can provide important insights into both the SJ and its vertebrate counterpart, the PSJ (Genova, 2003).

Neuroglian and Nrx are interdependent for their SJ localization and these proteins form a tripartite complex

Septate junctions (SJs) in epithelial and neuronal cells play an important role in the formation and maintenance of charge and the size of selective barriers. They form the basis for the ensheathment of nerve fibers in Drosophila and for the attachment of myelin loops to axonal surface in vertebrates. The cell-adhesion molecules NRX IV/Caspr/Paranodin (NCP1), contactin and Neurofascin-155 (NF-155) are all present at the vertebrate axo-glial SJs. Mutational analyses have shown that vertebrate NCP1 and its Drosophila homolog, Neurexin IV are required for the formation of SJs. The Drosophila homolog of vertebrate contactin, Contactin, has been genetically, molecularly, and biochemically characterized. Ultrastructural and dye-exclusion analyses of Cont mutant embryos show that Cont is required for organization of SJs and paracellular barrier function. Cont, Neuroglian (Nrg) (Drosophila homolog of NF-155) and Nrx IV are interdependent for their SJ localization and these proteins form a tripartite complex. Hence, these data provide evidence that the organization of SJs is dependent on the interactions between these highly conserved cell-adhesion molecules (Faivre-Sarrailh, 2004).

Analyses of cont, nrx IV and nrg mutants have shown that these genes display a common alteration of SJ phenotype and thus are required for the formation and/or organization of SJs. In addition, the immunohistochemical analysis of these mutants shows that Cont, Nrx IV and Nrg are dependent on each other for SJ localization. The phenotypic similarities raise the interesting issue of whether these proteins are part of a macromolecular protein complex that exists at SJs, and thus become dependent on each other for their localization. To determine whether these three proteins form a biochemical complex, microsomal NP-40 extracts were prepared from Drosophila embryos and used for co-immunoprecipitation experiments. Cont was efficiently co-immunoprecipitated with Nrx IV using either anti-Nrx IV or anti-Cont antibodies. Cont and Nrx IV were not co-precipitated by monoclonal anti-Nrg antibodies, owing to weak ability to immunoprecipitate Nrg complexes. However, by probing Western blots with the 3C1 anti-Nrg mAb, Nrg was detected in anti-Cont immunoprecipitates. Only the lower molecular weight Nrg167 isoform, which is expressed in epithelial cells, was co-immunoprecipitated with Cont. By contrast, the neuronal Nrg180 isoform could only be detected in the total NP-40 lysate. These results demonstrate that Cont, Nrx IV and Nrg are part of a protein complex and that the molecular interactions between these proteins may underlie the organization of the SJs (Faivre-Sarrailh, 2004).

Thus biochemical and immunohistochemical data show that Cont is part of a protein complex that includes Nrx IV and that Cont is mislocalized in nrx IV mutants. Furthermore, in these mutants, Cont protein is not expressed properly on the plasma membrane and is seen in intracellular vesicles in the epithelial cells. This raised the possibility that Nrx IV might be involved in the cell surface targeting or stabilization of Cont (Faivre-Sarrailh, 2004).

Since Drosophila S2 cells constitutively express Nrx IV and Cont, this question was addressed using neuroblastoma N2a cells as a heterologous expression system. In transfected N2a cells, Nrx IV is efficiently expressed at the cell surface. By contrast, in cells expressing Cont, the Cont protein appears to remain localized intracellularly and double-staining with the ER marker BiP indicates that Cont is retained in the ER. Immunofluorescence staining under permeabilizing conditions of N2a cells, which were co-transfected with Cont and Nrx IV, revealed that Cont is transported to and co-localized with Nrx IV at the plasma membrane. Similar results were obtained with intact living cells. Thus, these results indicate that Nrx IV mediates the cell-surface targeting of Cont. Next, Cont was co-immunoprecipitated with Nrx IV from co-transfected COS cell lysates, as further evidence that these proteins interact in cis. Such a cis-interaction has been reported for their vertebrate counterparts contactin and NCP1 (Peles, 1997; Faivre-Sarrailh, 2004).

Neuroglian: Biological Overview | Evolutionary Homologs | Developmental Biology | Effects of Mutation | References

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