Formation Of Septate Junctions Research Articles

Sinuous Is a Claudin Required for Locust Molt in Locusta migratoria.

The epidermal cells of insects are polarized epithelial cells that play a pivotal role in the insect's molting process. Sinuous, a pivotal structural protein involved in the formation of septate junctions among epithelial cells, is essential for its physiological function. In this study, to determine whether sinuous participates in the regulation of insect molting, we identified the sinuous gene, Lmsinu, in Locusta migratoria, which encodes a protein belonging to the claudin family and shares 62.6% identity with Drosophila's sinuous protein. Lmsinu is expressed in multiple tissues, and its expression level in the integument significantly increases prior to molting. Knockdown of Lmsinu in L. migratoria results in larval mortality during molting. Furthermore, hematoxylin and eosin and chitin staining demonstrate that the downregulation of Lmsinu led to a prolonged degradation process of the old cuticle during the molting process. Electron microscopy analysis further revealed that knockdown of Lmsinu disrupts the formation of septate junctions among epidermal cells, which are a monolayer of polarized epithelial cells, which may hinder the functionality of epidermal cells during the process of molting. In summary, these findings suggest that Lmsinu plays a role in nymph molting by regulating the formation of septate junctions among epidermal cells.

The Drosophila blood–brain barrier invades the nervous system in a GPCR-dependent manner

The blood–brain barrier (BBB) represents a crucial interface between the circulatory system and the brain. In Drosophila melanogaster, the BBB is composed of perineurial and subperineurial glial cells. The perineurial glial cells are small mitotically active cells forming the outermost layer of the nervous system and are engaged in nutrient uptake. The subperineurial glial cells form occluding septate junctions to prevent paracellular diffusion of macromolecules into the nervous system. To address whether the subperineurial glia just form a simple barrier or whether they establish specific contacts with both the perineurial glial cells and inner central nervous system (CNS) cells, we undertook a detailed morphological analysis. Using genetically encoded markers alongside with high-resolution laser scanning confocal microscopy and transmission electron microscopy, we identified thin cell processes extending into the perineurial layer and into the CNS cortex. Interestingly, long cell processes were observed reaching the glia ensheathing the neuropil of the central brain. GFP reconstitution experiments highlighted multiple regions of membrane contacts between subperineurial and ensheathing glia. Furthermore, we identify the G-protein-coupled receptor (GPCR) Moody as negative regulator of the growth of subperineurial cell processes. Loss of moody triggered a massive overgrowth of subperineurial cell processes into the CNS cortex and, moreover, affected the polarized localization of the xenobiotic transporter Mdr65. Finally, we found that GPCR signaling, but not septate junction formation, is responsible for controlling membrane overgrowth. Our findings support the notion that the Drosophila BBB is able to bridge the communication gap between circulation and synaptic regions of the brain by long cell processes.

CG11426 gene product negatively regulates glial population size in the Drosophila eye imaginal disc.

Glial cells play essential roles in the nervous system. Although glial populations are tightly regulated, the mechanisms regulating the population size remain poorly understood. Since Drosophila glial cells are similar to the human counterparts in their functions and shapes, rendering them an excellent model system to understand the human glia biology. Lipid phosphate phosphatases (LPPs) are important for regulating bioactive lipids. In Drosophila, there are three known LPP-encoding genes: wunen, wunen-2, and lazaro. The wunens are important for germ cell migration and survival and septate junction formation during tracheal development. Lazaro is involved in phototransduction. In the present study, we characterized a novel Drosophila LPP-encoding gene, CG11426. Suppression of CG11426 increased glial cell number in the eye imaginal disc during larval development, while ectopic CG11426 expression decreased it. Both types of mutation also caused defects in axon projection to the optic lobe in larval eye-brain complexes. Moreover, CG11426 promoted apoptosis via inhibiting ERK signaling in the eye imaginal disc. Taken together, these findings demonstrated that CG11426 gene product negatively regulates ERK signaling to promote apoptosis for proper maintenance of the glial population in the developing eye disc.

Non-Cell-Autonomous Function of the GPI-Anchored Protein Undicht during Septate Junction Assembly

Occluding cell-cell junctions are pivotal during the development of many organs. One example is septate junction (SJ) strands, which are found in vertebrates and invertebrates. Although several proteins have been identified that are responsible for septate junction formation in Drosophila, it is presently unclear how these structures are formed or how they are positioned in a coordinated manner between two neighboring cells and within the tissue. Here, we identified a GPI-anchored protein called Undicht required for septate junction formation. Clonal analysis and rescue experiments show that Undicht acts in a non-cell-autonomous manner. It can be released from the plasma membrane by the proteolytic activity of two related ADAM10-like proteases, Kuzbanian and Kuzbanian-like. We propose that juxtacrine function of Undicht coordinates the formation of septate junction strands on two directly neighboring cells, whereas paracrine activity of Undicht controls the formation of occluding junctions within a tissue.

Axonal wrapping in the Drosophila PNS is controlled by glia-derived neuregulin homolog Vein.

Efficient neuronal conductance requires that axons are insulated by glial cells. For this, glial membranes need to wrap around axons. Invertebrates show a relatively simple extension of glial membranes around the axons, resembling Remak fibers formed by Schwann cells in the mammalian peripheral nervous system. To unravel the molecular pathways underlying differentiation of glial cells that provide axonal wrapping, we are using the genetically amenable Drosophila model. At the end of larval life, the wrapping glia differentiates into very large cells, spanning more than 1 mm of axonal length. The extension around axonal membranes is not influenced by the caliber of the axon or its modality. Using cell type-specific gene knockdown we show that the extension of glial membranes around the axons is regulated by an autocrine activation of the EGF receptor through the neuregulin homolog Vein. This resembles the molecular mechanism employed during cell-autonomous reactivation of glial differentiation after injury in mammals. We further demonstrate that Vein, produced by the wrapping glia, also regulates the formation of septate junctions in the abutting subperineurial glia. Moreover, the wrapping glia indirectly controls the proliferation of the perineurial glia. Thus, the wrapping glia appears center stage to orchestrate the development of the different glial cell layers in a peripheral nerve.

The Drosophila blood-brain barrier: development and function of a glial endothelium.

The efficacy of neuronal function requires a well-balanced extracellular ion homeostasis and a steady supply with nutrients and metabolites. Therefore, all organisms equipped with a complex nervous system developed a so-called blood-brain barrier, protecting it from an uncontrolled entry of solutes, metabolites or pathogens. In higher vertebrates, this diffusion barrier is established by polarized endothelial cells that form extensive tight junctions, whereas in lower vertebrates and invertebrates the blood-brain barrier is exclusively formed by glial cells. Here, we review the development and function of the glial blood-brain barrier of Drosophila melanogaster. In the Drosophila nervous system, at least seven morphologically distinct glial cell classes can be distinguished. Two of these glial classes form the blood-brain barrier. Perineurial glial cells participate in nutrient uptake and establish a first diffusion barrier. The subperineurial glial (SPG) cells form septate junctions, which block paracellular diffusion and thus seal the nervous system from the hemolymph. We summarize the molecular basis of septate junction formation and address the different transport systems expressed by the blood-brain barrier forming glial cells.

The transmembrane protein Macroglobulin complement-related is essential for septate junction formation and epithelial barrier function inDrosophila

Occluding cell-cell junctions in epithelia form physical barriers that separate different membrane domains, restrict paracellular diffusion and prevent pathogens from spreading across tissues. In invertebrates, these functions are provided by septate junctions (SJs), the functional equivalent of vertebrate tight junctions. How the diverse functions of SJs are integrated and modulated in a multiprotein complex is not clear, and many SJ components are still unknown. Here we report the identification of Macroglobulin complement-related (Mcr), a member of the conserved α-2-macroglobulin (α2M) complement protein family, as a novel SJ-associated protein in Drosophila. Whereas α2M complement proteins are generally known as secreted factors that bind to surfaces of pathogens and target them for phagocytic uptake, Mcr represents an unusual α2M protein with a predicted transmembrane domain. We show that Mcr protein localizes to lateral membranes of epithelial cells, where its distribution overlaps with SJs. Several SJ components are required for the correct localization of Mcr. Conversely, Mcr is required in a cell-autonomous fashion for the correct membrane localization of SJ components, indicating that membrane-bound rather than secreted Mcr isoforms are involved in SJ formation. Finally, we show that loss of Mcr function leads to morphological, ultrastructural and epithelial barrier defects resembling mutants lacking SJ components. Our results, along with previous findings on the role of Mcr in phagocytosis, suggest that Mcr plays dual roles in epithelial barrier formation and innate immunity. Thus, Mcr represents a novel paradigm for investigating functional links between occluding junction formation and pathogen defense mechanisms.

Control of tissue morphology by Fasciclin III-mediated intercellular adhesion

Morphogenesis is dependent on the orchestration of multiple developmental processes to generate mature functional organs. However, the signalling pathways that coordinate morphogenesis and the mechanisms that translate these signals into tissue shape changes are not well understood. Here, we demonstrate that changes in intercellular adhesion mediated by the transmembrane protein Fasciclin III (FasIII) represent a key mediator of morphogenesis. Using the embryonic Drosophila hindgut as an in vivo model for organogenesis, we show that the tightening of hindgut curvature that normally occurs between embryonic stage 12 and 15 to generate the characteristic shepherd’s crook shape is dependent on localised JAK/STAT pathway activation. This localised pathway activity drives the expression of FasIII leading to its subcellular lateralisation at a stage before formation of septate junctions. Additionally, we show that JAK/STAT- and FasIII-dependent morphogenesis also regulates folds within the third instar wing imaginal disc. We show that FasIII forms homophilic intercellular interactions that promote intercellular adhesion in vivo and in cultured cells. To explore these findings, we have developed a mathematical model of the developing hindgut, based on the differential interfacial tension hypothesis (DITH) linking intercellular adhesion and localised surface tension. Our model suggests that increased intercellular adhesion provided by FasIII can be sufficient to drive the tightening of tube curvature observed. Taken together, these results identify a conserved molecular mechanism that directly links JAK/STAT pathway signalling to intercellular adhesion and that sculpts both tubular and planar epithelial shape.

A novel protein complex, mesh-ssk, is required for septate junction formation indrosophilamidgut

Septate junctions (SJs) are specialized intercellular junctions that restrict the free diffusion of solutes through the paracellular route in invertebrate epithelia. In arthropods, two morphologically different types of SJs have been reported: pleated SJs and smooth SJs (sSJs), which are found in ectodermally and endodermally derived epithelia, respectively. However, the molecular and functional differences between these SJ types have not been fully elucidated. Here, we report that a novel sSJ-specific component, a single-pass transmembrane protein, which we term 'Mesh' (encoded by CG31004), is highly concentrated in Drosophila sSJs. Compromised mesh expression causes defects in the organization of sSJs, in the localizations of other sSJ proteins, and in the barrier function of the midgut. Ectopic expression of Mesh in cultured cells induces cell-cell adhesion. Mesh forms a complex with Ssk, another sSJ-specific protein, and these proteins are mutually interdependent for their localization. Thus, a novel protein complex comprising Mesh and Ssk has an important role in sSJ formation and in intestinal barrier function in Drosophila.

Description of Triangulamyxa psittaca sp. nov. (Myxozoa: Myxosporea), a New Parasite in the Urinary Bladder of Colomesus psittacus (Teleostei) from the Amazon River, with Emphasis on the Ultrastructure of Plasmodial Stages

A fish-infecting myxosporean was found in the urinary bladder of the teleostean Colomesus psittacus, collected from the Amazon River, Brazil. Specimens were sampled in three different periods: May and June, with water temperature ranging from 18-23oC; August, with water temperature ranging from 24-28oC; and November and December, with water temperature ranging from 29-32oC. Upon observation, several fish displayed abnormal behaviour, consisting of erratic movements, and mortality was recorded among them. Necropsy of all sampled fishes revealed hypertrophy of the urinary bladder only among specimens previously displaying the irregular behaviour. Microscopic analysis of this organ confirmed the parasitic infection, resulting in the observation of spores floating free in the urine, and numerous plasmodia attached to the epithelium of the urinary bladder. Light and ultrastructural studies allowed recognition of the spores and plasmodia morphological characteristics. Coelozoic plasmodia were polysporic with varying organizational structure, according to the sampling period. Spores were equilaterally triangular with rounded ends in valvar view, measuring 8.8 ± 0.4 μm (n = 30) in length and 8.4 ± 0.5 μm (n = 30) in width, and displaying a ridge surface pattern. Two polar capsules were observed in the anterior end of the spores, measuring 3.1-3.2 μm in diameter. The spores were morphologically identified as belonging to the recently described genus Triangulamyxa. Further observation and comparison to the morphological features described for Triangulamyxa amazonica, the only other species within this genus, allowed us to conclude our parasite as a new species, herein named Triangulamyxa psittaca sp. nov. from the Amazon River, Brazil. Also, three different stages were distinguished in the plasmodium evolution, based on the observed morphological features at the three sampling periods. Fish sampled during May and June displayed small plasmodia (up to ~ 15-20 μm long), containing early stages of sporogenic development. Fish sampled during November and December presented larger plasmodia (up to ~ 850 μm long), which appeared flattened against and lining the urinary bladder epithelial cells and contained the later stages of sporogenic development, including some mature spores. Fish sampled during August presented plasmodia displaying intermediate morphological features between those observed in infected fish from the other sampling periods. Several immature and mature spores were among the different developmental stages. The parasite-host interface evolution is described throughout the different observed stages, with emphasis on the formation of septate junctions. Considering several previous reports, as well as the different environmental conditions during the sampling periods, the plasmodium development here described appears to be influenced by environmental factors, namely water temperature.

The CD59 Family Member Leaky/Coiled Is Required for the Establishment of the Blood–Brain Barrier inDrosophila

The blood-brain barrier of Drosophila is established by the subperineurial glial cells that encase the CNS and PNS. The subperineurial glial cells are thin, highly interdigitated cells with epithelial character. The establishment of extensive septate junctions between these cells is crucial for the prevention of uncontrolled paracellular leakage of ions and solutes from the hemolymph into the nervous system. In the absence of septate junctions, macromolecules such as fluorescently labeled dextran can easily cross the blood-brain barrier. To identify additional components of the blood-brain barrier, we followed a genetic approach and injected Texas-Red-conjugated dextran into the hemolymph of embryos homozygous for chromosomal deficiencies. In this way, we identified the 153-aa-large protein Coiled, a new member of the Ly6 (leukocyte antigen 6) family, as being crucially required for septate junction formation and blood-brain barrier integrity. In coiled mutants, the normal distribution of septate junction markers such as NeurexinIV, Coracle, or Discs large is disturbed. EM analyses demonstrated that Coiled is required for the formation of septate junctions. We further show that Coiled is expressed by the subsperineurial glial cells in which it is anchored to the cell membrane via a glycosylphosphatidylinositol anchor and mediates adhesive properties. Clonal rescue studies indicate that the presence of Coiled is required symmetrically on both cells engaged in septate junction formation.

The Drosophila Claudin Kune-kune Is Required for Septate Junction Organization and Tracheal Tube Size Control

The vertebrate tight junction is a critical claudin-based cell-cell junction that functions to prevent free paracellular diffusion between epithelial cells. In Drosophila, this barrier is provided by the septate junction, which, despite being ultrastructurally distinct from the vertebrate tight junction, also contains the claudin-family proteins Megatrachea and Sinuous. Here we identify a third Drosophila claudin, Kune-kune, that localizes to septate junctions and is required for junction organization and paracellular barrier function, but not for apical-basal polarity. In the tracheal system, septate junctions have a barrier-independent function that promotes lumenal secretion of Vermiform and Serpentine, extracellular matrix modifier proteins that are required to restrict tube length. As with Sinuous and Megatrachea, loss of Kune-kune prevents this secretion and results in overly elongated tubes. Embryos lacking all three characterized claudins have tracheal phenotypes similar to any single mutant, indicating that these claudins act in the same pathway controlling tracheal tube length. However, we find that there are distinct requirements for these claudins in epithelial septate junction formation. Megatrachea is predominantly required for correct localization of septate junction components, while Sinuous is predominantly required for maintaining normal levels of septate junction proteins. Kune-kune is required for both localization and levels. Double- and triple-mutant combinations of Sinuous and Megatrachea with Kune-kune resemble the Kune-kune single mutant, suggesting that Kune-kune has a more central role in septate junction formation than either Sinuous or Megatrachea.

Crooked, Coiled and Crimpled are three Ly6-like proteins required for proper localization of septate junction components

Cellular junction formation is an elaborate process that is dependent on the regulated synthesis, assembly and membrane targeting of constituting components. Here, we report on three Drosophila Ly6-like proteins essential for septate junction (SJ) formation. SJs provide a paracellular diffusion barrier and appear molecularly and structurally similar to vertebrate paranodal septate junctions. We show that Crooked (Crok), a small GPI-anchored Ly6-like protein, is required for septa formation and barrier functions. In embryos that lack Crok, SJ components are produced but fail to accumulate at the plasma membrane. Crok is detected in intracellular puncta and acts tissue-autonomously, which suggests that it resides in intracellular vesicles to assist the cell surface localization of SJ components. In addition, we demonstrate that two related Ly6 proteins, Coiled (Cold) and Crimpled (Crim), are required for SJ formation and function in a tissue-autonomous manner, and that Cold also localizes to intracellular vesicles. Specifically, Crok and Cold are required for correct membrane trafficking of Neurexin IV, a central SJ component. The non-redundant requirement for Crok, Cold, Crim and Boudin (Bou; another Ly6 protein that was recently shown to be involved in SJ formation) suggests that members of this conserved family of proteins cooperate in the assembly of SJ components, possibly by promoting core SJ complex formation in intracellular compartments associated with membrane trafficking.

The leading edge during dorsal closure as a model for epithelial plasticity: Pak is required for recruitment of the Scribble complex and septate junction formation

Dorsal closure (DC) of the Drosophila embryo is a model for the study of wound healing and developmental epithelial fusions, and involves the sealing of a hole in the epidermis through the migration of the epidermal flanks over the tissue occupying the hole, the amnioserosa. During DC, the cells at the edge of the migrating epidermis extend Rac- and Cdc42-dependent actin-based lamellipodia and filopodia from their leading edge (LE), which exhibits a breakdown in apicobasal polarity as adhesions are severed with the neighbouring amnioserosa cells. Studies using mammalian cells have demonstrated that Scribble (Scrib), an important determinant of apicobasal polarity that functions in a protein complex, controls polarized cell migration through recruitment of Rac, Cdc42 and the serine/threonine kinase Pak, an effector for Rac and Cdc42, to the LE. We have used DC and the follicular epithelium to study the relationship between Pak and the Scrib complex at epithelial membranes undergoing changes in apicobasal polarity and adhesion during development. We propose that, during DC, the LE membrane undergoes an epithelial-to-mesenchymal-like transition to initiate epithelial sheet migration, followed by a mesenchymal-to-epithelial-like transition as the epithelial sheets meet up and restore cell-cell adhesion. This latter event requires integrin-localized Pak, which recruits the Scrib complex in septate junction formation. We conclude that there are bidirectional interactions between Pak and the Scrib complex modulating epithelial plasticity. Scrib can recruit Pak to the LE for polarized cell migration but, as migratory cells meet up, Pak can recruit the Scrib complex to restore apicobasal polarity and cell-cell adhesion.

The leading edge during dorsal closure as a model for epithelial plasticity: Pak is required for recruitment of the Scribble complex and septate junction formation

Dorsal closure (DC) of the Drosophila embryo is a model for the study of wound healing and developmental epithelial fusions, and involves the sealing of a hole in the epidermis through the migration of the epidermal flanks over the tissue occupying the hole, the amnioserosa. During DC, the cells at the edge of the migrating epidermis extend Rac- and Cdc42-dependent actin-based lamellipodia and filopodia from their leading edge (LE), which exhibits a breakdown in apicobasal polarity as adhesions are severed with the neighbouring amnioserosa cells. Studies using mammalian cells have demonstrated that Scribble (Scrib), an important determinant of apicobasal polarity that functions in a protein complex, controls polarized cell migration through recruitment of Rac, Cdc42 and the serine/threonine kinase Pak, an effector for Rac and Cdc42, to the LE. We have used DC and the follicular epithelium to study the relationship between Pak and the Scrib complex at epithelial membranes undergoing changes in apicobasal polarity and adhesion during development. We propose that, during DC, the LE membrane undergoes an epithelial-to-mesenchymal-like transition to initiate epithelial sheet migration, followed by a mesenchymal-to-epithelial-like transition as the epithelial sheets meet up and restore cell-cell adhesion. This latter event requires integrin-localized Pak, which recruits the Scrib complex in septate junction formation. We conclude that there are bidirectional interactions between Pak and the Scrib complex modulating epithelial plasticity. Scrib can recruit Pak to the LE for polarized cell migration but, as migratory cells meet up, Pak can recruit the Scrib complex to restore apicobasal polarity and cell-cell adhesion.

Drosophila convoluted/dALS is an essential gene required for tracheal tube morphogenesis and apical matrix organization.

Insulin-like growth factors (IGFs) control cell and organism growth through evolutionarily conserved signaling pathways. The mammalian acid-labile subunit (ALS) is a secreted protein that complexes with IGFs to modulate their activity. Recent work has shown that a Drosophila homolog of ALS, dALS, can also complex with and modulate the activity of a Drosophila IGF. Here we report the first mutations in the gene encoding dALS. Unexpectedly, we find that these mutations are allelic to a previously described mutation in convoluted (conv), a gene required for epithelial morphogenesis. In conv mutants, the tubes of the Drosophila tracheal system become abnormally elongated without altering tracheal cell number. conv null mutations cause larval lethality, but do not disrupt several processes required for tracheal tube size control, including septate junction formation, deposition of a lumenal/apical extracellular matrix, and lumenal secretion of Vermiform and Serpentine, two putative matrix-modifying proteins. Clearance of lumenal matrix and subcellular localization of clathrin also appear normal in conv mutants. However, we show that Conv/dALS is required for the dynamic organization of the transient lumenal matrix and normal structure of the cuticle that lines the tracheal lumen. These and other data suggest that the Conv/dALS-dependent tube size control mechanism is distinct from other known processes involved in tracheal tube size regulation. Moreover, we present evidence indicating that Conv/dALS has a novel, IGF-signaling independent function in tracheal morphogenesis.

Nak regulates Dlg basal localization in Drosophila salivary gland cells

Protein trafficking is highly regulated in polarized cells. During development, how the trafficking of cell junctional proteins is regulated for cell specialization is largely unknown. In the maturation of Drosophila larval salivary glands (SGs), the Dlg protein is essential for septate junction formation. We show that Dlg was enriched in the apical membrane domain of proximal cells and localized basolaterally in distal mature cells. The transition of Dlg distribution was disrupted in nak mutants. Nak associated with the AP-2 subunit α-Ada and the AP-1 subunit AP-1γ. In SG cells disrupting AP-1 and AP-2 activities, Dlg was enriched in the apical membrane. Therefore, Nak regulates the transition of Dlg distribution likely through endocytosis of Dlg from the apical membrane domain and transcytosis of Dlg to the basolateral membrane domain during the maturation of SGs development.

The lateral mobility of cell adhesion molecules is highly restricted at septate junctions in Drosophila

BackgroundA complex of three cell adhesion molecules (CAMs) Neurexin IV(Nrx IV), Contactin (Cont) and Neuroglian (Nrg) is implicated in the formation of septate junctions between epithelial cells in Drosophila. These CAMs are interdependent for their localization at septate junctions and e.g. null mutation of nrx IV or cont induces the mislocalization of Nrg to the baso-lateral membrane. These mutations also result in ultrastructural alteration of the strands of septate junctions and breakdown of the paracellular barrier. Varicose (Vari) and Coracle (Cora), that both interact with the cytoplasmic tail of Nrx IV, are scaffolding molecules required for the formation of septate junctions.ResultsWe conducted photobleaching experiments on whole living Drosophila embryos to analyze the membrane mobility of CAMs at septate junctions between epithelial cells. We show that GFP-tagged Nrg and Nrx IV molecules exhibit very stable association with septate junctions in wild-type embryos. Nrg-GFP is mislocalized to the baso-lateral membrane in nrx IV or cont null mutant embryos, and displays increased mobile fraction. Similarly, Nrx IV-GFP becomes distributed to the baso-lateral membrane in null mutants of vari and cora, and its mobile fraction is strongly increased. The loss of Vari, a MAGUK protein that interacts with the cytoplasmic tail of Nrx IV, has a stronger effect than the null mutation of nrx IV on the lateral mobility of Nrg-GFP.ConclusionThe strands of septate junctions display a stable behavior in vivo that may be correlated with their role of paracellular barrier. The membrane mobility of CAMs is strongly limited when they take part to the multimolecular complex forming septate junctions. This restricted lateral diffusion of CAMs depends on both adhesive interactions and clustering by scaffolding molecules. The lateral mobility of CAMs is strongly increased in embryos presenting alteration of septate junctions. The stronger effect of vari by comparison with nrx IV null mutation supports the hypothesis that this scaffolding molecule may cross-link different types of CAMs and play a crucial role in stabilizing the strands of septate junctions.

HOW to Wrap Axons with Crooked Neck

How can RNA processing direct specific aspects of nervous system development? In this issue of Neuron, Edenfeld et al. identified a novel function for two regulators of mRNA splicing in Drosophila: peripheral glial cells require Crooked neck (Crn) and Held out wings (HOW) to mediate migration and ensheathment of peripheral axons.

Axonal Ensheathment and Septate Junction Formation in the Peripheral Nervous System ofDrosophila

Axonal insulation is critical for efficient action potential propagation and normal functioning of the nervous system. In Drosophila, the underlying basis of nerve ensheathment is the axonal insulation by glial cells and the establishment of septate junctions (SJs) between glial cell membranes. However, the details of the cellular and molecular mechanisms underlying axonal insulation and SJ formation are still obscure. Here, we report the characterization of axonal insulation in the Drosophila peripheral nervous system (PNS). Targeted expression of tau-green fluorescent protein in the glial cells and ultrastructural analysis of the peripheral nerves allowed us to visualize the glial ensheathment of axons. We show that individual or a group of axons are ensheathed by inner glial processes, which in turn are ensheathed by the outer perineurial glial cells. SJs are formed between the inner and outer glial membranes. We also show that Neurexin IV, Contactin, and Neuroglian are coexpressed in the peripheral glial membranes and that these proteins exist as a complex in the Drosophila nervous system. Mutations in neurexin IV, contactin, and neuroglian result in the disruption of blood-nerve barrier function in the PNS, and ultrastructural analyses of the mutant embryonic peripheral nerves show loss of glial SJs. Interestingly, the murine homologs of Neurexin IV, Contactin, and Neuroglian are expressed at the paranodal SJs and play a key role in axon-glial interactions of myelinated axons. Together, our data suggest that the molecular machinery underlying axonal insulation and axon-glial interactions may be conserved across species.