Neurexin IV
The gene is transcribed only zygotically , with its peak of expression between 6 and 18 hours after egg lay. NRX is first detected at late stage 11/early stage 12. NRX is localized to epithelial cells of ectodermal origin such as epidermis, the tracheal system, pharynx, esophagus, proventriculus, hindgut, salivary gland, and cells of the peripheral and central nervous systems. Staining in the PNS is localized to the scolopales of the lateral chordotonal orgtans and is restricted to glial cells. NRX positive cells in the CNS and along the peripheral nerves express glial-specific markers and not neuronal markers. Two types of epithelia do not express NRX: amnioserosa and Malpighian tubules. All tissues that express NRX in the embryo are characterized by the presence of pleated synaptic junctions.
In the chordotonal organs, NRX is expressed in cells surrounding the scolopales where synaptic junctions have been localized. In addition, NRX is distributed in a ring at the apical end of many cell types, as shown for synaptic junctions (Baumgartner, 1996).
The function of a complex nervous system depends on an intricate interplay between neuronal and glial cell types. One of the many functions of glial cells is to provide an efficient insulation of the nervous system and thereby allowing a fine tuned homeostasis of ions and other small molecules. This study presents a detailed cellular analysis of the glial cell complement constituting the blood-brain barrier in Drosophila. Using electron microscopic analysis and single cell-labeling experiments, different glial cell layers at the surface of the nervous system, the perineurial glial layer, the subperineurial glial layer, the wrapping glial cell layer, and a thick layer of extracellular matrix, the neural lamella, were characterized. To test the functional roles of these sheaths a series of dye penetration experiments were performed in the nervous systems of wild-type and mutant embryos. Comparing the kinetics of uptake of different sized fluorescently labeled dyes in different mutants led to the conclusion that most of the barrier function is mediated by the septate junctions formed by the subperineurial cells, whereas the perineurial glial cell layer and the neural lamella contribute to barrier selectivity against much larger particles (i.e., the size of proteins). The requirements of different septate junction components were compared for the integrity of the blood-brain barrier, and evidence is provided that two of the six Claudin-like proteins found in Drosophila are needed for normal blood-brain barrier function (Stork, 2008).
Fast neuronal conductance requires a tight electrical insulation of the axons and in the mammalian nervous system, myelin and saltatory conductance evolved. Arthropods have not evolved saltatory conductance, but they are nevertheless in need for fast electrical conductance. In this respect it is not surprising that in marine shrimps myelin-like structures have been described . Drosophila follows two different and seemingly independent strategies to ensure fast conductance. In some central neuronal networks large caliber axons develop, whereas in the peripheral nervous system axons are insulated by several glial sheaths to ensure insulation. Initially, at the beginning of larval life, the different sensory and motor axons are kept as separate fascicles within the segmental nerves, suggesting there might be some degree of electrical cross talk within the different modalities. As the larva matures, the inner wrapping glia starts to grow around single axons, which may allow more sophisticated movements of the wandering larvae (Stork, 2008).
Whereas the wrapping glia insulates individual axons, do perineurial and subperineurial glia insulate the entire nervous system and set up the blood-brain-barrier? Genetic experiments and ultrastructural studies have long indicated that septate junctions provide the most effective part of this barrier. Indeed, the subperineurial cell are formed early in development and these cells are connected by septate junctions from late embryonic stages onwards. Using Gal4 driver strains specific to the subperineurial cells as well as in vivo septate junction markers, this study confirms that during larval life the subperineurial cells do not divide but grow enormously large in size. Septate junctions formed by the subperineurial cells are mostly found in interdigitated zones of cell-cell contact. Cell division would likely require disintegration of septate junctions and thus result in a temporal opening of the blood-brain barrier, which would be deleterious for the animal. This is in agreement with previous findings that Gliotactin expressing cells, forming septate junctions, do not divide during larval live (Stork, 2008).
The outermost glial cell layer is formed by the perineurial cells. Although these cells have long been described, their origin is still a matter of debate. In EM micrographs of late embryonic staged peripheral nerves some perineurial cells can be detected apically to the subperineurial cells. These glial cells divide during larval life and generate a large number of fine cell protrusions that cover the subperineurial cells. One function of the perineurium might be to influence the development and/or the tightness of the subperineurial layer. A comparable cellular function has been attributed to the astrocytes in the mammalian nervous system. Alternatively, the perineurial glial cells might provide a cellular basis for the response to injury. Unfortunately, to date there is no specific driver strains that allow manipulation of this glial cell population. Interestingly, a reverse relationship between subperineurial and perineurial cells has been suggested previously as subperineurial expression of activated Ras or PI3K (phosphoinositide 3-kinase) resulted in an thickening of the perineurial sheath (Stork, 2008).
Additionally, the fray gene has been shown to be required for normal axonal ensheathment. Interestingly the mutant phenotype could be rescued by expressing fray using three different Gal4 drivers. After the analysis of the specificity of these drivers (Mz317, subperineurial glia and weak wrapping glia; Mz709, all glial cell types; gliotactinGal4, subperineurial glia), it is concluded that fray is expressed in subperineurial glia and controls axonal ensheathment of wrapping glia in a noncell autonomous manner (Stork, 2008).
Given the different cellular barriers described in this report, questions arise concerning the functional contributions of the different layers. Kinetic studies were performed that supported the importance of the septate junctions in particular for small components. Animals lacking septate junctions are as leaky to a 10 kDa dextran as animals lacking all glial cell layers. However, when it comes to larger molecules, the relevance of the other cell layers becomes obvious. Although a 500 kDa dextran can easily penetrate into the nervous system of a glial cells missing embryo, its leakage into the nervous system of a neurexinIV mutant lacking septate junctions is greatly reduced. Thus, the other layers contribute to the function of the blood-brain barrier. Because a continuous perineurium is not fully formed in first instar larvae, the barrier function has to be assigned to the neural lamella and the inner glial layer. There are several reports showing that the neural lamella can act as an efficient filter for heavy metal ions. Possibly, large molecules such as the 500 kDa dextran are also trapped in this ECM. Alternatively, large particles are stopped by the diffusion barrier established by the normal cell-cell contacts between subperineurial cells and inner glial cell types like wrapping glia in the peripheral nerves and cortex and neuropile glia in the CNS (Stork, 2008).
The diffusion barrier provided by glial cells or epithelial sheaths is generated by special junctional complexes that help to tightly associate the involved cells. Drosophila epithelia as well as glial cells are characterized by septate junctions. Quite similar structures are also found at the mammalian paranodal junctions, which provide the structural basis for the tight electrical insulation of the nerve. A core component of the mammalian axoglial septate junctions is the NeurexinIV homolog Caspr that together with its binding partners, Contactin and Neurofascin155, sets up a tripartite adhesion complex at the paranode (Stork, 2008).
The function of this complex appears conserved in Drosophila, although there are some notable differences. The Caspr homolog NeurexinIV is expressed by glial cells as are Contactin and the Neurofascin155 homolog Neuroglian. As a consequence, in the fly septate junctions are formed between glial cells, whereas they are formed between neuronal and glial membranes in the mammalian system. The Caspr/Contactin/Neurofascin155 complex seals the paranodal junction and a similar function has been attributed to this protein complex in the invertebrate blood-brain barrier. This study found a less pronounced function of Contactin compared with NeurexinIV for the blood-brain barrier establishment, corroborating findings made in embryonic epithelia (Stork, 2008).
Another prominent component of the junctional complexes are the Claudin proteins. In mammals, members of these four transmembrane domain proteins are associated with tight junctions that are often considered to be functionally equivalent to the invertebrate septate junctions. In Drosophila two Claudin-like proteins have been described to be required for formation of normal epithelial barrier formation. This study shows that both Sinuous and Megatrachea are also needed for the establishment of normal blood-brain barrier formation. Similarly, it was shown that mammalian claudin5 is a major component of tight junctions of brain endothelial cells. claudin5 mutant mice show no structural or ultrastructural deficits, but have an impaired blood-brain barrier. The association of Claudins integrated in opposing membranes is thought to provide pores that can control the paracellular diffusion of small molecules. Although Drosophila Sinuous and Megatrachea clearly contribute to the barrier function, it is inconceivable that fly Claudins traverse the 20 nm wide septate gap to form a Claudin pore as it is discussed for the vertebrate Claudins. It has also been suggested that invertebrate Claudins might have lost their pore-like functions and exert only signaling function to establish the barrier (Stork, 2008).
Such a signaling function may control the size selectivity of the barrier and indeed sinuous mutants show only a weak barrier phenotype comparable with moody mutants, correlating with reduced septate junctions. This study demonstrates that a loss of septate junctions associated with neurexinIV mutants results in breakdown of the blood-brain barrier comparable with what is observed in animals lacking all glial cells. However, additional mechanisms are in place to control the paracellular diffusion of larger particles. A 500 kDa dextran can easily penetrate the nervous system of a glial cells missing embryo but cannot enter a nerve cord only lacking septate junctions (Stork, 2008).
Postembryonically, in third-instar larvae, staining can also be detected in areas where synaptic junctions have been repeated: the subperineural sheath of the larval CNS, imaginal disc cells, and salivary glands (Baumgartner, 1996).
Mutants produce no adult survivors, and most alleles are embryonic lethal. Rare adult escapers exhibit rough eyes, notched wings with vein broadening and duplicate legs with malformations (Baumgartner, 1996).
Nrx mutants exhibit behavioral defects. Mutants exhibit a severe reduction of coordinate muscle propagation waves. In addition, tracheae do not fill with air. These movement defects are similar to those observed in other mutants in which neurotransmitter release is abolished (Baumgartner, 1996).
Axons are seen to cross segmental boundaries in 10-20% of mutant embryos. Thus there is no absolute requirement for NRX in axonal pathfinding, but its absence results in a higher propensity for neuronal connectivity defects.
About 10% of the neuromuscular junctions examined fail to respond to nerve stimulation, indicating that a fraction of synapses fail to form in Nrx mutants. In the other 90% of the NMJs examined, the mean amplitude of evoked synaptic transmission is reduced by 40-45% in mutant embryos. Ionotophoresis of L-glutamate (the excitatory neurotransmitter) into mutant NMJs results in essentially wild-type responses, indicating that the postsynaptic receptor field develops normally. Thus NRX function appears to be required presynaptically to facilitate transmission. Bursts of activity at NMJs, which correlate with muscle contractions that permit first-instar larvae to hatch from the egg case, are reduced in mutants, suggesting a suppression of endogenous activity in motoneurons. Interesting, some muscle contractions are observed in Nrx mutant embryos upon dissection in low potassium ion recording solutions. A similar effect occurs in gliotactin mutant embryos in which the blood-nerve barrier is broken down. It is concluded that NRX expression in glial septate junctions is required for proper axonal insulation and blood-nerve barrier formation (Baumgartner, 1996).
coracle mutants display a cell-migration defect affecting dorsal closure (Fehon, 1994). Severe Nrx alleles also cause a defect in dorsal closure, resulting in a small than normal dorsal hole, consistent with abnormal function of D4.1/Coracle in Nrx mutant embryos. Hence, septate junctions may play a role in targeting cytoskeletal components and contributing to intercellular communication and cell migration during Dorsal closure (Baumgartner, 1996).
In Nrx mutants, smooth septate junctions remain unaffected. However, pleated septate junctions (pSJs) with a typical ladder-like structure are absent in ectodermal cells and in perineural glia-ensheathing peripheral axons. Apposing membranes in these tissues have a smooth and electron-dense appearance; unstructured cleft material is present. Thus pSJs in Nrx mutant embryos lose their characteristic ladder-like septae that distinguish them from smooth septate junctions. The affected pSJs retain some characteristics of septate junctions but lack the regularly arranged septae, the principle features distinguishing pSJs from smooth septate junctions. Obliquely sectioned septate junctions appear as honeycomb-like structures in ectodermal pSJs of wild type, and as regularly spaced parallel lines (normally seen in smooth septate junctions) in the ectoderm of mutant embryos (Baumgartner, 1996).
Myelinated fibers are organized into distinct domains that are necessary for saltatory conduction. These domains include the nodes of Ranvier and the flanking paranodal regions where glial cells closely appose and form
specialized septate-like junctions with axons. These junctions contain a Drosophila Neurexin IV-related
protein, Caspr/Paranodin (NCP1). NCP1 contains an open reading frame of 1385 amino acids (~153 kDa) with 93% identity to rat Caspr/Paranodin. NCP1 contains an amino-terminal discoidin domain, a laminin G domain, two Neurexin domains, and a PGY-enriched segment in the extracellular region. The cytoplasmic domain
contains a band 4.1 binding motif and a proline-rich sequence with at least one consensus SH3 domain binding site. In contrast to Drosophila NRX IV and Caspr2, NCP1 lacks a PDZ binding domain consensus sequence at its carboxyl terminus.
Mice that lack NCP1 exhibit tremor, ataxia, and significant motor paresis. In
the absence of NCP1, normal paranodal junctions fail to form, and the organization of the paranodal loops is disrupted. Contactin is undetectable in the paranodes, and K+ channels are displaced from the juxtaparanodal into the paranodal domains. Loss of NCP1 also results in a severe decrease in peripheral nerve conduction velocity. These results show a critical role for NCP1 in the delineation of specific axonal domains and the axon-glia interactions required for normal saltatory conduction (Bhat, 2001).
Septate junctions form between the ensheathing glial cells that surround the nerve bundles in Drosophila and are essential for establishment of the blood-nerve barrier. The Drosophila NRX IV protein localizes to and is required for the formation of the ladder-like septae characteristic of these junctions. In nrx IV mutants, these septae are absent, and another component of these junctions, Coracle, a band 4.1 homolog, is mislocalized. The current studies demonstrate that the septate and paranodal junctions appear to serve conserved functions in maintaining the axonal milieu required for action potential propagation. However, the topology and localization of the proteins is clearly different. In Drosophila, NRX IV is expressed by and localized between glial cells. In mice and other vertebrates, NCP1 is expressed by neurons and localized between the axon and glial cells. Hence, even though there is functional conservation, significant changes in expression occurred during evolution (Bhat, 2001).
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. This study reports 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 allow visualization the glial ensheathment of axons. Individual or a groups 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. Neurexin IV, Contactin, and Neuroglian are coexpressed in the peripheral glial membranes and 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, these data suggest that the molecular machinery underlying axonal insulation and axon-glial interactions may be conserved across species (Banerjee, 2006a).
The localization of Nrx IV, Cont, and Nrg in the embryonic nervous system was studied. Nrx IV, Nrg, and Cont show colocalization at the nerve glial membranes. Nrx IV, Cont, and Nrg are interdependent for their epithelial SJ localization. Having established that Nrx IV, Cont, and Nrg colocalize in the peripheral nerves, whether these proteins are interdependent for their localization in the peripheral nerves was studied. The effect of the absence of each of these three proteins on the localization of the other two was studied in the embryonic peripheral nerves using nrx IV, cont, and nrg null mutants. nrx IV mutant embryos were stained with anti-Nrx IV, anti-Cont, and anti-Nrg. Cont and Nrg proteins showed a rather diffused localization when compared with their wild-type localization. In addition, Cont is present as puncta in the cytoplasm of the glial cells because of its failure to get targeted properly to the membrane in the absence of Nrx IV. Similarly, cont null mutant embryos also showed less defined distribution and reduction of Nrx IV and Nrg in the glial membrane. nrg null mutant embryos display significant reduction in Nrx IV and Cont localization in the glial membranes. These results demonstrate that Nrx IV, Cont, and Nrg are interdependent for their proper localization in the embryonic peripheral nerves (Banerjee, 2006a).
Ultrastructural analyses of the embryonic epithelia in nrx IV, cont, and nrg mutants showed that these genes are required for the formation and/or organization of epithelial SJs. Nrx IV, Cont, and Nrg are interdependent for their proper localization both in the epithelia and in the peripheral nerves. These phenotypic similarities raised an interesting possibility that these proteins are part of a macromolecular protein complex that exists in the nervous system. To determine whether these three proteins form a biochemical complex in the nervous system, Drosophila heads, which are a rich source of both neurons and glial cells, were used. Coimmunoprecipitation experiments using Nrx IV, Cont, and Nrg antibodies efficiently coprecipitated Nrx IV, Cont, and Nrg. Interestingly, both isoforms of Nrg (180 kDa neuronal and 167 kDa epithelial) were immunoprecipitated by Nrx IV and Cont antibodies, suggesting that both isoforms are part of a protein complex that includes Nrx IV and Cont. However, at this point, it is not possible to differentiate whether isoform-specific complexes are formed or whether both isoforms are in the same complex. In addition, sucrose density gradient analysis of the fly head lysates was performed to determine how Nrx IV, Cont, and Nrg distribute in buoyant density gradients. Nrx IV and Cont cosediment in overlapping fractions. Nrg shows distribution in the lighter sucrose density fractions that do not overlap with Nrx IV and Cont but partially overlaps with that of Nrx IV and Cont, indicating that these proteins are associated with subcellular structures of the same density and may associate into a biochemical complex that is partially maintained during subcellular fractionation (Banerjee, 2006a).
The immunofluorescence analysis of nrx IV, cont, and nrg null mutants showed that in each of these mutants, the other two proteins show qualitatively reduced fluorescence intensities under identical confocal settings. The possible explanation for reduced fluorescence intensity could be that loss of any of these proteins affects the localization or stability of the other proteins. Whether the levels of the other two proteins had changed in nrx IV, cont, and nrg null mutants was examined using immunoblot analysis. Nrx IV protein levels did not seem to be affected in cont and nrg mutants when compared with wild type. Cont protein levels were severely affected in nrx IV mutants compared with wild-type and nrg mutant embryos. Nrg protein levels were also affected in nrx IV mutants but showed no change in cont mutants. At this stage, it cannot be rule out whether the change in Cont and Nrg protein levels in nrx IV mutants are attributable to reduced stability or less synthesis of these proteins. Together, the biochemical data indicate that Nrx IV, Cont, and Nrg form a protein complex in the nervous system and that, in the absence of Nrx IV, the stability of Cont and Nrg is severely affected (Banerjee, 2006a).
Inner glial membrane processes are involved in the ensheathment of either an individual axon or a group of axons. This glial ensheathment not only provides insulation of axons but also generates unique junctions between either glial membranes or between axons and glial membranes. Nrx IV, Cont, and Nrg are involved in the establishment of glial–glial SJs. Although additional components involved in these interactions need to be identified, these findings provide a basis for additional analysis of neuronal SJs, which would be relevant to the understanding of their vertebrate counterparts: the paranodal axo-glial SJs (Bhat, 2003; Hortsch, 2003; Salzer, 2003; Banerjee, 2006a and references therein).
The fundamental basis of axonal ensheathment in any species is to faithfully transmit neuronal signals along the nerve fibers and optimize desired cellular responses. To maximize the speed of conduction and/or to minimize the loss of nerve signals, many species evolved mechanisms in which axonal lengths remained short (as seen in insects) by increasing the diameter of the axons or by clustering voltage-gated Na+ channels to discrete unmyelinated regions of the axon, the node of Ranvier, as seen in myelinated nerve fibers of vertebrates. Most invertebrate species use some type of glial cells to ensheath their axons without generating a myelin sheath. The insulation is contiguous without any breaks, suggesting that primitive nodal structures or clustering of voltage-gated Na+ channels may not exist in invertebrates. However, recent reports have challenged some of these notions. In copepod crustaceans, ultrastructural analysis of the first antenna and the CNS has revealed extensive myelination of sensory and motor axons. These studies have raised some fundamental questions about axonal insulation and origins of myelination (Banerjee, 2006a and references therein).
In invertebrates like Drosophila, two types of glial cells are involved in insulation. The inner peripheral glial cells are involved in axonal ensheathment, and the outer (perineurial) glial cells wrap around the inner glial cells to provide another level of ensheathment. This two-cell ensheathment in Drosophila peripheral nerves may be advantageous to ensure that high K+ containing hemolymph does not interfere with action potential propagation. Although the cellular aspects of axonal insulation are being unraveled, the molecular mechanisms underlying the axonal ensheathment remain to be investigated. Most importantly, what are the protein constituents of the insulating membranes, and whether some of the vertebrate myelin proteins have their homologs in invertebrates? A detailed molecular analysis of the nature of the glial cells that ensheath axons as in Drosophila or produce myelin-like structures and the type of myelin in copepods may provide insights into whether myelinating glial cells arose from a common ancestor. However, a genetic dissection of the axonal ensheathment in Drosophila will uncover some of the basic aspects of the neuron-glial interactions that lead to ensheathment of nerve fibers across species (Banerjee, 2006a).
Cell adhesion molecules play a pivotal role in establishing intercellular junctions [e.g., cadherins and associated catenins form a protein scaffold that establish adhesion contacts at the adherens junctions and link them to the actin cytoskeleton. Similarly, claudins and associated cellular scaffolding proteins are required for establishing TJs. In both of these examples, transmembrane proteins bring two opposing membranes together to establish junctions through homophilic and/or heterophilic interactions (Banerjee, 2006a and references therein).
The finding that nrg null mutant nerves display increased spacing between the outer and inner glial membranes suggest that Nrg may be involved in cell–cell interactions and cell–cell adhesion between glial membranes. In contrast, the observation that loss of nrx IV and cont does not affect the membrane spacing between the inner and outer glial membranes suggests that Nrx IV and Cont are not involved in membrane adhesion or bringing the glial membranes in close apposition. Together, these data suggest that Nrg is critical for both the adhesion and SJ formation, whereas Nrx IV and Cont are critical for the formation of the septa at SJs. In addition, the missing axonal fascicles in nrg mutant nerves could result from axon fasciculation defects or axonal degeneration as a secondary consequence resulting from the loss of glial support. Axonal fasciculation defects have been observed in nrg mutants. Alternatively, axonal loss in nrg mutants might result from a disruption in axonal cytoskeleton, because Nrg possesses domains that could potentially interact with and stabilize the axonal cytoskeleton. Based on Nrg expression in S2 cells, Nrg is predicted to recruit membrane skeleton assembly within specialized domains of neurons in response to cell adhesion. Both Nrg protein forms Nrg167 and Nrg180 contain a short cytoplasmic domain as a binding site for ankyrin. Ankyrins are linker proteins that connect various membrane proteins with the actin-spectrin network in the cell. Loss of axons observed in nrg mutants is clearly suggestive of axon-glial interdependence that may alter axonal survival. In vertebrates, axolemmal-myelin interactions are critical for the formation of the paranodal axo-glial SJs (see Garcia-Fresco, 2006). This raises an interesting possibility that axon-glial interactions in Drosophila may use similar interactive mechanism for establishing axo-glial SJs. Thus, the current studies on Nrx IV, Cont, and Nrg suggest that these proteins are critical for the formation and/or organization of the SJs between either glial cells and possibly between axons and glial cells, which remains to be further investigated (Banerjee, 2006a).
Invertebrate axons are insulated from their salt-rich environment through a glial-dependent BBB, which plays a crucial role in electrical and chemical insulation. Ultrastructural studies have demonstrated SJs between perineurial glial cells and inner glial cell membrane to form the structural basis of BBB of some insects. Absence of SJs in nrx IV, cont, and nrg mutants, and a compromised BNB in the PNS as evidenced by dye exclusion analyses, provide additional confirmation in support of SJs as a prerequisite for blood-nerve barrier (BNB) formation. Not just in the PNS, recent reports on the BBB formation and function also in the CNS of Drosophila underscore the importance of SJs in proper sealing and insulation of the nerve cord (Schwabe, 2005), thereby supporting that insulation and establishment of functional SJs in both PNS and CNS go hand in hand. Although, G-protein-coupled receptor signaling pathway members have been identified to establish the BBB in Drosophila CNS, the signaling mechanisms that operate in the PNS still remain to be established (Banerjee, 2006a).
The axo-glial SJs in the myelinated axons share many anatomical features similar to those of invertebrate SJs, especially the electron-dense ladder-like transverse septa (Bhat, 2003; Banerjee, 2006a). Ensheathment of Drosophila axons by perineurial glial cells in the absence of myelin-producing glial cells would have predicted that the molecular components of the invertebrate SJs would be different from those of the vertebrate axo-glial SJs. Surprisingly, the fly SJ molecular components are present at the vertebrate axo-glial SJs and not at the TJs, which serve similar functions. Thus, the molecular similarities reflect an evolutionarily conserved function of creating an ionic barrier both at Drosophila SJs and at axo-glial SJs in the paranodal region. More importantly, Drosophila nerves contain a large number of axons, which are collectively held together by glial membranes not only to maintain nerve fasciculation but also to maintain the insulation of individual axons. It would be of significant interest to establish whether axo-glial SJs are present in Drosophila nerves and to identify downstream components of these junctions in Drosophila that link these junctions to the glial or axonal cytoskeleton. Identification of such molecules will provide insights into whether these junctions play a much broader role in axon-glial signal transduction. In summary, the molecular and functional similarities between the Drosophila SJs and vertebrate axo-glial SJs should allow a genetic and molecular dissection to be undertaken of the formation and function of these junctions in Drosophila (Banerjee, 2006a).
Neurexins have been proposed to function as major mediators of the coordinated pre- and postsynaptic apposition. However, key evidence for this role in vivo has been lacking, particularly due to gene redundancy. Null mutations have been obtained in the single Drosophila Neurexin IV gene. Nrx loss of function prevents the normal proliferation of synaptic boutons at glutamatergic neuromuscular junctions, while Nrx gain of function in neurons has the opposite effect. Nrx mostly localizes to the active zone of presynaptic terminals. Conspicuously, Nrx null mutants display striking defects in synaptic ultrastructure, with the presence of detachments between pre- and postsynaptic membranes, abnormally long active zones, and increased number of T bars. These abnormalities result in corresponding alterations in synaptic transmission with reduced quantal content. Together, these results provide compelling evidence for an in vivo role of neurexins in the modulation of synaptic architecture and adhesive interactions between pre- and postsynaptic compartments (Li, 2007).
Although cell adhesion molecules have long been postulated and in several cases have been shown to be major participants in synapse development and plasticity, the impact of their function and the molecular mechanisms that they activate remain a puzzle. Particularly intriguing is the function of neurexins, which may provide clues to understanding of synapse organization. Null mutants were isolated in the single Drosophila dnrx gene. Nrx mutants were shown to have striking abnormalities in synapse development and function. A recent study reported that Drosophila neurexin is required for synapse formation in the adult CNS (Zeng, 2007). The current study demonstrates a primary role of Nrx in regulating the formation of synapses, and also reveal the crucial role of Nrx in the proper development of active zones and regulating synaptic function in an intact organism, thus providing insights into understanding the function of neurexins in vivo (Li, 2007).
These studies provide compelling evidence that Nrx plays a prime role during the expansion of the NMJ and, in particular, in defining the cytoarchitecture of the active zones within synaptic boutons. (1) In Nrx mutants, synaptic bouton proliferation is severely disrupted, and therefore NMJ expansion is significantly stunted. (2) Nrx gain of function promotes the formation of new boutons in a gene-dosage-dependent manner. (3) The ultrastructural analyses show that presynaptic densities (PRDs) are not apposed normally to PSDs displaying signs of abnormal adhesion to the postsynaptic density (PSD), although every PRD is exactly juxtaposed to the PSD. (4) In Nrx mutants, critical components of the presynaptic compartment, such as synaptic vesicle proteins and active zone components, are ectopically localized within axons. (5) The distribution of GluRs at the PSD is abnormally large, although this phenotype may arise as a consequence of the presynaptic defects observed in Nrx mutants (Li, 2007).
The great majority of abnormal phenotypes in Nrx mutants could be completely rescued by expressing a wild-type Nrx transgene in neurons; although in some instances the rescue was partial. However, even in the later case, expressing Nrx in both muscles and neurons did not further improve the residual abnormalities, suggesting that Nrx functions primarily if not exclusively in the presynaptic compartment (Li, 2007).
The partial rescue of some of the phenotypes, such as the defects in mEJPs and the morphology of active zones, might be due to the high sensitivity of these processes to the right levels and correct temporal expression of Nrx, which is not completely mimicked by the UAS/Gal4 system. This view is supported by the observation that overexpression of Nrx in a wild-type background also decreased quantal content, suggesting that increased Nrx dosage may have detrimental effects on synapse structure and/or function. However, the data strongly support that the abnormal phenotypes arise from the lack of Nrx: (1) all the experiments were carried out in mutants over a deficiency chromosome in an independent genetic background; (2) a precise excision of the P element did not show any of the mutant phenotypes. Together these data establish a specific role for Nrx in proper synaptic development (Li, 2007).
One of the important findings of this study is that Nrx mutants displayed defective active zones with larger PRD, and especially containing regions of detachment from the PSD. These detachment sites implicate Nrx as a mediator of cell adhesion between the pre- and the postsynaptic cell,. While a complete detachment of active zones is not observed, Nrx mutants have a significant decrease in the number of boutons. This raises the possibility that the phenotypes observed are from those boutons that are maintained and that a more drastic consequence is a failure to form synaptic boutons. Nevertheless, the lack of complete detachment of active zones in Nrx null mutants suggests that Nrx, although an important synapse-organization molecule, is not sufficient for trans-synaptic cell adhesion (Li, 2007).
Another notable phenotype in Nrx mutants was the presence of enlarged PRDs and increased number of T bars. A major feature of Drosophila larval NMJ is its ability to compensate for decreased postsynaptic responses by upregulating neurotransmitter release. For instance, a decrease in the number of postsynaptic GluRs results in an increase in neurotransmitter release, thus maintaining the amplitude of evoked responses. It is plausible that the enlarged PRDs and increase in number of T bars in Nrx mutants are a compensatory mechanism to adjust for defective presynaptic cell adhesion and/or reduced neurotransmitter release. In support of this notion, in Nrx mutants there was a 50% decrease in synaptic bouton number, but this was accompanied by a 2-fold increase in the number of T bars, such that the total number of T bars/NMJ remained constant, despite the changes in bouton number. Similarly, defective presynaptic cell adhesion and/or reduced neurotransmitter release could lead to an increase in GluR accumulation. In these studies, it was found that the length of the PSD was enlarged in Nrx mutants as well as the distribution of GluR clusters (Li, 2007).
The above structural abnormalities were accompanied by corresponding functional deficits. In Nrx mutants, the frequency of mEJPs was strikingly increased. Further, although the T bars were rescued by expression of a Nrx transgene, the length of the PRDs was not, and a similar lack of rescue was observed for mEJP frequency. Thus, there appears to be a notable correlation between the size of the PRD and spontaneous miniature excitatory potential (mEJP) frequency, perhaps due to increased probability of synaptic vesicle release with increased synapse size. In addition, a substantial increase was observed in mEJP amplitude. Two factors may contribute to this change; (1) the distribution of GluR clusters was enlarged, while the GluR intensity remained unchanged, suggesting that more GluRs were accumulated at mutant synapses; (2) an additional contributing factor is that mEJP frequency was increased, and instances of summation were observed (Li, 2007).
Overall, despite the increase in PRD size and the maintenance of overall T bar number, evoked events had a decrease in amplitude and quantal content. Recent studies have suggested that a major constituent of the T bars is Brunchpilot [BRP/CAST (Kittel, 2006; Wagh, 2006)]. In brp mutants, T bars fail to form, but PRDs appear unaltered. Further, although EJP amplitude is decreased, mEJP amplitude and frequency are normal. This has led to the model that T bars per se are not required for synaptic transmission but that they regulate the efficiency of transmission. In Nrx mutants, PRDs are disproportionately large, which could result in asynchronous release, leading to an EJP with decreased amplitude. It is also possible that the presynaptic membrane detachments observed in Nrx mutants could contribute to the functional impairment of neurotransmitter release (Li, 2007).
A recent study demonstrated that in Nrx mutant larvae associative learning is impaired in an olfactory choice paradigm (Zeng, 2007). However, in this study, larval locomotion was not assessed. The current study showing that locomotor behavior is impaired in Nrx mutants raises the possibility that the poor performance of mutant larvae in the conditioning assay might also result from the locomotor abnormalities. Zeng also reported that the number of T bars in the calyx of the mushroom bodies, the learning centers of the fly, was reduced in adult flies. In contrast, a significant increase was found in the number of T bar/bouton, and since Nrx mutants have fewer boutons, this translated in the maintenance of T bar number per NMJ. The differing results might be due to different mechanisms regulating T bar formation in the two tissues (Li, 2007).
The presence of a neurexin in Drosophila strengthened the view that neurexins are highly conserved across species. The synaptic Nrx expression pattern and its function show remarkable parallels with mammalian neurexins. Moreover, the proteins that have been shown to interact with mammalian neurexins also have homologs in Drosophila, which further supports the idea that the function of neurexins and underlying signaling mechanism are evolutionarily conserved. Among these, Drosophila neuroligin and/or Dystroglycan (Dg) might be potential Nrx ligands. Drosophila neuroligin transcription exhibits almost an identical temporal and spatial expression pattern as Nrx during embryonic stages. dg is highly expressed in the somatic musculature of embryos. dg mutants are embryonic lethal, and perturbation of Dg function by RNAi as well as genetic interaction studies suggest an involvement of Dg in muscle maintenance and axonal pathfinding in adult flies (Shcherbata; 2007). Future studies on the identification and characterization of Nrx binding partners in Drosophila should provide additional insights into the mechanisms by which neurexins function in synapse development and function (Li, 2007).
Extensive cell culture studies of neurexins and neuroligins and functional studies using α-neurexin knockout mice have established a central role for neurexins as synaptic adhesive and organizing molecules. Studies on Nrx provide evidence in an intact organism that neurexin is required for important aspects of synapse development and function. Gain-of-function analysis of Drosophila Nrx reveals that overexpression is sufficient to promote the formation of synaptic boutons in vivo, in agreement with the findings from cell culture studies suggesting that mammalian neurexin-neuroligin trans-synaptic complexes can induce pre- and postsynaptic differentiation and synapse formation. Moreover, the accumulations of synaptic vesicle and active zone proteins along axons of Nrx null mutants further support the notion that neurexins may recruit or organize synaptic proteins or organelles during presynaptic differentiation. Phenotypic analyses of α-neurexin knockout mice demonstrated that α-neurexin is essential for synaptic transmission in a process that depends on presynaptic voltage-dependent Ca2+ channels. However, triple-knockout mice have normal surface expression of Ca2+ channels. These findings have led to the hypothesis that neurexins regulate the coupling between Ca2+ channels and the neurotransmitter release machinery. Similarly, in Drosophila Nrx null mutants it was found that the Ca2+ sensitivity of evoked responses was abnormal, but the distribution or levels of presynaptic Ca2+ channel Cac was unchanged, consistent with the above hypothesis. Notably, Syt I, a synaptic vesicle protein that binds Ca2+ and has been proposed to function as a Ca2+ sensor during synaptic vesicle exocytosis, was partly mislocalized to axons in Drosophila Nrx mutants. Furthermore, the structure of active zones was impaired in these mutants. Therefore, the organization of active zone proteins including the assembly of neurotransmitter release machinery might be affected in Nrx mutants (Li, 2007).
In conclusion, these studies in Drosophila demonstrate that Nrx is required for both synapse development and function and, in particular, for proper formation of active zones. These studies provide compelling evidence for an in vivo role of neurexins in the modulation of synaptic architecture and adhesive interactions between pre- and postsynaptic compartments (Li, 2007).
A 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. For example, 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. Photobleaching experiments were conducted on whole living Drosophila embryos to analyze the membrane mobility of CAMs at septate junctions between epithelial cells. GFP-tagged Nrg and Nrx IV molecules were shown to 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. It is concluded that the 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 (see Organization of adhesion complexes at epithelial cell contacts in the wild-type and mutant embryos). 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 (Laval, 2008. Full text of article).
Neurexins are neuron-specific cell surface molecules thought to localize to presynaptic membranes. Recent genetic studies using Drosophila have implicated an essential role for the single Drosophila Neurexin in the proper architecture, development and function of synapses in vivo. However, the precise mechanisms underlying these actions are not fully understood. To elucidate the molecular mechanism of Neurexin in vivo, dnrx and caki mutant flies, combined with various methods, were used to analyze locomotion, synaptic vesicle cycling and neurotransmission of neuromuscular junctions. Dneurexin (DNRX) was found to be important for locomotion through a genetic interaction with the scaffold protein, CAKI/CMG, the Drosophila homolog of vertebrate CASK. Similar to its mammalian counterparts, DNRX is essential for synaptic vesicle cycling, which plays critical roles in neurotransmission at neuromuscular junctions (NMJ). However, this interaction appears not to be required for the synaptic targeting of DNRX, but may instead be needed for proper synaptic function, possibly by regulating the synaptic vesicle cycling process (Sun, 2009).
The integrity of polarized epithelia is critical for development and human health. Many questions remain concerning the full complement and the function of the proteins that regulate cell polarity. This study reports that the Drosophila FERM proteins Yurt (Yrt) and Coracle (Cora) and the membrane proteins Neurexin IV (Nrx-IV) and Na+,K+-ATPase are a new group of functionally cooperating epithelial polarity proteins. This 'Yrt/Cora group' promotes basolateral membrane stability and shows negative regulatory interactions with the apical determinant Crumbs (Crb). Genetic analyses indicate that Nrx-IV and Na+,K+-ATPase act together with Cora in one pathway, whereas Yrt acts in a second redundant pathway. Moreover, it was shown that the Yrt/Cora group is essential for epithelial polarity during organogenesis but not when epithelial polarity is first established or during terminal differentiation. This property of Yrt/Cora group proteins explains the recovery of polarity in embryos lacking the function of the Lethal giant larvae (Lgl) group of basolateral polarity proteins. It was also found that the mammalian Yrt orthologue EPB41L5 (also known as YMO1 and Limulus) is required for lateral membrane formation, indicating a conserved function of Yrt proteins in epithelial polarity (Laprise, 2009).
To clarify the mechanisms of epithelial polarization, the function was examined of the FERM-domain protein Yrt, which was shown to act as a negative regulatory component of the Crb complex in both Drosophila and vertebrates. Crb regulates epithelial apical basal polarity by promoting apical membrane and apical junctional complex formation. Crb also controls the growth of the apical membrane at late stages of epithelial differentiation. Yrt binds to the cytoplasmic tail of Crb and restricts Crb activity in apical membrane growth. However, Yrt is predominantly a basolateral protein that is recruited into the Crb complex only at late stages of epithelial differentiation. Embryos completely lacking maternal and zygotic Yrt (yrtM/Z) displayed polarity defects before Yrt is recruited into the Crb complex at late organogenesis. This raises the question whether Yrt has a function in epithelial organization as a basolateral protein (Laprise, 2009).
yrtM/Z mutants and double-mutant combinations of yrt and genes encoding basolateral proteins were examined for synergistic genetic interactions that could indicate functional cooperation. yrtM/Z embryos showed clear polarity defects at post-gastrula stages of development, as indicated by the basolateral mislocalization of Crb. In contrast, zygotic yrt mutants demonstrated only minor polarity defects in the trunk ectoderm. This indicates that yrt is required for polarity, and that zygotic yrt mutants could provide a sensitized background to reveal genetic interactions. Within the basolateral membrane, Yrt is enriched at the septate junction together with the Na+,K+-ATPase and other septate junction components from stage 14 onwards. Marked genetic interactions were found between yrt and Atpα (which encodes the α-subunit of Na+,K+-ATPase) and between yrt and Nrx-IV, but not between yrt and six other genes that encode septate junction transmembrane proteins. In contrast to wild type and single mutants, apical markers were mislocalized in yrt Atpα and yrt Nrx-IV double mutants similar to yrtM/Z embryos. Enhancement of the Nrx-IV null phenotype by yrt indicates that Nrx-IV and yrt have overlapping functions and do not operate in a linear pathway. The pathway relationship between yrt and Atpα remains uncertain as embryos completely devoid of Na+,K+-ATPase function cannot be analysed. These findings reveal previously unrecognized functions of the Na+,K+-ATPase and Nrx-IV in epithelial polarity (Laprise, 2009).
The developmental timing of the yrtM/Z and the yrt Atpα and yrt Nrx-IV polarity phenotypes is notable. These mutants show no polarity defects during cellularization when epithelial cells first form or during gastrulation when the apical junctional belt is assembled. Polarity defects are first seen at stage 11 or 12, and are most prominent at embryonic stage 13. In contrast to wild type, Crb and other apical markers are found throughout the plasma membrane and colocalize with basolateral markers such as Discs large (Dlg, also known as Dlg1) and Fasciclin 3 in yrtM/Z, yrt Atpα and yrt Nrx-IV mutants. During late embryogenesis, apical markers become restricted again to the apical membrane, which, however, remains abnormally extended and dome-shaped. Thus, Yrt, Na+,K+-ATPase and Nrx-IV cooperate and have critical functions in maintaining epithelial polarity during early organogenesis (stages 11-13), well before septate junction assembly is observed, and Yrt is recruited to the apical membrane at stage 14 (Laprise, 2009).
Na+,K+-ATPase and Nrx-IV are required for septate junction assembly. Because Yrt also accumulates at septate junctions, it was asked whether Yrt has a role in septate junction formation or function. Septate junctions are basal to the adherens junction and, like vertebrate tight junctions, prevent diffusion of solutes between cells. Dye injection assays show that paracellular barriers in epithelia of yrtM/Z embryos are compromised. However, yrtM/Z embryos showed a normal complement of septa when examined ultrastructurally whereas Atpα and Nrx-IV mutants lack septa. Immunoprecipitation experiments demonstrated FERM-domain-dependent interactions between Yrt and the septate junction transmembrane protein Neuroglian (Nrg), and the enrichment of Yrt at septate junctions was less pronounced in Nrg mutants. However, Nrg or Nrg yrt mutant embryos did not display overt defects in epithelial polarity. These findings indicate that Yrt is a bona fide septate junction component that has a function distinct from Nrx-IV and Na+,K+-ATPase because it is not required for septa formation but essential for barrier function (Laprise, 2009).
Next, the analysis extended to cytoplasmic adaptor proteins associated with septate junctions. yrt does not show genetic interactions with dlg or lgl, genes encoding conserved basolateral polarity proteins required for septate junction formation and apical/basal polarity. varicose (vari), which encodes a membrane-associated guanylate kinase, and cora, which encodes a FERM protein that is the Drosophila orthologue of mammalian erythrocyte protein band 4.1 and its paralogues, were examined. vari and cora null mutant embryos did not show defects in apical basal polarity. No functional interactions were seen between yrt and vari. In contrast, yrt cora double-mutant embryos displayed marked apicalization defects, with strongly expanded apical membranes and reduced basolateral membranes in all ectodermal epithelia including the epidermis. This phenotype is significantly stronger than the yrtM/Z or cora null mutant phenotypes indicating that Yrt and Cora have redundant functions in promoting epithelial polarity (Laprise, 2009).
Similar to yrtM/Z mutants, yrt cora double mutants did not show polarity defects during gastrulation. By stage 11, polarity defects were more prominent in yrt cora mutants than in yrtM/Z embryos, which correlates with Cora being expressed from stage 11 onwards. In yrt cora mutants, the most severe polarity defects characterized by overlapping distributions of apical and basolateral markers occurred during stage 13. However, by late embryogenesis, apical and basolateral markers were segregated, although expanded apical membranes and severe defects in tissue organization persisted. Interestingly, the yrt cora apicalization phenotype is very similar to the phenotype that results from high-level overexpression of Crb in both timeline and severity. The segregation of apical and basolateral markers in late-stage yrt cora mutants is also observed in late-stage yrtM/Z, yrt Atpα and yrt Nrx-IV embryos. Thus, polarization mechanisms are at play in late embryos that are not dependent on Yrt and Cora. To address the possibility that this repolarization is the result of redundancy between Yrt/Cora and Lgl-group proteins, which include Scribble (Scrib) as well as Lgl and Dlg, embryos lacking Yrt and Scrib (yrtM/Z scribM/Z) were examined. Apical and basolateral markers segregated in yrtM/Z scribM/Z embryos indicating that a basolateral polarity mechanism exists in late embryos that is independent of Yrt/Cora and Lgl-group proteins (Laprise, 2009).
Together, these findings suggest that Yrt, Cora, Nrx-IV and the Na+,K+-ATPase are a new functionally cooperating group of basolateral epithelial polarity proteins, which is refered to as the ‘Yrt/Cora group’. In contrast to the enhancement of the cora and Nrx-IV null phenotypes by yrt mutations, no phenotypic enhancement was seen in cora Nrx-IV or cora Atpα double mutants, indicating that the Yrt/Cora group is composed of two functionally overlapping pathways. Cora, Nrx-IV and the Na+,K+-ATPase belong to one pathway, which is consistent with previously documented biochemical and genetic interactions between these proteins, whereas Yrt defines a second redundant pathway (Laprise, 2009).
One critical aspect of polarity regulation is that apical and basolateral proteins act antagonistically to set up mutually exclusive membrane domains. In gastrulating Drosophila embryos, this interaction occurs between apical factors and basolateral Lgl-group proteins. Similar to lgl, dlg and scrib mutations, yrt mutations partially suppress the crb mutant phenotype. Because Yrt can bind Crb directly, it is suggested that Yrt negatively regulates Crb function as a component of the apical Crb complex in both apical basal polarity and the control of apical membrane size. The data reported in this study argue that Yrt opposes Crb function also as a basolateral polarity protein. To test this hypothesis further it was asked whether the loss of other Yrt/Cora-group genes could suppress the crb mutant phenotype. It was found that mutations in Atpα, Nrx-IV and cora ameliorate the epithelial defects of crb mutants to an extent similar to that of yrt mutations. Remarkably, yrt cora crb triple-mutant embryos not only showed a suppression of the crb mutant phenotype, which was much stronger than the one observed in yrt crb or cora crb double mutants, but also caused a strong suppression of the apicalization effect observed in yrt cora mutants. These findings further support the conclusion that Yrt and Cora act redundantly, and indicate that mutual competition of basolateral Yrt/Cora-group proteins and apical Crb organizes epithelial membrane domains (Laprise, 2009).
Epithelial differentiation during Drosophila embryogenesis can be subdivided into four phases that are characterized by distinct mechanisms governing epithelial polarity. (1) Initial cues for polarity are given before or during cellularization. (2) Fully polarized epithelial cells are established during gastrulation through the interplay of apical Par and Crb complexes and basolateral Lgl-group proteins. (3) The Yrt/Cora group acts during organogenesis to promote basolateral polarity and counteracts apical determinants, thereby functionally replacing the Lgl group. (4) The function of Yrt/Cora and Lgl groups does not account for the polarization of epithelial cells in late embryos, because normalization of polarity is observed in the absence of these factors. This implies the existence of a yet unknown mechanism that stabilizes basolateral polarity. Septate junctions form well after Lgl-group and Yrt/Cora-group proteins have contributed to epithelial polarity, indicating that polarity and septate junction formation are independent functions of these proteins. The identification of a novel group of polarity factors that acts in a discrete time window during development highlights the temporal complexity of the regulation of the epithelial phenotype and further explains why the loss of individual polarity proteins does not completely compromise polarity (Laprise, 2009).
The vertebrate Yrt orthologues Mosaic eyes (Moe, also known as Epb41l5) in zebrafish and EPB41L5 (also known as YMO1 and Limulus) and EPB41L4B (also known as EHM2) in mammals bind to Crb proteins and contribute to epithelial organization. However, as in Drosophila, vertebrate Yrt homologues are predominantly associated with the basolateral membrane and seem to be recruited to the apical membrane only at later stages of epithelial development. To test whether EPB41L5 is required for basolateral differentiation, RNA interference was used in MDCK cells. Transient depletion of EPB41L5 using shRNA resulted in a notable cell flattening and an expansion of the cell perimeter, indicating that lateral membranes were strongly reduced and apical and basal membranes were enlarged. Consistently, it was found that the lateral markers Na+,K+-ATPase, Scrib and E-cadherin were reduced or lost from the plasma membrane. The same effects were observed using siRNA oligonucleotides targeted to a distinct region of EPB41L5. MDCK cells were established that were stably transfected with EPB41L5 short hairpin RNA (shRNA), and lateral membrane formation was examined after Ca2+-switch. After re-addition of Ca2+, EPB41L5 shRNA cells displayed significant delays in lateral membrane formation and recruitment of E-cadherin to cell-cell contacts. At 8 h after Ca2+-switch, E-cadherin levels appeared similar in control and knockdown cells although experimental cells remained slightly flatter. Interestingly, by 24 h after Ca2+-switch, E-cadherin levels at the plasma membrane had decreased again and appeared lower than in control cells but similar to EPB41L5 shRNA cells before Ca2+-switch. EPB41L5 knockdown cells ultimately established normal cell shape. Whether the formation of a normal lateral membrane in EPB41L5 shRNA cells is due to residual EPB41L5 expression, or reflects a transient requirement of EPB41L5 for cell polarization as in Drosophila epithelia, remains unclear. It is concluded that the function of Yrt as a basolateral polarity protein is conserved in mammalian epithelial cells (Laprise, 2009).
The loss of basolateral membrane in Drosophila embryos that lack Yrt/Cora-group function and in MDCK cells depleted of EPB41L5 is reminiscent of the loss of basolateral membrane in MDCK cells depleted of the phosphoinositide PtdIns(3,4,5)P3 and of human bronchial epithelial cells depleted of β2-spectrin or ankyrin-G. Cora/band 4.1 and the Na+,K+-ATPase can associate with either spectrin or the spectrin adaptor ankyrin, and loss or ectopic expression of EPB41L5 can cause defects in basolateral actin. This raises the possibility that Yrt/Cora-group proteins act by stabilizing the lateral actin/spectrin membrane cytoskeleton. Analysis of vertebrate development in the absence of the function of the Yrt orthologues Moe in zebrafish and EPB41L5 in mice revealed defects in epithelial organization that reflect Crb-dependent and probably also Crb-independent functions of Yrt proteins. For example, the abnormal cell shape and multi-layering defects seen in the developing neuroepithelium of mutant mouse embryos could result from defects in basolateral polarity. Moreover, mouse embryos lacking EPB41L5 show defects in epithelial-mesenchymal transition of mesodermal cells during gastrulation. These surprising findings indicate that Yrt proteins are core regulators of animal tissue organization that can enhance epithelial or mesenchymal cell differentiation (Laprise, 2009).
Regulation of epithelial tube size is critical for organ function. However, the mechanisms of tube size control remain poorly understood. In the Drosophila trachea, tube dimensions are regulated by a luminal extracellular matrix (ECM). ECM organization requires apical (luminal) secretion of the protein Vermiform (Verm), which depends on the basolateral septate junction (SJ). This study shows that apical and basolateral epithelial polarity proteins interact to control tracheal tube size independently of the Verm pathway. Mutations in yurt (yrt) and scribble (scrib), which encode SJ-associated polarity proteins, cause an expansion of tracheal tubes but do not disrupt Verm secretion. Reducing activity of the apical polarity protein Crumbs (Crb) suppresses the length defects in yrt but not scrib mutants, suggesting that Yrt acts by negatively regulating Crb. Conversely, Crb overexpression increases tracheal tube dimensions. Reducing crb dosage also rescues tracheal size defects caused by mutations in coracle (cora), which encodes an SJ-associated polarity protein. In addition, crb mutations suppress cora length defects without restoring Verm secretion. Together, these data indicate that Yrt, Cora, Crb, and Scrib operate independently of the Verm pathway. These data support a model in which Cora and Yrt act through Crb to regulate epithelial tube size (Laprise, 2010).
The transmembrane protein Crumbs (Crb) acts as an apical determinant during establishment of epithelial apical-basal polarity. During later stages of epithelial differentiation, Crb promotes apical membrane growth independently of its role in apical-basal polarity. Crb activity is counteracted by different groups of basolateral polarity proteins including the Yrt/Cora group, which is composed of Yurt (Yrt), Coracle (Cora), Na+/K+-ATPase, and Neurexin IV (Nrx-IV). Loss of Yrt results in Crb-dependent apical membrane growth during late stages of epithelial cell maturation in Drosophila. Thus, the equilibrium between the activities of these polarity proteins is important to define the size of the apical domain. Precise control of the apical surface of tracheal cells is crucial to define epithelial tube size in the Drosophila respiratory system, suggesting a potential role for polarity proteins in tube morphogenesis. However, the contribution of polarity regulators to the regulation of epithelial tube shape and size is poorly understood. Controlling length and diameter of the lumen is important for organ function, as illustrated by the deleterious tubule enlargements that occur in polycystic kidney disease (Laprise, 2010).
To better understand the role of polarity regulators and apical membrane growth in epithelial tube morphogenesis, the role of Yrt was investigated in the formation of the Drosophila respiratory system, a network of interconnected tubules that delivers oxygen throughout the body. Yrt is mainly associated with the lateral membrane in tracheal cells and is enriched at septate junctions (SJs), as shown by its colocalization with the SJ marker Nrx-IV. Tracheal development was characterized in zygotic yrt mutants or yrt null embryos devoid of both maternal and zygotic yrt (yrtM/Z). Segmental tracheal placodes invaginated and established a normal branching pattern of tracheal tubes with intersegmental connections in both yrt and yrtM/Z mutants. yrtM/Z embryos display apical-basal polarity defects and irregularities in the tracheal epithelium at midembryogenesis. However, apical-basal polarity normalizes during terminal differentiation. In contrast, zygotic yrt mutants have only minor, if any, polarity defects in tracheal cells. The most apparent defect in
yrt mutant trachea was the presence of excessively long and convoluted dorsal trunks compared to the straight dorsal trunks seen wild-type. The average dorsal trunk length was 417 ± 12 μm in wild-type embryos, whereas it was 476 ± 22 μm in yrt and 470 ± 23 μm in yrtM/Z mutant embryos. Similar but milder tube length defects were observed in other tracheal branches. In addition, the diameter of dorsal trunks in yrt and yrtM/Z mutants was uniform, but wider than in wild-type embryos. The average dorsal trunk diameter was 9.1 ± 0.6 μm in tracheal segment seven of wild-type embryos compared to 12.2 ± 0.9 μm in yrt and 12.7 ± 0.6 μm in yrtM/Z mutant embryos. In smaller branches, some diameter expansions were also apparent. In addition, the smaller tracheal branches in yrtM/Z embryos showed frequent interruptions indicating either breaks or a failure in the luminal accumulation of the 2A12 antigen. These findings indicate that Yrt regulates the size of tracheal tubes and supports the integrity of segmental tracheal branches. Remarkably, despite the prominent differences in the apical-basal polarity defects between yrt and yrtM/Z mutant embryos, both mutants exhibit similar dorsal trunk elongation and diameter defects. This finding suggests that the transient loss of apical-basal polarity in yrtM/Z embryos is not the cause of dorsal trunk size defects and that Yrt therefore has distinct functions during early and late stages of tracheal morphogenesis (Laprise, 2010).
The enlargement of the tracheal tube lumen observed in yrt mutants could be caused by an increase in cell number or an increase in the dimension of the apical surface of tracheal cells that surround the lumen. To address this question, the number of dorsal trunk cells in yrt mutants and wild-type embryos was counted. No significant differences in cell numbers between wild-type and yrt mutant embryos were found, indicating that the enlargement of tracheal tubes observed in yrt and yrtM/Z mutants must be accompanied by an increase in the dimension of the apical surface of tracheal cells (Laprise, 2010).
Several other mutants display enlarged dorsal trunks similar to yrt. One group of genes required for limiting tube length encodes components of the SJ, including the Na+/K+-ATPase (α and β subunits), Cora, Nrx-IV, Scribble (Scrib), Lachesin (Lac), Sinuous (Sinu), Megatrachea (Mega), and Varicose (Vari). Among these SJ proteins, Na+/K+-ATPase, Cora, Nrx-IV, and Scrib also play a role as basolateral polarity proteins. In Drosophila and other invertebrates, SJs appear as a ladder-like group of septa basal to the cadherin-based adherens junctions. SJs have functions analogous to vertebrate tight junctions, because they provide a transepithelial diffusion barrier. Yrt is not required for normal septa formation or localization of SJ components such as Cora but is essential for the barrier function of SJs. Zygotic yrt mutants show only minor defects in paracellular barrier function, whereas barrier function is fully compromised in yrtM/Z embryos. This observation supports the notion that transepithelial barrier function and the regulation of tube dimension are independent functions of Yrt because yrt and yrtM/Z mutant embryos show similar tube size defects. This conclusion is consistent with previous findings suggesting that the regulation of tracheal tube elongation and the regulation of the paracellular diffusion barrier are distinct roles of SJ proteins (Laprise, 2010).
A second class of mutants exhibiting abnormally long tracheal tubes are defective in the genesvermiform (verm) and serpentine (serp), which encode enzymes predicted to modify the chitin-based luminal extracellular matrix (ECM), and mutants of which show structural defects in the luminal ECM. The chitin matrix filling the tracheal lumen is transiently present during lumen morphogenesis and is critical for determining lumen diameter and length. Interestingly, all of the mutations affecting SJ components tested so far are associated with a failure to secrete Verm into the tracheal lumen. This suggests that SJ proteins control tube size by regulating apical secretion and remodeling of the apical chitin matrix. Although Yrt is required for the barrier function of SJs, it was found that luminal secretion of Verm and Serp was normal in yrt and yrtM/Z mutants. In contrast, low but detectable levels of Verm were observed in cora mutants. This finding suggests that Yrt regulates tracheal tube length through a pathway that is independent of Verm and Serp (Laprise, 2010).
During late stages of epithelia maturation, Yrt is known to restrict apical membrane growth in epidermal and photoreceptor cells by limiting Crb activity. This interplay between Yrt and Crb governs apical membrane size in stage 14 and later embryos when tracheal tube size is defined. This raises the possibility that Crb-dependent apical membrane growth is responsible for dorsal trunk expansion in yrt mutant embryos. To test this hypothesis, crb dosage was reduced in yrt mutant embryos by introducing one copy of a crb null allele into a yrt mutant background. Loss of one copy of crb suppressed the dorsal trunk elongation defects seen in
yrt mutants, because the dorsal trunks appeared similar to wild-type in yrt crb/yrt + mutants. In addition, moderate Crb overexpression increased dorsal trunk length and diameter without interfering with the integrity of the tracheal epithelium or the secretion of Verm or Serp. These results show that Crb is required for promoting the expansion of tracheal tubes at late stages of embryogenesis. It was previously suggested that Crb also acts during early tracheal branch outgrowth in addition to its role in apical-basal polarity. Therefore, Crb plays a critical role at several steps of tracheal development. Together, these findings indicate that the antagonistic interactions between Yrt and Crb determine tracheal tube size (Laprise, 2010).
Verm levels in yrt crb/yrt + and in yrt/yrt mutants were indistinguishable, indicating that the minor reduction in Verm levels sometimes seen in yrt mutants are not the cause of the tracheal elongation defects. Accordingly, reduction of crb dosage in a verm or serp mutant background did not suppress tube size defects. Similarly, loss of one copy of crb in sinu mutant embryos, which fail to secrete Verm, had no impact on the length of dorsal trunks that remained enlarged as in sinu single mutants. These findings suggest that the apical secretion of matrix-modifying enzymes such as Verm and the control of Crb activity by Yrt are two independent and nonredundant modes of tracheal tube size regulation. The data also establish that epithelial tube size control by SJ-associated proteins involves Verm-dependent and Verm-independent mechanisms (Laprise, 2010).
To further characterize the function of SJ-associated polarity proteins in the regulation of tracheal tube size, tube elongation, the integrity of SJs, and the secretion of Verm in scrib, lethal giant larvae (lgl), and discs large (dlg) mutant embryos were examined. Zygotic loss of scrib, lgl, or dlg resulted in excessively long dorsal trunks, indicating that these genes are critical for tube size control. Zygotic loss of lgl expression caused fully penetrant defects in SJ paracellular barrier function, whereas zygotic scrib or dlg mutants did not have compromised transepithelial barriers. Luminal Verm deposition was not detected in lgl mutants but appeared near normal in dlg and in scrib mutants. Thus, Scrib and Dlg act like Yrt by controlling tracheal tube size through mechanisms distinct from Verm secretion (Laprise, 2010).
Further analysis concentrated on Scrib and it was asked whether this protein, like Yrt, controls tracheal tube size by negatively regulating Crb activity. Scrib together with Lgl and Dlg shows antagonistic interactions with Crb to regulate apical-basal epithelial polarity in early Drosophila embryos. However, the tracheal tube defects were not ameliorated in
scrib crb/scrib + embryos compared to scrib single mutants. Thus, in contrast to Yrt, Scrib does not seem to limit tube length by restricting Crb activity. Because Yrt and Scrib appear to control tracheal tube size through different mechanisms, tests were performed to see whether
yrt scrib double homozygous mutant embryos had a more severe phenotype. The double mutants had Verm levels that were lower compared to yrtM/Z and scrib mutants. Moreover, the tracheal defects also appeared more severe in yrt scrib mutants than in yrt null mutant embryos, particularly in the smaller-diameter branches. The defects in small-diameter branches of the yrt scrib double mutants are not likely caused by the reduction in Verm secretion, because the complete loss of Verm has only a mild effect on smaller branches. The enhanced severity of the yrt scrib double-mutant tracheal defects compared to the defects seen in yrt null embryos and the differences in the genetic interactions of scrib and yrt with crb suggest that Scrib and Yrt act in separate pathways to regulate the size of tracheal tubes and that Scrib does not act by modulating Crb activity. It is possible that Scrib acts through other proteins, such as proteins of the Par complex, that promote apical domain formation. Therefore, SJ-associated polarity proteins use at least two Verm-independent mechanisms to restrict the dimension of tracheal tubes (Laprise, 2010).
Cora is an SJ-associated protein required for optimal secretion of Verm. This suggests that Cora may control tracheal tube length through a Verm-luminal matrix pathway. However, Cora is also a basolateral polarity protein restricting the activity of Crb, which promotes Verm-independent expansion of the dorsal trunk. This led to an investigation of
the functional relationship between Cora and Crb in tracheal morphogenesis. In the epidermis, a striking redundancy between yrt and cora is observed in the regulation of apical-basal polarity. Similarly, it was found that tracheal cells in yrt cora double mutants show severe apicalization defects characterized by a broad expansion of the surface distribution of Crb. The antigen recognized by the monoclonal antibody 2A12 was found surrounding tracheal cells and was not confined to the luminal cavity. In addition, the 2A12 antigen was associated not only with tracheal cells but also with
epidermal cells. These tracheal defects seen in cora yrt mutant embryos mimic defects that result from high levels of Crb overexpression. This observation argues that the tracheal defects observed in
cora yrt double-mutant embryos result from strong Crb overactivation, which is associated with a loss of basolateral polarity and an expansion of apical membrane character. Because epidermal cells did not acquire expression of the tracheal cell marker Tango, it is unlikely that epidermal cells adopt a tracheal cell fate in cora yrt mutants. The association of 2A12 with epidermal cells is therefore presumably due to the apicalization of tracheal cells, which would consequently secrete the 2A12 antigen not only on the luminal side but all around their cell surface, allowing the 2A12 antigen to diffuse and bind to surrounding cells. Accordingly, cuticle deposition, taking place at the apical membrane, was seen at both luminal and abluminal sides of tracheal cells overexpressing Crb (Laprise, 2010).
The data indicate that Yrt and Cora cooperate to control apical-basal polarity of tracheal cells by limiting Crb to the apical cell pole, but they do not reveal whether Cora and Crb interact to control the length of tracheal tubes. To address this question, cora crb/cora + embryos were examined for a suppression of the tracheal size defects seen in
cora single mutants. Reduction of crb dosage suppresses tube overelongation defects resulting from the loss of Cora. This restriction of dorsal trunk elongation does not result from the restoration of Verm secretion, because the level of Verm present in the dorsal trunk lumen was as low in
cora crb/cora + embryos as in single cora mutants. Together, these data suggest that Crb overactivation is the primary cause of epithelial tube length defects observed in the absence of Cora. Thus, Cora and Yrt act independently from each other to counteract Crb activity and maintain the appropriate size of epithelial tubes. Because the reduction of crb dosage does not rescue the verm mutant phenotype, it is concluded that the residual amount of Verm found in cora mutants is sufficient to maintain Verm pathway activity (Laprise, 2010).
This analysis suggests that basolateral proteins that are enriched at SJs have several critical functions in determining the size of epithelial tubes in the Drosophila tracheal system. This study shows that the increase in tube size is not caused by an increase in cell number and therefore must be accompanied by an increase in the apical surface area of individual tracheal cells. Given that Crb is a well-known regulator of apical membrane size, these findings suggest that the interplay between Yrt, Cora, and Crb modulates the dimensions of the apical surface of tracheal cells to control tracheal tube size. Moreover, this mechanism acts independently of and in parallel to a previously proposed pathway depending on the apical secretion of the matrix-modifying enzymes Verm and Serp, which requires several SJ-associated proteins. Yet another mechanism is revealed by results demonstrating that
scrib mutants also have long trachea with normal Verm levels but that, in contrast to cora and yrt, tracheal defects in scrib mutants are not suppressed by loss of one copy of crb. Together, these findings suggest that basolateral proteins utilize at least three distinct mechanisms to regulate tube size in the Drosophila tracheal system. Unexpectedly, these mechanisms involve functional interactions between polarity proteins that appear to be different from those involved in establishing
apical-basal polarity at earlier stages of development. For example, in promoting apical-basal polarity, Yrt and Cora act redundantly so that
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