InteractiveFly: GeneBrief

sinuous: Biological Overview | References

Epithelial tubes of the correct size and shape are vital for the function of the lungs, kidneys, and vascular system, yet little is known about epithelial tube size regulation. Mutations in the Drosophila gene sinuous cause tracheal tubes to be elongated and have diameter increases. Genetic analysis using a sinuous null mutation suggests that sinuous functions in the same pathway as the septate junction genes neurexin and scribble, but that Nervana 2, convoluted, varicose, and cystic have functions not shared by sinuous. Molecular analyses reveal that sinuous encodes a claudin that localizes to septate junctions and is required for septate junction organization and paracellular barrier function. These results provide important evidence that the paracellular barriers formed by arthropod septate junctions and vertebrate tight junctions have a common molecular basis despite their otherwise different molecular compositions, morphologies, and subcellular localizations (Wu, 2004).

Major organs such as the lung, vascular system, and kidney are composed of endothelial and epithelial tubes that have reproducible sizes and shapes. Tubes that deviate from their normal sizes can cause medical conditions such as polycystic kidney disease and ischemia. However, the cell biological and molecular mechanisms by which cells assemble into tubes with highly regulated lengths and diameters is poorly understood. The Drosophila tracheal system provides a genetically tractable model system to investigate the molecular processes of tubulogenesis. The tracheal system is a network of ramifying epithelial tubes that forms the gas exchange organ of the fly, but it also functions analogously to the vertebrate vascular system by delivering oxygen directly to the tissues. The tracheal system is created from 20 clusters of ~80 epithelial cells each that undergo a series of coordinated movements and junctional rearrangements to produce a complex network of tubes. These tubes have characteristic diameters and lengths that undergo developmentally regulated size changes (Wu, 2004).

In a genetic screen to identify genes that are required for epithelial tube size control, mutations were isolated in sinuous (sinu) and seven other genes that cause defects in a specific increase in tracheal diameter that occurs over an ~2-h period late in embryogenesis. Embryos homozygous for mutations in these genes have normal trachea before this 'tube expansion,' but during tube expansion, the lengths and diameters of multicellular tracheal tubes become abnormally increased, resulting in tracheal tubes that follow tortuous pathways and have local diameter expansions. As the number of tracheal cells in these mutants is not increased, the increased tube sizes must result from changes in cell shape. Recently, one of these genes, nervana 2 (nrv2), was cloned and it was found to encodes a Na/K ATPase ß-subunit that is a component of pleated septate junctions (Paul, 2003). Further, it was found that the septate junction proteins Neurexin (Nrx) and Coracle (Cor), which are homologous to the vertebrate CASPR and band 4.1 proteins, respectively, are also required for tracheal tube expansion (Paul, 2003). This report shows that Sinuous encodes a claudin that plays a critical role in septate junction function (Wu, 2004).

The best-characterized function of invertebrate pleated septate junctions, hereafter referred to simply as septate junctions, is to form a barrier that prevents free diffusion of water and solutes between adjacent epithelial cells. The paracellular barrier in vertebrates is formed by tight junctions; however, septate and tight junctions have been considered analogous rather than homologous structures because of the dramatic morphological and molecular differences between them. Tight junctions lie apically to adherens junctions and have 'kissing points' where the intercellular space is periodically obliterated. In contrast, septate junctions lie basally to adherens junctions and have regularly spaced septa bridging an ~15-nm intercellular space. Both tight and septate junctions form continuous barriers around cells, but tight junctions appear as a network of anatomizing strands of particles, whereas arthropod septate junctions appear as ribbons that are tightly parallel to each other. In addition to barrier function, tight junctions also have a 'fence' function that is thought to create a boundary between apical and basolateral domains. Although possible fence functions of septate junctions have not been investigated in detail, mutations that compromise septate junction components such as Cor, Nrx or the Na/K ATPase do not cause mislocalization of apical epithelial markers (Wu, 2004).

At the molecular level, many Drosophila homologues of vertebrate tight junction proteins including DaPKC/aPKC, DmPar6/ASIP, Crumbs/CRB1, and polychaetoid/ZO 1-3 do not localize to the septate junction, but instead are found in the adherens junction or the marginal zone, which, like the tight junction, lies apically to the adherens junction. Furthermore, the Drosophila genome appears to lack the tight junction proteins occludin and junction adhesion molecule (Wu, 2004).

Vertebrate claudins are highly divergent, four-transmembrane domain proteins that homo- and heterophilically interact to form the paracellular barrier in the tight junctions (Tsukita, 2001; Tsukita, 2002). Claudins were first identified by Furuse (1998) and were subsequently shown to be a large multigene family (Morita, 1999) that currently has at least 19 human members (Kollmar, 2001). When expressed in nonadherent cells, several vertebrate claudins can mediate homophilic cell-cell adhesion and assemble strands reminiscent of those found at tight junction kissing points (Furuse, 1998; Kubota, 1999). Evidence that claudins directly form the paracellular barrier comes from the demonstrations that mice lacking claudin-1 or -5 display apparently normal tight junction ultrastructure, but show altered paracellular permeability (Furuse, 2002; Nitta, 2003). In addition, amino acids in the first extracellular loop of claudins determine the charge selectivity and resistance of tight junctions (Colegio, 2002; Colegio, 2003). Together, the divergent morphologies of septate and tight junctions, coupled with the apparent absence of claudins in Drosophila septate junctions, has supported the view that the epithelial barrier junctions in insects and vertebrates are analogous rather than homologous structures (Wu, 2004).

This study provides critical evidence that the paracellular barriers of septate and tight junctions have a common molecular basis. Sinuous is predicted to have the same membrane topology as vertebrate claudins, and has molecular similarity to canonical vertebrate claudins comparable to that of several more divergent vertebrate claudins. Furthermore, Sinuous localizes to Drosophila paracellular barrier junctions and is required for barrier function. Thus, Sinuous has both molecular and functional similarities to vertebrate claudins. In investigating the relationship between Sinuous and other septate junction components, it was determined that the localization of Sinuous to septate junctions depends on other septate junction components including Nrx, Cor, the claudin Megatrachea (Mega; Behr, 2003), and Nrv2. Further, sinuous appears to function in the same genetic pathway of septate junction-mediated tracheal tube size control as nrx and the cell polarity/septate junction gene scribble (scrib), but that the nrv2 Na/K ATPase ß-subunit and the as-yet-uncloned genes convoluted (conv), varicose (vari), and cystic have tube size control functions not shared by sinuous (Wu, 2004).

The finding that a Drosophila claudin is required for paracellular barrier function is significant because there has been minimal evidence of a conserved molecular basis for the barrier functions of invertebrate septate junctions and vertebrate tight junctions. In combination with the recent finding that C. elegans paracellular barrier function also requires claudins (Asano, 2003), the results raise the possibility that the different types of barrier junctions present in diverse species evolved from a common ancestral claudin-containing barrier junction. They also raise the possibility that vertebrate paranodal junctions, which have significant morphological, functional, and molecular similarities to septate junctions, may also contain claudins (Wu, 2004).

If paracellular barrier junctions all evolved from a common ancestor, there currently exists a remarkable number of differences between the subcellular localization, ultrastructure, and molecular composition of these junctions. For example, as detailed in the introduction, tight junctions are apical to the adherens junction, whereas septate junctions are basal. Tight junctions have a series of 'kissing points,' septate junctions have ladder-like septa, and C. elegans barrier junctions have neither kissing points nor ladder-like septa (Asano, 2003). One explanation for this variability may lie in the multiple distinct nonbarrier functions of these junctions. Although claudins are essential for barrier function, junctional morphology and localization may be dictated by components whose cellular functions, such as cell polarization, signaling, or directed secretion, are not directly involved in paracellular barrier formation. Thus, rather than trying to describe entire junctional complexes as being analogous or homologous, it may be more useful to define and compare aspects of these junctions in terms of particular functions and protein components (Wu, 2004).

In sinuous mutants, septate junction organization is disrupted at both the confocal and TEM levels. In all tissues examined, the number of septa is reduced and there are cell-cell contacts devoid of septa. Thus, Sinuous is required for normal septate junction organization. Surprisingly, in contrast to Nrx, Cor, Nrv2, Dlg, and Mega, Sinuous is not absolutely required for septa formation. This is reminiscent of the role of Gliotactin, which is required for ribbon organization, but not septa formation. However, in contrast to Gliotactin, the number of septa is reduced in sinuous mutants, indicating that Sinuous is required for both organization and formation on the normal number of septa. The ability of morphologically normal septa to form in the absence of Sinuous may result from septate junctions containing at least one other Drosophila claudin and at least five other transmembrane proteins. Whether the remaining septa in sinuous mutants have normal barrier function is an interesting but difficult to answer question, as the discontinuities in the septal ribbons breach the paracellular diffusion barrier and obscure the barrier properties of remaining septa (Wu, 2004).

An additional function of Sinuous that appears to be distinct from its role in barrier formation is that Sinuous is required for tracheal tube size control. The disruption of paracellular barriers in sinuous mutants is unlikely to be the cause of tracheal tube size defects, it was previously shown that mutations in different genes cause qualitatively different tracheal tube size defects despite causing equivalent barrier defects. For example, cystic mutants have severe diameter but no length defects, whereas mega mutants have length but almost no diameter defects. In addition, mutants can have barrier defects but minimal tracheal size defects (Wu, 2004).

These results extend the distinction between barrier and tube size functions by showing that the presence or absence of septa, which are thought to form the paracellular barrier, are not correlated with tracheal tube size defects. Specifically, although sinuous and nrx mutants have the same tracheal phenotypes, well-differentiated septa are present in sinuous but not nrx mutants. Furthermore, although septa are presumably absent in nrx sinu double mutants, an nrx null mutation does not enhance the sinuous null mutant phenotype. In contrast, mutations in the genes cystic and vari, which cause septate junction barrier defects (Paul, 2003), strongly enhance the phenotypes of both sinuous null mutants, which have septa, and of nrv2 null mutants, which have few or no septa (Genova, 2003; Paul, 2003). These observations imply that there are molecular complexes that control tracheal tube size that do not correspond to the septa visible by TEM. Although additional work is required to define the molecular composition and function of the septate junction-associated complexes that mediate tracheal tube size control, the conservation of the proteins that function in tracheal tube size control suggests that this mechanism of tube size control may also be conserved (Wu, 2004).

Behr (2003) reported that mega also encodes a Drosophila claudin, providing an opportunity to compare the in vivo functions of two claudins required for barrier formation in the same tissues. The lengths of multicellular tracheal tubes are increased in both mega and sinuous mutants, but the effects of sinuous and mega mutations on cellular and junctional architecture are very different. In sinuous mutants, morphologically normal but discontinuous septa are present, and apical cuticle patterning is abnormal. By contrast, in mega mutants, apical surface patterning appears normal, but no septa form, and instead unstructured intercellular material is seen (Behr, 2003). Overexpression of Mega (but not Sinuous) can mislocalize Cor and Nrx. Thus, although Sinuous and Mega may be partially redundant, they have different functions in septate junction formation. As the exact roles of claudins in formation and organization of tight junctions and septate junctions have not yet been fully determined, the molecular-genetic tools available in Drosophila coupled with the limited number of Drosophila claudins provide an excellent opportunity for understanding the multiple functions of claudins in barrier junction formation and function (Wu, 2004).

Organization and function of the blood-brain barrier in Drosophila

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).

Heterotrimeric G proteins regulate a noncanonical function of septate junction proteins to maintain cardiac integrity in Drosophila

The gene networks regulating heart morphology and cardiac integrity are largely unknown. The heterotrimeric G protein gamma subunit 1 (Ggamma1) has been shown to mediate cardial-pericardial cell adhesion in Drosophila. This study shows that G-oalpha47A and Gβ13F cooperate with Ggamma1 to maintain cardiac integrity. Cardial-pericardial cell (CC-PC) adhesion also relies on the septate junction (SJ) proteins Neurexin-IV (Nrx-IV), Sinuous, Coracle, and Nervana2, which together function in a common pathway with Ggamma1. Furthermore, Ggamma1 signaling is required for proper SJ protein localization, and loss of at least one SJ protein, Nrx-IV, induces cardiac lumen collapse. These results are surprising because the embryonic heart lacks SJs and suggest that SJ proteins perform noncanonical functions to maintain cardiac integrity in Drosophila. These findings unveil the components of a previously unrecognized network of genes that couple G protein signaling with structural constituents of the heart (Yi, 2008).

Since SJ formation requires the interdependent function of multiple SJ proteins, it was hypothesized that additional SJ proteins maintain CC-PC adhesion. Indeed, the bro phenotype was fully penetrant in embryos homozygous for sinuous (sinu), coracle (cora), nervana2 (nrv2), and contactin (cont) mutations. Similar to Nrx-IV mutants, a subset of cora mutants also showed mesoderm closure defects, presumably due to known dorsal closure defects in cora embryos. The bro phenotype was also observed at low penetrance (20%-30%) in embryos homozygous for mutations in Lachesin (Lac), Gliotactin (Gli), and Neuroglian (Nrg), which also encode SJ components. Since SJs themselves are absent from the embryonic heart, it is concluded that SJ proteins fulfill a noncanonical function outside of SJs to maintain CC-PC adhesion (Yi, 2008).

Double mutant analysis was used to test whether heterotrimeric G proteins and SJ proteins function in a common pathway. Heart morphology in Gγ1/Sinu, Gγ1/Cora, and Gγ1/Nrv2 double mutant embryos was comparable to that of each single mutant, except that Gγ1/Sinu and Gγ1/Cora double mutant embryos showed a higher frequency of mesoderm closure defects. It is concluded that Gγ1, Sinu, Cora, and Nrv2 operate in a single pathway to regulate CC-PC adhesion, but Gγ1, Sinu, and Cora function in separate pathways during dorsal closure (Yi, 2008).

The functional relationship between SJ proteins and the ECM in tracheal tube size control prompted an examination of whether the ECM protein Pericardin (Prc) maintains CC-PC adhesion. Prc mediates the attachment of the dorsal mesoderm to the ectoderm and prc null mutants do not complete mesoderm closure. Since hypomorphic prc alleles have not been identified, the requirement of prc for CC-PC adhesion was tested by knocking down Prc expression with double-stranded RNA. Injecting blastoderm embryos with 5 μM prc dsRNA recapitulated the mesoderm closure phenotype prc null mutants. However, injecting 0.5 μM prc dsRNA induced the bro phenotype. These findings support the conclusion that Prc functions, at least in part, to mediate CC-PC adhesion (Yi, 2008).

To understand the effect of the bro phenotype on cardiac function, heart rate was assessed in Stage 17 embryos homozygous for mutations in Gγ1 and loco, as well as the SJ components Nrx-IV, Cont, and sinu. Heart rate was found to be dramatically reduced in Gγ1 and loco embryos, and the SJ mutants had a similar reduction in heart rate. These results indicate that both heterotrimeric G protein signaling and SJ proteins are indispensable for proper cardiac performance (Yi, 2008).

Genetic studies of Cora, Sinu, and Nrv2 suggested these SJ proteins also carry out novel functions essential for CC-PC adhesion. Similar to Nrx-IV, both Cora and Sinu localize to CC and PC membranes. Two Nrv isoforms, Nrv1 and Nrv2, are recognized by the Nrv antibody, and Nrv1/2 also localize to CC and PC membranes. Since Nrv1 and Nrv2 have nonoverlapping subcellular localizations in other epithelial tissues, the expression pattern of the SJ specific isoform, Nrv2, may be more restricted than shown (Yi, 2008).

The results of this study show that the heterotrimeric G proteins G-oα47A, Gβ13F, and Gγ1 function together to maintain CC-PC adhesion during the late stage of heart formation in Drosophila. By mapping a new broken hearted (bro) mutant (Nrx-IV) and characterizing additional candidate genes, a noncanonical role was discovered for SJ proteins in mediating CC-PC and CC-CC adhesion outside SJs. Four SJ proteins, Nrx-IV, Sinu, Cora, and Nrv2, operate in a common pathway with Gγ1 to maintain cardiac integrity; these proteins require Gγ1 for proper subcellular localization in the heart. Mechanistically, the presence of SJ proteins in both CCs and PCs suggests that these proteins act in trans to maintain cell-cell adhesion in the dorsal vessel. A model is favored in which the extracellular domain of Nrx-IV engages in heterophilic interactions with SJ-proteins such as Neuroglian or Contactin, and that these interactions would be stabilized by ECM proteins such as Pericardin (Prc). Alternatively, the SJ proteins may directly interact with ECM proteins to provide a structural basis for cardiac integrity (Yi, 2008).

Heterotrimeric G proteins G-oα47A/G-iα65A, Gβ13F, and Gγ1 function with the GPCR moody and the RGS protein loco to regulate SJ formation in the Drosophila brain-blood barrier. Although loco mutant embryos show the bro heart phenotype, moody mutations do not induce a heart phenotype. A search of the Drosophila protein interaction map reveals that the GPCR CG32447 interacts with both the SJ protein Sinu and the RGS Kermit. Kermit also interacts with Loco, suggesting that the CG32447 GPCR participates in the control of cardiac integrity. However, a deficiency uncovering CG32447 does not induce the bro phenotype. Since the screen for bro mutants, visualized as a perturbation in the ordered expression pattern of Hand-GFP in cardial and pericardial cells, did not identify a GPCR that maintains cardiac integrity, it is concluded that the GPCR regulating cardiac integrity is either pleiotropic, with an early embryonic function that precludes its identification as a regulator of cardiac integrity, or is redundant to a second GPCR in the dorsal vessel (Yi, 2008).

Alternatively, cardiac integrity may be regulated by a GPCR-independent mechanism. In neuroblasts, G-iα65A, Gβ13F, Gγ1, and loco regulate mitotic spindle orientation, protein localization, and ultimately asymmetric cell division via a GPCR-independent signaling pathway. During neuroblast cell division, heterotrimeric G proteins are activated by the GTPase exchange factor (GEF) Ric-8, but not by GPCRs. However, the lethal mutation ric-8^G0397 does not induce the bro phenotype (Yi, 2008).

During blood-brain barrier formation, sequestering Gβγ or hyperactivating G-oα47A signaling in glial cells leads to SJ defects, whereas hyperactivating G-iα65A signaling does not affect SJ function. A similar relationship exists among heterotrimeric G proteins during asymmetric cell division in neuroblasts. In contrast, sequestering Gβγ in the dorsal vessel has no effect on cardiac integrity, while hyperactivating G-oα47A in the embryonic heart induces the bro phenotype. It is concluded that the bro phenotype in Gβ13F or Gγ1 mutants is caused by misregulation of G-oα47A signaling. This is in sharp contrast to the G proteins regulating blood-brain barrier formation and asymmetric cell division where Gβγ dimers activate a set of downstream effectors distinct from that of G-oα47A signals (Yi, 2008).

G protein signaling regulates SJ formation in Drosophila and tight junction formation in mammalian cells. Even though SJs are analogous to vertebrate tight junctions, it is striking that G protein signaling components colocalize with both SJ and tight junction proteins. In addition, Gα_s interacts with the tight junction protein ZO-1 throughout junction formation, suggesting that Gα subunits physically regulate tight junction assembly. Thus, septate/tight junction proteins appear to be direct targets of G proteins in both flies and vertebrates (Yi, 2008 and references therein).

Although the embryonic heart lacks SJs, the current results are consistent with the idea that SJ proteins are direct targets of G proteins in the dorsal vessel. G protein mutants phenocopy SJ-protein mutants and G proteins operate in a common pathway with SJ proteins to maintain cardiac integrity. In addition, proper localization of SJ proteins in the embryonic heart requires G protein signaling, and G proteins regulate at least one SJ protein at the posttranscriptional level. Finally, loss of G-oα47A signaling (G-oα47A mutants) and hyperactivation of G-oα47A signaling (overexpressing G-oα47A) both result in the bro phenotype; thus Gα signaling is localized to specific foci in cells of the dorsal vessel. It is proposed that an appropriate level of Gα signaling mediates SJ-protein localization, whereas loss or hyperactivation of Gα signaling mislocalizes SJ proteins leading to a loss in cardiac integrity (Yi, 2008).

Cell-cell adhesion plays an essential role during organ morphogenesis. In the Drosophila heart, cell-cell adhesion along three distinct CC membrane domains is required to maintain cardiac integrity. Medioni (2008) provide a detailed description of two CC domains participating in cell-cell adhesion: the adherent domain, positioned immediately dorsal and ventral to the cardiac lumen, promotes cell-cell adhesion between CCs on opposing sides of the heart, and the basal-lateral adherent domain, positioned along the lateral CC membrane, promotes cell-cell adhesion between neighboring CCs on one side of the heart. These studies suggest that a third CC membrane domain, referred to as the pericardial adherent domain, is positioned opposite to the luminal domain and promotes PC-CC adhesion. The loss of cell-cell adhesion along each of the three CC domains gives rise to a unique phenotype: luminal collapse (referred to hereafter as type-1), breaks between neighboring cardial cells (type-2), and loss of PC-CC adhesion (type-3), respectively. The unique nature of these three phenotypes can provide insight into the molecular pathways regulating cardiac integrity (Yi, 2008).

Loss of heterotrimeric G proteins or SJ proteins induces the type-3 (bro) phenotype, and mutations in at least one SJ-protein gene, Nrx-IV, leads to the type-1 phenotype. In addition, Sinu, Cora, and Nrv2 localize to the luminal and perhaps the adherent domains, suggesting that loss of these proteins will also cause the type 1 phenotype. The type 2 phenotype is observed in a subset of Gγ1 embryos, but not in any other heterotrimeric G protein or SJ-protein mutants. Thus, the pathways regulating cell-cell adhesion along the CC basal-lateral membrane may be distinct from those identified in this study (Yi, 2008).

The guidance ligand Slit has been shown to regulate multiple aspects of cardiogenesis in Drosophila, and mutations in slit induce luminal collapse (referred to hereafter as type-1), breaks between neighboring cardial cells (type-2), and likely loss of PC-CC adhesion (type-3) phenotypes. In addition, slit mutant embryos show mesoderm migration and CC polarity defects, however these defects are genetically separable from cardiac integrity defects. Slit signals through the Robo receptors and mutations in genes encoding downstream components of the Robo signaling pathway do not dominantly enhance slit mutations. In contrast, mutations in genes encoding integrins or integrin ligands, such as scab, mys, and Lan-A, dominantly enhance slit mutations and transheterozygous embryos show the type-2 phenotype. This study suggests that Slit activates two pathways during cardiogenesis: one pathway utilizes typical Robo signaling to regulate mesoderm migration and CC polarity while a second pathway uses atypical, or Robo-independent, signaling to regulate cell adhesion between neighboring CCs and likely between opposing CCs to promote lumen formation. Although the role of Slit in regulating PC-CC adhesion has not been studied in detail, one possibility is that Slit signals through G-oα47A/Gβ13F/Gγ1 to regulate CC-CC and even PC-CC adhesion (Yi, 2008).

SJ proteins are functionally interdependent and localization of Sinu to SJs requires Nrx-IV, Cora, and Nrv2 (Wu, 2004), while Nrx-IV, Cora, Cont, and Nrg are equally interdependent for localization to SJs. In addition, both Nrv2 and Nrx-IV are transmembrane proteins, and the extracellular domain of Nrv2 at least is required for SJ function. Since every SJ-protein mutant examined showed PC-CC adhesion defects, SJ proteins likely form interdependent complexes in PCs and CCs. The extracellular domains of SJ proteins may act in trans, either through direct interactions with SJ proteins along opposing membranes or through indirect interactions with ECM proteins such as Pericardin, to maintain cardiac integrity. A search of the Drosophila protein interaction map reveals an interaction between Pericardin and Sinu, supporting the latter possibility. Alternatively, SJ proteins could be required for the formation or function of adherens junctions in the dorsal vessel (Yi, 2008).

All of the bro genes have close vertebrate orthologs. Since the function of mevalonate pathway genes in heart development is conserved from Drosophila to vertebrates, it is speculated that G protein-mediated regulation of SJ proteins is also evolutionarily conserved. To date, the role of heterotrimeric G proteins in regulating vertebrate heart development has not been identified, but heterotrimeric G proteins do play a role in heart disease. In contrast, Sinu is a member of the Claudin protein family and even though this protein family is rather divergent (Wu, 2004), vertebrate Claudin-1 is required for normal heart looping in the chick. In addition, Claudin-5 localizes to the lateral membrane of cardiomyocytes and is associated with human cardiomyopathy. Lastly, mutations in the prc ortholog, collagen alpha-1(IV), cause vascular defects in mice and humans. Taken together, these studies raise the possibility that heterotrimeric G proteins and tight junction proteins ensure proper vertebrate cardiovascular morphogenesis (Yi, 2008).

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. This study identified 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, 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 (Nelson, 2010).

The Drosophila genome encodes seven predicted claudin-family molecules. Two of these, Mega and Sinu, have previously been characterized and were shown to be required for SJ organization and function. Sequence comparisons indicated that, although all seven claudin-like molecules show a large sequence divergence, CG1298 is more closely related to Sinu and Mega than the other Drosophila claudin-family members. Therefore, although many Drosophila claudin-family members are not required for barrier function, it was reasoned that CG1298 may play a role in SJ paracellular barrier formation. Accordingly, a detailed analysis of CG1298, which was named kune-kune (Japanese for 'wiggling like a snake,' pronounced koon-eh koon-eh and abbreviated kune) was characterized for its tracheal phenotype (Nelson, 2010).

The kune locus contains a single exon that codes for a protein of 264 amino acids. As is characteristic for claudins, the TMpred transmembrane algorith predicts Kune to have four transmembrane domains with intracellular N and C termini, a large initial extracellular loop, and two smaller loops. Kune also contains a W-GLW-C-C motif in the large extracellular loop and a C-terminal PDZ-binding motif, features that are found in almost all claudin family members. Notably, the PDZ-binding motif in Kune is a better match to consensus PDZ-binding motifs than the motif in Sinuous. Furthermore, in contrast to Mega and Sinu, whose N termini are 28 and 38 aa respectively, Kune has a short N terminus of 9 aa that is more typical of vertebrate claudins. Thus, Kune has features that more closely resemble vertebrate claudins than do the so far characterized Drosophila claudins Sinu and Mega (Nelson, 2010).

To determine the expression pattern of Kune, anti-Kune sera were generated and wild-type (WT) embryos were immunized. As with Mega and Sinu, Kune is highly expressed in ectodermally derived tissues, including the epidermis, salivary gland, trachea, hindgut, and foregut beginning at embryonic stage 13. In these tissues, Kune colocalizes with the SJ protein Coracle (Cor) and localizes basal to the adherens junction marker, DE-cadherin (E-cad), suggesting that Kune is a SJ protein. As with many other SJ proteins, Kune is also expressed in glial cells (Nelson, 2010).

Since most SJ proteins show interdependence for correct localization and junction function, it was asked whether localization of Kune depends on the presence of other SJ proteins. Indeed, Kune is mislocalized to more basal positions in the primary epithelia of mega, sinu, cor, and Atpα null mutants, providing strong evidence that Kune is a SJ protein (Nelson, 2010).

To directly assess the function of Kune during development, a PiggyBac insertion, PBac{3HPy}C309, in the 5'-untranslated region (UTR) of kune was identified as a putative null mutation. Embryos homozygous for the kune^C309 chromosome fail to hatch and completely lack Kune protein as assessed by immunohistochemistry. Expression of a UAS-kune construct using the ubiquitous da-Gal4 driver at 28° rescued the embryonic lethality of kune^C309 embryos, demonstrating that lethality was due to loss of Kune. Further, embryos trans-heterozygous for kune^C309 and Df(2R)BSC696 (which deletes the kune locus and also eliminates Kune staining) or homozygous for Df(2R)BSC696 fail to hatch and display tracheal and septate junction phenotypes that are indistinguishable from kune^C309 homozygotes. These results indicate that kune is an essential gene and that kune^C309 is a null or strong loss-of-function allele of kune (Nelson, 2010).

To determine if Kune is required for SJ organization and function, the subcellular localization of several SJ proteins was examined in kune mutant epithelia. As is seen in other SJ mutants, kune epithelia show a reduction and/or mislocalization of the SJ components Cor, Mega, Sinu, Atpα, Discs large (Dlg), and NeurexinIV (Nrx) to more basal locations in all primary epithelia. This phenotype is also seen in animals that express kune-RNAi using the ubiquitous da-Gal4 driver, although the phenotype is less severe, presumably due to incomplete knockdown. Consistent with the immunohistological evidence of SJ defects, a 10-kDa fluorescent dye injected into the body cavity of kune animals readily diffused into the lumen of the trachea and salivary gland, indicating a loss of the paracellular barrier. Expression of the UAS-kune construct with da-Gal4 rescued Cor localization and improved the barrier function of kune mutants. Thus, Kune is an essential component of SJs in primary epithelia (Nelson, 2010).

In addition to their roles in epithelial tissues, SJs are also required to establish the blood-brain barrier in flies. In the central nervous system (CNS), surface glial cells completely ensheath the ventral nerve cord and form SJs at glia-glia contacts. This generates a tight paracellular seal that separates the K⁺-rich hemolymph from neural cells, which is essential for generation of action potentials. This study found that Kune is expressed in glial cells, which is most clearly seen at the central midline. Dye injections revealed that Kune, like Sinu and Mega, is required for the CNS glial barrier, since the dye penetrated into the nerve cord of kune but not WT embryos. Taken together, the above results all identify Kune as a critical SJ component in multiple tissue types (Nelson, 2010).

Since almost all characterized SJ proteins are required for tracheal tube size control, the tracheal system of kune embryos was examined. Staining with the 2A12 lumenal marker demonstrated that the length of the DT of stage 16 kune embryos was significantly increased over WT controls and appeared tortuous (thus the name kune-kune). This phenotype was identical in both kune/Df(2R)BSC696 embryos and embryos expressing kune-RNAi using the da-Gal4 driver (Nelson, 2010).

It has been established that Sinu, Mega, and other SJ proteins are required for apical secretion of the putative chitin deacetylases Verm and Serp, which restrict tracheal tube length. Therefore lumenal accumulation of Verm and Serp and the organization of the chitin-based lumenal matrix were examined at embryonic stage 16. As is typical for a SJ component, kune mutant embryos and embryos expressing kune-RNAi do not accumulate Verm or Serp in the tracheal lumen. This secretion defect could be largely rescued by expression of UAS-kune with the da-Gal4 driver, although not to WT levels. Additionally, staining with a fluorescent chitin binding probe showed that, while the lumen of WT trachea contains a dense, fibrilar chitin cable, kune trachea have a diffuse, disorganized lumenal matrix. kune trachea also lack the gap between the chitin cable and the apical surface of the cells that is found in WT trachea (Nelson, 2010).

Since Kune is closely related to both Mega and Sinu and all three localize to the SJ, it was asked if these claudins are partially redundant in junction organization or tube size control. To test this, the trachea of mega, kune, and sinu single, double, and triple mutants were examined. If the claudins have redundant functions in tube size control, the phenotypes should be worse when multiple claudins are missing. However, the tracheal length defects of mega; kune and kune; sinu embryos appear no more severe than in any of the single mutants. Strikingly, even embryos lacking all three Kune-related claudins do not appear to have more severe tracheal length defects than single mutants. In contrast, previous work has shown that embryos containing mutations in both sinu and the SJ gene varicose (vari) have trachea that are more tortuous than either single mutant. These results suggest that, although some SJ proteins have redundant functions in restricting tracheal tube length, the Drosophila claudins Kune, Sinu, and Mega all function in the same pathway of tracheal tube size control (Nelson, 2010).

Given that tracheal tube length is only a limited readout of SJ function, the effects of single-, double-, and triple-mutant combinations of kune, sinu, and mega on SJ organization were compared using the subcellular localization of Cor as an assay. Focused was placed on the hindgut and salivary gland, since SJ organization is clearest in these columnar cells. Interestingly, the levels and localization of Cor are strikingly different between the three claudin mutations, suggesting that different claudins have unique functions. For example, Cor is completely mislocalized to basal positions in the hindgut of mega embryos, but the levels are not dramatically lower than in WT. On the other hand, the hindguts of sinu embryos show lower overall levels of Cor, but retain significant apicolateral enrichment where the SJ is normally found. In kune mutants, Cor is both reduced and completely mislocalized. Similar, more pronounced effects are seen in the salivary glands where loss of mega causes only slight basolateral mislocalization of Cor, loss of sinu causes almost no Cor mislocalization, and loss of kune strongly mislocalizes and reduces Cor staining. The localization and levels of the SJ markers Dlg and Atpα were also more severely disrupted in kune mutants than in sinu or mega mutants, indicating that the effects were not specific to Cor. Interestingly, the levels and localization of Cor are not obviously different between the kune single mutant and the double and triple mutants, indicating that the kune phenotype is the most severe. Together, these results suggest that Kune has a more central role in SJ organization than either Sinu or Mega. This possibility is particularly intriguing in light of the greater similarity of Kune to vertebrate claudins than either Sinu or Mega. Perhaps a more central role for Kune in barrier junction formation has constrained its evolution and thus Kune more closely resembles ancestral claudins than do Sinu and Mega, which may have evolved more specialized functions (Nelson, 2010).

It is curious that multiple nonredundant claudins are required for SJ organization and barrier function. The exact reason for this is unclear, but perhaps each claudin interacts independently with specific junctional molecules to establish a SJ scaffold. This would be consistent with their divergent protein sequences and the differences in their N and C termini. Importantly, vertebrate claudins also have nonredundant roles in TJ function. For example, paracellular barrier function is compromised in the epidermis of mice lacking claudin-1 despite the presence of claudin-4 at the TJ (Nelson, 2010).

Like other SJ components, Kune does not appear to be required for establishment of apical-basal polarity since the levels and localization of the apical marker, Crumbs (Crb), and the adherens junction marker, E-cad, were normal in kune embryos. However, it was recently shown that some SJ components have a role in a newly identified phase of Drosophila epithelial polarity that occurs between stages 11 and 13. Because of redundancy between SJ components involved in this polarity phase and the SJ component yrt, SJ proteins required for polarity can be identified only in a yrt zygotic mutant background. For example, single zygotic mutations in either the SJ gene Atpα or yrt show normal apical localization of Crb at stage 12. Atpα, yrt double mutants on the other hand show severe mislocalization of Crb, indicative of a loss of polarity. In contrast, neither kune single mutants nor kune; yrt double mutants display any obvious polarity defects, demonstrating that kune is not required for either establishment or maintenance of epithelial polarity (Nelson, 2010).

Previous work has shown that neither mega nor sinu are required for establishment of apical-basal polarity or for maintenance of epithelial polarity at mid-embryogenesis. Together with the findings in this article, the available evidence suggests that Drosophila claudins are not required for epithelial polarity. This parallels the situation in Caenorhabditis elegans where mutations in the claudin-like proteins CLC1-4 disrupt barrier function, but not epithelial polarity. Similarly, claudins do not appear to be required for epithelial polarity in mammalian epithelial cells, since Eph4 cells can establish normal polarity even when lacking claudin complexes and tight junction strands due to elimination of ZO-1 and ZO-2. The absence of a role for claudins in polarity in any characterized species is consistent with the proposal that barrier junctions arose after polarity during the evolution of metazoans (Nelson, 2010).

These results show that Kune is an essential claudin that is required in all examined tissues for the organization and function of SJs. Kune expression and localization overlaps with the Drosophila claudins Mega and Sinu, but it was found that all three claudins play unique roles in SJ organization. Importantly, Kune more closely resembles vertebrate claudins than either Mega or Sinu and appears to play a more central role in SJ organization. Further work is needed to establish the complete molecular organization of SJs, but such work will be facilitated by the presented characterization of Kune and its interaction with other SJ components (Nelson, 2010).

Search PubMed for articles about Drosophila Sinuous

Asano, A., et al. (2003). Claudins in Caenorhabditis elegans: their distribution and barrier function in the epithelium. Curr. Biol. 13: 1042-1046. PubMed ID: 12814550

Behr, M., Riedel, D. and Schuh, R. (2003). The claudin-like megatrachea is essential in septate junctions for the epithelial barrier function in Drosophila. Dev. Cell. 5: 611-620. PubMed ID: 14536062

Colegio, O. R., et al. (2002). Claudins create charge-selective channels in the paracellular pathway between epithelial cells. Am. J. Physiol. Cell Physiol. 283: C142-C147. PubMed ID: 12055082

Colegio, O. R., et al. (2003). Claudin extracellular domains determine paracellular charge selectivity and resistance but not tight junction fibril architecture. Am. J. Physiol. Cell Physiol. 284: C1346-C1354. PubMed ID: 12700140

Furuse, M., et al. (1998). A single gene product, claudin-1 or -2, reconstitutes tight junction strands and recruits occludin in fibroblasts. J. Cell Biol. 143: 391-401. PubMed ID: 9786950

Furuse, M., et al. (2002). Claudin-based tight junctions are crucial for the mammalian epidermal barrier: a lesson from claudin-1-deficient mice. J. Cell Biol. 156: 1099-1111. PubMed ID: 11889141 Genova, J. L. and Fehon. R. G. (2003). Neuroglian, Gliotactin, and the Na+/K+ ATPase are essential for septate junction function in Drosophila. J. Cell Biol. 161: 979-989. PubMed ID: 12782686

Kollmar, R., et al. (2001). Expression and phylogeny of claudins in vertebrate primordia. Proc. Natl. Acad. Sci. 98: 10196-10201. PubMed ID: 11517306

Kubota, K., et al. (1999). Ca2+-independent cell-adhesion activity of claudins, a family of integral membrane proteins localized at tight junctions. Curr. Biol. 9: 1035-1038. PubMed ID: 11517306

Morita, K., et al. (1999). Claudin multigene family encoding four-transmembrane domain protein components of tight junction strands. Proc. Natl. Acad. Sci. 96: 511-516. PubMed ID: 9892664

Nelson, K. S., Furuse, M., Beitel, G. J. (2010). The Drosophila Claudin Kune-kune is required for septate junction organization and tracheal tube size control. Genetics 185(3): 831-9. PubMed ID: 20407131

Nitta, T., et al. (2003). Size-selective loosening of the blood-brain barrier in claudin-5-deficient mice. J. Cell Biol. 161: 653-660. PubMed ID: 12743111

Paul, S. M., et al. (2003). The Na⁺/K⁺ ATPase is required for septate junction function and epithelial tube-size control in the Drosophila tracheal system. Development 130: 4963-4974. PubMed ID: 12930776

Stork, T., et al. (2008). Organization and function of the blood-brain barrier in Drosophila. J. Neurosci. 28(3): 587-597. PubMed ID: 18199760

Tsukita, S., Furuse, M. and Itoh, M. (2001). Multifunctional strands in tight junctions. Nat. Rev. Mol. Cell Biol. 2: 285-293. PubMed ID: 11283726

Tsukita, S. and Furuse. M. (2002). Claudin-based barrier in simple and stratified cellular sheets. Curr. Opin. Cell Biol. 14: 531-536. PubMed ID: 12231346

Wu, V. M., Schulte, J., Hirschi, A., Tepass, U. and Beitel, G. J. (2004). Sinuous is a Drosophila claudin required for septate junction organization and epithelial tube size control. J. Cell Biol. 164(2): 313-23. PubMed ID: 14734539

Yi, P., Johnson, A. N., Han, Z., Wu, J. and Olson, E. N. (2008). Heterotrimeric G proteins regulate a noncanonical function of septate junction proteins to maintain cardiac integrity in Drosophila. Dev. Cell 15(5): 704-13. PubMed ID: 19000835

date revised: 30 January 2010

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