Invertebrate Epithelia Research Articles

Septate junction proteins are required for cell shape changes, actomyosin reorganization and cell adhesion during dorsal closure in Drosophila.

Septate junctions (SJs) serve as occluding barriers in invertebrate epithelia. In Drosophila, at least 30 genes are required for the formation or maintenance of SJs. Interestingly, loss-of-function mutations in core SJ components are embryonic lethal, with defects in developmental events such as head involution and dorsal closure (DC) that occur prior to the formation of a mature SJ, indicating a role for these proteins in mid-embryogenesis independent of their occluding function. To understand this novel function in development, we examined loss-of-function mutations in three core SJ proteins during the process of DC. DC occurs during mid-embryogenesis to seal a dorsal gap in the epidermis following germ band retraction. Closure is driven by contraction of the extraembryonic amnioserosa cells that temporarily cover the dorsal surface and by cell shape changes (elongation) of lateral epidermal cells that bring the contralateral sheets together at the dorsal midline. Using live imaging and examination of fixed tissues, we show that early events in DC occur normally in SJ mutant embryos, but during later closure, coracle, Macroglobulin complement-related and Neurexin-IV mutant embryos exhibit slower rates of closure and display aberrant cells shapes in the dorsolateral epidermis, including dorsoventral length and apical surface area. SJ mutant embryos also show mild defects in actomyosin structures along the leading edge, but laser cutting experiments suggest similar tension and viscoelastic properties in SJ mutant versus wild type epidermis. In a high percentage of SJ mutant embryos, the epidermis tears free from the amnioserosa near the end of DC and live imaging and immunostaining reveal reduced levels of E-cadherin, suggesting that defective adhesion may be responsible for these tears. Supporting this notion, reducing E-cadherin by half significantly enhances the penetrance of DC defects in coracle mutant embryos.

Formation and remodelling of septate junctions in the epidermis of isopod Porcellioscaber during development.

Septate junctions (SJs) perform an occluding function in invertebrate epithelia and consist of parallel septa extending across the intercellular space between neighbouring cells. In addition, they are required for several morphogenetic processes in arthropods. The biogenesis of SJs during development is inadequately studied and it was characterised in detail only for various epithelia of Drosophilamelanogaster. This paper provides a detailed analysis of the ultrastructural differentiation of SJs in the epidermis of the terrestrial isopod Porcellioscaber during embryonic and postembryonic development. In this study, mid-stage embryo S13 was the earliest stage in which single septa were observed basally to the adherens junction (AJ). Differentiation of SJs during further development includes gradual elongation of septa arrays and formation of continuous arrays of septa. The enlargement of SJs in the epidermis is most pronounced at the transition from embryonic to postembryonic development and after the release of mancae from the marsupium. SJs of postmarsupial mancae are similar to those of adults, but are not yet as extensive. Comparison of the differentiation of SJs in the epidermis and hindgut of P.scaber, reveals a similar sequence of events. In addition, remodelling of SJs was observed in the epidermis of late marsupial mancae, the stage of cuticle renewal. Common features of SJs' biogenesis in P.scaber and D.melanogaster ectodermal epithelia are indicated.

Blending physiology and RNAseq to provide new insights into regulation of epithelial transport: switching between ion secretion and reabsorption.

This Review addresses the means by which epithelia change the direction of vectorial ion transport. Recent studies have revealed that insect Malpighian (renal) tubules can switch from secreting to reabsorbing K+. When the gut of larval lepidopterans is empty (during the moult cycle) or when the larvae are reared on K+-deficient diet, the distal ileac plexus segment of the tubule secretes K+ from the haemolymph into the tubule lumen. By contrast, in larvae reared on K+-rich diet, ions and fluid are reabsorbed from the rectal lumen into the perinephric space surrounding the cryptonephridial tubules of the rectal complex. Ions and fluid are then transported from the perinephric space into the lumen of the cryptonephridial tubules, thus supplying the free segments of the tubule downstream. Under these conditions, some of the K+ and water in the tubule lumen is reabsorbed across the cells of the distal ileac plexus, allowing for expansion of haemolymph volume in the rapidly growing larvae, as well as recycling of K+ and base equivalents. RNA sequencing data reveal large-scale changes in gene transcription that are associated with the switch between ion secretion and ion reabsorption by the distal ileac plexus. An unexpected finding is the presence of voltage-gated, ligand-gated and mechanosensitive ion channels, normally seen in excitable cells, in Malpighian tubules. Transcriptomic surveys indicate that these types of channels are also present in multiple other types of vertebrate and invertebrate epithelia, suggesting that they may play novel roles in epithelial cell signalling and regulation of epithelial ion transport.

An alternative mode of epithelial polarity in the Drosophila midgut.

Apical–basal polarity is essential for the formation and function of epithelial tissues, whereas loss of polarity is a hallmark of tumours. Studies in Drosophila have identified conserved polarity factors that define the apical (Crumbs, Stardust, Par-6, atypical protein kinase C [aPKC]), junctional (Bazooka [Baz]/Par-3), and basolateral (Scribbled [Scrib], Discs large [Dlg], Lethal [2] giant larvae [Lgl]) domains of epithelial cells. Because these conserved factors mark equivalent domains in diverse types of vertebrate and invertebrate epithelia, it is generally assumed that this system underlies polarity in all epithelia. Here, we show that this is not the case, as none of these canonical factors are required for the polarisation of the endodermal epithelium of the Drosophila adult midgut. Furthermore, like vertebrate epithelia but not other Drosophila epithelia, the midgut epithelium forms occluding junctions above adherens junctions (AJs) and requires the integrin adhesion complex for polarity. Thus, Drosophila contains two types of epithelia that polarise by fundamentally different mechanisms. This diversity of epithelial types may reflect their different developmental origins, junctional arrangement, or whether they polarise in an apical–basal direction or vice versa. Since knock-outs of canonical polarity factors in vertebrates often have little or no effect on epithelial polarity and the Drosophila midgut shares several common features with vertebrate epithelia, this diversity of polarity mechanisms is likely to be conserved in other animals.

Boudin trafficking reveals the dynamic internalisation of specific septate junction components in Drosophila.

The maintenance of paracellular barriers in invertebrate epithelia depends on the integrity of specific cell adhesion structures known as septate junctions (SJ). Multiple studies in Drosophila have revealed that these junctions have a stereotyped architecture resulting from the association in the lateral membrane of a large number of components. However, little is known about the dynamic organisation adopted by these multi-protein complexes in living tissues. We have used live imaging techniques to show that the Ly6 protein Boudin is a component of these adhesion junctions and can diffuse systemically to associate with the SJ of distant cells. We also observe that this protein and the claudin Kune-kune are endocytosed in epidermal cells during embryogenesis. Our data reveal that the SJ contain a set of components exhibiting a high membrane turnover, a feature that could contribute in a tissue-specific manner to the morphogenetic plasticity of these adhesion structures.

Identification of the septate junction protein gliotactin in the mosquito Aedes aegypti: evidence for a role in increased paracellular permeability in larvae.

Septate junctions (SJs) regulate paracellular permeability across invertebrate epithelia. However, little is known about the function of SJ proteins in aquatic invertebrates. In this study, a role for the transmembrane SJ protein gliotactin (Gli) in the osmoregulatory strategies of larval mosquito (Aedes aegypti) was examined. Differences in gli transcript abundance were observed between the midgut, Malpighian tubules, hindgut and anal papillae of A. aegypti, which are epithelia that participate in larval mosquito osmoregulation. Western blotting of Gli revealed its presence in monomer, putative dimer and alternatively processed protein forms in different larval mosquito organs. Gli localized to the entire SJ domain between midgut epithelial cells and showed a discontinuous localization along the plasma membranes of epithelial cells of the rectum as well as the syncytial anal papillae epithelium. In the Malpighian tubules, Gli immunolocalization was confined to SJs between the stellate and principal cells. Rearing larvae in 30% seawater caused an increase in Gli protein abundance in the anterior midgut, Malpighian tubules and hindgut. Transcriptional knockdown of gli using dsRNA reduced Gli protein abundance in the midgut and increased the flux rate of the paracellular permeability marker, polyethylene glycol (molecular weight 400 Da; PEG-400). Data suggest that in larval A. aegypti, Gli participates in the maintenance of salt and water balance and that one role for Gli is to participate in the regulation of paracellular permeability across the midgut of A. aegypti in response to changes in environmental salinity.

Making the connection - shared molecular machinery and evolutionary links underlie the formation and plasticity of occluding junctions and synapses.

The pleated septate junction (pSJ), an ancient structure for cell-cell contact in invertebrate epithelia, has protein components that are found in three more-recent junctional structures, the neuronal synapse, the paranodal region of the myelinated axon and the vertebrate epithelial tight junction. These more-recent structures appear to have evolved through alterations of the ancestral septate junction. During its formation in the developing animal, the pSJ exhibits plasticity, although the final structure is extremely robust. Similar to the immature pSJ, the synapse and tight junctions both exhibit plasticity, and we consider evidence that this plasticity comes at least in part from the interaction of members of the immunoglobulin cell adhesion molecule superfamily with highly regulated membrane-associated guanylate kinases. This plasticity regulation probably arose in order to modulate the ancestral pSJ and is maintained in the derived structures; we suggest that it would be beneficial when studying plasticity of one of these structures to consider the literature on the others. Finally, looking beyond the junctions, we highlight parallels between epithelial and synaptic membranes, which both show a polarized distribution of many of the same proteins - evidence that determinants of apicobasal polarity in epithelia also participate in patterning of the synapse.

A tetraspanin regulates septate junction formation in Drosophila midgut

Septate junctions (SJs) are membrane specializations that restrict the free diffusion of solutes through the paracellular pathway in invertebrate epithelia. In arthropods, two morphologically different types of septate junctions are observed; pleated (pSJs) and smooth (sSJs), which are present in ectodermally and endodermally derived epithelia, respectively. Recent identification of sSJ-specific proteins, Mesh and Ssk, in Drosophila indicates that the molecular compositions of sSJs and pSJs differ. A deficiency screen based on immunolocalization of Mesh identified a tetraspanin family protein, Tsp2A, as a newly discovered protein involved in sSJ formation in Drosophila Tsp2A specifically localizes at sSJs in the midgut and Malpighian tubules. Compromised Tsp2A expression caused by RNAi or the CRISPR/Cas9 system was associated with defects in the ultrastructure of sSJs, changed localization of other sSJ proteins, and impaired barrier function of the midgut. In most Tsp2A mutant cells, Mesh failed to localize to sSJs and was distributed through the cytoplasm. Tsp2A forms a complex with Mesh and Ssk and these proteins are mutually interdependent for their localization. These observations suggest that Tsp2A cooperates with Mesh and Ssk to organize sSJs.

The response of claudin-like transmembrane septate junction proteins to altered environmental ion levels in the larval mosquito Aedes aegypti.

Septate junctions (SJs) occlude the paracellular pathway and function as paracellular diffusion barriers within invertebrate epithelia. However, integral components of SJs and their contribution to barrier properties have received considerably less attention than those of vertebrate occluding junctions. In arthropods, SJ proteins have only been identified in Drosophila and among these are three integral claudin-like proteins, Megatrachea (Mega), Sinuous (Sinu) and Kune-kune (Kune), as well as a receptor-like transmembrane SJ protein known as Neurexin IV (Nrx IV). In this study, mega, sinu, kune and nrx IV are identified and characterized in aquatic larvae of the mosquito Aedes aegypti and a role for these proteins in ionoregulatory homeostasis is considered. Transcripts encoding Mega, Sinu, Kune and Nrx IV were found in iono/osmoregulatory tissues such as the midgut, Malpighian tubules, hindgut and anal papillae, but abundance was greater in the hindgut and anal papillae. Using immunohistochemical and western blot analysis it was found that Kune localized to the regions of intercellular contact between epithelial cells of the rectum and posterior midgut and in the apical membrane domain of the syncytial epithelium of anal papillae. To investigate a potential role for integral SJ proteins in larval A. aegypti iono/osmoregulation, abundance was examined in animals reared in freshwater or brackish water (30% seawater). In iono/osmoregulatory epithelia, larvae exhibited tissue-specific alterations in mega mRNA and Kune protein abundance, but not sinu or nrx IV mRNA. These studies provide a first look at the potential contribution of integral SJ components to iono/osmoregulatory homeostasis in an aquatic invertebrate.

A tetraspanin regulates septate junction formation in Drosophila midgut.

Septate junctions (SJs) are membrane specializations that restrict the free diffusion of solutes through the paracellular pathway in invertebrate epithelia. In arthropods, two morphologically different types of septate junctions are observed; pleated (pSJs) and smooth (sSJs), which are present in ectodermally and endodermally derived epithelia, respectively. Recent identification of sSJ-specific proteins, Mesh and Ssk, in Drosophila indicates that the molecular compositions of sSJs and pSJs differ. A deficiency screen based on immunolocalization of Mesh identified a tetraspanin family protein, Tsp2A, as a newly discovered protein involved in sSJ formation in Drosophila Tsp2A specifically localizes at sSJs in the midgut and Malpighian tubules. Compromised Tsp2A expression caused by RNAi or the CRISPR/Cas9 system was associated with defects in the ultrastructure of sSJs, changed localization of other sSJ proteins, and impaired barrier function of the midgut. In most Tsp2A mutant cells, Mesh failed to localize to sSJs and was distributed through the cytoplasm. Tsp2A forms a complex with Mesh and Ssk and these proteins are mutually interdependent for their localization. These observations suggest that Tsp2A cooperates with Mesh and Ssk to organize sSJs.

Occluding junctions of invertebrate epithelia.

Invertebrate diversity and architecture is immense. This is achieved by the organization and function of four tissue types found in most metazoan phyla-epithelial, connective, muscle and nervous tissue. Epithelial tissue is found in all extant animals (parazoan and metazoan alike). Epithelial cells form cellular sheets that cover internal or external surfaces and regulate the passage of material between separated compartments. The transepithelial movement of biological material between compartments can occur across the transcellular pathway (i.e. across cells) or the paracellular pathway (i.e. between cells) and the latter is regulated by occluding junctions that typically link cells in a subapical domain. In this review, information on occluding junctions of invertebrate epithelia is consolidated and discussed in the context of morphology, ultrastructure and physiology. In addition, an overview of what is currently known about invertebrate occluding junction proteins and their role in maintaining the integrity of invertebrate epithelia and regulating the barrier properties of these tissues is presented.

Pasiflora proteins are novel core components of the septate junction.

Epithelial sheets play essential roles as selective barriers insulating the body from the environment and establishing distinct chemical compartments within it. In invertebrate epithelia, septate junctions (SJs) consist of large multi-protein complexes that localize at the apicolateral membrane and mediate barrier function. Here, we report the identification of two novel SJ components, Pasiflora1 and Pasiflora2, through a genome-wide glial RNAi screen in Drosophila. Pasiflora mutants show permeable blood-brain and tracheal barriers, overelongated tracheal tubes and mislocalization of SJ proteins. Consistent with the observed phenotypes, the genes are co-expressed in embryonic epithelia and glia and are required cell-autonomously to exert their function. Pasiflora1 and Pasiflora2 belong to a previously uncharacterized family of tetraspan membrane proteins conserved across the protostome-deuterostome divide. Both proteins localize at SJs and their apicolateral membrane accumulation depends on other complex components. In fluorescence recovery after photobleaching experiments we demonstrate that pasiflora proteins are core SJ components as they are required for complex formation and exhibit restricted mobility within the membrane of wild-type epithelial cells, but rapid diffusion in cells with disrupted SJs. Taken together, our results show that Pasiflora1 and Pasiflora2 are novel integral components of the SJ and implicate a new family of tetraspan proteins in the function of these ancient and crucial cell junctions.

Cohesin controls planar cell polarity by regulating the level of the seven‐pass transmembrane cadherin Flamingo

Planar cell polarity (PCP) refers to the coordination of global organ axes and individual cell polarity in vertebrate and invertebrate epithelia. Mechanisms of PCP have been best studied in the Drosophila wing, in which each epidermal cell produces a single wing hair at the distal cell edge, and this spatial specification is mediated by redistribution of the core group proteins, including the seven-pass transmembrane cadherin Flamingo/Starry night (Fmi/Stan), to selective plasma membrane domains. Through genetic screening, we found that a mutation of the SMC3 gene caused dramatic misspecification of wing hair positions. SMC3 protein is one subunit of the cohesin complex, which regulates sister chromatid cohesion and also plays a role in transcriptional control of gene expression. In the SMC3 mutant cells, Fmi appeared to be upregulated by a posttranscriptional mechanism(s), and this elevation of Fmi was at least one cause of the PCP defect. In addition to the PCP phenotype, the loss of the cohesin function affected wing morphogenesis at multiple levels: one malformation was loss of the wing margin, and this was most likely a result of downregulation of the homeodomain protein Cut. At the cellular level, apical cell size and hexagonal packing were affected in the mutant wing. Dysfunction of cohesin in humans results in Cornelia de Lange syndrome (CdLS), which is characterized by various developmental abnormalities and mental retardation. Our analysis of cohesin in epithelia may provide new insight into cellular and molecular mechanisms of CdLS.

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

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

Cell Flow Reorients the Axis of Planar Polarity in the Wing Epithelium of Drosophila

Planar cell polarity (PCP) proteins form polarized cortical domains that govern polarity of external structures such as hairs and cilia in both vertebrate and invertebrate epithelia. The mechanisms that globally orient planar polarity are not understood, and are investigated here in the Drosophila wing using a combination of experiment and theory. Planar polarity arises during growth and PCP domains are initially oriented toward the well-characterized organizer regions that control growth and patterning. At pupal stages, the wing hinge contracts, subjecting wing-blade epithelial cells to anisotropic tension in the proximal-distal axis. This results in precise patterns of oriented cell elongation, cell rearrangement and cell division that elongate the blade proximo-distally and realign planar polarity with the proximal-distal axis. Mutation of the atypical Cadherin Dachsous perturbs the global polarity pattern by altering epithelial dynamics. This mechanism utilizes the cellular movements that sculpt tissues to align planar polarity with tissue shape.

Renal defects associated with improper polarization of the CRB and DLG polarity complexes in MALS-3 knockout mice

Kidney development and physiology require polarization of epithelia that line renal tubules. Genetic studies show that polarization of invertebrate epithelia requires the crumbs, partition-defective-3, and discs large complexes. These evolutionarily conserved protein complexes occur in mammalian kidney; however, their role in renal development remains poorly defined. Here, we find that mice lacking the small PDZ protein mammalian LIN-7c (MALS-3) have hypomorphic, cystic, and fibrotic kidneys. Proteomic analysis defines MALS-3 as the only known core component of both the crumbs and discs large cell polarity complexes. MALS-3 mediates stable assembly of the crumbs tight junction complex and the discs large basolateral complex, and these complexes are disrupted in renal epithelia from MALS-3 knockout mice. Interestingly, MALS-3 controls apico-basal polarity preferentially in epithelia derived from metanephric mesenchyme, and defects in kidney architecture owe solely to MALS expression in these epithelia. These studies demonstrate that defects in epithelial cell polarization can cause cystic and fibrotic renal disease.

Amiloride-sensitive sodium absorption is different in vertebrates and invertebrates

Amiloride-sensitive Na+ absorption is a well-described feature of numerous transporting epithelia in vertebrates. Yet, very little is known about this important physiological process regarding invertebrates. In the present paper, we compare vertebrate Na+ absorption mediated by the amiloride-sensitive epithelial Na+ channel (ENaC) and its invertebrate counterpart. We used the dorsal skin of the annelid Hirudo medicinalis as a model for the Na+ absorption of invertebrate epithelia. In applying electrophysiological, molecular, and biochemical techniques we found striking functional and structural differences between vertebrate and invertebrate amiloride-sensitive Na+ absorption. Using modified Ussing chambers, we analyzed the influence of different known blockers and effectors of vertebrate ENaC on leech epithelial Na+ absorption. We demonstrate that the serine protease trypsin had no effect on the Na+ transport across leech integument, while it strongly activates vertebrate ENaC. While protons, and the divalent cations Ni2+ and Zn2+ stimulate vertebrate ENaC, amiloride-sensitive Na+ currents in leech integument were substantially reduced. For molecular studies, we constructed a cDNA library of Hirudo medicinalis and screened it with specific ENaC antibodies. We performed numerous PCR approaches using a vast number of different degenerated and specific ENaC primers to identify ENaC-like structures. Yet, both strategies did not reveal any ENaC-like sequence in leech integument. From these data we conclude that amiloride-sensitive Na+ absorption in leech skin is not mediated by an ENaC-like Na+ channel but by a still unknown invertebrate member of the ENaC/DEG family that we termed lENaTP (leech epithelial Na+ transporting protein).

The Apical Determinants aPKC and dPatj Regulate Frizzled-Dependent Planar Cell Polarity in the Drosophila Eye

Planar cell polarity (PCP) is a common feature of many vertebrate and invertebrate epithelia and is perpendicular to their apical/basal (A/B) polarity axis. While apical localization of PCP determinants such as Frizzled (Fz1) is critical for their function, the link between A/B polarity and PCP is poorly understood. Here, we describe a direct molecular link between A/B determinants and Fz1-mediated PCP establishment in the Drosophila eye. We demonstrate that dPatj binds the cytoplasmic tail of Fz1 and propose that it recruits aPKC, which in turn phosphorylates and inhibits Fz1. Accordingly, components of the aPKC complex and dPatj produce PCP defects in the eye. We also show that during PCP signaling, aPKC and dPatj are downregulated, while Bazooka is upregulated, suggesting an antagonistic effect of Bazooka on dPatj/aPKC. We propose a model whereby the dPatj/aPKC complex regulates PCP by inhibiting Fz1 in cells where it should not be active.

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

One essential function of epithelia is to form a barrier between the apical and basolateral surfaces of the epithelium. In vertebrate epithelia, the tight junction is the primary barrier to paracellular flow across epithelia, whereas in invertebrate epithelia, the septate junction (SJ) provides this function. In this study, we identify new proteins that are required for a functional paracellular barrier in Drosophila. In addition to the previously known components Coracle (COR) and Neurexin (NRX), we show that four other proteins, Gliotactin, Neuroglian (NRG), and both the α and β subunits of the Na+/K+ ATPase, are required for formation of the paracellular barrier. In contrast to previous reports, we demonstrate that the Na pump is not localized basolaterally in epithelial cells, but instead is concentrated at the SJ. Data from immunoprecipitation and somatic mosaic studies suggest that COR, NRX, NRG, and the Na+/K+ ATPase form an interdependent complex. Furthermore, the observation that NRG, a Drosophila homologue of vertebrate neurofascin, is an SJ component is consistent with the notion that the invertebrate SJ is homologous to the vertebrate paranodal SJ. These findings have implications not only for invertebrate epithelia and barrier functions, but also for understanding of neuron–glial interactions in the mammalian nervous system.

Transport of amino acids by marine invertebrates

AbstractMarine invertebrates transport amino acids and other organic solutes across their body surfaces. This surface absorption, in some instances, may contribute significantly to the overall nutritional requirements for an organism. Amino acids are accumulated against gradients as high as 106:1 to 107:1 (intracellular:extracellular concentration). The transport mechanism that has been consistently observed to account for this process is Na dependent cotransport. A review of the general characteristics of these transport systems characterized in marine invertebrate epithelia indicates certain common features: Na dependency with coupling coefficients of 2:1 or 3:1 (Na:amino acid translocated), influx coupled to membrane potential, and low intracellular Na activity. Under these conditions Na cotransport can readily account for gradients approaching 107:1. These transport systems may play a role in acquisition of nutrients by marine invertebrates, but it has also been suggested that they may play additional roles in osmoregulation, nutrient conservation, and chemo‐reception. © 1993 Wiley‐Liss, Inc.