Anti-innexin 2 aptamers specifically inhibit the heterologous interaction of the innexin 2 and innexin 3 carboxyl-termini in vitro

We recently demonstrated that heteromerization of innexins 2 and 3 from Drosophila melanogaster (Dm) is crucial for epithelial organization and polarity of the embryonic epidermis. Both innexins are thought to interact via their C-terminal cytoplasmic domains during the assembly of heteromeric gap junction channels. However, the mechanisms that control heteromeric versus homomeric channel formation are still largely unknown. Here we report the isolation of both non-modified and 2'-fluoro-2'-deoxy-modified RNA anti-innexin 2 aptamers by in vitro selection. The aptamers bind to a proximal epitope on the carboxyl-tail of Dm innexin 2 protein and specifically inhibit the heterologous interaction of innexin 2 and innexin 3 carboxyl-termini in vitro. These domain-specific inhibitors represent the first step towards functional studies focusing on the activity of these domains in vivo.     

Developmental expression and molecular characterization of two gap junction channel proteins expressed during embryogenesis in the grasshopper Schistocerca americana

Gap junctions are membrane channels that directly connect the cytoplasm of neighboring cells, allowing the exchange of ions and small molecules. Two analogous families of proteins, the connexins and innexins, are the channel-forming molecules in vertebrates and invertebrates, respectively. In order to study the role of gap junctions in the embryonic development of the nervous system, we searched for innexins in the grasshopper Schistocerca americana. Here we present the molecular cloning and sequence analysis of two novel innexins, G-Inx(1) and G-Inx(2), expressed during grasshopper embryonic development. The analysis of G-Inx(1) and G-Inx(2) proteins suggests they bear four transmembrane domains, which show strong conservation in members of the innexin family. The study of the phylogenetic relationships between members of the innexin family and the new grasshopper proteins suggests that G-Inx(1) is orthologous to the Drosophila 1(1)-ogre. However, G-Inx(2) seems to be a member of a new group of insect innexins. We used in situ hybridization with the G-Inx(1) and G-Inx(2) cDNA clones, and two polyclonal sera raised against different regions of G-Inx(1) to study the mRNA and protein expression patterns and the subcellular localization of the grasshopper innexins. G-Inx(1) is primarily expressed in the embryonic nervous system, in neural precursors and glial cells. In addition, a restricted stripe of epithelial cells in the developing limb, involved in the guidance of sensory growth cones, expresses G-Inx(1). G-Inx(2) expression is more widespread in the grasshopper embryo, but a restricted expression is found in a subset of neural precursors. The generally different but partially overlapping expression patterns of G-Inx(1) and G-Inx(2) supports the combinatorial character of gap junction formation in invertebrates, an essential property to generate specificity in this form of cell-cell communication.     

DE-cadherin, a core component of the adherens junction complex modifies subcellular localization of the Drosophila gap junction protein innexin2

The Drosophila innexin multigene family of gap junction encoding proteins consists of eight family members whose function in epithelial morphogenesis is mostly unknown. We have recently shown that innexin2 plays a crucial role in the organization of embryonic epithelia. Innexin2 protein accumulates in the epidermis in the apico-lateral membrane domain and colocalizes with core proteins of adherens junctions, such as DE-cadherin and Armadillo, the ss -catenin homolog. Innexin2 localization is altered in both armadillo and DE-cadherin mutants Biochemical interaction studies point to a direct interaction of DE-cadherin and Armadillo with innexin2 suggesting a close link between gap junction and adherens junction biogenesis. We have used the Drosophila Schneider cell tissue culture system to further study the interaction of innexin2 with DE-cadherin. Our results provide evidence that DE-cadherin may be a key component to control trafficking, and localization of Innexin2 to the plasma membrane.     

Gap junctions in Drosophila: developmental expression of the entire innexin gene family

Invertebrate gap junctions are composed of proteins called innexins and eight innexin encoding loci have been identified in the now complete genome sequence of Drosophila melanogaster. The intercellular channels formed by these proteins are multimeric and previous studies have shown that, in a heterologous expression system, homo- and hetero-oligomeric channels can form, each combination possessing different gating characteristics. Here we demonstrate that the innexins exhibit complex overlapping expression patterns during oogenesis, embryogenesis, imaginal wing disc development and central nervous system development and show that only certain combinations of innexin oligomerization are possible in vivo. This work forms an essential basis for future studies of innexin interactions in Drosophila and outlines the potential extent of gap-junction involvement in development.     

Innexins in C. elegans

Innexins are functionally analogous to the vertebrate connexins, and the innexin family of gap junction proteins has been identified in many invertebrates, including Drosophila and C. elegans. The genome sequencing project has identified 25 innexins in C. elegans. We are particularly interested in the roles that gap junctions may play in embryonic development and in wiring of the nervous system. To identify the particular C. elegans innexins that are involved in these processes, we are examining their expression patterns using specific antibodies and translational GFP fusions. In addition we are investigating mutant, RNAi and overexpression phenotypes for many of these genes. To date, we have generated specific antibodies to the non-conserved carboxyl termini of 5 innexins. We have constructed GFP translational fusions for 17 innexins and observed expression patterns for 13 of these genes. In total we have characterized expression patterns representing 14 innexins. Mutations have been identified in 5 of these genes, and at least 3 others have RNAi mutant phenotypes. Generalities emerging from our studies include: 1) most tissues and many individual cells express more than one innexin, 2) some innexins are expressed widely, while others are expressed in only a few cells, and 3) there is a potential for functional pairing of innexins.     

Innexins get into the gap

Connexins were first identified in the 1970s as the molecular components of vertebrate gap junctions. Since then a large literature has accumulated on the cell and molecular biology of this multi-gene family culminating recently in the findings that connexin mutations are implicated in a variety of human diseases. Over two decades, the terms "connexin" and "gap junction" had become almost synonymous. In the last few years a second family of gap-junction genes, the innexins, has emerged. These have been shown to form intercellular channels in genetically tractable invertebrate organisms such as Drosophila melanogaster and Caenorhabditis elegans. The completed genomic sequences for the fly and worm allow identification of the full complement of innexin genes in these two organisms and provide valuable resources for genetic analyses of gap junction function.     

Gap junction gene and protein families: Connexins,innexins, and pannexins

Gap junction channels facilitate the intercellular exchange of ions and small molecules. While this process is critical to all multicellular organisms, the proteins that form gap junction channels are not conserved. Vertebrate gap junctions are formed by connexins, while invertebrate gap junctions are formed by innexins. Interestingly, vertebrates and lower chordates contain innexin homologs, the pannexins, which also form channels, but rarely (if ever) make intercellular channels. While the connexin and the innexin/pannexin polypeptides do not share significant sequence similarity, all three of these protein families share a similar membrane topology and some similarities in quaternary structure. This article is part of a Special Issue entitled: Gap Junction Proteins edited by Jean Claude Herve.     

Gap junction channel protein innexin 2 is essential for epithelial morphogenesis in the Drosophila embryo

Direct communication of neighboring cells by gap junction channels is essential for the development of tissues and organs in the body. Whereas vertebrate gap junctions are composed of members of the connexin family of transmembrane proteins, in invertebrates gap junctions consist of Innexin channel proteins. Innexins display very low sequence homology to connexins. In addition, very little is known about their cellular role during developmental processes. In this report, we examined the function and the distribution of Drosophila Innexin 2 protein in embryonic epithelia. Both loss-of-function and gain-of-function innexin 2 mutants display severe developmental defects due to cell death and a failure of proper epithelial morphogenesis. Furthermore, immunohistochemical analyses using antibodies against the Innexins 1 and 2 indicate that the distribution of Innexin gap junction proteins to specific membrane domains is regulated by tissue specific factors. Finally, biochemical interaction studies together with genetic loss- and gain-of-function experiments provide evidence that Innexin 2 interacts with core proteins of adherens and septate junctions. This is the first study, to our knowledge, of cellular distribution and protein-protein interactions of an Innexin gap junctional channel protein in the developing epithelia of Drosophila.     

Gastrointestinal Development in the Drosophila Embryo Requires the Activity of Innexin Gap Junction Channel Proteins

Cell to cell communication plays an essential role during pattern formation and morphogenesis of the diverse tissues and organs of the body. In invertebrates, such as the fruitfly Drosophila, the direct communication of closely apposed cells is mediated by gap junctions which are composed of oligomers of the innexin family of transmembrane channel proteins. Few data exist about the developmental role of the eight innexin genes which have been found in the Drosophila genome. We have investigated the role of the innexin 2 and ogre genes during gastrointestinal development of the fly embryo. Our findings suggest that innexins are involved in the formation of the proventriculus, an organ that develops at the foregut/midgut boundary by migration of primordial cells and subsequent infolding of epithelial tissue layers.     

DE-Cadherin, a Core Component of the Adherens Junction Complex Modifies Subcellular Localization of the Drosophila Gap Junction Protein Innexin2

The Drosophila innexin multigene family of gap junction encoding proteins consists of eight family members whose function in epithelial morphogenesis is mostly unknown. We have recently shown that innexin2 plays a crucial role in the organization of embryonic epithelia. Innexin2 protein accumulates in the epidermis in the apico-lateral membrane domain and colocalizes with core proteins of adherens junctions, such as DE-cadherin and Armadillo, the β -catenin homolog. Innexin2 localization is altered in both armadillo and DE-cadherin mutants Biochemical interaction studies point to a direct interaction of DE-cadherin and Armadillo with innexin2 suggesting a close link between gap junction and adherens junction biogenesis. We have used the Drosophila Schneider cell tissue culture system to further study the interaction of innexin2 with DE-cadherin. Our results provide evidence that DE-cadherin may be a key component to control trafficking, and localization of Innexin2 to the plasma membrane.     

Gastrointestinal development in the Drosophila embryo requires the activity of innexin gap junction channel proteins

Cell to cell communication plays an essential role during pattern formation and morphogenesis of the diverse tissues and organs of the body. In invertebrates, such as the fruitfly Drosophila, the direct communication of closely apposed cells is mediated by gap junctions which are composed of oligomers of the innexin family of transmembrane channel proteins. Few data exist about the developmental role of the eight innexin genes which have been found in the Drosophila genome. We have investigated the role of the innexin 2 and ogre genes during gastrointestinal development of the fly embryo. Our findings suggest that innexins are involved in the formation of the proventriculus, an organ that develops at the foregut/midgut boundary by migration of primordial cells and subsequent infolding of epithelial tissue layers.     

Cellular distribution of innexin 1 and 2 gap junctional channel proteins in epithelia of the Drosophila embryo

Invertebrate gap junctions are composed of Innexin channel proteins that are structurally and functionally analogous to the connexins in vertebrates. In situ hybridization experiments have shown that most of the eight known innexin genes in Drosophila are expressed in a complex and overlapping temporal and spatial profile, with several members showing high levels of expression in developing epithelia of the embryo. To further study the cellular roles of Innexins, we have generated antibodies against Innexins 1 and 2 and studied their protein distribution in the developing embryo. We find that both Innexins are co-expressed in a number of epithelial tissues including the epidermis, the gut and the salivary glands. On the cellular level, we find both proteins localized to the membranes of epithelial cells. Immunohistochemical analysis using cell polarity markers indicates that Innexin 1 is predominantly localized to the baso-lateral domain of epithelial cells, basal to septate junctions. In contrast, we find a variable positioning of Innexin 2 along the apico-basal axis of epithelial cells depending on the type of tissue and organ. Our findings suggest that the distribution of Innexin channel proteins to specific membrane domains of epithelial cells is regulated by tissue specific factors during the development of epithelia in the fly embryo.     

Genes, gene knockouts, and mutations in the analysis of gap junctions

Gop junctions are cell junctions found between most cells and tissues. They contain membrane channels that mediate the cell-to-cell diffusion of ions, metabolites, and small cell signaling molecules. Cell-cell communication mediated by gap junctions has been proposed to have a variety of functions, including roles in regulating events in development, cell differentiation, and cell growth and proliferation. The analysis of these possibilities has been confounded by the fact that there are over a dozen connexin genes encoding polypeptides that make up vertebrate gap junctions. This complexity, coupled with the fact that most cells express multiple connexin isotypes, likely explains why recent studies using reverse genetic and genetic approaches to disrupt connexin gene function have yielded only limited insights into the physiological roles of gap junctions. Nevertheless, studies in vivo and in vitro together have provided evidence for gap junctions being involved in the regulation of cell metabolism, growth, and differentiation in restricted cell and tissue types. Surprisingly, studies in invertebrates suggest that their gap junctions are encoded not by connexins, but by a family of proteins referred to as innexins. Analysis of various Drosophila and C. elegans mutants suggest that innexins may be functional homologs to the connexins. However, whether innexins are the elusive invertebrate gap junction proteins or, rather, accessory proteins that facilitate gap junction formation remains an open question. Given the rapid progress being made in the cloning and functional analysis of gap junctions in many diverse species, confusion and difficulties with nomenclature are coming to a head in this rapidly expanding field. It may be timely to form a Nomenclature Committee to establish a uniform classification scheme for naming gap junction proteins.     

Gap junctions in the ovary of <Emphasis Type="Italic">Drosophila melanogaster</Emphasis>: localization of innexins 1, 2, 3 and 4 and evidence for intercellular communication via innexin-2 containing channels

Background  

In the Drosophila ovary, germ-line and soma cells are interconnected via gap junctions. The main gap-junction proteins in invertebrates are members of the innexin family. In order to reveal the role that innexins play in cell-cell communication during oogenesis, we investigated the localization of innexins 1, 2, 3 and 4 using immunohistochemistry, and analyzed follicle development following channel blockade.     

Innexins: members of an evolutionarily conserved family of gap-junction proteins

Gap junctions are clusters of intercellular channels that provide cells, in all metazoan organisms, with a means of communicating directly with their neighbours. Surprisingly, two gene families have evolved to fulfil this fundamental, and highly conserved, function. In vertebrates, gap junctions are assembled from a large family of connexin proteins. Innexins were originally characterized as the structural components of gap junctions in Drosophila, an arthropod, and the nematode Caenorhabditis elegans. Since then, innexin homologues have been identified in representatives of the other major invertebrate phyla and in insect-associated viruses. Intriguingly, functional innexin homologues have also been found in vertebrate genomes. These studies have informed our understanding of the molecular evolution of gap junctions and have greatly expanded the numbers of model systems available for functional studies. Genetic manipulation of innexin function in relatively simple cellular systems should speed progress not only in defining the importance of gap junctions in a variety of biological processes but also in elucidating the mechanisms by which they act.     

Six Innexins Contribute to Electrical Coupling of C. elegans Body-Wall Muscle

C. elegans body-wall muscle cells are electrically coupled through gap junctions. Previous studies suggest that UNC-9 is an important, but not the only, innexin mediating the electrical coupling. Here we analyzed junctional current (I _j) for mutants of additional innexins to identify the remaining innexin(s) important to the coupling. The results suggest that a total of six innexins contribute to the coupling, including UNC-9, INX-1, INX-10, INX-11, INX-16, and INX-18. The I _j deficiency in each mutant was rescued completely by expressing the corresponding wild-type innexin specifically in muscle, suggesting that the innexins function cell-autonomously. Comparisons of I _j between various single, double, and triple mutants suggest that the six innexins probably form two distinct populations of gap junctions with one population consisting of UNC-9 and INX-18 and the other consisting of the remaining four innexins. Consistent with their roles in muscle electrical coupling, five of the six innexins showed punctate localization at muscle intercellular junctions when expressed as GFP- or epitope-tagged proteins, and muscle expression was detected for four of them when assessed by expressing GFP under the control of innexin promoters. The results may serve as a solid foundation for further explorations of structural and functional properties of gap junctions in C. elegans body-wall muscle.     

Two gap junction channel (innexin) genes of the Bombyx mori and their expression

Gap junctions are clusters of intercellular channels that are associated with embryonic development and neural signaling. Innexins, invertebrate gap junction proteins, have been identified in Drosophila and Caenorhabditis. Here, we report the isolation and characterization of two novel members of the insect innexin family, Bm inx2 and Bm inx4, from embryos of the silkworm, Bombyx mori, during the germ-band formation stage. Bm inx2 is a single copy gene with one exon, while Bm inx4 is a single copy gene with four exons and three introns. The predicted proteins show structural similarities with other innexin family members, including four transmembrane (TM) domains, two extracellular loops (ELs), one cytoplasmic loop (CL), and typical conserved amino acids. Bm inx2 is phylogenetically orthologous to the other insect inx2 genes, but Bm inx4 is not orthologous to any known innexin including Dm inx4. Interestingly, Northern blotting and in situ hybridization showed that Bm inx2 was variously expressed across all developmental stages and in various tissues, with high expression seen in the nervous system at the time of embryogenesis. In contrast, Bm inx4 was transiently expressed at the germ-band formation stage of embryogenesis, and was specifically expressed in the ovary and testis during the larval and pupal stages. The isolation and characterization of these novel genes should form the basis for further study of the functional events that occur during development and neuronal communication in B. mori.     

Two Classes of Gap Junction Channels Mediate Soma-Germline Interactions Essential for Germline Proliferation and Gametogenesis in Caenorhabditis elegans

In all animals examined, somatic cells of the gonad control multiple biological processes essential for germline development. Gap junction channels, composed of connexins in vertebrates and innexins in invertebrates, permit direct intercellular communication between cells and frequently form between somatic gonadal cells and germ cells. Gap junctions comprise hexameric hemichannels in apposing cells that dock to form channels for the exchange of small molecules. Here we report essential roles for two classes of gap junction channels, composed of five innexin proteins, in supporting the proliferation of germline stem cells and gametogenesis in the nematode Caenorhabditis elegans. Transmission electron microscopy of freeze-fracture replicas and fluorescence microscopy show that gap junctions between somatic cells and germ cells are more extensive than previously appreciated and are found throughout the gonad. One class of gap junctions, composed of INX-8 and INX-9 in the soma and INX-14 and INX-21 in the germ line, is required for the proliferation and differentiation of germline stem cells. Genetic epistasis experiments establish a role for these gap junction channels in germline proliferation independent of the glp-1/Notch pathway. A second class of gap junctions, composed of somatic INX-8 and INX-9 and germline INX-14 and INX-22, is required for the negative regulation of oocyte meiotic maturation. Rescue of gap junction channel formation in the stem cell niche rescues germline proliferation and uncovers a later channel requirement for embryonic viability. This analysis reveals gap junctions as a central organizing feature of many soma–germline interactions in C. elegans.