Heteromerization of innexin gap junction proteins regulates epithelial tissue organization in Drosophila

Gap junctions consist of clusters of intercellular channels, which enable direct cell-to-cell communication and adhesion in animals. Whereas deuterostomes, including all vertebrates, use members of the connexin and pannexin multiprotein families to assemble gap junction channels, protostomes such as Drosophila and Caenorhabditis elegans use members of the innexin protein family. The molecular composition of innexin-containing gap junctions and the functional significance of innexin oligomerization for development are largely unknown. Here, we report that heteromerization of Drosophila innexins 2 and 3 is crucial for epithelial organization and polarity of the embryonic epidermis. Both innexins colocalize in epithelial cell membranes. Innexin3 is mislocalized to the cytoplasm in innexin2 mutants and is recruited into ectopic expression domains defined by innexin2 misexpression. Conversely, RNA interference (RNAi) knockdown of innexin3 causes mislocalization of innexin2 and of DE-cadherin, causing cell polarity defects in the epidermis. Biochemical interaction studies, surface plasmon resonance analysis, transgenesis, and biochemical fractionation experiments demonstrate that both innexins interact via their C-terminal cytoplasmic domains during the assembly of heteromeric channels. Our data provide the first molecular and functional demonstration that innexin heteromerization occurs in vivo and reveal insight into a molecular mechanism by which innexins may oligomerize into heteromeric gap junction channels.     

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.     

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.     

Expression of a dominant negative mutant innexin in identified neurons and glial cells reveals selective interactions among gap junctional proteins

Neurons and glia of the medicinal leech CNS express different subsets of the 21 innexin genes encoded in its genome. We report here that the punctal distributions of fluorescently tagged innexin transgenes varies in a stereotypical pattern depending on the innexin expressed. Furthermore, whereas certain innexins colocalize extensively (INX1 and INX14), others do not (e.g., INX1 and INX2 or INX6). We then demonstrate that the mutation of a highly conserved proline residue in the second transmembrane domain of innexins creates a gap junction protein with dominant negative properties. Coexpressing the mutated INX1 gene with its wild type blocks the formation of fluorescent puncta and decouples the expressing neuron from its normal gap junction‐coupled network of cells. Similarly, expression of an INX2 mutant transgene (a glial cell innexin), blocks endogenous INX2 puncta and wild‐type transgene puncta, and decouples the glial cell from the other glial cells in the ganglion. We show in cell culture with dye‐uptake and plasma membrane labeling experiments that the mutant innexin transgene is not expressed on the cell membrane but instead appears to accumulate in the cell's perinuclear region. Lastly, we use these mutant innexin transgenes to show that the INX1 mutant transgene blocks not only INX1 puncta formation, but also puncta of INX14, with which INX1 usually colocalizes. By contrast, the formation of INX6 puncta was unaffected by the INX1 mutant. Together, these experiments suggest that leech innexins can selectively interact with one another to form gap junction plaques, which are heterogeneously located in cellular arbors. © 2013 Wiley Periodicals, Inc. Develop Neurobiol 73: 571–586, 2013     

Specificity of cellular expression of C. variopedatus polychaete innexin in the developing embryo: evolutionary aspects of innexins' heterogeneous gene structures

Innexins are a family of membrane proteins involved in the formation of gap junctions in invertebrates. They have been found to participate in several aspects of cell differentiation and in embryonic patterning through the formation of specific intercellular communication channels. We present here data showing that the recently identified innexin of the marine worm Chaetopterus variopedatus is expressed only in particular cells of the early stage, demonstrating cell specificity of innexin expression also in polychaete annelids. Phylogenetic analysis of all known innexins results in a phylogenetic tree clearly distinguishing insect, nematode, and other invertebrate innexins. Comparative analysis of proteins and known related genes shows that the apparent similarity of protein composition, overall structural organization, and specificity of cellular expression, typical of innexins of all studied organisms, correspond to highly heterogeneous gene structures even for genes that are in close contiguity on the same chromosome. A possible evolutionary motive producing this situation is discussed.     

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.     

Tryptophan scanning mutagenesis of the first transmembrane domain of the innexin Shaking-B(Lethal)

The channel proteins of gap junctions are encoded by two distinct gene families, connexins, which are exclusive to chordates, and innexins/pannexins, which are found throughout the animal kingdom. Although the relationship between the primary structure and function of the vertebrate connexins has been relatively well studied, there are, to our knowledge, no structure-function analyses of invertebrate innexins. In the first such study, we have used tryptophan scanning to probe the first transmembrane domain (M1) of the Drosophila innexin Shaking-B(Lethal), which is a component of rectifying electrical synapses in the Giant Fiber escape neural circuit. Tryptophan was substituted sequentially for 16 amino acids within M1 of Shaking-B(Lethal). Tryptophan insertion at every fourth residue (H27, T31, L35, and S39) disrupted gap junction function. The distribution of these sites is consistent with helical secondary structure and identifies the face of M1 involved in helix-helix interactions. Tryptophan substitution at several sites in M1 altered channel properties in a variety of ways. Changes in sensitivity to transjunctional voltage (Vj) were common and one mutation (S39W) induced sensitivity to transmembrane voltage (Vm). In addition, several mutations induced hemichannel activity. These changes are similar to those observed after substitutions within the transmembrane domains of connexins.     

Characterization of innexin gene expression and functional roles of gap-junctional communication in planarian regeneration

Planaria possess remarkable powers of regeneration. After bisection, one blastema regenerates a head, while the other forms a tail. The ability of previously-adjacent cells to adopt radically different fates could be due to long-range signaling allowing determination of position relative to, and the identity of, remaining tissue. However, this process is not understood at the molecular level. Following the hypothesis that gap-junctional communication (GJC) may underlie this signaling, we cloned and characterized the expression of the Innexin gene family during planarian regeneration. Planarian innexins fall into 3 groups according to both sequence and expression. The concordance between expression-based and phylogenetic grouping suggests diversification of 3 ancestral innexin genes into the large family of planarian innexins. Innexin expression was detected throughout the animal, as well as specifically in regeneration blastemas, consistent with a role in long-range signaling relevant to specification of blastema positional identity. Exposure to a GJC-blocking reagent which does not distinguish among gap junctions composed of different Innexin proteins (is not subject to compensation or redundancy) often resulted in bipolar (2-headed) animals. Taken together, the expression data and the respecification of the posterior blastema to an anteriorized fate by GJC loss-of-function suggest that innexin-based GJC mediates instructive signaling during regeneration.     

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.     

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.     

Regulation of intermuscular electrical coupling by the Caenorhabditis elegans innexin inx-6

The innexins represent a highly conserved protein family, the members of which make up the structural components of gap junctions in invertebrates. We have isolated and characterized a Caenorhabditis elegans gene inx-6 that encodes a new member of the innexin family required for the electrical coupling of pharyngeal muscles. inx-6(rr5) mutants complete embryogenesis without detectable abnormalities at restrictive temperature but fail to initiate postembryonic development after hatching. inx-6 is expressed in the pharynx at all larval stages, and an INX-6::GFP fusion protein showed a punctate expression pattern characteristic of gap junction proteins localized to plasma membrane plaques. Video recording and electropharyngeograms revealed that in inx-6(rr5) mutants the anterior pharyngeal (procorpus) muscles were electrically coupled to a lesser degree than the posterior metacorpus muscles, which caused a premature relaxation in the anterior pharynx and interfered with feeding. Dye-coupling experiments indicate that the gap junctions that link the procorpus to the metacorpus are functionally compromised in inx-6(rr5) mutants. We also show that another C. elegans innexin, EAT-5, can partially substitute for INX-6 function in vivo, underscoring their likely analogous function.     

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.     

Molecular Characterization,Localization, and Distribution of Innexins in the Silkworm, <Emphasis Type="Italic">Bombyx mori</Emphasis>

Gap junctions that allow for a direct exchange of second messenger and ions are the most conserved cellular structures in multicellular organisms. We have isolated and characterized a Bombyx mori gene innexin3 that encodes a new member of the innexin family required for the early embryonic development. The BmINX3 mRNA was 1,814 nucleotide residues in length, and the deduced amino acid sequence of BmInx3 shared 74% similarity with Apis melifera innexin3. The expression profile of the BmINX3 mRNA is similar to that of previously described BmINX2, expressed in ovary and testis after 5th instar larvae and in fat body after gut purge. However, during embryogenesis, the expression of BmINX3 mRNA is restricted to the blastokinesis stage. Microscopic observation of the BmInx2 and BmInx3 fused to fluorescent proteins showed an overlapping cytoplasmic expression, whereas the BmInx4 is accumulated in the cytoplasmic surface at which two cells have physical contact. This finding of innexins distribution in silkworm would provide an essential basis for future studies of the functions and interactions of innexins.     

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.     

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.     

The mammalian pannexin family is homologous to the invertebrate innexin gap junction proteins

We have cloned the genes PANX1, PANX2 and PANX3, encoding putative gap junction proteins homologous to invertebrate innexins, which constitute a new family of mammalian proteins called pannexins. Phylogenetic analysis revealed that pannexins are highly conserved in worms, mollusks, insects and mammals, pointing to their important function. Both innexins and pannexins are predicted to have four transmembrane regions, two extracellular loops, one intracellular loop and intracellular N and C termini. Both the human and mouse genomes contain three pannexin-encoding genes. Mammalian pannexins PANX1 and PANX3 are closely related, with PANX2 more distant. The human and mouse pannexin-1 mRNAs are ubiquitously, although disproportionately, expressed in normal tissues. Human PANX2 is a brain-specific gene; its mouse orthologue, Panx2, is also expressed in certain cell types in developing brain. In silico evaluation of Panx3 expression predicts gene expression in osteoblasts and synovial fibroblasts. The apparent conservation of pannexins between species merits further investigation.     

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.     

The Caenorhabditis elegans innexin INX-3 is localized to gap junctions and is essential for embryonic development

Innexins are the proposed structural components of gap junctions in invertebrates. Antibodies that specifically recognize the Caenorhabditis elegans innexin protein INX-3 were generated and used to examine the patterns of inx-3 gene expression and the subcellular sites of INX-3 localization. INX-3 is first detected in two-cell embryos, concentrated at the intercellular interface, and is expressed ubiquitously throughout the cellular proliferation phase of embryogenesis. During embryonic morphogenesis, INX-3 expression becomes more restricted. Postembryonically, INX-3 is expressed transiently in several cell types, while expression in the posterior pharynx persists throughout development. Through immuno-EM techniques, INX-3 was observed at gap junctions in the adult pharynx, providing supporting evidence that innexins are components of gap junctions. An inx-3 mutant was isolated through a combined genetic and immunocytochemical screen. Homozygous inx-3 mutants exhibit defects during embryonic morphogenesis. At the comma stage of early morphogenesis, variable numbers of cells are lost from the anterior of inx-3(lw68) mutants. A range of terminal defects is seen later in embryogenesis, including localized rupture of the hypodermis, failure of the midbody to elongate properly, abnormal contacts between hypodermal cells, and failure of the pharynx to attach to the anterior of the animal.     

Gap junctional proteins of animals: the innexin/pannexin superfamily

There has been some controversy as to whether vertebrate pannexins are related to invertebrate innexins. Using statistical, topological and conserved sequence motif analyses, we establish that these proteins belong to a single superfamily. We also demonstrate the occurrence of large homologues with C-terminal proline-rich domains that may have arisen by gene fusion events. Phylogenetic analyses reveal the orthologous and paralogous relationships of these homologues to each other. We show that different sets of orthologous paralogues underwent sequence divergence at markedly different rates, suggesting differential pressures through evolutionary time promoting or restricting sequence divergence. We further show that the first 2 TMS-containing halves of these homologues underwent sequence divergence more slowly than the second 2 TMS-containing halves and analyze these differences. These bioinformatic analyses should serve as useful guides for future studies of structure, function and evolutionary aspects of this important superfamily.