A hyperpolarizing neuron recruits undocked innexin hemichannels to transmit neural information in Caenorhabditis elegans.

While depolarization of the neuronal membrane is known to evoke the neurotransmitter release from synaptic vesicles, hyperpolarization is regarded as a resting state of chemical neurotransmission. Here, we report that hyperpolarizing neurons can actively signal neural information by employing undocked hemichannels. We show that UNC-7, a member of the innexin family in Caenorhabditis elegans, functions as a hemichannel in thermosensory neurons and transmits temperature information from the thermosensory neurons to their postsynaptic interneurons. By monitoring neural activities in freely behaving animals, we find that hyperpolarizing thermosensory neurons inhibit the activity of the interneurons and that UNC-7 hemichannels regulate this process. UNC-7 is required to control thermotaxis behavior and functions independently of synaptic vesicle exocytosis. Our findings suggest that innexin hemichannels mediate neurotransmission from hyperpolarizing neurons in a manner that is distinct from the synaptic transmission, expanding the way of neural circuitry operations.

Gap Junctions in Ctenophora.

Gap junction proteins form specialized intercellular communication channels, including electrical synapses, that regulate cellular metabolism and signaling. We present a molecular inventory of the gap junction proteins-innexins (INX-like) in ctenophores, focusing on two reference species, Pleurobrachia bachei and Mnemiopsis leidyi. Innexins were identified in more than 15 ctenophore species, including such genera as Euplokamis, Pukia, Hormiphora, Bolinopsis, Cestum, Ocyropsis, Dryodora, Beroe, benthic ctenophores, Coeloplana and Vallicula, and undescribed species of Mertensiidae. The observed diversity of innexins resulted from the independent expansion of this family from the common ancestor of ctenophores. Innexins show the conserved topology with four transmembrane domains connected by two extracellular loops, which bridge intracellular gaps. However, INX-like genes have highly diverse exon organization and low percentage identity for their amino acid sequences within the same species and between ctenophore species. Such a broad scope of molecular diversity differs from innexins in other phyla. We predicted posttranslational modifications in innexins: 249 and 188 for M. leidyi and P. bachei, respectively. Neither their number nor their locations were conserved within or between species. When the number of posttranslational modifications is factored into the innexins' radiation, the potential for molecular and physiological diversity within gap junctions of ctenophores is almost unfathomable. RNA-seq and in situ hybridization data revealed that innexins are expressed across embryogenesis, including early cleavage stages and gastrulation. They are abundant in all adult tissues, with the highest expression level in the aboral organ (the major integrative center and the gravity sensor in ctenophores), followed by tentacles and comb plates. Nevertheless, each organ and tissue has a unique combination of innexins, suggesting their involvement in complex integrative functions and behaviors of ctenophores.

A new look at the architecture and dynamics of the Hydra nerve net.

The Hydra nervous system is the paradigm of a 'simple nerve net'. Nerve cells in Hydra, as in many cnidarian polyps, are organized in a nerve net extending throughout the body column. This nerve net is required for control of spontaneous behavior: elimination of nerve cells leads to polyps that do not move and are incapable of capturing and ingesting prey (Campbell, 1976). We have re-examined the structure of the Hydra nerve net by immunostaining fixed polyps with a novel antibody that stains all nerve cells in Hydra. Confocal imaging shows that there are two distinct nerve nets, one in the ectoderm and one in the endoderm, with the unexpected absence of nerve cells in the endoderm of the tentacles. The nerve nets in the ectoderm and endoderm do not contact each other. High-resolution TEM (transmission electron microscopy) and serial block face SEM (scanning electron microscopy) show that the nerve nets consist of bundles of parallel overlapping neurites. Results from transgenic lines show that neurite bundles include different neural circuits and hence that neurites in bundles require circuit-specific recognition. Nerve cell-specific innexins indicate that gap junctions can provide this specificity. The occurrence of bundles of neurites supports a model for continuous growth and differentiation of the nerve net by lateral addition of new nerve cells to the existing net. This model was confirmed by tracking newly differentiated nerve cells.

Morphological Observation and Transcriptome Analysis of Ciliogenesis in Urechis unicinctus (Annelida, Echiura).

During the early development of marine invertebrates, planktic larvae usually occur, and their body surfaces often form specific types of cilia that are involved in locomotion and feeding. The echiuran worm Urechis unicinctus sequentially undergoes the formation and disappearance of different types of body surface cilia during embryonic and larval development. The morphological characteristics and molecular mechanisms involved in the process remain unclear. In this study, we found that body surface cilia in U. unicinctus embryos and larvae can be distinguished into four types: body surface short cilia, apical tufts, circumoral cilia and telotrochs. Further, distribution and genesis of the body surface cilia were characterized using light microscope and electron microscope. To better understand the molecular mechanism during ciliogenesis, we revealed the embryonic and larval transcriptome profile of the key stages of ciliogenesis in U. unicinctus using RNA-Seq technology. A total of 29,158 differentially expressed genes (DEGs) were obtained from 24 cDNA libraries by RNA-Seq. KEGG pathway enrichment results showed that Notch, Wnt and Ca2+ signaling pathways were significantly enriched during the occurrence of apical tufts and circumoral cilia. Furthermore, all DEGs were classified according to their expression pattern, and DEGs with similar expression pattern were grouped into a module. All DEG co-expression modules were correlated with traits (body surface short cilia, apical tufts, circumoral cilia and telotrochs) by WGCNA, the results showed DEGs were divided into 13 modules by gene expression patterns and that the genes in No. 7, No. 8 and No. 10 modules were to be highly correlated with the occurrence of apical tufts, circumoral cilia and telotrochs. The top 10 hub genes in the above three modules were identified to be highly correlated with ciliogenesis, including the reported cilium-related gene Cnbd2 and unreported cilium-related candidate genes FAM181B, Capsl, Chst3, TMIE and Innexin. Notably, Innexin was included in the top10 hub genes of the two modules (No. 7 and No. 8), suggesting that Innexin may play an important role in U. unicinctus apical tufts, circumoral cilia and telotrochs genesis. This study revealed the characteristics of ciliogenesis on the body surface of U. unicinctus embryos and larvae, providing basic data for exploring the molecular mechanism of ciliogenesis on the body surface.

Independent Innexin Radiation Shaped Signaling in Ctenophores.

Innexins facilitate cell-cell communication by forming gap junctions or nonjunctional hemichannels, which play important roles in metabolic, chemical, ionic, and electrical coupling. The lack of knowledge regarding the evolution and role of these channels in ctenophores (comb jellies), the likely sister group to the rest of animals, represents a substantial gap in our understanding of the evolution of intercellular communication in animals. Here, we identify and phylogenetically characterize the complete set of innexins of four ctenophores: Mnemiopsis leidyi, Hormiphora californensis, Pleurobrachia bachei, and Beroe ovata. Our phylogenetic analyses suggest that ctenophore innexins diversified independently from those of other animals and were established early in the emergence of ctenophores. We identified a four-innexin genomic cluster, which was present in the last common ancestor of these four species and has been largely maintained in these lineages. Evidence from correlated spatial and temporal gene expression of the M. leidyi innexin cluster suggests that this cluster has been maintained due to constraints related to gene regulation. We describe the basic electrophysiological properties of putative ctenophore hemichannels from muscle cells using intracellular recording techniques, showing substantial overlap with the properties of bilaterian innexin channels. Together, our results suggest that the last common ancestor of animals had gap junctional channels also capable of forming functional innexin hemichannels, and that innexin genes have independently evolved in major lineages throughout Metazoa.

The Caenorhabditis elegans innexin INX-20 regulates nociceptive behavioral sensitivity.

Organisms rely on chemical cues in their environment to indicate the presence or absence of food, reproductive partners, predators, or other harmful stimuli. In the nematode Caenorhabditis elegans, the bilaterally symmetric pair of ASH sensory neurons serves as the primary nociceptors. ASH activation by aversive stimuli leads to backward locomotion and stimulus avoidance. We previously reported a role for guanylyl cyclases in dampening nociceptive sensitivity that requires an innexin-based gap junction network to pass cGMP between neurons. Here, we report that animals lacking function of the gap junction component INX-20 are hypersensitive in their behavioral response to both soluble and volatile chemical stimuli that signal through G protein-coupled receptor pathways in ASH. We find that expressing inx-20 in the ADL and AFD sensory neurons is sufficient to dampen ASH sensitivity, which is supported by new expression analysis of endogenous INX-20 tagged with mCherry via the CRISPR-Cas9 system. Although ADL does not form gap junctions directly with ASH, it does so via gap junctions with the interneuron RMG and the sensory neuron ASK. Ablating either ADL or RMG and ASK also resulted in nociceptive hypersensitivity, suggesting an important role for RMG/ASK downstream of ADL in the ASH modulatory circuit. This work adds to our growing understanding of the repertoire of ways by which ASH activity is regulated via its connectivity to other neurons and identifies a previously unknown role for ADL and RMG in the modulation of aversive behavior.

Innexin-Mediated Adhesion between Glia Is Required for Axon Ensheathment in the Peripheral Nervous System.

Glia are essential to protecting and enabling nervous system function and a key glial function is the formation of the glial sheath around peripheral axons. Each peripheral nerve in the Drosophila larva is ensheathed by three glial layers, which structurally support and insulate the peripheral axons. How peripheral glia communicate with each other and between layers is not well established and we investigated the role of Innexins in mediating glial function in the Drosophila periphery. Of the eight Drosophila Innexins, we found two (Inx1 and Inx2) are important for peripheral glia development. In particular loss of Inx1 and Inx2 resulted in defects in the wrapping glia leading to disruption of the glia wrap. Of interest loss of Inx2 in the subperineurial glia also resulted in defects in the neighboring wrapping glia. Inx plaques were observed between the subperineurial glia and the wrapping glia suggesting that gap junctions link these two glial cell types. We found Inx2 is key to Ca2+ pulses in the peripheral subperineurial glia but not in the wrapping glia, and we found no evidence of gap junction communication between subperineurial and wrapping glia. Rather we have clear evidence that Inx2 plays an adhesive and channel-independent role between the subperineurial and wrapping glia to ensure the integrity of the glial wrap.SIGNIFICANCE STATEMENT Gap junctions are critical for glia communication and formation of myelin in myelinating glia. However, the role of gap junctions in non-myelinating glia is not well studied, yet non-myelinating glia are critical for peripheral nerve function. We found the Innexin gap junction proteins are present between different classes of peripheral glia in Drosophila. Here Innexins form junctions to facilitate adhesion between the different glia but do so in a channel-independent manner. Loss of adhesion leads to disruption of the glial wrap around axons and leads to fragmentation of the wrapping glia membranes. Our work points to an important role for gap junction proteins in mediating insulation by non-myelinating glia.

A new player in circadian networks: Role of electrical synapses in regulating functions of the circadian clock.

Several studies have indicated that coherent circadian rhythms in behaviour can be manifested only when the underlying circadian oscillators function as a well-coupled network. The current literature suggests that circadian pacemaker neuronal networks rely heavily on communication mediated by chemical synapses comprising neuropeptides and neurotransmitters to regulate several behaviours and physiological processes. It has become increasingly clear that chemical synapses closely interact with electrical synapses and function together in the neuronal networks of most organisms. However, there are only a few studies which have examined the role of electrical synapses in circadian networks and here, we review our current understanding of gap junction proteins in circadian networks of various model systems. We describe the general mechanisms by which electrical synapses function in neural networks, their interactions with chemical neuromodulators and their contributions to the regulation of circadian rhythms. We also discuss the various methods available to characterize functional electrical synapses in these networks and the potential directions that remain to be explored to understand the roles of this relatively understudied mechanism of communication in modulating circadian behaviour.

Innexin function dictates the spatial relationship between distal somatic cells in the Caenorhabditis elegans gonad without impacting the germline stem cell pool.

Gap-junctional signaling mediates myriad cellular interactions in metazoans. Yet, how gap junctions control the positioning of cells in organs is not well understood. Innexins compose gap junctions in invertebrates and affect organ architecture. Here, we investigate the roles of gap-junctions in controlling distal somatic gonad architecture and its relationship to underlying germline stem cells in Caenorhabditis elegans. We show that a reduction of soma-germline gap-junctional activity causes displacement of distal sheath cells (Sh1) towards the distal end of the gonad. We confirm, by live imaging, transmission electron microscopy, and antibody staining, that bare regions-lacking somatic gonadal cell coverage of germ cells-are present between the distal tip cell (DTC) and Sh1, and we show that an innexin fusion protein used in a prior study encodes an antimorphic gap junction subunit that mispositions Sh1. We determine that, contrary to the model put forth in the prior study based on this fusion protein, Sh1 mispositioning does not markedly alter the position of the borders of the stem cell pool nor of the progenitor cell pool. Together, these results demonstrate that gap junctions can control the position of Sh1, but that Sh1 position is neither relevant for GLP-1/Notch signaling nor for the exit of germ cells from the stem cell pool.

Connexins evolved after early chordates lost innexin diversity.

Gap junction channels are formed by two unrelated protein families. Non-chordates use the primordial innexins, while chordates use connexins that superseded the gap junction function of innexins. Chordates retained innexin-homologs, but N-glycosylation prevents them from forming gap junctions. It is puzzling why chordates seem to exclusively use the new gap junction protein and why no chordates should exist that use non-glycosylated innexins to form gap junctions. Here, we identified glycosylation sites of 2388 innexins from 174 non-chordate and 276 chordate species. Among all chordates, we found not a single innexin without glycosylation sites. Surprisingly, the glycosylation motif is also widespread among non-chordate innexins indicating that glycosylated innexins are not a novelty of chordates. In addition, we discovered a loss of innexin diversity during early chordate evolution. Most importantly, lancelets, which lack connexins, exclusively possess only one highly conserved innexin with one glycosylation site. A bottleneck effect might thus explain why connexins have become the only protein used to form chordate gap junctions.

Contribution of large-pore channels to inflammation induced by microorganisms.

Plasma membrane ionic channels selectively permeate potassium, sodium, calcium, and chloride ions. However, large-pore channels are permeable to ions and small molecules such as ATP and glutamate, among others. Large-pore channels are structures formed by several protein families with little or no evolutionary linkages including connexins (Cxs), pannexins (Panxs), innexin (Inxs), unnexins (Unxs), calcium homeostasis modulator (CALHMs), and Leucine-rich repeat-containing 8 (LRRC8) proteins. Large-pore channels are key players in inflammatory cell response, guiding the activation of inflammasomes, the release of pro-inflammatory cytokines such as interleukin-1 beta (IL-1ß), and the release of adenosine-5'-triphosphate (ATP), which is considered a danger signal. This review summarizes our current understanding of large-pore channels and their contribution to inflammation induced by microorganisms, virulence factors or their toxins.

Molecular and morphological approach to study the innexin gap junctions in Rhynchosciara americana.

Gap junctions mediate communication between adjacent cells and are fundamental to the development and homeostasis in multicellular organisms. In invertebrates, gap junctions are formed by transmembrane proteins called innexins. Gap junctions allow the passage of small molecules through an intercellular channel, between a cell and another adjacent cell. The dipteran Rhynchosciara americana has contributed to studying the biology of invertebrates and the study of the interaction and regulation of genes during biological development. Therefore, this paper aimed to study the R. americana innexin-2 by molecular characterization, analysis of the expression profile and cellular localization. The molecular characterization results confirm that the message is from a gap junction protein and analysis of the expression and cellular localization profile shows that innexin-2 can participate in many physiological processes during the development of R. americana.

ßPS-Integrin acts downstream of Innexin 2 in modulating stretched cell morphogenesis in the Drosophila ovary.

During oogenesis, a group of specialized follicle cells, known as stretched cells (StCs), flatten drastically from cuboidal to squamous shape. While morphogenesis of epithelia is critical for organogenesis, genes and signaling pathways involved in this process remain to be revealed. In addition to formation of gap junctions for intercellular exchange of small molecules, gap junction proteins form channels or act as adaptor proteins to regulate various cellular behaviors. In invertebrates, gap junction proteins are Innexins. Knockdown of Innexin 2 but not other Innexins expressed in follicle cells attenuates StC morphogenesis. Interestingly, blocking of gap junctions with an inhibitor carbenoxolone does not affect StC morphogenesis, suggesting that Innexin 2 might control StCs flattening in a gap-junction-independent manner. An excessive level of ßPS-Integrin encoded by myospheroid is detected in Innexin 2 mutant cells specifically during StC morphogenesis. Simultaneous knockdown of Innexin 2 and myospheroid partially rescues the morphogenetic defect resulted from Innexin 2 knockdown. Furthermore, reduction of ßPS-Integrin is sufficient to induce early StCs flattening. Taken together, our data suggest that ßPS-Integrin acts downstream of Innexin 2 in modulating StCs morphogenesis.

Polydnavirus Innexins Disrupt Host Cellular Encapsulation and Larval Maturation.

Polydnaviruses are dsDNA viruses associated with endoparasitoid wasps. Delivery of the virus during parasitization of a caterpillar and subsequent virus gene expression is required for production of an amenable environment for parasitoid offspring development. Consequently, understanding of Polydnavirus gene function provides insight into mechanisms of host susceptibility and parasitoid wasp host range. Polydnavirus genes predominantly are arranged in multimember gene families, one of which is the vinnexins, which are virus homologues of insect gap junction genes, the innexins. Previous studies of Campoletis sonorensis Ichnovirus Vinnexins using various heterologous systems have suggested the four encoded members may provide different functionality in the infected caterpillar host. Here, we expressed two of the members, vnxG and vnxQ2, using recombinant baculoviruses in susceptible host, the caterpillar Heliothis virescens. Following intrahemocoelic injections, we observed that >90% of hemocytes (blood cells) were infected, producing recombinant protein. Larvae infected with a vinnexin-recombinant baculovirus exhibited significantly reduced molting rates relative to larvae infected with a control recombinant baculovirus and mock-infected larvae. Similarly, larvae infected with vinnexin-recombinant baculoviruses were less likely to survive relative to controls and showed reduced ability to encapsulate chromatography beads in an immune assay. In most assays, the VnxG protein was associated with more severe pathology than VnxQ2. Our findings support a role for Vinnexins in CsIV and more broadly Ichnovirus pathology in infected lepidopteran hosts, particularly in disrupting multicellular developmental and immune physiology.

Presynaptic coupling by electrical synapses coordinates a rhythmic behavior by synchronizing the activities of a neuron pair.

Electrical synapses are specialized structures that mediate the flow of electrical currents between neurons and have well known roles in synchronizing the activities of neuronal populations, both by mediating the current transfer from more active to less active neurons and by shunting currents from active neurons to their less active neighbors. However, how these positive and negative functions of electrical synapses are coordinated to shape rhythmic synaptic outputs and behavior is not well understood. Here, using a combination of genetics, behavioral analysis, and live calcium imaging in Caenorhabditis elegans, we show that electrical synapses formed by the gap junction protein INX-1/innexin couple the presynaptic terminals of a pair of motor neurons (AVL and DVB) to synchronize their activation in response to a pacemaker signal. Live calcium imaging reveals that inx-1/innexin mutations lead to asynchronous activation of AVL and DVB, due, in part, to loss of AVL-mediated activation of DVB by the pacemaker. In addition, loss of inx-1 leads to the ectopic activation of DVB at inappropriate times during the cycle through the activation of the L-type voltage-gated calcium channel EGL-19. We propose that electrical synapses between AVL and DVB presynaptic terminals function to ensure the precise and robust execution of a specific step in a rhythmic behavior by both synchronizing the activities of presynaptic terminals in response to pacemaker signaling and by inhibiting their activation in between cycles when pacemaker signaling is low.

On the molecular nature of large-pore channels.

Membrane transport is a fundamental means to control basic cellular processes such as apoptosis, inflammation, and neurodegeneration and is mediated by a number of transporters, pumps, and channels. Accumulating evidence over the last half century has shown that a type of so-called "large-pore channel" exists in various tissues and organs in gap-junctional and non-gap-junctional forms in order to flow not only ions but also metabolites such as ATP. They are formed by a number of protein families with little or no evolutionary linkages including connexin, innexin, pannexin, leucine-rich repeat-containing 8 (LRRC8), and calcium homeostasis modulator (CALHM). This review summarizes the history and concept of large-pore channels starting from connexin gap junction channels to the more recent developments in innexin, pannexin, LRRC8, and CALHM. We describe structural and functional features of large-pore channels that are crucial for their diverse functions on the basis of available structures.

Analysis of Hemichannels and Gap Junctions: Application and Extension of the Passive Transmembrane Ion Transport Model.

Electrical synaptic transmission is an essential form of interneuronal communication which is mediated by gap junctions that permit ion flow. Three gene families (connexins, innexins, and pannexins) have evolved to form gap junctional channels. Each gap junctional channel is formed by the docking of the hemichannel of one cell with the corresponding hemichannel of an adjacent cell. To date, there has been a lack of study models to describe this structure in detail. In this study, we demonstrate that numerical simulations suggest that the passive transmembrane ion transport model, based on the generality of ion channels, also applies to hemichannels in non-junctional plasma membranes. On this basis, we established a gap junctional channel model, which describes hemichannels' docking. We simulated homotypic and heterotypic gap junctions formed by connexins, innexins, and pannexins. Based on the numerical results and our theoretical model, we discussed the physiology of hemichannels and gap junctions, including ion blockage of hemichannels, voltage gating of gap junctions, and asymmetry and delay of electrical synaptic transmission, for which the numerical simulations are first comprehensively realized.

Integration of Migratory Cells into a New Site In Vivo Requires Channel-Independent Functions of Innexins on Microtubules.

During embryonic development and cancer metastasis, migratory cells must establish stable connections with new partners at their destinations. Here, we establish the Drosophila border cells as a model for this multistep process. During oogenesis, border cells delaminate from the follicular epithelium and migrate. When they reach their target, the oocyte, they undergo a stereotypical series of steps to adhere to it, then connect with another migrating epithelium. We identify gap-junction-forming innexin proteins as critical. Surprisingly, the channel function is dispensable. Instead, Innexins 2 and 3 function within the border cells, and Innexin 4 functions within the germline, to regulate microtubules. The microtubule-dependent border cell-oocyte interaction is essential to brace the cells against external morphogenetic forces. Thus, we establish an experimental model and use genetic, thermogenetic, and live-imaging approaches to uncover the contributions of Innexins and microtubules to a cell-biological process important in development and cancer.

Intracellular dynamics of polydnavirus innexin homologues.

Polydnaviruses associated with ichneumonid parasitoid wasps (Ichnoviruses) encode large numbers of genes, often in multigene families. The Ichnovirus Vinnexin gene family, which is expressed in parasitized lepidopteran larvae, encodes homologues of Innexins, the structural components of insect gap junctions. Here, we have examined intracellular behaviours of the Campoletis sonorensis Ichnovirus (CsIV) Vinnexins, alone and in combination with a host Innexin orthologue, Innexin2 (Inx2). QRT-PCR verified that transcription of CsIV vinnexins occurs contemporaneously with inx2, implying co-occurrence of Vinnexin and Inx2 proteins. Confocal microscopy demonstrated that epitope-tagged VinnexinG (VnxG) and VinnexinQ2 (VnxQ2) exhibit similar subcellular localization as Spodoptera frugiperda Inx2 (Sf-Inx2). Surface biotinylation assays verified that all three proteins localize to the cell surface, and cytochalasin B and nocodazole that they rely on actin and microtubule cytoskeletal networks for localization. Immunomicroscopy following co-transfection of constructs indicates extensive co-localization of Vinnexins with each other and Sf-Inx2, and live-cell imaging of mCherry-labelled Inx2 supports that Vinnexins may affect Sf-Inx2 distribution in a Vinnexin-specific fashion. Our findings support that the Vinnexins may disrupt host cell physiology in a protein-specific manner through altering gap junctional intercellular channel communication, as well as indirectly by affecting multicellular junction characteristics.

Analysing bioelectrical phenomena in the Drosophila ovary with genetic tools: tissue-specific expression of sensors for membrane potential and intracellular pH, and RNAi-knockdown of mechanisms involved in ion exchange.

BACKGROUND: Changes in transcellular bioelectrical patterns are known to play important roles during developmental and regenerative processes. The Drosophila follicular epithelium has proven to be an appropriate model system for studying the mechanisms by which bioelectrical signals emerge and act. Fluorescent indicator dyes in combination with various inhibitors of ion-transport mechanisms have been used to investigate the generation of membrane potentials (Vmem) and intracellular pH (pHi). Both parameters as well as their anteroposterior and dorsoventral gradients were affected by the inhibitors which, in addition, led to alterations of microfilament and microtubule patterns equivalent to those observed during follicle-cell differentiation. RESULTS: We expressed two genetically-encoded fluorescent sensors for Vmem and pHi, ArcLight and pHluorin-Moesin, in the follicular epithelium of Drosophila. By means of the respective inhibitors, we obtained comparable effects on Vmem and/or pHi as previously described for Vmem- and pHi-sensitive fluorescent dyes. In a RNAi-knockdown screen, five genes of ion-transport mechanisms and gap-junction subunits were identified exerting influence on ovary development and/or oogenesis. Loss of ovaries or small ovaries were the results of soma knockdowns of the innexins inx1 and inx3, and of the DEG/ENaC family member ripped pocket (rpk). Germline knockdown of rpk also resulted in smaller ovaries. Soma knockdown of the V-ATPase-subunit vha55 caused size-reduced ovaries with degenerating follicles from stage 10A onward. In addition, soma knockdown of the open rectifier K+channel 1 (ork1) resulted in a characteristic round-egg phenotype with altered microfilament and microtubule organisation in the follicular epithelium. CONCLUSIONS: The genetic tool box of Drosophila provides means for a refined and extended analysis of bioelectrical phenomena. Tissue-specifically expressed Vmem- and pHi-sensors exhibit some practical advantages compared to fluorescent indicator dyes. Their use confirms that the ion-transport mechanisms targeted by inhibitors play important roles in the generation of bioelectrical signals. Moreover, modulation of bioelectrical signals via RNAi-knockdown of genes coding for ion-transport mechanisms and gap-junction subunits exerts influence on crucial processes during ovary development and results in cytoskeletal changes and altered follicle shape. Thus, further evidence amounts for bioelectrical regulation of developmental processes via the control of both signalling pathways and cytoskeletal organisation.