InteractiveFly: GeneBrief

optic ganglion reduced & Innexin 2: Biological Overview | References

Gene name - optic ganglion reduced & Innexin 2

Synonyms - Inx-1 for Ogre

Cytological map position - 6E4-6E4 and 6E4-6E4

Function - Surface transmembrane

Keywords - gap junction proteins; blood-brain barrier glia - mediate the influence of metabolic changes on stem cell behavior; response of glia nutritional signals; Inx-2 functions in oogenesis, in visual transmission in lamina glia and in proventriculus development; Innexins 2 and 3 are crucial for epithelial organization and polarity of the embryonic epidermis

Symbol - ogre & Inx2

FlyBase ID: FBgn0004646 & FBgn0027108

Genetic map position - chrX:6,868,049-6,875,731 & chrX:6,892,696-6,897,547

Classification - Innexins

Cellular location - surface transmembrane

NCBI link for Ogre/Inx-1: EntrezGene
NCBI link for Inx-2: EntrezGene
Ogre orthologs: Biolitmine
Inx2 orthologs: Biolitmine
Recent literature
Richard, M. and Hoch, M. (2015). Drosophila eye size is determined by Innexin 2-dependent Decapentaplegic signalling. Dev Biol [Epub ahead of print]. PubMed ID: 26455410
Organogenesis relies on specific genetic and molecular programmes, which orchestrate growth and cellular differentiation over developmental time. This is particularly important during Drosophila eye development in which cell-cell inductive events and long-range signalling have to be integrated to regulate proper cell proliferation, differentiation and morphogenesis. How these processes are coordinated is still not very well understood. This study identified the gap junction protein Innexin2 (Inx2) as an important regulator of eye development. Depleting inx2 during eye development reduces eye size whereas elevating inx2 levels increases eye size. Loss- and gain-of-function experiments demonstrate that inx2 is required functionally in larval eye disc cells where it localises apico-laterally. inx2 regulates disc cell proliferation as well as morphogenetic furrow movement and as a result the amount of differentiated photoreceptors. inx2 interacts genetically with the Dpp pathway, and proper activation of the Dpp pathway transducer Mad at the furrow and expression of Dpp receptors Thickveins and Punt in the anterior disc compartment were found to require inx2. It was further shown that inx2 is required for the transcriptional activation of dpp and punt in the eye disc. Our results highlight the crucial role of gap junction proteins in regulating morphogen-dependent organ size determination.
Sahu, A., Ghosh, R., Deshpande, G. and Prasad, M. (2017). A gap junction protein, Inx2, modulates calcium flux to specify border cell fate during Drosophila oogenesis. PLoS Genet 13: e1006542. PubMed ID: 28114410
Intercellular communication mediated by gap junction (GJ) proteins is indispensable during embryogenesis, tissue regeneration and wound healing. This study reports functional analysis of a gap junction protein, Innexin 2 (Inx2), in cell type specification during Drosophila oogenesis. Data reveal a novel involvement of Inx2 in the specification of Border Cells (BCs), a migratory cell type, whose identity is determined by the cell autonomous STAT activity. Inx2 influences BC fate specification by modulating STAT activity via Domeless receptor endocytosis. Furthermore, detailed experimental analysis uncovers that Inx2 also regulates a calcium flux that transmits across the follicle cells. The study proposes that Inx2 mediated calcium flux in the follicle cells stimulates endocytosis by altering Dynamin (Shibire) distribution which is in turn critical for careful calibration of STAT activation and, thus for BC specification. Together these data provide unprecedented molecular insights into how gap junction proteins can regulate cell-type specification.

Richard, M., Bauer, R., Tavosanis, G. and Hoch, M. (2017). The gap junction protein Innexin3 is required for eye disc growth in Drosophila. Dev Biol [Epub ahead of print]. PubMed ID: 28390801
The Drosophila compound eye develops from a bilayered epithelial sac composed of an upper peripodial epithelium layer and a lower disc proper, the latter giving rise to the eye itself. The gap junction protein Innexin2 (Inx2) has been shown to be crucial for early larval eye disc growth. By analysing the contribution of other Innexins to eye size control, this study identified Innexin3 (Inx3) as an important growth regulator. Depleting inx3 during larval eye development reduces eye size, while elevating inx3 levels increases eye size, thus phenocopying the inx2 loss- and gain-of-function situation. As demonstrated previously for inx2, inx3 regulates disc cell proliferation and interacts genetically with the Dpp pathway, being required for the proper activation of the Dpp pathway transducer Mad at the furrow and the expression of Dpp receptor Punt in the eye disc. At the developmental timepoint corresponding to eye disc growth, Inx3 colocalises with Inx2 in disc proper and peripodial epithelium cell membranes. In addition, Inx3 protein levels were shown to critically depend on inx2 throughout eye development, and inx3 was shown to modulates Inx2 protein levels in the larval eye disc. Rescue experiments demonstrate that Inx3 and Inx2 cooperate functionally to enable eye disc growth in Drosophila. Finally, it was demonstrated that expression of Inx3 and Inx2 is not only needed in the disc proper but also in the peripodial epithelium to regulate growth of the eye disc. These data provide a functional demonstration that putative Inx2/Inx3 heteromeric channels regulate organ size.
Wu, L., Dong, A., Dong, L., Wang, S. Q. and Li, Y. (2019). PARIS, an optogenetic method for functionally mapping gap junctions. Elife 8. PubMed ID: 30638447
Cell-cell communication via gap junctions (see Optic ganglion reduced & Innexin 2) regulates a wide range of physiological processes by enabling the direct intercellular electrical and chemical coupling. However, the in vivo distribution and function of gap junctions remain poorly understood, partly due to the lack of non-invasive tools with both cell-type specificity and high spatiotemporal resolution. This study developed PARIS (pairing actuators and receivers to optically isolate gap junctions), a new fully genetically encoded tool for measuring the cell-specific gap junctional coupling (GJC). PARIS successfully enabled monitoring of GJC in several cultured cell lines under physiologically relevant conditions and in distinct genetically defined neurons in Drosophila brain, with ~10-sec temporal resolution and sub-cellular spatial resolution. These results demonstrate that PARIS is a robust, highly sensitive tool for mapping functional gap junctions and study their regulation in both health and disease.
Schotthofer, S. K. and Bohrmann, J. (2020). 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. BMC Dev Biol 20(1): 15. PubMed ID: 32635900
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. Two genetically-encoded fluorescent sensors for V(mem) and pH(i), ArcLight and pHluorin-Moesin, were expressed in the follicular epithelium of Drosophila. 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. The genetic tool box of Drosophila provides means for a refined and extended analysis of bioelectrical phenomena. Tissue-specifically expressed V(mem)- and pH(i)-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.
Peterson, N. G., Stormo, B. M., Schoenfelder, K. P., King, J. S., Lee, R. R. and Fox, D. T. (2020). Cytoplasmic sharing through apical membrane remodeling. Elife 9. PubMed ID: 33051002
Multiple nuclei sharing a common cytoplasm are found in diverse tissues, organisms, and diseases. Yet, multinucleation remains a poorly understood biological property. Cytoplasm sharing invariably involves plasma membrane breaches. In contrast, this study discovered cytoplasm sharing without membrane breaching in highly resorptive Drosophila rectal papillae. During a six-hour developmental window, 100 individual papillar cells assemble a multinucleate cytoplasm, allowing passage of proteins of at least 62 kDa throughout papillar tissue. Papillar cytoplasm sharing does not employ canonical mechanisms such as incomplete cytokinesis or muscle fusion pore regulators. Instead, sharing requires gap junction proteins Ogre (Inx1), Inx2, and Inx3 (normally associated with transport of molecules < 1 kDa), which are positioned by membrane remodeling GTPases. This work reveals a new role for apical membrane remodeling in converting a multicellular epithelium into a giant multinucleate cytoplasm.
Ho, K. Y. L., Khadilkar, R. J., Carr, R. L. and Tanentzapf, G. (2021). A gap-junction-mediated, calcium-signaling network controls blood progenitor fate decisions in hematopoiesis. Curr Biol. PubMed ID: 34480855
Stem cell homeostasis requires coordinated fate decisions among stem cells that are often widely distributed within a tissue at varying distances from their stem cell niche. This requires a mechanism to ensure robust fate decisions within a population of stem cells. This study shows that, in the Drosophila hematopoietic organ, the lymph gland (LG), gap junctions (see Drosophila Ogre) form a network that coordinates fate decisions between blood progenitors. Using live imaging of calcium signaling in intact LGs, it was found that blood progenitors are connected through a signaling network. Blocking gap junction function disrupts this network, alters the pattern of encoded calcium signals, and leads to loss of progenitors and precocious blood cell differentiation. Ectopic and uniform activation of the calcium-signaling mediator CaMKII restores progenitor homeostasis when gap junctions are disrupted. Overall, these data show that gap junctions equilibrate cell signals between blood progenitors to coordinate fate decisions and maintain hematopoietic homeostasis.
Das, M., Cheng, D., Matzat, T. and Auld, V. J. (2023). Innexin-Mediated Adhesion between Glia Is Required for Axon Ensheathment in the Peripheral Nervous System. J Neurosci 43(13): 2260-2276. PubMed ID: 36801823
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 this study investigated the role of Innexins in mediating glial function in the Drosophila periphery. Of the eight Drosophila Innexins, it was 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. Inx2 is key to Ca(2+) pulses in the peripheral subperineurial glia but not in the wrapping glia, and no evidence was found of gap junction communication between subperineurial and wrapping glia. Rather there is 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.
Petsakou, A., Liu, Y., Liu, Y., Comjean, A., Hu, Y., Perrimon, N. (2023). Cholinergic neurons trigger epithelial Ca(2+) currents to heal the gut. Nature, 623(7985):122-131. PubMed ID: 37722602 ID:
A fundamental and unresolved question in regenerative biology is how tissues return to homeostasis after injury. Answering this question is essential for understanding the aetiology of chronic disorders such as inflammatory bowel diseases and cancer. This study used the Drosophila midgut to investigate this and discovered that during regeneration a subpopulation of cholinergic neurons triggers Ca(2+) currents among intestinal epithelial cells, the enterocytes, to promote return to homeostasis. It was found that downregulation of the conserved cholinergic enzyme Acetylcholine esterase in the gut epithelium enables acetylcholine from specific Eiger (TNF in mammals)-sensing cholinergic neurons to activate nicotinic receptors in innervated enterocytes. This activation triggers high Ca(2+), which spreads in the epithelium through Innexin2-Innexin7 gap junctions, promoting enterocyte maturation followed by reduction of proliferation and inflammation. Disrupting this process causes chronic injury consisting of ion imbalance, Yki (YAP in humans) activation, cell death and increase of inflammatory cytokines reminiscent of inflammatory bowel disease. Altogether, the conserved cholinergic pathway facilitates epithelial Ca(2+) currents that heal the intestinal epithelium. These findings demonstrate nerve- and bioelectric-dependent intestinal regeneration and advance current understanding of how a tissue returns to homeostasis after injury.
Tu, R., Tang, X. A., Xu, R., Ping, Z., Yu, Z., Xie, T. (2023). Gap junction-transported cAMP from the niche controls stem cell progeny differentiation. Proc Natl Acad Sci U S A, 120(35):e2304168120 PubMed ID: 37603749
The niche has been shown to control stem cell self-renewal in different tissue types and organisms. Recently, a separate niche has been proposed to control stem cell progeny differentiation, called the differentiation niche. However, it remains poorly understood whether and how the differentiation niche directly signals to stem cell progeny to control their differentiation. In the Drosophila ovary, inner germarial sheath (IGS) cells contribute to two separate niche compartments for controlling both germline stem cell (GSC) self-renewal and progeny differentiation. This study shows that IGS cells express Inx2 protein, which forms gap junctions (GJs) with germline-specific Zpg protein to control stepwise GSC lineage development, including GSC self-renewal, germline cyst formation, meiotic double-strand DNA break formation, and oocyte specification. Germline-specific Zpg and IGS-specific Inx2 knockdowns cause similar defects in stepwise GSC development. Additionally, secondary messenger cAMP is transported from IGS cells to GSCs and their progeny via GJs to activate PKA signaling for controlling stepwise GSC development. Therefore, this study demonstrates that the niche directly controls GSC progeny differentiation via the GJ-cAMP-PKA signaling axis, which provides important insights into niche control of stem cell differentiation and highlights the importance of GJ-transported cAMP in tissue regeneration. This may represent a general strategy for the niche to control adult stem cell development in various tissue types and organisms since GJs and cAMP are widely distributed.
Luedke, K. P., Yoshino, J., Yin, C., Jiang, N., Huang, J. M., Huynh, K., Parrish, J. Z. (2023). Dendrite intercalation between epidermal cells tunes nociceptor sensitivity to mechanical stimuli in Drosophila larvae. bioRxiv PubMed ID: 37745567
An animal's skin provides a first point of contact with the sensory environment, including noxious cues that elicit protective behavioral responses. Nociceptive somatosensory neurons densely innervate and intimately interact with epidermal cells to receive these cues. This study identified a role for dendrite intercalation between epidermal cells in tuning sensitivity of Drosophila larvae to noxious mechanical stimuli. In wild-type larvae, dendrites of nociceptive class IV da neurons intercalate between epidermal cells at apodemes, which function as body wall muscle attachment sites. From a genetic screen miR-14 was identified as a regulator of dendrite positioning in the epidermis: miR-14 is expressed broadly in the epidermis but not in apodemes, and miR-14 inactivation leads to excessive apical dendrite intercalation between epidermal cells. miR-14 regulates expression and distribution of the epidermal Innexins ogre and Inx2 and that these epidermal gap junction proteins restrict epidermal dendrite intercalation. Finally, it was found that altering the extent of epidermal dendrite intercalation had corresponding effects on nociception: increasing epidermal intercalation sensitized larvae to noxious mechanical inputs and increased mechanically evoked calcium responses in nociceptive neurons, whereas reducing epidermal dendrite intercalation had the opposite effects. Altogether, these studies identify epidermal dendrite intercalation as a mechanism for mechanical coupling of nociceptive neurons to the epidermis, with nociceptive sensitivity tuned by the extent of intercalation.


Neural stem cells in the adult brain exist primarily in a quiescent state but are reactivated in response to changing physiological conditions. How do stem cells sense and respond to metabolic changes? In the Drosophila CNS, quiescent neural stem cells are reactivated synchronously in response to a nutritional stimulus. Feeding triggers insulin production by blood-brain barrier glial cells, activating the insulin/insulin-like growth factor pathway in underlying neural stem cells and stimulating their growth and proliferation. This study shows that gap junction proteins, Inx1 and Inx2, in the blood-brain barrier glia mediate the influence of metabolic changes on stem cell behavior, enabling glia to respond to nutritional signals and reactivate quiescent stem cells. It is proposed that gap junctions in the blood-brain barrier are required to translate metabolic signals into synchronized calcium pulses and insulin secretion (Speder, 2014).

Changes in environmental conditions can have a significant impact on the development and function of the brain. Neural stem cells (NSCs) integrate both local and systemic signals to modulate their rate and extent of proliferation to meet the needs of the organism. Most NSCs in the vertebrate adult brain exist in a mitotically dormant state. These quiescent NSCs are reactivated in response to a variety of metabolic stimuli. Understanding how systemic and metabolic signals are sensed by the brain and converted into specific neural stem cell behaviors is essential to deciphering how the brain adapts to a changing environment (Speder, 2014).

In Drosophila, NSCs enter quiescence at the end of embryogenesis and are reactivated during early larval life in response to feeding. Amino acid availability is sensed by the fat body, the functional equivalent of the mammalian liver and adipose tissue. The fat body sends an as-yet-unidentified signal, or signals, to the brain to induce the production and secretion of insulin-like peptides (dIlps) by blood-brain barrier (BBB) glial cells. dIlps act locally to trigger the insulin/insulin-like growth factor receptor pathway in underlying NSCs. Consequently, the NSCs enlarge and re-enter the cell cycle (Speder, 2014).

NSC reactivation occurs synchronously in all neurogenic zones of the CNS, suggesting that BBB glial cells and/or NSCs are linked by an intercellular signaling mechanism. Gap junctions are intercellular channels formed by the juxtaposition of connexin hexamers. They enable the propagation and amplification of signals within or between cell populations. Gap junctions are found throughout the mammalian brain and are important regulators of stem cell behavior, controlling self-renewal, survival, and aging. This study shows that gap junction proteins play a key role in the nutrient-dependent reactivation of dormant neural stem cells in the Drosophila brain. Interestingly, gap junction proteins are required in the BBB glia, but not in neural stem cells, for reactivation. This study shows that gap junction proteins coordinate nutrient-dependent calcium oscillations within the BBB and are required for the production and secretion of insulin-like peptides. Gap junction proteins thus enable the synchronous reactivation of quiescent stem cells throughout the CNS (Speder, 2014).

To assess whether gap junctions play a role in NSC reactivation, this study systematically targeted each of the eight members of the innexin (Inx) family (Bauer, 2005), the Drosophila functional equivalents of connexins and pannexins, by RNAi in either NSCs or glia. Interestingly, no detectable phenotype was observed when innexins were knocked down in NSCs. However, knockdown of innexin 1 (inx1) or innexin 2 (inx2) in glia gave a striking phenotype in which brain size is dramatically reduced without affecting overall body size. This suggests that the inx phenotype is not the result of a systemic growth defect but that inx1 and inx2 have a specific role in the brain. The specificy of inx1RNAi and inx2RNAi was checked out using in silico methods, which predict no off-targets. The data are consistent with the recent results of Holcroft (2013), who showed that targeted RNAi against inx1 (ogre) or inx2 in glia disrupts development of the larval nervous system and leads to adult behavioral phenotypes. This study demonstrates that innexins are not required to link NSCs either to each other or to glial cells. Instead, Inx1 and Inx2 are required within the glial population alone for brain development (Speder, 2014).

To understand how glial gap junctions regulate growth in the CNS, NSC behavior was examined after inx1 or inx2 knockdown at different time points during the process of NSC reactivation. Knockdown of inx1 or inx2 in glia did not affect the number of NSCs in the ventral nerve cord (VNC), demonstrating that the phenotype is not due to the loss of NSCs prior to NSC reactivation (0 hr after larval hatching, ALH0). Next, cell diameter was assessed because one of the earliest events in NSC exit from quiescence is cell enlargement. It was found that NSC diameter is markedly reduced (ALH24) after inx1 or inx2 knockdown in glia. Finally, NSC proliferation was assessed after inx knockdown. The mitotic marker phosphohistone H3 (PH3) was assessed before NSC reactivation (ALH0), just after reactivation (ALH48) and at a time when wild-type NSCs are cycling actively (ALH72). Knockdown of either inx1 or inx2 in glial cells resulted in a severe reduction in the number of dividing NSCs at all times. It was found that NSC enlargement and entry into mitosis were also dramatically impaired in inx1 and inx2 mutants, and that reactivation could be rescued in inx2 mutants by glial expression of inx2. It is concluded that inx1 and inx2 are required in the glia for NSC exit from quiescence (Speder, 2014).

Gap junction proteins (connexins, pannexins, or innexins) are classically involved in forming intercellular channels or hemichannels, which enable exchange between the cytoplasm and the extracellular medium. Evidence also exists for channel-independent roles, such as cell adhesion and direct gene regulation. To test if channel function is important for NSC reactivation, brains were treated in culture with carbenoxolone, a classic blocker of gap junction channels and hemi-channels. Carbenoxolone completely blocked NSC reactivation, implying a channel role for Inx1 and/or Inx2, the only innexins required for NSC reactivation. It was also found that protein fusions that interfere with the folding of the innexin N-terminal domain (GFP-Inx1 and RFP-Inx2), which is essential for channel formation (Nakagawa, 2010), act as dominant-negative mutants. This suggests that the function of Inx1 and Inx2 in glia is channel-based (Speder, 2014).

Inx1 and Inx2 could be part of the same channel or form two distinct channels, performing different functions that are both required for NSC reactivation. Gap junction channels are formed by the apposition of connexons (innexons in Drosophila) on adjacent cells. Connexons can be homomeric, formed from six molecules of a single subtype of connexin, or heteromeric, formed from different subtypes. In the larval VNC prior to NSC reactivation (ALH7), Inx1 and Inx2 were strongly expressed in glia and colocalized in plaques typical of gap junctions (Segretain, 2004). Super-resolution microscopy further demonstrated the tight association of Inx1 and Inx2, using tagged fusion proteins. This close association is present from hatching and is not lost under starvation conditions, demonstrating that formation of the complex is not driven by nutrition. Most Inx1 staining was lost after knockdown of inx2 in glia, and vice versa. This suggests that Inx1 and Inx2 localization is interdependent and that Inx1 and Inx2 form heteromeric innexons (Segretain, 2004) rather than independent gap junction channels (Lehmann, 2006). Inx1 and Inx2 have been shown to form functional heteromeric channels in paired Xenopus oocytes (Holcroft, 2013). It is concluded, therefore, that Inx1 and Inx2 form heteromeric channels or hemi-channels in the glia (Speder, 2014).

Interestingly, a change was observed in Inx1/Inx2 colocalization over time. By ALH24, when reactivation has taken place, Inx1 and Inx2 are still expressed but they no longer colocalize, suggesting that formation of Inx1/Inx2 channels is temporally regulated. Consistent with this observation, it was discovered that the temporal requirement for inx1 and inx2 function in NSC reactivation is between ALH0 and ALH24. Therefore, the formation and maintenance of Inx1 and Inx2 heteromeric channels are developmentally regulated and coincide with the time when the innexins are required for NSC reactivation (Speder, 2014).

Inx1/Inx2 channels are required in glia to transmit nutritional stimuli to quiescent NSCs, They are likely to be found, therefore, in cells situated between the NSCs and the exterior of the brain. To determine in which glial cells Inx1/Inx2 are required, Inx1/Inx2 was knocked down in different glial populations using subtype- restricted GAL4 drivers to drive RNAi or express dominant-negative constructs. inx function was found to be necessary within the subperineurial glia because knockdown in this glial subtype alone phenocopies knockdown in the entire glial population, preventing NSC reactivation (Speder, 2014).

The subperineurial glia and the perineurial glia constitute the Drosophila BBB (Stork, 2008). In vertebrates, the BBB consists of a single layer of vascular endothelium closely associated with astrocytic glia. The BBB shields the brain from the external environment owing to tight junctions between endothelial cells. It acts as a selective sieve to reject potentially neurotoxic factors but allow the passage of nutrients, ions, or other signals to maintain brain homeostasis (Speder, 2014).

The Drosophila BBB exhibits similar neuroprotective strategies to its vertebrate counterpart, including a layer that limits the diffusion of neurotoxic factors, and an array of conserved transporters that regulates BBB permeability (Daneman, 2005; De-Salvo, 2011; Mayer, 2009). The subperineurial glia are large, flat polyploid (Unhavaithaya, 2012) cells that envelop the brain and are closely apposed to the NSCs. They isolate the brain from the hemolymph (the Drosophila equivalent of blood) by virtue of lateral septate junctions. Knockdown of inx did not disrupt the septate junctions, and no change was seen in Dextran dye penetration. Although weak permeability defects are difficult to detect at this stage and cannot be excluded, they do not prevent NSC reactivation (moodyD17 mutant) (Bainton, 2005). These data suggest that the inx mutant phenotype is not due to an impaired, leaky BBB. Using super-resolution microscopy, Inx1 and Inx2 were detected along the BBB membranes and the septate junctions lining the lateral cell membranes. It is concluded that Inx1/Inx2 channels are required autonomously in the BBB glial cells for NSC reactivation (Speder, 2014).

NSC reactivation requires the expression and secretion of insulin- like peptides, dIlps, by BBB glial cells (Chell, 2010). Of the eight identified insulin-like peptides in Drosophila, dIlp6 transcription was shown to increase dramatically in the CNS upon feeding. Furthermore, when larvae were starved, forced expression of dIlp6 in the glia was able to rescue NSC reactivation (Chell, 2010). dIlp6 binds to the insulin receptor (InR) on NSCs, activating the PI3K/Akt pathway and inducing exit from quiescence (Chell, 2010; Speder, 2014 and references therein).

Whether gap junction proteins within the BBB are required for insulin signaling was assayed. A significant decrease was found in dIlp6 transcription after knocking down both inx1 and inx2 in the glia. Next, dIlp6 secretion was assayed. In the absence of an effective dIlp6 antiserum, a tagged, functional version of dIlp6 (dIlp6-FLAG) was expressed in the BBB glia. dIlp6 secretion from the BBB glia was found to be strongly impaired in inx1 loss-of-function mutants. Secretion of dIlp6 was similarly impaired upon starvation. Therefore, both the expression and secretion of dIlp6 are regulated by nutrition and depend on gap junction proteins (Chell, 2010; Speder, 2014 and references therein).

NSC reactivation in inx mutants was rescued by forced expression of dIlp6 in glia, as shown by the recovery of brain volume (80% of the brains) and of NSC diameter. Direct activation of the PI3K/Akt pathway in NSCs also resulted in rescue of brain volume and NSC enlargement and entry into mitosis. It is concluded that gap junction proteins in the BBB glia are required to activate insulin signaling and induce NSC reactivation (Speder, 2014).

Secretion of insulin by the pancreas is induced by glucose, leading to synchronized calcium oscillations within gap junction- coupled beta cells and insulin exocytosis (MacDonald, 2006). Gap junctions enable the passage of secondary messengers that either trigger the release of calcium from intracellular stores or the influx of calcium from the extracellular environment. Blocking gap junctions inhibits coordinated intercellular calcium signaling (Leybaert, 2012; Orellana, 2012). Gap junction proteins are thus an important means of transmitting calcium waves (Speder, 2014). To investigate whether calcium signaling plays a role in gap junction-mediated NSC reactivation, a calcium sensor, GCaMP3, was expressed in the BBB glia of living larvae. Before reactivation (ALH7) the BBB glia of feeding larvae exhibited clear calcium oscillations. The BBB glia pulsed simultaneously, suggesting that calcium oscillations are coordinated across the entire CNS. Individual cell tracking showed that glial calcium oscillations exhibited striking synchrony in all brains analyzed (Speder, 2014).

To further assess the extent of calcium oscillation coordination within the BBB glia under these conditions, correlation analysis was performed for 20 regions of interest (ROI) chosen at random within the BBB glial layer. In fed larvae before reactivation, the central correlation peak demonstrates the synchronicity of calcium oscillations within the BBB layer. Additional peaks on each side reveal that this synchronicity repeats (Speder, 2014).

Next, calcium dynamics were monitored in inx1 mutants. None of the mutant brains showed coordinated calcium oscillations. Instead, BBB glial cells pulse independently, with no coordination between neighboring cells. It is concluded that Inx1/Inx2 gap junctions are required to coordinate synchronous calcium oscillations within the BBB glia. In accordance with this observation, the graph of correlation coefficient for inx1 mutants established the total absence of synchronicity between BBB glial cells, showing that gap junctions are required for propagating calcium oscillations within the BBB (Speder, 2014).

To assess whether calcium oscillations in the BBB are induced by a nutritional stimulus, calcium dynamics were first assayed in the BBB glia of newly hatched larvae (ALH0), before they started to feed. The calcium oscillations differed both in extent and frequency from those seen in fed larvae. Correlation analysis revealed a partial coordination within the BBB glia, strengthening the idea that nutrition is important for extending and establishing robust calcium synchronicity in the BBB (Speder, 2014).

Next, calcium dynamics were assessed after starvation, specifically the absence of essential amino acids. Calcium dynamics in the BBB of starved larvae resembled inx1 mutant brains. Synchronous calcium oscillations were completely abolished in all brains examined. Individual BBB cells displayed some calcium pulses, but with different profiles to those seen in fed larvae. In addition, correlation analysis of starved larvae showed very weak synchronicity. This suggests that the synchronous, nutrition-dependent, calcium oscillations are lost upon starvation. Importantly, neither Inx1 nor Inx2 is lost under starvation conditions. It is concluded that nutrition, in particular essential amino acids, shape calcium dynamics. Upon feeding, calcium oscillations are amplified and synchronized across the BBB (Speder, 2014).

Whether glial calcium oscillations arise from the release of intracellular calcium, or from the influx of extracellular calcium, was assayed, along with how these influence NSC reactivation. First, the importance of the inositol-triphosphate (IP3) pathway was assessed. The IP3 pathway triggers calcium release from intracellular stores. Stimulation of G-coupled receptors by a wide range of signals activates phospholipase C, leading to the production of IP3 from cleaved PIP2. IP3 then binds to its receptor (Ins3PR), a ligand-gated Ca2+ channel found on the surface of the ER, releasing intracellular calcium. Ins3PR (itpr in Drosophila) was knocked down in the BBB glia by RNAi. Both NSC enlargement and proliferation were strongly impaired. It is concluded that NSC reactivation depends on IP3-mediated release of calcium from intracellular stores (Speder, 2014).

Next, the importance of calcium influx was assessed. Membrane depolarization triggers the entry of extracellular calcium via voltage-gated calcium channels, whereas hyperpolarization prevents it. BBB membranes were hyperpolarized by expressing the inward-rectifying potassium channel, kir2.1. BBB hyperpolarization blocks NSC reactivation dramatically, as revealed by the complete failure of both enlargement and mitotic re-entry. Interestingly, the mushroom body neuroblasts, a small group of central brain NSCs that do not undergo quiescence and reactivation, are not affected by BBB hyperpolarization, suggesting that nutrition-dependent NSC reactivation is specifically affected (Speder, 2014).

BBB hyperpolarization both decreases dIlp6 mRNA levels and dIlp6 secretion, similar to what is seen during starvation or gap junction loss-of- function. In support of the role of calcium oscillations in reactivating NSCs, it was found that overexpressing the calcium-binding protein, calmodulin, prevents NSC reactivation. These results show that intracellular and extracellular calcium both contribute to NSC reactivation (Speder, 2014).

Gap junction proteins within the BBB glia are required for insulin expression and secretion, a prerequisite for NSC exit from quiescence. This study demonstrates that gap junction proteins coordinate glial calcium oscillations that are required for NSC reactivation. Both intracellular calcium stores and calcium influx contribute to reactivation. Membrane depolarization is known to regulate exocytosis via calcium signaling, which controls stimulus-secretion coupling in secretory cells, such as in endocrine cells. Conditions that block calcium oscillation in the BBB glia (the loss of gap junction proteins, starvation) also impair insulin secretion (Speder, 2014).

The sequence of events leading to glial secretion of insulin bears a striking resemblance to the diet-induced release of insulin by the beta cells of the pancreas. In the pancreas, a nutritional stimulus is sensed by gap junction-coupled beta cells, inducing depolarization resulting in synchronized calcium oscillation and insulin secretion. Loss of gap junction coupling results in uncoordinated calcium pulses. In Drosophila inx loss of function mutants, individual subperineurial BBB glial cells oscillate independently of one another. Compared to starvation, in which the nutritional signal is absent and NSC reactivation cannot occur, the scattered signals from individual BBB cells are able to induce delayed, asynchronous, reactivation in a small number of NSCs. It is proposed that gap junction function within the BBB enables glial insulin release to reach a threshold high enough to trigger NSC reactivation throughout the central nervous system (Speder, 2014).

In both beta cells and BBB cells, membrane depolarization is crucial for generating calcium oscillations. Failure to depolarize or an active block to depolarization prevents insulin release. However, sustained depolarization of β cells can lead to desensitization and a decline in insulin release. Interestingly, this study found that forced depolarization of BBB glia only mildly enhances NSC reactivation. This could be due to desensitization or it may be that the system is already maximally active (Speder, 2014).

Insulin mRNA levels are decreased after gap junction knockdown, both in pancreatic islets (Bosco, 2011) and in the BBB glia. In both cases it remains to be determined if calcium oscillations can directly affect gene expression, as has been shown in other systems (Speder, 2014).

Insulin produced by the pancreas is distributed via the circulatory system, whereas glial insulin is secreted locally, directly to underlying NSCs. Glial insulin signaling is thus contained within the brain, enabling local, differential regulation of this organ. The BBB acts both as a niche and as a protective barrier, providing specific factors directly to the stem cell while shielding the brain from unwelcome systemic regulation. In the context of NSC reactivation, these two roles are conveniently complementary. In the vertebrate BBB, similar functions may be split between endothelial cells and astrocytic glia. The vascular endothelium provides the barrier function, while astrocytic glia have a regulatory role in sensing and adjusting barrier permeability to various stimuli. BBB endothelial cells can secrete cytokines, chemokines, and prostaglandins, suggesting that the BBB behaves like an endocrine tissue. Interestingly, calcium oscillations have been observed in cultured vertebrate BBB endothelial cells, but their function is largely unknown (Speder, 2014).

Gap junction communication can influence stem cell behavior by directly coupling stem cells to each other or to supporting cells, such as found in a stem cell niche. In the brain, connexon-mediated communication has been reported to occur between progenitor cells, within astrocytic networks, and between radial glia and neurons or progenitor cells and astrocytes (Giaume, 2010, Lacar, 2011, Nakase, 2004, Lo Turco, 1991). The proliferation of neural progenitors and the formation of cortical layers in the mouse brain depend on an intercellular gap junction network (Malmersjö, 2013), and grafted human NSCs integrate into organotypic cultures through connexin coupling (Jäderstad, 2010). This study shows that gap junction function within a niche, the BBB, can also influence NSC behavior (Speder, 2014).

The Drosophila BBB is a protective and selective barrier as well as a signaling center that orchestrates major developmental and physiological events. Here we show that gap junction communication enables cells within the BBB to act as a concerted unit, leading to coordinated calcium signaling and insulin release. Similarities between the BBB in vertebrates and invertebrates suggest that our findings are likely to have broader significance (Speder, 2014).

Bi-directional gap junction-mediated Soma-Germline communication is essential for spermatogenesis

Soma-germline interactions play conserved essential roles in regulating cell proliferation, differentiation, patterning, and homeostasis in the gonad. In the Drosophila testis, secreted signalling molecules of the JAK-STAT, Hedgehog, BMP, and EGF pathways are used to mediate germline-soma communication. This study demonstrates that gap junctions may also mediate direct, bi-directional signalling between the soma and germline. When gap junctions between the soma and germline are disrupted, germline differentiation is blocked and germline stem cells are not maintained. In the soma, gap junctions are required to regulate proliferation and differentiation. Localization and RNAi-mediated knockdown studies reveal that gap junctions in the fly testis are heterotypic channels containing Zpg/Inx4 and Inx2 on the germline and the soma side, respectively. Overall, the results show that bi-directional gap junction-mediated signalling is essential to coordinate the soma and germline to ensure proper spermatogenesis in Drosophila. Moreover, this study shows that stem cell maintenance and differentiation in the testis are directed by gap junction-derived cues (Smendziuk, 2015).

The work presented in this study demonstrates that gap junctions between the soma and germline are essential for fly spermatogenesis. Previous work showing an essential role for Zpg in the fly gonads raised the possibility that signals either from the soma or from other germ cells travel through gap junctions to regulate germline survival and differentiation. Subsequent work in the fly ovaries showed that Zpg was also required for GSC maintenance. This analysis supports and extends these conclusions by finding a cell-autonomous requirement for Zpg in GSC maintenance in the fly testis and demonstrates a role for Inx2 in the soma. Furthermore, it was found that gap junction-mediated signals from the germline also play unique and essential roles in the soma during spermatogenesis, independent of general germline defects. In particular, gap junctions are required to control the proliferation of CySCs and promote the differentiation of their daughters. This work illustrates that the main type of gap junction between the soma and the germline in the fly testis is a heterotypic channel coupling Inx2 in the soma and Zpg in the germline. Importantly, disrupting gap junctions in the soma by knocking down Inx2 phenocopies the zpg mutant phenotype in both the germline and soma. Therefore gap junction-mediated soma-germline regulation in the testis is bi-directional (Smendziuk, 2015).

Gap junctions contribute to stem cell regulation in the testis Recent work has highlighted the importance of gap junctions in stem cell regulation in a number of systems. In line with results from other stem cell models, the data illustrates a specific role for gap junctions in both GSCs and CySCs. The role of gap junctions in stem cell regulation in the testes was illustrated by the requirement for Zpg in the germline and Inx2 in the soma for GSC maintenance. Moreover, loss of Zpg or somatic knockdown of Inx2 also affected CySC proliferation. Furthermore, ultrastructural analysis revealed the presence of gap junctions between GSCs and CySCs. These results, as a whole, suggest a requirement for gap junction-mediated soma-germline communication in both stem cell populations and at the earliest stages of sperm differentiation (Smendziuk, 2015).

Gap junctions facilitate signalling between the soma and germline Following the stem cell stage, strong expression and co-localization of Zpg and Inx2 was consistently detected starting at the 4-cell cyst stage. Expression of Zpg and Inx2 began to diminish after the early spermatocyte stages and was not detected past meiotic stages. The timing at which Inx2 and Zpg expression were most prominent corresponds to a period during which niche signals such as BMP are lost. Loss of these signals causes the germline to undergo rapid differentiation and specialization. It has been shown that as somatic cells move away from the niche and begin differentiating, the soma forms a permeability barrier around the germline, isolating the germline from the outside environment. This transition corresponds with a switch occurs whereby soma-germline communication shifts from predominantly exocrine to juxtacrine signalling. Thus, as the germline becomes increasingly isolated, it becomes more dependent on differentiation signals that arrive via gap junctions from the soma. Once the germline becomes isolated, gap junctions may also play an important nutritive role and permit the movement of essential small metabolites between the germline and soma. Similarly, the soma requires gap junction-mediated signals to allow it to accommodate the increasingly expanded, differentiated, and specialized germline (Smendziuk, 2015).

The observations that gap junctions regulate germline differentiation and soma proliferation are in line with studies from both vertebrate and invertebrate models. In C. elegans, it was recently shown that gap junction-mediated signals are required to maintain GSCs in the niche and for germline differentiation (Starich, 2014). Similarly, work in vertebrates has shown that loss of gap junction-mediated signalling in the soma increased proliferation in post-mitotic Sertoli cells. It is therefore likely that an early role for gap junctions in coordinating soma-germline differentiation is an evolutionarily-conserved mechanism. One recurring feature of germline-soma gap junctions is the expression of different gap junction proteins, resulting in heterotypic gap junctions, exemplified by the Inx2-Zpg gap junctions observed in flies. A key problem in understanding the role of gap junctions in mediating soma-germline communication is identifying the transported signalling cargos. Some possible signals are cAMP, Ca2+, and cGMP, which have been implicated in regulating meiosis in the germline. Attempts to study cAMP and Ca2+ in the testis have proven inconclusive. However, recent work in Drosophila ovaries has suggested that somatic gap junctions may play roles in regulating pH, membrane potential, and ion transport. Overall, multiple signals are likely exchanged between the soma and germline through gap junctions and elucidating their respective functions is a complex task that should be further studied (Smendziuk, 2015).

Based on the results presented in this study, the following model is proposed: GSCs receive multiple cues that control their behaviour, with gap junctions mostly provide a supporting role, allowing the passage of cues from the soma that facilitate long-term GSC maintenance. After stem cell division germline undergoes rapid differentiation. The germline becomes increasingly isolated from the outside environment, and a permeability barrier is formed by the soma. As outside signals from the niche are lost, the germline relies more heavily on gap junctions to allow the passage of small molecules and metabolites from the soma to promote differentiation and provide nourishment. To ensure coordinated growth and differentiation of the soma and germline, signals pass from the germline through the gap junctions into the soma. Taken together, this work defines gap junction-mediated juxtacrine signalling as an additional signalling mechanism in the fly testis. Furthermore, this study provides a clear illustration of the bi-directional regulatory action of soma-germline gap junctions. As this study demonstrates, disrupting innexins in the soma or germline leads to a specific regulatory effect in the other tissue. Therefore bi-directional gap junction-mediated signalling plays a vital role in ensuring proper coordination of the soma and germline during spermatogenesis (Smendziuk, 2015).

Gap junction-mediated signaling from motor neurons regulates motor generation in the central circuits of larval Drosophila

This study used the peristaltic crawling of Drosophila larvae as a model to study how motor patterns are regulated by central circuits. An experimental system was constructed that allows simultaneous application of optogenetics and calcium imaging to the isolated ventral nerve cord (VNC). Next, the effects of manipulating local activity of motor neurons (MNs) on fictive locomotion were observed as waves of MN activity propagating along neuromeres. Optical inhibition of MNs with halorhodopsin3 (NpHR3) in a middle segment (A4, A5 or A6), but not other segments, dramatically decreases the frequency of the motor waves. Conversely, local activation of MNs with channelrhodopsin2 (ChR2) in a posterior segment (A6 or A7) increases the frequency of the motor waves. Since peripheral nerves mediating sensory feedback are severed in the VNC preparation, these results indicate that MNs send signals to the central circuits to regulate motor pattern generation. These results also indicate segmental specificity in the roles of MNs in motor control. The effects of the local MN activity manipulation are lost in shakB2 or ogre2, gap-junction mutations in Drosophila, or upon acute application of the gap junction blocker CBX, implicating electrical synapses in the signaling from MNs. Cell-type specific RNAi suggests shakB and ogre function in MNs and interneurons, respectively, during the signaling. These results not only reveal an unexpected role for MNs in motor pattern regulation but also introduce a powerful experimental system that enables examination of the input-output relationship among the component neurons in this system (Matsunaga, 2017).

Animal movement is accomplished by spatially and temporally coordinated contraction of various muscles throughout the body. It is generally thought that a neuronal network composed of premotor interneurons generates a motor pattern, and this network sequentially activates different classes of motor neurons (MNs). In this view, MNs play only passive roles in pattern generation, relaying the information they receive from upstream interneuronal networks to muscles. By contrast, there is some evidence that MNs themselves contribute to the motor pattern generation. In the crustacean stomatogastric ganglion and in leech swimming circuits, MNs are part of the pattern-forming network. In mammalian spinal cords, MNs send a collateral to innervate Renshaw cells, which in turn convey feedback signals to MNs. However, whether and how MNs regulate motor pattern generation during animal movements remains largely unexplored (Matsunaga, 2017).

Larval Drosophila is emerging as an excellent model system for studying motor pattern generation since one can apply powerful genetic tools including a large collection of Gal4-drivers to study the function of individual component neurons in a numerically simple nervous system. Furthermore, previous development of a platform for electronic microscope (EM) image data reconstruction of the entire nervous system of the larval CNS now allows mapping of the circuit structure that mediates specific behaviors. The larval ventral nerve cord (VNC) consists of three thoracic neuromeres (T1, T2, and T3) and eight abdominal neuromeres (A1-A8). Larval peristaltic crawling is accomplished by successive bilateral muscle contraction that propagates from tail to head. Muscle contraction in each segment is in turn regulated by sequential activation of MNs in the corresponding neuromere of the VNC. Although recent studies have begun to identify several types of premotor interneurons that regulate aspects of movement such as the speed of locomotion and left-right or intersegmental coordination, how a motor pattern is generated by the neural circuits remains largely unknown (Matsunaga, 2017).

In a previous study, halorhodopsin (NpHR) was used to locally and transiently inhibit MN activity in one or a few segments; local activity perturbation was found to halt the propagation of the peristaltic wave at the site of manipulation. This suggests that MNs are part of the neural circuits that generate the peristaltic wave. However, how information is retrogradely transmitted from MNs to the central circuits remained unknown. Furthermore, since muscle contraction was usedt as a measure of the motor outputs, changes in the activity dynamics in the CNS could not be studied. That study was extended by constructing a new experimental system in which the effects of local optogenetic manipulation of MNs on global motor activity could be studied in the VNC. Optical inhibition of MNs in a middle segment (A4, A5, or A6) decreased the motor frequency. Conversely, photoactivation of MNs in a posterior segment (A6 or A7) increased the frequency of the motor wave. These results indicate that the local activity level of MNs impacts the global outputs of the motor circuits in a segment-specific manner. It was also show that gap junctions are involved in this process. While this manuscript was in preparation, a study in zebrafish reported that motor neurons retrogradely influence the activity level of the premotor V2a interneurons via gap junctions and regulate motor generation. Thus, regulation by gap junction-mediated retrograde MN signaling appears to be a common mechanism of motor control (Matsunaga, 2017).

In a previous study, MN activity was locally and transiently inhibited in one or a few segments during peristalsis of dissected larvae, and activity manipulation was shown to halt peristalsis. This indicates that MN activity is required for the motor activity wave to propagate along the VNC and suggests the presence of retrograde signaling from MNs to the central circuits. However, since dissected larvae were used, the possibility that the signaling was instead mediated via the sensory feedback of muscular contraction could not be excluded. Furthermore, the mechanism of the retrograde signaling remained unknown (Matsunaga, 2017).

A new experimental system was built that allow studying the direct causal relationship between the manipulation of MN activity and changes in neural dynamics in the motor circuits with superior spatial and temporal resolution. Optical perturbations were applied for a longer period and in a more systematic manner than in the previous study and their effects on the global circuit activity was analyzed. Using the new experimental system, the previous study was extended by showing that (1) MN outputs within the CNS, not mediated by sensory feedback, are critical for motor wave regulation, (2) there is segmental difference in the role of the MN outputs, and (3) the MN signaling is mediated by gap junctions (Matsunaga, 2017).

This study shows that manipulation of motor neuronal activity in just one segment robustly affects the output of the entire motor network in Drosophila larvae. Optical inhibition or activation of MNs in a single segment decreased or increased, respectively, the calcium level of MNs in distant neuromeres. Furthermore, these perturbations strongly affected the frequency of motor waves. Thus, changes in MN activity in one segment affect the activity level and wave generation of the entire motor system (Matsunaga, 2017).

It should be noted that in the isolated VNC preparation used in this study, peripheral nerves with motor activity output and sensory feedback input were severed. Local changes in MN activity therefore influenced the activity of distant MNs through intersegmental neural connections within the CNS, not via sensory feedback. Thus, the results establish the presence of retrograde signaling from MNs that is critical for motor pattern regulation. The identity of the synaptic connections mediating the signaling is currently unknown. They could be direct MN-MN connections, or they may also involve coupling between MNs and interneurons (Matsunaga, 2017).

Electrical synapses are commonly found in the nervous systems of vertebrates and invertebrates. In particular, electrical coupling mediated by gap junctions has been implicated in motor pattern control in various systems. This study has showed that gap junctions are involved in the retrograde MN signaling controlling motor wave frequency in Drosophila larvae. Local photomanipulation of MNs that would normally increase or decrease wave frequency had no effect in shakB2 and ogre2 mutants. This suggests that electrical synapses including ShakB and Ogre mediate the MN signaling controlling motor frequency. In contrast, CBX administration but not shakB2 or ogre2 mutation abolished the calcium level changes of distant MNs induced by the activity manipulation, suggesting that innexins other than those deleted in shakB2 or ogre2 mediate this aspect of motoneuronal communication (eight innexin genes are present in the Drosophila genome). It should also be noted that wave generation normally occurred in the isolated VNCs of shakB2 and ogre2 mutants. There was also no obvious abnormality in the locomotion of the shakB2 or ogre2 larvae. These observations suggest that ShakB and Ogre-mediated MN signaling is part of redundant pathway(s) regulating motor waves. Only upon optical perturbation are the role of MN signaling in wave generation and involvement of ShakB and Ogre manifested (Matsunaga, 2017).

Previous work has reported the existence of electrical coupling between MNs and the premotor excitatory V2a interneurons, a neuronal class that provides a major drive for MNs during locomotion in zebrafish. Hyperpolarizing or depolarizing MNs decreased or increased the firing activity of V2a interneurons. Furthermore, selective inhibition of MNs during locomotion interrupted the recruitment of V2a interneurons and decreased the frequency of locomotion. Thus, control of locomotor circuits by gap junction-mediated retrograde MN signaling may be an evolutionarily conserved mechanism used in both invertebrates and vertebrates (Matsunaga, 2017).

An interesting feature of MN signaling revealed in this study is segment specificity. On the one hand, inhibition of MNs in A4, A5, or A6, but not other segments, reduced the motor wave frequency. On the other hand, activation of MNs in A6 or A7, but not other segments, increased the frequency of the wave. This segmental discord with regard to the MN signaling may contribute to the regulation of the wave initiation. How can gap junctions selectively mediate one type of activity change but not another? For example, how can the decline but not the elevation in activity level of MNs in the A4 or A5 segment affect the wave frequency? One possibility is the involvement of rectifying electrical synapses. Rectifying electrical synapses have been found in both vertebrates and invertebrates and can mediate unidirectional synaptic transmission in a voltage-dependent manner. Rectifying electrical synapses are often composed of a heteromeric assembly of gap junction proteins on each side of the apposing neurons. A role of ShakB in rectification has been shown in the giant fiber system of adult Drosophila. Two splicing forms of ShakB, ShakB(N) and ShakB(N+16), are expressed in the presynaptic and postsynaptic sites of the giant synapse, respectively. When expressed in neighboring oocytes, these two ShakB variants form heterotypic channels that are asymmetrically gated by voltage. Since RNAi knockdown experiments showed that shakB, but not ogre, is required in MNs to mediate the retrograde signaling, an interesting possibility is the involvement of heterotypic channels composed of ShakB in MNs and Ogre in interneurons. Future studies are necessary first to identify the target neurons that receive the retrograde MN signaling and then to study whether the relevant electrical synapses are indeed rectified. Revealing the information flow mediating the MN retrograde signaling will provide valuable insights on how intersegmentally coordinated motor patterns are generated in this and other systems. The experimental system established in this study can also be applied more generally to study the input-output relationship among the component neurons in this system. While the GAL4/UAS system alone was sufficient to express both GCaMP/RGECO and NpHR/ChR2 in MNs in this study, the introduction of another expression system such as the LexA system will allow expression of GCaMP/RGECO and NpHR/ChR2 in different classes of neurons, including interneurons. This will allow study of the the influence of the optogenetic manipulation of one class of neurons on the activity of others. The functional analyses may also be combined with the circuit diagram elucidated by ongoing EM reconstruction. It is anticipated that such systematic analyses will elucidate fundamental mechanisms of how central circuits coordinate intersegmental movements (Matsunaga, 2017).

Innexins Ogre and Inx2 are required in glial cells for normal postembryonic development of the Drosophila central nervous system

Innexins are one of two gene families that have evolved to permit neighbouring cells in multicellular systems to communicate directly. Innexins are found in prechordates and persist in small numbers in chordates as divergent sequences termed pannexins. Connexins are functionally analogous proteins exclusive to chordates. Members of these two families of proteins form intercellular channels, assemblies of which constitute gap junctions. Each intercellular channel is a composite of two hemichannels, one from each of two apposed cells. Hemichannels dock in the extracellular space to form a complete channel with a central aqueous pore that regulates the cell-cell exchange of ions and small signalling molecules. Hemichannels can also act independently by releasing paracrine signalling molecules. optic ganglion reduced (ogre) is a member of the Drosophila innexin family, originally identified as a gene essential for postembryonic neurogenesis. This study demonstrates, by heterologous expression in paired Xenopus oocytes, that Ogre alone does not form homotypic gap-junction channels; however, co-expression of Ogre with Innexin2 (Inx2) induces formation of functional channels with properties distinct from Inx2 homotypic channels. In the Drosophila larval central nervous system, Inx2 partially colocalises with Ogre in proliferative neuroepithelia and in glial cells. Downregulation of either ogre or inx2 selectively in glia, by targeted expression of RNA interference transgenes, leads to a significant reduction in the size of the larval nervous system and behavioural defects in surviving adults. It is concluded that these innexins are crucially required in glial cells for normal postembryonic development of the central nervous system (Holcroft, 2013).

The genome of Drosophila has eight innexin-encoding loci, transcripts from seven of which, shakB, ogre, inx2, inx3, inx5, inx6 and inx7, are found at some stage in the nervous system. Knowledge of precise spatial and temporal patterns of expression and functions of the individual genes is far from complete. ShakB, Inx6 and Inx7 mediate electrical signalling in defined adult neural circuits. These and the other innexins are widely and dynamically expressed during development and previous studies have highlighted roles for Ogre (Lipshitz, 1985; Curtin, 2002a), ShakB (Curtin, 2002b) and Inx7 (Ostrowski, 2008) in neural development. The key findings of this study are that Ogre and Inx2 are required in glia for normal postembryonic development of the CNS. Their expression patterns in these cells partially overlap and, in vitro, channel activity of one protein is influenced by the presence of the other. Thus, the proteins could act independently and/or in concert (Holcroft, 2013).

Paired Xenopus oocytes have been used widely as a heterologous system to examine the ability of proteins to form gap-junction channels. In this system, Inx2 forms homotypic channels. Previous studies demonstrated that the properties of these channels are influenced by co-expression of Inx3, which itself is not competent to form intercellular channels (Stebbings, 2000). This study demonstrate that Ogre, like Inx3, does not form intercellular channels independently, but when co-expressed also modifies intercellular conductance in Inx2-expressing cells. Specifically, co-expression of Ogre with Inx2 in both cells of a pair reduces the voltage sensitivity, without affecting the mean level, of intercellular conductance. When expressed in single oocytes, magnitude of hemichannel currents varies with Ogre+Inx2>Inx2>Ogre. There are a number of possible mechanisms to explain these findings. Trafficking of Ogre to the membrane might be aided by Inx2; once at the membrane, Ogre, like Inx2, might form homomeric hemichannels and homotypic intercellular channels. Recordings from co-expressing cells would then reflect the presence of two distinct populations of channels. Alternatively, Ogre and Inx2 might preferentially assemble heteromeric hemichannels that dock to form intercellular channels with properties distinct from Inx2 homotypic channels. The latter is favored because even at high levels of RNA ogre did not induce homotypic intercellular channels although increasing the amount of RNA injected did induce non-junctional currents. There is also some precedent for such a mechanism as Inx2 and Inx3 have been shown (Lehmann, 2006) to form hetero-oligomers (Holcroft, 2013).

Previous studies demonstrated that Ogre is expressed in the larval optic lobe proliferation centres (Watanabe, 1992). This is confirmed in this study, and it was found that Inx2 is also expressed in the optic anlage. The two innexins show significant overlap particularly in the opc and to a lesser extent in the ipc. Neither innexin is found in postmitotic neurons. By contrast, both proteins are extensively expressed, in a largely overlapping pattern, in several populations of glial cells in the larval brain lobes and ventral ganglion. The presence of two Inx proteins in highly overlapping patterns in different cell types in the developing CNS prompts several questions. Are both required for normal development? If so, in which cell types do they act and what is the mechanism of action (Holcroft, 2013)?

This study has demonstrated, by cell-specific knockdown, that Ogre and Inx2 are critically required in glial cells for normal development of the postembryonic CNS. Downregulation of the expression of either protein specifically in glia leads to a marked reduction in the size of the larval CNS. In the case of Inx2, the flies die as larvae. Flies with reduced glial Ogre expression, in contrast, develop to eclosion and survive briefly despite a small and morphologically abnormal CNS. Loss of ogre function in surviving adults leads to defective locomotor and sensorimotor activity. The behavioural phenotypes presumably are due to the failure of adult neural circuitry to develop and are reminiscent of those seen in various neural degeneration or neural wiring mutants. Circling behaviour was first described in pirouette mutants and associated with degeneration of the brain, particularly the optic lobes. Abnormal grooming is seen in flies with mutations in various molecules involved in axon growth and synaptogenesis. Flies with mutations in the cyclin-dependent kinase activator, p35, have defects in axon patterning at early developmental stages and as adults exhibit a supine phenotype (inability to right themselves) (Holcroft, 2013).

This is the first study to implicate inx2 [which is involved in epithelial morphogenesis (Bauer, 2002; Bauer, 2004)] in neural development. ogre has a known role in postembryonic neurogenesis from early studies of Lipshitz and Kankel that characterised the phenotype of various mutations at the ogre locus (Lipshitz, 1985). The major defect was a reduction in the size of the CNS, particularly the optic lobes, the extent of which correlated with the severity of the mutation. Hypomorphic mutants survived to adulthood but with highly abnormal neuronal architecture, at least in the visual system, which was the focus of analysis. The CNS of putative null mutants was even smaller than that of hypomorphs and these flies died as late larvae or pupae. The site of ogre activity was mapped to the CNS but not to specific cell types therein (Lipshitz, 1985). The strong expression of the protein in the wild-type optic proliferation centres and scattered neuroblasts (Watanabe, 1992) (also this study), coupled with the profound reduction in the size of the nervous system in ogre mutants (Lipshitz, 1985; Singh, 1989), was consistent with a requirement for the gene in the neural precursors. Interestingly, genetic mosaic analysis provided some evidence of cellular non-autonomy of the phenotype within the optic lobes (Lipshitz, 1985). The current data support the latter in demonstrating that downregulation of ogre in glial cells largely reproduces the ogre mutant phenotype. Downregulation of inx2 produces a remarkably similar phenotype. The accumulated data lead to the conclusion that these two innexins act in glial cells to regulate postembryonic neurogenesis and, quite possibly, subsequent stages of neuronal development (Holcroft, 2013).

Classically, gap-junction proteins act by forming intercellular channels that permit direct transfer of molecules between coupled cells. Increasingly, there is evidence that these proteins also have additional functions ranging from the formation of functional hemichannels that release signalling molecules such as ATP to acting as cell adhesion molecules. In considering how glial-expressed Ogre and Inx2 might regulate neuronal development it is possible to hypothesize both junctional and non-junctional mechanisms, which are not mutually exclusive. Intercellular and/or hemichannel communication among neighbouring glial cells might be crucial for development and maintenance of the extensive glial network, which, in turn, supports neurons. Alternatively, the primary role of glial innexins might be in transfer of signals from these cells to developing neurons. The former is an attractive hypothesis because glial cells are known to regulate neuronal development through provision of signalling molecules including Anachronism and Perlecan, E-cadherin and TGF-β family member Myoglianin. However, both scenarios are possible, and distinguishing these are key questions for future studies (Holcroft, 2013).

Long-distance mechanism of neurotransmitter recycling mediated by glial network facilitates visual function in Drosophila

Neurons rely on glia to recycle neurotransmitters such as glutamate and histamine for sustained signaling. Both mammalian and insect glia form intercellular gap-junction networks, but their functional significance underlying neurotransmitter recycling is unknown. Using the Drosophila visual system as a genetic model, this study shows that a multicellular glial network transports neurotransmitter metabolites between perisynaptic glia and neuronal cell bodies to mediate long-distance recycling of neurotransmitter. In the first visual neuropil (lamina), which contains a multilayer glial network, photoreceptor axons release histamine to hyperpolarize secondary sensory neurons. Subsequently, the released histamine is taken up by perisynaptic epithelial glia and converted into inactive carcinine through conjugation with beta-alanine for transport. In contrast to a previous assumption that epithelial glia deliver carcinine directly back to photoreceptor axons for histamine regeneration within the lamina, both carcinine and beta-alanine were detected in the fly retina, where they are found in photoreceptor cell bodies and surrounding pigment glial cells. Downregulating Inx2 gap junctions within the laminar glial network causes beta-alanine accumulation in retinal pigment cells and impairs carcinine synthesis, leading to reduced histamine levels and photoreceptor synaptic vesicles. Consequently, visual transmission is impaired and the fly is less responsive in a visual alert analysis compared with wild type. These results suggest that a gap junction-dependent laminar and retinal glial network transports histamine metabolites between perisynaptic glia and photoreceptor cell bodies to mediate a novel, long-distance mechanism of neurotransmitter recycling, highlighting the importance of glial networks in the regulation of neuronal functions (Chaturvedi, 2014).

Rescue of Notch signaling in cells incapable of GDP-L-fucose synthesis by gap junction transfer of GDP-L-fucose in Drosophila

Notch (N) is a transmembrane receptor that mediates cell-cell interactions to determine many cell-fate decisions. N contains EGF-like repeats, many of which have an O-fucose glycan modification that regulates N-ligand binding. This modification requires GDP-L-fucose as a donor of fucose. The GDP-L-fucose biosynthetic pathways are well understood, including the de novo pathway, which depends on GDP-mannose 4,6 dehydratase (Gmd) and GDP-4-keto-6-deoxy-D-mannose 3,5-epimerase/4-reductase (Gmer). However, the potential for intercellularly supplied GDP-L-fucose and the molecular basis of such transportation have not been explored in depth. To address these points, the genetic effects were studied of mutating Gmd and Gmer on fucose modifications in Drosophila. These mutants functioned cell-nonautonomously, and that GDP-L-fucose was supplied intercellularly through gap junctions composed of Innexin 2. GDP-L-fucose was not supplied through body fluids from different isolated organs, indicating that the intercellular distribution of GDP-L-fucose is restricted within a given organ. Moreover, the gap junction-mediated supply of GDP-L-fucose was sufficient to support the fucosylation of N-glycans and the O-fucosylation of the N EGF-like repeats. These results indicate that intercellular delivery is a metabolic pathway for nucleotide sugars in live animals under certain circumstances (Ayukawa, 2012).

Innexin2 gap junctions in somatic support cells are required for cyst formation and for egg chamber formation in Drosophila

Germ cells require intimate associations with surrounding somatic cells during gametogenesis. During oogenesis, gap junctions mediate communication between germ cells and somatic support cells. However, the molecular mechanisms by which gap junctions regulate the developmental processes during oogenesis are poorly understood. This study has identified a female sterile allele of innexin2 (inx2), which encodes a gap junction protein in Drosophila. In females bearing this inx2 allele, cyst formation and egg chamber formation are impaired. In wild-type germaria, Inx2 is strongly expressed in escort cells and follicle cells, both of which make close contact with germline cells. inx2 function in germarial somatic cells is required for the survival of early germ cells and promotes cyst formation, probably downstream of EGFR pathway, and that inx2 function in follicle cells promotes egg chamber formation through the regulation of DE-cadherin and Bazooka (Baz) at the boundary between germ cells and follicle cells. Furthermore, genetic experiments demonstrate that inx2 interacts with the zero population growth (zpg) gene, which encodes a germline-specific gap junction protein. These results indicate a multifunctional role for Inx2 gap junctions in somatic support cells in the regulation of early germ cell survival, cyst formation and egg chamber formation. Inx2 gap junctions may mediate the transfer of nutrients and signal molecules between germ cells and somatic support cells, as well as play a role in the regulation of cell adhesion (Mukai, 2011).

The isolation of a female-sterile mutation in inx2 has allowed study of the requirement of Inx2 gap junctions in oogenesis and of the mechanisms that regulate germline development. The results have advanced understanding of the function of Inx2 gap junction protein in two different somatic support cells. First, it was shown that Inx2 in IGS cells is required for survival of early germ cells and as well as for cyst formation. Second, it was demonstrated that Inx2 in follicle cells promotes egg chamber formation through the regulation of DE-cadherin. It has not been tested whether Inx2 forms functional gap junctions during early oogenesis. However, previous studies have demonstrated that gap junctions are formed between germ cells and somatic support cells including IGS cells. It is believed that the most plausible interpretation of these data is that Inx2 expressing in the somatic support cells forms functional gap junctions in order to regulate germline development. The findings strongly support a multifunctional role for Inx2 gap junctions in regulating these important developmental processes during early oogenesi (Mukai, 2011).

Inx2 gap junction protein in inner germarial sheath (IGS) cells is required for survival of early germ cells and for cyst formation. From analysis of inx2 expression and function, it is clear that inx2 acts in IGS cells but not in germ cells. The genetic interaction between inx2 and zpg therefore supports the view that Inx2/Zpg gap junctions between germ cells and IGS cells are required for survival of early germ cells. Inx2 protein in follicle cells is in close proximity to Zpg protein in germline cells (Bohrmann, 2008). Inx2 protein has been found to form the functional, voltage-gated, heterotypic gap junctions with Zpg in the paired Xenopus oocyte system. These data are consistent with the idea that Inx2 in IGS cells forms functional gap junctions with Zpg in germ cells. The mutant phenotypes of inx2FA42 are mainly associated with defects in cystoblast differentiation, whereas the phenotypes in zpg mutants are mostly associated with cystoblast survival. Thus, Inx2 expressed in IGS cells may also form a heterotypic junction with other Innexins expressed in germ cells to promote cyst formation. To investigate this possibility, further study will be needed in order to explore the function of other Innexins in germ cells (Mukai, 2011).

The requirement of inx2 for survival of early germ cells and for cyst formation indicates that Inx2 gap junctions may supply differentiating early germ cells with nutrients and signal molecules, to support their survival and differentiation into cysts, respectively. As cyst formation is affected in a weak allele of inx2 (inx2FA42) but the survival of early germ cells is not, it is concluded that inx2 activity required for cyst formation is higher than that for survival. Gap junctions are intercellular channels that allow passage of small molecules of up to 3 kDa in arthropods (Bohrmann, 2008). In some cases, even much larger molecules, such as calmodulin and siRNAs, are able to pass through gap junctions. The signal molecules involved in cyst formation may be larger than nutrient molecules. Moreover, these findings imply that the regulation of permeability of Inx2 gap junctions may be involved in cyst formation (Mukai, 2011).

A functional EGFR signaling pathway in escort cells is required for cyst formation. This study has shown that the expression of EGFR-CA is able to restore cyst formation in inx2FA42 mutants. This result is consistent with the idea that Inx2 in escort cells may act downstream of the EGFR/MAP-kinase pathway. In mammalian cells, MAP-kinase regulates gap junction permeability by phosphorylation of Connexins. Moreover, in mammalian ovaries, luteinizing hormone (LH), which acts on ovarian follicle cells to regulate oocyte development, causes MAP kinase-dependent phosphorylation and closure of Connexin 43 gap junctions in follicles. The EGFR/MAP-kinase pathway may regulate the permeability of Inx2 gap junctions between the escort cells and germ cells in order to control the transfer of signal molecules, which promote cyst formation. However, it is also plausible that EGFR signaling in escort cells is facilitated by inx2 function (Mukai, 2011).

The results raise the possibility that Inx2 gap junctions may mediate local signaling from germ cells to prefollicular cells in order to promote egg chamber formation. Because egg chamber formation is blocked by the large inx2- FSC clones, but not by the small FSC clones, it is concluded that inx2 function in a part of follicle cells is sufficient for egg chamber formation. This implies that egg chamber formation might occur through discrete steps: in a first step, Inx2 gap junctions mediate local signaling between the germ cells and prefollicular cells to trigger egg chamber formation; in the next step, other signaling among follicle cells coordinates the movement of follicle cells to implement egg chamber formation. As the expression of DE-cadherin is able to restore the association of follicle cells with germ cells in inx2FA42 mutants, Inx2 gap junctions may mediate signaling from germ cells into prefollicular cells to regulate DE-cadherin. The molecular nature of the signaling remains elusive (Mukai, 2011).

The finding that Inx2 gap junction protein promotes egg chamber formation through the regulation of DE-cadherin might have important implications regarding the mechanism of egg chamber formation. This is the first evidence of the functional link between the intercellular communication mediated by gap junctions and cell adhesion during egg chamber formation. Different concentrations of DE-cadherin drive a cell sorting process, which promotes the association of an oocyte with follicle cells. By analogy, inx2 may control a cell sorting process that facilitates the association of germ cells with follicle cells via accumulation of DE-cadherin. However, the mechanism by which Inx2 gap junctions regulate the behavior of DE-cadherin is unclear; both direct and indirect mechanisms are possible. The direct interaction between Inx2 and DE-cadherin may play a role in the accumulation of DE-cadherin at germ cell/follicle cell boundaries. The cytoplasmic loop of Inx2 protein directly interacts with the cytoplasmic domain of DE-cadherin. DE-cadherin is specifically co-immunoprecipitated by anti-Inx2 antibody from embryonic extracts (Bauer, 2004). But the interaction may be transiently required for the regulation of DE-cadherin in germarium region 2b, as inx2 is dispensable for apical localization of DE-cadherin in egg chamber follicle cells. Alternatively, Inx2 gap junctions may mediate signaling between the germ cells and prefollicular cells in order to regulate the cellular events that increase the amount of DE-cadherin. The argument in favor of a role of inx2 in signaling leading to DE-cadherin accumulation comes from mosaic analysis in which inx2 function in a subset of follicle cells is sufficient for egg chamber formation. Moreover, this study shows that Baz is a target gene of inx2. Baz interacts with Armadillo (Arm), which binds directly to DE-cadherin, and plays a key role in positioning the AJ. A signal mediated by Inx2 may regulate Baz accumulation at the junction in order to position DE-cadherin. The molecular mechanism by which Inx2 controls Baz remains to be investigated. As the expression of DE-cadherin is not sufficient for restoring egg chamber formation, the process must require the function of other genes, acting downstream of inx2 (Mukai, 2011).

This study shows inx2 expression in follicle cells in the germarium region, and reveals the requirement of inx2 for egg chamber formation. inx2 is also expressed in the precursor cells of the proventriculus, and is required for the folding and invagination of the proventriculus. Both wingless (wg) and hedgehoge (hh) signaling pathways activate inx2 expression in the proventriculus. These signaling pathways may regulate inx2 expression in the germarium region of the ovary, because these signaling pathways are involved in the formation of egg chambers. A small deletion was identified in the intron of the inx2 gene of inx2FA42 mutants. Signaling pathways such as wg and hh may activate inx2 transcription in germaria via a cis-element located in the intron of inx2; the identification and characterization of such a cis-element remains to be investigated (Mukai, 2011).

This study shows the requirement for inx2 function in cyst formation and egg chamber formation. However, whether functional gap junctions are required for the developmental processes was not demonstrated. It is worth noting that the action of inx2 on the processes might be independent of gap junction function. The studies using chemical inhibitors and activators of gap junction permeability and the identification of direct interactor proteins of Inx2 will facilitate understanding of the molecular mechanisms by which inx2 regulate these processes (Mukai, 2011).

In many animals, gametes develop within gonads, in which germ cells are linked with the surrounding somatic cells via gap junctions. Such gap junctions are essential for gametogenesis in both vertebrates and invertebrates. The results suggest that Inx2 gap junctions in somatic support cells control important developmental processes during early oogenesis through the regulation of intercellular communication and cell adhesion. It is anticipated that these results will facilitate better understanding of the molecular mechanisms that regulate gametogenesis (Mukai, 2011).

Cross regulation of intercellular gap junction communication and paracrine signaling pathways during organogenesis in Drosophila

The spatial and temporal coordination of patterning and morphogenesis is often achieved by paracrine morphogen signals or by the direct coupling of cells via gap junctions. How paracrine signals and gap junction communication cooperate to control the coordinated behavior of cells and tissues is mostly unknown. This study found that Hedgehog signaling is required for the expression of wingless and of Delta/Notch target genes in a single row of boundary cells in the foregut-associated proventriculus organ of the Drosophila embryo. These cells coordinate the movement and folding of proventricular cells to generate a multilayered organ. hedgehog and wingless regulate gap junction communication by transcriptionally activating the innexin2 gene, which encodes a member of the innexin family of gap junction proteins. In innexin2 mutants, gap junction-mediated cell-to-cell communication is strongly reduced and the proventricular cell layers fail to fold and invaginate, similarly as in hedgehog or wingless mutants. It was further found that innexin2 is required in a feedback loop for the transcriptional activation of the hedgehog and wingless morphogens and of Delta in the proventriculus primordium. It is proposed that the transcriptional cross regulation of paracrine and gap junction-mediated signaling is essential for organogenesis in Drosophila (Lechner, 2007).

In both vertebrates and invertebrates, the posterior foregut constitutes a center of organogenesis from which gut-associated organs such as the lung in vertebrates or the proventriculus in Drosophila develop. Proventriculus development involves the folding and invagination of epithelial cell layers to generate a multiply-folded organ. Two cell populations, the anterior and the posterior boundary cells, were shown previously to control cell movement and the folding of the proventriculus organ. In the posterior boundary cells, which organize the endoderm rim of the proventriculus, the JAK/STAT signaling cascade cooperates with Notch signaling to control the expression of the gene short stop encoding a cytoskeletal crosslinker protein of the spectraplakin superfamily. Thereby the Notch signaling pathway is connected to cytoskeletal organization in the posterior boundary cells, which have to provide a stiffness function to enable the invagination of the ectodermal foregut cells. The findings in this paper provide evidence that hedgehog is essential for the Notch signaling-dependent allocation of the anterior boundary cells. In amorphic hedgehog mutants, evagination and the formation of the constriction at the ectoderm/endoderm boundary are not affected, however, the inward movement of the anterior boundary cells is not initiated at the keyhole stage. The lack of cell movement of the ectodermal proventricular cells is consistent with the finding that hedgehog specifically controls Notch target gene activity in the anterior boundary cells. Genetic experiments further identify wingless as a target gene of hedgehog in the anterior boundary cells. wingless, in turn, controls the transcription of the innexin2 gene, which is expressed in the invaginating proventricular cells. When wingless is re-supplied in the genetic background of hedgehog mutants, innexin2 expression is rescued, providing further evidence that innexin2 is a target gene of wingless in the proventriculus primordium. Innexin2 encodes a member of the innexin family of gap junction proteins and is essential for the development of epithelial tissues (Bauer, 2004). In the proventriculus, innexin2 mRNA is initially expressed in the early evagination stage in a broad domain covering both the ectodermal and endodermal precursor cells of the proventriculus primordium. When the ectodermal cells start to invaginate into the proventricular endoderm, innexin2 expression is upregulated in the ectodermal cell layer. Invagination of the ectodermal cells fails in hedgehog, wingless and kropf mutant proventriculi and dye tracer injection experiments demonstrate that hedgehog and kropf mutants show a strong reduction of gap junction communication. These data suggest that the direct coupling of cells via Innexin2-containing gap junctions, which are induced in response to hedgehog and wingless activities, is important for the coordinated movement of the ectodermal cell layer. It is known from extensive studies in mammals that the coupling of cells and tissues via gap junctions enables the diffusion of second messengers, such as Ca2+, inositol-trisphosphate (IP3) or cyclic nucleotides to allow the rapid coordination of cellular behavior during morphogenetic processes such as cell migration and growth control. Cell movement and folding involves a modulation of cell adhesion and of cytoskeletal architecture of the proventricular cells. A functional interaction of innexin2 with the cell adhesion regulator DE-cadherin, which is a core component of adherens junctions has been shown recently by co-immunoprecipitation, yeast two-hybrid studies, and genetic analysis (Bauer, 2004). In mutants of DE-cadherin, Innexin2 is mislocalized and vice versa suggesting that the regulation of cell adhesion and gap junction-mediated communication may be linked. Similar evidence (Wei, 2005) for a coordinated regulation of connexin activity and N-cadherin has been obtained in mammals during migration of neural crest cells (Lechner, 2007).

In kropf mutants or innexin2 knockdown animals, hedgehog, wingless and Delta transcription is strongly reduced as shown by in situ hybridization and by quantitative RT PCR experiments using mRNAs isolated from staged embryos. Furthermore, hedgehog, wingless and Delta are ectopically expressed and their mRNA is upregulated in embryos in which innexin2 is overexpressed. In summary, these experiments provide strong support that the gap junction protein Innexin2 plays an essential role enabling or promoting transcriptional activation of hedgehog, wingless and Delta. These data point towards an essential requirement of gap junction communication for the transcriptional activation of morphogen-encoding genes activating evolutionary conserved signaling cascades essential for patterning in animals. It is of note that gap junctions are established at very early stages of embryonic development, correlating with a maternal and zygotic expression of innexin2 and other innexin family members. kropf mutant animals, which are devoid of maternal and zygotic innexin2 expression are early embryonic lethal and develop no epithelia (Bauer, 2004), consistent with a fundamental role of gap junctions in development, on top of which pattern formation of tissues and organs may occur. It has been shown previously that gap junctions are essential for C. elegans, Drosophila, and vertebrate embryogenesis from early stages onwards (Bauer, 2005 and Wei, 2004; Lechner, 2007 and references therein).

In the nematode C. elegans, a transient network formed by the innexin gap junction protein NSY-5 was recently shown to coordinate left-right asymmetry in the developing nervous system (Chuang, 2007). Previous findings in chick and Xenopus laevis embryos have suggested an essential role of connexin43-mediated gap junction for the determination of the left-right asymmetry of the embryos (Levin, 1999). Treatment of cultured chick embryos with lindane, which results in a decreased gap junctional communication, frequently unbiased normal left-right asymmetry of Sonic hedgehog and Nodal gene expression, causing the normally left-sided program to be recapitulated. An important role of connexin43 (Cx43)-dependent gap junction communication for sonic hedgehog expression was also observed in limb patterning of the chick wing (Law, 2002). Additionally, modulation of gap junctions in Xenopus embryos by pharmacological agents specifically induced heterotaxia involving mirror-image reversals of the heart, gut, and gall bladder. These data in combination with the current findings indicate that the transcriptional regulation of hedgehog and other morphogen-encoding genes by gap junction proteins may be evolutionary conserved between deuterostomes (vertebrates) and protostomes (Drosophila), although the Drosophila innexin gap junction genes share very little sequence homology with the connexin genes. The molecular mechanism underlying innexin2-mediated transcriptional regulation of hedgehog, wingless and Delta is not clear. It has been proposed that the nuclear localization of the carboxy-tail of connexin43 may exert effects on gene expression and growth in cardiomyocytes and HeLa cells. This would infer a cleavage of connexin43 to release the C-terminus, however, in vivo evidence for this event is still lacking. Sequence analysis reveals a nuclear receptor recognition motif within the C-terminus of Innexin2. It has been demonstrated that this recognition motif mediates the interaction of coactivators with nuclear receptors. However, there is no immunohistochemical evidence for a nuclear localization of Innexin2 or the Innexin2 C-terminus in Drosophila embryonic cells indicating that a direct involvement of Innexin2 in regulating transcription of target genes may not occur. The direct association of a transcription factor with gap junctions has been recently proposed for the mouse homolog of ZO-1-associated nucleic acid-binding protein (ZONAB). This transcription factor binds to ZO-1, which is associated with oligodendrocyte, astrocyte and retina gap junctions. It is possible that innexin2-dependent transcriptional regulation may involve a similar type of mechanism: a still unknown transcriptional regulator associated with the C-terminus of innexin2-containing gap junctions could be released upon modulation of gap junction composition thereby modulating the transcription of innexin2-dependent target genes (Lechner, 2007).

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. This study reports 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. These 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 (Lehmann, 2006).

Gap junctions in the ovary of Drosophila melanogaster: localization of innexins 1, 2, 3 and 4 and evidence for intercellular communication via innexin-2 containing channels

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, this study investigated the localization of innexins 1, 2, 3 and 4 using immunohistochemistry, and follicle development was analyzed following channel blockade. Innexin 1 predominantly localized to the baso-lateral domain of follicle cells, whereas innexin 2 is positioned apico-laterally as well as apically between follicle cells and germ-line cells. Innexin 3 was observed laterally in follicle cells and also in nurse cells, and innexin 4 was detected in the oolemma up to stage 8 and in nurse-cell membranes up to stage 12. In order to test whether innexins form channels suitable for intercellular communication, innexin antibodies were microinjected in combination with a fluorescent tracer into the oocyte of stage-10 follicles. Dye-coupling between oocyte and follicle cells was largely reduced by Innexin-2 antibodies directed against the intracellular C-terminus as well as against the intracellular loop. Analyzing in vitro, between stages 10 and 14, the developmental capacities of follicles following microinjections of Innexin-2 antibodies revealed defects in follicle-cell differentiation, nurse-cell regression, oocyte growth and choriogenesis. These results suggest that all analyzed innexins are involved in the formation of gap junctions in the ovary. While innexins 2 and 3 are colocalized between soma cells, Innexins 2 and 4 are colocalized between soma and germ-line cells. Innexin 2 is participating in cell-cell communication via hemichannels residing in the oolemma. It is obvious that gap-junctional communication between germ-line and soma cells is essential for several processes during oogenesis (Bohrmann, 2008).

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

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. This study reports 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 (Knieps, 2007).

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. innexin2 has been shown to play 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. This study used the Drosophila Schneider cell tissue culture system to further study the interaction of Innexin2 with DE-cadherin. The results provide evidence that DE-cadherin may be a key component to control trafficking, and localization of Innexin2 to the plasma membrane (Bauer, 2006).

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. The function and the distribution of Drosophila Innexin 2 protein was examined 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 of cellular distribution and protein-protein interactions of an Innexin gap junctional channel protein in the developing epithelia of Drosophila (Bauer, 2004).

The Drosophila gap junction channel gene innexin 2 controls foregut development in response to Wingless signalling

In invertebrates, the direct communication of neighbouring cells is mediated by gap junctions, which are composed of oligomers of the innexin family of transmembrane proteins. Studies of the few known innexin mutants in Drosophila and C. elegans have shown that innexin proteins, which are structurally analogous to the connexins in vertebrates, play a major structural role as gap junctional core components in electric signal transmission. This study shows that Drosophila innexin 2 mutants display a feeding defect that originates from a failure of epithelial cells to migrate and invaginate during proventriculus organogenesis. The proventriculus is a valve-like organ that regulates food passage from the foregut into the midgut. Immunohistological studies indicate that innexin 2 is functionally required to establish a primordial structure of the proventriculus, the keyhole, during the regionalisation of the embryonic foregut tube, which is under the control of Wingless and Hedgehog signalling. Genetic lack- and gain-of-function studies, and experiments in Drosophila tissue culture cells provide strong evidence that innexin 2 is a target gene of Wingless signalling in the proventricular cells. This is the first evidence that an invertebrate gap junction gene controls epithelial tissue and organ morphogenesis in response to the conserved WNT signalling cascade (Bauer, 2002).

Gap junction proteins are not interchangeable in development of neural function in the Drosophila visual system

Gap junctions (GJs) are composed of proteins from two distinct families. In vertebrates, GJs are composed of connexins; a connexin hexamer on one cell lines up with a hexamer on an apposing cell to form the intercellular channel. In invertebrates, GJs are composed of an unrelated protein family, the innexins. Different connexins have distinct properties that make them largely non-interchangeable in the animal. Innexins are also a large family with high sequence homology, and some functional differences have been reported. The biological implication of innexin differences, such as their ability to substitute for one another in the animal, has not been explored. Recently, it has been shown that GJ proteins are necessary for the development of normal neural transmission in the Drosophila visual system. Mutations in either of two Drosophila GJ genes (innexins), shakB and ogre, lead to a loss of transients in the electroretinogram (ERG), which is indicative of a failure of the lamina to respond to retinal cell depolarization. Ogre is required presynaptically and shakB(N) postsynaptically. Both act during development. This study asked if innexins are interchangeable in their role of promoting normal neural development in flies. Specifically, several innexins were tested for their ability to rescue shakB2 and ogre mutant ERGs; by and large innexins were found to be noninterchangeable. The protein regions required for this specificity were mapped by making molecular chimeras between shakBN and ogre and testing their ability to rescue both mutants. Each chimera rescued either shakB or ogre but never both. Sequences in the first half of each protein are necessary for functional specificity. Potentially crucial residues include a small number in the intracellular loop as well as a short stretch just N-terminal to the second transmembrane domain. Temporary GJs, possibly between the retina and lamina, may play a role in final target selection and/or chemical synapse formation in the Drosophila visual system. In that case, specificity in GJ formation or function could contribute, directly or indirectly, to chemical synaptic specificity by regulating which neurons couple and what signals they exchange. Cells may couple only if their innexins can mate with each other. The partially overlapping expression patterns of several innexins make this 'mix and match' model of GJ formation a possibility (Curtin, 2002a).

Gap junction proteins expressed during development are required for adult neural function in the Drosophila optic lamina

Evidence is provided that gap junction proteins, expressed during development, are necessary for the formation of normally functioning connections in the Drosophila optic lamina. Flies with mutations in the gap junction genes (innexins), shakingB, and ogre have normal photoreceptor potentials but a defective response of the postsynaptic cells in the optic lamina. This is indicated by a reduction in, or absence of, transients in the electroretinogram. Ogre is required in the presynaptic retinal photoreceptors. ShakingB(N) is, at a minimum, required in postsynaptic lamina neurons. Transgenic expression of the appropriate innexins during pupal development (but not later) rescues connection defects. Transient gap junctions have been observed to precede chemical synapse formation and have been hypothesized to play a role in connectivity and synaptogenesis; however, no causal role has been demonstrated. This study shows that developmental gap junction genes can be required for normally functioning neural connections to form (Curtin, 2002b).

Two Drosophila innexins are expressed in overlapping domains and cooperate to form gap-junction channels

Members of the innexin protein family are structural components of invertebrate gap junctions and are analogous to vertebrate connexins. This study investigated two Drosophila innexin genes, Dm-inx2 and Dm-inx3 and shows that they are expressed in overlapping domains throughout embryogenesis, most notably in epidermal cells bordering each segment. The gap-junction-forming capabilities of the encoded proteins was explored. In paired Xenopus oocytes, the injection of Dm-inx2 mRNA results in the formation of voltage-sensitive channels in only approximately 40% of cell pairs. In contrast, Dm-Inx3 never forms channels. Crucially, when both mRNAs are coexpressed, functional channels are formed reliably, and the electrophysiological properties of these channels distinguish them from those formed by Dm-Inx2 alone. These in vitro data were related to in vivo studies. Ectopic expression of Dm-inx2 in vivo has limited effects on the viability of Drosophila, and animals ectopically expressing Dm-inx3 are unaffected. However, ectopic expression of both transcripts together severely reduces viability, presumably because of the formation of inappropriate gap junctions. It is concluded that Dm-Inx2 and Dm-Inx3, which are expressed in overlapping domains during embryogenesis, can form oligomeric gap-junction channels (Stebbings, 2000).

Specificity of gene action during central nervous system development in Drosophila melanogaster: analysis of the lethal (1) optic ganglion reduced locus

A newly defined genetic locus designated lethal (1) optic ganglion reduced (l(1)ogre: 1-18.8, 6E1/2-6E4/5) is characterized. Four alleles have been isolated, one organismal viable and three organismal lethals. Histological analyses of these mutants at the light microscopic level have detected defects only in the developing and adult central nervous system (CNS). Examination of genetic mosaics suggests that the wild-type product of this locus may function specifically in the CNS. Analyses of staged material show that abnormalities first become apparent early in the larval period, indicating that the l(1)ogre+ gene product normally acts at or before this stage. No maternal effects were detectable. Determination of the temperature-sensitive period for lethality, of a temperature-sensitive heteroallelic combination, indicates that the l(1)ogre+ gene product also acts late in the larval period. These results show that the time of l(1)ogre+ gene action overlaps the period during which growth and assembly of the imaginal CNS occurs and are consistent with the hypothesis that l(1)ogre may act specifically in the imaginal CNS during its morphogenesis (Lipshitz, 1985).


Search PubMed for articles about Drosophila Inx-1 or Inx-2

Ayukawa, T., Matsumoto, K., Ishikawa, H. O., Ishio, A., Yamakawa, T., Aoyama, N., Suzuki, T. and Matsuno, K. (2012). Rescue of Notch signaling in cells incapable of GDP-L-fucose synthesis by gap junction transfer of GDP-L-fucose in Drosophila. Proc Natl Acad Sci U S A 109: 15318-15323. PubMed ID: 22949680

Bainton, R. J., Tsai, L. T., Schwabe, T., DeSalvo, M., Gaul, U. and Heberlein, U. (2005). moody encodes two GPCRs that regulate cocaine behaviors and blood-brain barrier permeability in Drosophila. Cell 123: 145-156. PubMed ID: 16213219

Bauer, R., Lehmann, C., Fuss, B., Eckardt, F. and Hoch, M. (2002). The Drosophila gap junction channel gene innexin 2 controls foregut development in response to Wingless signalling. J Cell Sci 115: 1859-1867. PubMed ID: 11956317

Bauer, R., Lehmann, C., Martini, J., Eckardt, F. and Hoch, M. (2004). Gap junction channel protein innexin 2 is essential for epithelial morphogenesis in the Drosophila embryo. Mol Biol Cell 15: 2992-3004. PubMed ID: 15047872

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Biological Overview

date revised: 18 February 2024

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