inx2 is one of the most extensively analysed of the Drosophila innexins using immunocytochemical, developmental, behavioural and electrophysiological techniques. It lies at chromosomal band position 6E4 in an innexin gene cluster including ogre and inx7 near the distal end of the X chromosome. Serendipitously, the region upstream of inx2 appears to be a hotspot for transposable element insertion and this has facilitated the generation of mutants such as the inx2kropf (Bauer et al, 2002) alleles studied using immunocytochemical and developmental techniques and inx2 homozygous-viable lines used for olfactory behavioural analysis (Sambandan et al, 2006). Inx2 is expressed at all stages of the flies life, from germ line cells (Tazuke et al, 2002, Stebbings et al, 2002) to embryo (Bauer et al, 2002), larva, pupa and adult (Sambandan et al, 2008, Inx in adult brain glia). In addition to germline and zygotic expression, a maternal contribution of inx2 mRNA is essential for embryonic development (Bauer et al, 2004). Embryos (inx2null) lacking the maternally inherited complement exhibit strong defects in the epidermis and many internal organs and display increased levels of cell death (Bauer et al, 2004).
The subcellular distribution if Inx2 protein is tissue dependant; it is primarily detected apico-laterally in follicle cells (Bohrmann and Zimmermann, 2008) and embryonic hindgut epithelia (Bauer et al, 2004) and baso-laterally in embryonic salivary gland cells (Bauer et al, 2004)….although, it looks more apically restricted in our embryonic salivary gland preps. The location (as well as the expression levels) of Inx2 within cells is dynamic. For example, Inx2 protein in Nurse cells is distributed within the cytoplasm but is transported into the oocyte after stage 11 as the nurse cells regress (Bohrmann and Zimmermann, 2008) and in pupal salivary glands Inx2 becomes much more basally-located than in embryonic salivary glands. inx2 mRNA is also spatially restricted in certain cells (apically in proventricular cells, Bauer et al, 2002) although the biological significance of this is currently not known.
Based on the subcellular distribution of Inx2 it has been proposed to interact with Zpg/Inx4 at the oolemma/follicle cell interface (Bohrmann and Zimmermann, 2008, Inx distribution in the follicle) and it has been reported (as a personal communication) that Inx2 expressing oocytes can couple to Zpg expressing oocytes in the Xenopus oocyte model (Tazuke et al, 2002) forming heterotypic holochannels. Inx2 also colocalises with Inx3 in a large number of tissues and the evidence suggests that together they form heteromeric channels; in the Xenopus oocyte model oocytes expressing both subunits couple, whereas Inx3 alone does not produce coupling (Inx2 can also form homomeric homotypic channels but these are electrophysiologically distinguishable from Inx2/Inx3 heteromeric channels) (Stebbings et al, 2000). Only a subset of the available Inx2 and Inx3 protein within a cell appears to colocalise (Bauer et al, 2004 and Lehmann et al, 2006) so it is likely that Inx2, and possibly Inx3 as well, interact with other innexin subunits to form channels with different conductivity (..or function?) at the plasma membrane. Alternatively, they could be "otherwise engaged" - interacting with other cell-cell junction components and not functioning as classical gap junctions…but it's anyones guess until more information becomes available. Inx2 and Inx3 are mutually dependent on each other for correct subcellular targeting. Knockdown of Inx3 protein levels via a specific snap-back RNAi results in cytoplasmic accumulation of Inx2, and Inx3 protein becomes distributed over the cell membrane (and possibly within the cytoplasm) when Inx2 is removed by mutation in inx2kropf embryos (Lehmann et al, 2006, Inx3 protein distribution in the posterior embryonic gut of an inx2null mutant). Two pieces of evidence suggest that Inx2 and Inx3 interact via their carboxy-termini; firstly, a surface plasmon resonance measurement technique reveals that their C-termini strongly interact and, secondly, a C-terminal-GFP-tagged Inx2 protein fails to colocalise with Inx3 (Lehmann et al, 2006), presumably due to steric interference between the C-tails. Broadly speaking, it appears that the targeting of Inx2 (and Inx3) to the plasma membrane and the formation of plaques are tied to the heteromerization of Inx2 and Inx3 via their C-termini. However, analysis of homo- (and hemi-) -zygous dominant Inx2 mutants possessing single substitutions in the second extracellular domains reveals similar defects in Inx2 and Inx3 targeting and plaque formation to those seen in inx2null and Inx3 RNAi knockdown experiments. Inx2 protein produced from Inx2TA181 and Inx2UA104 (originally referred to as Stout alleles; Florence and McGinnis, 1998) is full-length (it's detected by an antibody recognising the C-terminus) and is targeted to the plasma membrane (see images: Inx2 protein distribution in Inx2TA181 and Inx2UA104 and Inx3 protein in Inx2TA181 mutant ) but fails to form/or maintain plaques. Presumably, Inx2 and Inx3 subunits can still heteromerise (this is an assumption) in these mutants via their normal C-termini and they appear to reach the plasma membrane yet the protein distribution pattern is clearly abnormal (compared to wild type Inx2 and Inx3). Inx2 in known to interact with components of other cell-cell junctions such as the adherens junction-associated β-catenin (armadillo), DE-cadherin (shotgun) and a septate junction-associated membrane skeleton scaffolding protein 4.1 (coracle) in addition to components of the cytoskeleton (β-tubulin) (Bauer et al, 2004). Loss of, or defective, inx2 by mutation could prevent these interactions, resulting in failure of hemichannels to incorporate into junctions or form stable junctions and subsequent diffusion of innexin protein over the cell surface.
There is no reported biochemical or genetic evidence that Inx2 interacts with Ogre/Inx1 despite the numerous co-immunoprecipitation experiments reported by the Hoch lab (Bauer et al, 2004) but these two innexins are expressed in many overlapping tissues (Stebbings et al, 2002, Bohrmann and Zimmermann, 2008). Also, a myc-tagged Ogre protein colocalises with Inx2 in putative annular junctions observed in pupal salivary glands (Inx2/Ogre colocalisation in annular junctions) and in putative plaques located in neuropile glial cells of the adult brain (Inx2/Ogre in the pedunculus glial sheath). So, the potential for interaction between these two innexin subunits exists.
Analysis of inx2 mutants has identified a degree of crosstalk between gap junction and ligand/receptor based intercellular communication pathways at the transcriptional level (Lechner et al, 2007). In cells of the proventriculus, hedgehog (hh) induces wingless (wg) expression, which in turn can activate β-catenin (armadillo) to mediate inx2 expression (Bauer et al, 2002). Inx2 protein is then generated which promotes wg expression in the same cells (Lechner et al, 2007). Components of three paracrine signalling pathways (wg, hh and delta) can be transcriptionally up-regulated by overexpressing UAS-inx2 and downregulated by Inx2 knockdown using a UAS-snapback RNAi construct suggesting that the underlying mechanism permits crosstalk between gap junction signalling and potentially a whole range of paracrine pathways. The expression of Inx2 in embryonic salivary gland cells, and possibly most tissues, does not require hh. So, the crosstalk mechanism may allow coordinated regulation of gap junction- mediated and ligand/receptor pathway -mediated developmental processes (morphogenetic movement in the case of the proventriculus) in cells that are actively undergoing such processes, but the mechanism is apparently not "active" all cells. The intermediate molecules involved in the positive feedback loop are currently unknown and, although Lechner et al showed a decrease of gap junctional dye transfer in proventriculus cells lacking Inx2 protein (in inx2kropf and hh mutants), there is no direct evidence that a signal that passes through a channel is required, or that gap junction channel activity is involved, in the crosstalk process at all (…not that it would be an easy experiment…).
The proventriculus dye-transfer experiments are interesting in that they agree with observations made by Bohrmann and Zimmermann who subjected follicles to challenge using a variety of antibodies specific for different innexin subunits known to be expressed in the follicle and found that perturbing Inx2 led to the greatest decrease in Lucifer yellow dye transfer from the oocyte into neighbouring cells. This is despite the presence of other subunits that could theoretically form channels that could compensate for the loss of Inx2. Could Inx2 be a subunit of central importance for channel formation (at least in all the cell types analysed so far)? It would be interesting to perform similar experiments in a tissue where more cells were coupled with gap junctions (providing more extensive potential for dye transfer and the possibility of quantifying areas), such as the embryonic epithelium, to see if removal of any innexin subunit could permit a greater level of dye transfer than expected - essentially asking the question whether there are inhibitory innexin subunits with roles similar to the inhibitory connexin subunits (Carette et al, 2009).
The phenotypes arising from loss of Inx2 protein in inx2 mutants, and overexpression of UAS-inx2, have been studied at the cellular and whole-organism level. Embryos that lack both maternal and zygotic inx2 fail to produce normal epithelia and internal organs and have no cuticle. Epithelial cells become rounded as though they've lost polarity -this can be phenocopied by expressing UAS-inx2 in the epithelium (Bauer et al, 2004). The interaction between gap junction and adherens/septate junction subunits may underlie a loss of polarity particularly in Inx2 overexpression experiments where excess Inx2 could sequester molecules that it interacts with away from their normal target, indeed Bauer et al report that cadherin, β-catenin and coracle have altered subcellular distribution when Inx2 is lost or overexpressed. The cause of the phenotype is less easy to conceptualize in the inx2kropf null mutant as the other junctional molecules are free to form their respective structures (are not sequestered away from their appropriate targets). Bauer et al propose that Inx2 and the other junctional components it interacts with might be transported, and targeted, to the plasma membrane as a multiprotein structure. If this turns out to be true it would certainly help to explain why Inx2 protein loss, and Inx2 overexpression, can produce similar phenotypes (mislocalisation of cadherin, β-catenin and coracle and disrupted polarity). Connexins are also intimately involved with the formation/maintenance of tight junctions (the vertebrate analogue of septate junctions)( Maass et al, 2004, Morita et al, 2004) so there could be an evolutionarily conserved process involved. The phenotype of embryos lacking only zygotic inx2 expression are quite weak suggesting that maternally inherited Inx2 can perdure to quite late in embryogenesis (although, almost none is detected in wholemount antibody-stained preps; Inx2 in inx2null embryo). The mild phenotypes reported include failure of proventriculus morphogenesis (Bauer et al, 2002), slight aberration of ventricle denticle belts and embryonic head skeleton ( Florence and McGinnis, 1998 where Stout = Inx2dominant alleles), and late embryo/early larval lethality. Bauer et al (2004) also report the existence of holes in the inx2kropf embryonic epidermis, which is plausible given the phenotype observed in maternal+zygotic mutant embryos, however, these aren't obvious in the inx2F43 null allele (make your own decision - Inx3 in inx2F43 embryo). The embryonic phenotype of the dominant Inx2TA181 and Inx2UA104 alleles (Stout mutants in Florence and McGinnis, 1998; also see Inx2TA181 and Inx2UA104 substitution sites) appear to be identical to those of inx2 nulls. Inx2TA181/+ and Inx2UA104/+ heterozygous adults consistently display clubbed aristae and arched wings…which doesn't give much away about the function of the mutant proteins. However, an enhancer/suppressor screen for loci that genetically interact has identified an interaction between the dominant Inx2 alleles and mutants of the Na+/K+ ATPase α and β subunits suggesting that ionic homeostasis might be compromised by the mutant Inx2 proteins. Alternatively, this might turn out to functionally reveal an interaction between gap junctions and the components of other types of cellular junctions (Bauer et al (2004) has already described interactions of Inx2 protein with many sub-component proteins of septate and adherens junctions). The Na+/K+ ATPase is required for formation/maintenance of Drosophila septate junctions (Paul et al, 2003, Genova et al, 2003), although, the ion-pump activity does not seem to be involved in this process (Paul et al, 2007). Interestingly, the Na+/K+ ATPase inhibitor Ouabain exerts a strong influence on connexin-mediated gap junctional intercellular communication in in vitro experiments (Martin et al, 2004) so the relationship between gap junctions and the sodium pump may be evolutionarily conserved.
It is notable that some of adult phenotypes revealed by genetic interaction of dominant Inx2 and sodium pump mutants resemble weak Notch pathway mutant phenotypes (rough eyes, wing serration, aberrant leg joints). This is intriguing for three reasons; 1/ a functional link has already been made between paracrine (eg. Notch) and gap junction signalling in the embryo (Lechner et al, 2007), 2/ Inx2TA181 and Inx2UA104 display a complex genetic interaction with both the Notch receptor (Inx2TA181 interaction with N264-39 in the wing) and one of its ligands, Serrate (Inx2TA181 interaction with Ser1 in the wing), 3/ expression of UAS-inx2 in developing legs and wings induces phenotypes that also resemble Notch pathway mutants (UAS-inx2 expression in the wing, UAS-inx2 expression in the leg). It would come as no surprise that crosstalk between gap junction and paracrine signalling pathways occurs extensively throughout development, but currently there is little understanding of the underlying mechanism whereby genetic nulls, overexpression or gain-of-function (dominant alleles) can produce similar phenotypes. Is there is single “unifying” mechanism?...not necessarily, if there was, one might then expect different identified elements to genetically interact but there’s no obvious genetic interaction between UAS-inx2 induced phenotypes and Na+/K+ ATPase mutants.
Although inx2 is reportedly not expressed in the embryonic nervous system (Stebbings et al, 2002) it is found in the larval, pupal and adult central nervous system. Up to now (September 2009) there’s very little information on its role in this tissue, despite the functional similarities between innexin- and connexin-based gap junctions and hence the possibility to model connexin-based neurological diseases (Bergoffen et al, 1993) in the fly. inx2 mutant adult flies exhibit a decreased odor avoidance response when exposed to benzaldehyde (Sambandan et al, 2006) compared to isogenic control flies suggesting some role for inx2 in olfactory perception/processing. Wholemount anti-Inx2 antibody staining of adult brains reveals that Inx2 protein is mainly present in glial cells of the central nervous system but not in neurons (…so far, anyway…). It can be observed in the olfactory nerve connecting primary olfactory sensillae to the olfactory lobes, although it’s not known whether peripheral nervous system staining is of neuronal or glial origin. Inx2 is also detected in neuropile glia that ensheath the mushroom bodies (MBs) (Inx2 in adult brain glia). The MBs are higher order neural structures involved in olfactory learning (Connolly et al, 1996) and possibly other olfactory processing. inx2 mutants could have defects in glial development (similar to those observed in connexin deficient glia, Neuberg et al, 1998 and Menichella et al, 2003) which could exert secondary effects on the neural circuit underlying olfactory avoidance behaviour in Drosophila. Glia have a role in axonal guidance, ensuring neurons extend in the correct direction to synapse with their appropriate postsynaptic targets during the formation of neural circuits (Hidalgo et al, 2003) and are also essential for the correct activity of neurons (eg. by regulating glutamate flux (Rival et al, 2004, Figiel et al, 2007)). There is no evidence that inx2 is essential for glial function/development, but ogre definitely is (see; MB glia in ogre mutant, MB neurons in ogre mutant ) and there is a possibility that Inx2 and Ogre interact in glia based on their co-localization (Inx2 and Ogre colocalise in MB glia). Homozygous viable inx2 reduction-of-function mutants appear to behave in a ‘normal’ manner, broadly speaking (apart from olfactory avoidance behaviour), but heterozygotes of the dominant alleles, Inx2TA181/+ and Inx2UA104/+, are quite sluggish and these may be useful in analysing the role(s) of Inx2 in brain function.