ogre is located at chromosomal position 6E4 on the X chromosome in an innexin gene cluster alongside inx2 and inx7. It is expressed throughout development: in oocyte follicle cells (Bohrmann and Zimmermann, 2008), embryonic epidermis, gut, salivary glands and ventral nerve chord (Stebbings et al, 2002), in larval wing and leg discs, in the pupal central nervous system (CNS) (Stebbings et al, 2002) and in the adult peripheral (retinal neurons - Curtin et al, 2002) and central nervous system (Ogre protein in the adult brain). Ogre protein may be required in adults as it is easily detectable using anti-Ogre antibodies in the CNS of five day old adults. The expression pattern reported by antisense-RNA probe in situ’s does not tally exactly with that obtained using Ogre-specific antibody – mRNA is detected in both the oocyte chamber and follicle cells (Stebbings et al, 2002) but anti-Ogre antibody reveals Ogre protein only in follicle cells (Bohrmann and Zimmermann, 2008)(Ogre distribution in the follicle). Also, ogre mRNA was not detected in photoreceptor neurons (Stebbings et al, 2002) but these cells express a reporter construct under the control of an ogre-GAL4 driver element containing 4.5kb of ogre upstream regulatory sequence (Curtin et al, 2002). The latter observations are from pharates and adults, respectively, and that could possibly explain the difference in expression.
Ogre cannot form channels on its own in the Xenopus oocyte system (Phelan and Starich et al, 2001). Assuming this reflects the situation in vivo then Ogre must interact with other members of the innexin protein family. Its expression pattern overlaps with those of Inx2+Inx3 protein in follicle cells (..although its subcellular distribution is restricted, which may keep it separate from other innexins), salivary glands (Innexins in salivary glands: Ogre vs. Inx2) and in the central nervous system (Innexins in the CNS: Ogre versus Inx2), however, other innexins may also be expressed in these tissues (….transcripts from all innexin genes are apparently present throughout larval discs (Stebbings et al, 2000)) so identifying Ogre’s channel-forming partners may not be all that simple. Further complicating the issue is the observation that, even within cells of a given Ogre-expressing tissue, its subcellular distribution can be restricted to specific regions of the plasma membrane that do not correspond to regions where co-expressed innexins reside. For example, in follicle cells Ogre is predominantly found at the baso-lateral end of cells whereas Inx2 is mostly apico-lateral (Bohrmann and Zimmermann, 2008, Distribution of innexins in the follicle ) and in embryonic hindgut epithelial cells Ogre is also baso-lateral but Inx2 is more apical (Bauer et al, 2004). Despite these differences, one observation indicates that Ogre may interact with Inx2 (….and Inx3, given that Inx2 and Inx3 appear to be mutually dependent (Lehmann et al, 2006)):- a myc-tagged Ogre protein colocalises with Inx3 and Inx2 (Ogre-myc + Inx2 colocalization) in salivary gland cells and both Ogre-myc and Inx2 co-localize in adult brain neuropile glia. No research group as yet reported any changes in Ogre subcellular distribution - similar to those seen for Inx3 (Lehmann et al, 2006) - in inx2 mutants, even though this might be expected if Ogre really does interact with Inx2. Another observation suggesting some type of link between Ogre and Inx2 has been reported (Curtin et al, 2002). Expression of UAS-Ogre in the eye can cause a rough eye phenotype using some driver lines (eg. GMR-GAL4) but co-expression of UAS-inx2 can suppress this phenotype. Unfortunately, experiments using the GAL4/UAS system can be difficult to interpret with regard to innexins, given different strengths of driver and expressor lines and the potential for innexins to induce cell morphology defects during development (Bauer et al, 2004, Hazards of overexpressing innexins using UAS/GAL4)…..without even taking into account interactions of the overexpressed proteins with each other and with endogenous innexins. Looking at wing development, co-expression of UAS-Ogre and UAS-inx2 in the wing margin leads to a very slight enhancement of a wing serration phenotype.
Despite the extensive distribution of Ogre expression throughout development (described above), no external defects have been reported for the ogreviable, or escapers from ogrelethal, mutants ie. wings and legs appear to be normal even though Ogre is present in potentially all disc cells (wing, leg, eye and (Stebbings et al, 2002)). Ogre may therefore be dispensable, or redundant (its loss can be compensated for by the activity of another innexin), during the development of discs. Currently it’s not known which of these possibilities explains the lack of external morphological phenotypes. The molecular basis of most ogre mutations is unknown and, since the focus of most published Ogre research has been on its role in neurodevelopment, it is not known whether Ogre expression remains in tissues other than the nervous system in the studied mutants. For example, the ogrejnl3 / Df(1)Sxlbt (An adult viable ogre genotype) genotype generated nervous system defects very similar to those described for ogrelethal escapers but retains some endogenous Ogre expression in a subset of nervous system cells (Ogre expression in an ogre mutant). Expression of Ogre in larval discs of this genotype hasn’t been looked at, but it might be present, and the adults display no external phenotypes. If one assumes that Ogre is not dispensable then what innexin(s) could compensate for its loss? Curtin et al, (2002) attempted to rescue an electroretinogram (ERG) phenotype displayed by ogre mutants by expressing various innexin family members in retinal and postsynaptic neurons of an ogre mutant using the GAL4/UAS system. They found that UAS-inx7 can rescue the phenotype to almost the same extent as UAS-ogre. Inx7 transcript is detected in larval discs and could possibly compensate for loss of Ogre, assuming the ERG rescue translates to other tissues - which isn’t known. The finding that one innexin could functionally replace another isn’t unanticipated, but could lead to problems in the future, as rescue experiments similar to that described above for the ERG phenotype are often used to demonstrate that a particular protein is required in a given cell type…as evidence in developmental studies for example….since we now know that innexins can be interchangeable in certain respects such studies will require more detailed analysis. The fact that only Inx7 can replace Ogre in the ERG system is slightly surprising though, given the 36% identity (based on an Ensembl blastp alignment) between these proteins. Also, for UAS-ogre to effect rescue it must be full length …including the C-terminal tail (Curtin et al, 2002). The C-tail of Inx7 has ten predicted phosphorylation sites and a PDZ domain whereas Ogre has only two predicted phosphorylation sites and no PDZ domain (Bauer et al, 2005).
One tissue in which Ogre is absolutely required for normal development, and perhaps for maintaining tissue function, is the nervous system where Ogre has been associated with a number of important, and distinct, processes;- cell proliferation (or possibly survival), a potential glial role and formation of transient electrical synapses in neurons prior to the establishment of chemical synapses. Ogre transcript is detectable in the embryonic ventral nerve chord (Stebbings et al, 2002) but its role at this stage has not been extensively studied and no defects are reported. Putative null alleles are lethal at the pharate stage (Lipshitz and Kankel, 1985) (…or late larval stage depending on who you read (Phelan and Starich et al, 2001)) and the first reported neurodevelopmental process requiring Ogre is in a population of neuroblasts in the larval optic lobe proliferation centers. The role of Ogre in these cells seems to be to promote proliferation or survival of the neuroblasts or their offspring as ogre mutants display severely reduced optic lobes (in fact the whole ogre brain is much smaller than wild type) as pharates and adults. No CNS defects are reported at all before the larval stage. To date (2009) no group appears to have asked whether Ogre’s function at this stage is primarily as a promoter of proliferation or cell survival. Interestingly, study of the germline-specific innexin, Inx4, suggests that germ-line stem cells (GSCs) can receive, and respond to, a proliferation-stimulating signal (Dpp) but their observed failure to increase in number results from cell death as cells move away from the stem cell niche (Gilboa et al, 2003). If the small optic lobes phenotype of ogre mutants is found to also be a cell survival, rather than cell proliferation issue, it would help to explain some of the other phenotypes observed in the ogre mutant nervous system such as the holes observed in the brain and ventral nerve chord of ogrelethal escapers (Lipshitz and Kankel, 1985).
A survival-promoting role for Ogre might also explain why neuropile glia in the adult brain, and the neuronal populations associated with them, are severely reduced in ogre mutant flies. In wild type animals Ogre protein (and Inx2 and Inx3) is abundant in cortex glia and neuropile glia (Adult brain glia: Ogre versus Inx2) and clusters of innexin-immunopositive staining are particularly strong in neuropile glial processes that ensheath discrete neuronal structures such as the central complex and mushroom bodies (Glia ensheathing the mushroom body pedunculus: Inx2 versus Ogre). When Ogre is genetically removed from these glia, for example in flies of genotype ogrejnl3 / Df(1)Sxlbt (and presumably in other ogre mutants), neuropile glial processes are essentially absent and those that remain look like they’re disintegrating. Also, the neuronal tracts that they would otherwise ensheath become reduced in size and exhibit abnormal morphology (Glia and neurons in the mushroom body pedunculus: ogre mutant versus wild type). These phenotypes can be rescued when UAS-ogre is expressed in glia driven by nrv2GAL4 (Sun et al,1999) ( Rescue of optic lobe glia, Rescue of brain glia and neuronal structure). Interestingly, the cortex glia are affected to a much lesser extent than neuropile glia, although we don’t know why. In the vertebrate brain the equivalent cell types to Drosophila neuropile glia are the oligodendrocytes and astrocytes which are coupled by gap junctions and form a pan-glial syncytium that may have a role in shuttling ions and neurotransmitters from areas of high concentration to areas of low concentration, hence preventing their build-up to toxic levels – a process referred to as ionic buffering (Wallraff et al, 2006). The extensive glial distribution of Ogre (…and Inx2 + Inx3 and who knows what other members of the innexin family…) suggests that a similar mechanism might exist in flies. Whether gap junctional coupling is essential for glial survival or only for glial processes like ionic buffering is an unanswered question but we already know that removal of just a single function from glia – neurotransmitter uptake – can lead to signs of neurodegeneration in associated neurons (microvacuolization and mitochondrial swelling, Rival et al, 2004) so loss of the whole glial network due to ogre mutation could theoretically underlie the severe phenotypes seen in associated neurons (Mushroom body neuronal morphology in an ogre mutant). It is noteworthy that Ogre protein or transcript has not been reported in developing mushroom body neurons, where neuronal defects have been mostly examined, so ogre mutant neural aberration is likely a secondary response to glial defects. The glial and neuronal defects could partly explain the reduced size of the brain and optic lobes of ogre mutant flies when considered in addition to the optic lobe proliferation/cell survival role discussed earlier. Indeed, if the promotion of cell survival is the primary function of Ogre then this might help our understanding of the many phenotypes observed in the ogre nervous system, - why the optic lobe cells don’t appear to proliferate; why glia degenerate (leading to subsequent loss of associated neurons); why holes appear in the nervous system and why only 1/54 ogre mosiac gynanders displayed holes (Lipshitz and Kankel, 1985)…if Ogre promotes cell survival then the non-mutant cells in the mosaic animal would outcompete the ogre mutant cells. One caveat of the proposed function for Ogre in glia is that we do not know if the nrv2GAL4 driver line used to effect rescue drives expression in the neuroblasts that give rise to neurons and glia. If it is, then the phenotypes observed in adult ogre brains most likely to stem from the proliferation defect.
The third reported role for Ogre in the nervous system is to form transient electrical synapses between neurons before the establishment of long lasting chemical synapses. Curtin et al, (2002) found that ogre mutant animals require Ogre expression during larval and pupal neural development ….but importantly, not afterwards… in order to rescue an electrophysiologically recorded phenotype that monitors chemical synapse activity between retinal neurons and their postsynaptic partners in adult flies. Rescue of the phenotype was achieved through UAS-ogre expression driven by the pan-neural line elavGAL4 or ogreGAL4 (created using 4.5kb of ogre upstream sequence). Ogre protein has not been observed specifically in neurons of the central nervous system (…with the proviso that if it is extremely transient then it might be missed) and a UAS-lacZ reporter driven by ogreGAL4 leaves no detectable β-galactosidase in the lamina cortex where postsynaptic neurons of the ERG circuit lie - despite lacZ perduring even when expression is only transient. The evidence so far suggests that Ogre is required transiently and is only expressed in neurons of the peripheral nervous system.