Innexin Gap Junctions

Rescue of the aberrant neuronal architecture phenotype exhibited in ogre mutants



Confocal images of wild type and ogre mutant brains visualising the mushroom body lobes using anti-fasII antibodies

Figure legend: The mushroom body lobe structures (area of the shaded box in the brain diagram) can be visualised by antibody staining to detect neuronal-specific proteins such as FasII. Left panel: FasII-stained (purple) mushroom body lobes of a wild type Drosophila brain showing normal lobe morphology. Right panel: FasII-stained lobes in an ogre mutant exhibit similar structural phenotypes to those identified using the 201Y-GAL4 / UAS-mCD8:GFP reporter genotype (see webpage ogre mutant - neuronal defects). These phenotypes include; weak reporter (FasII) staining in many of the lobe axons (hence the high background), thin α lobes, irregularly shaped β/γ lobes (arrowheads), punctate distribution of FasII in axons (more obvious in the thin α lobe and at the base of this lobe), β/γ-lobe axons cross the brain midline.



Rescue of ogre mutant neuronal and glial mushroom body phenotypes by transgenic expression of Ogre protein in glia

Figure legend: The structural phenotypes observed in ogre mutant mushroom body (MB) neurons and glia can be rescued by transgenically expressing wild type Ogre protein in glial cells. Left panel: Mushroom body lobes visualised by FasII antibody staining (purple) in an ogre mutant. The severe neuronal defects regularly observed in ogre mutants (Image: ogre mutant MB defects) are almost completely rescued due to the expression of UAS-ogre in glial cells under the transcriptional direction of the nrv2-GAL4 enhancer trap line (Sun et al, 1999). Occasionally, some mutant phenotypes still appear such as β/γ-lobe axons crossing the brain midline (arrowhead). Right panel: ogre mutant glia transgenically expressing wild type Ogre protein ensheath the mushroom body lobes (arrows). In the absence of transgenically provided Ogre protein these glia fail to ensheath mushroom body axonal tracts in ogre mutants. This is esier to visualise when focusing on specific brain parts, such as the pedunculus (Image: Rescue of glia ensheathing the pedunculus in ogre mutants).



The observation that an endogenous, neuronal-specific, cell adhesion protein such as FasII ( Fasciclin II, homologue of N-CAM Kurusu et al. 2002) exhibits aberrant expression and subcellular distribution in neurons in an ogre mutant (image at top of this page) suggests that loss of ensheathing glia from the mushroom body axonal tracts has dire consequences for the general cell biology of de-sheathed neurons. Many questions remain about the nature of glial and neuronal defects observed in ogre mutant animals:

  • What is the role of innexins such as Ogre in glia? Is it required for cell adhesion? Or formation of gap junctions that mediate spatial buffering of ions (Wallraff et al. 2006) via a pan-glial syncytium network?...Or something else?
  • If Ogre is required only in glial cells, why do some rescued brains (expressing wild type Ogre in the glia of ogre mutants) occasionally show structural defects? Here are three, out of many, possibilities: 1, There may be undetectably low levels of innexins in neurons that are essential for normal development/function. This theory could be addressed by expressing wild type Ogre protein in neurons (or both neurons and glia together?) of ogre mutants and looking for phenotypic rescue. 2, Alternatively, the level of Ogre expressed using the GAL4/UAS system may be variable and relative innexin subunit levels could be a very important determinant in mediating rescue. Attempts at rescuing the electroretinogram (ERG) of an ogre mutant by GAL4/UAS -mediated expression of wild type Ogre also notably achieved only partial rescue (Curtin et al, 2002). 3, Expressing transgenic innexins can generate phenotypes that could mask rescue. For example, overexpressing UAS-inx2 can disrupt cell polarity in embryonic epithelia (Bauer et al, 2004) and also causes defects in larval disc cells (Image: Hazards of innexin overexpression).
  • Inx2 and Inx3 are also expressed in glia. Will they prove to be as essential as Ogre for normal glial and neuronal structure?
  • Dominant gap junction mutants exist, both innexin (see Inx2 summary) and connexin. Presumably they would cause defects by a different mechanism than loss-of-function mutants (...the ogre mutant is essentially loss-of-function in neuropile glial cells due to its absence). What effects do dominant innexin mutants have on nervous system development?...and are these comparable phenotypically and mechanistically to those in vertebrates?
  • ogre mutant brains are much smaller than those of wild type animals. Are innexins essential for the proliferation/survival of neurons and/or glia? Obviously neuropile glia remain in the ogre mutant (Image: ogre mutant genotype) used in these studies...but cell numbers were not quantified and there could have been fewer than normal. What happens in other innexin (Inx2/Inx3) null mutants?
  • Neuropile glial processes accumulate at the brain midline in the ogre mutant. If Ogre is required for cell adhesion or cell guidance/recognition does it have to exist in the plasma membrane as part of a channel?
  • Gap junctions are required in vertebrate glia for the normal expression of other proteins such as glutamate transporters (Figiel et al. 2007). Is this also the case for innexins? Failure to remove excess glutamate from active glutamatergic neurons can result in neurotoxic effects - which might explain some of the structural phenotype observed in the ogre mutant (Images: glial defects, neuronal defects).

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This is the end of the Innexins in the nervous system section



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