Innexin Gap Junctions

Reduced Ogre protein expression in the brain of ogre mutant animals



Bright-field and confocal images of wild type and ogre mutant brains antibody stained for Ogre protein

Figure legend: ogre mutant (Image: ogre mutant genotype) brains are smaller than wild type brains and express less Ogre protein. Bright field photos of a wild type (top left) and an ogre mutant (top right) brain show that mutant brains and optic lobes (OL) (Image: ogre mutant optic lobe) are much smaller than those of normal animals. Antibody staining using an Ogre-specific rabbit polyclonal antibody reveals that Ogre protein (purple) is abundant near the outer surface of the wild type brain (bottom left) (Image: Innexins in putative surface glia). Focusing on the surface of an ogre mutant brain (bottom right) reveals that much less protein is present than in the wild type. The Ogre that remains in the mutant exists in a linear distribution corresponding to the outlines of perineurial cells. Within the mutant brain, Ogre protein cannot be detected.



Graph comparing the intensity of fluorescently detected Ogre protein in wild type and ogre mutant brains

Figure legend: The consistent difference in size between wild type and ogre mutant (Image: ogre mutant genotype) brains permitted an experiment where brains from both genotypes were mixed, antibody stained to detect Ogre protein, and then scanned using identical laser confocal microscope parameters. Post-scanning, the brains (and corresponding fluorescence measurements) of different genotypes could be distinguished based on brain size and morphology. Assuming that fluorescent intensity levels accurately reflect the quantity of Ogre protein present, then ogre mutant brains have approximately 50% less Ogre protein expressed near the brain surface than wild type brains (graph above). Most of the Ogre protein that remains in ogre mutant brains appears to be present in specific cells (right panel images). The outline of these cells indicates that they correspond to the large, flat, perineurial cells that cover the outer boundary of the brain and possibly contribute to the insect blood brain barrier (Carlson et al. 2000).



Optical sections through the midbrain of an ogre mutant and a wild type animal

Figure legend: Comparison of an optical section from an ogre mutant (left) and a wild type (right) brain. Both sections were scanned near the middle of the brains in the approximate area of the box-outline in the brain illustration above. The images reveal that Ogre protein (purple) is virtually absent from the brain interior of ogre mutants. Arrows point to spots of Ogre protein corresponding to cross-sections through perineurial cell boundaries - these appear to be the only cells in the mutant brain that retain high levels of Ogre expression and hence intense fluorescence (see image at top of page). Neuropile glial staining (green, nrv2-GAL4 / UAS-mCD8:GFP) is shown in the ogre mutant brain and reveals severely abnormal architecture (see ogre mutant - glial defects). Size bars = 20μm.


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