The organization of Drosophila wing epithelial cells after wing inflation
Upon emergence of Drosophila from the pupal case its wing exhibits a shriveled morphology. The wing tissue at this point is enclosed within a semi-constrained cuticle. As a final step in wing morphogenesis the fly pumps hemolymph into the compacted structure (Lehmacher et al. 2009) so that the inflation results in the formation of a wing blade.
It is possible to fix the epithelial cells of a freshly-inflated wing blade after creating small nicks along the wing margin. This permits immunolabeling procedures followed by the visualization of cellular details using confocal microscopy. Panel-A above shows the result of this "nick-approach" after which the fixed wing (4% Paraformaldehyde) was incubated with an anti-BetaPS-integrin MAb (6G11, DSHB, Univ. of Iowa, USA) first and then with AlexaFluor594 secondary antibody. This wing also expressed GFP (Kiger et al. 2007). Panel A'-A'': The betaPS-integrin adhesion sites localize to distinctive locations within the GFP-labeled epithelial cells. The numerous intercellular adhesion sites between the top and bottom cellular monolayers (Brower et al. 1995) are essential to preserve the structural integrity of the wing blade during the inflation process. Compromised adhesion can result in wing blisters (Walsh and Brown 1998).
Panel-B: Cellular arrangment within the monolayer of the new wing blade can also be revealed through visualizing the localization pattern of Neuroglian-GFP (Nrg-GFP), a protein-trap line (Morin et al. 2001). Neuroglian is an L1-type cell adhesion molecule. Its expression in the Manduca wing epithelial cells has been reported earlier, it localizes to the basal and lateral cell surfaces (Nardi 1994). The merged image above suggests that the pattern of Nrg-GFP-label has a noticeble level of correlation with the overtly visible hexagonal structures of the internal portions of the wing blade. The latter was visualized using phase-contrast illumination. Will mutants that exhibit abnormal NRG-GFP pattern result in the defective maturation of the wing blade structure?
Images above were obtained using Leica TCS SPE, SP2 confocal microscopes with a 40x (oil) objective.
Acknowledgement: Thanks to the Advanced Light Microscopy Unit, Center for Genomic Regulation (Barcelona, Spain) for the support and to Dr. Kiger (UC, Davis) and Dr. Cooley (Yale) for the kind donation of transgenic fly strains.
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