Spatial Expression Pattern and Cellular Organisation of Gap Junctions in Third Instar Wing Imaginal Discs of Drosophila melanogaster

The Drosophila wing imaginal disc serves as a powerful model to study intercellular communication during development. In our study, we report and discuss the expression pattern and cellular distribution of innexin-1, innexin-2 and innexin-3 in the cells of the wing imaginal discs. Our immunohistochemical data show that all three innexins are broadly expressed across the membranes of both the disc proper and peripodial epithelial cells of the wing disc. The stainings further reveal that, within the disc proper epithelium, junctional proteins are arranged in a clear apico-basal hierarchy: cadherins at the apical surface, followed by septate junction proteins, with innexins localised sub-apically beneath these components. All three innexins are enriched within this sub-apical domain, and are additionally detected at mid-and baso-lateral sites in varying levels. Notably, innexin-2 exhibits partial colocalization with coracle, a septate junction-associated protein, suggesting a functional association. In the peripodial epithelium, innexins are detected in distinct punctate patterns across cell membranes, implying heterogeneity in their molecular characteristics. To validate these expression patterns, we carried out tissue-specific RNAi-mediated knockdowns using the pannier-Gal4 driver targeting the notum, a structurally and functionally important but underexplored region in innexin research. Knockdown of innexin-2 and innexin-3 led to complete loss of their expression within this region. Notably, silencing of innexin-2 also affected the expression of septate junction associated proteins and innexin-3 knockdown was accompanied by a significant reduction in disc size and altered morphology. These findings depict and confirm the presence of innexins in the notum region and also indicate that individual innexins may have distinct or shared functional roles within the same tissue domain of expression. Their localization to specific membrane domains is likely to underlie their differential modes of action. Although previous studies have demonstrated the functional involvement of gap junctions in various aspects of normal wing development in Drosophila, a description of the arrangement of innexins on the third instar wing discs is required for better understanding of their roles. Our study addresses this gap by providing a comprehensive analysis of the cellular localisation and organisation of gap junctions, specifically innexin-1,-2 and-3, within the third instar wing discs, thereby supporting and extending existing knowledge.