Live imaging basement membrane assembly under the pupal notum epithelium

Basement membranes are sheet-like extracellular matrices containing Collagen IV, and they are conserved across the animal kingdom. Basement membranes usually line the basal surfaces of epithelia, where they contribute to structure, maintenance, and signaling. Although adult epithelia contact basement membranes, in early embryos the epithelia contact basement membranes only after basement membranes are assembled in embryogenesis. In Drosophila , the pupal notum epithelium is a useful model for live imaging epithelial cell behaviors, yet it is unclear when the basement membrane assembles in the pupa, as pupae are undergoing metamorphosis, similar to embryogenesis. To characterize the basement membrane in the pupal notum, we used spinning disk fluorescent microscopy to visualize Collagen IV subunit Vkg-GFP and adherens junction protein p120ctnRFP. Bright punctae of Vkg-GFP were observed in the X-Y plane, possibly representing Vkg-containing cells. We found that a thin continuous Vkg-containing basement membrane was evident at 14 h APF, which became more enriched with Vkg-GFP over the next 6 h, indicating the basement membrane is still assembling during that time. Live imaging of the pupal notum during this time could provide insight into formation, assembly, and repair of the basement membranes.