FIGURE 6.

Major O-linked glycans are enriched in the dorsal portion of the larval wing disc. Imaginal wing discs were harvested from wandering third instar larvae. At this stage of development, the wing disc is differentiated into distinct spatial compartments based on the expression of cellular differentiation markers. A, cells at the boundary between the dorsal and ventral compartments express the Wingless protein in response to activation of the Notch signaling pathway (DIC, differential interference contrast; anti-Wingless immunofluorescence). B, a total of 269 freshly harvested wing discs, including the associated peripodial membrane, were microdissected into dorsal and ventral halves roughly along the meridian defined by Wingless expression (pseudocolored for illustrative purposes). Following tissue delipidation, O-linked glycans were released by reductive β-elimination. Equal portions of the dorsal and ventral preparations were analyzed by NSI-MS and TIM. C, O-linked glycans are enriched in the dorsal portion of the wing disc. D, filtered TIM scans reveal that the O-Fuc trisaccharide (m/z = 722) is enriched by almost 4-fold in the dorsal disc compared with the ventral disc. Similar dorsal enrichment is detected for the glucuronylated linear core 1 trisaccharide (signature fragment at m/z = 477 from MS/MS at 752), but the branched isomer (signature fragment at m/z = 516 from MS/MS at 752) is enriched in the ventral disc.