Specific glycan expression is an essential characteristic of developing tissues. Our molecular characterization of a mutation that abolishes neural-specific glycosylation in the Drosophila embryo demonstrates that cellular interactions influence glycan expression. The HRP epitope is an N-linked oligosaccharide expressed on a subset of neuronal glycoproteins. Embryos homozygous for the TM3 balancer chromosome lack neural HRP-epitope expression. Genetic and molecular mapping of the relevant locus reveals that Tollo/Toll-8, a member of the Toll-like receptor family, is altered on the TM3 chromosome. In wild-type embryos, Tollo/Toll-8 is expressed by ectodermal cells that surround differentiating neurons and precedes HRP-epitope appearance. Re-introduction of Tollo/Toll-8 into null embryos rescues neural-specific glycan expression. Thus, loss of an ectodermal cell surface protein alters glycosylation in juxtaposed differentiating neurons. The portfolio of expressed oligosaccharides in a cell reflects its identity and also influences its interactions with other cells and with pathogens. Therefore, the ability to induce specific glycan expression complements the previously identified developmental and innate immune functions of Toll-like receptors.

Addition of fucose (Fuc) to glycoprotein N-linked glycans or in O-linkage directly to Ser/Thr residues modulates specific cell-cell interactions and cell signaling events. Vertebrates and invertebrates add Fuc in α6-linkage to the reducing terminal N-acetylglucosamine residue of N-glycans. In Drosophila and other invertebrates, Fuc can also be added in α3-linkage to the same residue. These difucosylated N-glycans are recognized by anti-horseradish peroxidase (anti-HRP) antisera, providing a well-established marker for insect neural tissue. To understand the mechanisms and consequences of tissue-specific glycan expression, we identified a single α3-fucosyltransferase (FucTA) that produces the anti-HRP epitope in Drosophila embryos. FucTA transcripts are temporally and spatially restricted to cells that express the anti-HRP epitope and are missing in a mutant that lacks neural α3-fucosylation. Transgenic expression of FucTA, but not of any other candidate α3-fucosyltransferase, rescues the anti-HRP epitope in the embryonic nervous system of this mutant. Mass spectrometric characterization of the N-glycans of Drosophila embryos overexpressing FucTA confirms that this enzyme is indeed responsible for the biosynthesis of difucosylated glycans in vivo. Whereas ectopic expression of FucTA in the larval wing disc produces mild wing notching, the heterochronic, pan-neural expression of FucTA in early differentiating neurons generates neurogenic and cell migration phenotypes; this latter effect is associated with reduced GDP-Fuc levels in the embryo and indicates that the diversion of fucosylation resources towards fucosylation of N-glycans has an impact on developmental signaling associated with O-fucosylation.

SUMMARYPrecise glycan structures on specific glycoproteins impart functionalities essential for neural development. However, mechanisms controlling embryonic neural-specific glycosylation are unknown. A genetic screen for relevant mutations in Drosophila generated the sugar-free frosting (sff) mutant that reveals a new function for protein kinases in regulating substrate flux through specific Golgi processing pathways. Sff is the Drosophila homolog of SAD kinase, which regulates synaptic vesicle tethering and neuronal polarity in nematodes and vertebrates. Our Drosophila sff mutant phenotype has features in common with SAD kinase mutant phenotypes in these other organisms, but we detect altered neural glycosylation well before the initiation of embryonic synaptogenesis. Characterization of Golgi compartmentation markers indicates altered colocalization that is consistent with the detected shift in glycan complexity in sff mutant embryos. Therefore, in analogy to synaptic vesicle tethering, we propose that Sff regulates vesicle tethering at Golgi membranes in the developing Drosophila embryo. Furthermore, neuronal sff expression is dependent on transcellular signaling through a non-neural toll-like receptor, linking neural-specific glycan expression to a kinase activity that is induced in response to environmental cues.

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