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We have devised a sensitive method for the isolation and structural analysis of glycosaminoglycans from two genetically tractable model organisms, the fruit fly, Drosophila melanogaster, and the nematode, Caenorhabditis elegans. We detected chondroitin/chondroitin sulfate-and heparan sulfate-derived disaccharides in both organisms. Chondroitinase digestion of glycosaminoglycans from adult Drosophila produced both nonsulfated and 4-O-sulfated unsaturated disaccharides, whereas only unsulfated forms were detected in C. elegans. Heparin lyases released disaccharides bearing N-, 2-O-, and 6-O-sulfated species, including mono-, di-, and trisulfated forms. We observed tissue-and stage-specific differences in both chondroitin sulfate and heparan sulfate composition in Drosophila. We have also applied these methods toward the analysis of tout-velu, an EXTrelated gene in Drosophila that controls the tissue distribution of the growth factor Hedgehog. The proteins encoded by the vertebrate tumor suppressor genes EXT1 and 2, show heparan sulfate co-polymerase activity, and it has been proposed that tout-velu affects Hedgehog activity via its role in heparan sulfate biosynthesis. Analysis of total glycosaminoglycans from tout-velu mutant larvae show marked reductions in heparan sulfate but not chondroitin sulfate, consistent with its proposed function as a heparan sulfate co-polymerase.Proteoglycans consisting of core proteins with glycosaminoglycan chains are abundant molecules, found both in the extracellular matrix and on the cell surface. These diverse molecules serve a wide range of functions, from affecting the compressive properties of cartilage to growth factor reception. Until recently, proteoglycans were studied principally in vertebrate systems. However, genetic experiments in the fruit fly, Drosophila melanogaster, established that proteoglycans, and their associated glycosaminoglycans, are required for normal development of this invertebrate model organism (reviewed in Ref. 1). A Drosophila member of the glypican family, division abnormally delayed (dally) 1 (2, 3), affects signaling mediated by two conserved growth factors, Wingless, a member of the Wnt family, and Decapentaplegic, a transforming growth factor-␤/bone morphogenetic protein-related protein (3, 4). Wnts and transforming growth factor-␤/bone morphogenetic proteins are important patterning molecules in vertebrate and invertebrate species, and studies of Drosophila and Caenorhabditis elegans have identified many of the evolutionarily conserved components of these signaling systems (5, 6).Mutations affecting genes encoding proteins related to known glycosaminoglycan biosynthetic enzymes have also been described in Drosophila. sugarless shows striking homology to UDP-glucose dehydrogenase (7-9) and affects signaling mediated by multiple growth factors, including Wingless, Decapentaplegic, and the fibroblast growth factor receptor-related proteins Heartless and Breathless (10). sulfateless encodes a protein similar to N-deacetylase/N-sulfotransferase an...

Glycosaminoglycans (GAGs) play a critical role in binding and activation of growth factors involved in cell signaling critical for developmental biology. The biosynthetic pathways for GAGs have been elucidated over the past decade and now analytical methodology makes it possible to determine GAG composition in as few as 10 million cells. A glycomics approach was used to examine GAG content, composition, and the level of transcripts encoding for GAG biosynthetic enzymes as murine embryonic stem cells (mESCs) differentiate to embryoid bodies (EBs) and to extraembryonic endodermal cells (ExE) to better understand the role of GAGs in stem cell differentiation. Hyaluronan synthesis was enhanced by 13-and 24-fold, most likely due to increased expression of hyaluronan synthase-2. Chondroitin sulfate (CS)/dermatan sulfate (DS) synthesis was enhanced by 4-and 6-fold, and heparan sulfate (HS) synthesis was enhanced by 5-and 8-fold following the transition from mESC to EB and ExE. Transcripts associated with the synthesis of the early precursors were largely unaltered, suggesting other factors account for enhanced GAG synthesis. The composition of both CS/DS and HS also changed upon differentiation. Interestingly, CS type E and highly sulfated HS both increase as mESCs differentiate to EBs and ExE. Differentiation was also accompanied by enhanced 2-sulfation in both CS/DS and HS families. Transcript levels for core proteins generally showed increases or remained constant upon mESC differentiation. Finally, transcripts encoding selected enzymes and isoforms, including GlcNAc-4,6-O-sulfotransferase, C5-epimerases, and 3-O-sulfotransferases involved in late GAG biosynthesis, were also enriched. These biosynthetic enzymes are particularly important in introducing GAG fine structure, essential for intercellular communication, cell adhesion, and outside-in signaling. Knowing the changes in GAG fine structure should improve our understanding the biological properties of differentiated stem cells.