1. Essentials of Glycobiology May 16, 2002 Jeff Esko Lecture 28 Developmental glycobiology in model organisms: Drosophila and C. elegans

2. Worms and Flies - Ideal Model Organisms  Entire genomes are now available  19,099 genes in C.elegans, 959 cells, all fate mapped elegans.swmed.edu www.sanger.ac.uk  ~13,600 genes in D. melanogaster, www.fruitfly.org/  Can study differentiation, morphogenesis, and behavior C. elegans D. melanogaster

3. Few structures, lots of biology In comparison to vertebrate systems, few glycan structures are currently known in model organisms Homologs for most vertebrate “glyco” genes have been described, but in only a few cases have corresponding activities been demonstrated in vitro In many cases, developmental biologists have stumbled into glycobiology Reverse genetic methods (mutations, RNAi) allow one to dissect the importance of these genes in development

4. In Comparison, Vertebrate Systems... …have lots of glycans of known structure …have hundreds of genes cloned and biochemically characterized…. But, fewer genetic tools are available Reverse genetics is possible in mice, which provide models for human disease

5. Some people say that the best organism in the world to work on is the fruitfly, Drosophila melanogaster

6. Drosophila melanogaster Easily studied developmental program Complex neural system Behavior Obviously discernable phenotypes Virtually all vertebrate pathways of glycosylation are present, except…. No sialic acids or sialic acid binding proteins No acquired immunity Higher order brain functions absent Let’s look at some examples…….

7. Example 1: O-Mannosylation In bilateral animals, the left and right sides of the body usually have asymmetric structures. In Drosophila melanogaster, mutations in the rotated abdomen (rt) locus cause a clockwise helical rotation of the body Cuticular phenotypes of rt mutations (A) Cuticle of a third instar rt 2 hemizygous larvae showing a correct alignment of cuticle landmarks. (B) Ventral view of abdominal segments of an adult fly of the same genotype showing the staggering of sternites along the anterior/posterior axis.

8. rotated abdomen (rt) encodes a putative integral membrane glycoprotein homologous to yeast mannosyltransferases (Pmts) that utilize dolichol- P-Man for protein O-mannosylation…. ….but the activity has not yet been demonstrated in vitro nor has the substrates been identified in vivo Hydropathy plot

9. Example 2: O-Fucose Glycans - Notch, a cell surface receptor, is part of a multicomponent signaling system that regulates cell differentiation along tissue borders - Notch is expressed on one cell and notch ligands (Delta, Serrate and Scabrous) are presented by adjacent cells

10. …and Fringe - Fringe (fng) and Fringe Connection (frc) also influence the wing margin. Fringe expression boundaries coincide with Notch-dependent patterning centers and with Notch-ligand expression boundaries. - Fringe adds GlcNAc  1,3 to O-linked fucose on the EGF modules of Notch. - Fringe Connection encodes a multifunctional UDP-sugar transporter (UDP-GlcA, UDP-GlcNAc) Moloney et al (2000) Nature 406:369 Bruckner et al (2000) Nature 406:411 Selva et al (2001) Nat Cell Biol 3:809

11. Example 3: Genome Comparisons - Compare genes involved in vertebrate fucosylation to the D. Melanogaster genome - Fruit fly GDP-fucose formed exclusively by the de novo pathway from GDP-mannose. No orthologs for salvage pathway enzymes - Two novel fucosyltransferases predicted to catalyze  1,3- and  1,6-specific linkages by sequence homology - No genes encoding  1,2-specific fucosyltransferases - A fucosidase enzyme discovered - Two novel human genes putatively coding for new fucosyltransferases identified Roos et al (2002) J.Biol.Chem. 277:3168

12. Example 4: Glycome Comparisons Drosophila makes numerous glycolipids Notice that the second sugar is mannose instead of galactose as in vertebrate glycolipids Predict new glycosyltransferases Seppo & Tiemeyer (2000) 10:751

13. wild-typewingless Example 5: Proteoglycans - The fly body plan is divided into segments. Normally, each segment contains an anterior denticle band, and a more posterior region of naked cuticle. - In wingless mutants, the naked cuticle is absent, replaced by a disordered array of denticles

14. dally/dallydally/+ x sgl/+ -note loss of naked cuticle Dally Dally mutations delay key cell division events required for maturation of the eye, antennae, wings, etc. Dally mutants also have a modest wg-like patterning defect Dally shows genetic interactions with sgl (UDP-Glc dehydrogenase) based on the enhanced cuticle phenotype. UDP-Glc DH makes UDP-GlcA. - phenotype accentuated

15. sgl, sfl, and ttv define essential steps in heparan sulfate synthesis, suggesting that wingless requires heparan sulfate to bind to its receptor Mutations that enhance wingless phenotype dallyGlypican proteoglycan sugarless (sgl)UDP-glucose dehydrogenase that makes UDP-GlcA sulfateless (sfl)GlcNAc N-deacetylase/N- sulfotransferase tout-velu (ttv)Heparan sulfate copolymerase

16. Dally acts as a coreceptor Notice the similarity of this model to one proposed for FGF-2 signaling in vertebrate cells Signaling Event Mitogenesis FGF Heparan sulfate Proteoglycan FGF

17. Selective Effects of Mutants Another Gene? Chondroitin sulfate might substitute for heparan sulfate? Another Proteoglycan?

18. Vertebrate Mutants

19. Example 6: Morphogen gradients Loss of tout-velu function does not abrogate the ability of cells to respond to Hh Instead it alters the distribution of Hh, preventing its dispersal across domains of 10-12 cell diameters Proteoglycans may facilitate the diffusion of the morphogen or permit transcytosis

20. Hedgehog is produced posteriorly to anterior- posterior (A/P) boundary in wing imaginal disc and diffuses to anterior side (Blue lines indicate anterior/posterior boundary). Hh induces Patched expression (green) anterior to A/P boundary. Patched expression V D PA ptc clone

21. ttv clone anterior ttv clone posterior V D PA Notice that Patched is not induced to the anterior side of the ttv clone. However, when the ttv clone is located posteriorly, patched is induced. The ttv clone is acting like a barrier to Hh signaling on the anterior side

22. This time Patched is green, and Hh is stained red. The lack of ttv expression in the clone prevents Hh diffusion. ptc clone ttv/ptc clone Hedgehog Patched V D PA

23. Example 7: C. elegans sqv mutations In 1999, Herman and Horvitz described a set of mutants defective in vulval development (sqv, squashed vulva). These mutations affect epithelial invagination (and other developmental events) Of 8 complementation groups, 6 genes have been cloned sqv-2, sqv-3, sqv-5, sqv-6, sqv-7, and sqv-8. wild-typesqv

24. sqv Mutations Affect GAG Biosynthesis [GalNAc-GlcA] n -GalNAc-GlcA-Gal-Gal-Xyl-O-Ser

25. wild-type sqv One theory of cellular invagination is that the adjacent epithelial cells may secrete a polyanion in a polarized fashion Hydration of the matrix might cause expansion and an inward curvature of the cell layer Could the missing link be a chondroitin sulfate proteoglycan?

26. Vertebrate Mutants

27. Zebrafish —Zebrafish have transparent embryos which allows one to visualize development —Saturation mutagenesis has been undertaken to identify developmental phenotypes —Excellent model for vertebrate development —Disadvantage: Reverse genetics, but morpholinos can be used to inactivate genes

28. Example 8: Chitin —DG42, originally identified in Xenopus, is expressed between midblastula/neurulation stages DG42 shows homology to nodC and has chitin synthase activity ([GlcNAc  1,4GlcNAc] n ) —DG42 also has HA synthase activity, suggesting the possibility that small chitin oligosaccharides may act as a primer for HA synthesis —nodZ is a rhizobial gene that adds fucose  1,3 to chitin oligosaccharides —Injection of nodZ, antibodies to DG42, or chitinase causes defects in trunk and tail development

29. Zebrafish express many of the same genes found in higher vertebrates Zebrafish - jekyll (UDP-Glc dehydrogenase) mutations cause cardiac valve malformation - knypek, glypican homolog. Defects impair cellular movements during convergent extension, but not cell fates) - biglycan is present - chondroitin sulfate and chondroitinases inhibit axon growth