Walker-Warburg syndrome, caused by mutations in protein O-mannosyltransferase-1 (POMT1), is an autosomal recessive disorder characterized by severe brain malformation, muscular dystrophy, and structural eye abnormalities. As humans have a second POMT, POMT2, we cloned each Drosophila ortholog of the human POMT genes and carried out RNA interference (RNAi) knockdown to investigate the function of these proteins in vivo. Drosophila POMT2 (dPOMT2) RNAi mutant flies showed a "twisted abdomen phenotype," in which the abdomen is twisted 30 -60°, similar to the dPOMT1 mutant. Moreover, dPOMT2 interacted genetically with dPOMT1, suggesting that the dPOMTs function in collaboration with each other in vivo. We expressed dPOMTs in Sf21 cells and measured POMT activity. dPOMT2 transferred a mannose to the dystroglycan protein only when it was coexpressed with dPOMT1. Likewise, dPOMT1 showed POMT activity only when coexpressed with dPOMT2, and neither dPOMT showed any activity by itself. Each dPOMT RNAi fly totally reduced POMT activity, despite the specific reduction in the level of each dPOMT mRNA. The expression pattern of dPOMT2 mRNA was found to be similar to that of dPOMT1 mRNA using whole mount in situ hybridization. These results demonstrate that the two dPOMTs function as a protein O-mannosyltransferase in association with each other, in vitro and in vivo, to generate and maintain normal muscle development.

Heparan sulfate proteoglycan plays an important role in developmental processes by modulating the distribution and stability of the morphogens Wingless, Hedgehog, and Decapentaplegic. Heparan and chondroitin sulfates share a common linkage tetrasaccharide structure, GlcA␤1,3Gal␤1, 3Gal␤1,4Xyl␤-O-Ser. In the present study, we identified Drosophila proteoglycan galactosyltransferase II (d␤3GalTII), determined its substrate specificity, and performed its functional analysis by using RNA interference (RNAi) mutant flies. The enzyme transferred a galactose to Gal␤1,4Xyl-pMph, confirming that it is the Drosophila ortholog of human proteoglycan galactosyltransferase II. Real-time PCR analyses revealed that d␤3GalTII is expressed in various tissues and throughout development. The d␤3GalTII RNAi mutant flies showed decreased amounts of heparan sulfate proteoglycans. A genetic interaction of d␤3GalTII with Drosophila ␤1,4-galactoslyltransferase 7 (d␤4GalT7) or with six genes that encode enzymes contributing to the synthesis of glycosaminoglycans indicated that d␤3GalTII is involved in heparan sulfate synthesis for wing and eye development. Moreover, d␤3GalTII knockdown caused a decrease in extracellular Wingless in the wing imaginal disc of the third instar larvae. These results demonstrated that d␤3GalTII contributes to heparan sulfate proteoglycan synthesis in vitro and in vivo and also modulates Wingless distribution.

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