In vivo activities of heparan sulfate differentially modified by NDSTs during development.

Heparan sulfate proteoglycans (HSPGs) serve as co-receptors for growth factor signaling during development. It is well known that the level and patterns of sulfate groups of heparan sulfate (HS) chains, or HS fine structures, have a major impact on HSPG function. On the other hand, the physiological significance of other structural features of HS, including NS/NA domain organization, remains to be elucidated. A blueprint of the HS domain structures is mainly controlled by HS N-deacetylase/N-sulfotransferases (NDSTs). To analyze in vivo activities of differentially modified HS, we established two knock-in (KI) Drosophila strains with the insertion of mouse Ndst1 (mNdst1) or Ndst2 (mNdst2) in the locus of sulfateless (sfl), the only Drosophila NDST. In these KI lines, mNDSTs are expressed from the sfl locus, in the level and patterns identical to the endogenous sfl gene. Thus, phenotypes of Ndst1 KI and Ndst2KI animals reflect the ability of HS structures made by these enzymes to rescue sfl mutation. Remarkably, we found that mNdst1 completely rescued the loss of sfl. mNdst2 showed a limited rescue ability, despite a higher level of HS sulfation compared to HS in mNdst1 KI. Our study suggests that independent of sulfation levels, additional HS structural features controlled by NDSTs play key roles during tissue patterning.

Differential heparan sulfate dependency of the Drosophila glypicans.

Heparan sulfate proteoglycans (HSPGs) are composed of a core protein and glycosaminoglycan (GAG) chains and serve as coreceptors for many growth factors and morphogens. To understand the molecular mechanisms by which HSPGs regulate morphogen gradient formation and signaling, it is important to determine the relative contributions of the carbohydrate and protein moieties to the proteoglycan function. To address this question, we generated ΔGAG alleles for dally and dally-like protein (dlp), two Drosophila HSPGs of the glypican family, in which all GAG-attachment serine residues are substituted to alanine residues using CRISPR/Cas9 mutagenesis. In these alleles, the glypican core proteins are expressed from the endogenous loci with no GAG modification. Analyses of the dallyΔGAG allele defined Dally functions that do not require heparan sulfate (HS) chains and that need both core protein and HS chains. We found a new, dallyΔGAG-specific phenotype, the formation of a posterior ectopic vein, which we have never seen in the null mutants. Unlike dallyΔGAG, dlpΔGAG mutants do not show most of the dlp null mutant phenotypes, suggesting that HS chains are dispensable for these dlp functions. As an exception, HS is essentially required for Dlp's activity at the neuromuscular junction. Thus, Drosophila glypicans show strikingly different levels of HS dependency. The ΔGAG mutant alleles of the glypicans serve as new molecular genetic toolsets highly useful to address important biological questions, such as molecular mechanisms of morphogen gradient formation.

Chondroitin sulfate is required for follicle epithelial integrity and organ shape maintenance in Drosophila.

Heparan sulfate (HS) and chondroitin sulfate (CS) are evolutionarily conserved glycosaminoglycans that are found in most animal species, including the genetically tractable model organism Drosophila. In contrast to extensive in vivo studies elucidating co-receptor functions of Drosophila HS proteoglycans (PGs), only a limited number of studies have been conducted for those of CSPGs. To investigate the global function of CS in development, we generated mutants for Chondroitin sulfate synthase (Chsy), which encodes the Drosophila homolog of mammalian chondroitin synthase 1, a crucial CS biosynthetic enzyme. Our characterizations of the Chsy mutants indicated that a fraction survive to adult stage, which allowed us to analyze the morphology of the adult organs. In the ovary, Chsy mutants exhibited altered stiffness of the basement membrane and muscle dysfunction, leading to a gradual degradation of the gross organ structure as mutant animals aged. Our observations show that normal CS function is required for the maintenance of the structural integrity of the ECM and gross organ architecture.

Regulation of morphogen pathways by a Drosophila chondroitin sulfate proteoglycan Windpipe.

Morphogens provide quantitative and robust signaling systems to achieve stereotypic patterning and morphogenesis. Heparan sulfate (HS) proteoglycans (HSPGs) are key components of such regulatory feedback networks. In Drosophila, HSPGs serve as co-receptors for a number of morphogens, including Hedgehog (Hh), Wingless (Wg), Decapentaplegic (Dpp) and Unpaired (Upd, or Upd1). Recently, Windpipe (Wdp), a chondroitin sulfate (CS) proteoglycan (CSPG), was found to negatively regulate Upd and Hh signaling. However, the roles of Wdp, and CSPGs in general, in morphogen signaling networks are poorly understood. We found that Wdp is a major CSPG with 4-O-sulfated CS in Drosophila. Overexpression of wdp modulates Dpp and Wg signaling, showing that it is a general regulator of HS-dependent pathways. Although wdp mutant phenotypes are mild in the presence of morphogen signaling buffering systems, this mutant in the absence of Sulf1 or Dally, molecular hubs of the feedback networks, produces high levels of synthetic lethality and various severe morphological phenotypes. Our study indicates a close functional relationship between HS and CS, and identifies the CSPG Wdp as a novel component in morphogen feedback pathways.