Physiology and Pathophysiology of Heparan Sulfate in Animal Models: Its Biosynthesis and Degradation.

Heparan sulfate (HS) is a type of glycosaminoglycan that plays a key role in a variety of biological functions in neurology, skeletal development, immunology, and tumor metastasis. Biosynthesis of HS is initiated by a link of xylose to Ser residue of HS proteoglycans, followed by the formation of a linker tetrasaccharide. Then, an extension reaction of HS disaccharide occurs through polymerization of many repetitive units consisting of iduronic acid and N-acetylglucosamine. Subsequently, several modification reactions take place to complete the maturation of HS. The sulfation positions of N-, 2-O-, 6-O-, and 3-O- are all mediated by specific enzymes that may have multiple isozymes. C5-epimerization is facilitated by the epimerase enzyme that converts glucuronic acid to iduronic acid. Once these enzymatic reactions have been completed, the desulfation reaction further modifies HS. Apart from HS biosynthesis, the degradation of HS is largely mediated by the lysosome, an intracellular organelle with acidic pH. Mucopolysaccharidosis is a genetic disorder characterized by an accumulation of glycosaminoglycans in the body associated with neuronal, skeletal, and visceral disorders. Genetically modified animal models have significantly contributed to the understanding of the in vivo role of these enzymes. Their role and potential link to diseases are also discussed.

Selective Inhibition of Heparan Sulphate and Not Chondroitin Sulphate Biosynthesis by a Small, Soluble Competitive Inhibitor.

The glycosaminoglycan, heparan sulphate (HS), orchestrates many developmental processes. Yet its biological role has not yet fully been elucidated. Small molecule chemical inhibitors can be used to perturb HS function and these compounds provide cheap alternatives to genetic manipulation methods. However, existing chemical inhibition methods for HS also interfere with chondroitin sulphate (CS), complicating data interpretation of HS function. Herein, a simple method for the selective inhibition of HS biosynthesis is described. Using endogenous metabolic sugar pathways, Ac4GalNAz produces UDP-GlcNAz, which can target HS synthesis. Cell treatment with Ac4GalNAz resulted in defective chain elongation of the polymer and decreased HS expression. Conversely, no adverse effect on CS production was observed. The inhibition was transient and dose-dependent, affording rescue of HS expression after removal of the unnatural azido sugar. The utility of inhibition is demonstrated in cell culture and in whole organisms, demonstrating that this small molecule can be used as a tool for HS inhibition in biological systems.

Spatial, temporal and cell-type-specific expression profiles of genes encoding heparan sulfate biosynthesis enzymes and proteoglycan core proteins.

Heparan sulfate (HS) is a linear polysaccharide found in almost all animal cells and plays an important role in various biological processes. HS functions mainly via covalently binding to core proteins to form HS proteoglycans (HSPGs), which are heterogeneous in the lengths of the HS chain, the modifications on HS and the core proteins. The molecular mechanisms underlying HSPG heterogeneity, although widely studied, are not yet fully defined. The expression profiles of HS biosynthesis enzymes and HSPG core proteins likely contribute to the HSPG heterogeneity, but these expression profiles remain poorly characterized. To investigate the expression profiles of genes encoding HS biosynthesis enzymes and HSPG core proteins, we systematically integrated the publicly available RNA sequencing data in mice. To reveal the spatial expression of these genes, we analyzed their expression in 21 mouse tissues. To reveal the temporal expression of these genes, we analyzed their expression at 17 time points during the mouse forebrain development. To determine the cell-type-specific expression of these genes, we obtained their expression profiles in 23 cell types in the mouse cerebral cortex by integrating single nucleus RNA sequencing data. Our findings demonstrate the spatial, temporal and cell-type-specific expression of genes encoding HS biosynthesis enzymes and HSPG core proteins and represent a valuable resource to the HS research community.

Heparan sulfate is essential for thymus growth.

Thymus organogenesis and T cell development are coordinated by various soluble and cell-bound molecules. Heparan sulfate (HS) proteoglycans can interact with and immobilize many soluble mediators, creating fields or gradients of secreted ligands. While the role of HS in the development of many organs has been studied extensively, little is known about its function in the thymus. Here, we examined the distribution of HS in the thymus and the effect of its absence on thymus organogenesis and T cell development. We found that HS was expressed most abundantly on the thymic fibroblasts and at lower levels on endothelial, epithelial, and hematopoietic cells. To study the function of HS in the thymus, we eliminated most of HS in this organ by genetically disrupting the glycosyltransferase Ext1 that is essential for its synthesis. The absence of HS greatly reduced the size of the thymus in fetal thymic organ cultures and in vivo, in mice, and decreased the production of T cells. However, no specific blocks in T cell development were observed. Wild-type thymic fibroblasts were able to physically bind the homeostatic chemokines CCL19, CCL21, and CXCL12 ex vivo. However, this binding was abolished upon HS degradation, disrupting the CCL19/CCL21 chemokine gradients and causing impaired migration of dendritic cells in thymic slices. Thus, our results show that HS plays an essential role in the development and growth of the thymus and in regulating interstitial cell migration.

The effect of electrospun scaffolds on the glycosaminoglycan profile of differentiating neural stem cells.

The use of electrospun scaffolds for neural tissue engineering applications allows a closer mimicry of the native tissue extracellular matrix (ECM), important for the transplantation of cells in vivo. Moreover, the role of the electrospun fiber mat topography on neural stem cell (NSC) differentiation remains to be completely understood. In this work REN-VM cells (NSC model) were differentiated on polycaprolactone (PCL) nanofibers, obtained by wet/wet electrospinning, and on flat glass lamellas. The obtained differentiation profile of NSCs was evaluated using immunofluorescence and qPCR analysis. Glycosaminoglycan (GAG) analysis was successfully emplyed to evaluate changes in the GAG profile of differentiating cells through the use of the highly sensitive liquid chromatography-tandem mass/mass spectrometry (LC-MS/MS) method. Our results show that both culture platforms allow the differentiation of REN-VM cells into neural cells (neurons and astrocytes) similarly. Moreover, LC-MS/MS analysis shows changes in the production of GAGs present both in cell cultures and conditioned media samples. In the media, hyaluronic acid (HA) was detected and correlated with cellular activity and the production of a more plastic extracellular matrix. The cell samples evidence changes in chondroitin sulfate (CS4S, CS6S, CS4S6S) and heparan sulfate (HS6S, HS0S), similar to those previously described in vivo studies and possibly associated with the creation of complex structures, such as perineural networks. The GAG profile of differentiating REN-VM cells on electrospun scaffolds was analyzed for the first time. Our results highlight the advantage of using platforms obtain more reliable and robust neural tissue-engineered transplants.

ER-Golgi dynamics of HS-modifying enzymes via vesicular trafficking is a critical prerequisite for the delineation of HS biosynthesis.

The cell surface and extracellular matrix polysaccharide, heparan sulfate (HS) conveys chemical information to control crucial biological processes. HS chains are synthesized in a non-template driven process mainly in the Golgi apparatus, involving a large number of enzymes capable of subtly modifying its substitution pattern, hence, its interactions and biological effects. Changes in the localization of HS-modifying enzymes throughout the Golgi were found to correlate with changes in the structure of HS, rather than protein expression levels. Following BFA treatment, the HS-modifying enzymes localized preferentially in COPII vesicles and at the trans-Golgi. Shortly after heparin treatment, the HS-modifying enzyme moved from cis to trans-Golgi, which coincided with increased HS sulfation. Finally, it was shown that COPI subunits and Sec24 gene expression changed. Collectively, these findings demonstrate that knowledge of the ER-Golgi dynamics of HS-modifying enzymes via vesicular trafficking is a critical prerequisite for the complete delineation of HS biosynthesis.

Heparan sulfate fine-tunes stromal-epithelial communication in the prostate gland.

Several studies reported the concerted and mutual communication between the prostate epithelium and stroma, which determines the final organ architecture and function, but gets awry in cancer. Deciphering the mechanisms involved in this communication is crucial to find new therapeutic strategies. HS sequesters a number of secreted growth factors and cytokines, controlling their bioavailability to the target cells, suggesting that HS is an important regulator of the extracellular matrix (ECM) and a key player in the cell-cell and cell-microenvironment communication during prostate morphogenesis and physiology. We propose that by controlling HS biosynthesis and sulfation pattern, as well as the cleavage of the HS chain and/or the shedding of proteoglycans, epithelial and stromal cells are able to precisely tune the availability of signaling molecules and modulate ligand-receptor interaction and intracellular signal transduction.

Chemical O-sulfation of N-sulfoheparosan: a route to rare N-sulfo-3-O-sulfoglucosamine and 2-O-sulfoglucuronic acid.

Heparosan, the capsular polysaccharide of E. coli K5 is currently used as the starting material in the chemoenzymatic synthesis of heparan sulfate and the structurally related anticoagulant drug heparin. Base hydrolysis of N-acetyl groups and their subsequent N-sulfonation, are used to prepare N-sulfoheparosan an intermediate of biosynthesis. In the present study, when excess sulfonation reagent was used during N-sulfonation, some O-sulfation also took place in the N-sulfoheparosan product. After a nearly full digestion, a hexasaccharide fraction exhibited resistance to heparin lyase II. Excessive digestion by heparin lyase II and structural identification by NMR and mass spectroscopy indicated that the resistant hexasaccharide fraction has two structures, ΔUA-GlcNS-GlcA2S-GlcNS-GlcA-GlcNS and ΔUA-GlcNS-GlcA- GlcNS3S-GlcA-GlcNS in similar amounts. The 2-sulfated structure exhibited partial resistance to heparin lyase II; however the structure of ΔUA-GlcNS-GlcA-GlcNS3S was completely resistant to heparin lyase II.

ZNF263 is a transcriptional regulator of heparin and heparan sulfate biosynthesis.

Heparin is the most widely prescribed biopharmaceutical in production globally. Its potent anticoagulant activity and safety makes it the drug of choice for preventing deep vein thrombosis and pulmonary embolism. In 2008, adulterated material was introduced into the heparin supply chain, resulting in several hundred deaths and demonstrating the need for alternate sources of heparin. Heparin is a fractionated form of heparan sulfate derived from animal sources, predominantly from connective tissue mast cells in pig mucosa. While the enzymes involved in heparin biosynthesis are identical to those for heparan sulfate, the factors regulating these enzymes are not understood. Examination of the promoter regions of all genes involved in heparin/heparan sulfate assembly uncovered a transcription factor-binding motif for ZNF263, a C2H2 zinc finger protein. CRISPR-mediated targeting and siRNA knockdown of ZNF263 in mammalian cell lines and human primary cells led to dramatically increased expression levels of HS3ST1, a key enzyme involved in imparting anticoagulant activity to heparin, and HS3ST3A1, another glucosaminyl 3-O-sulfotransferase expressed in cells. Enhanced 3-O-sulfation increased binding to antithrombin, which enhanced Factor Xa inhibition, and binding of neuropilin-1. Analysis of transcriptomics data showed distinctively low expression of ZNF263 in mast cells compared with other (non-heparin-producing) immune cells. These findings demonstrate a novel regulatory factor in heparan sulfate modification that could further advance the possibility of bioengineering anticoagulant heparin in cultured cells.

Heparan Sulfate Structure Affects Autophagy, Lifespan, Responses to Oxidative Stress, and Cell Degeneration in Drosophila parkin Mutants.

Autophagy is a catabolic process that provides cells with energy and molecular building blocks during nutritional stress. Autophagy also removes misfolded proteins and damaged organelles, a critical mechanism for cellular repair. Earlier work demonstrated that heparan sulfate proteoglycans, an abundant class of carbohydrate-modified proteins found on cell surfaces and in the extracellular matrix, suppress basal levels of autophagy in several cell types during development in Drosophila melanogaster In studies reported here, we examined the capacity of heparan sulfate synthesis to influence events affected by autophagy, including lifespan, resistance to reactive oxygen species (ROS) stress, and accumulation of ubiquitin-modified proteins in the brain. Compromising heparan sulfate synthesis increased autophagy-dependent processes, evident by extended lifespan, increased resistance to ROS, and reduced accumulation of ubiquitin-modified proteins in the brains of ROS exposed adults. The capacity of altering heparan sulfate biosynthesis to protect cells from injury was also evaluated in two different models of neurodegeneration, overexpression of Presenilin and parkin mutants. Presenilin overexpression in the retina produces cell loss, and compromising heparan sulfate biosynthesis rescued retinal patterning and size abnormalities in these animals. parkin is the fly homolog of human PARK2, one of the genes responsible for juvenile onset Parkinson's Disease. Parkin is involved in mitochondrial surveillance and compromising parkin function results in degeneration of both flight muscle and dopaminergic neurons in Drosophila Altering heparan sulfate biosynthesis suppressed flight muscle degeneration and mitochondrial dysmorphology, indicating that activation of autophagy-mediated removal of mitochondria (mitophagy) is potentiated in these animals. These findings provide in vivo evidence that altering the levels of heparan sulfate synthesis activates autophagy and can provide protection from a variety of cellular stressors.

By-Products of Heparin Production Provide a Diverse Source of Heparin-like and Heparan Sulfate Glycosaminoglycans.

Global production of pharmaceutical heparin (Hp) is increasing, and the production process from raw mucosal material results in large amounts of waste by-products. These contain lower sulfated Hp-like and heparan sulfate (HS), as well as other glycosaminoglycans, which are bioactive entities with pharmaceutical potential. Here we describe the first purification, structural and functional characterisation of Hp-like and HS polysaccharides from the four major by-product fractions of standard heparin production. Analysis of the by-products by disaccharide composition analysis and NMR demonstrated a range of structural characteristics which differentiate them from Hp (particularly reduced sulfation and sulfated disaccharide content), and that they are each distinct. Functional properties of the purified by-products varied, each displaying distinct anticoagulant profiles in different assays, and all exhibiting significantly lower global and specific inhibition of the coagulation pathway than Hp. The by-products retained the ability to promote cell proliferation via fibroblast growth factor receptor signalling, with only minor differences between them. These collective analyses indicate that they represent an untapped and economical source of structurally-diverse Hp-like and HS polysaccharides with the potential for enhancing future structure-activity studies and uncovering new biomedical applications of these important natural products.

Glycosaminoglycans are differentially involved in bacterial binding to healthy and cystic fibrosis lung cells.

BACKGROUND: Glycosaminoglycans (GAGs) are essential in many infections, including recurrent bacterial respiratory infections, the main cause of mortality in cystic fibrosis (CF) patients. METHODS: Using a cellular model of healthy and CF lung epithelium, a comparative transcriptomic study of GAG encoding genes was performed using qRT-PCR, and their differential involvement in the adhesion of bacterial pathogens analyzed by enzymatic degradation and binding competition experiments. RESULTS: Various alterations in gene expression in CF cells were found which affect GAG structures and seem to influence bacterial adherence to lung epithelium cells. Heparan sulfate appears to be the most important GAG species involved in bacterial binding. CONCLUSIONS: Adherence to lung epithelial cells of some of the main pathogens involved in CF is dependent on GAGs, and the expression of these polysaccharides is altered in CF cells, suggesting it could play an essential role in the development of infectious pathology.

Downstream Products are Potent Inhibitors of the Heparan Sulfate 2-O-Sulfotransferase.

Heparan Sulfate (HS) is a cell signaling molecule linked to pathological processes ranging from cancer to viral entry, yet fundamental aspects of its biosynthesis remain incompletely understood. Here, the binding preferences of the uronyl 2-O-sulfotransferase (HS2ST) are examined with variably-sulfated hexasaccharides. Surprisingly, heavily sulfated oligosaccharides formed by later-acting sulfotransferases bind more tightly to HS2ST than those corresponding to its natural substrate or product. Inhibition assays also indicate that the IC50 values correlate simply with degree of oligosaccharide sulfation. Structural analysis predicts a mode of inhibition in which 6-O-sulfate groups located on glucosamine residues present in highly-sulfated oligosaccharides occupy the canonical binding site of the nucleotide cofactor. The unexpected finding that oligosaccharides associated with later stages in HS biosynthesis inhibit HS2ST indicates that the enzyme must be separated temporally and/or spatially from downstream products during biosynthesis in vivo, and highlights a challenge for the enzymatic synthesis of lengthy HS chains in vitro.

Increased soluble heterologous expression of a rat brain 3-O-sulfotransferase 1 - A key enzyme for heparin biosynthesis.

Heparan sulfate (HS), is a glycosaminoglycan (GAG) involved in various biological processes, including blood coagulation, wound healing and embryonic development. HS 3-O-sulfotransferases (3-OST), which transfer the sulfo group to the 3-hydroxyl group of certain glucosamine residues, is a key enzyme in the biosynthesis of a number of biologically important HS chains. The 3-OST-1 isoform is one of the 7 known 3-OST isoforms and is important for the biosynthesis of anticoagulant HS chains. In this study, we cloned 3-OST-1 from the rat brain by reverse transcription-polymerase chain reaction (RT-PCR). After codon optimization and removal of the signal peptide, the recombinant plasmid was transformed into Escherichia coli BL21 (DE3) to obtain a His tagged-3-OST-1 fusion protein. SDS-PAGE analysis showed that the expressed 3-OST-1 was mainly found in inclusion bodies. The 3-OST-1 was purified by Ni affinity column and refolded by dialysis. The activity of obtained 3-OST-1 was 0.04 U/mL with a specific activity of 0.55 U/mg after renaturation. Furthermore, a co-expressed recombinant plasmid pET-28a-3-OST-1 with the chaperone expression system (pGro7) was constructed and transferred to E. coli BL21 (DE3) to co-express recombinant strain E. coli BL21 (DE3)/pET-28a-3-OST-1 + pGro7. The soluble expression of 3-OST-1 was significantly improved in the co-expressed recombinant strain, with enzyme activity reaching 0.06 U/mL and having a specific activity of 0.83 U/mg. N-sulfo, N-acetylheparosan (NSNAH) was modified by the recombinant expressed 3-OST-1 and the product was confirmed by 1H NMR showing the sulfo group was successfully transferred to NSNAH.

Controlled Chemoenzymatic Synthesis of Heparan Sulfate Oligosaccharides.

A chemoenzymatic approach has been developed for the preparation of diverse libraries of heparan sulfate (HS) oligosaccharides. It employs chemically synthesized oligosaccharides having a chemical entity at a GlcN residue, which in unanticipated manners influences the site of modification by NST, C5-Epi/2-OST and 6-OST1 /6-OST3 , thus resulting in oligosaccharides differing in N/O-sulfation and epimerization pattern. The enzymatic transformations defined fine substrate requirements of NST, C5-Epi, 2-OST, and 6-OST.

Exostosin-like 2 regulates FGF2 signaling by controlling the endocytosis of FGF2.

BACKGROUND: Heparan sulfate proteoglycans are ubiquitously expressed on cell surfaces and in extracellular matrices, and are engaged in heparin-binding growth factor-related signal transduction. Thus, changes in the amounts, structures, and chain lengths of heparan sulfate have profound effects on aspects of cell growth controlled by heparin-binding growth factors such as FGF2. Exostosin glycosyltransferases (EXT1, EXT2, EXTL1, EXTL2, and EXTL3) control heparan sulfate biosynthesis, and the expression levels of their genes regulate the amounts, chain lengths, and sulfation patterns of heparan sulfate. Unlike EXT1, EXT2, and EXTL3, EXTL2 functions chain termination of heparan sulfate. Here, we examined the importance of EXTL2 in FGF2-dependent signaling. METHODS: We investigated heparan sulfate biosynthesis and FGF2 signaling using four cell lines, EXT1-deficient cells, EXT2-, EXTL2-, or EXTL3-knockdown cells, by HPLC, qRT-PCR, flow cytometry, and western blotting. RESULTS: Reduced expression of either EXT1, EXT2, or EXTL3 decreased heparan sulfate biosynthesis, and consequently suppressed the FGF2-dependent proliferation of mouse L fibroblasts. In contrast, although knockdown of EXTL2 increased the amounts of heparan sulfate, FGF2-dependent proliferation was significantly inhibited because the increased heparan sulfate enhanced the incorporation of FGF2 into the cells. CONCLUSIONS: EXTL2 controls FGF2 signaling through regulation of heparan sulfate biosynthesis in a manner distinct from that of other exostosins. GENERAL SIGNIFICANCE: This study provides new insights into the regulatory mechanisms of FGF2 signaling by EXTL2.

Insights into the role of 3-O-sulfotransferase in heparan sulfate biosynthesis.

3-O-Sulfotransferase enzyme (sHS) from Litopenaeus vannamei was cloned and its substrate specificity was investigated against a number of GAG structures, including modified heparin polysaccharides and model oligosaccharides. For the heparin polysaccharides, derived from porcine intestinal mucosa heparin, sulfate groups were incorporated into glucosamine residues containing both N-sulfated and N-acetylated substitution within the regions of the predominant repeating disaccharide, either I-ANS or I-ANAc. However, the resulting polysaccharides did not stabilize antithrombin, which is correlated with anticoagulant activity. It was also shown that the enzyme was able to sulfate disaccharides, I2S-ANS and G-ANAc. The results further illustrate that 3-O-sulfation can be induced outside of the classical heparin-binding pentasaccharide sequence, show that 3-O-sulfation of glucosamine is not a sufficient condition for antithrombin stabilization and suggest that the use of this enzyme during HS biosynthesis may not occur as the final enzymatic step.

Unsuspected osteochondroma-like outgrowths in the cranial base of Hereditary Multiple Exostoses patients and modeling and treatment with a BMP antagonist in mice.

Hereditary Multiple Exostoses (HME) is a rare pediatric disorder caused by loss-of-function mutations in the genes encoding the heparan sulfate (HS)-synthesizing enzymes EXT1 or EXT2. HME is characterized by formation of cartilaginous outgrowths-called osteochondromas- next to the growth plates of many axial and appendicular skeletal elements. Surprisingly, it is not known whether such tumors also form in endochondral elements of the craniofacial skeleton. Here, we carried out a retrospective analysis of cervical spine MRI and CT scans from 50 consecutive HME patients that included cranial skeletal images. Interestingly, nearly half of the patients displayed moderate defects or osteochondroma-like outgrowths in the cranial base and specifically in the clivus. In good correlation, osteochondromas developed in the cranial base of mutant Ext1f/f;Col2-CreER or Ext1f/f;Aggrecan-CreER mouse models of HME along the synchondrosis growth plates. Osteochondroma formation was preceded by phenotypic alteration of cells at the chondro-perichondrial boundary and was accompanied by ectopic expression of major cartilage matrix genes -collagen 2 and collagen X- within the growing ectopic masses. Because chondrogenesis requires bone morphogenetic protein (BMP) signaling, we asked whether osteochondroma formation could be blocked by a BMP signaling antagonist. Systemic administration with LDN-193189 effectively inhibited osteochondroma growth in conditional Ext1-mutant mice. In vitro studies with mouse embryo chondrogenic cells clarified the mechanisms of LDN-193189 action that turned out to include decreases in canonical BMP signaling pSMAD1/5/8 effectors but interestingly, concurrent increases in such anti-chondrogenic mechanisms as pERK1/2 and Chordin, Fgf9 and Fgf18 expression. Our study is the first to reveal that the cranial base can be affected in patients with HME and that osteochondroma formation is amenable to therapeutic drug intervention.

Optimization of bioengineered heparin/heparan sulfate production for therapeutic applications.

Heparin has been used clinically as an anti-coagulant for more than 100 y and the major source of this therapeutic is still animal tissues. Contamination issues in some batches of heparin over 10 y ago have highlighted the need to develop alternative methods of production of this essential drug. 1 Bioengineering heparin by expressing serglycin in mammalian cells is a promising approach that was recently reported by the authors. 2 This addendum explores the approaches that the authors are taking to increase the yield of recombinantly expressed serglycin decorated with heparin/heparan sulfate focusing on cell culture and bioreactor conditions and proposes that the cell microenvironment is a key modulator of heparin biosynthesis.

Functional Requirements for Heparan Sulfate Biosynthesis in Morphogenesis and Nervous System Development in C. elegans.

The regulation of cell migration is essential to animal development and physiology. Heparan sulfate proteoglycans shape the interactions of morphogens and guidance cues with their respective receptors to elicit appropriate cellular responses. Heparan sulfate proteoglycans consist of a protein core with attached heparan sulfate glycosaminoglycan chains, which are synthesized by glycosyltransferases of the exostosin (EXT) family. Abnormal HS chain synthesis results in pleiotropic consequences, including abnormal development and tumor formation. In humans, mutations in either of the exostosin genes EXT1 and EXT2 lead to osteosarcomas or multiple exostoses. Complete loss of any of the exostosin glycosyltransferases in mouse, fish, flies and worms leads to drastic morphogenetic defects and embryonic lethality. Here we identify and study previously unavailable viable hypomorphic mutations in the two C. elegans exostosin glycosyltransferases genes, rib-1 and rib-2. These partial loss-of-function mutations lead to a severe reduction of HS levels and result in profound but specific developmental defects, including abnormal cell and axonal migrations. We find that the expression pattern of the HS copolymerase is dynamic during embryonic and larval morphogenesis, and is sustained throughout life in specific cell types, consistent with HSPGs playing both developmental and post-developmental roles. Cell-type specific expression of the HS copolymerase shows that HS elongation is required in both the migrating neuron and neighboring cells to coordinate migration guidance. Our findings provide insights into general principles underlying HSPG function in development.