On the Roles and Regulation of Chondroitin Sulfate and Heparan Sulfate in Zebrafish Pharyngeal Cartilage Morphogenesis

Katarina Holmborn,Judith Habicher,Zsolt Kasza,Anna S Eriksson,Beata Filipek-Gorniok,Sandeep Gopal,John R Couchman,Per E Ahlberg,Malgorzata Wiweger,Dorothe Spillmann,Johan Kreuger,Johan Ledin

doi:10.1074/jbc.m112.401646

Abstract

The present study addresses the roles of heparan sulfate (HS) proteoglycans and chondroitin sulfate (CS) proteoglycans in the development of zebrafish pharyngeal cartilage structures. uxs1 and b3gat3 mutants, predicted to have impaired biosynthesis of both HS and CS because of defective formation of the common proteoglycan linkage tetrasaccharide were analyzed along with ext2 and extl3 mutants, predicted to have defective HS polymerization. Notably, the effects on HS and CS biosynthesis in the respective mutant strains were shown to differ from what had been hypothesized. In uxs1 and b3gat3 mutant larvae, biosynthesis of CS was shown to be virtually abolished, whereas these mutants still were capable of synthesizing 50% of the HS produced in control larvae. extl3 and ext2 mutants on the other hand were shown to synthesize reduced amounts of hypersulfated HS. Further, extl3 mutants produced higher levels of CS than control larvae, whereas morpholino-mediated suppression of csgalnact1/csgalnact2 resulted in increased HS biosynthesis. Thus, the balance of the Extl3 and Csgalnact1/Csgalnact2 proteins influences the HS/CS ratio. A characterization of the pharyngeal cartilage element morphologies in the single mutant strains, as well as in ext2;uxs1 double mutants, was conducted. A correlation between HS and CS production and phenotypes was found, such that impaired HS biosynthesis was shown to affect chondrocyte intercalation, whereas impaired CS biosynthesis inhibited formation of the extracellular matrix surrounding chondrocytes.

Highlights

Chondroitin sulfate (CS) and heparan sulfate (HS) are important for cartilage formation
Analysis of Zebrafish Cartilage Development by Live Imaging of EGFP-expressing Chondrocytes—To study the roles of HS and chondroitin sulfate (CS) in pharyngeal development, we performed a systematic comparison of pharyngeal defects in fish having mutations in enzymes of the GAG biosynthetic pathway. ext2 and extl3 mutants, defective in enzymes directly involved in HS polymerization, were analyzed together with uxs1 and b3gat3 mutants, predicted to have defective formation of the PG tetrasaccharide linkage region, presumably with consequences for both HS and CS biosynthesis (Fig. 1) [11, 14, 20]
In this study we show that HS and CS biosynthesis in zebrafish larvae homozygous for mutations in either ext2, extl3, uxs1, or b3gat3 differs significantly from what could be hypothesized based on the results of previous studies and from the proposed positions of these enzymes in the GAG biosynthetic pathway (Fig. 1)

Summary

Background

Chondroitin sulfate (CS) and heparan sulfate (HS) are important for cartilage formation. A number of genes involved in PG biosynthesis have been shown to be crucial for the development of the jaw and the pharyngeal arches in zebrafish, including glypican 4 (gpc4/knypek) [9, 10], UDP-glucuronic acid decarboxylase 1 (uxs1) [11, 12], exostosin 2 (ext2/dackel) [12,13,14,15], exostoses (multiple)-like 3 (extl3/boxer) [15], udp-glucose dehydrogenase (ugdh/jekyll) [16, 17], and solute carrier family 35, member B2 (slc35b2/ pincher, reported as papst1) [13]. The uxs gene has been suggested to be crucial for both HS and CS synthesis and shown by Eames et al [11] to develop a cartilage phenotype similar to that found in ext mutants, which would suggest that removal of CS in addition to the lack of HS would not result in a strengthened phenotype. Ext2;uxs double mutants with severely reduced capacity to produce both HS and CS were shown to develop a stronger cartilage phenotype when compared with the individual mutants

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION