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Glycosaminoglycan Biosynthesis and Function in Zebrafish Development: Sugars Shaping Skeletons
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Organismal Biology, Evolution and Developmental Biology. Department of Biomedicine, University of Bergen, Norway.
2015 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Heparan sulfate (HS) and chondroitin/dermatan sulfate (CS/DS) proteoglycans are glycosylated proteins with important roles in animal development and homeostasis. HS and CS/DS are long, linear glycosaminoglycan (GAG) polysaccharides and attached to a core protein they form proteoglycans. GAGs on proteoglycans are often modified by sulfate groups and mainly found in the extracellular matrix or associated to the cell membrane. They interact with different proteins, for example signaling molecules, and influence developmental processes. Cells in cartilage produce a functionally specialized dense extracellular matrix, full of proteoglycans. Using the zebrafish as a model to study GAG biosynthesis we discovered that HS production is prioritized over CS/DS production, if the availability of link structures is restricted. We also found that the effects of removing HS and CS/DS biosynthetic enzymes in zebrafish larvae typically differ from what could be hypothesized solely from knowledge of the activity of each enzyme. These findings indicated a highly complex regulation of GAG biosynthesis and we thus proceeded to identify novel GAG biosynthetic enzymes in zebrafish and characterized their expression during early development. Notably, strong expression of CS/DS glycosyltransferases was found in cartilage structures, correlating with a drastic increase of CS/DS synthesis after two days of development, and high CS/DS deposition in cartilage. Finally, to understand how different GAG biosynthetic enzymes affect zebrafish development, we decided to use the CRISPR/Cas9 technology to generate new loss of function alleles for enzymes in HS and CS/DS biosynthesis. Some mutants show disturbed larval development or adult morphology, but we found many mutants to develop into adults without major morphological abnormalities, suggesting a high redundancy for GAG biosynthetic enzymes. Many GAG glycosyltransferases and modification enzymes have multiple isoforms, suggesting that a combination of mutations in one individual will become necessary to study the loss of specific modifications. To conclude, the zebrafish model gives new insights into the GAG machinery and the CRSIPR/Cas9 technology allows for swift production of new loss of function zebrafish lines with defective GAG biosynthesis.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2015. , 65 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 1266
National Category
Natural Sciences Biological Sciences Developmental Biology Biochemistry and Molecular Biology
Identifiers
URN: urn:nbn:se:uu:diva-259079ISBN: 978-91-554-9282-3 (print)OAI: oai:DiVA.org:uu-259079DiVA: diva2:843126
Public defence
2015-09-11, Friessalen, Norbyvägen 14, Uppsala, 09:15 (English)
Opponent
Supervisors
Available from: 2015-08-20 Created: 2015-07-27 Last updated: 2015-10-01
List of papers
1. On the Roles and Regulation of Chondroitin Sulfate and Heparan Sulfate in Zebrafish Pharyngeal Cartilage Morphogenesis
Open this publication in new window or tab >>On the Roles and Regulation of Chondroitin Sulfate and Heparan Sulfate in Zebrafish Pharyngeal Cartilage Morphogenesis
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2012 (English)In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 287, no 40, 33905-33916 p.Article in journal (Refereed) Published
Abstract [en]

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.

Keyword
Protein Linkage Region, Molecular-Cloning, Hspg Synthesis, Cell Polarity, Growth-Plate, Expression, Ext2, Proteoglycans, Glypican, Xylose
National Category
Natural Sciences
Identifiers
urn:nbn:se:uu:diva-185209 (URN)10.1074/jbc.M112.401646 (DOI)000309602100071 ()
Note

De två första författarna delar förstaförfattarskapet.

De två sista författarna delar sistaförfattarskapet.

Available from: 2012-11-22 Created: 2012-11-21 Last updated: 2017-12-07Bibliographically approved
2. Expression of chondroitin/dermatan sulfate glycosyltransferases during early zebrafish development
Open this publication in new window or tab >>Expression of chondroitin/dermatan sulfate glycosyltransferases during early zebrafish development
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2013 (English)In: Developmental Dynamics, ISSN 1058-8388, E-ISSN 1097-0177, Vol. 242, no 8, 964-975 p.Article in journal (Refereed) Published
Abstract [en]

Background: Chondroitin/dermatan sulfate (CS/DS) proteoglycans present in the extracellular matrix have important structural and regulatory functions. Results: Six human genes have previously been shown to catalyze CS/DS polymerization. Here we show that one of these genes, chpf, is represented by two copies in the zebrafish genome, chpfa and chpfb, while the other five human CS/DS glycosyltransferases csgalnact1, csgalnact2, chpf2, chsy1, and chsy3 all have single zebrafish orthologues. The putative zebrafish CS/DS glycosyltransferases are spatially and temporally expressed. Interestingly, overlapping expression of multiple glycosyltransferases coincides with high CS/DS deposition. Finally, whereas the relative levels of the related polysaccharide HS reach steady-state at around 2 days post fertilization, there is a continued relative increase of the CS amounts per larvae during the first 6 days of development, matching the increased cartilage formation. Conclusions: There are 7 CS/DS glycosyltransferases in zebrafish, which, based on homology, can be divided into the CSGALNACT, CHSY, and CHPF families. The overlap between intense CS/DS production and the expression of multiple CS/DS glycosyltransferases suggests that efficient CS/DS biosynthesis requires a combination of several glycosyltransferases.

Keyword
chondroitin sulfate, polymerase, CSGALNACT, CHSY, CHPF, zebrafish
National Category
Natural Sciences
Identifiers
urn:nbn:se:uu:diva-204834 (URN)10.1002/dvdy.23981 (DOI)000321843400008 ()
Note

De två (2) första författarna delar förstaförfattarskapet.

Available from: 2013-08-13 Created: 2013-08-12 Last updated: 2017-12-06Bibliographically approved
3. Chondroitin / Dermatan Sulfate Modification Enzymes in Zebrafish Development
Open this publication in new window or tab >>Chondroitin / Dermatan Sulfate Modification Enzymes in Zebrafish Development
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2015 (English)In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 10, no 3, e0121957Article in journal (Refereed) Published
Abstract [en]

Chondroitin/dermatan sulfate (CS/DS) proteoglycans consist of unbranched sulfated polysaccharide chains of repeating GalNAc-GlcA/IdoA disaccharide units, attached to serine residues on specific proteins. The CS/DS proteoglycans are abundant in the extracellular matrix where they have essential functions in tissue development and homeostasis. In this report a phylogenetic analysis of vertebrate genes coding for the enzymes that modify CS/DS is presented. We identify single orthologous genes in the zebrafish genome for the sulfotransferases chst7, chst11, chst13, chst14, chst15 and ust and the epimerase dse. In contrast, two copies were found for mammalian sulfotransferases CHST3 and CHST12 and the epimerase DSEL, named chst3a and chst3b, chst12a and chst12b, dsela and dselb, respectively. Expression of CS/DS modification enzymes is spatially and temporally regulated with a large variation between different genes. We found that CS/DS 4-O-sulfotransferases and 6-O-sulfotransferases as well as CS/DS epimerases show a strong and partly overlapping expression, whereas the expression is restricted for enzymes with ability to synthesize di-sulfated disaccharides. A structural analysis further showed that CS/DS sulfation increases during embryonic development mainly due to synthesis of 4-O-sulfated GalNAc while the proportion of 6-O-sulfated GalNAc increases in later developmental stages. Di-sulfated GalNAc synthesized by Chst15 and 2-O-sulfated GlcA/IdoA synthesized by Ust are rare, in accordance with the restricted expression of these enzymes. We also compared CS/DS composition with that of heparan sulfate (HS). Notably, CS/DS biosynthesis in early zebrafish development is more dynamic than HS biosynthesis. Furthermore, HS contains disaccharides with more than one sulfate group, which are virtually absent in CS/DS.

National Category
Evolutionary Biology
Identifiers
urn:nbn:se:uu:diva-252231 (URN)10.1371/journal.pone.0121957 (DOI)000352083900161 ()25793894 (PubMedID)
Available from: 2015-05-05 Created: 2015-05-04 Last updated: 2017-12-04Bibliographically approved
4. The Ndst Gene Family in Zebrafish: Role of Ndst1b in Pharyngeal Arch Formation
Open this publication in new window or tab >>The Ndst Gene Family in Zebrafish: Role of Ndst1b in Pharyngeal Arch Formation
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2015 (English)In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 10, no 3Article in journal (Refereed) Published
Abstract [en]

Heparan sulfate (HS) proteoglycans are ubiquitous components of the extracellular matrix and plasma membrane of metazoans. The sulfation pattern of the HS glycosaminoglycan chain is characteristic for each tissue and changes during development. The glucosaminyl N-deacetylase/N-sulfotransferase (NDST) enzymes catalyze N-deacetylation and N-sulfation during HS biosynthesis and have a key role in designing the sulfation pattern. We here report on the presence of five NDST genes in zebrafish. Zebrafish ndst1a, ndst1b, ndst2a and ndst2b represent duplicated mammalian orthologues of NDST1 and NDST2 that arose through teleost specific genome duplication. Interestingly, the single zebrafish orthologue ndst3, is equally similar to tetrapod Ndst3 and Ndst4. It is likely that a local duplication in the common ancestor of lobe-finned fish and tetrapods gave rise to these two genes. All zebrafish Ndst genes showed distinct but partially overlapping expression patterns during embryonic development. Morpholino knockdown of ndst1b resulted in delayed development, craniofacial cartilage abnormalities, shortened body and pectoral fin length, resembling some of the features of the Ndst1 mouse knockout.

National Category
Evolutionary Biology Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
Identifiers
urn:nbn:se:uu:diva-251802 (URN)10.1371/journal.pone.0119040 (DOI)000351277500060 ()25767878 (PubMedID)
Available from: 2015-04-28 Created: 2015-04-24 Last updated: 2017-12-04Bibliographically approved
5. Large-scale generation of zebrafish mutants with defective glycosaminoglycan biosynthesis
Open this publication in new window or tab >>Large-scale generation of zebrafish mutants with defective glycosaminoglycan biosynthesis
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(English)Manuscript (preprint) (Other academic)
National Category
Developmental Biology Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:uu:diva-258588 (URN)
Available from: 2015-07-27 Created: 2015-07-16 Last updated: 2015-11-10

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