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On the Roles and Regulation of Chondroitin Sulfate and Heparan Sulfate in Zebrafish Pharyngeal Cartilage Morphogenesis
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology. Uppsala University, Science for Life Laboratory, SciLifeLab.
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Organismal Biology, Evolution and Developmental Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology. Uppsala University, Science for Life Laboratory, SciLifeLab.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology. Uppsala University, Science for Life Laboratory, SciLifeLab.
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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.

Place, publisher, year, edition, pages
2012. Vol. 287, no 40, 33905-33916 p.
Keyword [en]
Protein Linkage Region, Molecular-Cloning, Hspg Synthesis, Cell Polarity, Growth-Plate, Expression, Ext2, Proteoglycans, Glypican, Xylose
National Category
Natural Sciences
Identifiers
URN: urn:nbn:se:uu:diva-185209DOI: 10.1074/jbc.M112.401646ISI: 000309602100071OAI: oai:DiVA.org:uu-185209DiVA: diva2:571339
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
In thesis
1. Syndecan - Regulation and Function of its Glycosaminoglycan Chains
Open this publication in new window or tab >>Syndecan - Regulation and Function of its Glycosaminoglycan Chains
2013 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The cell surface is an active area where extracellular molecules meet their receptors and affect the cellular fate by inducing for example cell proliferation and adhesion. Syndecans and integrins are two transmembrane molecules that have been suggested to fine-tune these activities, possibly in cooperation. Syndecans are proteoglycans, i.e. proteins with specific types of carbohydrate chains attached. These chains are glycosaminoglycans and either heparan sulfate (HS) or chondroitin sulfate (CS). Syndecans are known to influence cell adhesion and signaling. Integrins in turn, are important adhesion molecules that connect the extracellular matrix with the cytoskeleton, and hence can regulate cell motility. In an attempt to study how the two types of glycosaminoglycans attached to syndecan-1 can interact with integrins, a cell based model system was used and functional motility assays were performed. The results showed that HS, but not CS, on the cell surface was capable of regulating integrin-mediated cell motility.

Regulation of intracellular signaling is crucial to prevent abnormal cellular behavior. In the second part of this thesis, the aim was to see how the presentation of glycosaminoglycan chains to the FGF signaling complex could affect the cellular response. When attached to the plasma membrane via syndecan-1, CS chains could support the intracellular signaling, although not promoting as strong signals as HS. When glycosaminoglycans were attached to free ectodomains of syndecan-1, both types of chains sequestered FGF2 from the receptors to the same extent, pointing towards functional overlap between CS and HS.

To further study the interplay between HS and CS, their roles in the formation of pharyngeal cartilage in zebrafish were established. HS was important during chondrocyte intercalation and CS in the formation of the surrounding extracellular matrix. Further, the balance between the biosynthetic enzymes determined the ratio of HS and CS, and HS biosynthesis was prioritized over CS biosynthesis.

The results presented in this thesis provide further insight into the regulation of HS biosynthesis, as well as the roles of both HS and CS on the cell surface. It is evident, that in certain situations there is a strict requirement for a certain HS structure, albeit in other situations there is a functional overlap between HS and CS.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2013. 54 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 884
Keyword
heparan sulfate, chondroitin sulfate, integrin, cell motility, fibroblast growth factor signaling, pharyngeal cartilage, biosynthesis regulation
National Category
Cell and Molecular Biology
Research subject
Medical Science
Identifiers
urn:nbn:se:uu:diva-197691 (URN)978-91-554-8637-2 (ISBN)
Public defence
2013-05-17, A1:107a, BMC, Husargatan 3, Uppsala, 13:15 (English)
Opponent
Supervisors
Available from: 2013-04-25 Created: 2013-04-02 Last updated: 2018-01-11Bibliographically approved
2. The role of the extracellular matrix in zebrafish cartilage development
Open this publication in new window or tab >>The role of the extracellular matrix in zebrafish cartilage development
2013 (English)Licentiate thesis, comprehensive summary (Other academic)
Place, publisher, year, edition, pages
Uppsala: Department of Organismal Biology, Uppsala University, 2013
National Category
Natural Sciences
Research subject
Biology with specialization in Animal Development
Identifiers
urn:nbn:se:uu:diva-219406 (URN)
Presentation
2013-12-06, 16:14 (English)
Opponent
Supervisors
Available from: 2014-03-03 Created: 2014-02-28 Last updated: 2014-03-03Bibliographically approved
3. Glycosaminoglycan Biosynthesis and Function in Zebrafish Development: Sugars Shaping Skeletons
Open this publication in new window or tab >>Glycosaminoglycan Biosynthesis and Function in Zebrafish Development: Sugars Shaping Skeletons
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:nbn:se:uu:diva-259079 (URN)978-91-554-9282-3 (ISBN)
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
4. Glycosaminoglycan Biosynthesis in Zebrafish
Open this publication in new window or tab >>Glycosaminoglycan Biosynthesis in Zebrafish
2015 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Proteoglycans (PGs) are composed of highly sulfated glycosaminoglycans chains (GAGs) attached to specific core proteins. They are present in extracellular matrices, on the cell surface and in storage granules of hematopoietic cells. Heparan sulfate (HS) and chondroitin/dermatan sulfate (CS/DS) GAGs play indispensable roles in a wide range of biological processes, where they can serve as protein carriers, be involved in growth factor or morphogen gradient formation and act as co-receptors in signaling processes. Protein binding abilities of GAGs are believed to be predominantly dependent on the arrangement of the sugar modifications, sulfation and epimerization, into specific oligosaccharide sequences. Although the process of HS and CS/DS assembly and modification is not fully understood, a set of GAG biosynthetic enzymes have been fairly well studied and several mutations in genes encoding for this Golgi machinery have been linked to human genetic disorders.

This thesis focuses on the zebrafish N-deacetylase/N-sulfotransferase gene family, encoding key enzymes in HS chain modification, as well as glycosyltransferases responsible for chondroitin/dermatan sulfate elongation present in zebrafish. Our data illustrates the strict spatio-temporal expression of both the NDST enzymes (Paper I) and CS/DS glycosyltransferases (Paper II) in the developing zebrafish embryo. In Paper III we took advantage of the four preexisting zebrafish mutants with defective GAG biosynthesis. We could demonstrate a relation between HS content and the severity of the pectoral fin defects, and additionally correlate impaired HS biosynthesis with altered chondrocyte intercalation. Interestingly, altered CS biosynthesis resulted in loss of the chondrocyte extracellular matrix. One of the main findings was the demonstration of the ratio between the HS biosynthesis enzyme Extl3 and the Csgalnact1/Csgalnact2 proteins, as a main factor influencing the HS/CS ratio. In Paper IV we used the newly developed CRISPR/Cas9 technique to create a collection of zebrafish mutants with defective GAG biosynthetic machineries. Lack of phenotypes linked to null-mutations of most of the investigated genes is striking in this study.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2015. 54 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 1143
Keyword
Heparan sulfate, chondroitin/dermatan sulfate, biosynthesis, development, N-deacetylase N-sulfotransferase, glycosyltransferases, morpholino, CRISPR-Cas9
National Category
Biochemistry and Molecular Biology Developmental 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-264269 (URN)978-91-554-9368-4 (ISBN)
Public defence
2015-11-27, C8:305, BMC, Husargatan 3, Uppsala, 13:15 (English)
Opponent
Supervisors
Available from: 2015-11-05 Created: 2015-10-08 Last updated: 2015-11-10

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Holmborn, KatarinaHabicher, JudithKasza, ZsoltEriksson, Anna S.Gorniok, Beata FilipekAhlberg, Per ErikSpillmann, DorotheKreuger, JohanLedin, Johan

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