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Large-scale generation of zebrafish mutants with defective glycosaminoglycan biosynthesis
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Organismal Biology.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
Translational and Functional Genomics Branch, National Human Genome Research Institute, National Institutes of Health, Bethesda, MD 20892, USA.
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Organismal Biology.
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(English)Manuscript (preprint) (Other academic)
National Category
Developmental Biology Biochemistry and Molecular Biology
Identifiers
URN: urn:nbn:se:uu:diva-258588OAI: oai:DiVA.org:uu-258588DiVA: diva2:843123
Available from: 2015-07-27 Created: 2015-07-16 Last updated: 2015-11-10
In thesis
1. 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
2. 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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