Digital Comprehensive Summaries of Uppsala Dissertations
from the Faculty of Medicine 1143
Glycosaminoglycan Biosynthesis in
ISSN 1651-6206
ISBN 978-91-554-9368-4
Dissertation presented at Uppsala University to be publicly examined in C8:305, BMC,
Husargatan 3, Uppsala, Friday, 27 November 2015 at 13:15 for the degree of Doctor of
Philosophy (Faculty of Medicine). The examination will be conducted in English. Faculty
examiner: Associate Professor Kay Grobe (Institute for Physiological Chemistry and
Pathobiochemistry, University of Münster).
Filipek-Górniok, B. 2015. Glycosaminoglycan Biosynthesis in Zebrafish. Digital
Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine 1143.
54 pp. Uppsala: Acta Universitatis Upsaliensis. ISBN 978-91-554-9368-4.
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.
Keywords: Heparan sulfate, chondroitin/dermatan sulfate, biosynthesis, development, Ndeacetylase N-sulfotransferase, glycosyltransferases, morpholino, CRISPR-Cas9
Beata Filipek-Górniok, Department of Medical Biochemistry and Microbiology, Box 582,
Uppsala University, SE-75123 Uppsala, Sweden.
© Beata Filipek-Górniok 2015
ISSN 1651-6206
ISBN 978-91-554-9368-4
urn:nbn:se:uu:diva-264269 (http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-264269)
Nothing that comes to easy has any significance.
Jacek Hugo-Bader, journalist and reporter
Lena Kjellén, Professor
Department of Medical Biochemistry and Microbiology
Uppsala University
Uppsala, Sweden
Johan Ledin, Researcher
Department of Organismal Biology
Uppsala University
Uppsala, Sweden
Faculty opponent:
Kay Grobe, Associate Professor
Institute for Physiological Chemistry and Pathobiochemistry
University of Münster
Münster, Germany
Examining Committee:
Dan Larhammar, Professor
Department of Neuroscience, Pharmacology
Uppsala University
Uppsala, Sweden
Hiroshi Nakato, Associate Professor
Department of Genetics, Cell Biology and Development
University of Minnesota
Minnesota, US
Sally Stringer, Honorary Lecturer/Editorial team leader
Cardiovascular Division
University of Manchester/HealthCare21 Communications Ltd,
Manchester, UK
Jin-ping Li, Professor
Department of Medical Biochemistry and Microbiology
Uppsala University
Uppsala, Sweden
Cover: Adult female zebrafish. Drawing by Beata Filipek-Górniok, 2015.
List of Papers
This thesis is based on the following papers, which are referred to in the text
by their Roman numerals.
Filipek-Górniok B., Carlsson P., Haitina T., Habicher J., Ledin
J., Kjellén L. (2015) The NDST gene family in zebrafish: role
of NDST1b in pharyngeal arch formation. PLoS One,
10(3):e0119040. doi: 10.1371/journal.pone.0119040.
Filipek-Górniok B., Holmborn K., Haitina T., Habicher J.,
Oliveira M.B., Hellgren C., Eriksson I., Kjellén L., Kreuger J.,
Ledin J. (2013) Expression of chondroitin/dermatan sulfate
glycosyltransferases during early zebrafish development.
Holmborn K., Habicher J., Kasza Z., Eriksson A.S., FilipekGórniok B., Gopal S., Couchman J.R., Ahlberg P.E., Wiweger
M., Spillmann D., Kreuger J., Ledin J. (2012) On the roles and
regulation of chondroitin sulfate and heparan sulfate in
zebrafish pharyngeal cartilage morphogenesis. The Journal of
biological chemistry, 287(40):33905-16.
Habicher J., Filipek-Górniok B., Varshney G., Ahlberg P.E.,
Burgess S., Kjellén L., Ledin J. (2015) Large-scale generation
of zebrafish mutants with defective glycosaminoglycan
biosynthesis. Manuscript.
Reprints were made with permission from the respective publishers.
Additional Publications
Fisher S., Filipek-Górniok B., Ledin J. (2011) Zebrafish Ext2 is
necessary for Fgf and Wnt signaling, but not for Hh signaling. BMC
Developmental Biology, 11:53. doi: 10.1186/1471-213X-11-53.
II Dagälv A., Lundequist A., Filipek-Górniok B., Dierker T., Eriksson I.,
Kjellén L. (2015) Heparan sulfate structure: methods to study Nsulfation and NDST action. Methods in molecular biology, 1229:189200. doi: 10.1007/978-1-4939-1714-3_17.
Introduction ...................................................................................................13 Background ...................................................................................................14 Proteoglycans and glycosaminoglycans ...................................................14 Heparan sulfate proteoglycans .............................................................15 Chondroitin/dermatan sulfate proteoglycans .......................................16 Heparan sulfate biosynthesis................................................................16 Chondroitin/dermatan sulfate biosynthesis..........................................19 …and one to rule them all: PAPS.............................................................21 Regulation of the glycosaminoglycans biosynthesis – a puzzle not
yet solved. ............................................................................................22 Glycosaminoglycans in biological processes.......................................23 Zebrafish as a model system.....................................................................28 Zebrafish in glycosaminoglycan research............................................30 Method considerations..............................................................................32 Overexpression studies ........................................................................32 Rise and fall of the morpholino technology.........................................32 Selected methods for targeted genome editing in zebrafish ................33 Present investigation .....................................................................................36 Aim ...........................................................................................................36 Paper I ..................................................................................................36 Paper II.................................................................................................37 Paper III ...............................................................................................38 Paper IV ...............................................................................................39 Concluding remarks and future perspectives ................................................41 Populärvetenskaplig sammanfattning ...........................................................43 Acknowledgments.........................................................................................44 References .....................................................................................................46 Abbreviations
Adenosine diphosphate
Adenosine 5’-phosphosulfate
Adenosine triphosphate
Glucuronyltransferase I
Bone morphogenetic protein
Chondroitin sulfate 4-O-sulfotransferase
Chondroitin sulfate 6-O-sulfotransferase 1
Chondroitin sulfate/dermatan sulfate 2-O-sulfotransferase
Chondroitin polymerization factor
Chondroitin polymerization factor 2
Chondroitin synthase 1
Chondroitin synthase 3
Chondroitin sulfate
Chondroitin sulfate N-acetyl galactosaminyl transferase
Dermatan sulfate
Fibroblast growth factor
Glucuronc acid
Hereditary multiple exostoses
Heparan sulfate
Iduronic acid
Keratan sulfate
Protein 53 (Tumor protein 53)
3’-Phosphoadenosine 5’-phosphosulfate
PAPS synthase
PAPS transporter
Transforming growth factor
UDP-glucuronic acid decarboxylase 1
Wingless-related MMTV integration site
Xylosyltransferase 1
Glycosaminoglycans (GAGs), located in the extracellular matrix and on the
cell surface, have a fundamental role in many aspects of sustaining
vertebrate life. Due to their negative charge, they have an ability to interact
with a large number of proteins, including matrix components, morphogens
and signaling molecules, modifying their functions and availability.
Although knowledge about these extended carbohydrate polymers is not as
great as that of DNA and protein, the crucial role of GAGs in biological
systems gives them a place among the most fascinating biomolecules.
This work is focused on two types of glycosaminoglycans, heparan
sulfate and chondroitin/dermatan sulfate, the complex enzymatic machinery
needed for their biosynthesis, and the functions they play in early
development. All investigations included in this thesis are based on studies
of the zebrafish model.
Proteoglycans and glycosaminoglycans
Linear, sulfated and therefore negatively charged GAGs are produced by
virtually all vertebrate cells. Sulfated GAG chains, covalently attached to
certain serine residues within so called core proteins, form proteoglycans
Depending on their structure and degree of sulfation, GAGs can be
further divided into heparan sulfate (HS), heparin, chondroitin sulfate (CS),
dermatan sulfate (DS) and keratan sulfate (KS). Hyaluronan (HA), differs
from the other GAG family members, as it consist of a single non-sulfated
and core protein-free GAG chain (Fig. 1).
Figure 1. Glycosaminoglycan disaccharide structures. Heparin is a highly
modified and sulfated version of heparan sulfate. Positions that may be sulfated are
marked in red, except for the fully modified heparin disaccharide structure.
GAG-resembling polysaccharides, lacking core proteins, have been
identified in some bacterial strains where their function seems to be
connected to infection processes. In the animal kingdom, GAGs in the form
of CS and HS are present already in the platyhelminthes (Yamada, Sugahara
et al. 2011).
PGs are present mostly on the cell surface and in the extracellular matrix,
but also inside the storage granules of cells of hematopoietic origin and,
tentatively, inside the nuclei of some cells (Kjellen and Lindahl 1991;
Richardson, Trinkaus-Randall et al. 2001). Some PGs are present in a wide
range of tissues, whereas other members, such as neurocan and serglycin
appear to be, tissue or cell type specific, respectively (Sugahara and Mikami
2007; Esko, Kimata et al. 2009).
The various biological functions of PGs are possible to maintain due to
their high diversity. While the largest PG, aggrecan, contains more than
hundred CS chains, HSPGs at the cell surface often display 3-5 GAG chains.
Complex sulfation patterns of the GAG molecules allow interactions with a
range of cell surface and matrix proteins. Some interactions may be based on
more specific sulfation motifs with high binding affinities, whereas, for
others, only a certain degree of sulfation seems to be required (Gama, Tully
et al. 2006).
Heparan sulfate proteoglycans
The basic building blocks of HS are N-acetyl-D-glucosamine (GlcNAc) and
glucuronic acid (GlcA) units. Similar to CS/DS, HS chains are linked to the
core protein through the GlcA-Gal-Gal-Xyl tetrasaccharide sequence. HS
can be further modified by epimerization of a portion of the GlcA to iduronic
acid (IdoA), and sulfation of both glucosamine and IdoA at specific
Within HS chains, domains with various degree of sulfation can be
distinguished. N-acetylated domains (NA-domains) are virtually unmodified
and NS-domains, in most cases 4-12 residues long, are characterized by fully
N-sulfated GlcN residues (Turnbull and Gallagher 1991; Gallagher, Turnbull
et al. 1992). Epimerization, 3-O-, 6-O- and 2-O-sulfation are also typical for
NS-domains, whereas within NA/NS-domains, modifications are present
with lower density (Westling and Lindahl 2002).
HSPGs can be divided into four main groups, including the two types of
plasma membrane bound PGs syndecans and glypicans. Perlecan, agrin and
collagen XVIII form another group of PGs. Members of this group are
secreted and participate in extracellular matrix and basement membrane
organization (Iozzo 1998). Serglycin, carrying heparin, and in some cases
CS chains, is stored in the mast cell intracellular granules (Fig.2).
In the extracellular matrix, HSPGs can bind growth factors and store
morphogens designed for further use and membrane bound, they can serve
as co-receptors, modulate both signaling events and cell adhesion (Bernfield,
Gotte et al. 1999). Interestingly, HS chain composition and domain
organization have been shown to be tissue specific, despite the lack of
template for their biosynthesis (Ledin, Staatz et al. 2004).
Figure 2. Examples of PGs. PGs consist of a core protein (dark gray) and one ore
more covalently attached GAG chain: CS (black), HS (gray) or heparin (light grey).
The membrane bound proteoglycans syndecans, may contain both CS and HS chains.
Serglycin with attached heparin and in some cases also CS chains is stored in granules
of hematopoietic cells.
Chondroitin/dermatan sulfate proteoglycans
CS/DSPGs are abundant in the extracellular matrix and are essential both for
cartilage development and functionality. They are important players of
various processes, including inflammatory response, neuronal development
and cellular proliferation (Prabhakar and Sasisekharan 2006). Most of the
functions of CSPGs are believed to involve the CS chains, with core proteins
serving mostly as a scaffold (Sugahara and Kitagawa 2000; Sugahara,
Mikami et al. 2003).
CS chains are formed by 40, to more than 100 linearly aligned
disaccharide units of glucuronic acid (GlcA) and N-acetyl-D-galactosamine
(GalNAc). In DS chains, some of the GlcA residues are epimerized to IdoA
(Prabhakar and Sasisekharan 2006). Sulfation of CS includes 4-O-sulfation
and/or 6-O-sulfation of the GalNAc and occasional 2-O-sulfation or 3-Osulfation of the GlcA residue. DS sulfation patterns can involve 4-Osulfation of GalNAc residue, together with 6-O-sulfation of GalNAc and 2O-sulfation of the IdoA (Sugahara, Mikami et al. 2003). Aggrecan contains
CS side-chains, whereas DS is more common in versican and the small
leucine-rich PGs (Iozzo 1999).
Heparan sulfate biosynthesis
CS/DS and HS GAGs share a common linkage region (GlcAβ1-3Galβ13Galβ1-4Xylβ1-O-Ser) composed of four monosaccharides and covalently
attached to serine residues of the core proteins (Fig. 1,3) (Sugahara and
Kitagawa 2000; Sugahara and Kitagawa 2002).
In some cases, when the same serine residue can carry either type of GAG
chains, addition of the first residue to the linkage region will determine the
type of synthesized GAG chain. This information seems to be core protein
dependent and the reaction itself is accomplished by either
CSGALNACT1/CSGALNACT2 resulting in CS biosynthesis, or EXTL3
resulting in HS biosynthesis (Lindahl and Jin-ping 2009). Although
modifications (phosphorylation and sulfation) of the GAG linkage region
have been proposed to influence the choice between HS or CS attachment,
the exact mechanisms stay unclear (Prydz 2015).
Figure 3. Schematic outline of heparan sulfate and chondroitin/dermatan sulfate
biosynthesis. Enzymes with sulfotransferase activity, marked with red lines, are
dependent on the availability of 3’-phosphoadenosine 5’-phosphosulfate (PAPS).
Single mutations in the PAPS synthase or transporter can therefore affect the activity
of multiple enzymes. Adapted from (Häcker, Nybakken et al. 2005).
In mammals, the EXT1/EXT2 copolymerase is proposed to play the major
role in the further HS chain polymerization, although, as many as five
glycosyltransferases belonging to the exostosin family (EXT) have been
reported (Fig. 3). The mutations within EXT1 and EXT2 genes are linked to
Hereditary Multiple Exostoses (HME). The remaining members of this
family, EXTL1, EXTL2 and EXTL3, were identified based on sequence
homology to the HME linked genes, and are also proposed to have roles in
HS biosynthesis (Busse-Wicher, Wicher et al. 2014).
Further modifications of the HS chain, including GlcA epimerization to
IduA, and O-sulfation at various sites, occur in the N-sulfated regions
(Lindahl, Kusche-Gullberg et al. 1998). Epimerization is carried out by the
single glucuronyl C5-epimarase (GLCE). Iduronic acid residues, resulting
from GLCE action, can be further 2-O-sulfated by the single 2-Osulfotransferase. The 2-OST enzyme acts predominantly on IdoA residues,
however 2-O-sulfation of GlcA units can also occur, but at much lower rates
(Rong, Habuchi et al. 2001; Li, Gong et al. 2003).
As many as three different 6-O-sulfotransferases and seven 3-Osulfotransferases have been reported. 6-OST1 is the primary 6-Osulfotransferase in the majority of the tissues (Habuchi, Nagai et al. 2007).
Interestingly, the 3-O-sulfotransferases, the largest family among the HS
modifying enzymes, are responsible for the least frequently occurring
sulfation type (Thacker, Xu et al. 2014).
In HS, 40-50%, and in heparin, 80-90%, of the glucosamine residues are Nsulfated. In mammals, the N-sulfation process is catalyzed by the four
bifunctional N-deacetylase/N-sulotransferase (NDST1-4) enzymes, with the
use of 3’-phosphoadenosine 5’-phosphosulfate (PAPS) as sulfate donor
(Kusche-Gullberg, Eriksson et al. 1998; Aikawa and Esko 1999; Aikawa,
Grobe et al. 2001).
NDST is the first enzyme modifying the HS chain by removing acetyl
groups and then adding sulfate groups to the previously deacetylated
glucosamine residues. The NDSTs play a crucial role during the HS
modification process, since most of the sulfate groups added by Osulfotransferases will be located to residues in the N-sulfated regions of the
HS chain where also epimerization occurs (Holmborn, Ledin et al. 2004).
The four isoforms differ in their enzymatic activities. While murine
recombinant NDST1, 2 and 3 have relatively high N-deacetylase activities,
NDST4 seems to be primarily an N-sulfotransferase. In contrast, the Nsulfotransferase activty of NDST3 is very low, whereas the three other
NDSTs have more comparable activities (Aikawa, Grobe et al. 2001).
Both Drosophila and C. elegans express a single ndst gene. The
Drosophila Ndst mutant sulfateless (sfl) displays a phenotype manifested as
impaired segmentation during embryonic development, caused by
production of unmodified HS chains, unable to fulfill their regular function
(Lin, Buff et al. 1999). In mice and humans, NDST1 and NDST2 are
expressed at different levels from early embryonic stages and through
adulthood. The NDST3 and NDST4 isoforms on the other hand, are
expressed mostly during embryonic development and later in adult brain
tissues (Grobe, Ledin et al. 2002).
NDST1 null-mutant mice die prenatally or shortly after birth. They
display lung and craniofacial defects which make their phenotype the most
severe of all investigated NDST knockouts (Ringvall and Kjellen 2010).
Interestingly, the phenotype of the NDST2-/- mouse is very mild. The lack of
functional NDST2 results in reduced number of mast cells containing large,
empty vacuoles, in contrast to wild type mast cells, where granules are fully
packed with proteoglycans and inflammatory mediators (Grobe, Ledin et al.
2002). NDST3-/- mice are characterized by a minor undersulfation of HS and
phenotypically, by small behavioral and hematological defects, suggesting
that other isoforms might compensate for the lack of NDST3 (Pallerla,
Lawrence et al. 2008).
In a recent study, Reuter et. al have described patients carrying NDST1
missense mutations of conserved amino acids within the sulfotransferase
domain, leading to changes in the substrate binding abilities and/or structural
changes of this domain. These changes altering protein conformations, have
been linked to several clinical features, ranging from intellectual disability,
epilepsy, muscular hypotomia to deficiency in postnatal growth (Reuter,
Musante et al. 2014). Additionally, point mutations within the regulatory
region of NDST3 have been correlated with schizophrenia and bipolar
disorder. Postmortem cerebellar tissues from patients carrying risk allele
were found to express higher levels of NDST3 as compared with
heterozygotes (Lencz, Guha et al. 2013).
Chondroitin/dermatan sulfate biosynthesis
Similar to HS, CS/DS chain polymerization and modification take place in
the Golgi apparatus. After the initial step of GalNAc transfer to the linkage
region, further CS chain elongation is catalyzed by CS polymerases (see
In mammals, modification of the CS chain includes sulfation of the
GalNAc residue by the three chondroitin sulfate 4-O-sulfotransferases
(C4ST1-3) and chondroitin sulfate 6-O-sulfotransferases 1 and 2 (C6ST1-2).
As in HS biosynthesis, selected GlcA residues can undergo epimerization into
IdoA as a result of the activity of the two DS epimerases (Pacheco,
Malmstrom et al. 2009). The IdoA-GalNAc disaccharides can be further
sulfated at the C-4 position of the GalNAc residue, carried out by the dermatan
4-O-sulfotransferase (Evers, Xia et al. 2001). CS/DS chain sulfation is
considered to proceed at the same time as the chain undergoes elongation. It
involves 2-O-sulfotransferase action, leading to C-2 GlcA/IdoA sulfation of
the previously 4-O-sulfated or 6-O-sulfated disaccharides. Finally, the C-6
position of a previously C-4 sulfated GalNAc residue can become sulfated by
the GalNAc4S-6-O-sulfotransferase (Prabhakar and Sasisekharan 2006). A
majority of the polymers obtained as a result of the elongation and
modification processes consists of mixed GlcA and IdoA residues, and
therefore is often referred to as CS/DS (Fig. 3, Tab. 1).
Chondroitin/dermatan sulfate glycosyltransferases
Six CS glycosyltransferases have been identified until now. Each of them
have been known under several names, all listed here; chondroitin sulfate Nacetylgalactosaminyltransferase-1 (GalNAcT1,CSGALNACT1) (Gotoh,
Sato et al. 2002), chondroitin sulfate N-acetylgalactosaminyltransferase-2
(GalNAcT2, CSGALNACT2) (Sato, Gotoh et al. 2003), chondroitin
synthase 1 (CHSY1, ChSy, ChSy-1) (Kitagawa, Uyama et al. 2001),
chondroitin synthase 3 (CHSY3, CSS3, ChSy-2), chondroitin polymerization
factor (CHPF) (Kitagawa, Izumikawa et al. 2003) and chondroitin
polymerization factor 2 (CHPF2, CSGlcAT, ChSy-3) (Izumikawa, Koike et
al. 2008).
The specific role of each of these enzymes is still under debate, although
some information regarding their activities is available. Although the
CHSY1, CHPF2 and CHSY3 enzymes have been shown to possess both
GlcAT-II and GalNAcT-II activities, it was also demonstrated that only coexpression of two synthases, or any of the synthases together with one of the
polymerization factors, can lead to CS polymerization in vitro (Kitagawa,
Izumikawa et al. 2003; Izumikawa, Uyama et al. 2007; Izumikawa, Koike et
al. 2008). The remaining two enzymes, CSGALNACT1 and
CSGALNACT2, are thought to be the main enzymes responsible for both
CS chain initiation and elongation (Sato, Gotoh et al. 2003).
The importance of CSPG glycosyltransferases have been shown in several
models, starting from C. elegans, producing mainly the non-sulfated form of
CS. Animals with a defect in the sqv-5 gene, the nematode ortholog of
CHSY, exhibited defective vulva formation (Hwang, Olson et al. 2003). The
same phenotype was observed in C. elegans with a mutation in mig-22 (pfc1), the nematode CHPF ortholog.
Mutation in the human CHSY1 gene was reported to cause short posture
and skeletal defects including brachydactyly and hearing loss (Tian, Ling et
al. 2010). Zebrafish morpholino knockdown of Chsy1 results in defective
development of the jaw, pectoral fins and eyes (Tian, Ling et al. 2010),
whereas mouse with null-mutations in CHSY1 were reported to exhibit
deformations of the joints and digits (Wilson, Phamluong et al. 2012). Taken
together, this data would suggest that CHSY1 plays important roles in the
skeletal development including digit patterning.
Also CSGALNACT1 knockdown mice exhibit a cartilage phenotype,
including shortened axial skeleton and limbs, as well as cartilage
abnormalities. In these mice the CS content in cartilage is reduced to less
than half of the wild type amount (Watanabe, Takeuchi et al. 2010). Also
human missense mutations in CSGALNACT1 have been linked to hereditary
motor and sensory neuropathies (Saigoh, Izumikawa et al. 2011).
…and one to rule them all: PAPS
3’-phosphoadenosine 5’-phosphosulfate (PAPS) is synthesized in a two-step
reaction carried out by PAPS syntase in the cell cytosol. PAPS is an
universal sulfate donor for all sulfotransferase reactions both in the cytosol
(estrogen, steroid hormone, neurotransmitter, drug and xenobiotic sulfation)
and in the Golgi apparatus (tyrosine and GAG sulfation) (Besset, Vincourt et
al. 2000; Venkatachalam 2003) Therefore all sulfation reactions are
dependent on PAPS availability (Esko and Selleck 2002; Bishop, Schuksz et
al. 2007; Nadanaka and Kitagawa 2008). Additionally, in vitro studies have
shown that PAPS concentration during HS biosynthesis can affect NDST
enzyme performance (Carlsson, Presto et al. 2008).
Animal PAPS synthase combines ATP sulfurylase and APS kinase activity
in a single protein, whereas in bacteria, fungi, yeast and plants these enzyme
activities are found separately in two polypeptides chains. ATP sulfurylase
catalyzes the reaction in which inorganic sulfate is combined with ATP to
form APS. In the second step of the reaction, PAPS is formed from APS
through phopshorylation by APS kinase (Besset, Vincourt et al. 2000).
In D. melanogaster and C. elegans, only one PAPS synthase is present,
whereas in human and mouse two PAPSS isoforms, PAPSS1 and PAPSS2,
have been identified (Jullien, Crozatier et al. 1997; Dejima, Seko et al. 2006).
The zebrafish genome contains a single papss1 gene and two gene copies
encoding papss2a and papss2b.
Interestingly, in vitro studies revealed nuclear localization of PAPSS1
suggesting that it has a role in the nucleus, whereas PAPSS2b was found to be
located in the cytoplasm. It has also been suggested that PAPSS1 and PAPSS2
can form high-affinity heterodimers, allowing transport of PAPSS2 into the
cell nucleus (Besset, Vincourt et al. 2000). However both PAPSS1 and
PAPSS2 contain conserved nuclear localization motifs, the one present in
PAPSS1 is presumed to be more potent. This finding is not in agreement with
the common opinion that PAPS synthesis is exclusively localized to the
cytosol, but could explain why these two isoforms do not appear to
compensate for each other (Besset, Vincourt et al. 2000; Schroder, Gebel et al.
PAPSS1 is ubiquitously expressed in adult human tissues, whereas
PAPSS2 appears to be mainly expressed in the cartilage growth plate (Fuda,
Shimizu et al. 2002). Murine Papss2 expression was observed in elements
of forming cartilage, heart, tongue, kidney and neuronal tissue.
Brachymorphic mice, identified as PAPSS2 mutants, are characterized by
dwarfism and undersulfation of chondroitin sulfate localized in the
extracellular matrix (Orkin, Pratt et al. 1976; Stelzer, Brimmer et al. 2007).
PAPS needed in the Golgi apparatus for sulfation of glycoproteins,
proteoglycans and glycolipids is transported from the cytoplasm by 3’phosphoadenosine 5’-phosphosulfate transporters (PAPST). Two of these
specific PAPS transporters have been identified in vertebrates including
zebrafish. Both are localized to the Golgi membrane (Kamiyama, Suda et al.
2003; Kamiyama, Sasaki et al. 2006). The zebrafish papst1 mutant
(pinscher/slc35b2) has been identified based on its pectoral fin, cartilage and
axon miss-sorting phenotype. Pinscher mutants have been reported with
greatly reduced amounts of total GAGs (Wiweger, Avramut et al. 2011).
Regulation of the glycosaminoglycans biosynthesis – a puzzle
not yet solved.
One of the most challenging questions in the GAG field is the biosynthesis
regulation. Assembly and modification of the GAG chains is carried out by a
large set of enzymes. Spatiotemporally regulated expression, makes them
capable of producing organ-specific, or presumably, even cell-specific
GAGs epitopes (Ledin, Staatz et al. 2004). This finding suggests that similar
to the genome and proteome, also the glycome might be tightly regulated.
In spite of the intensive hunt for a model, which could explain the
formation of the GAG fine structure, their specific “sulfation fingerprint”
and chain length, this field still leaves a lot to be discovered (Little, Ballinger
et al. 2008; Victor, Nguyen et al. 2009). The existence of a “GAGosome”, a
set of Golgi membrane-bound enzymes arranged into a GAG biosynthetic
machinery, is a popular theory within the proteoglycan biosynthesis field
(Esko and Selleck 2002).
Several studies supporting this hypothesis for HS biosynthesis have been
published. Data presenting interactions between extostosin 1 (EXT1) and
exostosin 2 (EXT2), C5 epimerase and 2-O-sulfotransferase and finally Ndeacetylase N-sulfotransferase 1 and EXT2 are available (Kobayashi,
Morimoto et al. 2000; McCormick, Duncan et al. 2000; Pinhal, Smith et al.
2001; Presto, Thuveson et al. 2008). An additional study from 1974 reports
on the interaction between the linkage region enzymes, xylosyltransferase
and galactosyltransferase-1 (Schwartz, Roden et al. 1974).
Catalytic activity of the enzymes involved in the GAG biosynthetic
pathway can be restricted by their substrate preferences. Experimental
evidence suggests that HS modifications are tightly organized, where
epimerization occurs after N-sulfation (Esko and Selleck 2002; Carlsson,
Presto et al. 2008) and is followed by 2-O-sulfation, as N-sulfated and
epimerized substrate tends to be favored by the 2OST (Rong, Habuchi et al.
2001). Also 6OSTs and 3OSTs have certain substrate preferences (Habuchi,
Tanaka et al. 2000). Sulfation by the 3OSTs is considered to be the last
modification and 3OSTs preferentially act on previously modified residues,
where 2-O-sulfation on the non-reducing side of the target glucosamine can be
either inhibitory or preferred by different isoforms (Thacker, Xu et al. 2014).
Although these are only a few examples of the substrate specificities, they
exemplify how the diversity of GAG structures could be obtained. Tight
spatiotemporal regulation of the enzyme expression, their preferences
towards specific substrate, also in complexes with other enzymes, can
tentatively explain the occurrence of tissue-specific “sulfation fingerprints”.
Also, availability of PAPS and UDP-sugars, translational levels of GAG
biosynthetic enzymes, posttranslational modifications of the enzymes and
finally desulfation of HS by the extracellular endosulfatases capable of
remodeling HS chains after biosynthesis, play roles in shaping the final
GAG structure (Kreuger and Kjellen 2012).
Glycosaminoglycans in biological processes
A growing number of GAG related studies reveals the importance of GAGs
in a broad range of biological processes. GAGs are known to be involved in
cell adhesion, lipid metabolism, angiogenesis and inflammatory responses
(Bishop, Schuksz et al. 2007). In some cases, only particular modifications
of the GAG oligosaccharide sequences can support binding between a GAG
and its ligand, such as is case for the heparin/HS pentasaccharide interaction
with antithrombin. For other interactions, the specificity is less obvious, with
several possible oligasaccharides supporting a certain interaction. The beststudied example of this kind of GAG-protein interaction, is fibroblast growth
factor (FGF) and FGF receptor binding with HS/heparin (Kreuger,
Spillmann et al. 2006).
Morphogen gradient formation is another intriguing role of GAGs.
Binding to HS can protect proteins from degradation, but also regulate their
availability. Growth factors such as Wnt, Hedgehog and BMPs are
responsible for tissue patterning during development and HSPGs have been
shown to bind them, restrict their diffusion, and therefore form morphogen
gradients (Lander, Nie et al. 2002; Tabata and Takei 2004; Yan and Lin
2009). Additionally, HS can create signaling platforms where extended HS
chains interact with cell-surface receptors and signaling molecules.
Examples include FGF2 and its receptor, or BMP2 and BMP4 signal
transduction (Quarto and Amalric 1994; Yan and Lin 2009; Kuo, Digman et
al. 2010).
Finally, GAGs have been linked to a number of pathological states.
Table 1 contains a list of HS and CS/DS biosynthetic enzymes with
associated human disorders, phenotypes of mouse and zebrafish mutants and
zebrafish morpholino knock-downs.
Table 1. List of enzymes relevant for HS and CS/DS biosynthesis and known
disorders connected to each of the genes in human, mouse and zebrafish. OMIM
numbers represent entries listed at the Online Mendeian Inheritance in Men database.
Enzyme name
synthase 1
transporter 1
UDP-xylose synthase
1 //
decarboxylase 1
M: Prenatal death.
(Blake, Bult et al. 2014)
H: Point mutation associated
with the risk of psoriasis
(Yin, Cheng et al. 2014)
H: Spondyloepimetaphyseal
dysplasia, Pakistani type;
M: Brachymorphic mouse;
dwarfism, cartilage defects.
(Lane and Dickie 1968)
Human (H) disease and
OMIM #, (M) mouse mutant
Zebrafish gene(s), mutations
(-/-), morpholino knock-down
(MO) phenotypes
papst1/ slc35b2-/-: Cartilage
defects. (Clement, Wiweger et
al. 2008)
papst2/ slc35b3
uxs1-/-: Craniofacial cartilage
and bone defects, shorter
pectoral fins. (Eames, Singer et
al. 2010)
fam20b-/-: Cartilage defects.
(Eames, Yan et al. 2011)
H: Pseudoxanthoma
elasticum: #264800
H: Pseudoxanthoma
elasticum; #264800
M: Polycystic liver and
kidney disease at the age of
4-5 months. (Condac, SilasiMansat et al. 2007)
H: Ehlers-Danlos syndrome,
progeroid type1; #130070
H: Spondyloepimetaphyseal
dysplasia; #271640;
Ehlers-Danlos syndrome
progeroid type2; #615349
H: Multiple joint
dislocations, short stature,
craniofacial dysmorphism,
and congenital heart defects;
H: Chondrosarcoma,
Hereditary multiple
exostoses type1; #133700
M: Gastrulation failure,
death at E7.5. (Lin, Wei et al.
xylt1-/-: Cartilage defects.
(Eames, Yan et al. 2011)
b4galt7-/-: Cartilage defects.
(Amsterdam, Nissen et al.
2004; Nissen, Amsterdam et al.
b3gat3-/-; Craniofacial cartilage
bone defects, shorter pectoral
fins. (Amsterdam, Nissen et al.
2004; Wiweger, Avramut et al.
M: Normal development with
impaired liver regeneration.
(Nadanaka, Kagiyama et al.
2013; Nadanaka, Zhou et al.
2013; Nadanaka and
Kitagawa 2014)
M: Embryonic lethal at E9.
(Takahashi, Noguchi et al.
H: Mental retardation;
M: Prenatal death – lung,
brain and craniofacial
(Fan, Xiao et al. 2000;
Ringvall, Ledin et al. 2000;
Grobe, Inatani et al. 2005)
M: Reduced number of mast
cell with empty storage
vacuoles. (Forsberg, Pejler et
al. 1999; Humphries, Wong
et al. 1999)
H: Mutation in the regulatory
region, linked to
schizophrenia and bipolar
disorder. (Lencz, Guha et al.
M: Mild behavioral and
hematological changes.
(Pallerla, Lawrence et al.
Glucuronyl C5epimerase
H: Hereditary multiple
exostoses type2; #133701.
Seizures-scoliosismacrocephaly syndrome
(Farhan, Wang et al. 2015).
M: Mutants – gastrulation
failure, death at E6-7.5.
Heterozygous adults have
skeleton abnormalities.
(Stickens, Zak et al. 2005)
M: Lack of kidneys, lung
failure, skeletal defects with
aberrant chondrocyte
proliferation (Li, Gong et al.
2003; Dierker, Bachvarova et
al. 2015)
ext2-/-: Defects in craniofacial
cartilage, axon sorting, lack of
pectoral fins. (Lee, von der
Hardt et al. 2004; Clement,
Wiweger et al. 2008;
Holmborn, Habicher et al.
extl3-/-: Craniofacial cartilage
defects, shorter pectoral fins.
(Schilling, Piotrowski et al.
1996; Lee, von der Hardt et al.
2004; Clement, Wiweger et al.
ndst1a MO: Aberrant blood
circulation and vessel
(Harfouche, Hentschel et al.
ndst1b MO: Aberrant
craniofacial skeleton/ pectoral
fins. (Filipek-Gorniok,
Carlsson et al. 2015)
glcea (MO): Expansion of
ventral structures during
development. (Ghiselli and
Farber 2005)
glceb (MO): Expansion of
ventral structures during
development. (Ghiselli and
Farber 2005)
Hexuronyl 2-Osulfotransferase
Glucosaminyl 6-Osulfotransferase
M: Renal agenesis, overmineralized skeletons,
retardation of eye
development, neonatal death.
(Bullock, Fletcher et al.
H: Hypogonadotropic
hypogonadism; #614880
M: Increased embryonic
lethality and decreased
growth (Habuchi, Nagai et al.
Glucosaminyl 3-Osulfortansferase
Chondroitin Synthase
Polymerizing factor
H: Temtamy preaxial
brachydactyly syndrome; #
M: Limb patterning and
skeletal defects including
chondrodysplasia (Wilson,
Phamluong et al. 2012)
M: Normal. (Wilson,
Phamluong et al. 2012)
H: A heterozygous mutation
may be associated with
peripheral neuropathies.
(Saigoh, Izumikawa et al.
M: Affected blood
morphology. (Block, Ley et
al. 2012)
hs2st1a (MO): Epiboly
initiation failure. (Cadwalader,
Condic et al. 2012)
hs6st2 (MO): Abnormal
vascular development (Chen,
Stringer et al. 2005)
hs3st1l1/hs3st7 (MO): Loss of
the ventrical contraction
(Samson, Ferrer et al. 2013)
hs3st3x/ hs3st3b1a
hs3st3z/ hs3st3b1a
chsy1 (MO): Atrioventricular
abnormalities (Peal, Burns et
al. 2009); Impaired skeletal,
pectoral fin and retinal
development (Tian, Ling et al.
csgalnact1a (MO): In
combination with csgalnact2
MO decreased CS and
increased HS amounts.
(Holmborn, Habicher et al.
csgalnact2 (MO): In
combination with csgalnact1a
MO; decreased CS, increased
HS amounts. (Holmborn,
Habicher et al. 2012)
Dermatan sulfate
Dermatan sulfate
(chondroitin 6)
sulfotransferase 3
Carbohydrate (Nacetylglucosamine 6O) sulfotransferase 7
(chondroitin 4)
H: Ehlers-Danlos syndrome,
Musculocontractural type 2;
M: Viable, smaller, with no
changes in major organs,
alterations seen in the skin.
(Maccarana, Kalamajski et
al. 2009)
H: Spondyloepiphyseal
dysplasia with congenital
joint dislocation; #603799
M: Reduced cell number in
the lymph nodes, reduced
fertility (Orr, Le et al. 2013)
Carbohydrate (Nacetylgalactosamine
4-0) sulfotransferase
(chondroitin 4)
sulfotransferase 15
M: Severe dwarfism, death at
6h after birth from
respiratory distress. Bone and
cartilage defects. (Kluppel,
Wight et al. 2005)
chst11 (MO): Bent trunk,
curled and/or kinky tail,
impaired muscle development
and axon guidance.
(Mizumoto, Mikami et al.
H: Ehlers-Danlos syndrome,
musculocontractural type 1;
Notably, many of the listed human disorders involve either muscle, skeletal
or neuronal defects.
The extracellular matrix content of cartilage is close to 90% (dry weight),
and is known to be particularly rich in PGs. These are known to greatly
contribute to cartilage biomechanical properties, extracellular matrix water
retention, which together encounters for the resistant, yet elastic load-bearing
structure. The collagen-aggrecan-hyaluronan network is of a special
importance, providing cartilage with osmotic properties, making it pressure
resistant and giving it a turbid nature (Roughley and Lee 1994; Knudson and
Knudson 2001).
Agrrecans can form aggregates with hyaluronan, further stabilized by the
cartilage link proteins (Iozzo 1998). Other proteoglycans like decorin,
biglycan, fibromodulin and perlecan are commonly found components of the
cartilage, responsible for tissue integrity and metabolism (Roughley and Lee
1994; Knudson and Knudson 2001). The main signaling molecules involved
in bone formation belong to growth factors families, including BMP, IGF,
TGF-β, FGF, PDGH, EGF and Wnts. These growth factors participate in
bone and cartilage growth and repair processes, and a majority of them is
known to bind to HS (Bernfield, Gotte et al. 1999).
As already described, knockout mice for either CHSY1, CHPF or
CSGALNACT1, although viable and fertile, are characterized by reduced
CS content, and display chondrodysplasia and digit-patterning defects. Also
the C6ST-1 mutation in humans and the PAPS synthase mutation in mouse,
lead to severe chondroysplasia and brachymorphism, respectively. These
results may suggest that to fully support cartilage functionality, a specific
GAG sulfation pattern is needed (Orkin, Pratt et al. 1976; Yada, Gotoh et al.
2003; Watanabe, Takeuchi et al. 2010; Wilson, Phamluong et al. 2012).
Unlike cartilage, brain ECM tissue is presumed to be free from
fibronectin and collagen, frequently occurring in other organs. In contrast,
hyaluronan, HSPGs (including glypican and syndecan) and CS
proteoglycans like versican, brevican and neurocan are present in large
amounts in the intracellular spaces between neurons and glia (Bandtlow and
Zimmermann 2000; Bonneh-Barkay and Wiley 2009).
Either bound to the cell surface or as components of the ECM,
proteoglycans can influence neuronal growth, migration and synaptic
remodeling. CS proteoglycans seem to be of special importance also during
development of the central nervous system, playing roles in the formation of
the neuronal boundaries and limiting synaptic plasticity (Dyck and KarimiAbdolrezaee 2015). Zebrafish, described in the chapter below, is one of the
popular animal models, suitable for studies of the formation of cartilage and
the nervous system patterning.
Zebrafish as a model system
Zebrafish (Danio rerio), a teleost tropical freshwater fish, is a valuable
vertebrate model organism used in biological research. Adult individuals can
grow up to 6 cm (4 cm in captivity) and owe their name to five blue and
silver vertical stripes present on the side of their body.
Embryo fertilization and further development proceed externally in a
transparent chorion, enabling their observation (Fig. 4). Furthermore, to
facilitate imaging, pigmentation, normally occurring from about 24 hours
post fertilization (hpf), can be chemically inhibited.
Figure 4. Early zebrafish development. A. Zygote period (0-0.75
-0.75 hpf): the chorion is
separated from the fertilized egg cell and the non-yolk cytoplasm undergoes
movements towards the animal pole, B. Cleavage period (0.75-2.25 hpf): the cells
undergo six cell divisions in 15 min intervals, reaching a total number of 64 cells, C.
Blastula period (2.025-5.25 hpf): the epiboly process begins. The yolk syncytial layer
forms and midblastula transition starts, D. Gastrula period (5.25-10 hpf): when as a
result of involution, convergence and extension movements, primary germ layers and
embryonic axis arise, E. Segmentation period (10-24 hpf): first body movements
occur, groups of cells start to differentiate, formation of the primary organs is initiated.
Somites develop starting from the anterior end of the embryo, continuing towards its
posterior end, F. Pharyngula period (24-48 hpf): the embryo is organized bilaterally,
somite segmentation and notochord formation is finished. Development of the
pharyngeal arches, hatching gland, pigment cells, fins, heart with the first heart beats
and vasculature take place and the nervous system is composed of five lobes, G.
Hatching period (48-72 hpf): embryos will hatch, pectoral fins, jaw and gills develop
from the first two and posterior five pharyngeal arches, respectively. H. Early larval
period (3-7 days): the swimming bladder and the mouth appear. The larvae begin to
actively swim and feed. Adapted with permission from Charles Kimmel. Reprinted
with permission from (Kimmel, Ballard et al. 1995; Nusslein-Volhard and Dahm
2005), John Wiley and Sons.
The zebrafish genome is comprised of 25 chromosomes. For approximately
20% of all zebrafish genes, two homologues of a single mammalian gene are
present (Howe, Clark et al. 2013). These gene paralogs arose as a result of a
whole genome duplication which took place early in the evolution, most
likely before the origin of teleosts, in the ray-fin fish lineage. Investigations
carried out on paralogs in zebrafish suggest that ancestral gene function may
be divided between the duplicated genes as both their time and localization
of expression in some cases differ significantly (Nusslein-Volhard and Dahm
2005). The gene duplication may create some problems in the course of
studies, but the fully sequenced zebrafish genome and careful genomic
analyses diminish the risk of overlooking additional gene copies.
As a consequence of the relatively close evolutionary relation between
zebrafish and humans, the two species share many genetic and anatomical
features. Zebrafish display most of the human organs systems and structures,
making it a valuable model to study biological mechanisms underlying some
of the human diseases like muscular dystrophaty, melanoma or tuberculosis
(Bassett, Bryson-Richardson et al. 2003; Swaim, Connolly et al. 2006; Ceol,
Houvras et al. 2011).
Zebrafish in glycosaminoglycan research
A number of zebrafish mutants affecting GAG synthesis as well as
proteoglycans core proteins have been studied. Previously described
mutations alter proteoglycan integrity at many levels, affecting availability
of sugar nucleotide precursor (uxs1/hi954, udgh/jek), GAG chain
polymerization (ext2/dak, extl3/box, xylt1, b3gat3/hi307), enzymes
important for GAG sulfation (papst1/pic) and finally core protein availability
(gpc4/knypek) (Topczewski, Sepich et al. 2001; LeClair, Mui et al. 2009).
The zebrafish genes ext2 and extl2 involved in HS polymerization were
found to be important for optic-tract sorting as well as pectoral fin and jaw
development (Schilling, Piotrowski et al. 1996; Lee and Chien 2004). ext2
and extl3 mutant phenotypes have been associated with alterations in the
signaling pathways, in particular those responding to Wnt and Fgf (Norton,
Ledin et al. 2005). Mutants in both ext2 and papst1 (PAPS transporter 1)
have been shown to develop cartilage defects strongly resembling those
observed in humans with Hereditary Multiply Exostoses disease (HME)
(Clement, Wiweger et al. 2008).
Furthermore, studies of a zebrafish mutant lacking the core protein of
Glypican 4, knypek, have shown that this proteoglycan is involved in the
modulation of convergent extension movement during gastrulation
(Topczewski, Sepich et al. 2001). Also three available lines carrying
mutations in UDP-glucuronic acid decarboxylase 1 (uxs1) producing UDPxylose from UDP-glucuronic acid, xylotransferase 1 (xylt1) and
glucuronyltransferase I (b3gat3) exhibit impaired cartilage and bone
formation. These enzymes are all important for the formation of the GAG
linkage region (Eames, Yan et al. 2011; Wiweger, Avramut et al. 2011;
Holmborn, Habicher et al. 2012).
Zebrafish pharyngeal cartilage development
Several aspects of the zebrafish pharyngeal cartilage development, including
correct chondrocyte stacking and pharyngeal cartilage element maturation
are GAG structure dependent.
Formation of cartilaginous elements is initiated with the precartilage
condensation. Already at 2 days post fertilization, the first elements of the
cartilaginous skeleton begin to appear. This process gives rise to the
perichondrium filled with chondrocytes and extensive amounts of matrix.
With time, chondrocytes undergo flattening and intercalation forming
elongated, slender structures, whereas other chondrocytes assemble to form
flattened single cell layer elements. Soon after, cartilage is replaced by bone,
in contrast to dermal bones which develops without any intermediate
cartilage stage.
Development of the zebrafish cranial skeleton involves seven pharyngeal
arches where the two first arches serve as supportive structures for the jaw
and operculum. The remaining pharyngeal arches (3rd-7th) are known as
branchial arches (1st-5th), in adulthood functioning as a skeletal base for gills
and teeth (Kimmel, Miller et al. 1998) (Fig. 5).
Figure 5. Craniofacial cartilage of the zebrafish larvae at 5 dpf. A. A ventral view
of the Tg(fli1:EGFP) head region of a living zebrafish larvae. Blood vessels and
cartilage elements express GFP. B. Schematic outline of the craniofacial cartilage.
Abbreviations used: bh, basihyal; ch, ceratohyal; hs, hyosymplectic; ih, interhyal; m,
Meckels; pq, palatoquadrate.
Taken together, zebrafish are easy to maintain and culture, giving them a
strong advantage over other popular vertebrate models like mouse or rat.
Numerous clutches, reaching up to 300 embryos from a pair, can be obtained
weekly. All these advantages, together with their rapid embryonic
development, makes zebrafish suitable for many types of studies, including
toxicology screens, neurobiological investigations, behavioral studies, cancer
research or for generation of human disease models.
Method considerations
During the past few years, immense progress in the zebrafish reverse genetic
approaches has been made. Our work on glycosaminoglycan biosynthetic
enzymes have taken advantage of both old and newly established
technologies. Learning and applying these have been a considerable part of
my PhD work. A short description of the principles, advantages and
disadvantages of methods used during the course of my studies can be found
Overexpression studies
One of the biggest advantages of using zebrafish in research is the wellestablished microinjection procedure, commonly used to manipulate
zebrafish gene expression. Microinjection into fertilized eggs at the 1-2 cell
stage is relatively fast, gives low mortality rate and highly reproducible
results. Short-term expression of high levels of protein can be obtained by
injection of either DNA constructs or mRNA.
Additionally, DNA constructs driving tissue specific expression, can be
integrated into the genome. When combined with advanced microscopic
visualization methods, a wide range of dynamic processes taking place in the
developing embryo can be analyzed (Nusslein-Volhard and Dahm 2005).
Rise and fall of the morpholino technology
Morpholino antisense nucleotides (MO) have been widely used in studies of
gene function at the early stages of zebrafish development since the
beginning of the century (Corey and Abrams 2001). Transient “knockdown”
of translation or miss-splicing of a gene of interest can be obtained by
injecting syntetic MOs characterized by their high affinity for mRNA.
Complete penetration of the MO phenotype in the morphant fish (MOinjected) is usually observed during the first 2 days post fertilization, as the
concentration of the MO in the cells is getting lower with every cell division
(Nusslein-Volhard and Dahm 2005; Eisen and Smith 2008).
Translational blocking MOs result in inhibition of both the maternally
contributed transcripts and of mRNA from genes acting early during
development. Splice-inhibiting MOs, on the other hand, are designed to
cause either skipping of the exon or inhibition of pre-mRNA splicing. Their
big advantage is the possibility of quantification of MO efficiency, but
unfortunately splice-inhibiting MOs will not eliminate maternally
contributed transcripts.
After the initial burst of articles involving MO as a main technique to
achieve gene “knock-down” without fully established control protocol, a
growing need for monitoring possible off-target effects and knock-down
efficiency have been extensively discussed in the research community
(Robu, Larson et al. 2007; Eisen and Smith 2008). To make MO derived
results more reliable, several control experiments are recommended.
Multiple target sites (translational-blocking and splice-blocking MOs), allow
for confirmation of an obtained phenotype. Injections of the same amounts
of mismatch and control MO as phenotype-giving MO followed by test for
MO toxicity is one of the control experiment. If available, the most valuable
control is comparison of MO phenotype with mutants carrying a mutation in
the investigated gene. Also, MO blocking the most commonly up-regulated
off-target pathways (p53 MO) and finally, MO phenotype rescue with RNA
encoding for gene-of-interest orthologues or isoforms are suggested.
Use of MO has been linked to increased levels of apoptotic neural death
and to craniofacial cartilage development defects caused by off-target
activation of the p53 pathway. These false positive MO results are reported
to include close to 20% of total MO used in zebrafish (Robu, Larson et al.
2007). This bar has been now raised even higher by a study claiming that
only about 20% of the already reported MO phenotypes can be reproduced
in mutant fish created with CRISPR-Cas9 or TALENs technology (Kok,
Shin et al. 2015). Yet, the MO technique, used wisely, remains a powerful
instrument in zebrafish developmental studies.
A number of MO based functional studies concerning the role of GAG
biosynthetic genes in zebrafish including glce, hs2st, ndst1b, chsy1,
csgalnact1 and chst11 have been published. Zebrafish morphants for the
zebrafish glce (glcea and/or glceb) exhibit mild to severe early ventralization
defects (Ghiselli and Farber 2005), whereas the phenotype of Glce mouse
null-mutants is different, as they lack kidneys, and their lungs are poorly
inflated. Pups die immediately after birth. In addition, skeletal abnormalities,
with prominent defects in chondrocyte proliferation, are present (Li, Gong et
al. 2003; Dierker, Bachvarova et al. 2015).
This discrepancy between two different model organisms, points out the
need for a trustworthy genetic method to verify MO phenotypes, which
would be an important point of reference for any future studies using the MO
Selected methods for targeted genome editing in zebrafish
Transcription Activator-Like Effector Nucleases (TALENs)
In 2011 a new technique for targeted mutagenesis was reported (Huang,
Xiao et al. 2011). Transcription Activator-Like Effectors (TALEs) were
originally discovered as proteins produced by plant pathogens, where, after
entering the host cell, they bind to genomic DNA and affect transcription.
In genetic engineering, modified TALE peptides have the ability to
selectively, and with high affinity bind to DNA. Such peptides combined
with the nonspecific FokI endonuclease domain, have been presented as
promising tool to introduce locus-specific double strand DNA breaks (DSB)
(Dahlem, Hoshijima et al. 2012; Joung and Sander 2013). Depending on the
conditions, either non-homologous end-joining (NHEJ) or homology
directed repair (HDR) can then be used by the cell to repair damages.
Imprecise correction of the DSBs by the NHEJ system can lead to small,
insertions or/and deletions (indels) of nucleotides at the DSB site. Mistakes
created within open reading frames, can cause frame shift and result in the
generation of a premature stop codon, leading to gene disruption and
production of truncated protein (Joung and Sander 2013).
FokI nucleases work only as a dimer. Therefore, TALENS are designed to
bind two unique targets separated from each other by a short spacer. TALE
consists of sequence blocks of 33-35 amino acid repeats followed by a final
shorter repeat. The amino acids at positions 12 and 13 within each TALE
building block, determines to which of the four DNA bases the TALE will
bind (Cermak, Doyle et al. 2011).
Typically, working on a small scale with this technique involves two
rounds of complicated cloning steps and purification of as many as 60
different plasmids which can be both time consuming and troublesome.
Power of the simplicity, Clustered Regularly Interspaced Short
Palindromic Repeats (CRISPR) and CRISPR associated protein 9
The latest innovation in the field of genome editing technologies came in
2013, when the first work regarding usage of the CRISPR-Cas system in C.
elegans, Drosophila, zebrafish and many other systems, was published
(Bassett, Tibbit et al. 2013; Friedland, Tzur et al. 2013; Hwang, Fu et al.
2013). CRISPR (Clustered Regularly Interspaced Short Palindromic
Repeats) together with CRISPR-associated (Cas) genetic elements were first
discovered as a form of unique immune system against viral infections,
evolved in bacteria and archaea.
The CRISPR-Cas9 system, used as a reverse genetic tool, is based on its
ability to create targeted DSBs. In zebrafish, the CRISPR-Cas9 system is
based on two RNA components which are injected into the zebrafish embryo
at the 1-cell stage. The RNA molecules encode Cas9 endonuclease and a
recombinant single guide RNA, directing the Cas9 nuclease to the specific
site in the genome (Fig. 6).
Valuable applications of the CRISPR-Cas9 method are not limited to
generation of null-mutants. Combining the CRISPR-Cas9 system with single
strand DNA carrying homology blocks towards DNA sequences framing the
predicted DSB location, can be used for site specific DNA insertions or
substitutions. These developments of the CRISPR-Cas9 technology illustrate
the possibilities to use it to model human genetic disorders and in the future
for treatment of single-gene hereditary diseases (Doudna 2015).
Figure 6. The CRISPR-Cas9 system. Cas9 is guided by the RNA molecule
containing sequence matching the targeted site within the genome. The PAM site is
required by the CRISPR-Cas9 complex for target recognition. DBS are generated as a
result of Cas9 action and are then repaired by the cell by either homologous
recombination or by the NHEJ process. Adapted from (Charpentier and Doudna
Taken together, in comparison with other available targeted genome editing
tools, the CRISPR-Cas9 technique has the advantage to be much easier when
it comes to the target design and method implementation. High genome
editing efficiency together with relatively low toxicity make the CRISPRCas9 system possible to use also for targeting of several sites in the genome
at the same time, defined as multiplexing.
Present investigation
The overall aim of this work was to use the zebrafish model to study the role
of GAGs in embryonic development. Basic knowledge regarding several of
the zebrafish GAG biosynthesis enzymes was lacking, including information
about number of isoforms and their temporal and spatial expression during
development. The first objective was therefore to fill this gap. The second
objective to generate GAG biosynthesis zebrafish mutants for our studies
was greatly aided by the recent development of the CRISPR-Cas9 genetic
Paper I
The NDST gene family in zebrafish: role of NDST1b in pharyngeal arch
Much previous work in our group has concentrated on NDSTs, their
enzymatic properties and interactions with other HS biosynthetic enzymes
and the way by which the NDSTs affect GAG chain composition, length and
amounts. In vivo functions of NDSTs have also been the subject of previous
publications from our group including the characterization of mice lacking
functional NDST1 and NDST2 enzyme (Forsberg, Pejler et al. 1999;
Ringvall, Ledin et al. 2000; Ledin, Staatz et al. 2004).
When the present study was initiated, only fragmentary information
regarding NDST orthologues present in zebrafish was available. With our
study we aimed to complete the analysis of the zebrafish heparan sulfate
biosynthetic enzymes. We have identified, cloned and sequenced the five
members of the zebrafish NDST gene family which includes two
orthologues of NDST1 and NDST2, ndst1a, ndst1b, ndst1b, ndst2b, and a
single ndst3 orthologue (Filipek-Gorniok, Carlsson et al. 2015). Our results
indicate that NDST3 and NDST4, present in mouse and human and located
in close proximity to each other, arose as a result of a local gene duplication.
A knockout of the single ndst3 orthologue in zebrafish could possibly
mimic the effect of a NDST3/NDST4 double knockout in mice (FilipekGorniok, Carlsson et al. 2015).
All zebrafish ndsts were shown to be maternally contributed and were
expressed in the otic capsule and pectoral fins. Additionally, most of the
genes were expressed in the different brain parts, whereas in the spinal cord,
only ndst3 transcript was detected.
Attempts were also made to knock-down each of the zebrafish isoforms to
study their effects on zebrafish embryonic development. Despite our
strenuous attempts, we were not able to obtain confirmed, stable phenotypes
for all but the ndst1b morphants.
Two transgenic zebrafish lines were used to better visualize craniofacial
cartilage defects of the ndst1b morphants and the observed phenotype
showed resemblance but was somewhat milder than that of the Ndst1-/mouse. Ndst1b morphants had shortened pectoral fins and body axis. The
most prominent phenotypic change was observed in the second pharyngeal
arch, which was reverted in about 40% and perpendicular in approximately
10% of investigated morphant larvae.
Paper II
Expression of chondroitin/dermatan sulfate glycosyltransferases during
early zebrafish development.
In this work, we studied a previously not characterized group of enzymes
involved in CS/DS chain polymerization. Protein sequence comparison of
the CS/DS polymerizing enzymes revealed the existence of seven
glycosyltransferases, which can be further divided into the CSGALNACT,
CHSY and CHPF families. The single CHPF human gene was found to be
duplicated in zebrafish.
Whole mount in situ hybridization of the genes demonstrated their
overlapping expression in the notochord, the pectoral fin, the otic capsule
and in cranial cartilage including the branchial arches. These results suggest,
that the cartilaginous elements of the developing zebrafish embryo may be
especially rich in CS/DS. This hypothesis was confirmed by antibody
staining with the CS-specific antibody (CS-56) as well as by staining with
alcian blue, showing intense colouring in the pharyngeal cartilage elements
and notochord.
Most interestingly, the levels of HS were rather stable through all
analyzed developmental stages (1hpf-6dpf), whereas CS/DS levels increased
dramatically from 48hpf onwards, closely following the time course of
cartilage formation. Our results indicate that HS and CS/DS present in the
early embryo is produced by the zygote, rather than being of maternal origin.
Paper III
On the roles and regulation of chondroitin sulfate and heparan sulfate in
zebrafish pharyngeal cartilage morphogenesis.
In this study, we addressed the role of HS and CS proteoglycans in zebrafish
development using existing mutants of two enzymes important for linkage
region formation (uxs1, b3gat3) and two for HS chain initiation and
polymerization (extl3, ext2).
All investigated mutants exhibited severe defects and were lethal at larval
stage. We focused on the pharyngeal cartilage structures, as they were the
ones predominantly affected by altered GAG biosynthesis. To visualize and
analyze early stages of the craniofacial cartilage development, mutant
embryos of the fli:EGFP transgenic background were used. Interestingly,
lack of functional uxs1 enzyme caused a dramatic effect on the development
of the zebrafish larvae ceratohyal (pharyngeal cartilage element) leading to
its nearly complete loss.
We speculate that the craniofacial defects present in the mutant embryos
might be a consequence of a disruption of the extracellular matrix
physiochemical properties as a result of the loss of GAGs. The described
mutations did not change the expression of PG core proteins except for
biglycan, as analyzed by quantitative PCR.
Main phenotypic changes observed in mutant embryos are summarized
below (Tab. 2).
Table 2. Main findings. ‘+’; severity of the defect, ‘/’; not included in the study.
Pharyngeal cartilage
Chondrocyte stacking
Reduction of the ECM
Shortened pectoral fin
GAG content
HS; 52%
(in % of the control)
CS; 5%
HS; 55%
CS; 11%
HS; 44%
HS; 18%
CS; 88%
HS; 13%
CS; 16%
Additionally, based on the data obtained by analysis of the GAG content of
the extl3 mutant and the csgalnact1;csgalnact2 morphant fish and data
derived from the extl3 siRNA-treated cells, we propose that the balance
between expression of these genes affects the HS/CS content.
As shown in this work, functional ext2, extl3, uxs1 and b3gat3 enzymes
are required to provide normal HS and CS/DS levels in the developing
zebrafish larvae.
Paper IV
glycosaminoglycan biosynthesis.
In papers I and III our attempts to study the biological roles of the GAG
biosynthetic enzymes in the developing zebrafish embryo were highly
restricted by the lack of adequate tools to create new zebrafish mutants.
Moreover, the MO technique, used to knock-down expression of GAG
biosynthetic enzymes have several disadvantages, as discussed in the chapter
on method considerations.
In this ongoing study, we decided to take advantage of the rapidly
developing CRISPR-Cas9 method, which in a short time has revolutionized
the field of targeted mutagenesis in zebrafish. In close collaboration with
Shawn Burgess group, (NIH, US) we used a CRISPR-Cas9 based approach,
published by Varshney et. al, to create zebrafish mutants with defective HS
and CD/DS biosynthesis (Varshney, Pei et al. 2015) (Fig. 7).
I focused on the five zebrafish ndst genes as well as the two genes
encoding zebrafish papss2. Combining recent advantages of the CRISPRCas9 system, including multiplexing and genotyping protocols, we have
managed to identify 18 new zebrafish mutants of genes important for GAG
Lack of obvious morphological phenotypes linked to the null-mutations
of most of the investigated genes is the main finding of this study. For
example, no obvious morphological abnormalities were apparent in the adult
homozygous chsy1 mutants, where earlier published zebrafish chsy1
morphants show strong malformations during development (Peal, Burns et
al. 2009; Tian, Ling et al. 2010).
By creating vertebrate models with alterations in GAG stucture, we hope
to answer some of the questions regarding the roles of HS and CS/DS
biosynthetic enzymes in zebrafish development and homeostasis. Studying
function and effects of loss of one or more genes involved in GAG
biosynthesis in a vertebrate model system such as zebrafish, have never
before been so attainable.
Figure 7. Overview of the CRISPR/Cas9 zebrafish mutant pipeline. Two sgRNAs
are recommended for each targeted gene. In our set up, we have targeted up to four
genes at the same time by coinjection of maximally 8 sgRNAs with Cas9 mRNA into
a 1-cell stage embryo. The chimeric embryos were raised to generate founder fish,
incrossed and F1 offspring was screened for the mutations. Although identification of
several individuals carrying uniform mutant alleles is possible, in the vast majority of
cases, additional outcross steps were required to obtain a sufficient number of
heterozygous individuals. Correlation between phenotype and genotype was tested in
F3 embryos.
Concluding remarks and future perspectives
This thesis summarizes efforts made to elucidate functional aspects of GAG
biosynthesis using the zebrafish as a model system.
We demonstrate a strict spatiotemporal expression of both the members
of the NDST gene family (Paper I) and the CS/DS glycosyltransferases
(Paper II) in the developing zebrafish embryo. Paper III takes advantage of
four preexisting zebrafish mutants, defective in GAG biosynthesis.
Taken together, the results obtained in this paper demonstrate a
correlation between HS content and the severity of the pectoral fin defect.
Additionally, impaired HS biosynthesis was shown to be associated with
altered chondrocyte intercalation. Interestingly, defect CS biosynthesis
resulted in loss of the chondrocyte extracellular matrix in the investigated
mutant fish. One of the main findings in this study is that the relative
expression levels of Extl3 and Csgalnact1/Csgalnact2 may influence the
HS/CS ratio.
The importance of GAGs during embryonic development have been
studied in several model organisms and a number of human genetic diseases
have been linked to mutations in genes encoding HS and CS/DS biosynthetic
enzymes. Intervention of HS and CS/DS biosynthesis may therefore be a
future therapeutic approach to cure GAG-associated disorders.
In Paper IV, we took advantage of the development of the CRISPR-Cas9
technique. Here we aimed to create a collection of zebrafish mutants with
defected GAG biosynthetic machineries. The vast majority of the newly
created null-mutants did not exhibit any obvious aberrant phenotype.
The complexity of GAG structure in vertebrates may correlate with the
increasing number of enzymes involved in GAG assembly. Gene
duplications in the vertebrate lineage can, in some cases, be the reason why
efficient reduction of GAG synthesis is not achieved with
knockot/knockdown of only one of the gene copies Also, contribution of the
mRNA encoding the correct version of the protein deposited in the embryo
by the heterozygous mother could be sufficient for early development of
zebrafish (Kok, Shin et al. 2015). Additionally, other poorly understood
compensation mechanisms might be activated in the cell affected by the gene
knock-out. In a recent study by Rossi et al. it is pointed out that
transcriptome changes of the mutant, not present in morphant fish, can
compensate for the null mutations (Rossi, Kontarakis et al. 2015). Such
changes would be very interesting to study in the CRISPR-Cas9 mutants we
have generated.
Two of the promising candidate genes linked to human genetic disorders
and of special interest to me are NDST3 and PAPSS2, for which we are
currently raising fish carrying null mutations. Our preliminary results from
the morpholino studies of the papss2a gene, suggest that it is of importance
for muscle fiber organization as well as for notochord development.
Populärvetenskaplig sammanfattning
Förutom DNA, RNA och proteiner kan också kolhydrater bära information som
behövs för att möjliggöra liv. Proteoglykaner består av en proteinkärna till
vilken en eller flera långa ogrenade kolhydratkedjor (s k glykosaminoglykaner)
är fastsatta. Glykosaminoglykanena är negativt laddade, där ofta mer än hälften
av laddningarna beror på sulfatgrupper utspridda längs glykosaminoglykanerna i
mönster som känns igen av proteiner med positivt laddade aminosyror på sin
yta. Olika mönster attraherar olika molekyler och ett sätt för celler att
kommunicera med omgivningen är med hjälp av proteoglykanerna.
Mitt arbete har fokuserat på två typer av glykosaminoglykaner, heparansulfat
(HS) och chondroitin/dermatansulfat (CS/DS). Jag har intresserat mig för deras
biosyntes och de roller de har under embryonalutvecklingen. Mina studier har
gjorts på zebrafisk, som tillsammans med möss, råttor, fruktflugor (Drosophila
melanogaster) och nematoder (C. elegans) är populära biologiska modellsystem.
Zebrafiskar är ryggradsdjur och innehåller motsvarigheter till alla huvudsakliga
organ hos människor. De har använts i många typer av studier bl a för att göra
modeller för att efterlikna sjukdomar hos människa. Zebrafisken är en liten
tropisk sötvattensfisk. I fångenskap blir den upp till 4 cm lång. Befruktning och
fortsatt utveckling sker utanför honan och de många embryonerna utvecklas
innanför en genomskinlig fosterhinna. Detta gör det lätt att följa embryots
Mitt första arbete i avhandlingen fokuserar på NDST-genfamiljen. Enzymer i
denna familj deltar i biosyntes av HS och har en nyckelroll när det gäller att
bestämma hur sulfatmönstret på den färdiga HS-kedjan ska se ut. Vi fann att
zebrafiskar tillverkar fem sådana enzymer istället för människans fyra. Den
andra artikeln i avhandlingen handlar om de sju enzymer i zebrafisk som bygger
CS/DS-kedjorna. Dessa två första studier där vi kartlagt var och när enzymerna
tillverkas utgör en viktig bas för fortsatta studier av glykosaminoglykaners roll
under embryonalutvecklingen. I den tredje studien där vi använde oss av
mutanter med defekt syntes av glykosaminoglykaner kunde vi bl a visa att
CS/DS ibland kan kompensera för HS när dess syntes inte fungerar som den
ska. I det fjärde arbetet som är en preliminär studie, använde vi oss av nyligen
utvecklade genetiska metoder för att inaktivera zebrafiskgener. Detta har tills
nyligen varit svårt att göra. Vi har generarat mutanter för många av de enzymer
som deltar i biosyntes av både HS och CS/DS och påbörjat studier av hur detta
påverkar embryonalutvecklingen.
Starting from the earliest years, my interest in biology was encouraged by
the extraordinary teachers - Mr. Bogunia in the primary school; Ms. Dorda
and Zamarska in the high school; a group of passionate academics, including
Ms. Zięba, Mr. Szklarczyk and Mr. Jasienski at the University in Krakow;
and finally Lena Kjellén and Johan Ledin at Uppsala University.
In my wildest dreams, I never expected to be enrolled in a PhD program.
This work could never be completed without the support and guidance I
have had over the years. Thus, I would like to express my sincere gratitude
to all who made this exciting journey possible and enjoyable.
My supervisor Lena, thank you for all the support, time, attention and
patience you gave me! I could not wish for a better supervisor. You
permitted me to develop as a researcher, have shared your infinite
knowledge and have been an excellent role model as a scientist, but also as a
human being.
My co-supervisor Johan, without you, from the start until the end, none of
this would have been possible. Your help and support are always of the
greatest value, whether it regards professional or personal issues.
I would like to thank Jonas von Hofsten from Umeå University, Sweden,
Shawn Burgess and Gaurav Varshney from NIH, US for giving me the
great opportunity of getting the new perspectives by working in their labs.
To all present and former members of the Kjellen group, Inger, Pernilla,
Anders & Anders (Lunde), Audrey, Dagmar, Anh-Tri, Cathy and all the
project and master students, for sharing your knowledge, support, but also
for creating such a friendly atmosphere throughout these years. Additionally,
a special thanks to Tabea, who many times has proven to be an excellent
travel companion and cat-caretaker.
My lovely zebrafish gang, Judith, Katarina and more recently Ling, you
have been irreplaceable company whatever we did, or wherever we went.
Thank you all for your support, advice and friendship! Tatjana and other
collaborators, thank you for fruitful cooperation and sharing your
Office-team, you have filled my time with engaging conversations regarding
politics, sport, history, geography, music, books, movies and science! If I
needed help or company, there was someone to answer my questions or just
grab a cup of coffee with. I missed you all this last year!
Members of the D9:4 corridor, IMBIM and EvoDevo EBC colleges, it was
a pleasure and an inspiration to share my time with you.
Administrative workers in IMBIM and EBC, for solving all my requests,
support and friendly attitude.
To all my dear Uppsala friends through the years, Riccardo & Niroj,
Sebastian, Maja, Annette, Zsuzska & Juri, Prune, Zhenechka, Steffano,
Nhi & Enzo, Nick & Lisa, Åsa & Matan, Gabi & Alina, Veronique &
Benq, Mats & Harriet, Anna & Andrei. I am lucky to have such great
people around me!
Special thanks to all of you involved in Sputnik-Katushka-KalashnikovBalkan-Gyspy-Punk-Ska-More Palinka-Dubstep Madness! As long as
you will play, I will always be there.
Wszystkim byłym i obecnym członkom polskiej ekipy w Uppsali, za nasze
spotkania, wycieczki, domowe obiadki, imprezy, wsparcie, pomoc i
niańczenie Emilki i Mumrika. Ekipie campingowo-wypadowej, za
zapewnianie wybornej zabawy na wyjazdach!
Kabot, Didi, Lala i Wronia – dziękuję, że jesteście! Nie potrafię wyobrazić
sobie życia, bez waszej w nim obecności! Agatka, Maria i Ania – za stare,
dobre czasy na Akademi Rolniczej, za to, że zawsze znajdujecie czas na
krakowskie ploteczki i odwiedziny w Szwecji.
Moim rodzicom i rodzinie, za to, że nigdy nie zwątpili w moje możliwości.
Chociaż nasze życiowe drogi są różne, zawsze akceptowaliście moje wybory
i podróżniczą pasję. Dziękuję za wasze wsparcie i pomoc w każdej sytuacji!
Dziękuję Zosi i Henrykowi, za przyjęcie mnie do swojej rodziny,
wychowanie najlepszego w świecie męża i za pomoc w opiece nad Emilką.
Łukaszowi i Emilce, za to że jesteście Ziemią pod moimi stopami i Niebiem
nad moją głową.
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Acta Universitatis Upsaliensis
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