Publications by authors named "Malgorzata Wiweger"

22 Publications

  • Page 1 of 1

-Deficient Zebrafish Reproduce Neurological and Inflammatory Symptoms of Niemann-Pick Type C Disease.

Front Cell Neurosci 2021 27;15:647860. Epub 2021 Apr 27.

Laboratory of Neurodegeneration, International Institute of Molecular and Cell Biology in Warsaw, Warsaw, Poland.

Niemann-Pick type C (NPC) disease is an autosomal recessive lysosomal storage disease that is caused by a mutation of the or gene, in which un-esterified cholesterol and sphingolipids accumulate mainly in the liver, spleen, and brain. Abnormal lysosomal storage leads to cell damage, neurological problems, and premature death. The time of onset and severity of symptoms of NPC disease are highly variable. The molecular mechanisms that are responsible for NPC disease pathology are far from being understood. The present study generated and characterized a zebrafish mutant that lacks Npc2 protein that may be useful for studies at the organismal, cellular, and molecular levels and both small-scale and high-throughput screens. Using CRISPR/Cas9 technology, we knocked out the zebrafish homolog of . Five-day-old mutants were morphologically indistinguishable from wildtype larvae. We found that live larvae exhibited stronger Nile blue staining. The larvae exhibited low mobility and a high anxiety-related response. These behavioral changes correlated with downregulation of the (mitochondrial calcium uniporter) gene, (calcineurin) gene, and genes that are involved in myelination ( and ). Histological analysis of adult zebrafish revealed that pathological changes in the nervous system, kidney, liver, and pancreas correlated with inflammatory responses (i.e., the upregulation of , κβ, and ; i.e., hallmarks of NPC disease). These findings suggest that the mutant zebrafish may be a model of NPC disease.
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http://dx.doi.org/10.3389/fncel.2021.647860DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8111220PMC
April 2021

Caffeine-Cyclodextrin Complexes as Solids: Synthesis, Biological and Physicochemical Characterization.

Int J Mol Sci 2021 Apr 18;22(8). Epub 2021 Apr 18.

International Institute of Molecular and Cell Biology in Warsaw, Księcia Trojdena 4, 02-109 Warsaw, Poland.

Mechanochemical and in-solution synthesis of caffeine complexes with α-, β-, and γ-cyclodextrins was optimized. It was found that short-duration, low-energy cogrinding, and evaporation (instead of freeze-drying) are effective methods for the formation and isolation of these complexes. The products obtained, their pure components, and their mixtures were examined by powder X-ray diffraction (PXRD), differential scanning calorimetry (DSC), FT-IR and Raman spectroscopy. Moreover, molecular modeling provided an improved understanding of the association process between the guest and host molecules in these complexes. The complexes were found to exhibit high toxicity in zebrafish () embryos, in contrast to pure caffeine and cyclodextrins at the same molar concentrations. HPLC measurements of the caffeine levels in zebrafish embryos showed that the observed cytotoxicity is not caused by an increased caffeine concentration in the body of the organism, as the concentrations are similar regardless of the administered caffeine form. Therefore, the observed high toxicity could be the result of the synergistic effect of caffeine and cyclodextrins.
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http://dx.doi.org/10.3390/ijms22084191DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8073077PMC
April 2021

An improved method for precise genome editing in zebrafish using CRISPR-Cas9 technique.

Mol Biol Rep 2021 Feb 22;48(2):1951-1957. Epub 2021 Jan 22.

International Institute of Molecular and Cell Biology in Warsaw, Ks. Trojdena Str. 4, 02-109, Warsaw, Poland.

Current methods of CRISPR-Cas9-mediated site-specific mutagenesis create deletions and small insertions at the target site which are repaired by imprecise non-homologous end-joining. Targeting of the Cas9 nuclease relies on a short guide RNA (gRNA) corresponding to the genome sequence approximately at the intended site of intervention. We here propose an improved version of CRISPR-Cas9 genome editing that relies on two complementary guide RNAs instead of one. Two guide RNAs delimit the intervention site and allow the precise deletion of several nucleotides at the target site. As proof of concept, we generated heterozygous deletion mutants of the kcng4b, gdap1, and ghitm genes in the zebrafish Danio rerio using this method. A further analysis by high-resolution DNA melting demonstrated a high efficiency and a low background of unpredicted mutations. The use of two complementary gRNAs improves CRISPR-Cas9 specificity and allows the creation of predictable and precise mutations in the genome of D. rerio.
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http://dx.doi.org/10.1007/s11033-020-06125-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7925485PMC
February 2021

Glomerular permeability is not affected by heparan sulfate glycosaminoglycan deficiency in zebrafish embryos.

Am J Physiol Renal Physiol 2019 11 28;317(5):F1211-F1216. Epub 2019 Aug 28.

Department of Pathology, Leiden University Medical Center, Leiden, The Netherlands.

Proteinuria develops when specific components in the glomerular filtration barrier have impaired function. Although the precise components involved in maintaining this barrier have not been fully identified, heparan sulfate proteoglycans are believed to play an essential role in maintaining glomerular filtration. Although in situ studies have shown that a loss of heparan sulfate glycosaminoglycans increases the permeability of the glomerular filtration barrier, recent studies using experimental models have shown that podocyte-specific deletion of heparan sulfate glycosaminoglycan assembly does not lead to proteinuria. However, tubular reabsorption of leaked proteins might have masked an increase in glomerular permeability in these models. Furthermore, not only podocytes but also glomerular endothelial cells are involved in heparan sulfate synthesis in the glomerular filtration barrier. Therefore, we investigated the effect of a global heparan sulfate glycosaminoglycan deficiency on glomerular permeability. We used a zebrafish embryo model carrying a homozygous germline mutation in the gene. Glomerular permeability was assessed with a quantitative dextran tracer injection method. In this model, we accounted for tubular reabsorption. Loss of anionic sites in the glomerular basement membrane was measured using polyethyleneimine staining. Although mutant animals had significantly fewer negatively charged areas in the glomerular basement membrane, glomerular permeability was unaffected. Moreover, heparan sulfate glycosaminoglycan-deficient embryos had morphologically intact podocyte foot processes. Glomerular filtration remains fully functional despite a global reduction of heparan sulfate.
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http://dx.doi.org/10.1152/ajprenal.00126.2019DOI Listing
November 2019

Novel calcineurin A (PPP3CA) variant associated with epilepsy, constitutive enzyme activation and downregulation of protein expression.

Eur J Hum Genet 2019 01 25;27(1):61-69. Epub 2018 Sep 25.

Department of Medical Genetics, Medical University of Warsaw, Warsaw, Poland.

PPP3CA encodes calmodulin-binding catalytic subunit of calcineurin, a ubiquitously expressed calcium/calmodulin-regulated protein phosphatase. Recently de novo PPP3CA variants were reported as a cause of disease in 12 subjects presenting with epileptic encephalopathy and dysmorphic features. We describe a boy with similar phenotype and severe early onset epileptic encephalopathy in whom a novel de novo c.1324C>T (p.(Gln442Ter)) PPP3CA variant was found by whole exome sequencing. Western blot experiments in patient's cells (EBV transformed lymphocytes and neuronal cells derived through reprogramming) indicate that despite normal mRNA abundance the protein expression level is strongly reduced both for the mutated and wild-type protein. By in vitro studies with recombinant protein expressed in E. coli we show that c.1324C>T (p.(Gln442Ter)) results in constitutive activation of the enzyme. Our results confirm the role of PPP3CA defects in pathogenesis of a distinct neurodevelopmental disorder including severe epilepsy and dysmorphism and provide further functional clues regarding the pathogenic mechanism.
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http://dx.doi.org/10.1038/s41431-018-0254-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6303256PMC
January 2019

[email protected] core/shell nanoparticles for biomedical applications: Physicochemical, in vitro and in vivo characterization.

Mater Sci Eng C Mater Biol Appl 2017 Nov 11;80:603-615. Epub 2017 Jul 11.

NanoBioMedical Centre, Adam Mickiewicz University, Umultowska 85, 61-614 Poznan, Poland,. Electronic address:

The chemical composition of nanoparticles (NPs) may be so designed as to provide measurability for numerous imaging techniques in order to achieve synergistic advantages. Innovative and unique structure of the core/shell [email protected] NPs possesses luminescent and magnetic properties, and is expected that they will become a new generation of contrast agents for Magnetic Resonance Imaging (MRI) and nanocarriers for theranostics. Thus, by surface biofunctionalization, it is possible to indicate particular nanoparticle compositions which provide efficient imaging, targeted drug delivery, and biocompatibility. Novel [email protected] NPs were synthesized and biofunctionalized by folic acid (FA) and doxorubicin (Doxo) to provide target and anticancer functions. Physicochemical analyses of the nanoparticles were performed. The biological study included a cytotoxicity in vitro, cellular distribution evaluation, as well as toxicity analyses, performed for the first time, on the in vivo zebrafish (Danio rerio) model. Nanoparticles were found to be effective double-function biomarkers (MRI T contrast agents, fluorescent imaging). The biological study showed that [email protected] and [email protected]@[email protected] NPs are biocompatible in a particular concentration ranges. Conjugation with folic acid and/or doxorubicin resulted in effective drug delivery targeting. The in vivo results described the toxicology profile toward the zebrafish embryo/larvae, including new data concerning the survival, hatching ratio, and developmental malformations.
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http://dx.doi.org/10.1016/j.msec.2017.07.009DOI Listing
November 2017

Be Healthy as a Fish Educational Program at the International Institute of Molecular and Cell Biology in Warsaw, Poland.

Zebrafish 2016 08 30;13(4):266-71. Epub 2016 Mar 30.

International Institute of Molecular and Cell Biology in Warsaw , Warsaw, Poland .

The purpose of the Be Healthy as a Fish educational program that is organized by the International Institute of Molecular and Cell Biology (IIMCB) in Warsaw, Poland, is to educate children about the ways in which zebrafish can be used as a model organism to help scientists understand the way the human body works. We introduce Be Healthy as a Fish workshops to children in fourth to sixth grades of primary school (9-11 years old), together with two kinds of materials under the same title: a book and a movie. We focus on the field of biology in a way that complements the children's classroom curriculum and encourages them to broaden their interests in biology in the future. The Be Healthy as a Fish educational program was inaugurated in 2014 at the Warsaw Science Festival. As of October 31, 2015, 526 primary school students participated in 27 workshops. Approximately 2000 people have received the book and nearly 1700 people have watched the movie. Be Healthy as a Fish: Origin of the Title There is a popular saying in Poland that someone is "healthy as a fish" meaning that one enjoys good health. Does this imply that fish are really that healthy? Obviously, some fish may not be healthy. Just like other animals and humans, they can and do get sick. However, this common and deceptive impression of "healthy fish" results from the fact that people hardly ever have an opportunity to observe a fish that is sick. Why does our educational program have such a possibly misleading title that may not always be true? We took advantage of this provocative title and commonly known expression and assigned to it a completely new meaning: fish can get sick, but they are important for human health. Notably, this catchy sentence intrinsically combines two keywords-health and fish-which, in our opinion, makes it a good title for a successful educational program.
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http://dx.doi.org/10.1089/zeb.2015.1195DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4931308PMC
August 2016

Testing tuberculosis drug efficacy in a zebrafish high-throughput translational medicine screen.

Antimicrob Agents Chemother 2015 Feb 10;59(2):753-62. Epub 2014 Nov 10.

IBL, Leiden University, Leiden, The Netherlands

The translational value of zebrafish high-throughput screens can be improved when more knowledge is available on uptake characteristics of potential drugs. We investigated reference antibiotics and 15 preclinical compounds in a translational zebrafish-rodent screening system for tuberculosis. As a major advance, we have developed a new tool for testing drug uptake in the zebrafish model. This is important, because despite the many applications of assessing drug efficacy in zebrafish research, the current methods for measuring uptake using mass spectrometry do not take into account the possible adherence of drugs to the larval surface. Our approach combines nanoliter sampling from the yolk using a microneedle, followed by mass spectrometric analysis. To date, no single physicochemical property has been identified to accurately predict compound uptake; our method offers a great possibility to monitor how any novel compound behaves within the system. We have correlated the uptake data with high-throughput drug-screening data from Mycobacterium marinum-infected zebrafish larvae. As a result, we present an improved zebrafish larva drug-screening platform which offers new insights into drug efficacy and identifies potential false negatives and drugs that are effective in zebrafish and rodents. We demonstrate that this improved zebrafish drug-screening platform can complement conventional models of in vivo Mycobacterium tuberculosis-infected rodent assays. The detailed comparison of two vertebrate systems, fish and rodent, may give more predictive value for efficacy of drugs in humans.
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http://dx.doi.org/10.1128/AAC.03588-14DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4335901PMC
February 2015

Possible effects of EXT2 on mesenchymal differentiation--lessons from the zebrafish.

Orphanet J Rare Dis 2014 Mar 14;9:35. Epub 2014 Mar 14.

Department of Pathology, Leiden University Medical Center, Leiden, The Netherlands.

Background: Mutations in the EXT genes disrupt polymerisation of heparan sulphates (HS) and lead to the development of osteochondroma, an isolated/sporadic- or a multifocal/hereditary cartilaginous bone tumour. Zebrafish (Danio rerio) is a very powerful animal model which has shown to present the same cartilage phenotype that is commonly seen in mice model and patients with the rare hereditary syndrome, Multiple Osteochondroma (MO).

Methods: Zebrafish dackel (dak) mutant that carries a nonsense mutation in the ext2 gene was used in this study. A panel of molecular, morphological and biochemical analyses was used to assess at what step bone formation is affected and what mechanisms underlie changes in the bone formation in the ext2 mutant.

Results: During bone development in the ext2-/- zebrafish, chondrocytes fail to undergo terminal differentiation; and pre-osteoblasts do not differentiate toward osteoblasts. This inadequate osteogenesis coincides with increased deposition of lipids/fats along/in the vessels and premature adipocyte differentiation as shown by biochemical and molecular markers. Also, the ext2-null fish have a muscle phenotype, i.e. muscles are shorter and thicker. These changes coexist with misshapen bones. Normal expression of runx2 together with impaired expression of osterix and its master regulator--xbp1 suggest that unfolded protein responses might play a role in MO pathogenesis.

Conclusions: Heparan sulphates are required for terminal differentiation of the cartilaginous template and consecutive formation of a scaffold that is needed for further bone development. HS are also needed for mesenchymal cell differentiation. At least one copy of ext2 is needed to maintain the balance between bone and fat lineages, but homozygous loss of the ext2 function leads to an imbalance between cartilage, bone and fat lineages. Normal expression of runx2 and impaired expression of osterix in the ext2-/- fish indicate that HS are required by osteoblast precursors for their further differentiation towards osteoblastic lineage. Lower expression of xbp1, a master regulator of osterix, suggests that HS affect the 'unfolded protein response', a pathway that is known to control bone formation and lipid metabolism. Our observations in the ext2-null fish might explain the musculoskeletal defects that are often observed in MO patients.
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http://dx.doi.org/10.1186/1750-1172-9-35DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4004154PMC
March 2014

Robotic injection of zebrafish embryos for high-throughput screening in disease models.

Methods 2013 Aug 11;62(3):246-54. Epub 2013 Jun 11.

Department of Molecular Cell Biology, Institute of Biology, Leiden University, The Netherlands.

The increasing use of zebrafish larvae for biomedical research applications is resulting in versatile models for a variety of human diseases. These models exploit the optical transparency of zebrafish larvae and the availability of a large genetic tool box. Here we present detailed protocols for the robotic injection of zebrafish embryos at very high accuracy with a speed of up to 2000 embryos per hour. These protocols are benchmarked for several applications: (1) the injection of DNA for obtaining transgenic animals, (2) the injection of antisense morpholinos that can be used for gene knock-down, (3) the injection of microbes for studying infectious disease, and (4) the injection of human cancer cells as a model for tumor progression. We show examples of how the injected embryos can be screened at high-throughput level using fluorescence analysis. Our methods open up new avenues for the use of zebrafish larvae for large compound screens in the search for new medicines.
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http://dx.doi.org/10.1016/j.ymeth.2013.06.002DOI Listing
August 2013

On the roles and regulation of chondroitin sulfate and heparan sulfate in zebrafish pharyngeal cartilage morphogenesis.

J Biol Chem 2012 Sep 6;287(40):33905-16. Epub 2012 Aug 6.

Department of Medical Biochemistry and Microbiology, Science for Life Laboratory, Uppsala University, Uppsala, Sweden.

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.
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http://dx.doi.org/10.1074/jbc.M112.401646DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3460485PMC
September 2012

HSPG-deficient zebrafish uncovers dental aspect of multiple osteochondromas.

PLoS One 2012 11;7(1):e29734. Epub 2012 Jan 11.

Department of Pathology, Leiden University Medical Centre, Leiden, The Netherlands.

Multiple Osteochondromas (MO; previously known as multiple hereditary exostosis) is an autosomal dominant genetic condition that is characterized by the formation of cartilaginous bone tumours (osteochondromas) at multiple sites in the skeleton, secondary bursa formation and impingement of nerves, tendons and vessels, bone curving, and short stature. MO is also known to be associated with arthritis, general pain, scarring and occasional malignant transformation of osteochondroma into secondary peripheral chondrosarcoma. MO patients present additional complains but the relevance of those in relation to the syndromal background needs validation. Mutations in two enzymes that are required during heparan sulphate synthesis (EXT1 or EXT2) are known to cause MO. Previously, we have used zebrafish which harbour mutations in ext2 as a model for MO and shown that ext2⁻/⁻ fish have skeletal defects that resemble those seen in osteochondromas. Here we analyse dental defects present in ext2⁻/⁻ fish. Histological analysis reveals that ext2⁻/⁻ fish have very severe defects associated with the formation and the morphology of teeth. At 5 days post fertilization 100% of ext2⁻/⁻ fish have a single tooth at the end of the 5(th) pharyngeal arch, whereas wild-type fish develop three teeth, located in the middle of the pharyngeal arch. ext2⁻/⁻ teeth have abnormal morphology (they were shorter and thicker than in the WT) and patchy ossification at the tooth base. Deformities such as split crowns and enamel lesions were found in 20% of ext2⁺/⁻ adults. The tooth morphology in ext2⁻/⁻ was partially rescued by FGF8 administered locally (bead implants). Our findings from zebrafish model were validated in a dental survey that was conducted with assistance of the MHE Research Foundation. The presence of the malformed and/or displaced teeth with abnormal enamel was declared by half of the respondents indicating that MO might indeed be also associated with dental problems.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0029734PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3256178PMC
May 2012

Peripheral chondrosarcoma progression is associated with increased type X collagen and vascularisation.

Virchows Arch 2012 Jan 25;460(1):95-102. Epub 2011 Nov 25.

Department of Pathology, Leiden University Medical Center, RC Leiden, The Netherlands.

Endochondral bone formation requires a cartilage template, known as the growth plate, and vascular invasion, bringing osteoblasts and osteoclasts. Endochondral chondrocytes undergo sequences of cell division, matrix secretion, cell hypertrophy, apoptosis, and matrix calcification/mineralisation. In this study, two critical steps of endochondral bone formation, the deposition of collagen X-rich matrix and blood vessel attraction/invasion, were investigated by immunohistochemistry. Fourteen multiple osteochondromas and six secondary peripheral chondrosarcomas occurring in patients with multiple osteochondromas were studied and compared to epiphyseal growth plate samples. Mutation analysis showed all studied patients (expect one) to harbour a germ-line mutations in either EXT1 or EXT2. Here, we described that homozygous mutations in EXT1/EXT2, which are causative for osteochondroma formation, are likely to affect terminal chondrocyte differentiation and vascularisation in the osteocartilaginous interface. Contrastingly, terminal chondrocyte differentiation and vascularisation seem to be unaffected in secondary peripheral chondrosarcoma. In addition, osteochondromas with high vascular density displayed a higher proliferation rate. A similar apoptotic rate was observed in osteochondromas and secondary peripheral chondrosarcomas. Recently, it has been shown that cells with functional EXT1 and EXT2 are outnumbering EXT1/EXT2 mutated cells in secondary peripheral chondrosarcomas. This might explain the increased type X collagen production and blood vessel attraction in these malignant tumours.
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http://dx.doi.org/10.1007/s00428-011-1168-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3267032PMC
January 2012

Growth plate regulation and osteochondroma formation: insights from tracing proteoglycans in zebrafish models and human cartilage.

J Pathol 2011 Jun 19;224(2):160-8. Epub 2011 Apr 19.

Department of Pathology, Leiden University Medical Centre, Leiden, The Netherlands.

Proteoglycans are secreted into the extracellular matrix of virtually all cell types and function in several cellular processes. They consist of a core protein onto which glycosaminoglycans (e.g., heparan or chondroitin sulphates), are attached. Proteoglycans are important modulators of gradient formation and signal transduction. Impaired biosynthesis of heparan sulphate glycosaminoglycans causes osteochondroma, the most common bone tumour to occur during adolescence. Cytochemical staining with positively charged dyes (e.g., polyethyleneimine-PEI) allows, visualisation of proteoglycans and provides a detailed description of how proteoglycans are distributed throughout the cartilage matrix. PEI staining was studied by electron and reflection contrast microscopy in human growth plates, osteochondromas and five different proteoglycan-deficient zebrafish mutants displaying one of the following skeletal phenotypes: dackel (dak/ext2), lacking heparan sulphate and identified as a model for human multiple osteochondromas; hi307 (β3gat3), deficient for most glycosaminoglycans; pinscher (pic/slc35b2), presenting with defective sulphation of glycosaminoglycans; hi954 (uxs1), lacking most glycosaminoglycans; and knypek (kny/gpc4), missing the protein core of the glypican-4 proteoglycan. The panel of genetically well-characterized proteoglycan-deficient zebrafish mutants serves as a convincing and comprehensive study model to investigate proteoglycan distribution and the relation of this distribution to the model mutation status. They also provide insight into the distributions and gradients that can be expected in the human homologue. Human growth plate, wild-type zebrafish and fish mutants with mild proteoglycan defects (hi307 and kny) displayed proteoglycans distributed in a gradient throughout the matrix. Although the mutants pic and hi954, which had severely impaired proteoglycan biosynthesis, showed no PEI staining, dak mutants demonstrated reduced PEI staining and no gradient formation. Most chondrocytes from human osteochondromas showed normal PEI staining. However, approximately 10% of tumour chondrocytes were similar to those found in the dak mutant (e.g., lack of PEI gradients). The cells in the reduced PEI-stained areas are likely associated with loss-of-function mutations in the EXT genes, and they might contribute to tumour initiation by disrupting the gradients.
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http://dx.doi.org/10.1002/path.2886DOI Listing
June 2011

Cartilage ultrastructure in proteoglycan-deficient zebrafish mutants brings to light new candidate genes for human skeletal disorders.

J Pathol 2011 Mar 5;223(4):531-42. Epub 2011 Jan 5.

Department of Pathology, Leiden University Medical Centre, The Netherlands.

Proteoglycans are molecules consisting of protein cores onto which sugar chains, i.e., glycosaminoglycans (GAGs) such as heparan or chondroitin sulphates, are attached. Proteoglycans are produced by nearly all cells, and once secreted they become a major component of the extracellular matrix. Cartilage is particularly rich in proteoglycans, and changes in the structure and composition of GAGs have been found in osteochondromas and osteoarthritis. The zebrafish (Danio rerio) exhibits fast development, a growth plate-like organization of its craniofacial skeleton and an availability of various mutants, making it a powerful model for the study of human skeletal disorders with unknown aetiology. We analysed skeletons from five zebrafish lines with known mutations in genes involved in proteoglycan synthesis: dackel (dak/ext2), lacking heparan sulphate; hi307 (β3gat3), deficient for most GAGs; pinscher (pic/slc35b2), presenting defective sulphation of GAGs and other molecules; hi954 (uxs1), lacking Notch and most GAGs due to impaired protein xylosylation; and knypek (kny/gpc4), missing the protein core of the Glypican-4 proteoglycan. Here we show that each mutant displays different phenotypes related to: (a) cartilage morphology; (b) composition of the extracellular matrix; (c) ultrastructure of the extracellular matrix; and (d) the intracellular ultrastructure of chondrocytes, proving that sulphated GAGs orchestrate the cartilage intra- and extracellular ultrastructures. The mild phenotype of the hi307 mutant suggests that proteoglycans consisting of a protein core and a short sugar linker might suffice for proper chondrocyte stacking. Finally, knypek supports the involvement of Glypican-4 in the craniofacial phenotype of Simpson-Golabi-Behmel syndrome and suggests GPC4 as a modulator of the overgrowth phenotype that is associated with this syndrome and is primarily caused by a mutation in GPC3. Moreover, we speculate on the potential involvement of SLC35B2, β3GAT3 and UXS1 in skeletal dysplasias. This work promotes the use of zebrafish as a model of human skeletal development and associated pathologies.
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http://dx.doi.org/10.1002/path.2824DOI Listing
March 2011

Tiling resolution array-CGH shows that somatic mosaic deletion of the EXT gene is causative in EXT gene mutation negative multiple osteochondromas patients.

Hum Mutat 2011 Feb 7;32(2):E2036-49. Epub 2010 Dec 7.

Department of Molecular Cell Biology, Leiden University Medical Center, Leiden, the Netherlands.

Multiple osteochondromas (MO) is a hereditary skeletal disorder characterized by the presence of cartilage capped bony outgrowths at bone surface. Causative mutations in EXT1 or EXT2 genes have been described in 85-90 % of MO cases. However, in about 10-15 % of the MO cases, genomic alterations can not be detected, implying the potential role of other alterations. We have designed a custom-made Agilent oligonucleotide-based microarray, containing 44,000 probes, with tiling coverage of EXT1/2 genes and addition of 68 genes involved in heparan sulfate biosynthesis and other related pathways. Out of the 17 patient samples with previously undetected mutations, a low level of deletion of the EXT1 gene in about 10-15% of the blood cells was detected in two patients and mosaic deletion of the EXT2 was detected in one patient. Here we show that for the first time somatic mosaicism with large genomic deletions as the underlying mechanism in MO formation was identified. We propose that the existence of mosaic mutations and not alterations of other heparan sulfate biosynthesis related genes play a significant role in the development of MO in patients who are tested negative for mutations in Exostosins.
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http://dx.doi.org/10.1002/humu.21423DOI Listing
February 2011

Primary cilia organization reflects polarity in the growth plate and implies loss of polarity and mosaicism in osteochondroma.

Lab Invest 2010 Jul 26;90(7):1091-101. Epub 2010 Apr 26.

Department of Pathology, Leiden University Medical Center, Leiden, The Netherlands.

Primary cilia are specialized cell surface projections found on most cell types. Involved in several signaling pathways, primary cilia have been reported to modulate cell and tissue organization. Although they have been implicated in regulating cartilage and bone growth, little is known about the organization of primary cilia in the growth plate cartilage and osteochondroma. Osteochondromas are bone tumors formed along the growth plate, and they are caused by mutations in EXT1 or EXT2 genes. In this study, we show the organization of primary cilia within and between the zones of the growth plate and osteochondroma. Using confocal and electron microscopy, we found that in both tissues, primary cilia have a similar formation but a distinct organization. The shortest ciliary length is associated with the proliferative state of the cells, as confirmed by Ki-67 immunostaining. Primary cilia organization in the growth plate showed that non-polarized chondrocytes (resting zone) are becoming polarized (proliferating and hypertrophic zones), orienting the primary cilia parallel to the longitudinal axis of the bone. The alignment of primary cilia forms one virtual axis that crosses the center of the columns of chondrocytes reflecting the polarity axis of the growth plate. We also show that primary cilia in osteochondromas are found randomly located on the cell surface. Strikingly, the growth plate-like polarity was retained in sub-populations of osteochondroma cells that were organized into small columns. Based on this, we propose the existence of a mixture ('mosaic') of normal lining (EXT(+/-) or EXT(wt/wt)) and EXT(-/-) cells in the cartilaginous cap of osteochondromas.
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http://dx.doi.org/10.1038/labinvest.2010.81DOI Listing
July 2010

Running GAGs: myxoid matrix in tumor pathology revisited: what's in it for the pathologist?

Virchows Arch 2010 Feb 25;456(2):181-92. Epub 2009 Aug 25.

Department of Pathology, Leiden University Medical Center, L1Q, Leiden, The Netherlands.

Ever since Virchow introduced the entity myxoma, abundant myxoid extracellular matrix (ECM) has been recognized in various reactive and neoplastic lesions. Nowadays, the term "myxoid" is commonly used in daily pathological practice. But what do today's pathologists mean by it, and what does the myxoid ECM tell the pathologist? What is known about the exact composition and function of the myxoid ECM 150 years after Virchow? Here, we give an overview of the composition and constituents of the myxoid ECM as known so far and demonstrate the heterogeneity of the myxoid ECM among different tumors. We discuss the possible role of the predominant constituents of the myxoid ECM and attempt to relate them to differences in clinical behavior. Finally, we will speculate on the potential relevance of this knowledge in daily pathological practice.
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http://dx.doi.org/10.1007/s00428-009-0822-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2828560PMC
February 2010

The first crystal structures of a family 19 class IV chitinase: the enzyme from Norway spruce.

Plant Mol Biol 2009 Oct 23;71(3):277-89. Epub 2009 Jul 23.

Department of Molecular Biology, Biomedical Center, Swedish University of Agricultural Sciences, 751 24 Uppsala, Sweden.

Chitinases help plants defend themselves against fungal attack, and play roles in other processes, including development. The catalytic modules of most plant chitinases belong to glycoside hydrolase family 19. We report here x-ray structures of such a module from a Norway spruce enzyme, the first for any family 19 class IV chitinase. The bi-lobed structure has a wide cleft lined by conserved residues; the most interesting for catalysis are Glu113, the proton donor, and Glu122, believed to be a general base that activate a catalytic water molecule. Comparisons to class I and II enzymes show that loop deletions in the class IV proteins make the catalytic cleft shorter and wider; from modeling studies, it is predicted that only three N-acetylglucosamine-binding subsites exist in class IV. Further, the structural comparisons suggest that the family 19 enzymes become more closed on substrate binding. Attempts to solve the structure of the complete protein including the associated chitin-binding module failed, however, modeling studies based on close relatives indicate that the binding module recognizes at most three N-acetylglucosamine units. The combined results suggest that the class IV enzymes are optimized for shorter substrates than the class I and II enzymes, or alternatively, that they are better suited for action on substrates where only small regions of chitin chain are accessible. Intact spruce chitinase is shown to possess antifungal activity, which requires the binding module; removing this module had no effect on measured chitinase activity.
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http://dx.doi.org/10.1007/s11103-009-9523-9DOI Listing
October 2009

Regulation of zebrafish skeletogenesis by ext2/dackel and papst1/pinscher.

PLoS Genet 2008 Jul 25;4(7):e1000136. Epub 2008 Jul 25.

MRC Centre for Developmental and Biomedical Genetics, University of Sheffield, Sheffield, United Kingdom.

Mutations in human Exostosin genes (EXTs) confer a disease called Hereditary Multiple Exostoses (HME) that affects 1 in 50,000 among the general population. Patients with HME have a short stature and develop osteochondromas during childhood. Here we show that two zebrafish mutants, dackel (dak) and pinscher (pic), have cartilage defects that strongly resemble those seen in HME patients. We have previously determined that dak encodes zebrafish Ext2. Positional cloning of pic reveals that it encodes a sulphate transporter required for sulphation of glycans (Papst1). We show that although both dak and pic are required during cartilage morphogenesis, they are dispensable for chondrocyte and perichondral cell differentiation. They are also required for hypertrophic chondrocyte differentiation and osteoblast differentiation. Transplantation analysis indicates that dak(-/-) cells are usually rescued by neighbouring wild-type chondrocytes. In contrast, pic(-/-) chondrocytes always act autonomously and can disrupt the morphology of neighbouring wild-type cells. These findings lead to the development of a new model to explain the aetiology of HME.
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http://dx.doi.org/10.1371/journal.pgen.1000136DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2453328PMC
July 2008

The molecular characterization of PaHB2, a homeobox gene of the HD-GL2 family expressed during embryo development in Norway spruce.

J Exp Bot 2003 May;54(386):1343-50

Swedish University of Agricultural Sciences, Department of Forest Genetics, Box 7027, S-750 07 Uppsala Sweden.

PaHB1 (for Picea abies Homeobox1), an evolutionarily conserved HD-GL2 homeobox gene, specifically expressed in the protoderm during somatic embryogenesis in the gymnosperm Norway spruce has been reported previously. An additional HD-GL2 gene designated PaHB2 is reported here. During somatic embryogenesis, the PaHB2 gene is uniformly ex pressed in proembryogenic masses and in early somatic embryos, but it is not detectably transcribed at the beginning of maturation. In mature embryos, PaHB2 expression was essentially detected in the outermost layer of the cortex and the root cap. A similar PaHB2 expression is detected post-embryonically in both the primary root and the hypocotyl. Phylogenetic reconstructions and intron pattern analyses revealed that the PAHB proteins fall within two distinct subclasses comprising highly similar angiosperm homologues. The PAHB1 subclass consists of protoderm/epiderm-specific members. By contrast, the PAHB2 subclass gathers homologues with a subepidermal and protodermal/epidermal activity. This study suggests that at least two distinct HD-GL2 genes with a layer-specific expression already existed in the last common ancestor of angiosperms and gymnosperms. The conserved protodermal/epidermal and subepidermal expression of HD-GL2 genes could be used to study embryo radial pattern formation across seed plants.
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http://dx.doi.org/10.1093/jxb/erg145DOI Listing
May 2003

Endogenous Nod-factor-like signal molecules promote early somatic embryo development in Norway spruce.

Plant Physiol 2002 Feb;128(2):523-33

Department of Chemistry, Swedish University of Agricultural Sciences, S-750 07 Uppsala, Sweden.

Embryogenic cultures of Norway spruce (Picea abies) are composed of pro-embryogenic masses (PEMs) and somatic embryos of various developmental stages. Auxin is important for PEM formation and proliferation. In this report we show that depletion of auxin blocks PEM development and causes large-scale cell death. Extracts of the media conditioned by embryogenic cultures stimulate development of PEM aggregates in auxin-deficient cultures. Partial characterization of the conditioning factor shows that it is a lipophilic, low-molecular-weight molecule, which is sensitive to chitinase and contains GlcNAc residues. On the basis of this information, we propose that the factor is a lipophilic chitin oligosaccharide (LCO). The amount of LCO correlates to the developmental stages of PEMs and embryos, with the highest level in the media conditioned by developmentally blocked cultures. LCO is not present in nonembryogenic cultures. Cell death, induced by withdrawal of auxin, is suppressed by extra supply of endogenous LCO or Nod factor from Rhizobium sp. NGR234. The effect can be mimicked by a chitotetraose or chitinase from Streptomyces griseus. Taken together, our data suggest that endogenous LCO acts as a signal molecule stimulating PEM and early embryo development in Norway spruce.
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http://dx.doi.org/10.1104/pp.010547DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC148915PMC
February 2002