Abstract: 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.
Abstract: 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.
Abstract: 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.
Abstract: 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.
Abstract: 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.
Abstract: 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.
Abstract: 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.
Abstract: 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.
Abstract: 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.
Abstract: 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.
Abstract: The developmental pathway of somatic embryogenesis in Norway spruce involves proliferation of proembryogenic masses (PEMs), PEM-to-somatic embryo transition and further development of the somatic embryos. It has previously been shown that extracellular signal molecules, including arabinogalactan proteins, lipo-chitooligosaccharides and chitinases, regulate somatic embryogenesis. The Chia4-Pa1 gene from Norway spruce is described here. The Chia4-Pa1 encodes a typical basic class IV chitinase, although the intron-exon organization of this gymnosperm chitinase is different from that in angiosperm class IV chitinases. The Chia4-Pa1 belongs to a small gene family with highly similar members, and the expression pattern of Chia4-Pa1 cannot be distinguished from that of other Chia4-Pa members. Upon withdrawal of plant growth regulators, i.e. during a treatment that stimulates PEM-to-somatic embryo transition and massive programmed cell death, a significant increase in transcription and translation of Chia4-Pa genes takes place. The expression pattern analysis revealed that Chia4-Pa genes are expressed in a subpopulation of proliferating cells and at the base of the somatic embryo. Furthermore, in seeds, Chia4-Pa genes are expressed in the megagametophyte in the single cell-layered zone surrounding the corrosion cavity. Taken together these results suggest that the Chia4-Pa expressing cells have a megagametophyte signalling function and that CHIA4-Pa stimulates programmed cell death and promotes PEM-to-somatic embryo transition.
Abstract: 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.
