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

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 ext2 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.Animal science

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

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

HSPG-Deficient Zebrafish Uncovers Dental Aspect of Multiple Osteochondromas

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 5th 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

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

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

Signal molecules in embryogenesis of Norway spruce

Signaling molecules regulating embryo development have been described in angiosperms, but very little is known about how embryogenesis is controlled in gymnosperms. In this work we show that lipophilic low molecular weight molecule(s) with GlcNAc residues that are sensitive to chitinase are secreted by embryogenic cultures of Norway spruce. These data indicate that lipo-chitooligosaccharides (LCOs), homologous with rhizobial Nod factors, are present in plants. Interestingly, developmentally blocked lines secrete more LCOs than normally developing lines do. Endogenous LCOs from spruce and rhizobial Nod factors suppress programmed cell death (PCD) and stimulate proliferation of proembryogenic masses and somatic embryo formation but not further embryo development. LCOs are known to be degraded by chitinases. The Chia4-Pa1 gene, encoding for class IV chitinase, was isolated and characterised. The Chia4-Pa1 gene belongs to a small family with highly similar members. The expression of Chia4-Pa genes increases significantly after withdrawal of plant growth regulators, i.e. during a treatment that triggers PCD and stimulates the switch from proliferation of proembryogenic masses to somatic embryo differentiation. Based on the spatial expression pattern of Chia4-Pa, I propose that chitinase-expressing cells have a megagametophyte signaling function. The localisation of the CHIA4-Pa proteins does not correspond to the expression pattern of the encoding genes. I suggest that the CHIA4-Pa proteins are targeted to places where the substrates are localised. Furthermore, chitinases might act on arabinogalactan proteins (AGPs) thereby causing cell wall loosening and cell elongation. In our laboratory, work is being carried out to identify markers specific for different developmental stages of embryo development in Norway spruce. The level of endogenous LCOs secreted to a medium might be used as an indicator of the embrogenic potential of the proliferating cultures, while the increased level of Chia4-Pa transcript coincides with massive PCD and differentiation of somatic embryos. In addition, the PaHB2 gene, a member of the sub-group of the HD-GL2 family with subepiderm- and protoderm/epiderm-specificity, was isolated and characterised

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

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

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

MTG

Dental defects are present in 20% of adult <i>ext2^+/−</i> mutant fish.

<p>Lateral view of two ventral teeth stained with Alizarin red. In most cases, WT-like teeth were present (A, D). However, on few occasions we also observed: enamel malformation (B, E, F) or misshapen crowns (C, C′). Teeth start to calcify from the tip toward the base; hence the lack of staining at the base of 2V is most likely reflects uncompleted ossification of a recent replaced tooth – see black arrowhead (C). Teeth from MO patients (G, H). Note extra buckle in H (arrow head) which resembles split crown observed in <i>ext2^−/−</i> fish. C′ is a higher magnification of C. White arrows indicate lesions. Scale bars correspond to 0.1 mm.</p