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

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

<i>ext2^−/−</i> mutant displays severe tooth phenotype.

<p>Ventral view of alizarin-red-stained craniofacial skeleton and teeth at 6 dpf (A, B) and dissected and flat mounted 5^th pharyngeal arches with teeth (A′, B′) reveals the presence on each pharyngeal arch of 3 teeth in siblings (A, A′) and only one misshapen tooth in <i>ext2^−/−</i> larvae (B, B′). Note that the rod shaped branchial arch 5 to which the teeth attach is also ossified. Arrows point incomplete ossification of the mutant tooth. Tooth phenotype consisting of one misshapen tooth was observed in all (n>500) analysed <i>ext2^−/−</i> embryos whereas heterozygote fish were indistinguishable from WT. Tooth lengths varies between 3V¹, 4V¹ and 5V¹ in siblings (P<0.003). Each of those teeth was significantly longer then <i>dak-</i>tooth (P<0.0001) (C). Tooth widths of 3V¹ and 5V¹ were similar between siblings, and both were significantly narrower than 4V¹ (D). <i>ext2^−/−</i>-tooth was significantly broader than any of the siblings teeth (3V¹, P<0.0001; 4V¹, P = 0.023 and 5V¹, P = 0.0001) (D). White boxes, siblings; grey boxes, homozygote mutant. Scale bar = 0.1 mm.</p

FGF8 stimulates growth of additional tooth-bud-like structures in <i>ext2^−/−</i> mutant.

<p>Beads were implanted at 36–39 hpf on one side of the body into an area in between the heart, ear and pectoral fin, where the teeth start to form. At 5 dpf, fish were fixed and stained with Alizarin red. Tooth-buds-like structures were formed on the pharyngeal arch neighboured by FGF-coated bead (arrowhead). Opposite arch was not affected. The tooth-bud-like structures were observed on each side of <i>ext2^−/−</i>-tooth. Asterisk indicate position of the bead.</p

Summary of the expression pattern of dental markers during early tooth development.

<p>Results from this study (a), were juxtaposed to information available from: Ablooglu <i>et al</i>, 2007 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029734#pone.0029734-Ablooglu1" target="_blank">[14]</a>, (b); Jackman <i>et al</i>, 2004 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029734#pone.0029734-Jackman1" target="_blank">[15]</a>, (c); and Borday-Birraux <i>et al</i>.,2006 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029734#pone.0029734-BordayBirraux1" target="_blank">[12]</a>, (d). 4V¹ is the first tooth formed. The development of 4V¹ is closely followed by the formation of neighbouring teeth. The development of 3V¹ on the medial side of 4V¹ slightly precedes the formation of 5V¹ on the lateral side of 4V¹. The expression pattern of <i>dlx2a, dlx2b</i>, <i>connexin 43</i> and <i>osterix</i> does not clarify the identity of <i>ext2^−/−</i> tooth. However, comparison of the expression patterns in <i>ext2^−/−</i> and <i>slc35b2^−/−</i> indicate loss of 5V¹ in both mutants. Week expression is indicated by brackets; -, gene expression was not detected; na, not analysed.</p

Summary of the tooth phenotypes found in various developmental mutants.

<p>Mutant were raised till 6 dpf, fixed and stained with alizarin red. Dissected and flat mounted pharyngeal arches were analysed for number of the attached teeth, number of ossified teeth and abnormalities in tooth shape.</p

The attachment of the 1^st tooth occurs on time in <i>ext2^−/−</i> mutant.

<p>The expression of the <i>osterix</i> gene at 96 hpf underlines the pharyngeal arches in sibling (A, A′) and <i>ext2^−/−</i> mutant (B, B′). At this stage, <i>osterix</i> is also expressed in the sibling in the tooth germs of 3V¹ and 5V¹, but lost in the 4V¹. Mineralised dentine outlines the first to develop and attach – the 4V¹ tooth. Note that single tooth that does not express <i>osterix</i> is also attached into <i>osterix</i>-positive pharyngeal arch in the <i>ext2^−/−</i> mutant. A′ and B′ are higher magnification images of A and B. Scale bar = 0.1 mm.</p