Chapter 3 50 prevent proteinuria despite reduced glomerular permeability.(25) To test this hypothesis, we performed quantitative analyses using a zebrafish embryo model in which we could account for tubular reabsorption capacity. Materials and methods Animals Zebrafish (Danio rerio H) were maintained as described by Westfield (1995). Embryos were collected from natural crosses and kept at 28.5°C in E3 medium. Wild-type (WT) AB/TL strain zebrafish embryos and dackel mutant (dak/ext2to273b, referred to as “dak/ ext2”) zebrafish embryos were used for this study. Homozygous dak/ext2 mutants contain a biallelic premature stop codon in the ext2 gene. This mutation globally reduces zygotic ext2 expression in homozygous dak/ext2 mutants, resulting in impaired HS-GAG chain polymerization. Thus, HSPGs in homozygous mutants have truncated, functionally impaired HS-GAG side chains that are neither sulfated nor negatively charged.(9) This model has been characterized previously with respect to HSPG distribution.(21, 3032) The total decrease of HS-GAGs in dak/ext2 zebrafish embryos has previously been reported to be over 80%.(21, 30) Homozygous dak/ext2 mutants were sorted at 3 days post-fertilization (dpf) based on cranofacial, ear, and fin phenotypes as described previously.(33, 34) Embryos from separate crosses were used for each experiment. All experiments were performed on embryos prior to the free-feeding stage and therefore did not fall under the animal experimentation law in accordance with to EU Animal Protection Directive 2010/63/EU. Electron microscopy WT and homozygous dak/ext2 embryos at 3 and 5 dpf were anesthetized with 4% tricaine methanesulfonate (4 mg/ml), chemically fixed for one hour in 1.5% glutaraldehyde in 0.1 M sodium cacodylate buffer (pH 7.4), rinsed twice with 0.1 M sodium cacodylate, incubated for 1 hour in 1% osmium tetroxide in 0.1 M sodium cacodylate buffer, dehydrated sequentially in 70%, 80%, 90%, and 100% ethanol, and then immersed in 1:1 propylene oxide:epon LX112 solution for 1 hour. The fish were then washed, infiltrated with pure epon for 2 hours, embedded in epon LX-112, and polymerized at 60°C for 2 days. Ultrathin sections (100 nm) were mounted on copper slot grids (Storck Veco B.V., Eerbeek, The Netherlands), covered with formvar film and carbon layer, and then stained
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