M. Van Gils, A. Willaert, P. Coucke, O. M. Vanakker
Center for Medical Genetics Ghent, Ghent University Hospital
Introduction: Pseudoxanthoma elasticum (PXE) is a rare autosomal recessive multisystemic connective tissue disorder, in which mineral depositions of hydroxyapatite disrupt and destroy the elastic fibers. Patients have a very heterogeneous phenotype with skin, ocular and cardiovascular manifestations. To date, no therapeutic options are available, limiting management to controlling the complications of PXE, e.g. preserving visual acuity through regular check ups and anti-VEGF injections. Furthermore, identification of putative therapeutics for PXE is cumbersome. Animal studies have been performed in rodents. However, testing even a single compound in such studies costs considerable resources and time. Therefore, we had previously developed and characterized 3 abcc6a zebrafish models (Sa963, Cmg52 and Morpholino) to expedite the compound screening process. We demonstrated that spinal hypermineralization in these fish is an early and quantifiable phenotype. Here, we present our workflow as well as proof-of-concept data that were obtained for vitamin K1 and sodium thiosulfate (STS) in CRISPR/Cas9-mediated abcc6a knockout zebrafish larvae (Cmg52). Both compounds have been implicated in PXE and/or related mineralization disorders.
Materials & methods: Cmg52 heterozygote incrosses are performed and wild type, heterozygous and mutant embryos are collected. At 2-3 days post-fertilization (dpf) embryos are dechorionated and distributed randomly per 20 in baskets. Starting 3dpf up to 10dpf, baskets are transferred to 6-well plates. Per compound, 60 embryos (or 3 baskets) are incubated with 8ml compound in 1x E3-medium, which is refreshed daily. At 10dpf embryos are euthanized with 25x Tricain and fixated for 1 hour with 4% paraformaldehyde in 0.4M PO4-buffer. They are then bleached for 30 minutes, treated overnight in 0.1% alizarin red S-solution, destained in 30% glycerol and gradually transferred to 100% glycerol. Larvae are then positioned similarly under a fluorescence (or light) microscope and images are taken of every larvae using identical settings. Following imaging, DNA is extracted from the larvae for genotyping. Via a multiple steps process using ImageJ software, photos are analysed to quantify the alizarin red-S signal. Results are clustered based on genotype for statistical analysis.
Results: Our results indicate that 80µM Vitamin K1 can significantly reduce mineralization in Cmg52 abcc6a knockout mutants ((Mean ± SD) controls:19.371 ± 12.794; vitamin K1: 10.322 ± 5.519; P<0.05). Larvae treated with 30µM STS did not have any spinal mineralization or higher mortality compared to untreated embryos. However 33% of STS-treated embryos, regardless of genotype, displayed spotty abdominal mineralization, indicating putative compound toxicity.
Conclusions: This proof-of-concept study confirms that the previously described mineralization phenotype in abcc6a knockout zebrafish is a good read-out for compound screening. Preliminary data suggest a potential therapeutic role for vitamin K1 though repeat experiments on Cmg52 and Sa963 embryos are required. These data also provide additional evidence that Vitamin K plays a role in abcc6-related mineralization. The long term developmental effects of the compound on spinal hypermineralization remain to be evaluated. The STS data underline the importance of proper controls and that results from compound screening studies should be interpreted with caution.