The zebrafish as a model for recessive types of Osteogenesis Imperfecta.

Osteogenesis imperfecta (OI) is a hereditary brittle bone disorder that is mostly (>90%) caused by dominant mutations in the COL1A1 or COL1A2 genes, encoding the proα1 and proα2 chain of type I (pro)collagen, respectively. Recently, also genes for autosomal recessive forms of OI have been identified, which are mainly involved in collagen biosynthesis, secretion and processing, osteoblast differentiation and bone mineralization. To study human OI, zebrafish (ZF, Danio rerio) models are increasingly being used as a valuable complementation to the traditional murine models. Their unique attributes, such as the rapid development, large offspring numbers and ease and speed in generating mutant lines, accommodates the parallel analysis of multiple mutant models. Zebrafish bone mutants also tend to survive into adulthood far easier than corresponding mouse models. To investigate both the phenotypic and molecular characteristics of different recessive types of OI in zebrafish (ZF), we obtained zebrafish knock-out models for tmem38b, plod2, fkbp10a/b, tapt1a/b, wnt1 and bmp1 either via the Zebrafish Mutation Project or using CRISPR/Cas9-mediated genome editing. The knock-out effect of the germline mutations was analyzed by qPCR expression analysis in whole larvae or adult fins, which showed significantly decreased levels of mRNA expression of the affected genes. Initial characterization of the skeleton of ZF models was performed by whole mount Alizarin red staining on larvae and adults, revealing abnormalities in the vertebral column such as scoliosis, kyphosis, fusions, compressions and fractures of the ribs. This illustrates the high phenotypic similarity of these zebrafish mutants with human recessive OI. Additionally, high-resolution Micro Computed Tomography (µCT) of adults was performed and data was analyzed using ‘Fishcut’ software, which allows quantitative analysis of hundreds of morphological and densitometric traits in large sets of zebrafish skeletal mutants. This showed, significant differences in Tissue Mineral Density (TMD) and bone volume in different mutants. Interestingly, there is the relatively large phenotypical variability present within each genotype, a finding which is also frequently observed in human OI patients even when carrying the same mutation. We argue that, due to their outbred nature, zebrafish closely reflect the phenotypic variability of the human disease state. In conclusion, we present several zebrafish models for recessive types of OI and show the potential for zebrafish to aid in identifying unknown genetic modifiers and mechanisms underlying the phenotypic variability in OI and related disorders. Preliminary experiments using exome and transcriptome analysis to identify these factors in the described zebrafish models are ongoing