Zebrafish models for ectopic mineralization disorders: practical issues from morpholino design to post-injection observations

Abstract

Zebrafish (ZF, Danio rerio) has emerged as an important and popular model species to study different human diseases. Key regulators of skeletal development and calcium metabolism are highly conserved between mammals and ZF. The corresponding orthologs share significant sequence similarities and an overlap in expression patterns when compared to mammals, making ZF a potential model for the study of mineralization-related disorders and soft tissue mineralization. To characterize the function of early mineralization-related genes in ZF, these genes can be knocked down by injecting morpholinos into early stage embryos. Validation of the morpholino needs to be performed and the concern of aspecific effects can be addressed by applying one or more independent techniques to knock down the gene of interest. Post-injection assessment of early mineralization defects can be done using general light microscopy, calcein staining, Alizarin red staining, Alizarin red-Alcian blue double staining, and by the use of transgenic lines. Examination of general molecular defects can be done by performing protein and gene expression analysis, and more specific processes can be explored by investigating ectopic mineralization-related mechanisms such as apoptosis and mitochondrial dysfunction. In this paper, we will discuss all details about the aforementioned techniques; shared knowledge will be very useful for the future investigation of ZF models for ectopic mineralization disorders and to understand the underlying pathways involved in soft tissue calcification.

The zebrafish operculum: A powerful system to assess osteogenic bioactivities of molecules with pharmacological and toxicological relevance

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Revisiting in vivo staining with alizarin red S - a valuable approach to analyse zebrafish skeletal mineralization during development and regeneration

“…For live imaging, bone development can be tracked with radiographs in large specimens [20], but for small sized species, such as zebrafish, the use of radiographic and μCT approaches to visualize the skeleton is restricted due to resolution constraints [1, 21, 22]. Thus, there is a need for reliable and non-toxic in vivo imaging techniques to allow continuous monitoring of skeletal development in living individual zebrafish.…”

Persistence of the ABCC6 genes and the emergence of the bony skeleton in vertebrates

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