Zebrafish: A Resourceful Vertebrate Model to Investigate Skeletal Disorders

Abstract

Animal models are essential tools for addressing fundamental scientific questions about skeletal diseases and for the development of new therapeutic approaches. Traditionally, mice have been the most common model organism in biomedical research, but their use is hampered by several limitations including complex generation, demanding investigation of early developmental stages, regulatory restrictions on breeding, and high maintenance cost. The zebrafish has been used as an efficient alternative vertebrate model for the study of human skeletal diseases, thanks to its easy genetic manipulation, high fecundity, external fertilization, transparency of rapidly developing embryos, and low maintenance cost. Furthermore, zebrafish share similar skeletal cells and ossification types with mammals. In the last decades, the use of both forward and new reverse genetics techniques has resulted in the generation of many mutant lines carrying skeletal phenotypes associated with human diseases. In addition, transgenic lines expressing fluorescent proteins under bone cell-or pathway-specific promoters enable in vivo imaging of differentiation and signaling at the cellular level. Despite the small size of the zebrafish, many traditional techniques for skeletal phenotyping, such as x-ray and microCT imaging and histological approaches, can be applied using the appropriate equipment and custom protocols. The ability of adult zebrafish to remodel skeletal tissues can be exploited as a unique tool to investigate bone formation and repair. Finally, the permeability of embryos to chemicals dissolved in water, together with the availability of large numbers of small-sized animals makes zebrafish a perfect model for high-throughput bone anabolic drug screening. This review aims to discuss the techniques that make zebrafish a powerful model to investigate the molecular and physiological basis of skeletal disorders.

Appendage Regeneration in Vertebrates: What Makes This Possible?

“…The recently established CRISPR-Cas9 gene editing additionally increased the value of this model. Zebrafish are also particularly suitable for small-molecule drug application, which, in skeletal field, takes enormous advantage of tail regeneration [ 47 ].…”

“…Zebrafish is not only a fascinating example of vertebrate regeneration, but also represents a well-known vertebrate model for disease and has already contributed to several examples of successful phenotype-based drug discovery, providing a tool with many different applications in biomedical research, especially in the field of bone biology. Indeed, zebrafish skeleton conserves the general basis of development, gene expression and cell types that are found in mammals [ 47 ]. In this scenario, the remarkably fast regeneration of zebrafish caudal fin provides an excellent tool to easy monitor bone formation over time in healthy and pathological conditions [ 47 ].…”

“…Indeed, zebrafish skeleton conserves the general basis of development, gene expression and cell types that are found in mammals [ 47 ]. In this scenario, the remarkably fast regeneration of zebrafish caudal fin provides an excellent tool to easy monitor bone formation over time in healthy and pathological conditions [ 47 ]. The evaluation of bone regeneration rate can be performed in combination with drug administration to discover new molecules promoting regenerative and/or osteogenic activity.…”

Opportunities and Challenges in Functional Genomics Research in Osteoporosis: Report From a Workshop Held by the Causes Working Group of the Osteoporosis and Bone Research Academy of the Royal Osteoporosis Society on October 5th 2020

“…Zebrafish have several advantages over mice, making experiments quicker and less expensive: they are highly fecund, laying up to 300 eggs a week; phenotypes may be evident at the larval stage (skeletal elements develop by four days); embryos develop externally, enabling genetic manipulation at the single cell stage. In addition, larvae are translucent, allowing dynamic visualization of skeletal cell behavior, for which several transgenic reporter lines are available ( 77 , 78 ). Embryonic lethality is rare with fish able to survive despite mutations leading to a complete absence of bone tissue, as they are supported by water as they swim, which limits loading of malformed skeletal elements ( 79 ).…”

Tissue-Nonspecific Alkaline Phosphatase—A Gatekeeper of Physiological Conditions in Health and a Modulator of Biological Environments in Disease

“…Although, endochondral ossification is not as frequent as in mammals and additionally, intramembranous and perichondral ossification is present in zebrafish [ 79 ]. Investigation of TNAP function in zebrafish is still fragmented but may open up the possibility to investigate more basic developmental aspects due to its biological features, like extracorporeal fertilization, optical transparency, and numerous transgenic fluorescent reporter lines [ 77 , 80 ]. Genetic analyses of alpl gene evolution in vertebrates and their expression in zebrafish clarified that non-mammalian species possess a number of additional alpl genes due to genome duplication events [ 8 ].…”