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.
Highlights
Preclinical animal models can be used to elucidate gene and protein function in ways often impossible in humans, by means of genome sequencing, advances in DNA manipulation and high resolution live-imaging [1]
In the last decade the zebrafish has emerged as a unique model to investigate common and rare human skeletal disorders
The versatile and cheap CRISPR/Cas9 system has found a wide use in many laboratories and undergone a series of optimizations allowing an increasingly specific and error-free gene editing
Summary
Preclinical animal models can be used to elucidate gene and protein function in ways often impossible in humans, by means of genome sequencing, advances in DNA manipulation and high resolution live-imaging [1]. Intramembranous ossification starts with mesenchymal cell condensation and differentiation into osteoblasts, without the need of a cartilage template (Figure 2Bi) [45] This type of ossification occurs in the skull, for example in the cranial roof and opercular bones, in the vertebral column, where most of the vertebral body is formed by this type of ossification, in scales and in the fin rays [45]. Endochondral ossification, which is the main type of ossification in mammals, is uncommon in teleosts In this type of ossification, mesenchymal cells condense and differentiate into chondroblasts and chondrocytes, which secrete an extracellular cartilage matrix that functions as a template that is replaced by bone matrix (Figure 2Biii). Tg(osterix:mCherry-NTRo)pd Tg(osx:Kaede)pd Tg(osx:CFP-NTR) Tg(osx:H2A-mCherry)pd310 Tg(osterix:Lifeact-mCherry)◦u2032 Tg(Col10a1BAC:mCitrine)hu7050 Tg(-2.2col10a1a:GFP)ck Tg(Hsa.RUNX2Mmu.Fos:EGFP)zf259 Tg(RUNX2:egfp) Tg(Ola.bglap.1:EGFP)hu4008 TgBAC(entpd5a:YFP)hu5939 TgBAC(entpd5a:Kaede)hu6867 Tg(col1a1:EGFP)zf195 Tg(rankl:HSE:CFP) Tg(Ola.sp7:N1aICD)cy TgBAC(ctsk:Citrine)zf336 Tg(ctsk:YFP) Tg(ctsk:DsRed) Tg(CTSK-DsRed) Tg(Ola.ctsk:EGFP)zf305 Tg(ctsk:mEGFP) Tg(TRAP:GFP) Tg(trap:GFP-CAAX)◦u2031 Tg(Bre:GFP)p77
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