zebrafish in vivo scale regeneration

在体内再生过程中斑马鱼软骨鳞片的收获和可视化

Bone is a hard connective tissue that forms a major part of the skeleton, enabling locomotion and acting as a mineral reserve in the body. In order to maintain healthy bone, an exquisite balance between bone formation and degradation is essential via the coupled activity of osteoblasts (which are anabolic) and osteoclasts (which resorb bone). This balance is disrupted by aging or hormonal imbalance, often leading to bone fragility diseases such as osteoporosis1. Although existing drugs have been approved to target bone fragility diseases, many have side effects; therefore, there is a need for new therapeutics1. Thus, there remains a need for abundant sources of biologically relevant bone tissue that can be used to test potential therapeutic compounds.
Traditionally, rodent models and cell culture systems have been used to study bone biology. However, zebrafish are increasingly becoming another model of choice. While not a mammalian system, zebrafish offer certain advantages for bone research over rodents; these include their fecundity and the translucency of the larvae; even in adulthood, some skeletal tissues, including the scales and the fins, remain translucent, allowing high-resolution in vivo imaging and the increased availability of skeletal mutants2,3. Both the fins and scales of zebrafish are capable of complete regeneration after removal. Skeletal regeneration and injury repair of zebrafish fins have been studied extensively4,5, while zebrafish scales are a newer bone model in the field but offer advantages for ex vivo culture6.
Scales are highly abundant, with at least 300 scales on each fish that serve as a protective covering for the fish. Each scale is a small mineralized plate consisting of bone-forming osteoblasts and bone-resorbing osteoclasts of a collagen-rich skeletal matrix7. The ossification process of both zebrafish scales and human bones requires the differentiation of mesenchymal stem cells into osteoblasts to form the mineralized matrix. Zebrafish scales offer a great advantage for skeletal research with their strong regenerative ability that can be used to study bone regeneration and repair. However, despite the presence of both osteoblasts and osteoclasts, zebrafish scales lack osteocytes that are important for human bone remodeling and mechanosensation; the superficial location of the scales means that they can be easily removed with a pair of forceps. Upon removal of the scale, a cascade of events occurs, and scale regeneration begins8,9. There are various staining and imaging options available to visualize the activity of osteoblasts and osteoclasts and the mineralization of the scales, as shown in Figure 1. Additionally, the availability of many relevant fluorescent&#160;transgenic reporter lines of zebrafish means that one can visualize cell dynamics during regeneration7,10,11. This process allows one to gain more understanding of de novo&#160;bone formation by observing the early patterning of scale regeneration on the flank of the fish to study morphology, cellular activity, and genetic profiles of these regenerated scales. The biology of scale formation and regeneration has been well characterized. Importantly, scales can show a good predictive ability for therapeutically relevant compounds12 and treatment of fish with glucocorticoids leads to a scale that regenerates to show osteoporotic phenotypes13. The transcriptome of the regenerating scales shows that genes activated in scale regeneration are enriched for those linked to human skeletal diseases, further demonstrating their relevance as a model system6,14.
Finally, these scales can be cultured ex vivo for up to 7 days. Compared to cell line cultures that are typically composed of a single cell type, ex vivo scale culture provides in vitro bone study opportunities within its natural environment containing both osteoblasts and osteoclasts with its natural extracellular matrix8,12,15,16.
Scale culture also allows us to perform drug screening for novel osteoanabolic targets. The abundance of scales on the fish means that one can fill at least two plates of the 96-well plate from just a single fish, allowing for compound screening in a multiwell format where every single well contains one scale along with its natural niche of cells. Additionally, since the scales are thin, drug absorption is predictable12. In summary, the elasmoid scales of zebrafish have great potential in skeletal research and can help us gain more insight into the cellular events during bone formation and repair. Here, we describe protocols for harvesting scales to follow regeneration in vivo and culture the scales ex vivo.

作者聚焦：使用鱼鳞片进行骨重塑研究 – 离体成像和解剖细胞相互作用的进展

斑马鱼鳞片再生 在多多 和 离体 鳞片培养中

One of the advantages of using fish scales for research is that they contain both osteoblast and osteoclast, which are essential for bone remodeling. When the scales are cultured ex vivo, we can study the interactions of these two cell populations along with the skeletal matrix, unlike traditional cell culture where there's typically only one type of cell. An advantage of using fish scales for research is that they are abundant on the fish, readily harvested, and they are translucent, allowing better ex vivo imaging.As the scales can completely regenerate, they allow us to study bone regeneration which rarely occurs in mammalian models. The protocols we present here allow us to dissect cell interactions better. This will allow us to test how the different cell types respond to aging and to changes in circadian rhythm.

Watch this Scientific Journal Video about Zebrafish In Vivo Scale Regeneration at JoVE.com

斑马鱼体内鳞片再生

首先将斑马鱼从主水箱转移到各个水箱中，成像后将它们放回水箱中。将斑马鱼浸入0.05%三卡因甲烷磺酸盐中，使鱼麻醉。使用相机，在尺子旁边拍摄每条鱼的图像，以记录大小、长度和健康状况。然后，将麻醉的鱼放在带有湿组织的培养皿上。将鱼侧放，并使用体视显微镜以适当的放大倍率进行侧翼成像。在体视显微镜下使用细镊子和镊子收获鳞片，并将它们转移到1.5或2毫升收集管中，以便以后染色。要进行碱性磷酸酶和 von Kossa 染色，请将鳞片转移到装有去离子 Milli-Q 水的试管中。在将单个收获的鳞片转移到试管中之前捕获它们的图像。在再生的第 2 天可以看到新形成的鳞片的早期图案。与遗传鳞片上的第 0 天相比，再生鳞片显示出更高的 OSX 表达。

Research

JoVE Journal

Biology

Zebrafish In Vivo Scale Regeneration

463 次观看.  University of Bristol. 首先将斑马鱼从主水箱转移到各个水箱中，成像后将它们放回水箱中。将斑马鱼浸入0.05%三卡因甲烷磺酸盐中，使鱼麻醉。使用相机，在尺子旁边拍摄每条鱼的图像，以记录大小、长度和健康状况。然后，将麻醉的鱼放在带有湿组织的培养皿上。将鱼侧放，并使用体视显微镜以适当的放大倍率进行侧翼成像。在体视显微镜下使用细镊子和镊子收获鳞片，并将它们转移到1....

视频: 斑马鱼体内鳞片再生

Zebrafish Scale Regeneration In Toto and Ex Vivo Culture of Scales

Skeletal diseases are often complex in their etiology and affect millions of people worldwide. Due to the aging population, there is a need for new therapeutics that could ease the burden on healthcare systems. As these diseases are complex, it is difficult and expensive to accurately model bone pathophysiology in a lab setting. The challenge for the field is to establish a cost-effective, biologically relevant platform for modeling bone disease that can be used to test potential therapeutic compounds. Such a platform should ideally allow dynamic visualization of cell behaviors of bone-building osteoblasts and bone-degrading osteoclasts acting in their mineralized matrix environment. Zebrafish are increasingly used as models due to the availability of genetic tools, including transgenic reporter lines, and the fact that some skeletal tissues (including the scales) remain translucent to adulthood, allowing dynamic imaging options. Since zebrafish scales have both osteoblasts and osteoclasts and are highly abundant, they provide an easily accessible and abundantly available resource of independent bone units. Moreover, once removed, adult zebrafish scales fully regenerate, therefore offering a way to study the spatiotemporal growth of mineralized tissue in vivo. Here, we detail protocols for harvesting and tracking the regeneration of the scales. Lastly, a protocol for stable culture of scales ex vivo for a week and following the healing response after controlled damage to the mineralized matrix of the scale over time is also presented.

This protocol describes the harvesting and visualization of elasmoid scales of zebrafish during in vivo regeneration. In addition, the ex vivo culture of these scales for up to 7 days after harvest is presented.

Skeletal diseases are often complex in their etiology and affect millions of people worldwide. Due to the aging population, there is a need for new ...