Smith Laura L, Beggs Alan H, Gupta Vandana A
Division of Genetics and Genomics, Manton Center for Orphan Disease Research, Boston Children's Hospital, Harvard Medical School.
J Vis Exp. 2013 Dec 13(82):e50925. doi: 10.3791/50925.
Zebrafish (Danio rerio) have become a particularly effective tool for modeling human diseases affecting skeletal muscle, including muscular dystrophies, congenital myopathies, and disruptions in sarcomeric assembly, due to high genomic and structural conservation with mammals. Muscular disorganization and locomotive impairment can be quickly assessed in the zebrafish over the first few days post-fertilization. Two assays to help characterize skeletal muscle defects in zebrafish are birefringence (structural) and touch-evoked escape response (behavioral). Birefringence is a physical property in which light is rotated as it passes through ordered matter, such as the pseudo-crystalline array of muscle sarcomeres. It is a simple, noninvasive approach to assess muscle integrity in translucent zebrafish larvae early in development. Wild-type zebrafish with highly organized skeletal muscle appear very bright amidst a dark background when visualized between two polarized light filters, whereas muscle mutants have birefringence patterns specific to the primary muscular disorder they model. Zebrafish modeling muscular dystrophies, diseases characterized by myofiber degeneration followed by repeated rounds of regeneration, exhibit degenerative dark patches in skeletal muscle under polarized light. Nondystrophic myopathies are not associated with necrosis or regenerative changes, but result in disorganized myofibers and skeletal muscle weakness. Myopathic zebrafish typically show an overall reduction in birefringence, reflecting the disorganization of sarcomeres. The touch-evoked escape assay involves observing an embryo's swimming behavior in response to tactile stimulation. In comparison to wild-type larvae, mutant larvae frequently display a weak escape contraction, followed by slow swimming or other type of impaired motion that fails to propel the larvae more than a short distance. The advantage of these assays is that disease progression in the same fish type can be monitored in vivo for several days, and that large numbers of fish can be analyzed in a short time relative to higher vertebrates.
由于与哺乳动物在基因组和结构上具有高度保守性，斑马鱼（Danio rerio）已成为一种特别有效的工具，用于模拟影响骨骼肌的人类疾病，包括肌肉萎缩症、先天性肌病以及肌节组装的破坏。在受精后的头几天内，可以快速评估斑马鱼的肌肉紊乱和运动障碍。两种有助于表征斑马鱼骨骼肌缺陷的检测方法是双折射（结构）和触觉诱发逃避反应（行为）。双折射是一种物理特性，当光穿过有序物质（如肌肉肌节的假晶体阵列）时，光会发生旋转。这是一种简单、非侵入性的方法，用于在发育早期评估半透明斑马鱼幼虫的肌肉完整性。当在两个偏振光滤光片之间观察时，具有高度有组织的骨骼肌的野生型斑马鱼在黑暗背景中显得非常明亮，而肌肉突变体具有与其所模拟的原发性肌肉疾病特有的双折射模式。模拟肌肉萎缩症（一种以肌纤维变性随后反复再生为特征的疾病）的斑马鱼，在偏振光下骨骼肌中会出现退行性暗斑。非营养不良性肌病与坏死或再生变化无关，但会导致肌纤维紊乱和骨骼肌无力。患肌病的斑马鱼通常双折射总体降低，反映了肌节的紊乱。触觉诱发逃避试验包括观察胚胎对触觉刺激的游泳行为。与野生型幼虫相比，突变体幼虫经常表现出较弱的逃避收缩，随后是缓慢游泳或其他类型的运动障碍，无法推动幼虫游动超过很短的距离。这些检测方法的优点是，可以在体内对同一鱼型的疾病进展进行数天的监测，并且相对于高等脊椎动物，可以在短时间内分析大量的鱼。
