Jeong Sangyun
Department of Molecular Biology, Chonbuk National University;
J Vis Exp. 2017 Jun 16(124):55830. doi: 10.3791/55830.
The establishment of functional neuromuscular circuits relies on precise connections between developing motor axons and target muscles. Motor neurons extend growth cones to navigate along specific pathways by responding to a large number of axon guidance cues that emanate from the surrounding extracellular environment. Growth cone target recognition also plays a critical role in neuromuscular specificity. This work presents a standard immunohistochemistry protocol to visualize motor neuron projections of late stage-16 Drosophila melanogaster embryos. This protocol includes a few key steps, including a genotyping procedure, to sort the desired mutant embryos; an immunostaining procedure, to tag embryos with fasciclin II (FasII) antibody; and a dissection procedure, to generate filleted preparations from fixed embryos. Motor axon projections and muscle patterns in the periphery are much better visualized in flat preparations of filleted embryos than in whole-mount embryos. Therefore, the filleted preparation of fixed embryos stained with FasII antibody provides a powerful tool to characterize the genes required for motor axon pathfinding and target recognition, and it can also be applied to both loss-of-function and gain-of-function genetic screens.
功能性神经肌肉回路的建立依赖于发育中的运动轴突与靶肌肉之间的精确连接。运动神经元通过对周围细胞外环境中发出的大量轴突导向线索做出反应，延伸生长锥沿着特定路径导航。生长锥靶标识别在神经肌肉特异性中也起着关键作用。这项工作展示了一种标准免疫组织化学方法，用于可视化16期晚期黑腹果蝇胚胎的运动神经元投射。该方法包括几个关键步骤，包括基因分型程序，以筛选出所需的突变胚胎；免疫染色程序，用 fasciclin II（FasII）抗体标记胚胎；以及解剖程序，从固定的胚胎中制备去内脏标本。与整装胚胎相比，去内脏胚胎的扁平标本能更好地观察到外周的运动轴突投射和肌肉模式。因此，用FasII抗体染色的固定胚胎的去内脏标本为表征运动轴突寻路和靶标识别所需的基因提供了一个强大的工具，并且它还可应用于功能丧失和功能获得的基因筛选。
