Department of Neurobiology andHoward Hughes Medical Institute, University of Massachusetts Medical School, Worcester, MA 01605; and.
Dr. John T. Macdonald Foundation Department of Human Genetics and John P. Hussman Institute for Human Genomics, University of Miami Miller School of Medicine, Miami, FL 33136.
Proc Natl Acad Sci U S A. 2014 Jul 8;111(27):9965-70. doi: 10.1073/pnas.1406230111. Epub 2014 Jun 23.
Axons damaged by acute injury, toxic insults, or neurodegenerative diseases execute a poorly defined autodestruction signaling pathway leading to widespread fragmentation and functional loss. Here, we describe an approach to study Wallerian degeneration in the Drosophila L1 wing vein that allows for analysis of axon degenerative phenotypes with single-axon resolution in vivo. This method allows for the axotomy of specific subsets of axons followed by examination of progressive axonal degeneration and debris clearance alongside uninjured control axons. We developed new Flippase (FLP) reagents using proneural gene promoters to drive FLP expression very early in neural lineages. These tools allow for the production of mosaic clone populations with high efficiency in sensory neurons in the wing. We describe a collection of lines optimized for forward genetic mosaic screens using MARCM (mosaic analysis with a repressible cell marker; i.e., GFP-labeled, homozygous mutant) on all major autosomal arms (∼95% of the fly genome). Finally, as a proof of principle we screened the X chromosome and identified a collection eight recessive and two dominant alleles of highwire, a ubiquitin E3 ligase required for axon degeneration. Similar unbiased forward genetic screens should help rapidly delineate axon death genes, thereby providing novel potential drug targets for therapeutic intervention to prevent axonal and synaptic loss.
轴突在急性损伤、毒性损伤或神经退行性疾病的影响下，会执行一条定义不明确的自毁信号通路，导致广泛的碎片化和功能丧失。在这里，我们描述了一种在果蝇 L1 翅脉中研究 Wallerian 变性的方法，该方法允许在体内以单轴突分辨率分析轴突退行性表型。该方法允许对特定亚群的轴突进行横断，然后同时检查未受伤的对照轴突的渐进性轴突变性和碎片清除。我们使用神经前体细胞基因启动子开发了新的翻转酶（FLP）试剂，以在神经谱系中非常早期驱动 FLP 表达。这些工具允许在翅膀中的感觉神经元中以高效率产生具有高突变频率的嵌合体克隆群体。我们描述了一系列针对所有主要常染色体臂（约 95%的果蝇基因组）的 MARCM（可抑制细胞标记的马赛克分析；即 GFP 标记的纯合突变体）正向遗传马赛克筛选优化的品系。最后，作为原理验证，我们筛选了 X 染色体，鉴定出了八个 recessive 和两个 dominant 等位基因的 highwire，highwire 是一种泛素 E3 连接酶，对于轴突变性是必需的。类似的无偏正向遗传筛选应该有助于快速描绘出轴突死亡基因，从而为预防轴突和突触丧失的治疗干预提供新的潜在药物靶点。
