使用亲脂荧光染料的果蝇胚胎运动神经元的逆行追踪
Summary
我们描述了一种使用亲脂荧光染料逆行追踪果蝇胚胎运动神经元的方法。
Abstract
我们描述了一种在果蝇中逆行标记运动神经元的技术。我们使用一种油溶解的亲脂染料，并通过显微注射器将一小滴滴输送到胚胎圆角制剂中。其膜被滴接触的每个运动神经元可以迅速标记。单个运动神经元被连续标记，使精细的结构细节清晰地可视化。鉴于亲脂染料有多种颜色，该技术还提供了一种获取相邻神经元以多色标记的方法。因此，这种追踪技术对于研究果蝇运动神经元系统的神经元形态发生和突触连接非常有用。
Introduction
果蝇的胚胎运动神经元系统提供了一个强大的实验模型来分析中枢神经系统（CNS）1、2、3的发展机制。运动神经元系统适用于生化、遗传、成像和电生理技术。利用这些技术，基因操作和功能分析可以在单运动神经元2，4，5，6的水平进行。
在神经系统的早期发育过程中，神经细胞分裂并产生大量的胶质和神经元。神经细胞的分层和基因表达特征之间的时空关系，以前曾详细研究过7、8、9。在运动神经元系统的情况下，胚胎神经肌肉结（NMJ）的形成已经被广泛研究使用aCC（前角细胞），RP2（生虾2），和RP5运动神经元2，10。例如，当RP5运动神经元形成新生的突触结时，突触前和突触后filopodia被混合11，12，13。这种直接的蜂窝通信对于启动NMJ的形成至关重要。与我们所知道的周围神经分支相反，我们对运动树突如何在CNS内启动突触连接的知识仍然是原始的。
在这份报告中，我们提出了一种技术，允许通过微移液介导的亲脂染料的传递，在胚胎中逆行标记运动神经元。这项技术使我们能够在产卵（AEL）14后15小时，追踪38个运动神经元在15小时内，在30个身体壁肌肉中各进行内蚀。通过利用这一技术，我们小组彻底调查了许多功能增益/功能损失等位基因15，16，17。我们最近解开了驱动运动树突连接启动的分子机制，并证明Dscam1-Dock-Pak相互作用定义了aCC运动神经元17中树突生长的部位。一般来说，这种技术适用于野生类型或突变菌株中任何胚胎运动神经元的型态分析，增强了我们对果蝇神经系统功能设计提供新见解的能力。
Protocol
1. 设备和用品 收集胚胎和培训成人产卵的材料 通过切断 50 mL 管并切开盖上的孔来设置带 100 μm（材料表）的网状过滤器，从而在管和盖之间设置滤芯，从而准备过滤设备。注：或者，具有100μm孔（材料表）的细胞过滤器可用于胚胎收集的过滤步骤。 根据列出的说明，用葡萄琼脂预混料（材料表）制作琼脂板。简单，轻轻搅拌1包粉末…
Representative Results
图3C显示了aCC和RP3运动神经元的代表性图像，以演示在15小时AEL下运动神经元的多色标记。它们的树突形态在胚胎之间基本上都是不变的。使用抗HRP抗体获得的染色模式以灰色显示。一小滴DiO或DiD分别沉积在肌肉1或6/7的NMJ上。图 4演示了定量测量兴趣表型的能力。我们计算了野生类型的树突尖总数，与突变体（例如，dscam1-/-?…
Discussion
与遗传细胞标记技术而言，使用染料标记来研究神经元形态学具有几个优点。染料标记技术可以最大限度地减少标记和成像运动神经元形态所需的时间。染料标记过程相当快，因为它需要不到2小时，使我们能够定义神经元投影的轮廓。作为替代方案，可以通过选择在aCC中表达酵母GAL4转录因子的GAL4线来可视化aCC运动神经元，并穿过由上游活化序列（UAS）21控制的绿色荧光蛋白（GF…
Disclosures
The authors have nothing to disclose.
Acknowledgements
我们感谢金刚山实验室的成员对手稿的评论。这项工作得到了NIH R01 NS107558（到麻省理工学院、K.B.和D.K.）的支持。
Materials
| 10x objective lens | Nikon | Plan | |
| 40x water-immersion lens | Nikon | NIR Apo | |
| Capillary tubing | Frederick Haer&Co | 27-31-1 | |
| Confocal microscope | Andor | N/A | Dragonfly Spinning disk confocal unit |
| Cover glass | Corning | 22×22 mm Square #1 | |
| DiD | ThermoFisher | V22886 | |
| DiI | ThermoFisher | V22888 | |
| DiO | ThermoFisher | V22887 | |
| Dissecting microscope | Nikon | N/A | SMZ-U |
| Double Sided Tape | Scotch | 665 | |
| Dow Corning High-Vacuum Grease | Fisher Sci. | 14-635-5D | |
| Dumont #5 Forceps | Fine Science Tools | 11252-20 | |
| Egg collection cage | FlyStuff | 59-100 | |
| FemtoJet 5247 | Eppendorf | discontinued | FemtoJet 4i (Cat No. 5252000021) |
| ImageJ | NIH | Image processing software | |
| Micromanipulator | Sutter | MP-225 | |
| Micropipette beveler | Sutter | BV-10-B | |
| Needle puller | Narishige | PC-100 | |
| Nutri-Fly Grape Agar Powder Premix Packets | FlyStuff | 47-102 | |
| Nylon Net Filter | Millipore | ||
| Paraformaldehyde 16% Solution, EM grade | Electron Microscopy Sciences | 15710 | Any EM grades |
| PBS | Roche | 11666789001 | Sold on sigmaaldrich, boxed 10x solution |
| Photo-Flo 200 | Kodak | 146 4510 | Wetting agent |
| Upright fluorescence microscope | Nikon | N/A | Eclipse Ci with a LED light source |
| Vinyl Electrical Tape | Scotch | 6143 | |
| VWR Cell Strainers | VWR | 10199-659 | |
| Yeast | FlyStuff | 62-103 | Active dry yeast (RED STAR) |
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Cite This Article
Inal, M. A., Banzai, K., Kamiyama, D. Retrograde Tracing of Drosophila Embryonic Motor Neurons Using Lipophilic Fluorescent Dyes. J. Vis. Exp. (155), e60716, doi:10.3791/60716 (2020).