Name: retrograde tracing of drosophila embryonic motor neurons using lipophilic fluorescent dyes
Uploaded: 2020-01-12T16:00:00
Description: Watch this Scientific Journal Video about Retrograde Tracing of Drosophila Embryonic Motor Neurons Using Lipophilic Fluorescent Dyes at JoVE.com

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。

1. 设备和用品
<ol><li>收集胚胎和培训成人产卵的材料<ol>
<li>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。</li>
		<li>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...

<ol>
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P., Vasenkova, I., Kamiyama, D., Rosado, Y., Chiba, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Slit+and+Robo+control+the+development+of+dendrites+in+Drosophila+CNS.">Slit and Robo control the development of dendrites in Drosophila CNS.</a> Development. 134 (21), 3795-3804 (2007).</li><li>Kamiyama, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Specification+of+Dendritogenesis+Site+in+Drosophila+aCC+Motoneuron+by+Membrane+Enrichment+of+Pak1+through+Dscam1.">Specification of Dendritogenesis Site in Drosophila aCC Motoneuron by Membrane Enrichment of Pak1 through Dscam1.</a> Developmental Cell. 35 (1), 93-106 (2015).</li><li>Campos-Ortega, J. A., Hartenstein, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The embryonic development of Drosophila melanogaster. , (1985).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drosophila+Ringer's+solution.">Drosophila Ringer's solution.</a> Cold Spring Harbor Protocols. 2007 (4), (2007).</li><li>Rickert, C., Kunz, T., Harris, K. -. L., Whitington, P., Technau, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Labeling+of+single+cells+in+the+central+nervous+system+of+Drosophila+melanogaster.">Labeling of single cells in the central nervous system of Drosophila melanogaster.</a> Journal of Visualized Experiments. (73), e50150 (2013).</li><li>Fujioka, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Even-skipped,+acting+as+a+repressor,+regulates+axonal+projections+in+Drosophila.">Even-skipped, acting as a repressor, regulates axonal projections in Drosophila.</a> Development. 130 (22), 5385-5400 (2003).</li><li>Sink, H., Rehm, E. J., Richstone, L., Bulls, Y. M., Goodman, C. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=sidestep+encodes+a+target-derived+attractant+essential+for+motor+axon+guidance+in+Drosophila.">sidestep encodes a target-derived attractant essential for motor axon guidance in Drosophila.</a> Cell. 105 (1), 57-67 (2001).</li><li>Furrer, M. 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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...

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。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>10x objective lens</td>
			<td>Nikon</td>
			<td></td>
			<td>Plan</td>
		</tr>
		<tr>
			<td>40x water-immersion lens</td>
			<td>Nikon</td>
			<td></td>
			<td>NIR Apo</td>
		</tr>
		<tr>
			<td>Capillary tubing</td>
			<td>Frederick Haer&#38;Co</td>
			<td>27-31-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Confocal microscope</td>
			<td>Andor</td>
			<td>N/A</td>
			<td>Dragonfly Spinning disk confocal unit</td>
		</tr>
		<tr>
			<td>Cover glass</td>
			<td>Corning</td>
			<td></td>
			<td>22x22 mm Square #1</td>
		</tr>
		<tr>
			<td>DiD</td>
			<td>ThermoFisher</td>
			<td>V22886</td>
			<td></td>
		</tr>
		<tr>
			<td>DiI</td>
			<td>ThermoFisher</td>
			<td>V22888</td>
			<td></td>
		</tr>
		<tr>
			<td>DiO</td>
			<td>ThermoFisher</td>
			<td>V22887</td>
			<td></td>
		</tr>
		<tr>
			<td>Dissecting microscope</td>
			<td>Nikon</td>
			<td>N/A</td>
			<td>SMZ-U</td>
		</tr>
		<tr>
			<td>Double Sided Tape</td>
			<td>Scotch</td>
			<td>665</td>
			<td></td>
		</tr>
		<tr>
			<td>Dow Corning High-Vacuum Grease</td>
			<td>Fisher Sci.</td>
			<td>14-635-5D</td>
			<td></td>
		</tr>
		<tr>
			<td>Dumont #5 Forceps</td>
			<td>Fine Science Tools</td>
			<td>11252-20</td>
			<td></td>
		</tr>
		<tr>
			<td>Egg collection cage</td>
			<td>FlyStuff</td>
			<td>59-100</td>
			<td></td>
		</tr>
		<tr>
			<td>FemtoJet 5247</td>
			<td>Eppendorf</td>
			<td>discontinued</td>
			<td>FemtoJet 4i (Cat No. 5252000021)</td>
		</tr>
		<tr>
			<td>ImageJ</td>
			<td>NIH</td>
			<td></td>
			<td>Image processing software</td>
		</tr>
		<tr>
			<td>Micromanipulator</td>
			<td>Sutter</td>
			<td>MP-225</td>
			<td></td>
		</tr>
		<tr>
			<td>Micropipette beveler</td>
			<td>Sutter</td>
			<td>BV-10-B</td>
			<td></td>
		</tr>
		<tr>
			<td>Needle puller</td>
			<td>Narishige</td>
			<td>PC-100</td>
			<td></td>
		</tr>
		<tr>
			<td>Nutri-Fly Grape Agar Powder Premix Packets</td>
			<td>FlyStuff</td>
			<td>47-102</td>
			<td></td>
		</tr>
		<tr>
			<td>Nylon Net Filter</td>
			<td>Millipore</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde 16% Solution, EM grade</td>
			<td>Electron Microscopy Sciences</td>
			<td>15710</td>
			<td>Any EM grades</td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Roche</td>
			<td>11666789001</td>
			<td>Sold on sigmaaldrich, boxed 10x solution</td>
		</tr>
		<tr>
			<td>Photo-Flo 200</td>
			<td>Kodak</td>
			<td>146 4510</td>
			<td>Wetting agent</td>
		</tr>
		<tr>
			<td>Upright fluorescence microscope</td>
			<td>Nikon</td>
			<td>N/A</td>
			<td>Eclipse Ci with a LED light source</td>
		</tr>
		<tr>
			<td>Vinyl Electrical Tape</td>
			<td>Scotch</td>
			<td>6143</td>
			<td></td>
		</tr>
		<tr>
			<td>VWR Cell Strainers</td>
			<td>VWR</td>
			<td>10199-659</td>
			<td></td>
		</tr>
		<tr>
			<td>Yeast</td>
			<td>FlyStuff</td>
			<td>62-103</td>
			<td>Active dry yeast (RED STAR)</td>
		</tr>
	</tbody>
</table>

retrograde tracing of drosophila embryonic motor neurons using lipophilic fluorescent dyes

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。

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-/-?...

Watch this Scientific Journal Video about Retrograde Tracing of Drosophila Embryonic Motor Neurons Using Lipophilic Fluorescent Dyes at JoVE.com

Retrograde Tracing of Drosophila Embryonic Motor Neurons Using Lipophilic Fluorescent Dyes

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。

使用亲脂荧光染料的果蝇胚胎运动神经元的逆行追踪

We describe a technique for retrograde labeling of motor neurons in Drosophila. We use an oil-dissolved lipophilic dye and deliver a small droplet to an ...

本协议中描述的追踪技术有助于我们研究神经元形态发生，并捕捉果蝇运动神经元系统中的连接性。亲脂染料可以迅速扩散到细胞膜的每一部分，密度极高。这就是为什么我们的协议有效地解析，以找到一个标记神经元的结构。如果使用注射微移子可以进入轴孔终端，则该技术可应用于苍蝇幼虫和成年阶段的神经元或其他生物体中的任何神经元。如果您没有解剖或使用微显微器对试样进行精确操作的经验，可能会具有挑战性。了解胚胎的解剖结构，将有助于指导正确部分工具进行解剖或结节。首先，准备和布局所有胚胎收集材料。通过切断50毫升的管子，并在盖子上切开一个孔来准备过滤装置。然后设置一个网状过滤器，在管和盖之间有 100 微米的孔隙。从同一毛细管中准备染料注射微管和解剖针，内径为 1.2 毫米。用微管拉管，从 170 伏最大输出 7%拉拔器，形成长约 0.4 厘米的尖针。要准备染料喷射微管，请用润湿剂浸泡研磨机，然后将针头放在 25 至 30 度的微移子夹上。然后将尖端从斜面中心降到半径的三分之二。研磨针头，而注射器与管道推动空气进入它。用细尖永久标记标记微管，以指示斜角后开口的位置，因为定位以一定角度形成的狭窄开口可能具有挑战性。允许苍蝇在室温下过夜产卵，早上用鸡蛋收集管子。要收集胚胎，用50%漂白剂将盘子上的卵子去氯5分钟。一旦氯离子清除，通过过滤装置或细胞过滤器倒入板内的东西，以分离胚胎。使用挤压瓶水稀释盘子上留下的漂白剂，通过将混合物倒入过滤器中来收集尽可能多的胚胎。用水清洗胚胎三到四次，直到漂白气味消散。从设备中取下过滤器，然后用水将其洗到另一个干净的盘子上。然后从胚胎上的新盘子中抽取水。使用精细钳子在产卵后 15 小时选择 5 到 10 个胚胎，并将它们放在双面胶带上，侧朝上。将昆虫环环盐水添加到解剖池中，以保护胚胎免受干燥。在解剖显微镜下使用玻璃针将胚胎从胶带中膜中拖出玻璃，注意不要损坏胚胎的内部组织。然后切开单个胚胎的中线，从它的后端到前端。从中心翻转上皮组织，将表皮边缘连接到玻璃滑梯表面。使用管连接针，尖端开口约 300 微米，以吸气或吹气，以去除后置纵向气管树干以及任何剩余的肠道。使用4%PFA和PBS在轨道摇床的室温下固定胚胎5分钟。然后用 PBS 洗三次。用一微升的抗马萝卜过氧化物酶抗体与氰氨酸3染料和200微升PBS在轨道摇床上染色一小时。并在 PBS 中重复洗涤。要填充注射微管，请将注射微管放入毛细管支架中。接下来，将染料幻灯片放在舞台上，并使用微操纵器将其定位在幻灯片上。然后调整舞台，将微管放在染料上。通过将喷射压力设置为 200 至 500 次，将喷射时间设置为 0.1 至 0.5 秒，将补偿压力设置为 5 分钟，收集微管中的染料。收集染料后，取出染料幻灯片，将样品放在显微镜阶段。然后将补偿压力增加至 30 至 60 hectopascal 的范围，并降低微管进入样品。使用10倍的客观透镜定位胚胎，使微管与胚胎对齐。将客观透镜更改为水浸镜头 40 倍。将镜头淹没到 PBS 中。使用荧光显微镜检查以抗HRP Cy3标记的神经元形态并确定注射部位。当胚胎处于焦点时，改变微管的位置，与感兴趣的轴翁尖端进行温和接触。然后在注射过程中使用亮场显微镜查看染料液滴。将染料在 aCC 或 RP3 的神经肌肉交界处与 DID 或 DIO 一起将染料滴在右腹部半段。使用手动控制释放染料并拆下微管。然后转到下一个注射部位。在成像前在轨道摇床上将样品在室温下孵育一小时。使用精细钳子，从玻璃幻灯片上取下胶带。要安装样品，请准备在四个角落用少量真空润滑脂进行盖滑。小心地放在样品上，确保避免气泡。向下推盖滑，以调整样品中的空间，以便允许客观透镜和幻灯片之间的工作距离。使用任务擦除去除任何多余的 PBS。用指甲油完全密封盖滑的边缘。使用共体显微镜以 10 倍和 100 倍的放大倍率拍摄图像，并使用 imageJ 软件处理图像。该协议用于逆行标签的AC运动神经元内维肌肉1和RP3运动神经元内维肌肉6和7。来自 ACC 和 RP3 运动神经元的树突分支显示广泛的重叠。两个神经元是双极的，建立两个不同的树突种群。为了演示这种方法的定量能力，在野生类型和Dscam1突变体中计算了树突尖的总数。aCC 的异构树突显示为野生类型，但很少观察到 Dscam1 突变体的异构树突。尝试此过程时，重要的是要记住染料液滴的大小至关重要，约为 10 到 20 微米。测试双面胶带上的大小，然后将其应用于注射部位。利用这个程序，我们可以利用DID探头的光可切换性能获得等离子膜的超分辨率图像。这样，我们可以研究神经肌肉结的突触膜的超结构表征。我们点击了。我们对突变菌株中的 aCC 树突进行了表型分析，发现 Dscam1-Dock-Pak 注射定义了 aCC 中树突的位点。

Research

JoVE Journal

Developmental Biology

Retrograde Tracing of Drosophila Embryonic Motor Neurons Using Lipophilic Fluorescent Dyes

Analysis of Cardiomyocyte Development using Immunofluorescence in Embryonic Mouse Heart

在胚胎小鼠心脏采用免疫荧光心肌发展分析

During heart development, the generation of myocardial-specific structural and functional units including sarcomeres, contractile myofibrils, intercalated ...

科研

发育生物学

A Method for Lineage Tracing of Corneal Cells Using Multi-color Fluorescent Reporter Mice

方法对角膜细胞的谱系追踪使用多色荧光记者小鼠

Lineage tracing experiments define the origin, fate and behavior of cells in a specific tissue or organism. This technique has been successfully applied ...

Transcriptome Profiling of In-Vivo Produced Bovine Pre-implantation Embryos Using Two-color Microarray Platform

Transcriptome Profiling of In-Vivo Produced Bovine Pre-implantation Embryos Using Two-color Microarray Platform

转录组分析]<em&gt;活体</em&gt;生产牛胚胎植入前胚胎采用双色微阵列平台

Early embryonic loss is a large contributor to infertility in cattle. Moreover, bovine becomes an interesting model to study human preimplantation embryo ...

Dissection and Immunofluorescent Staining of Mushroom Body and Photoreceptor Neurons in Adult Drosophila melanogaster Brains

Dissection and Immunofluorescent Staining of Mushroom Body and Photoreceptor Neurons in Adult Drosophila melanogaster Brains

成人果蝇脑内蘑菇体和感光神经元的解剖和荧光染色

Nervous system development involves a sequential series of events that are coordinated by several signaling pathways and regulatory networks. Many of the ...

In Vivo Tracking of Human Adipose-derived Mesenchymal Stem Cells in a Rat Knee Osteoarthritis Model with Fluorescent Lipophilic Membrane Dye

In Vivo Tracking of Human Adipose-derived Mesenchymal Stem Cells in a Rat Knee Osteoarthritis Model with Fluorescent Lipophilic Membrane Dye

在体内荧光亲脂膜染料对大鼠膝骨关节炎模型中人脂肪间充质干细胞的追踪

In order to support the clinical application of human adipose-derived mesenchymal stem cell (haMSC) therapy for knee osteoarthritis (KOA), we examined the ...

Tracing Gene Expression Through Detection of &#946;-galactosidase Activity in Whole Mouse Embryos

Tracing Gene Expression Through Detection of ÃƒÆ’Ã…Â½Ãƒâ€šÃ‚Â²-galactosidase Activity in Whole Mouse Embryos

通过检测全小鼠胚胎β乳糖酶活性追踪基因表达

The Escherichia coli LacZ gene, encoding &#946;-galactosidase, is largely used as a reporter for gene expression and as a tracer in cell lineage studies. ...

Visualize Drosophila Leg Motor Neuron Axons Through the Adult Cuticle

Visualize Drosophila Leg Motor Neuron Axons Through the Adult Cuticle

通过成人角质层可视化果蝇腿运动神经元轴突

The majority of work on the neuronal specification has been carried out in genetically and physiologically tractable models such as C. elegans, Drosophila ...

Thawing, Culturing, and Cryopreserving Drosophila Cell Lines

Thawing, Culturing, and Cryopreserving Drosophila Cell Lines

解冻、培养和冷冻保存的嗜酸性粒细胞系

There are currently over 160 distinct Drosophila cell lines distributed by the Drosophila Genomics Resource Center (DGRC). With genome engineering, the ...

Clonal Genetic Tracing using the Confetti Mouse to Study Mineralized Tissues

使用纸屑小鼠研究矿化组织的克隆遗传追踪

Labeling an individual cell in the body to monitor which cell types it can give rise to and track its migration through the organism or determine its ...

Efficient Neural Differentiation using Single-Cell Culture of Human Embryonic Stem Cells

利用人类胚胎干细胞的单细胞培养进行高效神经分化

In vitro differentiation of human embryonic stem cells (hESCs) has transformed the ability to study human development on both biological and molecular ...