Name: visualization of the axonal projection pattern of embryonic motor neurons in drosophila
Uploaded: 2017-06-16T14:00:00
Description: Watch this Scientific Journal Video about Visualization of the Axonal Projection Pattern of Embryonic Motor Neurons in Drosophila at JoVE.com

我感谢Alex L. Kolodkin，因为我在他的实验室里学到了这个填写的准备方案。我也非常感谢杨宏宏的技术援助。本研究得到NRF-2013R1A1A4A01011329（SJ）的支持。

准备<ol><li>通过加入0.5g牛血清白蛋白（BSA）和0.5mL叔辛基苯氧基聚乙氧基乙醇（参见材料表），向具有叔辛基苯氧基聚乙氧基乙醇（PBT）溶液的500ml磷酸盐缓冲盐水（PBS）中制备500mL的1X PBS并搅拌至少30分钟。储存于4°C。使用时比较新鲜，并将溶液储存在干净的瓶子里。 </li><li>通过向6.5mL去离子水中加入2.5mL 16％的多聚甲醛溶液和1mL的10x PBS，制成10mL的4％多聚甲醛。储存于4°C并在一周内使用。 <br ...

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运动轴突指导缺陷的细节通过DAB染色的胚胎的切片制备比通过荧光标记的激光扫描共焦显微镜更快，更准确。因此，固定和1D4染色的胚胎的切片制备最适合于指导提示分子的功能表征。指导线索包括网络，狭缝，信号素（Semas）和水母以及他们的同源受体在蠕虫，苍蝇和脊椎动物中具有进化上保守的作用。在作为狭缝受体的迂回（Robo）家族分子中， 果蝇 Robo在CNS 6,21中首先被确定为调解排?...

胚胎发育期间运动轴突和目标肌肉之间的精确和选择性联系对于果蝇幼虫的正常运动是必不可少的 。每个腹部疾病A2-A7中的30个肌纤维的胚胎图案是由阶段16 1建立的 。在腹侧神经线中产生的36只运动神经元将其轴突延伸到外周以支配特定的目标肌肉2 。通过抗体（小鼠单克隆抗体1D4）3,4免疫组织化学可以观察到运动轴索寻路和靶标识别。在野生型胚胎中的运动轴突投影图案的多个图像可在网络5上获得 。 1D4抗体标记胚胎中枢神经系统（CNS）中线每侧的所有运动轴突和三个纵向轴突束</s， 6 （ 图1C和图2A ）。因此，FasII抗体的免疫组化提供了一个强大的工具，用于识别神经肌肉连接所需的基因，以证明运动轴突指导和靶标识别的分子机制。 在每个腹部A2-A7中，运动轴突突出并选择性地成形为两个主要神经分支，节段神经（SN）和节段间神经（ISN） 2,4和一个小神经分支，横向神经（TN ） 7 。 SN选择性地消弧以产生称为SNa和SNc的两个神经分支，而ISN分裂成称为ISN，ISNb和ISNd 2,4的三个神经分支。其中，ISN，ISNb和SNa运动轴突当晚期16期胚胎用FasII抗体染色时，投影图案最精确地可视化，并被切片（ 图1C和图2A ）。 ISN运动神经元延伸其轴突以支配1,2,3,4,9,10,11,18,19和20,2,4（ 图2A ）的背部肌肉。 ISNb运动神经元支配腹侧肌6,7,12,13,14,28和30,24（ 图2A和2B）。 SNa神经分支突出支配5,8,21,22,23和24,2,4（ 图2A ）的侧肌。由两个运动轴突组成的TN沿着节段边界向内侧投射以支配肌肉25，并且在侧向双极树突状神经元（LBD）中突触周边7 （ 图2A ）。这些目标肌肉神经支配不仅需要在特定选择点的运动轴突的选择性阻塞，而且还需要靶向肌肉识别。此外，在ISN和SNa途径中都发现了一些作为中间靶点的推定的中胚层路标细胞，但不是沿着ISNb途径4 。这可能表明与ISN和SNa运动轴突指导相比，ISNb运动轴索寻路可以以明显的方式进行调节，并且还表明外周运动轴突指导提供了一个有吸引力的实验模型来研究单个指导提示的差异或保守作用分子8 。 这项工作提出了一种标准方法，可视化果蝇中胚胎运动神经元的轴突投影模式。所描述的方案包括如何解剖用1D4a染色的固定胚胎在3,3&#39;-二氨基联苯胺（DAB）中加工用于切片制剂。固定胚胎的扁平制剂的一个关键优点是周边轴突突起和肌肉模式的更好的可视化。此外，这项工作还显示了如何使用LacZ染色法将固定的胚胎基因型分类为所需的突变体胚胎。

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			<td height="63" style="height:63px;width:284px;">Anti-FasciculinII Antibody</td>
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			<td height="40" style="height:40px;width:284px;">Slide Glass</td>
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</table>

visualization of the axonal projection pattern of embryonic motor neurons in drosophila

功能性神经肌肉回路的建立依赖于发育中的运动轴突和目标肌肉之间的精确连接。运动神经元通过响应大量来自周围细胞外环境的轴突指导线索，延长生长锥体以沿着特定途径导航。生长锥目标识别也在神经肌肉特异性中起关键作用。这项工作提出了一个标准的免疫组织化学方案，可视化晚期16 号果蝇黑色素瘤胚胎的运动神经元投影。该方案包括几个关键步骤，包括基因分型程序，以排序所需的突变体胚胎;免疫染色程序，用fasciclin II（FasII）抗体标记胚胎;和解剖程序，从固定胚胎产生鱼片制剂。周边的运动轴突突出物和肌肉图案在圆形胚胎的扁平制剂中比在wh中更好地可视化油籽胚胎。因此，用FasII抗体染色的固定胚胎的切片制备为表征运动轴突寻路和靶标识别所需的基因提供了有力的工具，也可以应用于功能丧失功能遗传屏障。

神经发育期间运动轴突和目标肌肉之间的精确连接取决于在特定选择点的选择性轴突 - 轴突排斥和目标识别。在果蝇中 ，运动轴突之间的选择性排斥部分由第1类和第2类信号素（Semas）（包括Sema-1a，Sema-2a和Sema-2b）8,14,15,16,17,18的组合作用。因此， Sema-1a功能的丧失常常导致ISNb轴突在特定选择点处失效，导致其表现出异常浓厚的形态（ 图2C中的<...

Watch this Scientific Journal Video about Visualization of the Axonal Projection Pattern of Embryonic Motor Neurons in Drosophila at JoVE.com

Visualization of the Axonal Projection Pattern of Embryonic Motor Neurons in Drosophila

这项工作详细描述了一个标准的免疫组织化学方法，可视化晚期16 号果蝇黑色素瘤胚胎的运动神经元投影。用FasII抗体染色的固定胚胎的切片制备提供了一个强大的工具，用于表征神经发育期间运动轴索寻路和靶标识别所需的基因。

胚胎运动神经元轴突投影模式的可视化<em&gt;果蝇</em

The establishment of functional neuromuscular circuits relies on precise connections between developing motor axons and target muscles. Motor neurons ...

Research

JoVE Journal

Neuroscience

Visualization of the Axonal Projection Pattern of Embryonic Motor Neurons in Drosophila

Dissection and Culture of Commissural Neurons from Embryonic Spinal Cord

从胚胎脊髓​​连合神经的解剖和文化

Commissural neurons have been widely used to investigate the mechanisms underlying axon guidance during embryonic spinal cord development. The cell bodies ...

科研

神经科学

Visualization of the Embryonic Nervous System in Whole-mount Drosophila Embryos

Visualization of the Embryonic Nervous System in Whole-mount Drosophila Embryos

在全安装的胚胎神经系统的可视化果蝇胚胎

The Drosophila embryo is an attractive model system for investigating the cellular and molecular basis of neuronal development. Here we describe the ...

Mechanical Manipulation of Neurons to Control Axonal Development

机械操纵控制神经元轴突的发展

Cell manipulations and extension of neuronal axons can be accomplished with calibrated glass micro-fibers capable of measuring and applying forces in the ...

DiI-Labeling of DRG Neurons to Study Axonal Branching in a Whole Mount Preparation of Mouse Embryonic Spinal Cord

DII的标签DRG神经元的研究轴突分支在小鼠胚胎脊髓整装准备

Here we  present a technique to label the trajectories of small groups of DRG neurons  into the embryonic spinal cord by diffusive staining using the ...

Visualization of Proprioceptors in Drosophila Larvae and Pupae

Visualization of Proprioceptors in Drosophila Larvae and Pupae

可视化本体感受器<em&gt;果蝇</em&gt;幼虫和蛹

Proprioception is the ability to sense the motion, or position, of body parts by responding to stimuli arising within the body. In fruitflies and other ...

Extracellularly Identifying Motor Neurons for a Muscle Motor Pool in Aplysia californica

Extracellularly Identifying Motor Neurons for a Muscle Motor Pool in Aplysia californica

运动神经元细胞外识别的肌肉运动游泳池<em&gt;海兔夜蛾</em

In animals with large  identified neurons (e.g. mollusks), analysis of motor pools is done using  intracellular techniques1,2,3,4. Recently,  we developed ...

Real-time Imaging of Axonal Transport of Quantum Dot-labeled BDNF in Primary Neurons

初级量子神经元轴突运输的实时成像点标记的BDNF

BDNF plays an important role in several facets of neuronal survival, differentiation, and function. Structural and functional deficits in axons are ...

The Swimmeret System of Crayfish: A Practical Guide for the Dissection of the Nerve Cord and Extracellular Recordings of the Motor Pattern

小龙虾的游泳足系统：实用指南为神经线及电机模式外记录的解剖

Here we demonstrate the dissection of the crayfish abdominal nerve cord. The preparation comprises the last two thoracic ganglia (T4, T5) and the chain of ...

Why Quantification Matters: Characterization of Phenotypes at the Drosophila Larval Neuromuscular Junction

Why Quantification Matters: Characterization of Phenotypes at the Drosophila Larval Neuromuscular Junction

为什么量化事项：表型的表征在<em&gt;果蝇</em&gt;幼虫神经肌肉接头

Most studies on morphogenesis rely on qualitative descriptions of how anatomical traits are affected by the disruption of specific genes and genetic ...

Retrograde Neuroanatomical Tracing of Phrenic Motor Neurons in Mice

小鼠膈运动神经元逆行神经解剖学追踪的研究

Phrenic motor neurons are cervical motor neurons originating from C3 to C6 levels in most mammalian species. Axonal projections converge into phrenic ...