Name: visualization of motor axon navigation and quantification of axon arborization in mouse embryos using light sheet fluorescence microscopy
Uploaded: 2018-05-11T15:00:00
Description: Watch this Scientific Journal Video about Visualization of Motor Axon Navigation and Quantification of Axon Arborization In Mouse Embryos Using Light Sheet Fluorescence Microscopy at JoVE.com

LSFM 实验和数据分析部分采用先进的光学显微镜, 在中央研究院仪器服务部门, 并在舒沈女士的协助下进行。我们感谢 IMB 的成像核心设施, Imaris 图像分析的大量技术援助苏女士。IMB 的科学英语编辑核心审查了手稿。这项工作由中央研究院职业发展奖 (CDA-107-L05)、多数 (104-2311-001-030-MY3) 和人权机构 (NHRI-EX106-10315NC) 资助。

所有活的动物都保存在一个特定的病原体免费 (SPF) 动物设施, 批准和监督的 IACUC 研究院。
1. 固定
<ol><li>收集胚胎12.5 天 (E12.5) 然后斩首和阉割他们。</li>
	<li>在1x 磷酸缓冲盐水 (PBS) 一夜之间, 将胚胎单独固定在24井板上, 1 毫升/井的新鲜制备的4% 多聚甲醛。在一个振动筛上孵化4摄氏度。</li>
	<li>将固定的胚胎至少洗 3x, 每5-10 分钟, 用1毫升 1x PBS, 并在4摄氏度在一夜床上孵化, ?...

<ol>
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M., Lee, D. H., Liu, J. P., Dasen, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hox+Proteins+Coordinate+Motor+Neuron+Differentiation+and+Connectivity+Programs+through+Ret/Gfralpha+Genes.">Hox Proteins Coordinate Motor Neuron Differentiation and Connectivity Programs through Ret/Gfralpha Genes.</a> Cell Rep. 14, 1901-1915 (2016).</li><li>Bonanomi, 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=Ret+is+a+multifunctional+coreceptor+that+integrates+diffusible-+and+contact-axon+guidance+signals.">Ret is a multifunctional coreceptor that integrates diffusible- and contact-axon guidance signals.</a> Cell. 148, 568-582 (2012).</li><li>Tung, Y. T., 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=Mir-17+approximately+92+Governs+Motor+Neuron+Subtype+Survival+by+Mediating+Nuclear+PTEN.">Mir-17 approximately 92 Governs Motor Neuron Subtype Survival by Mediating Nuclear PTEN.</a> Cell Rep. 11, 1305-1318 (2015).</li><li>Svoboda, K., Yasuda, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Principles+of+two-photon+excitation+microscopy+and+its+applications+to+neuroscience.">Principles of two-photon excitation microscopy and its applications to neuroscience.</a> Neuron. 50, 823-839 (2006).</li><li>Jonkman, J., Brown, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Any+Way+You+Slice+It-A+Comparison+of+Confocal+Microscopy+Techniques.">Any Way You Slice It-A Comparison of Confocal Microscopy Techniques.</a> J Biomol Tech. 26, 54-65 (2015).</li><li>Reynaud, E. G., Peychl, J., Huisken, J., Tomancak, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Guide+to+light-sheet+microscopy+for+adventurous+biologists.">Guide to light-sheet microscopy for adventurous biologists.</a> Nat Methods. 12, 30-34 (2015).</li><li>Santi, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Light+sheet+fluorescence+microscopy:+a+review.">Light sheet fluorescence microscopy: a review.</a> J Histochem Cytochem. 59, 129-138 (2011).</li><li>Hanley, O., 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=Parallel+Pbx-Dependent+Pathways+Govern+the+Coalescence+and+Fate+of+Motor+Columns.">Parallel Pbx-Dependent Pathways Govern the Coalescence and Fate of Motor Columns.</a> Neuron. 91, 1005-1020 (2016).</li><li>De Marco Garcia, N. V., Jessell, T. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Early+motor+neuron+pool+identity+and+muscle+nerve+trajectory+defined+by+postmitotic+restrictions+in+Nkx6.1+activity.">Early motor neuron pool identity and muscle nerve trajectory defined by postmitotic restrictions in Nkx6.1 activity.</a> Neuron. 57, 217-231 (2008).</li><li>Li, C. J., 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=MicroRNA+filters+Hox+temporal+transcription+noise+to+confer+boundary+formation+in+the+spinal+cord.">MicroRNA filters Hox temporal transcription noise to confer boundary formation in the spinal cord.</a> Nat Commun. 8, 14685 (2017).</li><li>Swoger, J., Pampaloni, F., Stelzer, E. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Light-sheet-based+fluorescence+microscopy+for+three-dimensional+imaging+of+biological+samples.">Light-sheet-based fluorescence microscopy for three-dimensional imaging of biological samples.</a> Cold Spring Harb Protoc. 2014, 1-8 (2014).</li><li>Pantazis, P., Supatto, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advances+in+whole-embryo+imaging:+a+quantitative+transition+is+underway.">Advances in whole-embryo imaging: a quantitative transition is underway.</a> Nat Rev Mol Cell Biol. 15, 327-339 (2014).</li><li>Reynaud, E. G., Tomancak, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Meeting+report:+first+light+sheet+based+fluorescence+microscopy+workshop.">Meeting report: first light sheet based fluorescence microscopy workshop.</a> Biotechnol J. 5, 798-804 (2010).</li></ol>

在某些情况下, 议定书中的若干步骤可能会受到变化。例如, 固定时间取决于胚胎的年龄, 从2小时到1天或更多的时候使用新鲜的多聚甲醛。由于固定是在整个染色之前进行的, 对于蛋白质交联敏感的抗体, 甲醇可以用作替代的固液剂。对于染色时的高信噪比, 必须优化洗涤剂浓度 (0.1-0.5%)。抗体孵化前后的额外洗涤步骤也可以增加信噪比。此外, 组织清除步骤对于确保在整个深轴突投影中的光路畅通?...

脊髓的 MNs 是中枢神经系统的一部分, 但支配周围的肌肉来控制运动。在发育中的脊髓中, 根据脊索和相邻胚胎背部发出的信号建立锰祖细胞 (pMNs)。所有分化后有丝分裂后的 MNs, 然后产生从 pMNs, 最终产生一系列锰亚型沿 rostrocaudal 轴的脊髓1,2。脊髓的地形和解剖良好组织。它们的形态学排列与各自目标在外围3中的位置相关。在达到他们的肌肉目标, 轴突接收细胞和外源性神经营养因子, 促使他们扩大和分支进一步进入肌肉。神经支配和分支缺陷可能导致无法形成肌电结 (NMJ)。例如, 神经胶质源性神经营养因子 (GDNF) 诱发的 Pea3 是必不可少的轴突树枝状到皮肤阔肌 (CM) 和后背筋 (LD) 肌肉 4, 5.此外,进餐挖空小鼠胚胎显示膈神经有缺陷的树枝状在隔膜, 导致呼吸衰竭和死亡后立即出生 6, 7.因此, 这最后一步的锰成熟 (即, 轴突投射和分支) 是至关重要的, 以确保神经元之间的沟通和目标细胞。
为了查看树枝状模式, 研究人员通常进行共焦成像或双光子荧光显微镜的切片或全挂载样本 8, 9, 10.这两种显微技术都能产生可接受的分辨率和深度穿透力。双光子荧光光镜涉及显影的激发, 同时吸收两个低能光子11。由于双光子激励使用近红外辐射, 减少的激发频率有助于减少散射和更好的组织穿透多达1毫米的组织, 从而使成像更深入。共焦显微镜通过过滤出焦点信号并在焦点平面12中收集光来消除。通过这种方法, 可以将来自不同焦平面的样本图像组合起来, 通过 Z 堆栈函数生成三维 (3D) 图像。然而, 由于大部分光线被堵塞, 而且高数值孔径掩盖了景深, 信号强度降低。更重要的是, 这两种技术都有助于严重的光损伤和毒性, 因为整个标本接受照明, 即使只有一架飞机是在给定的时间成像。
为了规避这些缺点, LSFM 已成为一种受青睐的替代方案, 具有快速、高效、光毒性13、14等优点.此外, LSFM 还允许多视图成像。这种方法特别适合于可视化电机轴突及其分散终端, 因为它们分布在3D 空间。LSFM 优于其他两个选项, 因为示例安装在允许围绕垂直轴旋转和沿 x、y 和 z 轴移动的舞台上。这种设置不仅允许对样本进行最小的屏蔽, 而且还可以选择理想的光照路径、双光子和共焦显微镜的缺点, 这两种方法都需要在平面滑动上安装样品。因此, LSFM 是最合适的工具, 3D 成像的轴突树枝状和量化的运动神经终端的小鼠胚胎。

<table>
	<tbody>
		<tr>
			<td>Hb9::GFP</td>
			<td>The Jackson labortory</td>
			<td>005029</td>
			<td>Collect embryos of embryonic day 13.5 (E13.5)</td>
		</tr>
		<tr>
			<td>4% Paraformaldehyde (PFA)</td>
			<td></td>
			<td></td>
			<td>For 200ml: Add 20ml 10X PBS, 8g PFA in ddH2O. Adjust pH to 7.4 with NaOH (10N). Filter sterilize and store at -20 &#176;C.</td>
		</tr>
		<tr>
			<td>Phosphate buffer saline 10X (PBS 10X)</td>
			<td></td>
			<td></td>
			<td>For 1L: Add 80 g NaCl, 2 g KCl,14.4g Na2HPO4, 2.4 g KH2PO4 and top up with ddH2O. Autoclave and store at RT.</td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma</td>
			<td>X100-500ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>ThermoFisher</td>
			<td>26140079</td>
			<td></td>
		</tr>
		<tr>
			<td>Sheep polyclonal anti-GFP</td>
			<td>AbD Serotec</td>
			<td>4745-1051</td>
			<td>1:1000</td>
		</tr>
		<tr>
			<td>Alexa Fluor 488 donkey anti-sheep</td>
			<td>Invitrogen</td>
			<td>A-11015</td>
			<td>1:1000</td>
		</tr>
		<tr>
			<td>RapiClear 1.47 clearing reagent</td>
			<td>SunJin Lab</td>
			<td>RC147001</td>
			<td></td>
		</tr>
		<tr>
			<td>1.5ml micro tube</td>
			<td>Sarstedt</td>
			<td>72.690.001</td>
			<td></td>
		</tr>
		<tr>
			<td>24 wells plate</td>
			<td>ThermoFisher</td>
			<td>142475</td>
			<td></td>
		</tr>
		<tr>
			<td>5 SA Tweezer</td>
			<td>ideal-tek</td>
			<td>3480641</td>
			<td></td>
		</tr>
		<tr>
			<td>Iris Scissors striaght sharp/sharp</td>
			<td>Aesculap</td>
			<td>BC110R</td>
			<td></td>
		</tr>
		<tr>
			<td>Microsurgery Scalpels, single use</td>
			<td>Aesculap</td>
			<td>BA365</td>
			<td></td>
		</tr>
		<tr>
			<td>Dissecting microscope</td>
			<td>Nikon</td>
			<td>SMZ800</td>
			<td></td>
		</tr>
		<tr>
			<td>Shaker</td>
			<td>TKS</td>
			<td>RS-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Lightsheet Z.1 microscope</td>
			<td>Carl Zeiss Microscopy</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Imaris 8.4.0 image analysis software</td>
			<td>Bitplane, Zurich, Switzerland</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>B6.Cg-Tg(Hlxb9-GFP)1Tmj/J (Hb9::GFP mice)</td>
			<td>The Jackson Laboratory</td>
			<td>005029</td>
			<td></td>
		</tr>
	</tbody>
</table>

visualization of motor axon navigation and quantification of axon arborization in mouse embryos using light sheet fluorescence microscopy

脊髓运动神经元 (MNs) 扩展其轴突, 以与其支配目标进行沟通, 从而控制脊椎动物的运动和复杂任务。因此, 在发育和变性过程中, 揭示运动神经轴突如何定位、arborize 和支配其周围肌肉靶点的分子机制至关重要。尽管转基因的Hb9::GFP鼠标线长期用于可视化胚胎发育过程中的马达轴突轨迹, 但由于模式复杂, 对轴突终端树枝状的详细描述仍然不完整,目前光学显微镜的局限性。在这里, 我们描述了一个改进的协议, 结合光片荧光显微镜 (LSFM) 和稳健的图像分析, 定性和定量可视化发展电机轴突。该系统可以很容易地用于交叉遗传突变体或锰疾病模型与Hb9::GFP线, 揭示了新的分子机制, 导致缺陷的电机轴突导航和树枝状。

LSFM 提供详细的3D 可视化的锰弹道和轴突树枝状在小鼠胚胎。在明亮的领域, 在被浸泡在商业清除试剂之后, 组织显得完全透明。无收缩或肿胀的样本被发现后, 一个星期的存储在清除试剂成像前。在荧光通道下, 电机神经元被标记为 transgenically 表达的 GFP (影片 1)。在某些情况下, 轴突结构的细节被破坏。例如,图 2表示低质量映像。有几个?...

Watch this Scientific Journal Video about Visualization of Motor Axon Navigation and Quantification of Axon Arborization In Mouse Embryos Using Light Sheet Fluorescence Microscopy at JoVE.com

Visualization of Motor Axon Navigation and Quantification of Axon Arborization In Mouse Embryos Using Light Sheet Fluorescence Microscopy

在这里, 我们描述了一个协议, 以可视化的运动神经元投射和轴突树枝状在转基因 Hb9::GFP 小鼠胚胎. 在对运动神经元进行染色后, 我们用光片荧光显微镜对图像胚胎进行了定量分析。本协议适用于中枢神经系统的其它神经元导航过程。

用光片荧光显微镜观察运动轴突导航和小鼠胚轴突树枝状的定量化

Spinal motor neurons (MNs) extend their axons to communicate with their innervating targets, thereby controlling movement and complex tasks in ...

Research

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研究胶质细胞轴突的生长异质性的影响，使用一个新的共培养法

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Micron-scale Resolution Optical Tomography of Entire Mouse Brains with Confocal Light Sheet Microscopy

整个老鼠大脑与光共聚焦显微镜板微米级分辨率光学层析成像

Understanding the architecture of mammalian  brain at single-cell resolution is one of the key issues of neuroscience.  However, mapping neuronal soma and ...

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The visualization of full-length neuronal projections in embryos is essential to gain an understanding of how mammalian neuronal networks develop. Here we ...

The Neuromuscular Junction: Measuring Synapse Size, Fragmentation and Changes in Synaptic Protein Density Using Confocal Fluorescence Microscopy

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The neuromuscular junction (NMJ) is the large, cholinergic relay synapse through which mammalian motor neurons control voluntary muscle contraction. ...

Imaging Ca2+ Dynamics in Cone Photoreceptor Axon Terminals of the Mouse Retina

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Retinal cone photoreceptors (cones) serve daylight vision and are the basis of color discrimination. They are subject to degeneration, often leading to ...

Utilizing Combined Methodologies to Define the Role of Plasma Membrane Delivery During Axon Branching and Neuronal Morphogenesis

采用组合运用各种方法，定义质膜交付在轴突分支和神经元形态发生中的作用

During neural development, growing axons extend to multiple synaptic partners by elaborating axonal branches. Axon branching is promoted by extracellular ...

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The mouse olfactory system is often used to study mechanisms of neural circuit formation&#12288;because of its simple anatomical structure. An Olfactory ...

Visualization of Thalamocortical Axon Branching and Synapse Formation in Organotypic Cocultures

脊髓 Cocultures 丘脑-皮质轴突分支和突触形成的可视化研究

Axon branching and synapse formation are crucial processes for establishing precise neuronal circuits. During development, sensory thalamocortical (TC) ...

Investigating Mammalian Axon Regeneration: In Vivo Electroporation of Adult Mouse Dorsal Root Ganglion

Investigating Mammalian Axon Regeneration: In Vivo Electroporation of Adult Mouse Dorsal Root Ganglion

哺乳动物轴突再生: 成年小鼠背根神经节体内电穿孔的研究

Electroporation is an essential non-viral gene transfection approach to introduce DNA plasmids or small RNA molecules into cells. A sensory neuron in the ...

In Situ Visualization of Axon Growth and Growth Cone Dynamics in Acute Ex Vivo Embryonic Brain Slice Cultures

In Situ Visualization of Axon Growth and Growth Cone Dynamics in Acute Ex Vivo Embryonic Brain Slice Cultures

原位 急性 离体 胚胎脑切片培养物中轴突生长和生长锥动力学的可视化

During neuronal development, axons navigate the cortical environment to reach their final destinations and establish synaptic connections. Growth cones ...