Name: visualization of motor axon navigation and quantification of axon arborization in mouse embryos using light sheet fluorescence microscopy
Uploaded: 2018-05-11T15:00:00.000+00:00
Duration: 8 min 56 s
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, 以除去残留的多聚甲醛。</li>
</ol>2. 全芒染色
<ol><li>Permeabilize 每个胚在1毫升 0.5% PBS-Triton-X-100 (PBST) 过夜在4°c 在一个振动筛。</li>
	<li>在 1x PBS 中, 每晚在4摄氏度的一个振动筛上, 用1毫升10% 胎的样品来阻断每个样本。</li>
	<li>用1毫升的抗 GFP 原抗体 (1:1, 000 稀释10% 血清) 在4摄氏度上孵化胚胎, 持续晃动以强化运动神经神经的 GFP 信号。</li>
	<li>在主抗体孵化后, 用1毫升 0.5% PBST, 在1-2 天的时间内, 在4摄氏度的振动筛上冲洗, 改变 0.5% PBST 至少三次。</li>
	<li>应用1毫升二次抗体 (1:1, 000 稀释在10% 血清) 和孵化夜间在黑暗中在4°c 与恒定的震动。</li>
	<li>洗涤3x 与1毫升 0.5% PBST 在4°c 在一个振动筛在2-3 天期间。将样品保持在 1x PBS 的4摄氏度, 直到成像前一天。 注意: 胚胎在清除前可以解剖成较小的部分, 这取决于胚胎的哪个部分会被成像。前肢是保存在这个实验方法 (图 1A)。</li>
	<li>在一个具有1.49 和1.5 毫升管的折射率的商业清除试剂中孵化胚胎, 在室温下防止夜间的光线, 从而使胚胎成像透明。 注: 清除试剂的体积约为样品体积的五倍。</li>
</ol>3. LSFM (2–3 h)
<ol><li>
示例设置
	<ol>
<li>设置 5 x/0.1 照明光学和 5 x/0.16 检测光学。组装样品持有人和样品毛细管 (1.5 毫米内径, 绿色), 并把它们放到显微镜。 注: 请参阅显微镜的操作手册, 以进行详细的设置程序。</li>
		<li>安装胚胎如下:<ol>
<li>P200 吸管尖端的切割, 使其适合毛细管直径, 并删除尖部 (~ 3 毫米) 的样品附件。</li>
			<li>用小火焰融化吸管尖端的钝端。</li>
			<li>熄灭火焰, 并迅速将胚胎垂直附着在融化的末端上。</li>
			<li>将吸管尖端的上部与样品毛细管配合使用。</li>
		</ol>
</li>
		<li>允许室缓冲与胚胎平衡1分钟清除碎片或气泡。</li>
		<li>使用成像软件, 在定位选项卡下选择定位毛细管, 并调整 x、y、z 轴以定位毛细管。</li>
		<li>选择定位示例并缩放到0.6X 以关注胚胎。旋转胚胎以确保成像肢体的长轴与两个光源 (图 1B) 的光路径对齐。</li>
	</ol>
</li>
	<li>
图像采集
	<ol>
<li>在获取选项卡下, 定义光路径参数, 如检测目标、激光阻挡过滤器、光束分配器、照相机和激光器。 注: GFP 通道用于本实验 (激发波长: 488 nm; 发射过滤器: BP 505 至 545 nm, 分束: SPS LP 560)。</li>
		<li>选中 "透视扫描" 复选框以减少阴影。</li>
		<li>定义采集设置: 位深度, 16 位;变焦, 0.36-0, 7 x;单侧照明 (左/右) 或双侧照明。</li>
		<li>单击连续并设置激光强度、曝光时间、激光功率和光片位置以获取更清晰的图像。 注: 激光功率保持尽可能低, 以最小化光损伤。</li>
		<li>按停止结束图像获取。</li>
	</ol>
</li>
	<li>
多维获取
	<ol>
<li>通过移动第一个和最后一个图像的 z 位置来定义 z 堆栈。单击最佳设置切片编号。</li>
		<li>单击开始实验获取选定的 z 堆栈。</li>
		<li>完成后, 将图像保存为. czi 格式。</li>
	</ol>
</li>
	<li>
图像处理 (可选)
	<ol>
<li>如果应用了双侧光照, 则在Lightsheet 处理通道下进行双侧融合。如果获取多视图来组合多个角度的图像, 则需要Multiview 处理。</li>
		<li>使用最大强度投影通道中沿投影轴的最高强度像素的数据创建2D 图像。</li>
		<li>在复制通道下, 单击子集从原始集选择图像子集。</li>
	</ol>
</li>
</ol>4. 轴突树枝状的定量 (每个人神经30分钟)
<ol><li>在成像分析软件中打开图像文件 (默认情况下, 包含 XYZ 数据的图像将以超越模式打开, 并作为3维渲染视图)。</li>
	<li>使用显示调整窗口调整图像颜色、亮度和对比度 (编辑 |显示调整) 以根据局部强度对比度检测花丝。</li>
	<li>单击添加新细丝图标, 然后在下拉菜单中选择Autopath (无循环)算法。</li>
	<li>选择感兴趣的区域 (在这种情况下, 细分轴的兴趣)。完成后, 单击下一步。</li>
	<li>通过指定最大和最薄直径测量来定义起始和种子点, 可以使用切片模式对其进行测量。</li>
	<li>在感兴趣区域的边缘分配一个起始点。要实现手动添加或删除起始点, 请首先更改指针模式 (导航 |选择), 然后Shift + 右键单击在感兴趣的点。</li>
	<li>为种子点选择手动阈值, 以确保标记了所有可见的树枝状。更改指针模式 (导航 |选择), 然后Shift + 左键单击在感兴趣的点, 以手动添加或删除种子点。 注意: 手动删除周围神经的背景噪声和信号是很重要的。</li>
	<li>选中在起始点周围移除种子点复选框。</li>
	<li>检查删除断开连接的段以允许排除距离太远且可能表示背景噪音的点。指示下一步骤中的最大间隙长度, 以定义排除的上限。</li>
	<li>调整背景减法的阈值, 使用高斯滤波器估计每个体素的背景强度。</li>
	<li>使用近似截面面积算法设置枝晶直径的自动阈值。</li>
	<li>跳过脊柱计算的步骤。</li>
	<li>完成该过程并选择所需的样式和颜色。</li>
	<li>取消选中属性中的卷框以仅查看重建的轴突。</li>
	<li>在统计选项卡下, 选择详细。使用灯丝号。枝晶末端点量化电机神经终端作为电机轴突树枝状的指示器。 注意: 如果需要, 可以添加统计注释。</li>
	<li>将重建后轴突的图像导出为. tif 文件。</li>
</ol>

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作者没有什么可透露的。

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 12.5 (E12.5)</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 &amp;deg;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 1.49 clearing reagent</td><td>SunJin Lab</td><td>RC149001</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 ...

visualization-motor-axon-navigation-quantification-axon-arborization

Research

JoVE Journal

Neuroscience

7.7K 次观看.  National Defense Medical Center. 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。

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

In vivo Visualization of Synaptic Vesicles Within Drosophila Larval Segmental Axons

Elucidating the mechanisms of axonal transport has shown to be very important in determining how defects in long distance transport affect different neurological diseases. Defects in this essential process can have detrimental effects on neuronal functioning and development. We have developed a dissection protocol that is designed to expose the Drosophila larval segmental nerves to view axonal transport in real time. We have adapted this protocol for live imaging from the one published by Hurd and Saxton (1996) used for immunolocalizatin of larval segmental nerves. Careful dissection and proper buffer conditions are critical for maximizing the lifespan of the dissected larvae. When properly done, dissected larvae have shown robust vesicle transport for 2-3 hours under physiological conditions. We use the UAS-GAL4 method 1 to express GFP-tagged APP or synaptotagmin vesicles within a single axon or many axons in larval segmental nerves by using different neuronal GAL4 drivers. Other fluorescently tagged markers, for example mitochrondria (MitoTracker) or lysosomes (LysoTracker), can be also applied to the larvae before viewing. GFP-vesicle movement and particle movement can be viewed simultaneously using separate wavelengths.

In vivo Visualization of Synaptic Vesicles Within Drosophila Larval Segmental Axons

This protocol discusses the live dissection of Drosophila larvae for the purpose of imaging the movement of GFP tagged axonal vesicles on microtubule tracks.

在果蝇幼虫分部轴突在体内的突触小泡的可视化

Elucidating the mechanisms of axonal transport has shown to be very important in determining how defects in long distance transport affect different ...

科研

神经科学

Light Sheet-based Fluorescence Microscopy of Living or Fixed and Stained Tribolium castaneum Embryos

The red flour beetle Tribolium castaneum has become an important insect model organism in developmental genetics and evolutionary developmental biology. The observation of Tribolium embryos with light sheet-based fluorescence microscopy has multiple advantages over conventional widefield and confocal fluorescence microscopy. Due to the unique properties of a light sheet-based microscope, three dimensional images of living specimens can be recorded with high signal-to-noise ratios and significantly reduced photo-bleaching as well as photo-toxicity along multiple directions over periods that last several days. With more than four years of methodological development and a continuous increase of data, the time seems appropriate to establish standard operating procedures for the usage of light sheet technology in the Tribolium community as well as in the insect community at large. This protocol describes three mounting techniques suitable for different purposes, presents two novel custom-made transgenic Tribolium lines appropriate for long-term live imaging, suggests five fluorescent dyes to label intracellular structures of fixed embryos and provides information on data post-processing for the timely evaluation of the recorded data. Representative results concentrate on long-term live imaging, optical sectioning and the observation of the same embryo along multiple directions. The respective datasets are provided as a downloadable resource. Finally, the protocol discusses quality controls for live imaging assays, current limitations and the applicability of the outlined procedures to other insect species.
This protocol is primarily intended for developmental biologists who seek imaging solutions that outperform standard laboratory equipment. It promotes the continuous attempt to close the gap between the technically orientated laboratories/communities, which develop and refine microscopy methodologically, and the life science laboratories/communities, which require 'plug-and-play' solutions to technical challenges. Furthermore, it supports an axiomatic approach that moves the biological questions into the center of attention.

Light Sheet-based Fluorescence Microscopy of Living or Fixed and Stained Tribolium castaneum Embryos

Imaging the morphogenesis of insect embryos with light sheet-based fluorescence microscopy has become state of the art. This protocol outlines and compares three mounting techniques appropriate for Tribolium castaneum embryos, introduces two novel custom-made transgenic lines well suited for live imaging, discusses essential quality controls and indicates current experimental limitations.

光片为主的生活的荧光显微镜或固定和染色<em&gt;赤拟谷盗</em&gt;胚胎

The red flour beetle Tribolium castaneum has become an important insect model organism in developmental genetics and evolutionary developmental biology. ...

发育生物学

Time-lapse Imaging of Neuroblast Migration in Acute Slices of the Adult Mouse Forebrain

There is a substantial body of evidence indicating that new functional neurons are constitutively generated from an endogenous pool of neural stem cells in restricted areas of the adult mammalian brain. Newborn neuroblasts from the subventricular zone (SVZ) migrate along the rostral migratory stream (RMS) to their final destination in the olfactory bulb (OB)1. In the RMS, neuroblasts migrate tangentially in chains ensheathed by astrocytic processes2,3 using blood vessels as a structural support and a source of molecular factors required for migration4,5. In the OB, neuroblasts detach from the chains and migrate radially into the different bulbar layers where they differentiate into interneurons and integrate into the existing network1, 6.
In this manuscript we describe the procedure for monitoring cell migration in acute slices of the rodent brain. The use of acute slices allows the assessment of cell migration in the microenvironment that closely resembling to in vivo conditions and in brain regions that are difficult to access for in vivo imaging. In addition, it avoids long culturing condition as in the case of organotypic and cell cultures that may eventually alter the migration properties of the cells. Neuronal precursors in acute slices can be visualized using DIC optics or fluorescent proteins. Viral labeling of neuronal precursors in the SVZ, grafting neuroblasts from reporter mice into the SVZ of wild-type mice, and using transgenic mice that express fluorescent protein in neuroblasts are all suitable methods for visualizing neuroblasts and following their migration. The later method, however, does not allow individual cells to be tracked for long periods of time because of the high density of labeled cells. We used a wide-field fluorescent upright microscope equipped with a CCD camera to achieve a relatively rapid acquisition interval (one image every 15 or 30 sec) to reliably identify the stationary and migratory phases. A precise identification of the duration of the stationary and migratory phases is crucial for the unambiguous interpretation of results. We also performed multiple z-step acquisitions to monitor neuroblasts migration in 3D. Wide-field fluorescent imaging has been used extensively to visualize neuronal migration7-10. Here, we describe detailed protocol for labeling neuroblasts, performing real-time video-imaging of neuroblast migration in acute slices of the adult mouse forebrain, and analyzing cell migration. While the described protocol exemplified the migration of neuroblasts in the adult RMS, it can also be used to follow cell migration in embryonic and early postnatal brains.

We describe a protocol for real-time videoimaging of neuronal migration in the mouse forebrain. The migration of virally-labeled or grafted neuronal precursors was recorded in acute live slices using wide-field fluorescent imaging with a relatively rapid acquisition interval to study the different phases of cell migration, including the durations of the stationary and migration phases and the speed of migration.

急性片的成年小鼠前脑的神经细胞迁移的延时成像

There is a substantial body of evidence indicating that new functional neurons are constitutively generated from an endogenous pool of neural stem cells ...

Live Imaging of Mitosis in the Developing Mouse Embryonic Cortex

Although of short duration, mitosis is a complex and dynamic multi-step process fundamental for development of organs including the brain. In the developing cerebral cortex, abnormal mitosis of neural progenitors can cause defects in brain size and function. Hence, there is a critical need for tools to understand the mechanisms of neural progenitor mitosis. Cortical development in rodents is an outstanding model for studying this process. Neural progenitor mitosis is commonly examined in fixed brain sections. This protocol will describe in detail an approach for live imaging of mitosis in ex vivo embryonic brain slices. We will describe the critical steps for this procedure, which include: brain extraction, brain embedding, vibratome sectioning of brain slices, staining and culturing of slices, and time-lapse imaging. We will then demonstrate and describe in detail how to perform post-acquisition analysis of mitosis. We include representative results from this assay using the vital dye Syto11, transgenic mice (histone H2B-EGFP and centrin-EGFP), and in utero electroporation (mCherry-&#945;-tubulin). We will discuss how this procedure can be best optimized and how it can be modified for study of genetic regulation of mitosis. Live imaging of mitosis in brain slices is a flexible approach to assess the impact of age, anatomy, and genetic perturbation in a controlled environment, and to generate a large amount of data with high temporal and spatial resolution. Hence this protocol will complement existing tools for analysis of neural progenitor mitosis.

Neural progenitor mitosis is a critical parameter of neurogenesis. Much of our understanding of neural progenitor mitosis is based on analysis of fixed tissue. Live imaging in embryonic brain slices is a versatile technique to assess mitosis with high temporal and spatial resolution in a controlled environment.

有丝分裂中的不同发育阶段小鼠胚胎皮层实时成像

Although of short duration, mitosis is a complex and dynamic multi-step process fundamental for development of organs including the brain. In the ...

Live Imaging of Cell Motility and Actin Cytoskeleton of Individual Neurons and Neural Crest Cells in Zebrafish Embryos

The zebrafish is an ideal model for imaging cell behaviors during development in vivo. Zebrafish embryos are externally fertilized and thus easily accessible at all stages of development. Moreover, their optical clarity allows high resolution imaging of cell and molecular dynamics in the natural environment of the intact embryo. We are using a live imaging approach to analyze cell behaviors during neural crest cell migration and the outgrowth and guidance of neuronal axons.
Live imaging is particularly useful for understanding mechanisms that regulate cell motility processes. To visualize details of cell motility, such as protrusive activity and molecular dynamics, it is advantageous to label individual cells. In zebrafish, plasmid DNA injection yields a transient mosaic expression pattern and offers distinct benefits over other cell labeling methods. For example, transgenic lines often label entire cell populations and thus may obscure visualization of the fine protrusions (or changes in molecular distribution) in a single cell. In addition, injection of DNA at the one-cell stage is less invasive and more precise than dye injections at later stages.
Here we describe a method for labeling individual developing neurons or neural crest cells and imaging their behavior in vivo. We inject plasmid DNA into 1-cell stage embryos, which results in mosaic transgene expression. The vectors contain cell-specific promoters that drive expression of a gene of interest in a subset of sensory neurons or neural crest cells. We provide examples of cells labeled with membrane targeted GFP or with a biosensor probe that allows visualization of F-actin in living cells1.
Erica Andersen, Namrata Asuri, and Matthew Clay contributed equally to this work.

This protocol describes imaging of individual neurons or neural crest cells in living zebrafish embryos. This method is used to examine cellular behaviors and actin localization using fluorescence confocal time-lapse microscopy.

在斑马鱼胚胎的单个神经元和神经嵴细胞的细胞运动和肌动蛋白细胞骨架的实时成像

The zebrafish is an ideal model for imaging cell behaviors during development in vivo. Zebrafish embryos are externally fertilized and thus easily ...

生物学

Whole-mount Imaging of Mouse Embryo Sensory Axon Projections

The visualization of full-length neuronal projections in embryos is essential to gain an understanding of how mammalian neuronal networks develop. Here we describe a method to label in situ a subset of dorsal root ganglion (DRG) axon projections to assess their phenotypic characteristics using several genetically manipulated mouse lines. The TrkA-positive neurons are nociceptor neurons, dedicated to the transmission of pain signals. We utilize a TrkAtaulacZ mouse line to label the trajectories of all TrkA-positive peripheral axons in the intact mouse embryo. We further breed the TrkAtaulacZ line onto a Bax null background, which essentially abolishes neuronal apoptosis, in order to assess growth-related questions independently of possible effects of genetic manipulations on neuronal survival. Subsequently, genetically modified mice of interest are bred with the TrkAtaulacZ/Bax null line and are then ready for study using the techniques described herein. This presentation includes detailed information on mouse breeding plans, genotyping at the time of dissection, tissue preparation, staining and clearing to allow for visualization of full-length axonal trajectories in whole-mount preparation.

We present here an optimized protocol to genotype, stain and prepare fetal mice for the imaging of peripheral nociceptor axon projections in the whole animal, as an effective method to assess sensory axon growth phenotypes in developing genetically engineered mice.

小鼠胚胎感觉轴突预测整个安装成像

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

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

The establishment of functional neuromuscular circuits relies on precise connections between developing motor axons and target muscles. Motor neurons extend growth cones to navigate along specific pathways by responding to a large number of axon guidance cues that emanate from the surrounding extracellular environment. Growth cone target recognition also plays a critical role in neuromuscular specificity. This work presents a standard immunohistochemistry protocol to visualize motor neuron projections of late stage-16 Drosophila melanogaster embryos. This protocol includes a few key steps, including a genotyping procedure, to sort the desired mutant embryos; an immunostaining procedure, to tag embryos with fasciclin II (FasII) antibody; and a dissection procedure, to generate filleted preparations from fixed embryos. Motor axon projections and muscle patterns in the periphery are much better visualized in flat preparations of filleted embryos than in whole-mount embryos. Therefore, the filleted preparation of fixed embryos stained with FasII antibody provides a powerful tool to characterize the genes required for motor axon pathfinding and target recognition, and it can also be applied to both loss-of-function and gain-of-function genetic screens.

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

This work details a standard immunohistochemistry method to visualize motor neuron projections of late stage-16 Drosophila melanogaster embryos. The filleted preparation of fixed embryos stained with FasII antibody provides a powerful tool to characterize the genes required for motor axon pathfinding and target recognition during neural development.

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 ...

Time-Lapse Imaging of Neuronal Arborization using Sparse Adeno-Associated Virus Labeling of Genetically Targeted Retinal Cell Populations

Discovering mechanisms that pattern dendritic arbors requires methods to visualize, image, and analyze dendrites during development. The mouse retina is a powerful model system for the investigation of cell type-specific mechanisms of neuronal morphogenesis and connectivity. The organization and composition of retinal subtypes are well-defined, and genetic tools are available to access specific types during development. Many retinal cell types also constrain their dendrites and/or axons to narrow layers, which facilitates time-lapse imaging. Mouse retina explant cultures are well suited for live-cell imaging using confocal or multiphoton microscopy, but methods optimized for imaging dendrite dynamics with temporal and structural resolution are lacking. Presented here is a method to sparsely label and image the development of specific retinal populations marked by the Cre-Lox system. Commercially available adeno-associated viruses (AAVs) used here expressed membrane-targeted fluorescent proteins in a Cre-dependent manner. Intraocular delivery of AAVs in neonatal mice produces fluorescent labeling of targeted cell types by 4-5 days post-injection (dpi). The membrane fluorescent signals are detectable by confocal imaging and resolve fine branch structures and dynamics. High-quality videos spanning 2-4 h are acquired from imaging retinal flat-mounts perfused with oxygenated artificial cerebrospinal fluid (aCSF). Also provided is an image postprocessing pipeline for deconvolution and three-dimensional (3D) drift correction. This protocol can be used to capture several cellular behaviors in the intact retina and to identify novel factors controlling neurite morphogenesis. Many developmental strategies learned in the retina will be relevant for understanding the formation of neural circuits elsewhere in the central nervous system.

Here, we present a method for investigating neurite morphogenesis in postnatal mouse retinal explants by time-lapse confocal microscopy. We describe an approach for sparse labeling and acquisition of retinal cell types and their fine processes using recombinant adeno-associated virus vectors that express membrane-targeted fluorescent proteins in a Cre-dependent manner.

使用稀疏腺相关病毒标记遗传靶向视网膜细胞群的神经元树化延时成像

Discovering mechanisms that pattern dendritic arbors requires methods to visualize, image, and analyze dendrites during development. The mouse retina is a ...

可视化发育中的小鼠大脑中的神经元和神经胶质细胞动力学

Time-Lapse Imaging of Migrating Neurons and Glial Progenitors in Embryonic Mouse Brain Slices

During the development of the cerebral cortex, neurons and glial cells originate in the ventricular zone lining the ventricle and migrate toward the brain surface. This process is crucial for proper brain function, and its dysregulation can result in neurodevelopmental and psychiatric disorders after birth. In fact, many genes responsible for these diseases have been found to be involved in this process, and therefore, revealing how these mutations affect cellular dynamics is important for understanding the pathogenesis of these diseases. This protocol introduces a technique for time-lapse imaging of migrating neurons and glial progenitors in brain slices obtained from mouse embryos. Cells are labeled with fluorescent proteins using in utero electroporation, which visualizes individual cells migrating from the ventricular zone with a high signal-to-noise ratio. Moreover, this in vivo gene transfer system enables us to easily perform gain-of-function or loss-of-function experiments on the given genes by co-electroporation of their expression or knockdown/knockout vectors. Using this protocol, the migratory behavior and migration speed of individual cells, information that is never obtained from fixed brains, can be analyzed.

作者聚焦：探索发育中的大脑中的细胞迁移和基因作用

During the development of the cerebral cortex, neurons and glial cells originate in the ventricular zone lining the ventricle and migrate toward the brain surface. Many genes are involved in this process. This protocol introduces the technique for the time-lapse imaging of migrating neurons and glial progenitors.

胚胎小鼠脑切片中迁移神经元和神经胶质祖细胞的延时成像

During the development of the cerebral cortex, neurons and glial cells originate in the ventricular zone lining the ventricle and migrate toward the brain ...

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

摘要

探索更多视频

此视频中的章节

在果蝇幼虫分部轴突在体内的突触小泡的可视化

光片为主的生活的荧光显微镜或固定和染色

急性片的成年小鼠前脑的神经细胞迁移的延时成像

有丝分裂中的不同发育阶段小鼠胚胎皮层实时成像

在斑马鱼胚胎的单个神经元和神经嵴细胞的细胞运动和肌动蛋白细胞骨架的实时成像

小鼠胚胎感觉轴突预测整个安装成像

胚胎运动神经元轴突投影模式的可视化

使用稀疏腺相关病毒标记遗传靶向视网膜细胞群的神经元树化延时成像

胚胎小鼠脑切片中迁移神经元和神经胶质祖细胞的延时成像

胚胎小鼠脑切片中迁移神经元和神经胶质祖细胞的延时成像