Name: antibody uptake assay for tracking notch/delta endocytosis during the asymmetric division of zebrafish radial glia progenitors
Uploaded: 2023-01-20T13:15:00
Description: Watch this Scientific Journal Video about Antibody Uptake Assay for Tracking Notch/Delta Endocytosis During the Asymmetric Division of Zebrafish Radial Glia Progenitors at JoVE.com

该项目得到了NIH R01NS120218，UCSF Mary Anne Koda-Kimble种子创新奖和Chan Zuckerberg Biohub的支持。

我们使用AB野生型系和转基因株系 Tg [ef1a：Myr-Tdtomato] 进行研究。所有动物实验均获得美国加州大学旧金山分校机构动物护理和使用委员会（IACUC）的批准（批准文号：AN179000）。
1. 斑马鱼胚胎的制备
<ol><li>在下午5：00之前设置鱼穿越水箱，使用分隔器将一条雌性野生型鱼和一条雄性Tg [ef1a：Myr-Tdtomato] 鱼分开。</li>
	<li>第二天早上上午 11：00 之前从?...

<ol>
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B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interaction+with+Notch+determines+endocytosis+of+specific+Delta+ligands+in+zebrafish+neural+tissue.">Interaction with Notch determines endocytosis of specific Delta ligands in zebrafish neural tissue.</a> Development. 136 (2), 197-206 (2009).</li><li>Kressmann, S., Campos, C., Castanon, I., F&#252;rthauer, M., Gonz&#225;lez-Gait&#225;n, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Directional+Notch+trafficking+in+Sara+endosomes+during+asymmetric+cell+division+in+the+spinal+cord.">Directional Notch trafficking in Sara endosomes during asymmetric cell division in the spinal cord.</a> Nature Cell Biology. 17 (3), 333-339 (2015).</li><li>Dong, Z., Yang, N., Yeo, S. -. 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我们已经开发了一种抗体摄取测定法，用于高效标记和成像斑马鱼径向胶质细胞祖细胞的内体Notch/Delta运输。与以前用于追踪果蝇SOP细胞7，8中标记的抗DeltaD抗体的方法相比，我们的方法使用显微注射而不是在偶联抗体中孵育样品。将荧光偶联的抗DLD抗体显微注射到后脑室;这种技术不会造成伤害，注射后胚胎的正常发育和完全恢复证明了这一点。胚胎脑...

Notch信号控制后生动物1发育过程中的细胞命运决定和模式，最近的研究表明，干细胞分裂中的Notch信号主要取决于内吞运输2，3。内吞Notch/Delta可以激活细胞核中的Notch信号传导并增强Notch靶基因4，5，6的转录。在果蝇感觉器官前体（SOP）细胞的不对称细胞分裂（ACD）过程中首次观察到定向Notch/Delta内体运输，导致pIIa中的Notch信号传导活性高于pIIb7，8。抗Delta和抗Notch抗体的抗体摄取测定已被应用于监测有丝分裂SOP细胞的内吞过程。Notch/DeltaD 内体在细胞分裂过程中与驱动蛋白一起移动到中心纺锤体，并且由于细胞分裂3，8 的最后一刻不对称中心纺锤体的反平行阵列而不对称地易位到 pIIa 细胞中。这些研究揭示了调节果蝇SOP细胞中不对称分裂的分子机制，但尚不清楚脊椎动物径向胶质细胞祖细胞（RGP）中是否发生类似的内吞过程。
此外，在脊椎动物RGP分裂过程中调节不对称Notch/DeltaD信号传导的分子机制尚不清楚。据报道，在斑马鱼中，Notch和Delta的相互作用促进了DeltaD配体9的内吞作用。目前尚不清楚DeltaD内吞作用是否会影响发育中的脊椎动物大脑中子细胞的细胞命运选择。最近的研究表明，将荧光偶联的抗DeltaD抗体注射到神经管中可以在神经上皮细胞中特异性标记Sara内体，并且含有Sara内体的抗DeltaD优先分离成增殖的子细胞10。有人提出，来自内体的Notch信号可以调节子细胞的命运。先前的结果表明，发育中的前脑中的大多数斑马鱼RGP细胞都会经历ACD，子细胞命运的确定取决于谱系内Notch/DeltaD信号传导11。为了阐明斑马鱼RGP中谱系内Notch/DeltaD信号传导的性质，我们开发了斑马鱼发育大脑中的抗DeltaD抗体摄取测定。使用该协议，我们已经成功地对有丝分裂RGP中的DeltaD内吞运输进行了活标记和成像。
荧光标记的抗 DeltaD 抗体沿前脑室有效内化到 RGP 中。它极大地促进了在不对称分裂的RGP中发现DeltaD内体的定向运输12，13。与以前为果蝇培养物和斑马鱼脊髓 10 开发的抗体摄取方案相比，该方案在脑室细胞层中实现了持久高效的抗 DeltaD 标记，特别是微量注射的抗体混合物少于10 nL。后脑脑室注射对于在发育中的大脑中应用抗体摄取测定非常方便，因为后脑脑室在斑马鱼胚胎中扩增良好，并在早期发育阶段14充满脑脊液。通过将抗体混合物注射到后脑心室而不损伤任何关键的发育组织，该方案尽可能减少了对前脑成像区域的可能损害。注射一抗剂量的减少也避免了干扰 体内内源性Delta-Notch信号传导的潜在副作用。该协议可以很容易地与其他药理学或遗传扰动相结合，用于不同的发育阶段，并且可能适用于成人大脑以及人类多能干细胞衍生的2D / 3D脑类器官。综上所述，该协议使得理解在ACD期间如何以及何时建立Notch信号不对称成为可能。成功实施该方案的主要挑战是根据特定的实验条件精确输送适当浓度的抗体。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>35mm glass bottom culture dish&#160;</td>
			<td>MatTek corporation</td>
			<td>P35GC-1.5-10-C</td>
			<td></td>
		</tr>
		<tr>
			<td>air pressure injector&#160;</td>
			<td>Narishige</td>
			<td>IM300</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-Mouse-IgG-Atto647N&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>50185</td>
			<td></td>
		</tr>
		<tr>
			<td>CaCl2.2H2O&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>C3306</td>
			<td></td>
		</tr>
		<tr>
			<td>Capillaries, 1.2 mm OD, 0.9 mm ID, with filament</td>
			<td>World Precision Instruments</td>
			<td>1B120F-6</td>
			<td></td>
		</tr>
		<tr>
			<td>CSU-W1 Spinning Disk/High Speed Widefield</td>
			<td>Nikin</td>
			<td>N/A</td>
			<td>Nikon&#160;Ti inverted fluorescence microscope with CSU-W1 large field of view confocal.&#160;</td>
		</tr>
		<tr>
			<td>Dumont Medical Tweezers Style 5</td>
			<td>Thomas Scientific</td>
			<td>72877-D</td>
			<td></td>
		</tr>
		<tr>
			<td>Flaming-Brown P897 puller</td>
			<td>Sutter Instruments</td>
			<td>N/A</td>
			<td>https://www.sutter.com/manuals/P-97-INT_OpMan.pdf</td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Millipore</td>
			<td>529552</td>
			<td></td>
		</tr>
		<tr>
			<td>MgSO4.7H2O</td>
			<td>Sigma-Aldrich</td>
			<td>M2773</td>
			<td></td>
		</tr>
		<tr>
			<td>micromanipulators</td>
			<td>World Precision Instruments</td>
			<td>WPI M3301R</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse anti-Dld&#160;</td>
			<td>Abcam</td>
			<td>AB_1268496</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse IgG blocking buffer from Zenon</td>
			<td>Thermofisher Scientific</td>
			<td>Z25008</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Sigma-Aldrich</td>
			<td>S3014</td>
			<td></td>
		</tr>
		<tr>
			<td>Phenol red</td>
			<td>Sigma-Aldrich</td>
			<td>P0290</td>
			<td></td>
		</tr>
		<tr>
			<td>Stemi 2000&#160;&#160;</td>
			<td>Zeiss&#160;</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Tricaine</td>
			<td>Sigma-Aldrich</td>
			<td>E10521</td>
			<td></td>
		</tr>
		<tr>
			<td>UltraPureTM low melting point agarose&#160;</td>
			<td>Invitrogen</td>
			<td>16520050</td>
			<td></td>
		</tr>
	</tbody>
</table>

antibody uptake assay for tracking notch/delta endocytosis during the asymmetric division of zebrafish radial glia progenitors

不对称细胞分裂（ACD）产生两个不同命运的子细胞，是产生细胞多样性的基础。在无脊椎动物和脊椎动物的发育器官中，不对称分裂的祖先产生一个Notchhi 自我更新和一个Notchlo 差异化的女儿。在胚胎斑马鱼的大脑中，径向神经胶质细胞祖细胞（RGP） - 主要的脊椎动物神经干细胞 - 主要经历ACD以产生一个RGP和一个分化神经元。斑马鱼胚胎的光学清晰度和易于接近性使其成为 体内 延时成像的理想选择，以直接可视化在ACD期间如何以及何时建立Notch信号的不对称性。最近的研究表明，Notch配体DeltaD的动态内吞作用在ACD期间的细胞命运决定中起着至关重要的作用，并且该过程由进化上保守的极性调节剂Par-3（也称为Pard3）和动力蛋白运动复合物调节。为了可视化有丝分裂RGP中Notch信号内体的 体内 运输模式，我们开发了这种抗体摄取测定。使用该测定法，我们揭示了RGP分裂过程中含DeltaD的内体的动力学。

在 图2A中，注射Atto647N的胚胎不与一抗结合，在脑室中显示出背景荧光。在细胞中可以观察到很少的吞噬荧光颗粒。注射抗Dld-Atto647N的斑马鱼胚胎在发育中的前脑的大多数细胞中显示出大量内化的荧光颗粒（图2A，右图）。放大以聚焦有丝分裂RGP后，如图 2B所示，我们能够记录细胞内抗Dld-Atto647N内体在细胞分裂过程中的动态运动（...

Watch this Scientific Journal Video about Antibody Uptake Assay for Tracking Notch/Delta Endocytosis During the Asymmetric Division of Zebrafish Radial Glia Progenitors at JoVE.com

Antibody Uptake Assay for Tracking Notch/Delta Endocytosis During the Asymmetric Division of Zebrafish Radial Glia Progenitors

这项工作开发了一种抗体摄取测定法，用于在胚胎斑马鱼大脑的径向胶质细胞祖细胞分裂中成像谱系内Notch/DeltaD信号传导。

用于追踪斑马鱼径向胶质细胞祖细胞不对称分裂期间缺口/δ内吞作用的抗体摄取测定

Asymmetric cell division (ACD), which produces two daughter cells of different fates, is fundamental for generating cellular diversity. In the developing ...

Notch信号控制后生动物发育过程中硫酸盐的决定和模式。我们开发了一种抗体摄取测定法，用于标记缺口配体 delta-D 并成像其在发育中的斑马鱼前脑中径向胶质细胞祖细胞的内吞运输。斑马鱼隐藏式脑室注射使脑室内衬的强直径向胶质细胞祖细胞选择性吸收抗Dld-Atto-647N具有高效率和持久的效果。该协议可以很容易地与在不同发育阶段使用的其他药理学或遗传扰动相结合，并可能适用于成人大脑以及人类多能干细胞衍生的2D或3D脑类器官。产生受精胚胎并孵育 18 到 20 小时后，将它们从培养箱中取出。首先使用白光在落射荧光显微镜下以20倍放大倍率观察它们。使用玻璃移液管丢弃在显微镜下看起来混浊或破裂的死胚胎。然后打开荧光灯并选择显微镜的RFP滤光片设置。选择具有强烈红色荧光的胚胎，并将它们转移到装有蛋水的新培养皿中。现在在白光下用两个细镊子手动去皮胚胎。用一对镊子握住绒毛膜，用其他镊子撕裂绒毛膜。用镊子小心地打开撕裂，并用其他镊子的尖端轻轻推动胚胎，使其足够大，使胚胎通过。将去皮的胚胎转移到带有新鲜胚胎培养基的新培养皿中，以便在显微注射前快速冲洗。在拉拔器上拉动细注射针后，在立体解剖显微镜下用镊子打开针尖。使尖端的直径约为 10 微米，锥角约为 30 度。现在将 0.5 微升抗 Dld 抗体与两微升抗小鼠 L G Atto-647N 抗体混合，移液 5 至 10 次以偶联它们并在室温下孵育至少 30 分钟。在孵育结束时，向抗体混合物中加入2.5微升封闭缓冲液和0.5微升0.5%酚红，并通过移液混合以封闭混合物中残留的任何未偶联抗体。接下来，在胚胎培养基中制备1%低熔点琼脂糖。在 70 摄氏度下加热含有琼脂糖的混合物，直到混合物变成透明。等分琼脂糖溶液后，将它们保持在 40 摄氏度的加热块中。在含有1%低熔点琼脂糖的试管中冲洗胚胎三秒钟。将胚胎放在带有单个琼脂糖滴的倒置塑料培养皿盖上，以分别安装每个胚胎。将胚胎横向放置在琼脂糖中并保持该位置，直到琼脂糖在室温下凝固。用蛋培养基覆盖琼脂糖中的所有镶嵌胚胎。将嵌入的胚胎放在体视显微镜下，并用蛋水覆盖琼脂糖。用微型机械手设置气压注射器，将它们靠近显微镜。使用高压下含有气态氮气的钢制气瓶作为空气资源。将胚胎安装在琼脂糖中后，打开气阀。在使用微量进样器的前填充模块时，将制备好的玻璃针头与两微升抗体混合物预先装入微量机械手上。现在将输入压力调整为每平方英寸 80 到 90 磅，将注射压力调整为每平方英寸 20 磅。在显微镜下使用千分尺校准注射体积。根据针开口的大小将调谐持续时间设置为10到120毫秒。轻触桨以输送每个注射脉冲，并将每次输送的注射量调整为 4 到 5 纳升。然后将微注射针的尖端戳穿后脑背顶板，向后至容地梅尔零一铰链点，在不撞击脑组织的情况下注射约10纳升抗体混合物。观察脑室中红色液体的流动。注射后，通过旋转微型机械手的旋钮迅速从胚胎中取出针尖。为了成功注射，注射混合物的红色染料稳定地保留在脑室中，而不会泄漏到周围的琼脂糖中。将安装板移动到显微镜下，将另一个安装的胚胎定位在合适的位置进行重复。注射六到八个胚胎后，用显微手术刀剥开琼脂糖，将胚胎从嵌入的琼脂糖中释放出来。将微注射的胚胎转移到装有30毫升胚胎培养基的新鲜培养皿中，并将其置于室温下进行下一步。30分钟后，将选定的胚胎转移到10毫升胚胎培养基中。为了安装胚胎，准备0.8%低熔点琼脂糖，在管中含有相同浓度的三烷。使用玻璃移液管将注射的胚胎浸入温热的琼脂糖中三秒钟。然后立即用相同的玻璃移液管从琼脂糖中取出胚胎，并将胚胎放在35毫米玻璃底部培养皿的中心，从管中取出一滴琼脂糖。用纤维探针或加载尖端轻轻地定向胚胎，以保持胚胎大脑的背侧尽可能靠近玻璃底部。轻轻转动胚胎位置以延长胚胎，而不会卷曲，因为琼脂糖逐渐凝固。然后，通过将玻璃底培养皿翻转过来检查胚胎位置。确保在显微镜下可以看到具有正确安装胚胎的整个背前脑。加入两到三毫升含有三烷的28.5摄氏度预热胚胎培养基以覆盖胚胎。将培养皿正确放置在共聚焦显微镜的温度控制载物台上，并将成像室的温度调节到28.5摄氏度。胚胎现在已准备好进行成像。注射Atto-647N的胚胎在脑室中显示出背景荧光。抗Dld-Atto-647N注射的斑马鱼胚胎在发育中的前脑的大多数细胞中显示出大量内化的荧光颗粒。延时成像显示细胞分裂过程中细胞内抗Dld-Atto-647N内体的运动。大多数有丝分裂径向神经胶质细胞祖细胞显示抗Dld-Atto-647N内体的前部或后部不对称分离为两个子细胞。不对称性在后期结束时稳定下来，导致子细胞之后不对称地遗传了缺口信号内体。确保针头已放入正确的位置隐藏的品牌心室，并检查显微注射后是否有任何泄漏。除了标记缺口δ信号传导外，如果细胞外结构域的良好抗体可用，该协议还适用于使用类似策略标记其他膜蛋白或细胞外蛋白。

Research

JoVE Journal

Developmental Biology

Ex Vivo Culture of Pharyngeal Arches to Study Heart and Muscle Progenitors and Their Niche

Ex Vivo Culture of Pharyngeal Arches to Study Heart and Muscle Progenitors and Their Niche

咽弓的体外培养研究心脏和肌肉祖细胞和自己的优势

The pharyngeal mesoderm of developing embryos contributes to broad regions of head and heart musculature. We have developed a novel method to study head ...

科研

发育生物学

Tracking Cells in GFP-transgenic Zebrafish Using the Photoconvertible PSmOrange System

在GFP-转基因斑马鱼使用Photoconvertible PSmOrange系统追踪单元

The rapid development of transparent zebrafish embryos (Danio rerio) in combination with fluorescent labelings of cells and tissues allows visualizing ...

Observing Mitotic Division and Dynamics in a Live Zebrafish Embryo

观察有丝分裂和动力学在现场斑马鱼胚胎

Mitosis is critical for organismal growth and differentiation. The process is highly dynamic and requires ordered events to accomplish proper chromatin ...

Directed Differentiation of Primitive and Definitive Hematopoietic Progenitors from Human Pluripotent Stem Cells

人多能干细胞对原始和最终造血祖细胞定向分化的研究

One of the major goals for regenerative medicine is the generation and maintenance of hematopoietic stem cells (HSCs) derived from human pluripotent stem ...

In Vivo Imaging of Transgenic Gene Expression in Individual Retinal Progenitors in Chimeric Zebrafish Embryos to Study Cell Nonautonomous Influences

In Vivo Imaging of Transgenic Gene Expression in Individual Retinal Progenitors in Chimeric Zebrafish Embryos to Study Cell Nonautonomous Influences

在嵌合斑马鱼胚胎的个别视网膜祖细胞转基因表达的体内成像来研究细胞非自治的影响

The genetic and technical strengths have made the zebrafish vertebrate a key model organism in which the consequences of gene manipulations can be traced ...

Histological Analyses of Acute Alcoholic Liver Injury in Zebrafish

斑马鱼急性酒精性肝损伤的组织学分析

Alcoholic Liver Disease (ALD) refers to damage to the liver due to acute or chronic alcohol abuse. It is among the leading causes of alcohol-related ...

Live Cell Imaging of Chromosome Segregation During Mitosis

有丝分裂过程中染色体分离的活体细胞成像

Chromosomes must be reliably and uniformly segregated into daughter cells during mitotic cell division. Fidelity of chromosomal segregation is controlled ...

Preparation of Drosophila Larval and Pupal Testes for Analysis of Cell Division in Live, Intact Tissue

Preparation of Drosophila Larval and Pupal Testes for Analysis of Cell Division in Live, Intact Tissue

用于活、完整组织中细胞分部分析的果蝇拉瓦尔和普帕尔睾试验的制备

Experimental analysis of cells dividing in living, intact tissues and organs is essential to our understanding of how cell division integrates with ...

Defining the Program of Maternal mRNA Translation during In vitro Maturation using a Single Oocyte Reporter Assay

使用单卵母细胞记者测定在体外成熟期间的母体 mRNA 翻译程序

Events associated with oocyte nuclear maturation have been well described. However, much less is known about the molecular pathways and processes that ...

In Situ Hybridization in Zebrafish Larvae and Juveniles during Mesonephros Development

In Situ Hybridization in Zebrafish Larvae and Juveniles during Mesonephros Development

原位 斑马鱼幼体和幼鱼在雌雄同体发育过程中的杂交

The zebrafish forms two kidney structures in its lifetime. The pronephros (embryonic kidney) forms during embryonic development and begins to function at ...