We thank F. Bouallague and the IBENS animal facility for excellent zebrafish care. Research reported in this publication was supported by the Fondation ARC pour la recherche sur le cancer, grants N&#176; SFI20111203770 and N&#176; PJA 20131200143.

注意: 图1呈现了协议的轮廓。 1.注射和移植针的制备方法注意:针可以在任何时间来制备和储存。让他们在培养皿中，在橡皮泥带。密封封口膜防尘保护的菜。 <ol><li>对于注射针，拉玻璃毛细管（外径1.0mm时，直径0.58毫米里面，没有灯丝（见材料清单））用微量拉马（见材料清单）。喜欢短而细的针（约5mm锥形部），因为它们是用于刺穿绒毛膜更有效。 注意:注射针是一次性使用的。 </li><li>针细胞移植<ol><li>拉玻璃毛细管（外径1.0mm时，直径0.78毫米里面，没有灯丝（见材料清单））用微量拉马。 </li><li> ü唱microforge（见的材料清单），切针的尖端的点处的内径为35微米（小于细胞的直径稍微待移植）。 </li><li>锥与一个microgrinder（见材料清单）45°角的毛细管的切削末端。 </li><li>任选地，拉使用microforge针的端部的倒钩。为此，触摸与锥的尖端的热丝，并迅速拉远，创造一个倒钩，可以帮助穿透胚胎。 </li><li>安装在附连到注射器针夹持器的针。使用注射器，冲洗，用2％氢氟酸针三倍的内侧，以消除玻璃残留物，然后用丙酮漂洗三次。 注意:氢氟酸是有毒的，操纵引擎盖下，带手套。 注:细胞移植针可以使用数次。 </li></ol></li></ol> 2.制备菜肴注射和细胞移植<ol><li>热火50毫升胚胎的介质11含有0.5g琼脂糖在满功率1分钟微波拿到1％琼脂糖胚胎网上平台。冷却该琼脂糖凝胶至约60℃，并倒入50毫升的90mm培养皿。在凝胶特殊设计的模具（见材料清单），无论是与注射菜行或以井细胞移植菜表面的地方。 <ol><li>观察琼脂糖模具浮动，如果琼脂糖是不是太热。琼脂糖凝胶被设置后，删除与镊子模具（ 图2A - B）。 注意:菜可以储存在4℃，直到几个星期。让他们在一个封闭的湿盒，以避免干燥。 </li></ol></li><li>在放置前使用28℃培养箱30分钟的菜。 </li></ol> 3.胚胎和注射的收藏<ol><li>注射液的制备 注:肌动蛋白结合域酵母肌动蛋白结合蛋白140（ABP-140）如Lifeact势必mCherry的（被称为ABP140-mCherry）用于在小区内可视聚合肌动蛋白，以分析凸起活性。添加另一个化合物测试的候选基因（ 例如显性阴性或组成型活性构建体的mRNA，或吗啉代寡核苷酸）的功能。为了评估在（ 图3），我们使用吗啉代寡核苷酸的结果描述SIN1的功能。作为对照，我们使用相同的SIN1啉但用于沿着序列分布使得该控制啉不匹配靶RNA 5个核苷酸的吗啉代。 <ol><li>解冻SIN1和5错配对照吗啉（2毫米储备液），并ABP140-mCherry的mRNA冰上。添加375毫微克的mRNA（75毫微克/μL最终），要么SIN1或5匹配控制啉的0.75微升（0.3毫米决赛）在Danieau缓冲液（58毫米氯化钠，0.7毫米氯化钾，0.4mM的MgSO 4上，0.6毫米的Ca（NO 3）2，5.0毫米的HEPES pH7.6）中，达到5微升的总体积。 </li></ol></li><li>胚胎收集 注:本实验建立捐助国和主机胚胎基因，表达goosecoid（GSC）的控制下启动的GFP，以可视化准脊索前板11。 <ol><li>收集的Tg（GSC:GFP）蛋。放置约80蛋90毫米的培养皿填充有胚胎培养基中并在28℃下孵育。 </li></ol></li><li>注射胚胎供体<ol><li>在立体显微镜，轻轻挤压收集胚胎钳放入盛有胚胎中期注射菜的线条。使用镊子，定向胚胎朝向顶部的细胞。放置在孵化器中注入培养皿在28℃，直至胚胎已达到4细胞期（1小时受精后）。 </li><li>加载的注射针与2微升的注射液。插入在被放置在一个微操纵器（见的材料清单）中，用聚四氟乙烯（PTFE）管（参见材料清单）连接到空气横向喷射器的针保持器（见的材料清单）的针（见的材料清单）。 </li><li>通过轻触用细镊子尖端打开毛细管。如果有必要，切断毛细管按捏其末端开放。 </li><li>进入四个单元与针中的一个，并在为了填补细胞体积（2 NL）的70％注入细胞内的溶液中。注资3000胚胎。 注意:获取针中的细胞可以是困难的，因为质膜是软的。针对小区和蛋黄之间的结便于针穿透。 </li><li>放置胚胎在28℃培养箱，直到他们达到球体阶段（4小时受精后）。 </li></ol></li></ol> 4.准备胚胎的细胞Transplantat离子<ol><li>准备20毫升胚胎培养基用200微升青霉素/链霉素（见的材料清单）（笔/链球菌EM）。 </li><li>在球体阶段胚胎Dechorionation（4小时后受精） 注:Dechorionated胚胎是十分脆弱的，并会与空气或塑料接触的情况下爆炸。因此需要将它们放置在琼脂糖包被的培养皿，用火抛光的玻璃巴斯德移液管转移。 <ol><li>用塑料巴斯德吸管，转让步骤3.2主机收集胚胎和胚胎供体在步骤3.3注入胚胎中涂以1％琼脂糖和充满笔/链球菌的EM两张35毫米的培养皿。保持主机胚胎和胚胎捐赠者在两个单独的菜肴。 </li><li>手动从他们用细镊子（见的材料清单）绒毛膜提取胚胎。 </li><li>放置在28℃培养箱胚胎，直到他们达到屏蔽阶段（6小时受精后）。 </li></ol></li></ol>5.细胞移植<ol><li>安排胚胎细胞移植。 <ol><li>下的荧光立体显微镜，选择供体胚胎的盾（绿色）内表达ABP140-mCherry（红色）。 </li><li>使用火抛光的巴斯德吸管，转移在充满笔/链球菌的EM细胞移植菜的井胚胎。放置在一排宿主胚胎和供体胚胎相邻排。 </li><li>使用睫毛，仔细定位与屏蔽起来的胚胎。 注意:在屏蔽的位置可以通过GFP表达或评估解剖。 </li></ol></li><li>移植系统设置。 注意:移植系统由一个针座（见的材料清单）用PTFE管（见材料清单）和管连接器（见材料清单）连接到装有微型驱动Hamilton注射器（见列表材料 ）。针托被安装在一个3路显微（参见材料清单）。 <ol><li>使用附连到注射器针夹持器，冲洗，用70％的乙醇细胞移植针。通过吸取空气进针干燥。不要被吹干，否则可能会导致灰尘被陷在针的锥形部。 </li><li>插入在连接到Hamilton注射器针座的针，以向上斜面。 </li></ol></li><li>盾对盾细胞移植<ol><li>输入与移植针供体胚胎的盾和制定标有ABP140-mCherry约10-20细胞。小心避免蛋黄绘制。 </li><li>移植细胞进入宿主胚的盾。重复，直到所有主机的胚胎被移植。 </li><li>用火抛光的巴斯德吸管取出任何损坏胚胎。放置在孵化器移植的胚胎在28℃，并让他们约30分钟恢复。 </li><li>使用针附连到注射器座，冲洗细胞移植针用水，并把它放回上橡皮泥的频带培养皿。密封封口膜的菜。 </li></ol></li></ol> 6.（可选）单细胞移植注意:在胚胎细胞彼此粘附，因此，它是很难得出只有一个小区在移植针。我们开发了一个修改的协议可以轻松地移植单脊索前板祖细胞。我们的想法是解离的细胞移植前。因为孤立脊索前板祖细胞往往会失去其身份，我们转强加他们的前瞻性脊索前板的身份，通过激活交点信号传导途径，在不存在的Sox32转录因子。我们已经证实，这些细胞诱导行为类似于内源性脊索前板祖细胞8。下面是进行单细胞移植的具体步骤。细胞dissocia化是由放置在胚胎无钙任氏11，解剖外植体和机械搅拌它来实现的。 <ol><li>在步骤2.1，准备在无钙任氏，而不是胚胎中期的1％琼脂糖移植菜。 </li><li>在步骤3.1中，除了ABP140-mCherry RNA和SIN1吗啉，加上编码节点受体Taram-A的活性形式（0.6纳克/μL最终）RNA的3纳克和1.25微升吗啉对sox32（原液2毫米，因此，0.5毫米决赛）。 </li><li>第4步，转移三个选定捐赠的胚胎在盘中的填充用火抛光的巴斯德吸管笔/链球菌无钙任氏细胞移植后。用细镊子，解剖含注射的细胞（ABP140-mCherry标记的细胞）外植体。迅速丢弃的胚胎的其余部分。 </li><li>随着睫毛，轻轻搅拌，直到外植体细胞分离。 </li><li>制定一个分离的细胞在移植针并移植于宿主胚胎的盾。 </li><li>使用火抛光的巴斯德吸管，转移在充满笔/链球菌的EM琼脂糖涂层培养皿胚胎。放置在孵化的胚胎在28℃30分钟。 </li></ol> 7.胚胎安装<ol><li>成像室<ol><li>方法1:使用带有4个孔（直径5.5毫米），用3毫米高边缘的一侧（ 图2C - D）扎一个塑料滑块（75 * 25 * 0.5mm）的组成的成像室。胶在背面玻璃盖玻片，用氰基丙烯酸酯胶（超级胶）。 注:在实验结束时，将在水腔室一晚以去除盖玻片并摆脱胶残基用砂纸的。 </li><li>方法2:用35毫米马蒂克玻璃底菜（见材料清单）为10毫米的孔。 </li></ol></li><li>胚胎安装<ol><li>填补玻璃小瓶与胚胎媒体1毫升热0.2％琼脂糖。保持在42℃的热块。 </li><li>下的荧光立体显微镜，选择的胚胎，其中红移植细胞是标记为绿色准脊索前板（ 即红色移植细胞是绿色的准脊索前板内，并已开始向动物极迁移）的一部分。 </li><li>传送所选胚胎成与火抛光的巴斯德吸移管将琼脂糖溶​​液。倒掉过剩从吸管胚胎培养基中。绘制胚胎在琼脂糖移液器，并传送胚胎在成像室中，淀积琼脂糖一滴。琼脂糖设置之前，东方与睫毛的胚胎。将准脊索前板向上成像与正置显微镜，或在玻璃底部倒置显微镜成像。等到琼脂糖在腔室的下一个井安装另一个胚胎之前设置（约1分钟，这取决于室温）。 注:成像茶MBER含4口井可安装多达4个胚胎。 </li><li>当琼脂糖设置，填写用钢笔/链球菌的EM室，以避免干燥。 </li></ol></li></ol> 8.实时成像<ol><li>使用40X水浸泡远距离镜头。使用适当的过滤器设置图像GFP和mCherry（GFP的:激励BP四十○分之四百七十○，分光器FT 510和排放BP五十零分之五百四十○;对于mCherry:激发BP二十一分之五百七十八，分光器FT 596，和发射LP 641 / 75）。调整曝光时间，以优化图像动态。 </li><li>到图像中的板的所有单元，获得的z栈，开始准脊索前板之上几微米（在外胚层）和结束准脊索前板下方几微米（在蛋黄）。用2微米的z步骤。为了优化采集速度，切换Z-栈之间，而不是帧之间的颜色。 注意:这可能导致在绿色和红色的图像之间的细微差别，但并不是因为没有准确的共同的问题是必需的绿色和红色信号之间-localization。进一步减少成像时间和暴露于光，绿色可获取仅一次，每5个时间点，这是足以识别脊索前板祖细胞。 </li><li>使用这样的设置，图像多达4胚胎这是必要的分析突起频率和方向的两分钟的时间间隔内。对于突一生的分析，缩短时间间隔30秒，以获得更高的时间分辨率。从60％到外包80％的外包图像。 </li></ol> 9.细胞动力学分析<ol><li>加载ImageJ的4D影像 注意:根据采集所用软件，图像被存储在不同的格式（每个图像的一个文件，每个Z堆叠一个文件，一个文件整个实验）。有些ImageJ的插件都可以在网上开4D栈。我们的数据存储为每Z堆叠一个文件。因为该数据集可以是大的，它可方便地打开它只有一部分（某些添ë点，或在z堆栈，或8位的一个子部分转换图像...）。我们使用一个特制的ImageJ的插件，这样做，我们会很乐意来分发要求。 </li><li>定位的分析和细胞突起频率<ol><li>使用GFP图像，以确定未来的脊索前板迁移的主要方向。以此作为细胞突起角测量的基准。旋转堆叠，使得该参考方向平行于该图像的一侧。 </li><li>按照一个单元格。选择角度工具。对于每一帧，每个突起，使用角度工具来测量突起和基准方向（ 图3A）之间的角度。使用分析/测量命令存储的值（或者按M键盘上）。将结果保存为txt文件。下一个单元格重复。 </li><li>将数据导入到R和情节角分布直方图。使用Kolmogorov-Smirnov检验（R中ks.test）来比较不同的条件。 注意:角工具提供包括在0°和180°之间的值，因而允许使用古典统计测试，而不是圆形的测试。 </li><li>与此数据集，计算出发射频率为每分钟每细胞的突起的数量。使用学生t检验（R中t.test）来比较不同的条件。 </li></ol></li><li>在细胞突起寿命分析<ol><li>为每个小区和每个突起，测量突起持续时间（帧数其间突起是帧之间存在×时间间隔）。 </li><li>将数据导入R.使用学生t检验（在读t.test）来比较不同的条件。 注:其他迁移功能可以是有趣为了表征细胞迁移分析。这些包括细胞轨道，速度，方向和持续的测量，或细胞形态。一些商业软件应用程序可用来测量这些功能，但大量开源软件APPLI的阳离子和ImageJ的插件也都具备，如例如适应分析地貌12，DiPer看细胞的轨迹和方向持久性13，CellTrack迁移14期间跟踪单元格边界。 </li></ol></li></ol>

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V., Way, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Open+source+software+for+quantification+of+cell+migration,+protrusions,+and+fluorescence+intensities.">Open source software for quantification of cell migration, protrusions, and fluorescence intensities.</a> The Journal of Cell Biology. 209 (1), 163-180 (2015).</li><li>Gorelik, R., Gautreau, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+and+unbiased+analysis+of+directional+persistence+in+cell+migration.">Quantitative and unbiased analysis of directional persistence in cell migration.</a> Nature Protocols. 9 (8), 1931-1943 (2014).</li><li>Sacan, A., Ferhatosmanoglu, H., Coskun, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CellTrack:+An+open-source+software+for+cell+tracking+and+motility+analysis.">CellTrack: An open-source software for cell tracking and motility analysis.</a> Bioinformatics. 24 (14), 1647-1649 (2008).</li><li>Tahinci, E., Symes, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distinct+functions+of+Rho+and+Rac+are+required+for+convergent+extension+during+Xenopus+gastrulation.">Distinct functions of Rho and Rac are required for convergent extension during Xenopus gastrulation.</a> Developmental biology. 259 (2), 318-335 (2003).</li><li>Zhang, T., Yin, C., 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=Stat3-Efemp2a+modulates+the+fibrillar+matrix+for+cohesive+movement+of+prechordal+plate+progenitors.">Stat3-Efemp2a modulates the fibrillar matrix for cohesive movement of prechordal plate progenitors.</a> Development. 141 (22), 4332-4342 (2014).</li><li>Riedl, J., Crevenna, A. H., 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=Lifeact:+a+versatile+marker+to+visualize+F-actin.">Lifeact: a versatile marker to visualize F-actin.</a> Nature methods. 5 (7), 605-607 (2008).</li><li>Burkel, B. M., Von Dassow, G., Bement, W. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Versatile+fluorescent+probes+for+actin+filaments+based+on+the+actin-binding+domain+of+utrophin.">Versatile fluorescent probes for actin filaments based on the actin-binding domain of utrophin.</a> Cell Motility and the Cytoskeleton. 64 (11), 822-832 (2007).</li><li>Yoo, S. K., Deng, Q., Cavnar, P. J., Wu, Y. I., Hahn, K. M., Huttenlocher, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+regulation+of+protrusion+and+polarity+by+PI3K+during+neutrophil+motility+in+live+zebrafish.">Differential regulation of protrusion and polarity by PI3K during neutrophil motility in live zebrafish.</a> Developmental cell. 18 (2), 226-236 (2010).</li><li>Spracklen, A. J., Fagan, T. N., Lovander, K. E., Tootle, T. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+pros+and+cons+of+common+actin+labeling+tools+for+visualizing+actin+dynamics+during+Drosophila+oogenesis.">The pros and cons of common actin labeling tools for visualizing actin dynamics during Drosophila oogenesis.</a> Developmental biology. 393 (2), 209-226 (2014).</li><li>Johnson, H. W., Schell, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuronal+IP3+3-kinase+is+an+F-actin-bundling+protein:+role+in+dendritic+targeting+and+regulation+of+spine+morphology.">Neuronal IP3 3-kinase is an F-actin-bundling protein: role in dendritic targeting and regulation of spine morphology.</a> Molecular biology of the Cell. 20 (24), 5166-5180 (2009).</li><li>Yi, J., Wu, X. S., Crites, T., Hammer, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Actin+retrograde+flow+and+actomyosin+II+arc+contraction+drive+receptor+cluster+dynamics+at+the+immunological+synapse+in+Jurkat+T+cells.">Actin retrograde flow and actomyosin II arc contraction drive receptor cluster dynamics at the immunological synapse in Jurkat T cells.</a> Molecular Biology of the Cell. 23 (5), 834-852 (2012).</li></ol>

The authors have nothing to disclose.

这个协议提供了一个简单的方法在体内 ，通过组合使用细胞移植实时成像嵌合体胚胎的创建来研究细胞迁移的候选基因的作用。 马赛克胚胎的创建研究细胞的动态要求其轮廓的可视化分析细胞质扩展。或不同标记 - - 环境，从而提供良好的视觉对比这可以通过标签在其它方面未标记的细胞分离来实现。 一种简单的方法，以随...

在多细胞生物体，细胞迁移都为在那里它确保细胞的组织成组织和器官，胚胎的发育和成年生活中，在那里它需要一部分组织稳态（伤口愈合）和免疫是至关重要的。除了这些生理功能，细胞迁移也是参与多种病理情况，包括，尤其是癌症转移。 细胞迁移已在体外进行了分析了几十年，提供的分子机制确保在平坦的表面细胞运动的全面理解。 体内然而，细胞通过更复杂的环境面对。它清楚地出现在过去的几年中，一个生物体内迁移可能由外部线索的影响，如细胞外基质中，相邻引导迁移细胞或分泌的趋化因子，并且驱动细胞迁移可能与已描述改变机制&lt;EM&gt;体外1,2。确保在体内细胞迁移的机制已经很少受到关注，到目前为止，主要是增加的技术难度，因为，相对于体外研究， 在体内特别是细胞迁移的分析需要对迁移细胞直接的光学连接，技术标签在独特的细胞为了看到其动力学和形态学，以及增益或功能丧失方法来测试候选基因的作用。迄今，携带这些特征仅几个模型系统已被用于在体细胞迁移3解剖。 我们最近使用准脊索前板的迁移早期斑马鱼胚胎作为一种新的方便的模型系统，以评估在体内细胞迁移4,5-控制候选基因的功能。准脊索前板（也称为前内胚层）是在GAST发作形成一组单元rulation对胚胎的背侧。在原肠胚形成该组共同迁移朝向胚胎6-8的动物极，以形成脊索前板，一个mesendodermal增厚，前方脊索和神经板底层。的脊索前板的前部将产生孵化腺体，而它的后部可能有助于头部中胚层9。由于外部发展和鱼胚胎的光学清晰度，细胞迁移，可直接和容易地在该结构中观察到。 细胞移植是一种非常有效的技术，它允许快速和方便地创建镶嵌胚10。表达在分离的细胞中的标记移植的细胞的结果的荧光骨架标记物，形态和动力学，其中可以容易地观察到。这个组合损失或功能增益接近许可证一罐的细胞自治功能分析didate基因。 所提出的协议描述了我们如何在体内 5控制细胞迁移和肌动蛋白动力学评估了TORC2 SIN1组件的功能。但是，如在结果中提到和进一步讨论的，它可用于分析任何候选基因的潜在含义在体内控制细胞迁移。

<table><tbody><tr><td>Glass capillaries (outside diameter 1.0 mm, inside diameter 0.58&amp;nbsp;mm)</td><td>Harvard Apparatus</td><td>300085</td><td>standard thickness</td></tr><tr><td>Glass capillaries (outside diameter 1.0 mm, inside diameter 0.78 mm)</td><td>Harvard Apparatus</td><td>300085</td><td>thin-walled</td></tr><tr><td>Penicillin-Streptomycin</td><td>Sigma-Aldrich</td><td>P4333</td><td>10&amp;nbsp;000&amp;nbsp;units penicillin and 10&amp;nbsp;mg streptomycin per ml</td></tr><tr><td>fine tweezers</td><td>Dumont Fine Science Tools</td><td>11254-20</td><td>5F</td></tr><tr><td>glass bottom dishes</td><td>MatTek</td><td>P35G-0-10-C</td><td></td></tr><tr><td>Air transjector</td><td>Eppendorf</td><td>5246</td><td></td></tr><tr><td>Micro-forge</td><td>Narishige</td><td>MF-900</td><td></td></tr><tr><td>Microgrinder</td><td>Narishige</td><td>EG-44</td><td></td></tr><tr><td>Micromanipulator (for injection)</td><td>Narishige</td><td>MN-151</td><td></td></tr><tr><td>Micromanipulator (for cell transplantation)</td><td>Leica</td><td>Leica Micromanipulator</td><td></td></tr><tr><td>Hammilton Syringe</td><td>Narishige</td><td>IM-9B</td><td></td></tr><tr><td>Micropipette puller</td><td>David Kopf Instruments</td><td>Model 720</td><td></td></tr><tr><td>Transplantation mold</td><td>Adapative Science Tools</td><td>PT-1</td><td></td></tr><tr><td>Needle holder</td><td>Narishige</td><td>HI-7</td><td></td></tr><tr><td>Tube connector</td><td>Narishige</td><td>CI-1</td><td></td></tr><tr><td>PTFE tubing</td><td>Narishige</td><td>CT-1</td><td></td></tr></tbody></table>

analyzing in vivo cell migration using cell transplantations and time-lapse imaging in zebrafish embryos

Cell migration is key to many physiological and pathological conditions, including cancer metastasis. The cellular and molecular bases of cell migration have been thoroughly analyzed in vitro. However, in vivo cell migration somehow differs from in vitro migration, and has proven more difficult to analyze, being less accessible to direct observation and manipulation. This protocol uses the migration of the prospective prechordal plate in the early zebrafish embryo as a model system to study the function of candidate genes in cell migration. Prechordal plate progenitors form a group of cells which, during gastrulation, undergoes a directed migration from the embryonic organizer to the animal pole of the embryo. The proposed protocol uses cell transplantation to create mosaic embryos. This offers the combined advantages of labeling isolated cells, which is key to good imaging, and of limiting gain/loss of function effects to the observed cells, hence ensuring cell-autonomous effects. We describe here how we assessed the function of the TORC2 component Sin1 in cell migration, but the protocol can be used to analyze the function of any candidate gene in controlling cell migration in vivo.

使用所提出的技术来分析SIN1，Tor的络合物2（TORC2）的核心组件中的一个的作用，在体内细胞迁移的控制。利用细胞移植允许分离的细胞和细胞自治效果分析的标签。电影S1示出了移植脊索前板祖细胞的迁移。与肌动蛋白ABP140标签允许富含肌动蛋白胞质突起的方便的可视化。我们测量了它们的频率和方向。野生型细胞产生的动物极的方向取向频繁大胞质突起， 即</...

Watch this Scientific Journal Video about Analyzing In Vivo Cell Migration using Cell Transplantations and Time-lapse Imaging in Zebrafish Embryos at JoVE.com

Analyzing In Vivo Cell Migration using Cell Transplantations and Time-lapse Imaging in Zebrafish Embryos

分析<em&gt;在体内</em&gt;细胞迁移使用细胞移植和时间推移成像在斑马鱼胚胎

Combining cell transplantation, cytoskeletal labeling and loss/gain of function approaches, this protocol describes how the migrating zebrafish prospective prechordal plate can be used to analyze the function of a candidate gene in in vivo cell migration.

Cell migration is key to many physiological and pathological conditions, including cancer metastasis. The cellular and molecular bases of cell migration ...

analyzing-vivo-cell-migration-using-cell-transplantations-time-lapse

Research

JoVE Journal

Developmental Biology

Analyzing In Vivo Cell Migration using Cell Transplantations and Time-lapse Imaging in Zebrafish Embryos

7.6K 次观看.  IBENS, Institut de Biologie de l'École Normale Supérieure. Combining cell transplantation, cytoskeletal labeling and loss/gain of function approaches, this protocol describes how the migrating zebrafish prospective prechordal plate can be used to analyze the function of a candidate gene in in vivo cell migration. 

视频: Analyzing In Vivo Cell Migration using Cell Transplantations and Time-lapse Imaging in Zebrafish Embryos

Imaging Subcellular Structures in the Living Zebrafish Embryo

In vivo imaging provides unprecedented access to the dynamic behavior of cellular and subcellular structures in their natural context. Performing such imaging experiments in higher vertebrates such as mammals generally requires surgical access to the system under study. The optical accessibility of embryonic and larval zebrafish allows such invasive procedures to be circumvented and permits imaging in the intact organism. Indeed the zebrafish is now a well-established model to visualize dynamic cellular behaviors using in vivo microscopy in a wide range of developmental contexts from proliferation to migration and differentiation. A more recent development is the increasing use of zebrafish to study subcellular events including mitochondrial trafficking and centrosome dynamics. The relative ease with which these subcellular structures can be genetically labeled by fluorescent proteins and the use of light microscopy techniques to image them is transforming the zebrafish into an in vivo model of cell biology. Here we describe methods to generate genetic constructs that fluorescently label organelles, highlighting mitochondria and centrosomes as specific examples. We use the bipartite Gal4-UAS system in multiple configurations to restrict expression to specific cell-types and provide protocols to generate transiently expressing and stable transgenic fish. Finally, we provide guidelines for choosing light microscopy methods that are most suitable for imaging subcellular dynamics.

Imaging the dynamic behavior of organelles and other subcellular structures in vivo can shed light on their function in physiological and disease conditions. Here, we present methods for genetically tagging two organelles, centrosomes and mitochondria, and imaging their dynamics in living zebrafish embryos using wide-field and confocal microscopy.

在活斑马鱼胚胎成像的亚细胞结构

In vivo imaging provides unprecedented access to the dynamic behavior of cellular and subcellular structures in their natural context. Performing such ...

科研

发育生物学

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

The genetic and technical strengths have made the zebrafish vertebrate a key model organism in which the consequences of gene manipulations can be traced in vivo throughout the rapid developmental period. Multiple processes can be studied including cell proliferation, gene expression, cell migration and morphogenesis. Importantly, the generation of chimeras through transplantations can be easily performed, allowing mosaic labeling and tracking of individual cells under the influence of the host environment. For example, by combining functional gene manipulations of the host embryo (e.g., through morpholino microinjection) and live imaging, the effects of extrinsic, cell nonautonomous signals (provided by the genetically modified environment) on individual transplanted donor cells can be assessed. Here we demonstrate how this approach is used to compare the onset of fluorescent transgene expression as a proxy for the timing of cell fate determination in different genetic host environments.
In this article, we provide the protocol for microinjecting zebrafish embryos to mark donor cells and to cause gene knockdown in host embryos, a description of the transplantation technique used to generate chimeric embryos, and the protocol for preparing and running in vivo time-lapse confocal imaging of multiple embryos. In particular, performing multiposition imaging is crucial when comparing timing of events such as the onset of gene expression. This requires data collection from multiple control and experimental embryos processed simultaneously. Such an approach can easily be extended for studies of extrinsic influences in any organ or tissue of choice accessible to live imaging, provided that transplantations can be targeted easily according to established embryonic fate maps.

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

Live tracking of individual WT retinal progenitors in distinct genetic backgrounds allows for the assessment of the contribution of cell non-autonomous signaling during neurogenesis. Here, a combination of gene knockdown, chimera generation via embryo transplantation and in vivo time-lapse confocal imaging was utilized for this purpose.

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 ...

Multi-Photon Time Lapse Imaging to Visualize Development in Real-time: Visualization of Migrating Neural Crest Cells in Zebrafish Embryos

Congenital eye and craniofacial anomalies reflect disruptions in the neural crest, a transient population of migratory stem cells that give rise to numerous cell types throughout the body. Understanding the biology of the neural crest has been limited, reflecting a lack of genetically tractable models that can be studied in vivo and in real-time. Zebrafish is a particularly important developmental model for studying migratory cell populations, such as the neural crest. To examine neural crest migration into the developing eye, a combination of the advanced optical techniques of laser scanning microscopy with long wavelength multi-photon fluorescence excitation was implemented to capture high-resolution, three-dimensional, real-time videos of the developing eye in transgenic zebrafish embryos, namely Tg(sox10:EGFP) and Tg(foxd3:GFP), as sox10 and foxd3 have been shown in numerous animal models to regulate early neural crest differentiation and likely represent markers for neural crest cells. Multi-photon time-lapse imaging was used to discern the behavior and migratory patterns of two neural crest cell populations contributing to early eye development. This protocol provides information for generating time-lapse videos during zebrafish neural crest migration, as an example, and can be further applied to visualize the early development of many structures in the zebrafish and other model organisms.

A combination of the advanced optical techniques of laser scanning microscopy with long wavelength multi-photon fluorescence excitation was implemented to capture high-resolution, three-dimensional, real-time imaging of neural crest migration in Tg(sox10:EGFP) and Tg(foxd3:GFP) zebrafish embryos.

多光子时间推移成像实时可视化发展: 迁移神经嵴细胞在斑马鱼胚胎中的可视化

Congenital eye and craniofacial anomalies reflect disruptions in the neural crest, a transient population of migratory stem cells that give rise to ...

Tissue Targeted Embryonic Chimeras: Zebrafish Gastrula Cell Transplantation

Certain fundamental questions in the field of developmental biology can only be answered when cells are placed in novel environments or when small groups of cells in a larger context are altered.  Watching how one cell interacts with and behaves in a unique environment is essential to characterizing cell functions.  Determining how the localized misexpression of a specific protein influences surrounding cells provides insightful information on the roles that protein plays in a variety of developmental processes.  Our lab uses the zebrafish model system to uniquely combine genetic approaches with classical transplantation techniques to generate genotypic or phenotypic chimeras.  We study neuron-glial cell interactions during the formation of forebrain commissures in zebrafish. This video describes a method that allows our lab to investigate the role of astroglial populations in the diencephalon and the roles of specific guidance cues that influence projecting axons as they cross the midline. Due to their transparency zebrafish embryos are ideal models for this type of ectopic cell placement or localized gene misexpression. Tracking transplanted cells can be accomplished using a vital dye or a transgenic fish line expressing a fluorescent protein. We demonstrate here how to prepare donor embryos with a vital dye tracer for transplantation, as well as how to extract and transplant cells from one gastrula staged embryo to another.  We present data showing ectopic GFP+ transgenic cells within the forebrain of zebrafish embryos and characterize the location of these cells with respect to forebrain commissures.  In addition, we show laser scanning confocal timelapse microscopy of Alexa 594 labeled cells transplanted into a GFP+ transgenic host embryo.  These data provide evidence that gastrula staged transplantation enables the targeted positioning of ectopic cells to address a variety of questions in Developmental Biology.

Zebrafish cell transplantation enables the combination of genetics and embryology to generate tissue specific chimeras. This video demonstrates gastrula staged cell transplantations that have allowed our lab to investigate the roles of astroglial populations and specific guidance cues during commissure formation in the forebrain.

组织有针对性的胚胎嵌合体：斑马鱼原肠胚细胞移植

Certain fundamental questions in the field of developmental biology can only be answered when cells are placed in novel environments or when small groups ...

生物学

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 ...

利用斑马鱼幼虫进行高效的体内肿瘤研究

A Simple, Rapid, and Effective Method for Tumor Xenotransplantation Analysis in Transparent Zebrafish Embryos

In vivo studies of tumor behavior are a staple of cancer research; however, the use of mice presents significant challenges in cost and time. Here, we present larval zebrafish as a transplant model that has numerous advantages over murine models, including ease of handling, low expense, and short experimental duration. Moreover, the absence of an adaptive immune system during larval stages obviates the need to generate and use immunodeficient strains. While established protocols for xenotransplantation in zebrafish embryos exist, we present here an improved method involving embryo staging for faster transfer, survival analysis, and the use of flow cytometry to assess disease burden. Embryos are staged to facilitate rapid cell injection into the yolk of the larvae and cell marking to monitor the consistency of the injected cell bolus. After injection, embryo survival analysis is assessed up to 7 days post injection (dpi). Finally, disease burden is also assessed by marking transferred cells with a fluorescent protein and analysis by flow cytometry. Flow cytometry is enabled by a standardized method of preparing cell suspensions from zebrafish embryos, which could also be used in establishing the primary culture of zebrafish cells. In summary, the procedure described here allows a more rapid assessment of the behavior of tumor cells in vivo with larger numbers of animals per study arm and in a more cost-effective manner.

作者聚焦:利用斑马鱼肿瘤异种移植推进癌症治疗

We describe a protocol for xenotransplantation into the yolk of transparent zebrafish embryos that is optimized by a simple, rapid staging method. Post-injection analyses include survival and assessing the disease burden of xenotransplanted cells by flow cytometry.

一种简单、快速、有效的透明斑马鱼胚胎肿瘤异种移植分析方法

In vivo studies of tumor behavior are a staple of cancer research; however, the use of mice presents significant challenges in cost and time. Here, we ...

癌症研究

Multicolor Time-lapse Imaging of Transgenic Zebrafish: Visualizing Retinal Stem Cells Activated by Targeted Neuronal Cell Ablation

High-resolution time-lapse imaging of living zebrafish larvae can be utilized to visualize how biological processes unfold (for review see 1). Compound transgenic fish which express different fluorescent reporters in neighboring cell types provide a means of following cellular interactions 2 and/or tissue-level responses to experimental manipulations over time. In this video, we demonstrate methods that can be used for imaging multiple transgenically labeled cell types serially in individual fish over time courses that can span from minutes to several days. The techniques described are applicable to any study seeking to correlate the "behavior" of neighboring cells types over time, including: 1) serial 'catch and release' methods for imaging a large number of fish over successive days, 2) simplified approaches for separating fluorophores with overlapping excitation/emission profiles (e.g., GFP and YFP), 3) use of hypopigmented mutant lines to extend the time window available for high-resolution imaging into late larval stages of development, 4) use of membrane targeted fluorescent reporters to reveal fine morphological detail of individual cells as well as cellular details in larger populations of cells, and 5) a previously described method for chemically-induced ablation of transgenically targeted cell types; i.e., nitroreductase (NTR) mediated conversion of prodrug substrates, such as metronidazole (MTZ), to cytotoxic derivatives 3,5.
As an example of these approaches, we will visualize the ablation and regeneration of a subtype of retinal bipolar neuron within individual fish over several days. Simultaneously we will monitor several other retinal cell types, including neighboring non-targeted bipolar cells and potential degeneration-stimulated retinal stem cells (i.e., M&#971;ller glia). This strategy is being applied in our lab to characterize cell- and tissue-level (e.g., stem cell niche) responses to the selective loss and regeneration of targeted neuronal cell types.

In this video, techniques for multicolor confocal time-lapse imaging and targeted cell ablation are provided. Time-lapse imaging is used to monitor the behavior of multiple cell types of interest in vivo. Targeted cell ablation facilitates the study neural circuit function and cell-specific neuronal regeneration paradigms.

多色成像定时转基因斑马鱼：可视化由针对性的神经细胞消融激活视网膜干细胞

High-resolution time-lapse imaging of living zebrafish larvae can be utilized to visualize how biological processes unfold (for review see 1). Compound ...

Live Imaging of Cell Extrusion from the Epidermis of Developing Zebrafish

Homeostatic maintenance of epithelial tissues requires the continual removal of damaged cells without disrupting barrier function. Our studies have found that dying cells send signals to their live neighbors to form and contract a ring of actin and myosin that ejects it out from the epithelial sheet while closing any gaps that might have resulted from its exit, a process termed cell extrusion1. The optical clarity of developing zebrafish provides an excellent system to visualize extrusion in living epithelia. Here we describe a method to induce and image extrusion in the larval zebrafish epidermis. To visualize extrusion, we inject a red fluorescent protein labeled probe for F-actin into one-cell stage transgenic zebrafish embryos expressing green fluorescent protein in the epidermis and induce apoptosis by addition of G418 to larvae. We then use time-lapse imaging on a spinning disc confocal microscope to observe actin dynamics and epithelial cell behaviors during the process of apoptotic cell extrusion. This approach allows us to investigate the extrusion process in live epithelia and will provide an avenue to study disease states caused by the failure to eliminate apoptotic cells.

Dying cells are extruded from epithelial tissues by concerted contraction of neighboring cells without disrupting barrier function. The optical clarity of developing zebrafish provides an excellent system to visualize extrusion in living epithelia. Here we describe methods to induce and image extrusion in the larval zebrafish epidermis at cellular resolution.

发展中国家斑马鱼的表皮细胞挤压实时成像

Homeostatic maintenance of epithelial tissues requires the continual removal of damaged cells without disrupting barrier function.  Our studies have found ...

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发展中国家斑马鱼的表皮细胞挤压实时成像