这项工作是由欧洲分子生物学组织的长期奖学金（ABW）的支持，美国国家科学基金会国际博士后研究奖学金（ABW），马克斯·普朗克促进协会（ABW，DS，JS，ACO），一个直销倍增-BB金（AO），而根据欧盟第七框架计划STG-207634欧洲研究委员会（DS，ACO）。我们感谢拉维德赛对稿件有用的意见。我们还要感谢MPI-CBG蛋白表达的设施进行生产的斑马鱼Fibronectin1片段，在MPI-CBG鱼设施的工作人员进行维护和保养我们的鱼线和MPI-CBG光镜设施成像支持。

1，解剖前<ol><li>上剥离的前一天，从斑马鱼对杂合的转基因等位基因的incross获得胚胎。 <ol><li>提高在E3媒体胚胎无亚甲蓝在28 O C，直到盾阶段（6小时后受精）。 </li><li>在没有亚甲基蓝，以20°CO / N的E3媒体转移胚胎在20℃时 ，胚胎就会形成每个小时1体节一旦达到tailbud阶段。经过17-20小时O / N为20℃，胚胎应该在5〜8体节期在第二天早上在解剖协议的开始。 </li></ol></li><li>装配工具和所需的解剖试剂。 <ol><li>火抛光的玻璃吸管的胚胎和组织转移</li><li>一双细镊子去除绒毛膜从胚胎</li><li>的Sylgard涂层35毫米的菜解剖 - 倾的Sylgard聚合物为35毫米的菜和治愈O / N在37°C。使夹层以及用针尖除去少量固化的聚合物。烹调，可清洗，随后再利用。 </li><li>削尖，扁平的PSM组织操纵钨丝工具</li><li>微型手术刀切割所需的片PSM的文化</li><li> 35毫米的塑料培养皿培养胰蛋白酶</li><li>含有10％胎牛血清的L15培养基</li><li> 0.25％胰蛋白酶/ EDTA溶液</li><li>为分散Sigmacoted凝胶加载提示</li><li>塑料培养皿中含有E3介质中无亚甲基蓝。 </li></ol></li><li>覆盖玻璃底培养皿的成像与Fibronectin1衬底（在PBS中的10微克/毫升）。离开盘上板凳外套夹层中。 </li><li>使用立体镜适当的荧光过滤器来识别和排序阳性转基因胚胎（5〜8体节期）。 <ol><li>通过检查presomitic中胚层（PSM）用于YFP的表达下荧光通道（ 图1A）鉴定转基因胚胎。荧光笑ULD可见在该地区从上形成体节的tailbud。选择胚胎各路信号;后代的25％应携带2份转基因的纯合子胚。需要确定胚胎的数量变化的基础上的实验。一个解剖tailbud片将产生1,000个细胞的平均值。通常情况下，一些额外的积极胚胎的情况下错误的解剖过程中进行了有益的。 </li><li>使用透射光作为位置基准来区分任何自发荧光，尤其是在卵黄细胞，从信号。 </li><li>积极转移胚胎到含有E3介质中无亚甲蓝一个单独的塑料培养皿中。 </li></ol></li></ol> 2，PSM解剖和扩散<ol><li>准备胚胎解剖。 <ol><li>在解剖显微镜下，用细镊子，小心地从在E3媒体每个胚胎取出绒毛。请务必不要损坏胚胎或破坏卵黄细胞。 </l我&gt; <li>填写的Sylgard涂层解剖盘与L15培养基中的血清。使用镊子或其他平坦的工具，从的Sylgard的表面除去任何气泡。 </li><li>用火抛光的玻璃吸管的解剖盘转移dechorionated胚胎。 </li><li>使用线工具，将所有的胚胎的解剖盘的一侧。 </li></ol></li><li>从单个胚胎解剖的PSM。 <ol><li>东方在其横向侧面的单个胚胎的小的解剖盘（ 图1B）内的Sylgard层以及制成。 </li><li>通过胚胎和卵黄细胞用微手术刀，切片只是前向后脑和通过胚胎（ 图1B，红色虚线）的腹杆。 </li><li>取下前片胚胎从井中，移开，在盘子的一边，然后用线工具从后段包括PSM刮去剩余卵黄细胞颗粒。 </li><li>一旦蛋黄已经被刮掉，压平及定向PSM与前端指向远离实验者和后部朝向实验者（ 图1C）。 </li><li>若该薄层外胚层尚未由这点被拉断，可使用线工具剥离它远离PSM组织的顶部。 </li><li>使用微型手术刀切开tailbud，PSM的最后尖过去脊索的末端，远离所述组织（ 图1C，红色虚线）的其余部分。注意：其他组织片从PSM，例如前PSM接近最后形成的体节，也可以考虑到培养，取决于实验问题。 </li><li>移动tailbud片的菜远离解剖区域的一角。使用线工具来清除解剖领域的任何碎片和不必要的胚胎组织。 </li><li>重复与下一个胚胎。 </li></ol></li><li>池tailbud件由多个电子mbryos，这取决于有多少细胞将需要进行实验。平均而言，单件tailbud组织产生1,000个细胞。 </li><li>填补空白的35毫米塑料盘具有体积小胰蛋白酶-EDTA的。 </li><li>用火抛光的玻璃吸管，从解剖盘到含有胰蛋白酶/ EDTA盘转移tailbud件。孵育tailbud条胰蛋白酶/ EDTA进行20分钟，在室温。 </li><li>当组织被培养在胰蛋白酶，从玻璃底培养皿的成像取出Fibronectin1溶液。 <ol><li>玻璃与超纯水冲洗液3次。使用抽吸去除每次洗涤，并确保菜是完全干燥。 </li></ol></li><li>分散tailbud枚介质成像。 <ol><li>吸取100微升L15培养基与血清成影像菜。 </li><li>使用凝胶涂层尖，从胰蛋白酶/ EDTA在尽可能小的体积尽可能去除tailbud件。 </li><li>移液器的tailbud片放入在培养基中成像菜。移液器件上下多次打破他们分开，并暂停在细胞培养基中。注意不要引入气泡。检查在显微镜下的细胞团块，并根据需要分散更多。 </li><li>允许分散的细胞在悬浮液中沉降到Fibronectin1被覆玻璃20分钟，在RT。 </li></ol></li><li>开始成像之前添加少量额外L15培养基用无血清的细胞。使用时注意不要破坏结算细胞。 </li><li>由于实验的问题，任何补充或药物治疗添加到文化。 </li></ol>分散PSM细胞3。成像<ol><li>设立成像盘中低温试验箱的时间推移成像显微镜。设置华纳室的实验所需的温度。让菜开始图像采集前平衡至少30分钟。交叉检验温度与外部温度探头放入盘中，如果需要的话。注：由于温度和体节形成率21稳定的温度控制之间的关系为周期的在单一的PSM细胞的精确测量是必不可少的。 </li><li>收购的荧光通道测试图像，检查曝光时间和增益，可提供强度的大动态范围，而不饱和度，以确保良好的信号噪声水平。使用该协议从我们的线路产生分散的细胞获得一个典型的荧光图像需要400毫秒和40毫秒的透射光图像与EMCCD相机在85的EM增益运行。另外，前置放大器增益和读出速度从摄像头也是必不可少的了最大化的噪声信号。 </li><li>采用透射光通道中，选择单元格的时间推移采集领域。 </li><li>运行的时间推移获取协议的时间所需要的长度。 <ol><li>获取一个透射光，每个字段的一个荧光图像。注：设置采集RO之间的间隔unds，将捕获时间动态没有光漂白过的细胞扩展成像或诱导毒性。该协议使用一个2分钟的间隔。 </li></ol></li><li>记录以确保电池保持在对焦，任何软件或硬件错误等时偶尔检查时间推移建立</li></ol> 4，图像的收购时间推移电影处理<ol><li>在图像处理软件，后天野外露天电影文件。注：斐济被用于所有的处理在此协议。 <ol><li>拆分透射光和荧光帧从一个字段创建2堆栈的图像。 </li></ol></li><li>跟踪单个细胞在透射光通道。 <ol><li>将一个圆形的ROI（感兴趣区域），选定的单元格周围在透射光通道的第一帧。注意：只有细胞，1是健康的在录音结束后，2不要领域的移动之外，和3不接触到其他CE接触。LLS被跟踪。 </li><li>除投资回报率每隔几帧的投资回报率经理。跟踪单元格，直到最后一帧。 </li></ol></li><li>使用上的荧光通道保存的ROI测量强度。 <ol><li>选择荧光堆栈和与保存的感兴趣区，使用定制的圆圈内插器插件和宏来测量跟踪的细胞随时间的强度。 </li><li>检查输出跟踪从宏观。如果跟踪定性地捕捉荧光延时功能，输出值到Excel工作表。 </li><li>除投资回报率清单的细胞。 </li></ol></li><li>重复跟踪传输信道并测量荧光信道用于其它细胞的字段。 </li><li>从实验重复其他字段的所有步骤。 </li></ol>

<ol>
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作者投稿： ABW发展和完善的扩散，文化，成像和细胞跟踪协议。 DS产生的转基因株系，并监督本协议中使用的延时荧光显微镜的设计。 AO率先验证性原则最初的实验离解和图像PSM细胞的培养。 JS在斐济写的圆圈内插器插件工具用于从定时短片测量荧光强度。 ABW和ACO写的稿子。

为了研究细胞过程，发生过胚胎发育过程中，生物学家通常使用的方法，在整个胚胎的上下文中。然而，要充分了解单细胞发育的时间框架内的行为，方法，研究和扰乱单个细胞的隔离也是非常有益的。通过生成PSM分散细胞培养使用转基因斑马鱼胚胎，我们现在要直接研究基因振荡的分割时钟的细胞自主性的定量方法的工具。有可能根据各种条件来测量在我们的荧光报道在数百个细胞的振荡的动力学。 单细胞的培养物所观察到的周期比在完整胚胎的体节形成期更长。我们观察到，在培养一个完整的PSM外植体也表现出较慢的振荡比完整胚（数据未显示），表明THA吨分离的细胞中观察到的长周期不只是由于从分散的损害。可变周期和振幅在我们的大多数单细胞时间序列的观察。这种变异的来源是未知的，但应该揭示分割时钟的节奏，使得电路的重要细节。 通过这种方法，我们可以学习分割在细胞水平上被挑战，研究在整个胚胎的遗传成分，和地址的问题。例如，通过控制加入在胚胎发育到我们的文化中找到已知的信号分子，我们可以审视自己在一个强大的和可重复的检测单PSM细胞振荡器的影响。我们的PSM培养体系打开大门的哪些因素，单独或结合，促进这些细胞振荡严格的评估，哪些因素抑制这些振荡，并测试这些分子之间的相互作用。有了这些工具，我们的目标是评估体节形成的那些基于公开的组织层次数据，以及在单个细胞中使用的结果来生成可以在整个胚胎被测试的预测现有模型。 据我们所知，这是第一个斑马鱼原代细胞培养协议，用于荧光单细胞急性延时录制;这个协议的进一步发展和完善是毫无疑问的可能。其他的协议经常使用斑马鱼胚胎生成可被转染与记者和用于长期成像27-29的稳定细胞株。而稳定的线成像，是不依赖于发展的时机，如生物钟的过程非常有用，胚胎分割的问题，必须立即成像，而细胞仍处于振荡，祖状态。一旦细胞停止振荡，他们承担了分化细胞的命运，而当在组织，将纳入一个体节。这是宝ssible，与右因子或本体外的因素，我们可以生成PSM状斑马鱼细胞培养系，将保持振荡，使显著较长期观察，多个连续的扰动，或高通量筛选。 我们预计，准备分散细胞的原代培养的时间推移成像的这种方法非常适合于发生发展的时间框架采用稳定的斑马鱼细胞系，是不是平易近人内的任何细胞过程的研究。不同的细胞类型从相应的转基因记者线条分明的发育阶段，或者通过剥离或通过流式细胞仪的分离胚胎分离后，可以提供起始细胞。的培养条件下，通过细胞的胚胎起源引导一些优化，可能需要。通过将这种灵活和敏感的协议与迅速增长的转基因斑马鱼的集合行，我们希望能促进体外发育生物学的方法，是相辅相成的经典遗传和胚胎的方法。

周期性形成沿脊椎动物体轴或体节形成段的，是一种空间和时间振荡器在胚胎发育的证据。青睐的机制控制体节形成是由一个“时钟和波前”的模式1，其中“钟”组成细胞的振荡器，现在认为是细胞内由一组环状基因2的有节奏的表达驱动，列举了形成概念描述的体节从presomitic中胚层（PSM）。随着胚胎发育，在PSM一个成熟的“波前”演唱会中与移动朝后的回归组织，减缓和逮捕细胞振荡器，因为它传递3。一起，这时空方式动态系统被称为分割时钟。目前的方法来研究分割时钟跨度组织三个层次增加从基因振荡器在单细胞局部耦合次在细胞和最后到的位置信息在集体PSM组织4全球监管之间发生。 以往的研究表明在斑马鱼的细胞自主分割振荡器由基因和蛋白质的产品从她/ HES转录factorfamily，这被认为是通过转录抑制5-7以形成负反馈回路。三角洲/ Notch信号通路同步邻近细胞之间振荡和调节人口的8-10集体经济时期。在tailbud产生FGF信号分子似乎建立在整个斑马鱼PSM的梯度，并由此推测有助于在前11趋缓和逮捕振荡细胞。到现在为止，这些分子在体节形成中的功能作用进行了研究基因突变，吗啉注射液，热休克过度表达，并拮抗药珍稀tment时钟部件和细胞5,7,10,12之间的信令。使用这些扰动，分割时钟功能已推断从体节缺陷的组织级别描述与均匀振荡的循环基因像HER1，her7，和 delta表达的损失C。然而，几乎所有这些数据都是从固定的胚胎，并不能准确地捕捉所固有的分割时钟的动态函数的变化。最近，多胚胎延时成像揭示具有改变振荡器的周期的第一个突变体，但这些观察结果也使在组织水平7,13。因此，体节形成过程中假设的细胞自主振荡器的行为没有被观察到。 体节形成的静态快照给一个不完整的情况，因为，本质上，这个过程是由一个振荡系统驱动。在小鼠和鸡胚细胞早期的工作表明，转录本水平和蛋白质兴衰，但取样约2小时振荡每30或45分钟一定限制收集到的数据，因此可以得出14,15的结论。研究其他生物振荡器，最值得注意的是，生物钟，已经远离基因和蛋白表达的阶段性测量，使用荧光灯和发光记者16,17实时监控。这些工具对于展示单细胞18时钟性能是必要的。该HES1基因循环的生物发光性报告已经制定并简要特征单一的鼠标PSM细胞19。平均周期和方差计算了一个小数目的细胞，显示出振荡持续几个周期体外 。然而，这些研究并没有定量地解决振荡器的频率和振幅的稳定性和鲁棒性，细胞是否能自发地进入或退出振荡以及如何将细胞维持其相位关系。此外，在胚胎上发现的细胞自主的时钟信号分子的影响并未直接测试。因此，单细胞振荡器的这些基本性能保持完全陌生的。 我们最近开发利用BAC重组工程20驱动金星（YFP）荧光HER1表达30记者转基因鱼线。这样的行利用完整的染色体位点中，它们被嵌入和概括的时空动态和HER1空间格局的监管线索。这一突破使得基因表达的体内显像斑马鱼胚胎的实时监控。为了研究细胞自主振荡的基本性质和如何这样表达的调节时间的推移，我们最近开发出一种可靠的方法，从PSM细胞在体外分离和记录。这个原山坳介绍了我们如何利用我们的转基因报告线产生分散的细胞培养，从中我们可以在单细胞表征斑马鱼分割时钟的振荡。我们可以借此解决该领域悬而未决的问题是，不是用静态或组织层面的分析访问，以及直接与信号分子和抑制剂操纵分割时钟在单细胞水平。

<table border="1">
 <tbody>
 <tr>
 <td>Name of Reagent/Material</td>
 <td>Company</td>
 <td>Catalog Number</td>
 <td>Comments</td>
 </tr>
 <tr>
 <td>Epifluorescence microscope</td>
 <td>Olympus</td>
 <td>Model: SZX16</td>
 <td ></td>
 </tr>
 <tr>
 <td>Epifluorescence microscope</td>
 <td>X-cite Illumination</td>
 <td>Series: 120Q</td>
 <td ></td>
 </tr>
 <tr>
 <td>Dissection microscope</td>
 <td>Olympus</td>
 <td>Model: SZX12</td>
 <td></td>
 </tr>
 <tr>
 <td>Fine forceps no. 55</td>
 <td>Fine Science Tools</td>
 <td>11295-51</td>
 <td></td>
 </tr>
 <tr>
 <td>Glass transfer pipettes</td>
 <td>Assistent</td>
 <td>567/2</td>
 <td></td>
 </tr>
 <tr>
 <td>35 mm plastic petri dishes</td>
 <td>Greiner</td>
 <td>627102</td>
 <td></td>
 </tr>
 <tr>
 <td>60 mm plastic petri dishes</td>
 <td>Greiner</td>
 <td>628102</td>
 <td></td>
 </tr>
 <tr>
 <td>Sylgard polymer</td>
 <td>SASCO</td>
 <td>266727</td>
 <td></td>
 </tr>
 <tr>
 <td>Manipulation tools</td>
 <td>Made in-house</td>
 <td></td>
 <td>For description of manipulation tools Ref. 22</td>
 </tr>
 <tr>
 <td>Microsurgical knife</td>
 <td>World Precision Instruments</td>
 <td>500249</td>
 <td></td>
 </tr>
 <tr>
 <td>L15 medium</td>
 <td>Invitrogen</td>
 <td>57322</td>
 <td></td>
 </tr>
 <tr>
 <td>Penicillin/streptomyocin</td>
 <td>PAA</td>
 <td>P11-010</td>
 <td></td>
 </tr>
 <tr>
 <td>Fetal bovine serum</td>
 <td>Invitrogen</td>
 <td>257322</td>
 <td></td>
 </tr>
 <tr>
 <td>0.05% trypsin / 0.02% EDTA</td>
 <td>PAA </td>
 <td>L11-004</td>
 <td></td>
 </tr>
 <tr>
 <td>Gel loading tips</td>
 <td>Fisher Scientific</td>
 <td>253188</td>
 <td></td>
 </tr>
 <tr>
 <td>Sigmacote</td>
 <td>Sigma Aldrich</td>
 <td>254589</td>
 <td></td>
 </tr>
 <tr>
 <td>Micropipette set</td>
 <td>Gilson International</td>
 <td>F167300</td>
 <td></td>
 </tr>
 <tr>
 <td>Zebrafish Fibronectin1 70 kD fragment</td>
 <td>MPI-CBG protein facility</td>
 <td></td>
 <td>Generated in-house from construct based on previously published work Ref. 23-25</td>
 </tr>
 <tr>
 <td>Glass bottom imaging dishes</td>
 <td>Mattek (single well)</td>
 <td>P35G-1.5-14-C</td>
 <td></td>
 </tr>
 <tr>
 <td>Glass bottom imaging dishes</td>
 <td>Greiner (CellView -multi-well)</td>
 <td>262502</td>
 <td></td>
 </tr>
 <tr>
 <td>E3 medium without methylene blue</td>
 <td>Made in-house</td>
 <td></td>
 <td>From The Zebrafish Book, 5th Ed. Ref. 26 </td>
 </tr>
 <tr>
 <td>Plastic transfer pipettes</td>
 <td>Ratiolab</td>
 <td>260011</td>
 <td></td>
 </tr>
 <tr>
 <td>Warner heating/cooling chamber</td>
 <td>Warner Instruments</td>
 <td>TC-324B/344B</td>
 <td></td>
 </tr>
 <tr>
 <td>EM-CCD camera</td>
 <td>Andor</td>
 <td>Model: iXOn 888</td>
 <td></td>
 </tr>
 <tr>
 <td>Wide-field fluorescence microscope with Venus filter set</td>
 <td>Zeiss</td>
 <td>Model: Axiovert 200M</td>
 <td></td>
 </tr>
 <tr>
 <td>Wide-field fluorescence microscope with Venus filter set</td>
 <td></td>
 <td>NeoFluor 40x, NA 0.75</td>
 <td></td>
 </tr>
 <tr>
 <td>Wide-field fluorescence microscope with Venus filter set</td>
 <td>Lumencor Light Engine</td>
 <td>Model: Spectra X</td>
 <td></td>
 </tr>
 <tr>
 <td>Wide-field fluorescence microscope with Venus filter set</td>
 <td></td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Wide-field fluorescence microscope with Venus filter set</td>
 <td>BrightLine HC 575/15</td>
 <td>F39-575</td>
 <td></td>
 </tr>
 </tbody>
</table>

generation of dispersed presomitic mesoderm cell cultures for imaging of the zebrafish segmentation clock in single cells

分割是在脊椎动物中的周期性和顺序形态发生过程。这个节奏形成组织的块称为体节沿身体轴线是遗传振荡器图案胚胎发育的证据。在斑马鱼中，细胞内的时钟驱动的分割是由组织成负反馈回路的她/住户开支统计调查的转录因子家族的成员。我们最近生成的环状基因HER1该概括振荡在presomitic中胚层（PSM）的空间-时间模式的转基因荧光报告线路。使用这些线，我们开发了一种在体外培养系统，它允许单个孤立的PSM细胞内的分割时钟振荡的实时分析。从转基因胚胎去除PSM组织，然后从振荡区域到玻璃底菜的分散细胞，我们产生适于荧光信号从延时成像培养个人生物钟细胞。这种方法提供了实验和概念框架的直接操纵分割时钟以前所未有的单细胞分辨率，允许其细胞自治和组织级属性加以区别和解剖。

这个协议产生可行的，分散的，单PSM细胞的培养物为荧光信号的延时成像（ 图2）。我们的转基因产生一个记者会发生相似的动力学的内源基因和蛋白在胚胎中，一个半小时的量级，其周期的生产和退化。由于其快速周转，在单细胞YFP的信号，应迅速检测，以尽量减少漂白和具有高时间分辨率来捕捉每一个振荡周期的特点。此外，考虑到信号的相对暗淡，文化和采集条件都经过精心调整，以确保敏感而稳定的结果。我们发现以下因素在产生最佳的PSM细胞培养成像是重要的：（1）用于涂覆玻璃底培养皿的细胞外基质基质。 2，加入血清的解剖和成像介质。 PSM 3。从交配HETE后所采取的确定胚胎解剖rozygous对，他们的后代可能携带2份转基因的。最佳放大倍数范围内4。图像采集，使用较高的NA物镜，以确保荧光信号的有效捕获。 5，照明与固态光源，以减少强度的波动，可能导致背景噪音。 6，信号检测具有高度灵敏的EM-CCD照相机以最大化信号的读出。 次优的培养将含有细胞不能保持圆形和健康整个记录。我们假设，我们的细胞外基质衬底，斑马鱼fibronectin1的片段保持细胞在未分化的PSM状态，相对于其他常用的基片一样多聚赖氨酸或层粘连蛋白。我们测试了其他底物引起的细胞变平放在玻璃板上并振荡荧光信号的损失在记录的过程中。我们还发现，加入血清的培养基中剥离时，扩散，和记录不仅是重要的淬火用于解离的胰蛋白酶，同时也增加了荧光强度超过背景，可能是由于改善细胞活力。为了确保最佳的信号捕获从单细胞，我们使用专门用于荧光成像（蔡司计划NeoFluor系列）设计了一个4​​0×物镜具有高数值孔径。我们还发现，固态光源比传统的汞灯，这是至关重要的，以减少背景波动，有助于噪声图像提供更稳定的照明。这些修改都是重要的，以确保稳定的结果。 使用此协议，我们期待的文化当中，多数细胞在某一领域是在录制过程中荧光在一些点。我们发现，荧光细胞在录音通常保持圆润，有时是相当能动。在录制过程中的一些细胞，包括荧光细胞，有可能成为细胞凋亡。这些细胞被排除在任何一个nalysis。我们还排除细胞，成与其他细胞接触或视场的外移动。平均来说，我们看到在每场的细胞数减少了10小时记录结束12％通过计数在透射光信道的健康细胞的数量来衡量。这种损失包括细胞死亡和迁出领域的细胞。鉴于这些警告，我们可以，平均每场的轨道5的荧光细胞保持活力和可见的，通常记录每个条件6场。例如，实验4的条件将有获得24总场，通常每个条件大约30跟踪的细胞。在标准成像条件用含有10％胎牛血清的L15培养基中，我们发现，PSM细胞（N = 101细胞从4个实验重复）可以在2和7之间的峰产生，以3±1的峰（2的平均值和标准偏差-3个周期）。平均峰值编号，以及25％百分位数，是2峰和75％百分位数我第3峰。使用我们的半自动化的跟踪和分析，我们可以快速生成荧光强度比单个细胞的PSM岁月的痕迹，用图2C所示的代表性细胞痕迹。这些原料的痕迹可以被用来使振荡PSM细胞，如频率，振幅，周期数，并且峰的定时性能的定量测量。 <img alt="图1" fo:content-width="4in" fo:src="/files/ftp_upload/50307/50307fig1highres.jpg" src="/files/ftp_upload/50307/50307fig1.jpg" /> 转基因斑马鱼胚胎表达YFP的荧光在〜5体节期的图1。鉴定其解剖转基因胚胎和原理图（A）横向视图。图片是透射光和荧光通道的叠加。箭头表示信号的开始在tailbud的尖端区域，在整个PSM，朝升<html> AST形成体节。插图显示了报告基因的示意图（关于其发展和体内行为的详细信息，请参阅Soroldoni 等 30。前剥离时先切断胚胎侧面观（二）示意图。红色虚线表示的首次下调整个后脑做，虽然卵黄细胞仅次于tailbud扁平的PSM以下去除卵黄颗粒和表皮层，沿其前后轴导向的（C）示意图。红色虚线表示第二切口去除尖端该tailbud为传播和文化。 <a href="https://www.jove.com/files/ftp_upload/50307/50307fig1highres.jpg" target="_blank">请点击这里查看这个数字的放大版本。</a> 07fig2.jpg“/&gt; 图2。从一个分散的PSM文化的单个单元格代表的图像和轨迹（A）蒙太奇在6 h的时间间隔录制发送一个PSM细胞的光图像。注意，该细胞保持圆形的和健康的整个记录过程（B）相应的荧光图像的蒙太奇从单一的PSM细胞。在细胞中的强度峰的编号在记录的过程中（C）的平均强度随着时间的推移从一个ROI放置在该小区测量。值取自从动画中的每一帧，每使用写在斐济圆插补器插件和自定义宏2分之一的帧速率。在强度峰再次编号在整个跟踪。 <a href="https://www.jove.com/files/ftp_upload/50307/50307fig2highres.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about Generation of Dispersed Presomitic Mesoderm Cell Cultures for Imaging of the Zebrafish Segmentation Clock in Single Cells at JoVE.com

Generation of Dispersed Presomitic Mesoderm Cell Cultures for Imaging of the Zebrafish Segmentation Clock in Single Cells

体节形成是有节奏的发育过程，从而在空间型态脊椎动物胚胎的体轴。以前，我们开发了使用荧光记者观察环状基因，推动这一进程的转基因斑马鱼线。在这里，我们培养从这些线和图像的振荡时间的推移在体外分散细胞。

的分散Presomitic中胚层细胞培养的斑马鱼分割时钟在单细胞成像代

Segmentation is a  periodic and sequential morphogenetic process in vertebrates. This rhythmic  formation of blocks of tissue called somites along the ...

generation-dispersed-presomitic-mesoderm-cell-cultures-for-imaging

Research

JoVE Journal

Biology

13.6K 次观看.  Max Planck Institute of Molecular Cell Biology and Genetics. 体节形成是有节奏的发育过程，从而在空间型态脊椎动物胚胎的体轴。以前，我们开发了使用荧光记者观察环状基因，推动这一进程的转基因斑马鱼线。在这里，我们培养从这些线和图像的振荡时间的推移在体外分散细胞。 

视频: Generation of Dispersed Presomitic Mesoderm Cell Cultures for Imaging of the Zebrafish Segmentation Clock in Single Cells

Single Cell Fate Mapping in Zebrafish

The ability to differentially label single cells has important implications in developmental biology. For instance, determining how hematopoietic, lymphatic, and blood vessel lineages arise in developing embryos requires fate mapping and lineage tracing of undifferentiated precursor cells. Recently, photoactivatable proteins which include: Eos1, 2, PAmCherry3, Kaede4-7, pKindling8, and KikGR9, 10 have received wide interest as cell tracing probes. The fluorescence spectrum of these photosensitive proteins can be easily converted with UV excitation, allowing a population of cells to be distinguished from adjacent ones. However, the photoefficiency of the activated protein may limit long-term cell tracking11. As an alternative to photoactivatable proteins, caged fluorescein-dextran has been widely used in embryo model systems7, 12-14. Traditionally, to uncage fluorescein-dextran, UV excitation from a fluorescence lamp house or a single photon UV laser has been used; however, such sources limit the spatial resolution of photoactivation. Here we report a protocol to fate map, lineage trace, and detect single labeled cells. Single cells in embryos injected with caged fluorescein-dextran are photoactivated with near-infrared laser pulses produced from a titanium sapphire femtosecond laser. This laser is customary in all two-photon confocal microscopes such as the LSM 510 META NLO microscope used in this paper. Since biological tissue is transparent to near-infrared irradiation15, the laser pulses can be focused deep within the embryo without uncaging cells above or below the selected focal plane. Therefore, non-linear two-photon absorption is induced only at the geometric focus to uncage fluorescein-dextran in a single cell. To detect the cell containing uncaged fluorescein-dextran, we describe a simple immunohistochemistry protocol16 to rapidly visualize the activated cell. The activation and detection protocol presented in this paper is versatile and can be applied to any model system. 
Note: The reagents used in this protocol can be found in the table appended at the end of the article.

A method is described to photoactivate single cells containing a caged fluorescent protein using two-photon absorption from a Ti:Sapphire femtosecond laser oscillator. To fate map the photoactivated cell, immunohistochemistry is used. This technique can be applied to any cell type.

单细胞在斑马鱼的命运映射

The ability to differentially label single cells has important implications in developmental biology. For instance, determining how hematopoietic, ...

科研

生物学

In vivo Electroporation of Morpholinos into the Adult Zebrafish Retina

Many devastating inherited eye diseases result in progressive and irreversible blindness because humans cannot regenerate dying or diseased retinal neurons. In contrast, the adult zebrafish retina possesses the robust ability to spontaneously regenerate any neuronal class that is lost in a variety of different retinal damage models, including retinal puncture, chemical ablation, concentrated high temperature, and intense light treatment 1-8. Our lab extensively characterized regeneration of photoreceptors following constant intense light treatment and inner retinal neurons after intravitreal ouabain injection 2, 5, 9. In all cases, resident M&uuml;ller glia re-enter the cell cycle to produce neuronal progenitors, which continue to proliferate and migrate to the proper retinal layer, where they differentiate into the deficient neurons. 
	We characterized five different stages during regeneration of the light-damaged retina that were highlighted by specific cellular responses. We identified several differentially expressed genes at each stage of retinal regeneration by mRNA microarray analysis 10. Many of these genes are also critical for ocular development. To test the role of each candidate gene/protein during retinal regeneration, we needed to develop a method to conditionally limit the expression of a candidate protein only at times during regeneration of the adult retina. 
	Morpholino oligos are widely used to study loss of function of specific proteins during the development of zebrafish, Xenopus, chick, mouse, and tumors in human xenografts 11-14. These modified oligos basepair with complementary RNA sequence to either block the splicing or translation of the target RNA. Morpholinos are stable in the cell and can eliminate or "knockdown" protein expression for three to five days 12. 
	Here, we describe a method to efficiently knockdown target protein expression in the adult zebrafish retina. This method employs lissamine-tagged antisense morpholinos that are injected into the vitreous of the adult zebrafish eye. Using electrode forceps, the morpholino is then electroporated into all the cell types of the dorsal and central retina. Lissamine provides the charge on the morpholino for electroporation and can be visualized to assess the presence of the morpholino in the retinal cells. 
	Conditional knockdown in the retina can be used to examine the role of specific proteins at different times during regeneration. Additionally, this approach can be used to study the role of specific proteins in the undamaged retina, in such processes as visual transduction and visual processing in second order neurons.

In vivo Electroporation of Morpholinos into the Adult Zebrafish Retina

A method to conditionally knockdown a target protein&#x2019;s expression in the adult zebrafish retina is described, which involves intravitreally injecting antisense morpholinos and electroporating them into the retina. The resulting protein is knocked down for several days, which allows testing the protein&#x2019;s role in the regenerating or intact retina.

在体内电穿孔Morpholinos到成年斑马鱼视网膜

Many devastating inherited eye diseases result in progressive and irreversible blindness because humans cannot regenerate dying or diseased retinal ...

In vivo Electroporation of Morpholinos into the Regenerating Adult Zebrafish Tail Fin

Certain species of urodeles and teleost fish can regenerate their tissues. Zebrafish have become a widely used model to study the spontaneous regeneration of adult tissues, such as the heart1, retina2, spinal cord3, optic nerve4, sensory hair cells5, and fins6.
The zebrafish fin is a relatively simple appendage that is easily manipulated to study multiple stages in epimorphic regeneration. Classically, fin regeneration was characterized by three distinct stages: wound healing, blastema formation, and fin outgrowth. After amputating part of the fin, the surrounding epithelium proliferates and migrates over the wound. At 33 &deg;C, this process occurs within six hours post-amputation (hpa, Figure 1B)6,7. Next, underlying cells from different lineages (ex. bone, blood, glia, fibroblast) re-enter the cell cycle to form a proliferative blastema, while the overlying epidermis continues to proliferate (Figure 1D)8. Outgrowth occurs as cells proximal to the blastema re-differentiate into their respective lineages to form new tissue (Figure 1E)8. Depending on the level of the amputation, full regeneration is completed in a week to a month.
The expression of a large number of gene families, including wnt, hox, fgf, msx, retinoic acid, shh, notch, bmp, and activin-betaA genes, is up-regulated during specific stages of fin regeneration9-16. However, the roles of these genes and their encoded proteins during regeneration have been difficult to assess, unless a specific inhibitor for the protein exists13, a temperature-sensitive mutant exists or a transgenic animal (either overexpressing the wild-type protein or a dominant-negative protein) was generated7,12. We developed a reverse genetic technique to quickly and easily test the function of any gene during fin regeneration.
Morpholino oligonucleotides are widely used to study loss of specific proteins during zebrafish, Xenopus, chick, and mouse development17-19. Morpholinos basepair with a complementary RNA sequence to either block pre-mRNA splicing or mRNA translation. We describe a method to efficiently introduce fluorescein-tagged antisense morpholinos into regenerating zebrafish fins to knockdown expression of the target protein. The morpholino is micro-injected into each blastema of the regenerating zebrafish tail fin and electroporated into the surrounding cells. Fluorescein provides the charge to electroporate the morpholino and to visualize the morpholino in the fin tissue.
This protocol permits conditional protein knockdown to examine the role of specific proteins during regenerative fin outgrowth. In the Discussion, we describe how this approach can be adapted to study the role of specific proteins during wound healing or blastema formation, as well as a potential marker of cell migration during blastema formation.

In vivo Electroporation of Morpholinos into the Regenerating Adult Zebrafish Tail Fin

We describe a method to conditionally knockdown the expression of a target protein during adult zebrafish fin regeneration. This technique involves micro-injecting and electroporating antisense oligonucleotide morpholinos into fin tissue, which allows testing the protein&#x2019;s role in various stages of fin regeneration, including wound healing, blastema formation, and regenerative outgrowth.

再生的成年斑马鱼尾鳍在吗啉代入体内电穿孔

Certain species of urodeles and teleost fish can regenerate their tissues. Zebrafish have become a widely used model to study the spontaneous regeneration ...

Generation of Mice Derived from Induced Pluripotent Stem Cells

The production of induced pluripotent stem cells (iPSCs) from somatic cells provides a means to create valuable tools for basic research and may also produce a source of patient-matched cells for regenerative therapies. iPSCs may be generated using multiple protocols and derived from multiple cell sources. Once generated, iPSCs are tested using a variety of assays including immunostaining for pluripotency markers, generation of three germ layers in embryoid bodies and teratomas, comparisons of gene expression with embryonic stem cells (ESCs) and production of chimeric mice with or without germline contribution2. Importantly, iPSC lines that pass these tests still vary in their capacity to produce different differentiated cell types2. This has made it difficult to establish which iPSC derivation protocols, donor cell sources or selection methods are most useful for different applications.
The most stringent test of whether a stem cell line has sufficient developmental potential to generate all tissues required for survival of an organism (termed full pluripotency) is tetraploid embryo complementation (TEC)3-5. Technically, TEC involves electrofusion of two-cell embryos to generate tetraploid (4n) one-cell embryos that can be cultured in vitro to the blastocyst stage6. Diploid (2n) pluripotent stem cells (e.g. ESCs or iPSCs) are then injected into the blastocoel cavity of the tetraploid blastocyst and transferred to a recipient female for gestation (see Figure 1). The tetraploid component of the complemented embryo contributes almost exclusively to the extraembryonic tissues (placenta, yolk sac), whereas the diploid cells constitute the embryo proper, resulting in a fetus derived entirely from the injected stem cell line.
Recently, we reported the derivation of iPSC lines that reproducibly generate adult mice via TEC1. These iPSC lines give rise to viable pups with efficiencies of 5-13%, which is comparable to ESCs3,4,7 and higher than that reported for most other iPSC lines8-12. These reports show that direct reprogramming can produce fully pluripotent iPSCs that match ESCs in their developmental potential and efficiency of generating pups in TEC tests. At present, it is not clear what distinguishes between fully pluripotent iPSCs and less potent lines13-15. Nor is it clear which reprogramming methods will produce these lines with the highest efficiency. Here we describe one method that produces fully pluripotent iPSCs and "all- iPSC" mice, which may be helpful for investigators wishing to compare the pluripotency of iPSC lines or establish the equivalence of different reprogramming methods.

Generating induced pluripotent stem cell (iPSC) lines produces lines of differing developmental potential even when they pass standard tests for pluripotency. Here we describe a protocol to produce mice derived entirely from iPSCs, which defines the iPSC lines as possessing full pluripotency1.

诱导多能干细胞来源于小鼠的生成

The production of induced pluripotent stem cells (iPSCs) from somatic cells provides a means to create valuable tools for basic research and may also ...

Monitoring Cell-autonomous Circadian Clock Rhythms of Gene Expression Using Luciferase Bioluminescence Reporters

In mammals, many aspects of behavior and physiology such as sleep-wake cycles and liver metabolism are regulated by endogenous circadian clocks (reviewed1,2). The circadian time-keeping system is a hierarchical multi-oscillator network, with the central clock located in the suprachiasmatic nucleus (SCN) synchronizing and coordinating extra-SCN and peripheral clocks elsewhere1,2. Individual cells are the functional units for generation and maintenance of circadian rhythms3,4, and these oscillators of different tissue types in the organism share a remarkably similar biochemical negative feedback mechanism. However, due to interactions at the neuronal network level in the SCN and through rhythmic, systemic cues at the organismal level, circadian rhythms at the organismal level are not necessarily cell-autonomous5-7. Compared to traditional studies of locomotor activity in vivo and SCN explants ex vivo, cell-based in vitro assays allow for discovery of cell-autonomous circadian defects5,8. Strategically, cell-based models are more experimentally tractable for phenotypic characterization and rapid discovery of basic clock mechanisms5,8-13.
Because circadian rhythms are dynamic, longitudinal measurements with high temporal resolution are needed to assess clock function. In recent years, real-time bioluminescence recording using firefly luciferase as a reporter has become a common technique for studying circadian rhythms in mammals14,15, as it allows for examination of the persistence and dynamics of molecular rhythms. To monitor cell-autonomous circadian rhythms of gene expression, luciferase reporters can be introduced into cells via transient transfection13,16,17 or stable transduction5,10,18,19. Here we describe a stable transduction protocol using lentivirus-mediated gene delivery. The lentiviral vector system is superior to traditional methods such as transient transfection and germline transmission because of its efficiency and versatility: it permits efficient delivery and stable integration into the host genome of both dividing and non-dividing cells20. Once a reporter cell line is established, the dynamics of clock function can be examined through bioluminescence recording. We first describe the generation of P(Per2)-dLuc reporter lines, and then present data from this and other circadian reporters. In these assays, 3T3 mouse fibroblasts and U2OS human osteosarcoma cells are used as cellular models. We also discuss various ways of using these clock models in circadian studies. Methods described here can be applied to a great variety of cell types to study the cellular and molecular basis of circadian clocks, and may prove useful in tackling problems in other biological systems.

Circadian clocks function within individual cells, i.e., they are cell-autonomous. Here, we describe methods for generating cell-autonomous clock models using non-invasive, luciferase-based real-time bioluminescence technology. Reporter cells provide tractable, functional model systems for studying circadian biology.

监测细胞自主的生物钟节律基因表达，利用荧光素酶生物发光记者

In mammals, many aspects of behavior and physiology such as sleep-wake cycles and liver metabolism are regulated by endogenous circadian clocks ...

Live Imaging of Apoptotic Cell Clearance during Drosophila Embryogenesis

The proper elimination of unwanted or aberrant cells through apoptosis and subsequent phagocytosis (apoptotic cell clearance) is crucial for normal development in all metazoan organisms. Apoptotic cell clearance is a highly dynamic process intimately associated with cell death; unengulfed apoptotic cells are barely seen in vivo under normal conditions. In order to understand the different steps of apoptotic cell clearance and to compare 'professional' phagocytes - macrophages and dendritic cells to 'non-professional' - tissue-resident neighboring cells, in vivo live imaging of the process is extremely valuable. Here we describe a protocol for studying apoptotic cell clearance in live Drosophila embryos. To follow the dynamics of different steps in phagocytosis we use specific markers for apoptotic cells and phagocytes. In addition, we can monitor two phagocyte systems in parallel: 'professional' macrophages and 'semi-professional' glia in the developing central nervous system (CNS). The method described here employs the Drosophila embryo as an excellent model for real time studies of apoptotic cell clearance.

Live Imaging of Apoptotic Cell Clearance during Drosophila Embryogenesis

Here we describe an effective method for studying dynamics of apoptotic cell clearance in vivo. This method employs live Drosophila embryos as a powerful model for monitoring phagocytosis of apoptotic cells using specific labeling of apoptotic cells and phagocytes.

凋亡细胞清除过程中实时成像<em&gt;果蝇</em&gt;胚胎发育

The  proper elimination of unwanted or aberrant cells through apoptosis and  subsequent phagocytosis (apoptotic cell clearance) is crucial for normal  ...

Efficient Generation Human Induced Pluripotent Stem Cells from Human Somatic Cells with Sendai-virus

A few years ago, the establishment of human induced pluripotent stem cells (iPSCs) ushered in a new era in biomedicine. Potential uses of human iPSCs include modeling pathogenesis of human genetic diseases, autologous cell therapy after gene correction, and personalized drug screening by providing a source of patient-specific and symptom relevant cells. However, there are several hurdles to overcome, such as eliminating the remaining reprogramming factor transgene expression after human iPSCs production. More importantly, residual transgene expression in undifferentiated human iPSCs could hamper proper differentiations and misguide the interpretation of disease-relevant in vitro phenotypes. With this reason, integration-free and/or transgene-free human iPSCs have been developed using several methods, such as adenovirus, the piggyBac system, minicircle vector, episomal vectors, direct protein delivery and synthesized mRNA. However, efficiency of reprogramming using integration-free methods is quite low in most cases.
Here, we present a method to isolate human iPSCs by using Sendai-virus (RNA virus) based reprogramming system. This reprogramming method shows consistent results and high efficiency in cost-effective manner.

Here, we present our established method to reprogram human somatic cells into transgene-free human iPSCs with Sendai virus, which shows consistent outcome and enhanced efficiency.

从人类体细胞与仙台病毒高效的新一代人类诱导多能干细胞

A few years ago, the establishment of human induced pluripotent stem cells (iPSCs) ushered in a new era in biomedicine. Potential uses of human iPSCs ...

Nephrotoxin Microinjection in Zebrafish to Model Acute Kidney Injury

The kidneys are susceptible to harm from exposure to chemicals they filter from the bloodstream. This can lead to organ injury associated with a rapid decline in renal function and development of the clinical syndrome known as acute kidney injury (AKI). Pharmacological agents used to treat medical circumstances ranging from bacterial infection to cancer, when administered individually or in combination with other drugs, can initiate AKI. Zebrafish are a useful animal model to study the chemical effects on renal function in vivo, as they form an embryonic kidney comprised of nephron functional units that are conserved with higher vertebrates, including humans. Further, zebrafish can be utilized to perform genetic and chemical screens, which provide opportunities to elucidate the cellular and molecular facets of AKI and develop therapeutic strategies such as the identification of nephroprotective molecules. Here, we demonstrate how microinjection into the zebrafish embryo can be utilized as a paradigm for nephrotoxin studies.

Renal injuries incurred from nephrotoxins, which include drugs ranging from antibiotics to chemotherapeutics, can result in complex disorders whose pathogenesis remains incompletely understood. This protocol demonstrates how zebrafish can be used for disease modeling of these conditions, which can be applied to the identification of renoprotective measures.

肾毒素显微注射斑马鱼到模型急性肾损伤

The kidneys are susceptible to harm from exposure to chemicals they filter from the bloodstream. This can lead to organ injury associated with a rapid ...

Analysis of Beta-cell Function Using Single-cell Resolution Calcium Imaging in Zebrafish Islets

Pancreatic beta-cells respond to increasing blood glucose concentrations by secreting the hormone insulin. The dysfunction of beta-cells leads to hyperglycemia and severe, life-threatening consequences. Understanding how the beta-cells operate under physiological conditions and what genetic and environmental factors might cause their dysfunction could lead to better treatment options for diabetic patients. The ability to measure calcium levels in beta-cells serves as an important indicator of beta-cell function, as the influx of calcium ions triggers insulin release. Here we describe a protocol for monitoring the glucose-stimulated calcium influx in zebrafish beta-cells by using GCaMP6s, a genetically encoded sensor of calcium. The method allows monitoring the intracellular calcium dynamics with single-cell resolution in ex vivo mounted islets. The glucose-responsiveness of beta-cells within the same islet can be captured simultaneously under different glucose concentrations, which suggests the presence of functional heterogeneity among zebrafish beta-cells. Furthermore, the technique provides high temporal and spatial resolution, which reveals the oscillatory nature of the calcium influx upon glucose stimulation. Our approach opens the doors to use the zebrafish as a model to investigate the contribution of genetic and environmental factors to beta-cell function and dysfunction.

Beta-cell functionality is important for blood-glucose homeostasis, which is evaluated at single-cell resolution using a genetically encoded reporter for calcium influx.

斑马鱼胰岛单细胞分辨率钙成像对β细胞功能的分析

Pancreatic beta-cells respond to increasing blood glucose concentrations by secreting the hormone insulin. The dysfunction of beta-cells leads to ...

Probing mRNA Kinetics in Space and Time in Escherichia coli using Two-Color Single-Molecule Fluorescence In Situ Hybridization

Single-molecule fluorescence in situ hybridization (smFISH) allows for counting the absolute number of mRNAs in individual cells. Here, we describe an application of smFISH to measure the rates of transcription and mRNA degradation in Escherichia coli. As smFISH is based on fixed cells, we perform smFISH at multiple time points during a time-course experiment, i.e., when cells are undergoing synchronized changes upon induction or repression of gene expression. At each time point, sub-regions of an mRNA are spectrally distinguished to probe transcription elongation and premature termination. The outcome of this protocol also allows for analyzing intracellular localization of mRNAs and heterogeneity in mRNA copy numbers among cells. Using this protocol many samples (~50) can be processed within 8 h, like the amount of time needed for just a few samples. We discuss how to apply this protocol to study the transcription and degradation kinetics of different mRNAs in bacterial cells.

Probing mRNA Kinetics in Space and Time in Escherichia coli using Two-Color Single-Molecule Fluorescence In Situ Hybridization

This protocol describes an application of single-molecule fluorescence in situ hybridization (smFISH) to measure the in vivo kinetics of mRNA synthesis and degradation.

利用两色单分子荧光在原位杂交中Escherichia coli探索大肠杆菌中空间和时间中的 mRNA 动力学

Single-molecule fluorescence in situ hybridization (smFISH) allows for counting the absolute number of mRNAs in individual cells. Here, we describe an ...