我们感谢陈J.提供的笼中的荧光素-葡聚糖合成协议。这项研究得到了由三月优生优育基金会奖5 - 09年78，美国心脏协会奖09BGIA2090075，美国国立卫生研究院/ NHLBI奖R01 - 01 HL107369到SS特别感谢他的显微镜协助您到C Closson。

1。笼荧光葡聚糖合成（改编自文献14。） <ol><li>准备的钠0.1 M的解决方案硼酸稀释DDH 2 O </li><li>测量10 kDa的aminodextran 4毫克，并把它添加到CMNB笼的荧光管。 </li><li>加入500μL准备硼酸钠溶液（步骤1.1）CMNB笼的荧光管。第30秒解散混合管和涡。 </li><li>让混合物在室温nutator过夜反应。封面混合物的接触，以防止环境光管与金属箔。 </li><li>获取一个Zeba脱盐离心柱断底帽和扭曲。马克在列上的点，这将用于东方列离心。置于15 mL锥形猎鹰管之列。注:在旋转列的解决方案是列的缓冲区。 </li><li>松开顶盖上淡化离心柱。将15 mL锥形猎鹰管房屋离心脱盐离心柱。东方自旋标记点列（步骤1.4）面临的离心机转子的中心。 1000在室温下2分钟XG的离心管。 </li><li>离心后，请从"猎鹰"管淡化离心柱。放弃通过收集到的流量和15 mL锥形管。获取一个新的15 mL锥形猎鹰管，并放置在新的Falcon管脱盐离心柱。注:列树脂床离心后，应该会出现压实。立即使用列。 </li><li>获得1.3步反应混合物（笼荧光素-葡聚糖）。取出离心柱帽，吸出反应混合物，并将其应用于慢慢淡化离心柱中心。应用反应混合物后，取代的自旋列第。 </li><li>将猎鹰管房屋离心脱盐离心柱。 1.5步，东方面临的离心机转子的中心标记点的自旋列。 1000在室温下2分钟XG的离心管。 </li><li>黄合剂（笼荧光素-葡聚糖）离心后，将收集（约400μL），15毫升猎鹰管。笼的荧光葡聚糖混合物转移到1.5 mL离心管。 </li><li>立即盖管与金属箔混合物的接触，以防止环境光线。 speedvac冻干笼荧光葡聚糖混合物。 350μL冻干应约4小时。 </li><li>完成后冻干，悬浮在DDH 2 O粉（约3 mg）至终浓度约为1％瓦特/ V。简要涡旋暂停粉混合。 </li><li>分装笼荧光葡聚糖，覆盖到单独的金属箔管，并将其储存在-20 ° C </li></ol> 2。收获的胚胎和微量注射笼荧光素-葡聚糖 *此过程使用斑马鱼作为动物模型系统。 <ol><li>在注射前一天，成立了斑马鱼杂交养殖分频器坦克无论是男性对女性或1:1 2:1男性，女性。 </li><li>第二天早晨，改变养殖鱼缸水和删除的分隔。立即准备5μL的1 / 10稀释笼荧光葡聚糖（见溶液的制备）在任1 + X Danieau媒体或DDH 2 O解决方案覆盖金属箔的解决方案，以防止暴露在光线下管工作。 </li><li>进针，移液器准备笼荧光葡聚糖溶液（步骤2.2）。解剖显微镜下将针尖在切割之前适当的大小，一块透明的黄色过滤器放置在前面的白色光源。黄色的过滤器，防止在注射自发荧光葡聚糖uncaging。当通过眼眼片，光源应该会出现黄色。 </li><li>收集胚胎显微注射模具托盘和吸管。设置显微注射时间3至4 NL提供笼荧光葡聚糖。解剖显微镜下，东方钳胚胎注入（3至4 NL）笼荧光葡聚糖的卵裂球细胞（卵裂球，蛋黄接口）的基础。注射胚胎阶段应该是1 - 2 - 细胞期。 </li><li>注射后，取出胚胎显微注射模具托盘，并放置在一个干净的培养皿包含新鲜的鱼水菜。亚甲蓝可能会增加鱼水，防止霉菌的生长，终浓度为0.02％W / V盖培养皿中，以防止金属箔笼荧光葡聚糖的注入uncaging。 </li><li>提高到一个阶段时需要被光活化细胞注射的胚胎。 photoactivation，准备在DDH 2 O 0.6％低熔点琼脂糖溶液根据胚胎阶段，Tricaine（MS222）可添加到低熔点琼脂糖。要做到这一点，淡化0.02％低熔点琼脂糖Tricaine的终浓度为0.0014％。 </li><li>只要胚胎达到适当的发展阶段，dechorionate在1 + X Danieau媒体与钳胚胎。当dechorionating，确保一个透明的黄色过滤器放在白色光源的路径（见步骤2.3）。 </li><li> 0.6％低熔点琼脂糖溶液加热至37 ° C。吸取1毫升低熔点琼脂糖成一个LabTek腔玻璃罩幻灯片以及。低熔点琼脂糖和东方他们要光敏细胞，向下面临着对底部的盖玻片钳小心地安装在3至4胚胎。 </li><li>允许琼脂糖巩固。凝固后，吸管1毫升1 × Danieau媒体通过琼脂糖。 </li><li>重复步骤2.8至2.9更多的胚胎。 </li></ol> 3。双光子激活笼荧光葡聚糖 *此过程假定胚胎表达GFP的记者。胚胎是使用氩离子（488 nm）的激光源，LSM 510 META非线性光学显微镜观看。选定一个单一的GFP阳性细胞和光敏使用耦合的LSM 510埋汰激光（飞秒激光）。的过程是相同的非荧光的胚胎。另外，白色光可以用来寻找被激活的细胞，激活使用埋汰激光源。 <ol><li>负载的LSM 510软件。白色的光束路径上放置一个透明的黄色过滤。 </li><li>选择扫描新形象，单击"开始"专家模式"。 </li><li>选择激光标签。打开氩离子（488纳米）和埋汰（飞秒）激光源。 </li><li>设置麦大波长为740纳米。 </li><li>选择配置选项卡。设置为氩离子和埋汰激光光路配置。 </li><li>选择"扫描"选项卡。改变客观20X/0.8。 </li><li>按一下最大的最大扫描速度。 </li><li>设置模式，方法，数量和线路，平均，和1。 </li><li>根据"通道"选项卡中取消选择的所有激光源除了埋汰。 </li><li>功率计放置在光束路径。 </li><li>选择连拍按钮来激活连续扫描。 </li><li>调整传输％，直到功率计读取平均激光功率为47毫瓦。 </li><li>通过选择"停止"按钮停止扫描。 </li><li>在"通道"选项卡取消选择"埋汰激光源和选择的氩离子（488纳米）。 </li><li>选择快速XY按钮。在"通道"选项卡，针孔检测器增益，放大器的失调，氩离子激光器和放大器增益调整，以达到最佳的图像质量。 </li><li>使用阶段控制器，找到并专注于细胞激活。 </li><li>点击"停止"按钮。 </li><li>使用激活细胞上的缩放工具，缩放，直到模式"标签下的缩放因子显示50至70。 </li><li>通过选择"停止"按钮停止扫描。 </li><li>根据"通道"选项卡中取消选择的氩离子激光（488纳米）和选择埋汰激光。 </li><li>在"模式"选项卡下设置，以31.46秒的扫描速度。 </li><li>选择单个按钮开始扫描笼荧光葡聚糖选定的单元格的双光子激活。 </li><li>激活后，取消选择"埋汰激光通道"选项卡下，并重新选择的氩离子激光器（488 nm）的。 </li><li>设置缩放因子，模式1下，单击标题设置扫描速度最快的速度下的最大按钮。 </li><li>选择快速XY审查光敏地区。检查，光敏地区是不是激光烧蚀激光烧蚀的更多信息，请参阅讨论部分 。 </li><li>其他胚胎重复上述步骤。 </li></ol> 4。 uncaged荧光素-葡聚糖的免疫检测（改编自文献16。） <ol><li> photoativati​​on后，放置在前面的白色光源的一个透明的黄色过滤器和使用尖锐镊子手动删除低熔点琼脂糖每个胚胎。放置在一个新的培养皿含有新鲜的1 + X Danieau媒体的菜全部拆除的胚胎。金属箔盖培养皿胚胎，以防止光线照射。 </li><li>如果需要的话，后面的胚胎发育阶段所需。在此期间，准备了4％多聚甲醛溶液（溶液的制备）。准备多聚甲醛的1.5毫升的离心管分装成。 </li><li>后胚胎已达到利益的发展阶段，吸进的离心管（步骤4.2）的胚胎。盖管与金属箔，以防止暴露在光线下。 </li><li> Nutate管一夜之间在4 ° C。为第二天准备分级在磷酸盐缓冲液（PBS）和DDH 2 O的乙醇（EtOH）解决方案; 30％乙醇:PBS，50％乙醇:PBS，70％乙醇:DDH 2 O，和100％乙醇。 </li><li>第二天，取出胚胎4 ° C。取出金属箔覆盖管和迅速吸第formaldehye（留下一个体积小，足以覆盖胚胎）。吸取1.5毫升30％乙醇:PBS管。重新覆盖金属箔管。 Nutate管在室温下10分钟。 </li><li>重复步骤4.5，使用50，70，和100％梯度乙醇解决方案。经过100％的乙醇洗，在100％乙醇冲洗胚胎，再次为10分钟。 </li><li>经过清洗，存放于-20 ° C过夜的胚胎为第二天准备磷酸盐缓冲吐温（PBT;见溶液的制备），5 ×封闭液（见制造商准备的协议），马来酸（协议作准备，可以发现在封闭液协议），10毫克/毫升蛋白酶K和一个10 ×抗体溶液（抗荧光素- AP;溶液的制备）。 </li><li>还准备2％，羔羊血清（LS溶液的制备）在PBT稀释。抗体保存在4 ° C过夜摇晃一个nutator。 2％LS可保持在4 ° C </li><li>第二天取出胚胎从-20 ° C取出金属箔覆盖管，迅速吸出100％的乙醇。管加入1.5毫升70％乙醇:DDH 2 O。重新覆盖金属箔管。 Nutate管在室温下10分钟。 </li><li>重复步骤4.8使用50和分级30％的乙醇解决方案，和100％PBT。后100％PBT洗，冲洗100％PBT的胚胎3次以上为10分钟。 </li><li>如果胚胎是24小时以上，蛋白酶K对待他们。如果没有，继续执行步骤4.13。要做到这一点，淡化准备的蛋白酶K PBT（步骤4.7）1:1000。孵育10分钟，或更长的时间，在蛋白酶K的胚胎，如果胚胎是老年人。保持与金属箔覆盖，以防止暴露在光线下的胚胎。 </li><li>蛋白酶K处理后，洗的胚胎在PBT的5倍，为10分钟。洗涤后，固定在4％多聚甲醛在室温下30分钟一个nutator的胚胎。随后，立即用10分钟的胚胎在PBT的5倍。保持与金属箔覆盖，以防止暴露在光线下的胚胎。 </li><li>马来酸缓冲液稀释5 ×封闭液1十，从PBT的取出胚胎和1 ×封闭液，封闭液。与金属箔覆盖的胚胎，并nutate在室温下5至6小时。 </li><li> 5至6小时的阻塞，热休克后的胚胎在65 ° C为15至20分钟。 </li><li>获取的抗体溶液和2％LS准备的前一天。离心机最高转速的抗体溶液。水相转移到2％LS管。倒置管组合。 </li><li>阻止缓冲区的LS -抗体混合物转移的胚胎。保持与金属箔覆盖，以防止暴露在光线下的胚胎。 Nutate胚胎在4 ° C过夜。 </li><li>第二天洗胚胎在室温下的6倍，在PBT 15分钟。准备AP缓冲区（见溶液的制备）。 </li><li>在AP缓冲液为10分钟，在室温下的2倍洗净的胚胎。准备NBT / BCIP开发解决方案（见溶液的制备）。 </li><li> NBT / BCIP显的胚胎转移。让污渍在黑暗中发展。 </li><li>洗涤胚胎在PBT的3倍，每5分钟停止发展。 </li><li>安装在0.6％的低熔点琼脂糖或3％甲基纤维素用于图像采集的胚胎，并获得使用复式显微镜倒置或直立的图像。 </li><li>光活化的样品可​​以储存，供日后分析（染色保持稳定），通过分级乙醇洗脱水。按照步骤4.4到4.7脱水样品。 </li></ol> 5。代表性的成果: <img src="/files/ftp_upload/3172/3172fig1.jpg" alt="图1"/> 图1。Photoactivation笼荧光葡聚糖。 （A，B）明场和一个侧视前photoactivati​​on 13/14-somite阶段的斑马鱼胚胎的荧光图像。 （C，D）胚胎the13/14-somite阶段光敏波长740纳米的体节（箭头），扫描时间为31秒，平均激光功率为47毫瓦。激活后，（四）从uncaged荧光葡聚糖荧光观察。 （右）方向:背，腹（左）。比例尺100微米。 <img src="/files/ftp_upload/3172/3172fig2.jpg" alt="图2"/> 图2。笼荧光免疫检测的葡聚糖。图2描绘了uncaged荧光素-葡聚糖在18/19 HPF斑马鱼胚胎的免疫。在10体节期胚胎的一个小区域被激活了在侧板中胚层，使用相同的激光参数在图1表示。使用免疫检测程序，箭头标识激活地区以最小的背景染色。方向:背（上），腹（底部）。比例尺100微米。

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H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Laser-Tissue Interactions: Fundamentals and Applications. , (2003).</li><li>Jowett, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+protein+and+gene+expression.">Analysis of protein and gene expression.</a> Methods in cell biology. 59, 63-85 (1999).</li></ol>

没有利益冲突的声明。

<html>本文介绍了一种单细胞的命运映射通用的方法的基础上笼荧光素-葡聚糖photoactivati​​on。在单细胞Uncaging笼荧光葡聚糖是使用耦合蔡司LSM 510 META激光共聚焦显微镜从埋汰飞秒激光双光子吸收。使用一个简单的免疫组化过程，在光敏细胞Uncaged荧光葡聚糖快速可视化。 在这个协议中的笼荧光葡聚糖photoactivati​​on双光子吸收波长为740纳米。实验测定波长为photoactivati​​ng笼荧光素右旋糖酐注射野生型胚胎。从600到800 nm激光激发波长不同，定性存在或没有荧光素的荧光激活后确定。我们发现，740 nm处产生荧光信号最强以下激活。理想的情况下，740 nm的所有模型系统的工作，但是，我们建议测试的波长范围内，为您的样品，以确定最优的双光子激发波长。 在笼荧光葡聚糖注射野生型胚胎中，每双光子激发波长测试，我们也各不相同的平均激光功率。对于激发波长为740纳米，平均激光功率47兆瓦在激活区域产生了较强的比较，以较低的平均激光功率的荧光信号。较高的平均激光功率不产生更强的荧光信号，但组织的诱导消融。由于飞秒激光脉冲烧蚀生物组织15的高效率，我们建议确定的阈值平均为photoactivation激光功率，以避免组织消融。 双光子吸收发生在一个小的互动量。为了确保正确的激活笼荧光葡聚糖，细胞必须被带入正确的焦点。在上述协议中，GFP阳性细胞同时成像在白光下，以确认细胞的焦点。因此，除了其他渠道的白光收购是可取的。经过确认细胞的焦点，我们的协议表明放大镜激活细胞。一个50至70的缩放因子是建议，但是，此值取决于所选择的单元格的大小。增加变焦分离从相邻的细胞激活​​细胞，并限制了双光子激活扫描区域。理想的情况下，扫描区域覆盖了大面积的细胞。 单活化细胞的检测要求是最小的背景染色。在上面的免疫检测协议，重要的是该样本是在5至6小时（步长4.13）阻塞缓冲区阻塞。当NBT / BCIP显进行开发，uncaged荧光葡聚糖应该成为可见在10至20分钟。如果背景染色是强的，我们建议一个较长的阻塞时间和额外的清洗，以去除抗体（步长4.17）。 图1和2提供代表photoactivati​​on和免疫检测结果。在图1中，早期的1 - 2 - 细胞期野生型胚胎注射笼荧光素-葡聚糖。胚胎提出了中期somitogenesis和低熔点琼脂糖安装在横向方向。图1（A，B）描绘photoactivati​​on前明和胚胎的荧光图像。胚胎仍然uncaged的证据证明荧光素的荧光图1（b）的情况下。与740 nm的波长为31秒，平均激光功率47毫瓦，图1（C;箭头）一个体节内的一个小区域被激活。激活后，双光子荧光葡聚糖uncaging发生荧光激活的区域，图1（D;箭头）所示。激活并没有伴随着激光烧蚀，somitic组织保持完好，图1（c）。图2展示了uncaged荧光葡聚糖的免疫检测。光敏外侧板中胚层的10体节阶段的胚胎中的一个小区域使用相同的激光参数在图1中提到的。使用步骤4.1通过4.20胚胎提出和处理。箭头在图2位于该地区的激活，表明由蓝色沉淀积累。只有激活的位置显然是无干扰背景染色检测。 解决方案的筹备工作: 1 + X PBT（1升） 100毫升10 × PBS 2克牛血清白蛋白 10毫升20％的吐温 10 × PBS（1升） 80克氯化钠 2克氯化钾 6.1克的Na 2 HPO 4 1.9克的KH 2 PO 4 DDH 2 O到1升 pH值至7.3 4％多聚甲醛（160毫升） 32％多聚甲醛（2个10 mL样品瓶） 8毫升20 × PBS 132毫升DDH 2 O jove_content"&gt; AP缓冲液（50毫升） 1毫升5 M氯化钠 2.5毫升1个M氯化镁2 5毫升的1 M Tris pH值9.5 250μL，20％的吐温 41.25毫升DDH 2 O 抗体溶液（1个样本） 5.5μL丙酮粉 40μL，PBT 0.8μL，LS 0.1μL的反荧光AP抗体 2％LS（1个样本360μL） 7.2μL，100％LS 352.8μL，PBT 1 + X Danieau媒体（1升）‡ 11.6毫升5 M氯化钠 700μL，1个中号氯化钾 400μL，1米硫酸镁4 600μL1米的Ca（NO 3） 2 5毫升1 M羟乙基 DDH 2 O到1升 pH值调整到7.6 ‡Danieau是一个理想的盐溶液，用于饲养dechorionated斑马鱼胚胎。可用于其他媒体，提供正确渗透压（斑马鱼〜300 mOsm）等渗解决方案 NBT股票（1毫升） 50毫克硝基四氮唑蓝 0.7毫升二甲基甲酰胺酐 0.3毫升DDH 2 O BCIP股票（1毫升） 50毫克5 - 溴-4 - 氯-3 - 吲哚基磷酸酯 1毫升二甲基甲酰胺酸酐 NBT / BCIP发展的解决方案 1毫升的AP缓冲 4.5μL的NBT股票 3.5μL，BCIP股票丙酮粉<ol><li>选择20名成年鱼和冻结他们在液氮。 </li><li>研磨与砂浆的冷冻鱼和杵成粉末。 </li><li>转移到50 mL锥形管的鱼胶粉。 </li><li>与100％的丙酮填充锥形管。涡旋管。 </li><li> 10分钟，最高转速旋转管。弃上清。 </li><li> 100％丙酮洗涤沉淀3倍和10分钟的最高转速旋转管。由最后洗的上清应该出现明确。 </li><li>再添加100％丙酮沉淀在室温让管站。更密集的粒子将落户，而将留在悬浮颗粒越细。 </li><li>倒出到一个新的管细颗粒。分装成单独的管粉末的解决方案，并并将它们存储在-20 ° C </li></ol>

<table border="1"><tbody><tr><th>试剂名称</th><th>公司</th><th>目录编号</th></tr><tr><td> CMNB笼荧光东南</td><td> Invitrogen公司</td><td> C20050 </td></tr><tr><td> 10 kDa的Aminodextran </td><td> Invitrogen公司</td><td> D1860 </td></tr><tr><td> Zeba脱盐离心柱</td><td>刺穿</td><td> 89889 </td></tr><tr><td>低熔化琼脂糖</td><td> AMRESCO </td><td> 0815 - 100G </td></tr><tr><td>硼酸钠</td><td> AMRESCO </td><td> 1131117 - 100G </td></tr><tr><td> Tricaine Methylsulfonate / MS222 </td><td>研究有机物</td><td> 1347A </td></tr><tr><td>抗荧光素- AP </td><td>罗氏公司</td><td> 11376622 </td></tr><tr><td>封闭液</td><td>罗氏应用科学</td><td> 11 096 176 001 </td></tr><tr><td>马来酸</td><td>西格玛</td><td> MO375 - 500G </td></tr><tr><td>多聚甲醛</td><td>电子显微镜科学</td><td> 15714 </td></tr><tr><td> NBT </td><td>西格玛</td><td> I8377 - 250毫克</td></tr><tr><td> BCIP </td><td>菲舍尔科学</td><td> PI - 34040 </td></tr><tr><td>微量</td><td>哈佛仪器</td><td> PLI - 2000 </td></tr><tr><td> LabTek腔玻璃罩幻灯片</td><td>国际Nalge NUNC </td><td> 155380 </td></tr><tr><td>蛋白酶K </td><td> Invitrogen公司</td><td> AM2544 </td></tr><tr><td>羔羊血清</td><td> Invitrogen公司</td><td> 16070096 </td></tr><tr><td> LSM 510 META NLO </td><td>蔡司</td><td></td></tr><tr><td>航空复消色差物镜20X 0.8 NA </td><td>蔡司</td><td></td></tr><tr><td>黄色透明过滤器</td><td>剧院"公司</td><td> Roscolux过滤器表颜色:312 </td></tr></tbody></table>

single cell fate mapping in zebrafish

的能力差异标签单细胞发育生物学的重要影响。例如，确定如何造血，淋巴管和血管的谱系出现在胚胎发育需要的命运映射和未分化的前体细胞的谱系跟踪。近日，photoactivatable蛋白质，其中包括:EOS 1，2，PAmCherry 3， 枫4-7，pKindling 8，和KikGR 9，10，收到细胞跟踪探针的广泛兴趣。可以很容易地转换这些光敏蛋白的荧光光谱与紫外线激发，使细胞人口从相邻的区分。然而，活化蛋白photoefficiency可能限制长期跟踪11细胞。笼荧光素-葡聚糖作为替代photoactivatable蛋白质，已广泛应用于胚胎的模型系统，7，12-14。传统上，从荧光灯的房子或单光子紫外激光的紫外光激发的uncage荧光葡聚糖，已被使用，然而，这样的来源限制的空间分辨率photoactivati​​on。在这里，我们报告一个协议命运的地图，谱系跟踪和检测单标记细胞。单笼荧光素-葡聚糖注射的胚胎细胞光敏钛蓝宝石飞秒激光产生的近红外激光脉冲。这种激光是在所有如本文采用LSM 510 META NLO显微镜的双光子共聚焦显微镜的习惯。由于生物组织是透明的近红外线照射15，激光脉冲可集中在胚胎深不uncaging高于或低于选定焦平面细胞。因此，非线性双光子吸收引起的，只有在几何焦点uncage在一个单细胞荧光葡聚糖。为了检测出含有荧光uncaged -葡聚糖的细胞，我们描述了一个简单的免疫组化协议16日至迅速激活细胞可视化。本文提出的激活和检测协议是通用的，可以适用于任何型号的系统。 注:在本议定书中所使用的试剂，可以发现在文章的末尾追加表。

Watch this Scientific Journal Video about Single Cell Fate Mapping in Zebrafish at JoVE.com

Single Cell Fate Mapping in Zebrafish

一种方法是描述photoactivate包含使用双光子吸收钛笼中的荧光蛋白单细胞:蓝宝石飞秒激光振荡器。命运地图的光敏细胞，免疫组化法是使用。这种技术可以应用到任何类型的细胞。

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, ...

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

13.4K 次观看.  Cincinnati Children's Hospital Medical Center. 一种方法是描述photoactivate包含使用双光子吸收钛笼中的荧光蛋白单细胞:蓝宝石飞秒激光振荡器。命运地图的光敏细胞，免疫组化法是使用。这种技术可以应用到任何类型的细胞。

视频: Single Cell Fate Mapping in Zebrafish

Transplantation of Whole Kidney Marrow in Adult Zebrafish

Hematopoietic stem cells (HSC) are a rare population of pluripotent cells that maintain all the differentiated blood lineages throughout the life of an organism. The functional definition of a HSC is a transplanted cell that has the ability to reconstitute all the blood lineages of an irradiated recipient long term. This designation was established by decades of seminal work in mammalian systems. Using hematopoietic cell transplantation (HCT) and reverse genetic manipulations in the mouse, the underlying regulatory factors of HSC biology are beginning to be unveiled, but are still largely under-explored. Recently, the zebrafish has emerged as a powerful genetic model to study vertebrate hematopoiesis. Establishing HCT in zebrafish will allow scientists to utilize the large-scale genetic and chemical screening methodologies available in zebrafish to reveal novel mechanisms underlying HSC regulation. In this article, we demonstrate a method to perform HCT in adult zebrafish. We show the dissection and preparation of zebrafish whole kidney marrow, the site of adult hematopoiesis in the zebrafish, and the introduction of these donor cells into the circulation of irradiated recipient fish via intracardiac injection. Additionally, we describe the post-transplant care of fish in an "ICU" to increase their long-term health. In general, gentle care of the fish before, during, and after the transplant is critical to increase the number of fish that will survive more than one month following the procedure, which is essential for assessment of long term (&lt;3 month) engraftment. The experimental data used to establish this protocol will be published elsewhere. The establishment of this protocol will allow for the merger of large-scale zebrafish genetics and transplant biology.

In this article, we demonstrate a method to perform HCT in adult zebrafish.

在成年斑马鱼的全肾骨髓移植

Hematopoietic stem cells (HSC) are a rare population of pluripotent cells that maintain all the differentiated blood lineages throughout the life of an ...

科研

生物学

Single-cell Suction Recordings from Mouse Cone Photoreceptors

Rod and cone photoreceptors in the retina are responsible for light detection. In darkness, cyclic nucleotide-gated (CNG) channels in the outer segment are open and allow cations to flow steadily inwards across the membrane, depolarizing the cell. Light exposure triggers the closure of the CNG channels, blocks the inward cation current flow, and thus results in cell hyperpolarization. Based on the polarity of photoreceptors, a suction recording method was developed in 1970s that, unlike the classic patch-clamp technique, does not require penetrating the plasma membrane 1. Drawing the outer segment into a tightly-fitting glass pipette filled with extracellular solution allows recording the current changes in individual cells upon test-flash exposure. However, this well-established "outer-segment-in (OS-in)" suction recording is not suitable for mouse cone recordings, because of the low percentage of cones in the mouse retina (3%) and the difficulties in identifying the cone outer segments. Recently, an inner-segment-in (IS-in) recording configuration was developed to draw the inner segment/nuclear region of the photoreceptor into the recording pipette 2,3. In this video, we will show how to record from individual mouse cone photoresponses using single-cell suction electrode.

We will show how to record flash responses from single mouse cones using a suction electrode.

单从小鼠锥感光细胞吸录音

Rod and cone photoreceptors in the retina are responsible for light detection. In darkness, cyclic nucleotide-gated (CNG) channels in the outer segment ...

A Practical Approach to Genetic Inducible Fate Mapping: A Visual Guide to Mark and Track Cells In Vivo

Fate maps are generated by marking and tracking cells in vivo to determine how progenitors contribute to specific structures and cell types in developing and adult tissue. An advance in this concept is Genetic Inducible Fate Mapping (GIFM), linking gene expression, cell fate, and cell behaviors in vivo, to create fate maps based on genetic lineage.
GIFM exploits X-CreER lines where X is a gene or set of gene regulatory elements that confers spatial expression of a modified bacteriophage protein, Cre recombinase (CreERT). CreERT contains a modified estrogen receptor ligand binding domain which renders CreERT sequestered in the cytoplasm in the absence of the drug tamoxifen. The binding of tamoxifen releases CreERT, which translocates to the nucleus and mediates recombination between DNA sequences flanked by loxP sites. In GIFM, recombination typically occurs between a loxP flanked Stop cassette preceding a reporter gene such as GFP.
Mice are bred to contain either a region- or cell type-specific CreER and a conditional reporter allele. Untreated mice will not have marking because the Stop cassette in the reporter prevents further transcription of the reporter gene. We administer tamoxifen by oral gavage to timed-pregnant females, which provides temporal control of CreERT release and subsequent translocation to the nucleus removing the Stop cassette from the reporter. Following recombination, the reporter allele is constitutively and heritably expressed. This series of events marks cells such that their genetic history is indelibly recorded. The recombined reporter thus serves as a high fidelity genetic lineage tracer that, once on, is uncoupled from the gene expression initially used to drive CreERT.
We apply GIFM in mouse to study normal development and ascertain the contribution of genetic lineages to adult cell types and tissues. We also use GIFM to follow cells on mutant genetic backgrounds to better understand complex phenotypes that mimic salient features of human genetic disorders.
This video article guides researchers through experimental methods to successfully apply GIFM. We demonstrate the method using our well characterized Wnt1-CreERT;mGFP mice by administering tamoxifen at embryonic day (E)8.5 via oral gavage followed by dissection at E12.5 and analysis by epifluorescence stereomicroscopy. We also demonstrate how to micro-dissect fate mapped domains for explant preparation or FACS analysis and dissect adult fate-mapped brains for whole mount fluorescent imaging. Collectively, these procedures allow researchers to address critical questions in developmental biology and disease models.

A Practical Approach to Genetic Inducible Fate Mapping: A Visual Guide to Mark and Track Cells In Vivo

Genetic Inducible Fate Mapping (GIFM) marks and tracks cells with fine spatial and temporal control in vivo and elucidates how cells from a specific genetic lineage contribute to developing and adult tissues. Demonstrated here are the techniques required to fate map E12.5 mouse embryos for epifluorescent and explant analysis.

一个实用的方法遗传诱导的命运映射：一个视觉引导标记和跟踪细胞在体内</em

Fate maps are generated by marking and tracking cells in vivo to determine how progenitors contribute to specific structures and cell types in developing ...

Fate Mapping of Human Embryonic Stem Cells by Teratoma Formation 

Human embryonic stem cells (hESCs) have an unlimited capacity for self-renewal, and the ability to differentiate into cells derived from all three embryonic germ layers (1). Directed differentiation of hESCs into specific cell types has generated much interest in the field of regenerative medicine (e.g., (2-5)), and methods for determining the in vivo fate of selected or manipulated hESCs are essential to this endeavor. We have adapted a highly efficient teratoma formation assay for this purpose. A small number of specifically selected hESCs is mixed with undifferentiated wild type hESCs and Phaseolus vulgaris lectin to form a cell pellet. This is grafted beneath the kidney capsule in an immunodeficient mouse. As few as 2.5 x 105 hESCs are needed to form a 16 cm3 teratoma within 8-12 weeks. The fate of the originally selected hESCs can then be determined by immunohistochemistry. This method provides a valuable tool for characterizing tissue-specific reagents for cell-based therapy.

Fate Mapping of Human Embryonic Stem Cells by Teratoma Formation

Directed differentiation of hESCs into specific cells has generated much interest in regenerative medicine. We provide a concise, step-by-step protocol for determining the in vivo fate of selected hESCs that provides a valuable tool for characterizing tissue-specific reagents for cell-based therapy.

人类胚胎干细胞畸胎瘤形成命运的映射

Human embryonic stem cells (hESCs) have an unlimited capacity for self-renewal, and the ability to differentiate into cells derived from all three ...

Single Cell Transfection in Chick Embryos

A central theme in developmental biology is the diversification of lineages and the elucidation of underlying molecular mechanisms. This entails a thorough analysis of the fates of single cells under normal and experimental conditions. To this end, transfection methods that target single progenitors are a prerequisite. We describe here a technically straightforward method for transfecting single cells in chicken tissues in-ovo, allowing reliable lineage tracing as well as genetic manipulation. Specific tissue domains are targeted within the somite or neural tube, and DNA is injected directly into the epithelium of interest, resulting in sporadic transfection of single cells. Using reporters, clonal populations may consequently be traced for up to three days, and behavior of genetically manipulated clonal populations can be compared with that of controls. This method takes advantage of the accessibility of the chick embryo along with emerging tools for genetic manipulation. We compare and discuss its advantages over the widely-used electroporation method, and possible applications and use in additional in-vivo models are also suggested. We advocate the use of this method as a significant addition and complement for existing lineage tracing and genetic interference tools.

Using fine tip micropipettes we inject plasmid DNA into subdomains of chicken somites or neural tubes. The concentration of the plasmid is adjusted to generate single transfected cells. We then allow the cells to develop into clonal populations.

单细胞转染鸡胚

A central theme in developmental biology is the diversification of lineages and the elucidation of underlying molecular mechanisms. This entails a ...

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 ...

Blastomere Explants to Test for Cell Fate Commitment During Embryonic Development

Fate maps, constructed from lineage tracing all of the cells of an embryo, reveal which tissues descend from each cell of the embryo. Although fate maps are very useful for identifying the precursors of an organ and for elucidating the developmental path by which the descendant cells populate that organ in the normal embryo, they do not illustrate the full developmental potential of a precursor cell or identify the mechanisms by which its fate is determined. To test for cell fate commitment, one compares a cell's normal repertoire of descendants in the intact embryo (the fate map) with those expressed after an experimental manipulation. Is the cell's fate fixed (committed) regardless of the surrounding cellular environment, or is it influenced by external factors provided by its neighbors? Using the comprehensive fate maps of the Xenopus embryo, we describe how to identify, isolate and culture single cleavage stage precursors, called blastomeres. This approach allows one to assess whether these early cells are committed to the fate they acquire in their normal environment in the intact embryo, require interactions with their neighboring cells, or can be influenced to express alternate fates if exposed to other types of signals.

The fate of an individual embryonic cell can be influenced by inherited molecules and/or by signals from neighboring cells. Utilizing fate maps of the cleavage stage Xenopus embryo, single blastomeres can be identified for culture in isolation to assess the contributions of inherited molecules versus cell-cell interactions.

卵裂球外植体测试胚胎发育过程中细胞命运的承诺

Fate maps, constructed from lineage tracing all of the cells of an embryo, reveal  which tissues descend from each cell of the embryo.  Although fate maps ...

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

Segmentation is a periodic and sequential morphogenetic process in vertebrates. This rhythmic formation of blocks of tissue called somites along the body axis is evidence of a genetic oscillator patterning the developing embryo. In zebrafish, the intracellular clock driving segmentation is comprised of members of the Her/Hes transcription factor family organized into negative feedback loops. We have recently generated transgenic fluorescent reporter lines for the cyclic gene her1 that recapitulate the spatio-temporal pattern of oscillations in the presomitic mesoderm (PSM). Using these lines, we developed an in vitro culture system that allows real-time analysis of segmentation clock oscillations within single, isolated PSM cells. By removing PSM tissue from transgenic embryos and then dispersing cells from oscillating regions onto glass-bottom dishes, we generated cultures suitable for time-lapse imaging of fluorescence signal from individual clock cells. This approach provides an experimental and conceptual framework for direct manipulation of the segmentation clock with unprecedented single-cell resolution, allowing its cell-autonomous and tissue-level properties to be distinguished and dissected.

Somitogenesis is a rhythmic developmental process that spatially patterns the body axis of vertebrate embryos. Previously, we developed transgenic zebrafish lines that use fluorescent reporters to observe the cyclic genes that drive this process. Here, we culture dispersed cells from these lines and image their oscillations over time in vitro.

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 ...

Identification of MyoD Interactome Using Tandem Affinity Purification Coupled to Mass Spectrometry

Skeletal muscle terminal differentiation starts with the commitment of pluripotent mesodermal precursor cells to myoblasts. These cells have still the ability to proliferate or they can differentiate and fuse into multinucleated myotubes, which maturate further to form myofibers. Skeletal muscle terminal differentiation is orchestrated by the coordinated action of various transcription factors, in particular the members of the Muscle Regulatory Factors or MRFs (MyoD, Myogenin, Myf5, and MRF4), also called the myogenic bHLH transcription factors family. These factors cooperate with chromatin-remodeling complexes within elaborate transcriptional regulatory network to achieve skeletal myogenesis. In this, MyoD is considered the master myogenic transcription factor in triggering muscle terminal differentiation. This notion is strengthened by the ability of MyoD to convert non-muscle cells into skeletal muscle cells. Here we describe an approach used to identify MyoD protein partners in an exhaustive manner in order to elucidate the different factors involved in skeletal muscle terminal differentiation. The long-term aim is to understand the epigenetic mechanisms involved in the regulation of skeletal muscle genes, i.e., MyoD targets. MyoD partners are identified by using Tandem Affinity Purification (TAP-Tag) from a heterologous system coupled to mass spectrometry (MS) characterization, followed by validation of the role of relevant partners during skeletal muscle terminal differentiation. Aberrant forms of myogenic factors, or their aberrant regulation, are associated with a number of muscle disorders: congenital myasthenia, myotonic dystrophy, rhabdomyosarcoma and defects in muscle regeneration. As such, myogenic factors provide a pool of potential therapeutic targets in muscle disorders, both with regard to mechanisms that cause disease itself and regenerative mechanisms that can improve disease treatment. Thus, the detailed understanding of the intermolecular interactions and the genetic programs controlled by the myogenic factors is essential for the rational design of efficient therapies.

MyoD is a myogenic transcription factor with a strong capacity to induce myogenic transdifferentiation of many fully differentiated non-muscle cell lines. The epigenetic mechanisms involved in this transdifferentiation are largely unknown. Here we describe a double-affinity purification method followed by mass spectrometry to exhaustively characterize MyoD partners.

使用串联亲和纯化的MyoD相互作用组鉴定质谱分析法

Skeletal muscle terminal differentiation starts with the commitment of pluripotent mesodermal precursor cells to myoblasts. These cells have still the ...