罗森布拉特实验室的科学讨论，建议和意见，我们感谢成员。我们还要感谢玛丽哈洛伦请提供质粒RFP - UtrCH和大卫格伦沃尔德提供的CK:GFP转基因斑马鱼。还要感谢格蕾琴国王和良好的维护和斑马鱼的护理工作人员在犹他州立大学的集中斑马鱼资源。这项工作是由美国国立卫生研究院NIGMS美国国立卫生研究院主任的新的创新奖1 DP2 OD002056 - 01至JR支持。 GTE公司是由美国国立卫生研究院癌症多学科培训项目资助5T32 CA03247 - 8的支持。

在发展中国家斑马鱼的表皮细胞挤压在肌动蛋白动力学的可视化的基本工作流程发展中国家斑马鱼的表皮是由两个不同的层，面层（或周皮）和基底层的细胞接触基底膜 2 。外表面层的细胞发生凋亡，并从组织消除挤出 3（图1） 。为了实时可视化这个过程中，我们注入的RNA编码红色荧光蛋白融合成单细胞阶段的转基因表达绿色荧光蛋白（GFP斑马鱼utrophin，肌动蛋白结合蛋白（RFP - UtrCH）4,5，调宁蛋白同源结构域）根据分层的上皮细胞启动子细胞角蛋白8月6 日（图2A） 。虽然在后的RNA注射的动物表达RFP - UtrCH是无处不在，我们集中表达RFP和GFP遵循的表皮（图2B）中的肌动蛋白动力学专上肤浅的表皮细胞。然后，我们把与G418幼虫诱导细胞凋亡的挤压和图像使用一个旋转盘共聚焦显微镜的表皮和微丝，并获得4D数据集。 实验开始之前<ol><li>使用mMessage mMachine SP6试剂盒（Ambion公司）从一个线性DNA模板体外转录RNA和净化使用的RNeasy试剂盒（QIAGEN）的上限核糖核酸。使用无菌水稀释的RNA〜60ng/ul，直到注射35μL分装并储存于-80 ° C。注意:避免重复冻结/解冻，以防止RNA的降解。 </li><li>制作模具在E3胚胎培养基（E3）的2％琼脂糖40ML浇筑100 × 15mm的培养板，此托盘放置在一个显微注射模具（自适应科学工具，＃I - 34）期间举行注射胚胎。一旦琼脂糖凝固后，取出模具，暴露在琼脂糖槽。储存在4 ° C，直到准备使用该板块。 </li><li>拉细丝（0.78外径1.0MM，编号，10公分长）和一个萨特的P - 97在以下设置（485，热拉50，速度60，延时/ 90，压力200移液器拖轮使用注射硼硅玻璃毛细管针）。注:不同类型的玻璃，将规定的上市条件的优化。 </li><li> 1％低熔点（LM）的琼脂糖在E3和存储毫升分装在42 ° C。要小心，因为从股票的E3蒸发可以增加琼脂糖浓度。 </li><li>保持标准的实验室条件下的成年斑马鱼，一个普通的光/暗周期14小时光照和10小时黑暗的 7 。前一天晚上的实验设置分频器分离坦克3名女性和2名男性，鱼交配。第二天早晨，改变水箱中的水和拉分压器，当灯光来。 </li></ol> 1。注射荧光标记的肌动蛋白结合蛋白编码RNA的<ol><li>在冰上解冻的RNA。 〜30ng/μl最终的RNA浓度为1:1的比例加入0.5％酚红的RNA装入一把拉住毛细管针回填加入1μl的解决方案。 </li><li>收集〜75 1 - 8细胞阶段CK:在E3 GFP的胚胎。吸管收集5胚胎的显微注射模具¾巴斯德吸管轻轻东方在与福斯特杜蒙＃5镊子的低谷胚胎。注意:小心迅速开展工作，以避免注射到多细胞阶段的胚胎，这可能会导致镶嵌注入的RNA表达。 </li><li>一个标准的实验室解剖立体显微镜下，使用压力控制微量（哈佛大学器械的PLI - 100的Pico -注射器）胚胎的卵黄注入的RNA。红点（酚红）应该在胚胎注射后可见。每个实验注入至少50个胚胎。 （见以前的Jove协议一个详细的RNA注射到斑马鱼的胚胎9协议）。注意: <ol type="A"><li> 〜2nl卷应使用的RNA注射到细胞阶段的胚胎。要确定注入的RNA的量，分配到校准微米幻灯片上的矿物油下降或比例尺内置目镜显微镜下的RNA丸。 RNA的液滴应该是一个完美的球体。计算所使用的方程交付量:卷（NL）=4/3πr3。调整通过改变喷油压力或时间上的仪器的交付量。 </li><li>也应采取注入RNA的最低数额，允许可视化的荧光，高浓度的RNA可以到发育中的胚胎有毒。要确定适当的表达水平，实验剂量曲线应进行运行实验之前&lt;/ LI&gt; </ol></li><li>胚胎进行排序，以消除任何未受精或损坏的鸡蛋和地方所有剩余的胚胎在28.5 ° C。 </li><li> 24小时后，使用荧光解剖显微镜选择，有一个高水平的绿色荧光蛋白（表皮）和RFP（肌动蛋白）荧光的斑马鱼胚胎。放置在选定的胚胎28.5 ° C，直到准备进行诱导细胞凋亡的药物治疗。 </li></ol> 2。在使用G418的斑马鱼幼虫诱导细胞挤出我们已经发现，暴露的氨基糖甙类抗生素G418的，或Geneticin，导致由一个未知的机制在发展中国家斑马鱼幼虫3的表皮细胞凋亡和挤压。更重要的是，这种治疗方法只幼虫，后4天施肥和老年人。我们发现，可以发现在任何给定的G418治疗幼虫斑马鱼的鳍上皮的时间挤出〜5-25细胞。由于挤出凋亡细胞的数量可能有所不同从鱼到渔，我们建议安装多种鱼类成像。 <ol><li>收集和地点4天的受精后（DPF）35 x 10mm的文化包含3毫升的E3（MLS）的菜都将参展GFP和RFP荧光的斑马鱼幼虫。可以治疗25幼虫在这种规模的培养皿。 </li><li>仔细取代含有1mg/ml的G418和孵化幼虫在药物4小时的E3 3mLs媒体在28.5 ° C需要注意的是G418的对光敏感，所以要小心，保持覆盖的培养皿或在黑暗中。 </li><li>删除媒体和预热28.5 ° C E3的媒体含有0.02％Tricaine的更换，并进行安装的幼虫。 </li></ol> 3。安装成像斑马鱼幼虫<ol><li> 3幼虫转移到1.5 mL Eppendorf管和后取出鱼沉到试管底部的E3。我们发现，3动物可以正确安装的琼脂糖凝固之前。 </li><li>在盖玻片上一个MatTek培养皿的底部使用一个吸管尖切进管，吸管1％LM -琼脂糖（42℃）120ul，混匀，然后轻轻吸管幼虫和琼脂糖混合物。 </li><li>使用插入针或钨针，快速定位的幼虫，所以他们对MatTek盘的玻璃罩平直，一个FST的针座。 注:我们通常使用直针第一东方幼虫，然后一个L形的针，以确保他们对玻璃罩持平，而防止损坏的标本。 </li><li>一旦琼脂糖凝固后，轻轻地填写E3含0.02％Tricaine的菜。注:另外，G418可以添加到成像盘继续诱导挤压E3，虽然长期暴露1m​​g/ml的G418的杀幼虫。 </li></ol> 4。住在细胞挤出肌动蛋白动力学和个人的上皮细胞行为的成像，使用一个旋转的光盘共聚焦显微镜我们发现，从发展的斑马鱼幼虫的表皮细胞凋亡挤出的整个过程大约需要20分钟3。在这里，我们将演示如何设置一个时间推移的成像实验，收集通过对斑马鱼表皮的单层一系列z平面可视化挤出过程。我们发现，造成重大典型的宽视场荧光显微镜的光漂白微丝和不允许timelapse成像。因此，最大限度地发挥我们的决议，并防止漂白，我们使用一个旋转盘共聚焦显微镜。下面我们介绍如何设置使用安道尔IQ软件与尼康显微镜的实验，虽然讨论的原则可以应用到类似显微镜调校。 <ol><li>首先，打开氩（488nm GFP的激发）和氦氖（RFP的激发561nm）激光器，显微镜，照相机和啶灯。 </li><li>在安道尔IQ软件（版本1.10.1），创建时间的一系列协议，其中包含你想要的波长图像，图像捕获和次数之间的间隔的频率应重复捕捉。 </li><li>轻轻将Ma​​tTek菜含有您的标本，在显微镜舞台上，并使用透射光20倍的目标，把重点放在幼虫。 </li><li>移动到正确位置的40倍的水浸泡目标（NA 0.8）。使用这个目标，我们可以拍摄约20％至25场，这让我们跟随在挤出过程中的细胞行为的同时，也解决了亚细胞的肌动蛋白动力学细胞。注意:相同的安装和成像策略可应用于直立显微镜，虽然40倍的水浸泡长工作距离的目标，必须使用。 </li><li>小心地40X目标上方的水下降，并迅速MatTek菜放在上面。不E:蔡司Immersol浸泡的解决方案也可用于水的蒸发是一个问题。 </li><li>确定使用阶段或DIC照明的标本，然后用488nm epifluorescence专门专注于GFP阳性表皮。 </li><li>切换到共焦扫描模式。调节激光的强度和曝光时间，为每个通道（488nm和561nm）作相应。照顾，平衡信号的最佳检测激光功率和曝光时间，并防止任何漂白或毒性。 </li><li>要确定挤出细胞，使用epifluorescence的可视化的绿色荧光蛋白标记的表皮和识别的细胞组中的一个经典的花环图案，中间一个小圆形细胞周围的细胞集合组成。使用共焦扫描模式，也应该有一个积累RFP标记的肌动蛋白作为一个环在中间，周围的小圆形细胞的细胞挤出的一个标志可见。一旦你已经确定了一个挤出细胞，迅速成立显微镜获取4D共聚焦数据集（XYZ时间）。 </li><li>设置在z平面的上部和下部的点看到一个单层表皮，包括挤出细胞，然后选择步长的大小。对于时间推移成像，图像采集和重复次数的时间间隔。例如，我们通常会获得一个Z系列横跨5 - 10微米，1微米的步长，每隔1-2分钟，30分钟。 </li><li>要查看数据，我们通常使Z系列的3 - D的预测，并保存为影片文件是兼容快速时间（苹果）设置的数据。在这种方式下，我们可以可视化的肌动蛋白环，随着时间的推移而死亡细胞周围发生的上皮行为的收缩。 </li></ol> 5。代表性的成果<img src="/files/ftp_upload/2689/2689fig1.jpg" alt="图1"/> 图1。细胞凋亡挤出示意图。微丝显示为红色，GFP阳性表皮细胞都是绿色的。 <img src="/files/ftp_upload/2689/2689fig2.jpg" alt="图2"/> 图2。实验工作流程。 A.实验的工作流程示意图， B。 RFP - UtrCH表达一个4dpf长江:绿色荧光斑马鱼的表皮。 <img src="/files/ftp_upload/2689/2689fig3.jpg" alt="图3"/> 图3。帧从一个时间推移电影，活肌动蛋白动力学在上皮细胞挤出 ​​的过程如下（Z -预测） 。箭头表示超过2分钟的过程中，合同和关闭邻近的细胞所形成的肌动蛋白环。

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动物福利保障在此使用的斑马鱼， 斑马鱼的协议执行的所有程序，已批准的机构动物护理和使用委员会（IACUC）在美国犹他州大学（动物福利保证＃10-07017） 。

这里所描述的协议演示了一个简单而直接的方法来可视化挤压过程，在现场的上皮组织。这种类型的实验使我们以前没有固定组织分析赞赏的肌动蛋白动力学研究中的细微之处，因此，常见的免疫方法的补充。我们可以利用这个协议在组合化学抑制剂或基因突变体，以便更好地分析删除和清除细胞凋亡，我们期望与故障相关的挤压过程和研究疾病状态下会导致炎症反应或受损细胞的积累，为见于?...

<table border="1"><tbody><tr><th>名称</th><th>类型</th><th>公司</th><th>产品编号</th><th>评论</th></tr><tr><td>的RNeasy迷你包</td><td>试剂</td><td> Qiagen公司</td><td> 74104 </td><td></td></tr><tr><td> mMessage mMachine SP6套件</td><td>试剂</td><td> Ambion公司</td><td> AM1340 </td><td></td></tr><tr><td>隧道坦克</td><td>工具</td><td> Thoren水产系统</td><td></td><td></td></tr><tr><td>吸管泵</td><td>工具</td><td>贝尔艺术产品</td><td> F3789 </td><td></td></tr><tr><td> 5 ¾巴斯德吸取</td><td>工具</td><td> VWR </td><td> 14672-608 </td><td></td></tr><tr><td>显微注射模具</td><td>工具</td><td>自适应科学工具</td><td>我34 </td><td></td></tr><tr><td> 100x15mm文化板</td><td>工具</td><td>费舍尔</td><td> 08-757-12 </td><td></td></tr><tr><td> Flamming /布朗Micropipet拖轮</td><td>工具</td><td>萨特仪器公司</td><td>型号P97 </td><td></td></tr><tr><td>与长丝的高硼硅玻璃毛细管</td><td>工具</td><td>萨特仪器公司</td><td> B1400 - 78 - 10 </td><td>外径1.0MM，编号0.78毫米，10公分长</td></tr><tr><td>电极储存罐</td><td>工具</td><td>世界精密仪器</td><td> E210 </td><td></td></tr><tr><td>酚红</td><td>试剂</td><td>西格玛</td><td> P0290 </td><td>在DPBS 0.5％ </td></tr><tr><td>压力控制的微量及微操作机器人</td><td>工具</td><td>哈佛器械</td><td> PLI - 100 </td><td></td></tr><tr><td> 35x10mm文化菜</td><td>工具</td><td>蜂星</td><td> 627-160 </td><td></td></tr><tr><td> G418（Geneticin） </td><td>试剂</td><td> GIBCO公司</td><td> 10131-035 </td><td> 50毫克/毫升，在卫生署 2 0 </td></tr><tr><td>杜蒙＃5镊子</td><td>工具</td><td>精细科学的工具</td><td> 11253-20 </td><td></td></tr><tr><td> 12厘米引脚持有人</td><td>工具</td><td>精细科学的工具</td><td> 26016-12 </td><td></td></tr><tr><td>插入销</td><td>工具</td><td>精细科学的工具</td><td> 26007-02和03 </td><td></td></tr><tr><td>玻璃底培养皿1.5玻璃罩</td><td>工具</td><td> MatTek </td><td> P35G - 1.5 - 10 - C </td><td></td></tr><tr><td>低熔体琼脂糖</td><td>试剂</td><td>费舍尔</td><td> BP1360 - 100 </td><td></td></tr><tr><td> Tricaine </td><td>试剂</td><td>西格玛</td><td> A - 5040 </td><td></td></tr><tr><td>解剖显微镜</td><td>显微镜</td><td>徕卡</td><td> MZ6 </td><td></td></tr><tr><td>解剖显微镜</td><td>显微镜</td><td>尼康</td><td> SMZ645 </td><tD&gt; </td></tr><tr><td>荧光解剖显微镜</td><td>显微镜</td><td>奥林巴斯</td><td> S2X12 </td><td></td></tr><tr><td>旋转的光盘共聚焦显微镜</td><td>显微镜</td><td>尼康</td><td> Eclipse的钛</td><td>配备一个Yokagawa转盘头</td></tr><tr><td> CCD相机</td><td>相机</td><td>安道​​尔</td><td> DV - 885 - VP </td><td> 1002x1004x8微米的正方形像素</td></tr></tbody></table>

live imaging of cell extrusion from the epidermis of developing zebrafish

稳态维持上皮组织需要不断清除而不破坏的屏障功能受损的细胞。我们的研究发现，死亡的细胞信号传递给他们住邻居的形式和合同的肌动蛋白和肌球蛋白环弹出上皮表，而关闭任何可能导致其退出的差距，这个过程被称为细胞挤出​​1。发展中国家斑马鱼的光学清晰度提供了一个极好的系统，以可视化挤出生活上皮。在这里，我们描述了一种方法来诱导和幼虫斑马鱼表皮的形象挤出。可视化挤出，注射F -肌动蛋白红色荧光蛋白标记的探针进入细胞阶段在表皮表达绿色荧光蛋白的转基因斑马鱼的胚胎和诱导凋亡的G418除了幼虫。然后，我们使用一个旋转的光盘共聚焦显微镜成像技术观察在细胞凋亡挤出过程中的肌动蛋白动力学和上皮细胞行为的时间推移。这种方法使我们能够调查上皮细胞在活的挤压过程中，将提供一个途径研究由故障消除细胞凋亡引起的疾病状态。

Watch this Scientific Journal Video about Live Imaging of Cell Extrusion from the Epidermis of Developing Zebrafish at JoVE.com

Live Imaging of Cell Extrusion from the Epidermis of Developing Zebrafish

死亡的细胞被挤压协调一致的收缩邻近的细胞，上皮组织，而不破坏的屏障功能。发展中国家斑马鱼的光学清晰度提供了一个极好的系统，以可视化挤出生活上皮。在这里，我们描述的方法来诱导和幼虫斑马鱼的表皮细胞决议形象挤压。

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 ...

live-imaging-cell-extrusion-from-epidermis-developing

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

14.6K 次观看.  University of Utah. 死亡的细胞被挤压协调一致的收缩邻近的细胞，上皮组织，而不破坏的屏障功能。发展中国家斑马鱼的光学清晰度提供了一个极好的系统，以可视化挤出生活上皮。在这里，我们描述的方法来诱导和幼虫斑马鱼的表皮细胞决议形象挤压。

视频: Live Imaging of Cell Extrusion from the Epidermis of Developing Zebrafish

Live Imaging of Innate Immune and Preneoplastic Cell Interactions Using an Inducible Gal4/UAS Expression System in Larval Zebrafish Skin

Here we describe a method to conditionally induce epithelial cell transformation by the use of the 4-Hydroxytamoxifen (4-OHT) inducible KalTA4-ERT2/UAS expression system1 in zebrafish larvae, and the subsequent live imaging of innate immune cell interaction with HRASG12V expressing skin cells. The KalTA4-ERT2/UAS system is both inducible and reversible which allows us to induce cell transformation with precise temporal/spatial resolution in vivo. This provides us with a unique opportunity to live image how individual preneoplastic cells interact with host tissues as soon as they emerge, then follow their progression as well as regression. Recent studies in zebrafish larvae have shown a trophic function of innate immunity in the earliest stages of tumorigenesis2,3. Our inducible system would allow us to live image the onset of cellular transformation and the subsequent host response, which may lead to important insights on the underlying mechanisms for the regulation of oncogenic trophic inflammatory responses. We also discuss how one might adapt our protocol to achieve temporal and spatial control of ectopic gene expression in any tissue of interest.

Studying the earliest events of preneoplastic cell progression and innate immune cell interaction is pivotal to understand and treat cancer. Here we describe a method to conditionally induce epithelial cell transformations and the subsequent live imaging of innate immune cell interaction with HRASG12V expressing skin cells in zebrafish larvae.

先天免疫和癌前细胞间的相互作用使用可诱导的Gal4 / UAS表达系统在斑马鱼幼体皮肤实时成像

Here we describe a method to conditionally induce epithelial cell transformation by the use of the 4-Hydroxytamoxifen (4-OHT) inducible KalTA4-ERT2/UAS ...

科研

发育生物学

Zebrafish Keratocyte Explants to Study Collective Cell Migration and Reepithelialization in Cutaneous Wound Healing

Due to their unique motile properties, fish keratocytes dissociated from explant cultures have long been used to study the mechanisms of single cell migration. However, when explants are established, these cells also move collectively, maintaining many of the features which make individual keratocytes an attractive model to study migration: rapid rates of motility, extensive actin-rich lamellae with a perpendicular actin cable, and relatively constant speed and direction of migration. In early explants, the rapid interconversion of cells migrating individually with those migrating collectively allows the study of the role of cell-cell adhesions in determining the mode of migration, and emphasizes the molecular links between the two modes of migration. Cells in later explants lose their ability to migrate rapidly and collectively as an epithelial to mesenchymal transition occurs and genes associated with wound healing and inflammation are differentially expressed. Thus, keratocyte explants can serve as an in vitro model for the reepithelialization that occurs during cutaneous wound healing and can represent a unique system to study mechanisms of collective cell migration in the context of a defined program of gene expression changes. A variety of mutant and transgenic zebrafish lines are available, which allows explants to be established from fish with different genetic backgrounds. This allows the role of different proteins within these processes to be uniquely addressed. The protocols outlined here describe an easy and effective method for establishing these explant cultures for use in a variety of assays related to collective cell migration.

Zebrafish keratocytes migrate in cell sheets from explants and provide an in vitro model for the study of the mechanisms of collective cell migration in the context of epithelial wound healing. These protocols detail an effective way to establish primary explant cultures for use in collective cell migration assays.

斑马鱼角膜植研究集体细胞迁移和再上皮化在皮肤伤口愈合

Due to their unique motile properties, fish keratocytes dissociated from explant cultures have long been used to study the mechanisms of single cell ...

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.

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

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.

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

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

Using Light Sheet Fluorescence Microscopy to Image Zebrafish Eye Development

Light sheet fluorescence microscopy (LSFM) is gaining more and more popularity as a method to image embryonic development. The main advantages of LSFM compared to confocal systems are its low phototoxicity, gentle mounting strategies, fast acquisition with high signal to noise ratio and the possibility of imaging samples from various angles (views) for long periods of time. Imaging from multiple views unleashes the full potential of LSFM, but at the same time it can create terabyte-sized datasets. Processing such datasets is the biggest challenge of using LSFM. In this protocol we outline some solutions to this problem. Until recently, LSFM was mostly performed in laboratories that had the expertise to build and operate their own light sheet microscopes. However, in the last three years several commercial implementations of LSFM became available, which are multipurpose and easy to use for any developmental biologist. This article is primarily directed to those researchers, who are not LSFM technology developers, but want to employ LSFM as a tool to answer specific developmental biology questions. 
Here, we use imaging of zebrafish eye development as an example to introduce the reader to LSFM technology and we demonstrate applications of LSFM across multiple spatial and temporal scales. This article describes a complete experimental protocol starting with the mounting of zebrafish embryos for LSFM. We then outline the options for imaging using the commercially available light sheet microscope. Importantly, we also explain a pipeline for subsequent registration and fusion of multiview datasets using an open source solution implemented as a Fiji plugin. While this protocol focuses on imaging the developing zebrafish eye and processing data from a particular imaging setup, most of the insights and troubleshooting suggestions presented here are of general use and the protocol can be adapted to a variety of light sheet microscopy experiments.

Light sheet fluorescence microscopy is an excellent tool for imaging embryonic development. It allows recording of long time-lapse movies of live embryos in near physiological conditions. We demonstrate its application for imaging zebrafish eye development across wide spatio-temporal scales and present a pipeline for fusion and deconvolution of multiview datasets.

采用轻质板材荧光显微镜到图像斑马鱼眼睛发育

Light sheet fluorescence microscopy (LSFM) is gaining more and more popularity as a method to image embryonic development. The main advantages of LSFM ...

Observing Mitotic Division and Dynamics in a Live Zebrafish Embryo

Mitosis is critical for organismal growth and differentiation. The process is highly dynamic and requires ordered events to accomplish proper chromatin condensation, microtubule-kinetochore attachment, chromosome segregation, and cytokinesis in a small time frame. Errors in the delicate process can result in human disease, including birth defects and cancer. Traditional approaches investigating human mitotic disease states often rely on cell culture systems, which lack the natural physiology and developmental/tissue-specific context advantageous when studying human disease. This protocol overcomes many obstacles by providing a way to visualize, with high resolution, chromosome dynamics in a vertebrate system, the zebrafish. This protocol will detail an approach that can be used to obtain dynamic images of dividing cells, which include: in vitro transcription, zebrafish breeding/collecting, embryo embedding, and time-lapse imaging. Optimization and modifications of this protocol are also explored. Using H2A.F/Z-EGFP (labels chromatin) and mCherry-CAAX (labels cell membrane) mRNA-injected embryos, mitosis in AB wild-type, auroraBhi1045, and esco2hi2865 mutant zebrafish is visualized. High resolution live imaging in zebrafish allows one to observe multiple mitoses to statistically quantify mitotic defects and timing of mitotic progression. In addition, observation of qualitative aspects that define improper mitotic processes (i.e., congression defects, missegregation of chromosomes, etc.) and improper chromosomal outcomes (i.e., aneuploidy, polyploidy, micronuclei, etc.) are observed. This assay can be applied to the observation of tissue differentiation/development and is amenable to the use of mutant zebrafish and pharmacological agents. Visualization of how defects in mitosis lead to cancer and developmental disorders will greatly enhance understanding of the pathogenesis of disease.

Mitosis is critical to every living organism and defects often lead to cancer and developmental disorders. Using this imaging protocol and zebrafish as a model system, researchers can visualize mitosis in a live vertebrate organism and the multitude of defects that arise when mitotic processes are defective.

观察有丝分裂和动力学在现场斑马鱼胚胎

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

A Versatile Mounting Method for Long Term Imaging of Zebrafish Development

Zebrafish embryos offer an ideal experimental system to study complex morphogenetic processes due to their ease of accessibility and optical transparency. In particular, posterior body elongation is an essential process in embryonic development by which multiple tissue deformations act together to direct the formation of a large part of the body axis. In order to observe this process by long-term time-lapse imaging it is necessary to utilize a mounting technique that allows sufficient support to maintain samples in the correct orientation during transfer to the microscope and acquisition. In addition, the mounting must also provide sufficient freedom of movement for the outgrowth of the posterior body region without affecting its normal development. Finally, there must be a certain degree in versatility of the mounting method to allow imaging on diverse imaging set-ups. Here, we present a mounting technique for imaging the development of posterior body elongation in the zebrafish D. rerio. This technique involves mounting embryos such that the head and yolk sac regions are almost entirely included in agarose, while leaving out the posterior body region to elongate and develop normally. We will show how this can be adapted for upright, inverted and vertical light-sheet microscopy set-ups. While this protocol focuses on mounting embryos for imaging for the posterior body, it could easily be adapted for the live imaging of multiple aspects of zebrafish development.

Here, we present a versatile mounting method that allows for the long-term time-lapse imaging of the posterior body development of live zebrafish embryos without perturbing normal development.

斑马鱼发展长期成像的多功能安装方法

Zebrafish embryos offer an ideal experimental system to study complex morphogenetic processes due to their ease of accessibility and optical transparency. ...

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 ...

Live Imaging of the Zebrafish Embryonic Brain by Confocal Microscopy

In this video, we demonstrate the method our lab has developed to analyze the cell shape changes and rearrangements required to bend and fold the developing zebrafish brain (Gutzman et al, 2008). Such analysis affords a new understanding of the underlying cell biology required for development of the 3D structure of the vertebrate brain, and significantly increases our ability to study neural tube morphogenesis. The embryonic zebrafish brain is shaped beginning at 18 hours post fertilization (hpf) as the ventricles within the neuroepithelium inflate. By 24 hpf, the initial steps of neural tube morphogenesis are complete. Using the method described here, embryos at the one cell stage are injected with mRNA encoding membrane-targeted green fluorescent protein (memGFP). After injection and incubation, the embryo, now between 18 and 24 hpf, is mounted, inverted, in agarose and imaged by confocal microscopy. Notably, the zebrafish embryo is transparent making it an ideal system for fluorescent imaging. While our analyses have focused on the midbrain-hindbrain boundary and the hindbrain, this method could be extended for analysis of any region in the zebrafish to a depth of 
80-100 &mu;m.

In this video, we demonstrate a method by which to analyze the developing vertebrate brain in live zebrafish embryos at single cell resolution by confocal microscopy. This includes the method by which we inject the single-cell zebrafish embryo and subsequently mount and image the developing brain.

现场共聚焦显微镜对斑马鱼的胚胎脑成像

In this video, we demonstrate the method our lab has developed to analyze the cell shape changes and rearrangements required to bend and fold the ...

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 ...

4-Dimensional Imaging of Zebrafish Optic Cup Morphogenesis

Visual system function requires the establishment of precise tissue and organ structures. In the vertebrate eye, structural defects are a common cause of visual impairment, yet mechanisms of eye morphogenesis are still poorly understood. The basic organization of the embryonic eye is conserved throughout vertebrates, thus live imaging of zebrafish embryos has become a powerful approach to directly observe eye development at real time under normal and pathological conditions. Dynamic cell processes including movements, morphologies, interactions, division, and death can be visualized in the embryo. We have developed methods for uniform labeling of subcellular structures and timelapse confocal microscopy of early eye development in zebrafish. This protocol outlines the method of generating capped mRNA for injection into the 1-cell zebrafish embryo, mounting embryos at optic vesicle stage (~12 hours post fertilization, hpf), and performing multi-dimensional timelapse imaging of optic cup morphogenesis on a laser scanning confocal microscope, such that multiple datasets are acquired sequentially in the same imaging session. Such an approach yields data that can be used for a variety of purposes, including cell tracking, volume measurements, three-dimensional (3D) rendering, and visualization. Our approaches allow us to pinpoint the cellular and molecular mechanisms driving optic cup development, in both wild type and genetic mutant conditions. These methods can be employed directly by other groups or adapted to visualize many additional aspects of zebrafish eye development.

This protocol describes an approach for in toto labeling and multidimensional imaging of zebrafish early eye development. We describe labeling, embedding, and four dimensional (4D) imaging using laser scanning confocal microscopy, and considerations for optimizing acquisition of datasets for dissecting mechanisms of optic cup morphogenesis.

斑马鱼光学杯形态形成的四维成像

Visual system function requires the establishment of precise tissue and organ structures. In the vertebrate eye, structural defects are a common cause of ...