Name: live imaging of cell extrusion from the epidermis of developing zebrafish
Uploaded: 2011-06-27T12:00:00
Description: Watch this Scientific Journal Video about Live Imaging of Cell Extrusion from the Epidermis of Developing Zebrafish at JoVE.com

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u...

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	<li>Rosenblatt, J., Raff, M. C., Cramer, L. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+epithelial+cell+destined+for+apoptosis+signals+its+neighbors+to+extrude+it+by+an+actin-+and+myosin-dependent+mechanism.">An epithelial cell destined for apoptosis signals its neighbors to extrude it by an actin- and myosin-dependent mechanism.</a> Curr Biol. 11, 1847-1857 (2001).</li><li>Le Guellec, D., Morvan-Dubois, G., Sire, J. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Skin+development+in+bony+fish+with+particular+emphasis+on+collagen+deposition+in+the+dermis+of+the+zebrafish+(Danio+rerio).">Skin development in bony fish with particular emphasis on collagen deposition in the dermis of the zebrafish (Danio rerio).</a> Int J Dev Biol. 48, 217-231 (2004).</li><li>Slattum, G., McGee, K. M., Rosenblatt, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=P115+RhoGEF+and+microtubules+decide+the+direction+apoptotic+cells+extrude+from+an+epithelium.">P115 RhoGEF and microtubules decide the direction apoptotic cells extrude from an epithelium.</a> J Cell Biol. 186, 693-702 (2009).</li><li>Burkel, B. M., Dassow, G. v. o. n., Bement, W. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Versatile+fluorescent+probes+for+actin+filaments+based+on+the+actin-binding+domain+of+utrophin.">Versatile fluorescent probes for actin filaments based on the actin-binding domain of utrophin.</a> Cell Motil Cytoskeleton. 64, 822-832 (2007).</li><li>Berndt, J. D., Clay, M. R., Langenberg, T., Halloran, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rho-kinase+and+myosin+II+affect+dynamic+neural+crest+cell+behaviors+during+epithelial+to+mesenchymal+transition+in+vivo.">Rho-kinase and myosin II affect dynamic neural crest cell behaviors during epithelial to mesenchymal transition in vivo.</a> Dev Biol. 324, 236-244 (2008).</li><li>Gong, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=fluorescent+protein+expression+in+germ-line+transmitted+transgenic+zebrafish+under+a+stratified+epithelial+promoter+from+keratin8.">fluorescent protein expression in germ-line transmitted transgenic zebrafish under a stratified epithelial promoter from keratin8.</a> Dev Dyn. 223, 204-215 (2002).</li><li>Westerfield, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The Zebrafish Book, A Guide for the Laboratory Use of Zebrafish (Danio rerio). , (2000).</li><li>Kimmel, C. B., Ballard, W. W., Kimmel, S. R., Ullmann, B., Schilling, T. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stages+of+embryonic+development+of+the+zebrafish.">Stages of embryonic development of the zebrafish.</a> Dev Dyn. 203, 253-310 (1995).</li><li>Yuan, S., Sun, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microinjection+of+mRNA+and+morpholino+antisense+oligonucleotides+in+zebrafish+embryos.">Microinjection of mRNA and morpholino antisense oligonucleotides in zebrafish embryos.</a> J Vis Exp. , (2009).</li><li>Curado, S., Stainier, D. Y., Anderson, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nitroreductase-mediated+cell/tissue+ablation+in+zebrafish:+a+spatially+and+temporally+controlled+ablation+method+with+applications+in+developmental+and+regeneration+studies.">Nitroreductase-mediated cell/tissue ablation in zebrafish: a spatially and temporally controlled ablation method with applications in developmental and regeneration studies.</a> Nat Protoc. 3, 948-954 (2008).</li></ol>

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 ...

Research

JoVE Journal

Biology

Two-photon axotomy and time-lapse confocal imaging in live zebrafish embryos

双光子现场斑马鱼的胚胎干切断和时间推移共焦成像

Zebrafish have long been utilized to study the cellular and molecular mechanisms of development by time-lapse imaging of the living transparent embryo.  ...

科研

生物学

Live Imaging of the Zebrafish Embryonic Brain by Confocal Microscopy

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 ...

Dissection of Organs from the Adult Zebrafish

剖析器官的成年斑马鱼

Over the last 20 years, the zebrafish has become a powerful model organism for understanding vertebrate development and disease.  Although experimental ...

Live Imaging of Cell Motility and Actin Cytoskeleton of Individual Neurons and Neural Crest Cells in Zebrafish Embryos

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 ...

Two-Photon-Based Photoactivation in Live Zebrafish Embryos

在现场的斑马鱼胚胎的基于双光子Photoactivati​​on

Photoactivation of target compounds in a living organism has proven a valuable approach to investigate various biological processes such as embryonic ...

Preparation of Developing and Adult Drosophila Brains and Retinae for Live Imaging

Preparation of Developing and Adult Drosophila Brains and Retinae for Live Imaging

发展和成人的制备果蝇大脑和视网膜实时成像

The Drosophila brain and visual system are widely utilized model systems to study neuronal development, function and degeneration. Here we show three ...

Live Imaging of Apoptotic Cell Clearance during Drosophila Embryogenesis

Live Imaging of Apoptotic Cell Clearance during Drosophila Embryogenesis

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  ...

Live Cell Imaging of Early Autophagy Events: Omegasomes and Beyond

活细胞成像早期自噬活动：Omegasomes与超越

Autophagy is a  cellular response triggered by the lack of nutrients, especially the absence of  amino acids. Autophagy is defined by the formation of ...

The Tomato/GFP-FLP/FRT Method for Live Imaging of Mosaic Adult Drosophila Photoreceptor Cells

The Tomato/GFP-FLP/FRT Method for Live Imaging of Mosaic Adult Drosophila Photoreceptor Cells

番茄/ GFP-FLP / FRT方法马赛克成人的实时成像<em&gt;果蝇</em&gt;感光细胞

The Drosophila eye is widely used as a model  for studies of development and neuronal degeneration. With the powerful mitotic  recombination technique, ...

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

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 ...