Name: live imaging of cell motility and actin cytoskeleton of individual neurons and neural crest cells in zebrafish embryos
Uploaded: 2010-02-03T16:25:00
Description: Watch this Scientific Journal Video about Live Imaging of Cell Motility and Actin Cytoskeleton of Individual Neurons and Neural Crest Cells in Zebrafish Embryos at JoVE.com

这项工作是支持由美国国立卫生研究院R01 NS042228妇幼保健奥林巴斯FV1000共聚焦与美国国立卫生研究院的共享仪器授予S10RR023717威斯康星大学动物学系（PI条例草案Bement）收购。 埃里卡Namrata Asuri，安德森，和马修粘土本文同等贡献。

1。大会注射的幻灯片和影像幻灯片注射用幻灯片： <ol><li>根据制造商的指示准备Sylgard有机硅弹性体。 </li><li>使用Sylgard债券三个标准玻璃显微镜幻灯片一起堆放其他两个幻灯片，这是安排在长边侧端的交集上一张幻灯片。顶部的幻灯片创建一个直角的角落或“墙”之间的顶部和底部的幻灯片。 </li><li>让Sylgard设置过夜。这些幻灯片是可重复使用的的。 </li></ol>?...

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	<li>Burkel, B. M., von Dassow, G., Bement, W. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Versatile+fluorescent+probes+for+actin+filaments+based+on+the+actin-binding+domain+of+utrophin.">Versatile fluorescent probes for actin filaments based on the actin-binding domain of utrophin.</a> Cell motility and the cytoskeleton. 64, 822-832 (2007).</li><li>Blader, P., Plessy, C., Strahle, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiple+regulatory+elements+with+spatially+and+temporally+distinct+activities+control+neurogenin1+expression+in+primary+neurons+of+the+zebrafish+embryo.">Multiple regulatory elements with spatially and temporally distinct activities control neurogenin1 expression in primary neurons of the zebrafish embryo.</a> Mech Dev. 120, 211-218 (2003).</li><li>Wada, N., Javidan, Y., Nelson, S., Carney, T. J., Kelsh, R. N., 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=Hedgehog+signaling+is+required+for+cranial+neural+crest+morphogenesis+and+chondrogenesis+at+the+midline+in+the+zebrafish+skull.">Hedgehog signaling is required for cranial neural crest morphogenesis and chondrogenesis at the midline in the zebrafish skull.</a> Development. 132, 3977-3988 (2005).</li><li>Cheo, D. L., Titus, S. A., Byrd, D. R., Hartley, J. L., Temple, G. F., Brasch, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Concerted+assembly+and+cloning+of+multiple+DNA+segments+using+in+vitro+site-specific+recombination:+functional+analysis+of+multi-segment+expression+clones.">Concerted assembly and cloning of multiple DNA segments using in vitro site-specific recombination: functional analysis of multi-segment expression clones.</a> Genome Res. 14, 2111-2120 (2004).</li><li>Kwan, K. M., Fujimoto, E., Grabher, C., Mangum, B. D., Hardy, M. E., Campbell, D. S., Parant, J. M., Yost, H. J., Kanki, J. P., Chien, C. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Tol2kit:+a+multisite+gateway-based+construction+kit+for+Tol2+transposon+transgenesis+constructs.">The Tol2kit: a multisite gateway-based construction kit for Tol2 transposon transgenesis constructs.</a> Dev Dyn. 236, 3088-3099 (2007).</li><li>O'Brien, G. S., Rieger, S., Martin, S. M., Cavanaugh, A. M., Portera-Cailliau, C., Sagasti, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two-photon+axotomy+and+time-lapse+confocal+imaging+in+live+zebrafish+embryos.">Two-photon axotomy and time-lapse confocal imaging in live zebrafish embryos.</a> J Vis Exp. 24, (2009).</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></ol>

注入的DNA的最佳浓度会有所不同规模和实力的推动者而兴建，并应凭经验确定。太多的DNA注射剂可能会导致不健康的胚胎具有广泛的细胞死亡，而太少会导致表达转基因注入胚胎的比例很小。 DNA的表达水平的荧光信号，从细胞到细胞的不同强度相关。在epifluorescence胚胎排序时，排除那些与表达非常高的水平。显得非常明亮的标记的细胞，通常也显示出明显的迹象，如细胞形态异常，标签分子misloc...

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<td>Tricaine (Ethyl 3-aminobenzoate methanesulfonate)</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>A5040-250G</td>
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<td>Sylgard Silicone Elastomer Kit</td>
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<td>Dow Corning Corporation</td>
<td>184</td>
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<td>QIAfilter Plasmid Midi Kit</td>
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<td>Qiagen Inc.</td>
<td>12243</td>
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<td>Low melting point agarose</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>15517-014</td>
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live imaging of cell motility and actin cytoskeleton of individual neurons and neural crest cells in zebrafish embryos

斑马鱼是一种理想的模型成像细胞在体内发育过程中的行为。斑马鱼的胚胎是外部受精，因而很容易在发展的各个阶段。此外，他们的光学清晰度，可在自然环境完整的胚胎细胞和分子动力学的高分辨率成像。我们使用的是一种实时成像方法，分析在神经嵴细胞迁移和神经轴突的生长和指导细胞的行为。 实时成像是了解调节细胞运动过程的机制，特别有用。要前伸活动和分子动力学，细胞运动的细节，如可视化，这是有利的标签单个细胞。在斑马鱼，质粒DNA注入产生一个短暂的马赛克的表达模式，并提供比其它细胞标记方法明显的好处。例如，转基因株系往往标签整个细胞群，从而有可能掩盖在一个单元格（或分子分布的变化）的罚款突起可视化。此外，在单细胞阶段的DNA注射，创伤小，更精确比染料注射在稍后阶段。 在这里，我们描述一个标签个别发展中国家的神经元或神经嵴细胞，其在体内的行为成像的方法。我们注入1 - 细胞期胚胎，结果马赛克转基因表达质粒DNA。载体含有细胞特异性启动子驱动的感觉神经元或神经嵴细胞的一个子集的兴趣的基因表达。我们提供膜有针对性的GFP标记的细胞或生物传感器探头，允许在活细胞1的F -肌动蛋白的可视化的例子。 埃里卡Namrata Asuri，安德森，和马修粘土贡献同样对这项工作。

Watch this Scientific Journal Video about Live Imaging of Cell Motility and Actin Cytoskeleton of Individual Neurons and Neural Crest Cells in Zebrafish Embryos at JoVE.com

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

本协议描述了生活的斑马鱼胚胎的单个神经元或神经嵴细胞成像。使用此方法来研究细胞行为和肌动蛋白定位使用荧光共焦时间推移显微镜。

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 ...

Research

JoVE Journal

Biology

Mouse Epidermal Neural Crest Stem Cell (EPI-NCSC) Cultures

Mouse Epidermal Neural Crest Stem Cell EPI-NCSC Cultures

小鼠表皮神经嵴干细胞（EPI的非公务员合约雇员）文化

EPI-NCSC are remnants of the embryonic neural crest in an adult location, the bulge of hair follicles. They are multipotent stem cells that have the ...

科研

生物学

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.  ...

Live Cell Imaging of F-actin Dynamics via Fluorescent Speckle Microscopy (FSM)

Live Cell Imaging of F-actin Dynamics via Fluorescent Speckle Microscopy FSM

F - actin的动力学通过荧光活细胞成像斑点显微镜（密克罗尼西亚）

In this protocol we describe the use of Fluorescent Speckle Microscopy (FSM) to capture high-resolution images of actin dynamics in PtK1 cells. A unique ...

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 ...

Transplantation of GFP-expressing Blastomeres for Live Imaging of Retinal and Brain Development in Chimeric Zebrafish Embryos

移植绿色荧光蛋白表达对视网膜和大脑发育的实时成像嵌合斑马鱼胚胎的卵裂球

Cells change extensively in their locations and property during embryogenesis. These changes are regulated by the interactions between the cells and their ...

Live-cell Imaging and Quantitative Analysis of Embryonic Epithelial Cells in Xenopus laevis

Live-cell Imaging and Quantitative Analysis of Embryonic Epithelial Cells in Xenopus laevis

活细胞成像和胚胎上皮细胞的定量分析非洲爪蟾</em

Embryonic epithelial cells serve as an ideal model to study morphogenesis where multi-cellular tissues undergo changes in their geometry, such as changes ...

Live Imaging of Cell Extrusion from the Epidermis of Developing Zebrafish

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 ...

Study of the Actin Cytoskeleton in Live Endothelial Cells Expressing GFP-Actin

表达GFP -肌动蛋白的内皮细胞的肌动蛋白细胞骨架的研究

The microvascular endothelium plays an important role as a selectively permeable barrier to fluids and solutes. The adhesive junctions between endothelial ...

4D Imaging of Protein Aggregation in Live Cells

在活细胞中蛋白质聚集的4D成像

One of the key tasks of any living cell is maintaining the proper folding of newly synthesized proteins in the face of ever-changing environmental ...

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  ...