Name: imaging cell shape change in living drosophila embryos
Uploaded: 2011-03-30T13:00:00
Description: Watch this Scientific Journal Video about Imaging Cell Shape Change in Living Drosophila Embryos at JoVE.com

我们非常感谢埃里克Wieschaus，提供了该协议开发的基础上。我们的工作是一个贝尔纳Marrs麦克莱恩部生物化学与分子生物学启动奖，贝勒医学院的支持。

1。装配胚胎收集杯<ol><li>切底关闭100 mL的烧杯用剃刀三玉米，尽可能顺利的边缘。杯子很容易处理，如果你还修剪关闭顶部的三个角，虽然这不是绝对必要的。 </li><li>切丝网（6厘米x 6厘米）的平方。在一个预热的热点板块，里面的通风橱，层丝网一块重型铝箔上的平方。推杯的削减，坚决到热网的底部边缘。等待几秒钟，并解除与现在连接的网状杯。如果箔也坚持，只是剥离其关闭。 </li><li>?...

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T., Peifer, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Abelson+kinase+(Abl)+and+RhoGEF2+regulate+actin+organization+during+cell+constriction+in+Drosophila.">Abelson kinase (Abl) and RhoGEF2 regulate actin organization during cell constriction in Drosophila.</a> Development. 134, 567-578 (2007).</li><li>Martin, A. C., Kaschube, M., Wieschaus, E. 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B., Adams, R. J., Arias, M. a. r. t. i. n. e. z., A, . <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanical+control+of+global+cell+behaviour+during+dorsal+closure+in+Drosophila.">Mechanical control of global cell behaviour during dorsal closure in Drosophila.</a> Development. 136, 1889-1898 (2009).</li><li>Solon, J., Kaya-Copur, A., Colombelli, J., Brunner, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pulsed+forces+timed+by+a+ratchet-like+mechanism+drive+directed+tissue+movement+during+dorsal+closure.">Pulsed forces timed by a ratchet-like mechanism drive directed tissue movement during dorsal closure.</a> Cell. 137, 1331-1342 (2009).</li><li>Bownes, 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+photographic+study+of+development+in+the+living+embryo+of+Drosophila+melanogaster.">A photographic study of development in the living embryo of Drosophila melanogaster.</a> J Embryol Exp Morphol. 33, 789-801 (1975).</li><li>Sokac, A. M., Wieschaus, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zygotically+controlled+F-actin+establishes+cortical+compartments+to+stabilize+furrows+during+Drosophila+cellularization.">Zygotically controlled F-actin establishes cortical compartments to stabilize furrows during Drosophila cellularization.</a> J Cell Sci. 121, 1815-1824 (2008).</li><li>Morin, X., Daneman, R., Zavortink, M., Chia, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+protein+trap+strategy+to+detect+GFP-tagged+proteins+expressed+from+their+endogenous+loci+in+Drosophila.">A protein trap strategy to detect GFP-tagged proteins expressed from their endogenous loci in Drosophila.</a> Proc Natl Acad Sci U S A. 98, 15050-15055 (2001).</li><li>Oda, H., Tsukita, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Real-time+imaging+of+cell-cell+adherens+junctions+reveals+that+Drosophila+mesoderm+invagination+begins+with+two+phases+of+apical+constriction+of+cells.">Real-time imaging of cell-cell adherens junctions reveals that Drosophila mesoderm invagination begins with two phases of apical constriction of cells.</a> J Cell Sci. 114, 493-501 (2001).</li><li>Kiehart, D. 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本文所述的协议将允许生活，在发展中国家果蝇胚胎的细胞形态变化的共焦成像。成像GFP的股票，可以由个人实验室（见表2）准备，但许多这类股票也公开如布卢明顿果蝇联合中心的中心在美国印第安纳大学<a href="http://flystocks.bio.indiana.edu">（</a> http://flystocks.bio.indiana.edu）和捕蝇草在美国耶鲁大学（ <a href="http://flytrap.med.yale.edu">http://flytrap.med.yale.edu</a> ）联合中心。一旦获得这些实时成像?...

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		<th>Catalogue Number</th>
		<th>Comment</th>
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<tr>
<td>Slides</td>
<td>&nbsp;</td>
<td>Fisherbrand/Fisher Scientific</td>
<td>12-550-343</td>
<td>&nbsp;</td>
<tr>
<td>Cover slips 25x25</td>
<td>&nbsp;</td>
<td>Fisher Scientific (Corning #2865-25)</td>
<td>1 2-524C</td>
<td>&nbsp;</td>
<tr>
<td>Squirt bottles (H2O)</td>
<td>&nbsp;</td>
<td>Fisher Scientific</td>
<td>02-897-11</td>
<td>&nbsp;</td>
<tr>
<td>50 ml Falcon tubes</td>
<td>&nbsp;</td>
<td>Fisher Scientific (BD# 352070)</td>
<td>14-432-22</td>
<td>&nbsp;</td>
<tr>
<td>Bulbs for small pipets, 1 mL</td>
<td>&nbsp;</td>
<td>Fisherbrand/Fisher Scientific</td>
<td>03-448-21</td>
<td>&nbsp;</td>
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<td>Scintillation vials with caps</td>
<td>&nbsp;</td>
<td>VWR (Wheaton #986546)</td>
<td>66021-533</td>
<td>&nbsp;</td>
<tr>
<td>Tri-Corn Beakers, 100 mL</td>
<td>&nbsp;</td>
<td>Electron Microscopy Sciences</td>
<td>60970</td>
<td>&nbsp;</td>
<tr>
<td>BD Falcon Petri dish 60x15mm </td>
<td>&nbsp;</td>
<td>Fisher Scientific (BD# 351007)</td>
<td>08757 100B</td>
<td>&nbsp;</td>
<tr>
<td>BD Falcon Cell strainer</td>
<td>&nbsp;</td>
<td>Fisher Scientific (BD#352350)</td>
<td>08-771-2</td>
<td>&nbsp;</td>
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<td>Yellow pipet tips</td>
<td>&nbsp;</td>
<td>Ranin</td>
<td>L200</td>
<td>&nbsp;</td>
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<td>Stainless steel mesh, 304, 12x24</td>
<td>&nbsp;</td>
<td>Small Parts</td>
<td>CX-0150-F-01</td>
<td>&nbsp;</td>
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<td>Glass 5&frac34; inch Pasteur Pipets</td>
<td>&nbsp;</td>
<td>Fisherbrand/Fisher Scientific</td>
<td>13-678-20B</td>
<td>&nbsp;</td>
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<td>P4 Filter paper</td>
<td>&nbsp;</td>
<td>Fisherbrand/Fisher Scientific</td>
<td>09-803-6F</td>
<td>&nbsp;</td>
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<td>Rubber bands</td>
<td>&nbsp;</td>
<td>Office Max</td>
<td>A620645</td>
<td>&nbsp;</td>
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<td>Scotch double-sided tape, &frac12; inch</td>
<td>&nbsp;</td>
<td>Office Max</td>
<td>A8137DM-2</td>
<td>&nbsp;</td>
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<td>Robert Simmons Expression paint brushes E85 round #2</td>
<td>&nbsp;</td>
<td>Jerry&#x2019;s Artarama</td>
<td>56460</td>
<td>&nbsp;</td>
<tr>
<td>Dumont #5 Forceps High Precision Inox</td>
<td>&nbsp;</td>
<td>Electron Microscopy Services</td>
<td>72701-DZ</td>
<td>&nbsp;</td>
<tr>
<td>Razor blades</td>
<td>&nbsp;</td>
<td>VWR (BD #214010)</td>
<td>55411-050</td>
<td>&nbsp;</td>
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<td>Halocarbon Oil 27</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>H 8773</td>
<td>&nbsp;</td>
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<td>Heptane</td>
<td>&nbsp;</td>
<td>Fisher Chemical/Fisher Scientific</td>
<td>H360-1</td>
<td>&nbsp;</td>
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<td>BD Bacto Agar</td>
<td>&nbsp;</td>
<td>VWR (BD# 214010)</td>
<td>90000-760</td>
<td>&nbsp;</td>
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<td>Sucrose</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>S7903</td>
<td>&nbsp;</td>
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<td>p-Hydroxybenzoic acid</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>H5501</td>
<td>&nbsp;</td>
<tr>
<td>Red Star Active Dry Yeast</td>
<td>&nbsp;</td>
<td>LeSaffre</td>
<td>15700</td>
<td>&nbsp;</td>
<tr>
<td>Paper towels, C-fold</td>
<td>&nbsp;</td>
<td>Kleenex</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
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<td>Heavy duty aluminum foil</td>
<td>&nbsp;</td>
<td>Reynolds Wrap</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Bleach</td>
<td>&nbsp;</td>
<td>Austin&#x2019;s A-1 Commercial</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
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<td>100% Apple juice</td>
<td>&nbsp;</td>
<td>Ocean Spray or Tree Top</td>
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imaging cell shape change in living drosophila embryos

发展的果蝇胚胎发生细胞形态的变化，非常适合现场共焦成像。在果蝇的细胞形状的变化类似于在高等生物的，他们驾驶的组织形态发生。因此，在许多情况下他们的研究有直接影响，为了解人类疾病（见表1）1-5 。规模在亚细胞，这些细胞形态的变化，产品范围从基因表达的信号转导，细胞极性，细胞骨架重塑和膜贩运活动。因此， 果蝇胚胎不仅提供了背景下，以评估细胞形状的变化，因为这涉及到组织的形态发生，但也提供了一个完全的生理环境研究的亚细胞形状的细胞的活动，。 这里描述的是该协议设计的图像称为cellularization一个特定的细胞形态变化。 Cellularization是一个戏剧性的质膜增长过程，并最终转换成合胞胚胎细胞胚。也就是说，在有丝分裂周期14相间，质膜同时invaginates〜6000 cortically挂靠核周围产生原发性上皮细胞表。 cellularization违反上述建议，是不是驱动由肌球蛋白收缩6，但燃料，而不是主要从7个内部存储的膜胞吐。因此，cellularization是一个优秀的系统研究在细胞形态的变化，需要质膜内陷或扩建，如细胞分裂或横管（T型肾小管）在肌肉形态发生，膜贩运。 请注意，这个协议是很容易应用到其他细胞形状的变化，在果蝇胚胎成像，并且只需要轻微的适应化修改，如改变胚胎收集的阶段，或使用“胚胎胶”，安装在一个特定的方向（表胚胎1）8-19。在所有情况下，工作流程基本上是相同的的（图1）。克隆和转基因果蝇的标准方法用于制备稳定的飞股票，表达利益的一种蛋白，融合，绿色荧光蛋白（GFP）或它的变种，和这些苍蝇提供了一种可再生的胚胎来源。另外，荧光蛋白/探针直接引入飞胚胎，通过简单的显微注射技术 9-10 。然后，根据发展的事件和细胞形状的变化进行成像，胚胎收集和形态解剖显微镜上演，并最终定位和安装时间推移共聚焦显微镜成像。

Watch this Scientific Journal Video about Imaging Cell Shape Change in Living Drosophila Embryos at JoVE.com

Imaging Cell Shape Change in Living Drosophila Embryos

果蝇，果蝇，早期发展的特点是由许多细胞形状的变化，以及成像方法适合。本文将描述活果蝇胚胎的共焦成像所需的基本工具和方法，将集中在细胞形态变化称为cellularization。

影像生活细胞的形状变化果蝇胚胎

The developing Drosophila melanogaster embryo undergoes a number of cell shape changes that are highly amenable to live confocal imaging.  Cell shape ...

Research

JoVE Journal

Biology

Imaging Cell Shape Change in Living Drosophila Embryos

Preparation of Neuronal Cultures from Midgastrula Stage Drosophila Embryos

从Midgastrula舞台果蝇胚胎神经文化的制备

This video illustrates the procedure for making primary neuronal cultures from midgastrula stage Drosophila embryos. The methods for collecting embryos ...

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.  ...

The Preparation of Drosophila Embryos for Live-Imaging Using the Hanging Drop Protocol

The Preparation of Drosophila Embryos for Live-Imaging Using the Hanging Drop Protocol

编制果蝇实时成像的胚胎使用悬滴协议

Green fluorescent protein (GFP)-based timelapse live-imaging is a powerful technique for studying the genetic regulation of dynamic processes such as ...

Preparation of embryos for Electron Microscopy of the Drosophila embryonic heart tube

Preparation of embryos for Electron Microscopy of the Drosophila embryonic heart tube

电子显微镜对胚胎的制备果蝇胚胎心管

The morphogenesis of the Drosophila  embryonic heart tube has emerged as a valuable model system for studying cell migration, cell-cell adhesion and cell ...

In-vivo Centrifugation of Drosophila Embryos

In-vivo Centrifugation of Drosophila Embryos

果蝇胚胎的体内离心

A major strategy for purifying and isolating different types of intracellular organelles is to separate them from each other based on differences in ...

Upright Imaging of Drosophila Embryos

Upright Imaging of Drosophila Embryos

直立成像果蝇胚胎

Several well-known morphogenetic gradients and cellular movements occur along the dorsal/ventral axis of the Drosophila embryo. However, the current ...

Primary Cell Cultures from Drosophila Gastrula Embryos

Primary Cell Cultures from Drosophila Gastrula Embryos

原代细胞培养从果蝇原肠胚

Here we describe a method for preparing and culturing primary cells dissociated from Drosophila gastrula embryos.   In brief, a large amount of staged ...

Isolation and Purification of Kinesin from Drosophila Embryos

Isolation and Purification of Kinesin from Drosophila Embryos

kinesin的分离和纯化果蝇胚胎

Motor proteins move cargos along microtubules, and transport them to specific sub-cellular locations. Because altered transport is suggested to underlie a ...

Studying Mitotic Checkpoint by Illustrating Dynamic Kinetochore Protein Behavior and Chromosome Motion in Living Drosophila Syncytial Embryos

Studying Mitotic Checkpoint by Illustrating Dynamic Kinetochore Protein Behavior and Chromosome Motion in Living Drosophila Syncytial Embryos

龙虎斗动粒蛋白的动态行为与染色体运动，在生活学习有丝分裂关卡<em&gt;果蝇</em&gt;合胞胚胎

The spindle assembly checkpoint (SAC) mechanism is an active signal, which monitors the interaction between chromosome kinetochores and spindle ...

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  ...