DP是从国家药物滥用研究所露丝Kirschstein奖1F32 DA021977 - 01A1收件人。这项工作是支持的玛格丽特M. Alkek基金会RHF。

<html>一，原理概述： <img src="/files/ftp_upload/old/1027_2009_1_22_2/Figure1.png" alt="图1" width="460" height="384" />本视频演示了在不同的步骤检测在鸡胚神经诱导。首先，主机的胚胎是explanted在新的文化[NI1]。然后，德纳胚胎是在生理盐水和恒胜的节点（在鸡的“组织者”）explanted与荧光染料DII标记[NI2]。恒胜的节点是从德纳胚胎切除[NI3]和主机的胚胎移植到[NI4，NI5]。主机的胚胎，然后长达22小时，培养后，期间德纳组织诱导异位轴在主机领域opaca [NI6]。 [网联科技]以下文化，主机胚胎是固定的，民建联存在的光氧化处理：通过这种化学反应，德纳源性细胞，FITC荧光下出现红色光氧化[NI7前，变成褐色与民建联以下反应。原位杂交胚胎处理与神经标记[网联科技];主机派生组织已形成存在德纳移植神经组织。这个组织显示为蓝色。 1：准备新的文化的主机的胚胎<ol><li>这一神经诱导实验协议，已培养汉堡和汉密尔顿第3阶段的鸡蛋开始 +（或HH3） 。这些鸡蛋已经奠定了他们一边在一个潮湿的孵化器孵育13小时。 </li><li>准备新的文化（步骤3.1至5.5 9）主机胚胎。 </li><li>主机胚胎表面覆盖200μL生理盐水。这将有利于以后的转移和定位德纳移植。 </li><li>最后，盖一个倒置的猎鹰35毫米培养皿大会。 </li></ol> 2：Explanting德纳胚胎在盐水<ol><li>打开攻壳钳和删除所有周围的壳壳件，蛋。拆下外壳的顶部和丢弃。 </li><li>删除与钳厚白蛋白，粗钳倾斜卵黄囊使胚胎朝上。 </li><li>用细剪刀，切割围绕胚胎的卵黄囊平方米。从蛋黄用勺子取出胚胎，并在含有PBS菜的地方。 </li><li>使用镊子，从区带分离德纳胚胎。 </li><li>德纳胚胎转移到Sylgard盖菜含有PBS。 </li></ol> 3：标签，切除和移植德纳移植<ol><li>使用微电极拉马，拉50μL玻璃microcapillary移液器。在显微镜下，削减微量用细镊子的尖端。放置在培养皿中的微管内衬的plasteline剪彩。 </li><li>准备染料溶液，将被用于标签移植前移植：carbocyanine染料DII（1,1&#39; - 双十八烷基- 3，3,3&#39;，3&#39; - tetramethylindocarbocyanine高氯酸盐）：首先，准备在1毫升一个DII股票在0.5％的无水乙醇。这个股票可以被存储在- 20C在黑暗中。在水浴42℃，混合预热40μLDII股票360μl0.3M蔗糖。水浴温度是必要的，以防止DII和形成不溶性晶体的沉淀。简要自旋对一个小型spectrafuge的解决方案（5-10秒）。 DII的解决方案现在已经可以使用。 </li><li>使用驱虫针，安全德纳胚胎Sylgard盖菜的底部。 </li><li>回到填补DII微量。通过运用柔和的压力使用吸引器管组装，适用于小的DII丸恒胜的节点。确保整个恒胜的节点标记。 </li><li>使用microcapillary吸管或microdissecting刀，切恒胜的节点（“组织者”，德纳移植）。 </li><li>标记的腹侧和胭脂红移植后结束。这将确保移植是适当的步骤4.3背腹方向定位。 </li><li>使用200μL吉尔森移液器，转让主机胚胎移植。 </li></ol> 4：保护主机胚胎移植和培养<ol><li>从主机胚胎中取出生理盐水，确保移植仍然定位在与主持人现场靠近。 </li><li>使用microcapillary吸管或microdissecting刀，在该地区的透明带/地区opaca边界地区的一个小切口（80 -100μm的），在恒胜的节点主机胚胎水平。 </li><li>使用一个microcapillary吸管或microdissecting刀，位置嫁接，使腹侧方对你和后部面临的是平行的主机胚胎相当于地区。 </li><li>去除剩余的生理盐水，并处理81-22小时（步骤6.1至6.6 9）为新的文化的主机胚胎。 </li><li>主机的胚胎，然后在4 ° C（7.1至7.7 9步骤）固定过夜。 </li></ol>E“&gt; 5：Photooxidise荧光下的移植<ol><li>通风柜下，准备一个DAB工作液：Tris缓冲液溶解民建联基板（3,3&#39; - 二氨基tetrahydrochloride）在500μg/ml的Tris缓冲液（100MM的Tris - HCl，pH值7.4）;保持在黑暗的解决方案，在冰上，。 </li><li>胚胎洗5分钟，两次在PBS每一次在Tris缓冲液为5分钟。 </li><li>将胚胎含有Tris缓冲液1毫升的玻璃腔幻灯片。 </li><li>更换Tris缓冲液，用1.5毫升民建联溶液。处理的Eppendorf提示中含有10％的漂白粉溶液，以净化民建联桶。将盖玻片上腔幻灯片的顶部。 </li><li> FITC（异硫氰酸荧光素）的荧光下，专注于移植（德纳细胞会出现红色荧光）显微镜的目标，直到所有的红色荧光细胞变成褐色（这DII的结果发生，暴露FITC荧光的荧光被photoconverted不溶性的棕色晶体，曝光FITC的激发波长（488nm）在民建联存在。，这个过程需要20分钟和2小时之间。 </li><li>氧化反应完成后，用细镊子取出盖玻片。胚胎转移到玻璃20毫升闪烁瓶中，含PBTw（或0.1％Tween - 20的PBS）。 </li><li>从盖玻片和玻璃腔滑停用民建联在10％的漂白粉溶液孵化。 </li></ol> 6：处理的胚胎在原位杂交技术，摄影和切片<ol><li>继续进行原位杂交（步骤3.7至6.5 9），孵化的胚胎只辛标记探针。 </li><li>继续拍摄（步骤8.2 9）和切片（8.3和8.4 9步骤）。 </li></ol>代表性的成果在下面的例子神经诱导实验，德纳移植诱导表达从主机派生组织的神经标记无尾。在这个特殊的例子中，我们评估收购胚胎地区一般不会有“组织者”的能力的神经诱导能力：德纳移植在其中的“组织者”的地区已经从德纳胚胎手术去除阶段的胚胎起源3 +德纳胚胎以下消融，是允许在新的文化发展，直到洞已愈合，并已形成一个结构类似恒胜的节点。这种结构与DII标记，从德纳胚胎切除，嫁接面积透明带/地区opaca主机胚胎的边界。新的文化，形成一个微型轴相邻主机胚胎：这种微型轴组成的红细胞杆（德纳派生）和一个头状结构（主机派生）：在（a），德纳衍生DiI标记FITC荧光显微镜下，细胞内固定前。这些德纳源性细胞增殖，形成一个微型的脊索，出现红细胞杆。 （二）以下光转​​化DII，这些德纳源性细胞出现棕黄色。 [转载]从6德纳移植诱导的神经组织形成从主机，这个组织包括大脑前体，因为它们的表达显示标记原位杂交无尾以下。 <img src="/files/ftp_upload/old/1027_2009_1_22_3/Figure 2.png" alt="图2" width="230" height="154" />注：在此过程中，我们用DiI标记的细胞，以区别于神经诱导德纳组织（红色/棕色）派生的“神经元”的组织（蓝色）的主机。而不是使用小鸡派生，DiI标记德纳移植，有些作者还使用德纳从鹌鹑胚胎，而不是小鸡的组织[如6]。这种替代的方法也可以让宿主组织，以区别于德纳。在这种情况下，移植（德纳组织）源于鹌鹑蛋代替​​鸡蛋。从协议中省略以下步骤4.5，主机是与特定的抗体，鹌鹑组织（QCP1）和步骤3.1到3.4和5.1至5.7整装免疫组织化学处理。这种替代过程中的一个例子如下所示：在这种情况下，我们使用相同的实验程序，在A，B，但我们用鹌鹑衍生德纳组织（表达QCPN抗体[棕色]所示）：再生从鹌鹑胚胎衍生的节点，引起泛神经标记Sox2基因在宿主的表达[转载]从6： <img src="/files/ftp_upload/old/1027_2009_1_22_4/Figure 3.png" alt="图3" width="154" height="230" />

<ol>
	<li>Waddington, C. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experiments+on+the+development+of+chick+and+duck+embryos,+cultivated+in+vitro.">Experiments on the development of chick and duck embryos, cultivated in vitro.</a> Phil. Trans. R. Soc. Lond. B. 221, 179-230 (1932).</li><li>Waddington, C. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+by+the+primitive+streak+and+its+derivatives+in+the+chick.">Induction by the primitive streak and its derivatives in the chick.</a> J. Exp. Biol. 10, 38-46 (1933).</li><li>Gallera, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Excision+et+transplantation+des+diff&amp;eacute;rentes+r&amp;eacute;gions+de+la+ligne+primitive+chez+le+poulet.">Excision et transplantation des diff&amp;eacute;rentes r&amp;eacute;gions de la ligne primitive chez le poulet.</a> C. R. Ass. Anat. 49, 632-639 (1964).</li><li>Gallera, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Primary+induction+in+birds.">Primary induction in birds.</a> Adv. Morph. 9. , 149-180 (1971).</li><li>Serbedzija, G. N., Fraser, S. E., Bronner-Fraser, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pathways+of+neural+crest+cell+migration+in+the+mouse+embryo+as+revealed+by+vital+dye+labeling.+Development+108.">Pathways of neural crest cell migration in the mouse embryo as revealed by vital dye labeling. Development 108.</a> , 605-612 (1990).</li><li>Psychoyos, D., Stern, C. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Restoration+of+the+organizer+after+radical+ablation+of+Hensen's+node+and+the+anterior+primitive+streak+in+the+chick+embryo.+Development+122.">Restoration of the organizer after radical ablation of Hensen's node and the anterior primitive streak in the chick embryo. Development 122.</a> , 3263-3273 (1996).</li><li>Joubin, K., Stern, C. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+interactions+continuously+define+the+organizer+during+the+cell+movements+of+gastrulation.">Molecular interactions continuously define the organizer during the cell movements of gastrulation.</a> Cell. 98, 559-571 (1999).</li><li>Streit, A., Berliner, A. J., Papanayotou, C., Sirulnik, A., Stern, C. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Initiation+of+neural+induction+by+FGF+signaling+before+gastrulation.">Initiation of neural induction by FGF signaling before gastrulation.</a> Nature. , 406-474 (2000).</li><li>Psychoyos, D., Finnell, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Method+for+Culture+of+Early+Chick+Embryos+ex+vivo+(New+Culture.">Method for Culture of Early Chick Embryos ex vivo (New Culture.</a> JoVE. 20, 10-3791 (2008).</li></ol>

本视频演示了不同的步骤执行检测神经诱导，这种检测基本上是公认的在小鸡的神经诱导分子的特性，因此可以使用一个广泛的应用， 范围从胚胎 micromanipulations 1-4，; 6日至揭开新的信号级联反应，7,8，所有旨在了解初步形成大脑和神经系统的其余步骤。

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<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Eggs</td>
<td>Animal</td>
<td>Charles River Laboratories</td>
<td>Premium Fertile</td>
<td>Fertilized, HH3+ (14 hr)</td>
</tr>
<tr>
<td>Stereomicroscope</td>
<td>Microscope</td>
<td>Leica Microsystems</td>
<td>MZ9.5 or similar</td>
<td>&nbsp;</td>
<tr>
<td>Hybridization Incubator</td>
<td>Equipment</td>
<td>Robbins Scientific</td>
<td>M1000</td>
<td>Use with inverted Pyrex dish and 500 ml ddH2O beaker</td>
</tr>
<tr>
<td>Marsh Automatic Incubator</td>
<td>Equipment</td>
<td>Lyon</td>
<td>RX</td>
<td>&nbsp;</td>
<tr>
<td>Pyrex dish </td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Watchmaker's glass 50mm</td>
<td>Tool</td>
<td>VWR</td>
<td>66112-060</td>
<td>&nbsp;</td>
<tr>
<td>Glass rings</td>
<td>Tool</td>
<td>Physical Plant facility</td>
<td>&nbsp;</td>
<td>cut 4 mm thick sections of glass tubing (27 mm outer diam, 25 mm inner diam). Do not fine polish.</td>
</tr>
<tr>
<td>Curved Forceps (1)</td>
<td>Tool</td>
<td>Electron Microscopy Sciences</td>
<td>72991-4C</td>
<td>&nbsp;</td>
<tr>
<td>Forceps (2)</td>
<td>Tool</td>
<td>Fine Science Tools</td>
<td>11002-13</td>
<td>blunt ended using sharpening Stone and 100ul mineral oil</td>
</tr>
<tr>
<td>Fine scissors</td>
<td>Tool</td>
<td>Fine Science Tools</td>
<td>14161-10</td>
<td>&nbsp;</td>
<tr>
<td>Plastic dishes</td>
<td>Tool</td>
<td>Falcon</td>
<td>353001</td>
<td>&nbsp;</td>
<tr>
<td>Rubber Bulb</td>
<td>Tool</td>
<td>Electron Microscopy Sciences</td>
<td>70980</td>
<td>&nbsp;</td>
<tr>
<td>DiI</td>
<td>Reagent</td>
<td>Invitrogen</td>
<td>D-282</td>
<td>&nbsp;</td>
<tr>
<td>Aspirator tube assembly</td>
<td>Tool</td>
<td>Sigma</td>
<td>A5177-5EA</td>
<td>&nbsp;</td>
<tr>
<td>Microelectrode puller</td>
<td>Equipment</td>
<td>Sutter Instruments</td>
<td>Sutter Instruments	P-97 Flaming/Brown Micropipette</td>
<td>&nbsp;</td>
<tr>
<td>Pasteur Capillary Pipette</td>
<td>Tool</td>
<td>Electron Microscopy Sciences</td>
<td>70950-12</td>
<td>round edge under flame</td>
</tr>
<tr>
<td>Culture chamber</td>
<td>Tool</td>
<td>Pioneer Plastics</td>
<td>030C</td>
<td>&nbsp;</td>
<tr>
<td>Microcapillary tube</td>
<td>Tool</td>
<td>Sigma</td>
<td>P1049-1PAK</td>
<td>&nbsp;</td>
<tr>
<td>Microdissecting knife</td>
<td>Tool</td>
<td>Fine Science Tools</td>
<td>10056-12</td>
<td>Use to puncture cavities prior to in situ hybridization</td>
</tr>
<tr>
<td>Minuten pins 0.2mm diam</td>
<td>Tool</td>
<td>Fine Science Tools</td>
<td>26002-20</td>
<td>Mix 1 part Curing Agent, 9 parts Base; set O/N at 37C</td>
</tr>
<tr>
<td>Diethylpyrocarbonate (depc)</td>
<td>Reagent</td>
<td>Electron Microscopy Sciences</td>
<td>15710</td>
<td>&nbsp;</td>
<tr>
<td>Sylgard 184 Silicon Elastomer Curing Agent and Base</td>
<td>&nbsp;</td>
<td>Dow Corning</td>
<td>0001986475</td>
<td>Mix 1 part Curing Agent, 9 parts Base; set O/N at 37C</td>
</tr>
<tr>
<td>Diethylpyrocarbonate (depc)</td>
<td>&nbsp;</td>
<td>Acros Organics</td>
<td>10025025</td>
<td>Add 1ml depc to 1l PBS; shake; autoclave</td>
</tr>
<tr>
<td>16% PFA</td>
<td>&nbsp;</td>
<td>Electron Microscopy Sciences</td>
<td>15710</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

assay for neural induction in the chick embryo

鸡胚是一个有价值的工具，在早期胚胎发育的研究。其透明度，可获得性和易于操纵，使其为研究神经系统的形成和最初的图案的理想工具。本视频演示了如何移植到主机组织者组织，恒胜小号节点的方法是，（在鸡胚的组织者）被嫁接到一个主机主管外胚层。主办移植指示覆NA已经组织采取通过神经诱导信号的神经命运。这一机制被称为神经诱导，并构成了脊椎动物的大脑和脊髓的形成初始步骤。这种方法实质上是公认的在小鸡的神经诱导分子的特性。本视频演示了在神经诱导实验的不同步骤，首先，德纳胚胎explanted和寄托一道菜。然后，主机胚胎准备为新的文化。移植是切除和移植到主机领域的透明带保证金。主机是培养18-22小时。大会是固定的，为进一步应用（例如原位杂交）处理。这种方法最初设计由沃丁顿<sup&gt; 1,2</sup&gt;和Gallera<sup&gt; 3,4</sup&gt;。

Watch this Scientific Journal Video about Assay for Neural Induction in the Chick Embryo at JoVE.com

Assay for Neural Induction in the Chick Embryo

神经诱导是在大脑形成的第一步。它是一种机制，使恒胜的节点（组织者），指示相邻的组织采用了神经的命运，即会引起神经系统。本视频演示了在鸡胚神经诱导实验。

在鸡胚神经诱导的含量为

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ...

assay-for-neural-induction-in-the-chick-embryo

Research

JoVE Journal

Biology

17.1K 次观看.  Texas A&M University (TAMU). 神经诱导是在大脑形成的第一步。它是一种机制，使恒胜的节点（组织者），指示相邻的组织采用了神经的命运，即会引起神经系统。本视频演示了在鸡胚神经诱导实验。

视频: Assay for Neural Induction in the Chick Embryo

Double Whole Mount in situ Hybridization of Early Chick Embryos

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ideal tool for studying  gene expression in brain, neural tube, somite and heart primordia formation. This video demonstrates the different steps in 2-color whole mount in situ hybridization; First, the embryo is dissected from the egg and fixed in paraformaldehyde. Second, the embryo is processed for prehybridization. The embryo is then hybridized with two different probes, one coupled to DIG, and one coupled to FITC.  Following overnight hybridization, the embryo is incubated with DIG coupled antibody. Color reaction for DIG substrate is performed, and the region of interest appears blue. The embryo is then incubated with FITC coupled antibody. The embryo is processed for color reaction with FITC, and the region of interest appears red. Finally, the embryo is fixed and processed for phtograph and sectioning. A troubleshooting guide is also presented.

This video demonstrates 2-color whole mount in situ hybridization, a method by which the spatial and temporal expression pattern of 2 different genes can be visualized in young chick embryos. This method was originally introduced by David Wilkinson, Domingos Henrique, Phil Ingham and David Ish -Horowicz.

双整装原位杂交技术在早期鸡胚

科研

生物学

Method for Whole Mount Antibody Staining in Chick

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ideal tool for studying antibody expression in developing brain, neural tube and somite. This video demonstrates the different steps in whole-mount antibody staining using HRP conjugated secondary antibodies; First, the embryo is dissected from the egg and fixed in paraformaldehyde. Second, endogenous peroxidase is inactivated; The embryo is then exposed to primary antibody. After several washes, the embryo is incubated with secondary antibody conjugated to HRP. Peroxidase activity is revealed using reaction with diaminobenzidine substrate. Finally, the embryo is fixed and processed for photography and sectioning. The advantage of this method over the use of fluorescent antibodies is that embryos can be processed for wax sectioning, thus enabling the study of antigen sites in cross section. This method was originally introduced by Jane Dodd and Tom Jessell 1.

This video demonstrates whole mount immunohistochemistry, a method by which the spatial and temporal expression pattern of an antigen can be visualized in young chick embryos. This method was originally introduced by Jane Dodd and Tom Jessell.

整装在鸡的抗体染色方法

Electrophysiological Recording in the Drosophila Embryo

Drosophila is a premier genetic model for the study of both embryonic development and functional neuroscience. Traditionally, these fields are quite isolated from each other, with largely independent histories and scientific communities. However, the interface between these usually disparate fields is the developmental programs underlying acquisition of functional electrical signaling properties and differentiation of functional chemical synapses during the final phases of neural circuit formation. This interface is a critically important area for investigation. In Drosophila, these phases of functional development occur during a period of &lt;8 hours (at 25&deg;C) during the last third of embryogenesis. This late developmental period was long considered intractable to investigation owing to the deposition of a tough, impermeable epidermal cuticle. A breakthrough advance was the application of water-polymerizing surgical glue that can be locally applied to the cuticle to enable controlled dissection of late-stage embryos. With a dorsal longitudinal incision, the embryo can be laid flat, exposing the ventral nerve cord and body wall musculature to experimental investigation. Whole-cell patch-clamp techniques can then be employed to record from individually-identifiable neurons and somatic muscles. These recording configurations have been used to track the appearance and maturation of ionic currents and action potential propagation in both neurons and muscles. Genetic mutants affecting these electrical properties have been characterized to reveal the molecular composition of ion channels and associated signaling complexes, and to begin exploration of the molecular mechanisms of functional differentiation. A particular focus has been the assembly of synaptic connections, both in the central nervous system and periphery. The glutamatergic neuromuscular junction (NMJ) is most accessible to a combination of optical imaging and electrophysiological recording. A glass suction electrode is used to stimulate the peripheral nerve, with excitatory junction current (EJC) recordings made in the voltage-clamped muscle. This recording configuration has been used to chart the functional differentiation of the synapse, and track the appearance and maturation of presynaptic glutamate release properties. In addition, postsynaptic properties can be assayed independently via iontophoretic or pressure application of glutamate directly to the muscle surface, to measure the appearance and maturation of the glutamate receptor fields. Thus, both pre- and postsynaptic elements can be monitored separately or in combination during embryonic synaptogenesis. This system has been heavily used to isolate and characterize genetic mutants that impair embryonic synapse formation, and thus reveal the molecular mechanisms governing the specification and differentiation of synapse connections and functional synaptic signaling properties.

Electrophysiological Recording in the Drosophila Embryo

Electrophysiological recordings from Drosophila embryos allow analyses of developing muscle and neuron electrical properties, as well as characterization of functional synaptogenesis at the glutamatergic neuromuscular junction and central cholinergic and GABAergic synapses.

在电生理记录果蝇胚胎

Drosophila is a premier genetic model for the study of both embryonic development and functional neuroscience. Traditionally, these fields are quite ...

Microinjection Techniques for Studying Mitosis in the Drosophila melanogaster Syncytial Embryo

This protocol describes the use of the Drosophila melanogaster syncytial embryo for studying mitosis1. Drosophila has useful genetics with a sequenced genome, and it can be easily maintained and manipulated. Many mitotic mutants exist, and transgenic flies expressing functional fluorescently (e.g. GFP) - tagged mitotic proteins have been and are being generated. Targeted gene expression is possible using the GAL4/UAS system2. 

The Drosophila early embryo carries out multiple mitoses very rapidly (cell cycle duration, &asymp;10 min). It is well suited for imaging mitosis, because during cycles 10-13, nuclei divide rapidly and synchronously without intervening cytokinesis at the surface of the embryo in a single monolayer just underneath the cortex. These rapidly dividing nuclei probably use the same mitotic machinery as other cells, but they are optimized for speed; the checkpoint is generally believed to not be stringent, allowing the study of mitotic proteins whose absence would cause cell cycle arrest in cells with a strong checkpoint. Embryos expressing GFP labeled proteins or microinjected with fluorescently labeled proteins can be easily imaged to follow live dynamics (Fig. 1). In addition, embryos can be microinjected with function-blocking antibodies or inhibitors of specific proteins to study the effect of the loss or perturbation of their function3. These reagents can diffuse throughout the embryo, reaching many spindles to produce a gradient of concentration of inhibitor, which in turn results in a gradient of defects comparable to an allelic series of mutants. Ideally, if the target protein is fluorescently labeled, the gradient of inhibition can be directly visualized4. It is assumed that the strongest phenotype is comparable to the null phenotype, although it is hard to formally exclude the possibility that the antibodies may have dominant effects in rare instances, so rigorous controls and cautious interpretation must be applied. Further away from the injection site, protein function is only partially lost allowing other functions of the target protein to become evident.

Microinjection Techniques for Studying Mitosis in the Drosophila melanogaster Syncytial Embryo

This protocol describes the use of microinjection and high resolution imaging in the Drosophila melanogaster syncytial embryo to study mitosis.

研究有丝分裂的显微注射技术果蝇合胞体胚胎

This protocol describes the use of the Drosophila melanogaster syncytial embryo  for studying mitosis1.  Drosophila has useful genetics with a sequenced ...

Chick ex ovo Culture and ex ovo CAM Assay: How it Really Works

Chicken eggs in the early phase of breeding are between in vitro and in vivo systems and provide a vascular test environment not only to study angiogenesis but also to study tumorigenesis. After the chick chorioallantoic membrane (CAM) has developed, its blood vessel network can be easily accessed, manipulated and observed and therefore provides an optimal setting for angiogenesis assays. Since the lymphoid system is not fully developed until late stages of incubation, the chick embryo serves as a naturally immunodeficient host capable of sustaining grafted tissues and cells without species-specific restrictions. In addition to nurturing developing allo- and xenografts, the CAM blood vessel network provides a uniquely supportive environment for tumor cell intravasation, dissemination, and vascular arrest and a repository where arrested cells extravasate to form micro metastatic foci.
For experimental purposes, in most of the recent studies the CAM was exposed by cutting a window through the egg shell and experiments were carried out in ovo, resulting in significant limitations in the accessibility of the CAM and possibilities for observation and photo documentation of effects. When shell-less cultures of the chick embryo were used1-4, no experimental details were provided and, if published at all, the survival rates of these cultures were low. We refined the method of ex ovo culture of chick embryos significantly by introducing a rationally controlled extrusion of the egg content. These ex ovo cultures enhance the accessibility of the CAM and chick embryo, enabling easy in vivo documentation of effects and facilitating experimental manipulation of the embryo. This allows the successful application to a large number of scientific questions: (1) As an improved angiogenesis assay5,6, (2) an experimental set up for facilitated injections in the vitreous of the chick embryo eye7-9, (3) as a test environment for dissemination and intravasation of dispersed tumor cells from established cell lines inoculated on the CAM10-12, (4) as an improved sustaining system for successful transplantation and culture of limb buds of chicken and mice13 as well as (5) for grafting, propagation, and re-grafting of solid primary tumor tissue obtained from biopsies on the surface of the CAM14.
In this video article we describe the establishment of a refined chick ex ovo culture and CAM assay with survival rates over 50%. Besides we provide a step by step demonstration of the successful application of the ex ovo culture for a large number of scientific applications. 
Daniel S. Dohle, Susanne D. Pasa, and Sebastian Gustmann contributed equally to this study.

Chick ex ovo Culture and ex ovo CAM Assay: How it Really Works

The chick chorioallantoic membrane (CAM) is a unique, naturally immunodeficient supportive culture environment to study angiogenesis and tumorigenesis. This video article demonstrates the different steps in chick ex ovo culture, application of potentially angiogenic substances and successful inoculation of tumor cells and tissues on the surface of the CAM.

小鸡前OVO文化前OVO CAM含量：它是如何真正起作用

Chicken eggs in the early phase of breeding are between in vitro and in vivo systems and provide a vascular test environment not only to study ...

Deciphering Axonal Pathways of Genetically Defined Groups of Neurons in the Chick Neural Tube Utilizing in ovo Electroporation

Employment of enhancer elements to drive expression of reporter genes in neurons is a widely used paradigm for tracking axonal projection. For tracking axonal projection of spinal interneurons in vertebrates, germ line-targeted reporter genes yield bilaterally symmetric labeling. Therefore, it is hard to distinguish between the ipsi- and contra-laterally projecting axons. Unilateral electroporation into the chick neural tube provides a useful means to restrict expression of a reporter gene to one side of the central nervous system, and to follow axonal projection on both sides 1 ,2-5. This video demonstrates first how to handle the eggs prior to injection. At HH stage 18-20, DNA is injected into the sacral level of the neural tube, then tungsten electrodes are placed parallel to the embryo and short electrical pulses are administered with a pulse generator. The egg is sealed with tape and placed back into an incubator for further development. Three days later (E6) the spinal cord is removed as an open book preparation from embryo, fixed, and processed for whole mount antibody staining. The stained spinal cord is mounted on slide and visualized using confocal microscopy.

Deciphering Axonal Pathways of Genetically Defined Groups of Neurons in the Chick Neural Tube Utilizing in ovo Electroporation

This video demonstrates how to visualize axonal pathways of genetically defined groups of neurons in the embryonic chick spinal cord utilizing in ovo electroporation of reporter genes under the control of specific enhancer elements.

利用鸡胚神经管破译基因定义的组的神经元轴突途径在OVO电穿孔

Employment of enhancer elements to drive expression of reporter genes in neurons is a widely used paradigm for tracking axonal projection. For tracking ...

Xenotransplantation of Human Stem Cells into the Chicken Embryo

The chicken embryo is a classical animal model for studying normal embryonic and fetal development and for xenotransplantation experiments to study the behavior of cells in a standardized in vivo environment. The main advantages of the chicken embryo include low cost, high accessibility, ease of surgical manipulation and lack of a fully developed immune system. Xenotransplantation into chicken embryos can provide valuable information about cell proliferation, differentiation and behavior, the responses of cells to signals in defined embryonic tissue niches, and tumorigenic potential. Transplanting cells into chicken embryos can also be a step towards transplantation experiments in other animal models. Recently the chicken embryo has been used to evaluate the neurogenic potential of human stem and progenitor cells following implantation into neural anlage1-6. In this video we document the entire procedure for transplanting human stem cells into the developing central nervous system of the chicken embryo. The procedure starts with incubation of fertilized eggs until embryos of the desired age have developed. The eggshell is then opened, and the embryo contrasted by injecting dye between the embryo and the yolk. Small lesions are made in the neural tube using microsurgery, creating a regenerative site for cell deposition that promotes subsequent integration into the host tissue. We demonstrate injections of human stem cells into such lesions made in the part of the neural tube that forms the hindbrain and the spinal cord, and into the lumen of the part of the neural tube that forms the brain. Systemic injections into extraembryonic veins and arteries are also demonstrated as an alternative way to deliver cells to vascularized tissues including the central nervous system. Finally we show how to remove the embryo from the egg after several days of further development and how to dissect the spinal cord free for subsequent physiological, histological or biochemical analyses.

In this paper we present a method for transplanting human stem cells into various regions of the central nervous system of the chicken embryo. This provides an in vivo model for assessing the proliferation and differentiation of various types of human stem cells in embryonic tissue environments.

人类干细胞异种移植到鸡胚

The chicken embryo is a classical animal model for studying normal embryonic and fetal development and for xenotransplantation experiments to study the ...

Clonogenic Assay: Adherent Cells

The clonogenic (or colony forming) assay has been established for more than 50 years; the original paper describing the technique was published in 19561. Apart from documenting the method, the initial landmark study generated the first radiation-dose response curve for X-ray irradiated mammalian (HeLa) cells in culture1. Basically, the clonogenic assay enables an assessment of the differences in reproductive viability (capacity of cells to produce progeny; i.e. a single cell to form a colony of 50 or more cells) between control untreated cells and cells that have undergone various treatments such as exposure to ionising radiation, various chemical compounds (e.g. cytotoxic agents) or in other cases genetic manipulation. The assay has become the most widely accepted technique in radiation biology and has been widely used for evaluating the radiation sensitivity of different cell lines. Further, the clonogenic assay is commonly used for monitoring the efficacy of radiation modifying compounds and for determining the effects of cytotoxic agents and other anti-cancer therapeutics on colony forming ability, in different cell lines. A typical clonogenic survival experiment using adherent cells lines involves three distinct components, 1) treatment of the cell monolayer in tissue culture flasks, 2) preparation of single cell suspensions and plating an appropriate number of cells in petri dishes and 3) fixing and staining colonies following a relevant incubation period, which could range from 1-3 weeks, depending on the cell line. Here we demonstrate the general procedure for performing the clonogenic assay with adherent cell lines with the use of an immortalized human keratinocyte cell line (FEP-1811)2. Also, our aims are to describe common features of clonogenic assays including calculation of the plating efficiency and survival fractions after exposure of cells to radiation, and to exemplify modification of radiation-response with the use of a natural antioxidant formulation.

The applicability of the clonogenic assay for evaluating reproductive viability has been established for more than 50 years. Here we demonstrate the general procedure for performing the clonogenic assay with adherent cells.

克隆形成含量：贴壁细胞

The clonogenic (or colony forming) assay has been established for more than 50 years; the original paper describing the technique was published in 19561. ...

Modified Mouse Embryonic Stem Cell based Assay for Quantifying  Cardiogenic Induction Efficiency

Differentiation of pluripotent stem cells is tightly controlled by temporal and spatial regulation of multiple key signaling pathways. One of the hurdles to its understanding has been the varied methods in correlating changes of key signaling events to differentiation efficiency. We describe here the use of a mouse embryonic stem (ES) cell based assay to identify critical time windows for Wnt/&beta;-catenin and BMP signal activation during cardiogenic induction. By scoring for contracting embryonic bodies (EBs) in a 96-well plate format, we can quickly quantify cardiogenic efficiency and identify crucial time windows for Wnt/&beta;-catenin and BMP signal activation in a time course following specific modulator treatments. The principal outlined here is not limited to cardiac induction alone, and can be applied towards the study of many other cell lineages. In addition, the 96-well format has the potential to be further developed as a high throughput, automated assay to allow for the testing of more sophisticated experimental hypotheses.

We describe the use of a mouse ES cell based assay to identify critical time windows for Wnt/&#946;-catenin and BMP signal activation during cardiogenic induction. The method provides a standardized platform that reliably quantifies cardiogenic efficiency, and it is applicable to the study of other cell lineages.

基因小鼠胚胎干细胞的基础含量为心源性诱导效率量化

Differentiation of pluripotent stem cells is tightly controlled by temporal and spatial regulation of multiple key signaling pathways.  One of the hurdles ...

Tracking Morphogenetic Tissue Deformations in the Early Chick Embryo

Embryonic epithelia undergo complex deformations (e.g. bending, twisting, folding, and stretching) to form the primitive organs of the early embryo. Tracking fiducial markers on the surfaces of these cellular sheets is a well-established method for estimating morphogenetic quantities such as growth, contraction, and shear. However, not all surface labeling techniques are readily adaptable to conventional imaging modalities and possess different advantages and limitations. Here, we describe two labeling methods and illustrate the utility of each technique. In the first method, hundreds of fluorescent labels are applied simultaneously to the embryo using magnetic iron particles. These labels are then used to quantity 2-D tissue deformations during morphogenesis. In the second method, polystyrene microspheres are used as contrast agents in non-invasive optical coherence tomography (OCT) imaging to track 3-D tissue deformations. These techniques have been successfully implemented in our lab to studythe physical mechanisms of early head fold, heart, and brain development, and should be adaptable to a wide range morphogenetic processes.

This article describes surface labeling and ex ovo tissue culture in the early chick embryo. Techniques amenable to time-lapse bright field, fluorescence, and optical coherence tomography imaging are presented. Tracking surface labels with high spatiotemporal resolution enables kinematic quantities such as morphogenetic strains (deformations) to be calculated in both two and three dimensions.

在早期鸡胚跟踪形态组织变形

Embryonic epithelia undergo complex deformations (e.g. bending, twisting, folding, and stretching) to form the primitive organs of the early embryo. ...