笔者想承认，这项工作是由加拿大自然科学和（NSERC）授予工程研究理事会的支持。

<ol>
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作者什么都没有透露。

本协议描述在胚胎发育中的使用各类显微镜的图像细胞分裂。特别是，该协议强调了如何将图像的神经母细胞的分裂，细胞，可促进表皮形态。后生动物的开发和C在细胞间的沟通对组织形成重要线虫是研究在体内组织形成一个很好的模型。一个事件，很好地描绘组织的相互作用是表皮形态，它涵盖了胚胎一层上皮细胞。特别是，腹侧外壳是其中腹侧表皮细胞迁移到包围胚胎的...

虽然有说明如何像细胞分裂初期C.协议线虫的胚胎，这个协议描述了如何像细胞分裂中期胚胎发育过程中组织内。其中一个在开发过程中的生物成像的主要挑战一直是他们的光毒性的敏感性。然而，增加无障碍旋转盘共聚焦显微镜或扫描显微镜领域已经允许更广泛的成像应用。这两个系统使用固态激光器和散射光，限制紫外线的生物体暴露于的电平。但是，广角台灯仍然可以用于成像在体内 ，尤其是当它们配备相机具有高灵敏度（ 例如 EMCCD），光圈控制和调光（ 例如发光二极管或可调汞灯泡）。本协议描述了如何使用一个基于共聚焦系统或宽视场系统，以图像的细胞分裂中开发C。线虫&lt;/ em&gt;的胚胎。作为一个例子，我们介绍了如何像神经细胞的细胞分裂。神经母细胞可以促进表皮细胞的形态通过提供化学或机械的线索来覆表皮细胞，并提供了细胞间通讯在组织中形成的重要性一个很好的例子。 秀丽隐杆线虫是一种理想的模式生物，由于其透明度和简单的组织机构1显微镜为基础的研究。此外，C。线虫是服从遗传方法和RNAi，并且由于它的许多基因具有人同系物，它可用于鉴定保守机制，组织形成2-5。在C线虫 ，形成表皮层的过程中胚发生时，当胚胎有&gt; 300个细胞。表皮形态包括几个主要阶段，在此期间，胚胎被封闭在一层表皮细胞的收缩和延伸，改造EMBR的哟从一个卵形形状成蜗杆6的细长形状。腹侧外壳描述了这些形态发生的事件之一，当腹侧的表皮细胞向腹侧中线迁移以覆盖胚胎的腹侧表面（ 图1）。首先，两对位于前前缘细胞的迁移对腹中线，在那里他们坚持和保险丝与对侧邻居6。其次是由后部位于口袋的细胞，从而形成楔形形状像创建腹侧口袋6-7的迁移。关闭所述口袋的机制还不是很清楚。一种可能性是，一个supracellular肌动球蛋白收缩结构领带口袋细胞一起在一个荷包喜欢时尚，类似伤口愈合8。有趣的是，一些袖珍细胞的迁移是由相关的神经母细胞9（神经元前体被发现的epidermi下方的特定亚群介导的秒; 图1B）。 以前的研究表明，神经母细胞调节腹侧表皮细胞的迁移和腹侧外壳。VAB-1（肝配蛋白受体）和VAB-2（肝配蛋白配体）高度表达在神经母细胞并彼此促进前部和后部的成神经细胞的分选，并在VAB-1或VAB-2引起腹外壳突变表型10-13。然而，启动子拯救实验表明，VAB-1，也需要在上覆的腹面的表皮细胞和受体的神经细胞分泌其它的指导线索被表达在腹侧表皮细胞9。虽然在任何这些受体的突变可导致腹侧壳体的表型，这是不明确的，如果出现缺陷，由于在成神经细胞的定位或者由于腹侧表皮细胞未能对引导反应的杆14的问题。改变神经母细胞无线网络的划分thout影响其分泌的指导线索的能力可能揭示神经细胞的作用，使她们在表皮形态提供机械输入能力的光。最近，人们发现，一个细胞分裂基因，ANI-1（的Anillin）高度表达在神经母细胞（ 图2A）和其耗尽导致神经细胞分裂缺陷。有趣的是，这些胚胎腹侧显示表型机柜（Fotopoulos，Wernike和Piekny，未发表意见）。 的Anillin是必需的细胞分裂，特别是对于细胞分裂，它描述了该过程，其中一个母细胞物理地划分成两个子细胞。胞质分裂是由肌动球蛋白收缩环，这需要进行严格控制在空间和时间，以确保它是正确加上姐妹染色单体偏析的形成驱动。后生动物胞质分裂的主调节器是RhoA蛋白（ 线虫 RHO-1），小GTP酶是一在莫如其GTP结合形式。环境基金Ect2/ECT-2激活的RhoA，在这之后的RhoA-GTP与形成收缩环并介导其侵入15下游效应相互作用。的Anillin是，通过其C-末端并通过其N-末端肌动蛋白和肌球蛋白结合的RhoA的多结构域蛋白。的Anillin需要稳定在哺乳动物或果蝇S2细胞16的收缩环的位置。的Anillin枯竭导致收缩环经过横向振荡，和胞质最终失败形成多核细胞17-19。有趣的是，尽管C。线虫ANI-1坐标肌动球蛋白收缩在早期胚胎，它不是对胞质分裂至关重要。然而，如上所述，ANI-1是在中胚（Fotopoulos，Wernike和Piekny，未发表的观察结果）所需的成神经细胞的胞质。胞质分裂失败会改变神经细胞的数量和位置，并可能影响到禄化学指导线索振动性，或者它可能会改变组织的机械性能。这两款机型突出对神经母细胞腹侧外壳的非自治作用，组织 - 组织沟通的胚胎发育过程中的重要性。 该实验方案介绍了如何像细胞分裂过程中C.线虫用荧光显微镜中胚。多数实验研究细胞分裂的机制是在内部培养皿（ 例如 ，海拉或S2细胞）的单细胞或早期胚胎细胞与数量有限的（ 如线虫的单细胞胚胎， 非洲爪蟾 ，或棘皮动物进行胚胎）。然而，重要的是要同时组织中研究细胞分裂，因为有外部线索可以影响分割平面的时机和位置。此外，细胞可以提供化学或机械线索来影响邻近组织的发展s，而它，了解细胞间的沟通有助于组织发展过程中，形成很重要。

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			<td height="21" style="height:21px;width:291px;">Agar</td>
			<td style="width:247px;">BioShop Canada Inc.</td>
			<td style="width:185px;">#AGR001.1</td>
			<td style="width:335px;">For making C. elegans NGM and RNAi plates</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Agar</td>
			<td>Bio Basic Inc.</td>
			<td>#9002-18-0</td>
			<td>For making bacteria LB agar plates</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Agarose</td>
			<td>BioShop Canada Inc.</td>
			<td>#AGA001.500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Anti-mouse Alexa 488 antibody</td>
			<td>Life Technologies Corporation (Invitrogen)</td>
			<td>#A11029</td>
			<td></td>
		</tr>
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			<td height="21" style="height:21px;">Anti-mouse anti-GFP antibody</td>
			<td>Roche Applied Science</td>
			<td>#11814460001</td>
			<td></td>
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			<td height="21" style="height:21px;">Anti-rabbit Alexa 568 antibody</td>
			<td>Life Technologies Corporation (Invitrogen)</td>
			<td>#A11011</td>
			<td></td>
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			<td height="21" style="height:21px;">Ampicillin</td>
			<td>BioShop Canada Inc.</td>
			<td>#AMP201.5</td>
			<td>Store powder at 4 &#176;C and dissolved ampicillin at -20 &#176;C</td>
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		<tr height="21">
			<td height="21" style="height:21px;">Bactopetone&#160; (peptone-A)</td>
			<td>Bio Basic Inc.</td>
			<td>#G213</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">CaCl2 (calcium chloride)</td>
			<td>BioShop Canada Inc.</td>
			<td>#C302.1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cholesterol</td>
			<td>BioShop Canada Inc.</td>
			<td>#CHL380.25</td>
			<td>Dissolve in ethanol</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DAPI&#160;</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>#D9542</td>
			<td>Use to stain nucleic acids (DNA)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Glycerol</td>
			<td>BioShop Canada Inc.</td>
			<td>#GLY001.1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">IPTG (isopropylthio-&#946;-galactoside)</td>
			<td>Bio Basic Inc.</td>
			<td>#367-93-1</td>
			<td>Store powder and dissolved IPTG at -20 &#176;C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">KH2PO4 (potassium phosphate, monobasic)</td>
			<td>BioShop Canada Inc.</td>
			<td>#PPM666.1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">K2HPO4 (potassium phosphate, dibasic)</td>
			<td>BioShop Canada Inc.</td>
			<td>#PPD303.1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">L4440&#160; (feeding vector)</td>
			<td>Addgene</td>
			<td>#1654</td>
			<td>Keep as glycerol stock at -80 &#176;C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MgSO4&#160;&#160; (magnesium sulfate)</td>
			<td>BioShop Canada Inc.</td>
			<td>#MAG511.500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NaCl (sodium chloride)</td>
			<td>Bio Basic Inc.</td>
			<td>#7647-14-5</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Na2HPO4 (sodium phosphate, dibasic)</td>
			<td>Bio Basic Inc.</td>
			<td>#7558-79-4</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Normal Donkey Serum (NDS)</td>
			<td>Wisent Bioproducts</td>
			<td>#035-110</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">n-propyl 3.4.5-trihydroxybenzoate (propyl gallate)</td>
			<td>Alfa Aesar</td>
			<td>#A10877</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Poly-L-lysine</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>#P8920</td>
			<td>For optimal results coat microscope slides three times&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Streptomycin</td>
			<td>BioShop Canada Inc.</td>
			<td>#STP101.50</td>
			<td>Store powder at 4 &#176;C and dissolved streptomycin at -20 &#176;C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tetracyclin</td>
			<td>BioShop Canada Inc.</td>
			<td>#TET701.10</td>
			<td>Store powder at 4 &#176;C and dissolved tetracycline at -20 &#176;C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tween-20</td>
			<td>Bio Basic Inc.</td>
			<td>CAS#9005-64-5</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tryptone</td>
			<td>BioShop Canada Inc.</td>
			<td>#TRP402.500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Yeast Extract</td>
			<td>Bio Basic Inc.</td>
			<td>#8013-01-2</td>
			<td></td>
		</tr>
	</tbody>
</table>

visualizing neuroblast cytokinesis during c. elegans embryogenesis

该协议描述了使用荧光显微镜的图像内开发秀丽隐杆线虫的胚胎分裂的细胞。特别是，这个协议的重点在于如何图像划分成神经细胞，这是发现了表皮细胞的下方和可以是用于表皮的形态很重要。组织的形成是后生动物发展是至关重要的，并依赖于外部线索来自邻近组织。C。线虫是一个很好的模式生物来研究组织形态发生在体内 ，由于其透明度和简单的组织，使得其组织容易通过显微镜来研究。腹侧外壳是其中胚胎的腹侧表面被上皮细胞单层的过程。此事件被认为是由底层的神经细胞，它提供化学品的指导线索进行调解覆上皮细胞的迁移提供便利。然而，成神经细胞是高度增殖和也可以充当作为机械基片的腹侧表皮细胞。使用此实验方案的研究可以在组织形成揭开间沟通的重要性，并且可以用来揭示发展组织中参与细胞分裂的基因的作用。

该实验方案描述了C。如何像细胞分裂线虫在胚胎发育中的胚胎。特别地，它描述了如何图象的成神经细胞，其可以促进表皮细胞的形态发生。表皮形态发生是由于表皮细胞形状的变化，迁移和黏附，而且还依赖于化学或机械线索来自相关神经母细胞（ 图1B）的组合。在神经母细胞分泌接收由受体腹表皮细胞，从而调节他们的迁移10,14,25的表面上的指导线索。此外...

Watch this Scientific Journal Video about Visualizing Neuroblast Cytokinesis During C. elegans Embryogenesis at JoVE.com

Visualizing Neuroblast Cytokinesis During C. elegans Embryogenesis

本协议描述了如何在图像秀丽隐杆线虫的胚胎组织中分裂的细胞。虽然一些协议描述了如何像细胞分裂在胚胎早期，这个协议描述了如何在图像中胚胎发育晚期一个发展中的组织内的细胞分裂。

可视化的神经细胞胞质分裂过程中<em&gt; C。线虫</em&gt;胚胎

This protocol describes the use of fluorescence microscopy to image dividing cells within developing Caenorhabditis elegans embryos. In particular, this ...

visualizing-neuroblast-cytokinesis-during-c-elegans-embryogenesis

Research

JoVE Journal

Neuroscience

Visualizing Neuroblast Cytokinesis During C. elegans Embryogenesis

11.7K 次观看.  Concordia University. 本协议描述了如何在图像秀丽隐杆线虫的胚胎组织中分裂的细胞。虽然一些协议描述了如何像细胞分裂在胚胎早期，这个协议描述了如何在图像中胚胎发育晚期一个发展中的组织内的细胞分裂。 