我们感谢马里奥de博诺 (分子生物学, 剑桥, 英国), 肯尼思 j. Kemphues (康奈尔大学, 伊萨卡, 美国), 米歇尔 Labouesse (Institut de Biologie 巴黎塞纳河, 大学皮埃尔和居里夫人, 巴黎, 法国),搜索米修 (大学de雷恩 1, 雷恩, 法国) 和 CGC, 由 NIH 研究基础设施项目 (P40 OD010440) 资助, 为菌株和抗体。这项工作得到了资助, NIH GM078653, MGH 是224570和223809至 iii

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作者声明他们没有竞争的金融利益。

本协议描述了如何将标准的功能遗传/rna 干扰和成像 (标记和显微) 方法结合起来, 利用线虫小肠上皮作为体内的视觉和分子解剖模型偏光膜和腔生物。
标签
本协议着重于抗体染色。原位抗体标记是一种高度特定的替代方法, 通过荧光融合蛋白 (在排泄管膜生物的附文中描述)2。虽然抗体染色不允许活体成?...

细胞及亚细胞不对称的产生, 如极化膜域的形成, 对于组织和器官的形态和功能是至关重要的1。极化膜生物在上皮细胞中的研究仍然是一个技术上的挑战, 因为在亚单位成分分配的方向性变化取决于多个连续和重合的胞外和胞内信号很难分离在大多数模型和强烈依赖于模型系统。这里提出的模型-单线虫线虫肠道-是一个精致的简单组织。与单细胞的c. 线虫排泄管 (见附纸的极化膜生物在c. 线虫排泄管)2, 它提供了几个独特的优势, 以识别和极化膜生物所需分子的表征。从酵母到人类的分子极性提示的保护使这个简单的无脊椎动物器官成为优秀的 "体内组织室", 以解决与人类系统直接相关的上皮极性问题, 这仍然是非常复杂的, 允许在单个单元格级别在体内对这些事件进行可视化剥离。
虽然多个保守的极性提示从 (1) 外矩阵, (2) 血浆膜及其交界处, (3) 胞内水泡贩运已被确定为3, 其基本的原理是它们在过程中集成极化上皮膜和组织生物不太了解4。经典的单细胞在体内模型 (e.g.S. 酵母和C. 线虫合子) 在确定极化细胞分裂和前后极性的原则方面发挥了重要作用, 并确定了关键膜相关的极性决定因素 (小 GTPases/CDC-42, 分区瑕疵的部)5,6, 但它们依赖于唯一的对称折断提示 (芽疤, 精子进入) 和缺乏连接保护apicobasal 膜域和, 想必, 相应的胞内 apicobasal 分拣机械。然而, 我们目前对组织的极化贩运上皮的知识, 主要依赖于哺乳动物2D 单一7, 缺乏生理细胞外和发展的线索, 可以改变膜的位置贩运轨道的领域和方向 (从2D 到3D 的一个开关,在体外培养系统仅能在 MDCK (Madin-达比犬肾脏) 细胞中逆转膜极性)8。在体内无脊椎动物模型生物体的上皮极性的发育研究最初在扁平上皮中进行, 例如在果蝇表皮, 在那里他们确定了关键的贡献为极性细胞迁移和细胞板料运动的接合动力学9, 并且吞贩卖为极端维护10。3D体外和体内分析了 MDCK 细胞中管状上皮的腔内形态发生, 并分别在线虫肠道中, 最近确定了细胞内贩运的要求. 从头(心尖) 域和流明生物和定位11,12,13。管状 (相对扁平) 上皮细胞的厚度是一个优势, 3D 分析的亚单位不对称, 因为它允许卓越的视觉区分顶端腔膜, apico 侧结, 侧膜, 和细胞器的位置。对于这些视觉优势, C. 线虫模型添加了体内设置、发育轴、透明度、人体计划的简单性、不变和定义的细胞谱系、分析 (遗传) 和下面描述的其他优点。
线虫本身是管状结构的蛔虫, 其透明和简单的结构使其同样管状的内部器官直接进入管和流明形态发生的可视化分析。它的肚腑的二十细胞 (偶尔地21或22个细胞)14从一个单一的祖细胞 (E) 获得, 并且从一个双层上皮发育成一个双侧对称的九 INT 环管 (四个细胞在第一圈;图 1示意图)14,15,16。肠道的谱系和组织分析, 最初由 Nomarski 光学通过核身份17和随后通过荧光显微镜通过标记的膜, 提供了重要的洞察力的形态发生,特别是细胞自治和细胞自治的要求为它的定向细胞分裂和运动 (例如, 插层, 左不对称, 前和后管旋转)14,18.早期胚细胞规范和基因调控网络控制该克隆模型器官的发育, 具有良好的特征19,20。然而, 这里的重点是分析单管细胞中的极化膜和腔生物, 以及伴随这个过程的 endomembranes、骨架结构和细胞器的胞内不对称。这一分析是便利的简单的这一管, 所有顶端膜 (在超微结构的水平, 由绒毛) 面对的流明和所有基底膜面对外管表面, 与侧膜接触对方,由接合处与顶端膜分离 (图 1示意图; 为C. 线虫特定组织的紧密和黏着连接组件的参考 (16,21)。顶端膜生物与腔内形态形成重合。此外, 成年小肠细胞的大小-这个小动物的最大的细胞 (除排泄细胞外)-近似哺乳动物细胞的大小, 允许在体内对亚单位元素的视觉跟踪,例如囊泡轨迹, 通常是在培养皿中尝试体外。
对于这种细胞和亚型分析的目的, 适当的标签是至关重要的。肠内-或等离子膜领域, 结, 骨架结构, 细胞核和其他亚细胞细胞器可以通过标记其特定的分子成分来可视化。许多此类组件已被定性并继续被发现 (表 1提供了几个示例并引用了资源)。例如, 不同的分子区分管状和/或水泡室的肠道膜系统, 从 ER 到高尔基通过后高尔基囊泡到等离子膜, 已被确定为22。特定的蛋白质 (以及脂质和糖) 可以直接标记, 或通过结合蛋白间接。本协议侧重于原位固定标本的抗体染色, 这是两种标准标记技术之一 (参见排泄运河 tubulogenesis 的附文, 用于描述其他技术2 -体内标记通过荧光蛋白融合-这是直接适用于肠道;Table2提供了一些肠道特异促进剂的例子, 可以用来驱动这种融合蛋白在肠道中的表达。双或多个标签的两种方法, 或结合额外的化学染色, 允许更大的 in-depth 视觉分辨率和检查的空间和时间的变化, 在共存和招募特定分子或亚细胞组分 (图 2)。本协议中描述的固定和染色程序支持在免疫过程中保存绿色荧光蛋白 (GFP) 标记。对于成像, 通过标准显微程序 (荧光解剖和共聚焦显微镜) 检测和表征 tubulogenesis 表型的关键点被描述(图 3, 4)。这些可以扩展到更高的分辨率成像方法, 例如超显微镜和透射电子显微镜 (这里没有描述)。
这个系统的一个关键力量是能够在不同发育阶段的个体细胞中分析极性, 从胚胎发育到成年。例如, 顶端膜领域和流明生物可以跟踪整个开发在单细胞水平通过标记与 ERM-1, 高度保守的膜肌动蛋白链接器的 Ezrin-Radixin-Moesin 家族23,24.ERM-1 形象化顶端膜生物 (1) 在胚胎管形态发生时, 当它伴随极化细胞分裂和迁移 (细胞移动顶部在流明期间在插层期间)15;(2) 在晚期胚胎和幼体管延长期间, 在没有细胞分裂或迁移的情况下进行;和 (3) 在成人肠道, 在那里的极化膜域保持 (图 1)。在扩张的有丝分裂幼虫上皮, 从头极化膜生物因此可以从极化组织形态发生分离, 这是不可能在大多数 体内和体外上皮极性模型,包括具有单细胞分辨率的 (例如3D MDCK 囊肿模型8)。对于其他成分的标签, 这个设置提供了机会 (特别是在 L1 幼虫阶段, 当细胞具有较高的细胞质/核比), 以区分这些细胞内的变化是特定的极化膜生物 (例如重新定向贩运的轨迹), 使之与极化细胞分裂和迁移所需的同时进行。
遗传多样性的线虫是众所周知的25, 并使它成为一个强大的模型系统的分子分析的任何生物问题。例如, 对形态发生的研究可以从一种野生型菌株开始, 这种转基因菌株的结构利益 (例如膜) 是用荧光标记标记的, 或者是有缺陷的功能突变体。结构.一个典型的反向遗传研究可能会产生一个突变体, 在系 (例如被目标删除) 中删除感兴趣的基因 (通常是产生点突变, 随之而来的损失、减少或增加功能的基因), 或者它的转录减少的 rna 干扰。在线虫26中, 通过喂养方便地进行 rna 干扰, 还有助于设计有针对性的屏幕, 以检查更大的一组感兴趣的基因。一个遗传模型生物体可以说是最大的力量是能够进行在体内前向屏幕 (如诱变, 系统或全基因组的 rna 干扰屏幕), 允许公正的调查分子原因的表型兴趣.例如, 一个无偏的视觉线虫rna 干扰 tubulogenesis 屏幕, 开始与 ERM-1-labeled 顶端膜的转基因动物, 发现了一个有趣的可逆性肠道极性转换和异位腔表型, 使用这里是这类分析的一个例子。这个屏幕确定了糖的损耗 (GSLs; 强制膜脂质, 通过他们的生物合成酶鉴定) 和囊泡涂层的组分格和它的 AP-1 适配器作为特定分子瑕疵导致这个极性转换表型, 从而将这些贩运分子定性为在体内提示心尖膜极性和流明定位12,13。当从特定的基因突变/形态发生表型开始时, 这样的屏幕 (或单一的基因/rna 干扰交互实验) 也可以检查两个或多个感兴趣的基因之间的功能相互作用 (参见随附的纸张排泄运河为这样分析的例子)2。该协议的重点是 rna 干扰, 除了它的能力, 以直接确定的基因, 其损失导致表型在前屏幕, 提供了具体的优势, 分析的形态发生。由于基因产物对形态发生的作用通常以剂量依赖性的方式起作用, 所以 rna 干扰通常是成功地生成了一个表型的频谱。产生信息性部分功能表型的能力也有助于解决大多数重要的 tubulogenesis 基因是必不可少的, 它们的损失导致不孕和早期胚胎致死的问题。该协议包括有条件的 rna 干扰策略, 以克服这一困难, 并建议的方法, 以优化生成更广泛的表型, 如位系列产生的突变。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr >
			<td>Antibody staining</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td>poly-L-lysine</td>
			<td>Sigma</td>
			<td>P5899</td>
			<td></td>
		</tr>
		<tr >
			<td>Methanol</td>
			<td>Fisher Scientific</td>
			<td>A452-4</td>
			<td></td>
		</tr>
		<tr >
			<td>Acetone</td>
			<td>Fisher Scientific</td>
			<td>A949SK-4</td>
			<td></td>
		</tr>
		<tr >
			<td>Tween</td>
			<td>Fisher Scientific</td>
			<td>50-213-612</td>
			<td></td>
		</tr>
		<tr >
			<td>Permount</td>
			<td>Fisher Scientific</td>
			<td>SP15-100</td>
			<td></td>
		</tr>
		<tr >
			<td>Powdered milk</td>
			<td>Sigma</td>
			<td>MT409-1BTL</td>
			<td></td>
		</tr>
		<tr >
			<td>Primary antibodies</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td>MH27 (mouse)</td>
			<td></td>
			<td></td>
			<td>Concentration: 1:20 Resources: Developmental Studies Hybridoma Bank.</td>
		</tr>
		<tr >
			<td>MH33 (mouse)</td>
			<td></td>
			<td></td>
			<td>Concentration: 1:10 Resources: Developmental Studies Hybridoma Bank.</td>
		</tr>
		<tr >
			<td>anti-ICB4 (rabbit)</td>
			<td></td>
			<td></td>
			<td>Concentration: 1:5 Resources: A gift from MariodeBono (Medical Research Council, England)</td>
		</tr>
		<tr >
			<td>anti-PAR-3 (rabbit)</td>
			<td></td>
			<td></td>
			<td>Concentration: 1:50 Resources: A gift from Kenneth J. Kemphues (Cornell University)</td>
		</tr>
		<tr >
			<td>Secondary antibodies</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td>Alexa Floor 568 (anti-rabbit)</td>
			<td>ABCam</td>
			<td>AB175471</td>
			<td>Concentration: 1:200</td>
		</tr>
		<tr >
			<td>Cy5 (anti-mouse)</td>
			<td>Life technologies</td>
			<td>A10524</td>
			<td>Concentration: 1:200</td>
		</tr>
		<tr >
			<td>TRITC (anti-rabbit)</td>
			<td>Invitrogen</td>
			<td>T2769</td>
			<td>Concentration: 1:200</td>
		</tr>
		<tr >
			<td>FITC (anti-mouse)</td>
			<td>Sigma</td>
			<td>F9006</td>
			<td>Concentration: 1:100</td>
		</tr>
		<tr >
			<td>Labeled chemicals</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td>Texas Red-Phalloidin</td>
			<td></td>
			<td></td>
			<td>Concentration: 1:100 Resources: Molecular Probes-T7471</td>
		</tr>
		<tr >
			<td>Materials</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td>Vacuum Grease Silicone</td>
			<td>Beckman</td>
			<td>335148</td>
			<td></td>
		</tr>
		<tr >
			<td>Microscope slides</td>
			<td>Fisher Scientific</td>
			<td>4448</td>
			<td></td>
		</tr>
		<tr >
			<td>Microscope coverslips (22x22-1)</td>
			<td>Fisher Scientific</td>
			<td>12-542-B</td>
			<td></td>
		</tr>
		<tr >
			<td>C. elegans related</td>
			<td></td>
			<td></td>
			<td>see reference29 for standardC. elegans culture and maintenance procedures.</td>
		</tr>
		<tr >
			<td>LB Medium and plates</td>
			<td></td>
			<td></td>
			<td>see reference29 for protocols.</td>
		</tr>
		<tr >
			<td>Tryptone</td>
			<td>Acros Organics</td>
			<td>611845000</td>
			<td></td>
		</tr>
		<tr >
			<td>Yeast Extract</td>
			<td>BD Biosciences</td>
			<td>212750</td>
			<td></td>
		</tr>
		<tr >
			<td>NaCl</td>
			<td>Sigma</td>
			<td>S7653</td>
			<td></td>
		</tr>
		<tr >
			<td>Bacto Agar</td>
			<td>BD Biosciences</td>
			<td>214040</td>
			<td></td>
		</tr>
		<tr >
			<td>Ampicillin</td>
			<td>Sigma</td>
			<td>A0116</td>
			<td></td>
		</tr>
		<tr >
			<td>Tetracycline</td>
			<td>Fisher Scientific</td>
			<td>BP912</td>
			<td></td>
		</tr>
		<tr >
			<td>M9 Medium</td>
			<td></td>
			<td></td>
			<td>see reference29 for protocols.</td>
		</tr>
		<tr >
			<td>NaCl</td>
			<td>Sigma</td>
			<td>S7653</td>
			<td></td>
		</tr>
		<tr >
			<td>KH2PO4</td>
			<td>Sigma</td>
			<td>P0662</td>
			<td></td>
		</tr>
		<tr >
			<td>Na2HPO4</td>
			<td>Sigma</td>
			<td>S7907</td>
			<td></td>
		</tr>
		<tr >
			<td>MgSO4</td>
			<td>Sigma</td>
			<td>M2773</td>
			<td></td>
		</tr>
		<tr >
			<td>NGM plates</td>
			<td></td>
			<td></td>
			<td>see reference29 for protocols.</td>
		</tr>
		<tr >
			<td>NaCl</td>
			<td>Sigma</td>
			<td>S7653</td>
			<td></td>
		</tr>
		<tr >
			<td>Peptone</td>
			<td>BD Biosciences</td>
			<td>211677</td>
			<td></td>
		</tr>
		<tr >
			<td>Tryptone</td>
			<td>Acros Organics</td>
			<td>611845000</td>
			<td></td>
		</tr>
		<tr >
			<td>Bacto Agar</td>
			<td>BD Biosciences</td>
			<td>214040</td>
			<td></td>
		</tr>
		<tr >
			<td>MgSO4</td>
			<td>Sigma</td>
			<td>M2773</td>
			<td></td>
		</tr>
		<tr >
			<td>CaCl2</td>
			<td>Sigma</td>
			<td>C3881</td>
			<td></td>
		</tr>
		<tr >
			<td>Cholesterol</td>
			<td>Sigma</td>
			<td>C8667</td>
			<td></td>
		</tr>
		<tr >
			<td>K2HPO4</td>
			<td>Sigma</td>
			<td>P3786</td>
			<td></td>
		</tr>
		<tr >
			<td>KH2PO4</td>
			<td>Sigma</td>
			<td>P0662</td>
			<td></td>
		</tr>
		<tr >
			<td>RNAi plates</td>
			<td></td>
			<td></td>
			<td>see reference30 for protocols.</td>
		</tr>
		<tr >
			<td>NaCl</td>
			<td>Sigma</td>
			<td>S7653</td>
			<td></td>
		</tr>
		<tr >
			<td>Peptone</td>
			<td>BD Biosciences</td>
			<td>211677</td>
			<td></td>
		</tr>
		<tr >
			<td>Tryptone</td>
			<td>Acros Organics</td>
			<td>611845000</td>
			<td></td>
		</tr>
		<tr >
			<td>Bacto Agar</td>
			<td>BD Biosciences</td>
			<td>214040</td>
			<td></td>
		</tr>
		<tr >
			<td>MgSO4</td>
			<td>Sigma</td>
			<td>M2773</td>
			<td></td>
		</tr>
		<tr >
			<td>CaCl2</td>
			<td>Sigma</td>
			<td>C3881</td>
			<td></td>
		</tr>
		<tr >
			<td>Cholesterol</td>
			<td>Sigma</td>
			<td>C8667</td>
			<td></td>
		</tr>
		<tr >
			<td>K2HPO4</td>
			<td>Sigma</td>
			<td>P3786</td>
			<td></td>
		</tr>
		<tr >
			<td>KH2PO4</td>
			<td>Sigma</td>
			<td>P0662</td>
			<td></td>
		</tr>
		<tr >
			<td>IPTG</td>
			<td>US Biological</td>
			<td>I8500</td>
			<td></td>
		</tr>
		<tr >
			<td>Carbenicillin</td>
			<td>Fisher Scientific</td>
			<td>BP2648</td>
			<td></td>
		</tr>
		<tr >
			<td>NaOH</td>
			<td>Fisher Scientific</td>
			<td>SS266-1</td>
			<td></td>
		</tr>
		<tr >
			<td>Sodium hypochlorite</td>
			<td>Fisher Scientific</td>
			<td>50371500</td>
			<td></td>
		</tr>
		<tr >
			<td>Bacteria</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td>OP50 bacteria</td>
			<td>CGC</td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td>HT115 bacteria</td>
			<td>CGC</td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td>Genome-wide RNAi libraries Ahringer genome-wide RNAi feeding library (ref 30,49,50)</td>
			<td>Source BioScience</td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td>C. elegans ORF-RNAi feeding library (ref51)</td>
			<td>Source BioScience</td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td>Imaging related</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td>Sodium azide</td>
			<td>Fisher Scientific</td>
			<td>BP9221-500</td>
			<td></td>
		</tr>
		<tr >
			<td>Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td>dissecting microscope</td>
			<td>Nikon</td>
			<td>SMZ-U</td>
			<td></td>
		</tr>
		<tr >
			<td>dissecting microscope equipped with a high-power stereo fluorescence attachment (Kramer Scientific), CCD camera with Q capture software and X-Cite fluorescent lamp (Photonic Solutions)</td>
			<td>Olympus</td>
			<td>SZX12</td>
			<td></td>
		</tr>
		<tr >
			<td>Laser-scanning confocal microscope</td>
			<td>Leica Microsystem</td>
			<td>TCS SL</td>
			<td></td>
		</tr>
		<tr >
			<td>laser-scanning confocal mounted on an ECLIPSE Ti-E inverted microscope</td>
			<td>Nikon</td>
			<td>C2</td>
			<td></td>
		</tr>
	</tbody>
</table>

the c. elegans intestine as a model for intercellular lumen morphogenesis and in vivo polarized membrane biogenesis at the single-cell level: labeling by antibody staining, rnai loss-of-function analysis and imaging

多细胞管, 所有内脏的基本单位, 由极化上皮或内皮细胞组成, 顶端膜内衬的流明和侧膜接触对方和/或细胞外基质。在器官形态发生过程中, 这种独特的膜不对称是如何建立和维持的仍然是一个尚未解决的细胞生物学问题。本协议将线虫小肠描述为一个模型, 用于分析管状形态发生中的极化膜生物, 重点是心尖膜和腔生物。C. 线虫二十细胞单肠上皮被安排成一个简单的双边对称管, 允许在单细胞水平上进行分析。在早期胚胎发生过程中, 细胞膜极化伴随着极化细胞分裂和迁移, 但当细胞不再增殖和移动时, de 从头极化膜生物继续贯穿幼虫生长。后一种设置允许一个分离亚细胞的变化, 同时调解这些不同的极化过程, 很难区分在大多数极性模型。顶端, 侧膜-, 连接, 骨架和膜成分可以标记和跟踪整个开发中的 GFP 融合蛋白, 或评估的原位抗体染色。与生物体的遗传多样性, C. 线虫小肠因此提供了一个独特的体内模型的视觉, 发展, 和分子遗传学分析极化膜和管生物。这里描述的具体方法 (所有标准) 包括如何: 通过抗体染色对肠亚细胞成分进行标记;通过功能 tubulogenesis 基因的研究, 分析极化膜生物中涉及的基因;评估不同发育阶段的极性缺陷;用荧光、差分干涉对比 (DIC) 和共聚焦显微镜解释表型;量化视觉缺陷。该协议可以用来分析任何通常高度保守的分子涉及上皮极性, 膜生物, 管和流明形态发生。

该协议描述了如何分子分析和可视化极化膜生物和流明形态发生在线虫肠道, 在单细胞和亚细胞水平。二十细胞单线虫小肠是由细胞分裂和迁移过程中形成的中期胚胎。在这个时候, 极化膜领域成为建立, 但de 从头极化膜生物继续在成熟, 但扩大上皮整个四幼体阶段, 直到成年, 允许集中分析极化膜生物 (图 1A)。
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Watch this Scientific Journal Video about The C. elegans Intestine As a Model for Intercellular Lumen Morphogenesis and In Vivo Polarized Membrane Biogenesis at the Single-cell Level: Labeling by Anti

The C. elegans Intestine As a Model for Intercellular Lumen Morphogenesis and In Vivo Polarized Membrane Biogenesis at the Single-cell Level: Labeling by Antibody Staining, RNAi Loss-of-function Analy

透明的C. 线虫小肠可作为 "体内组织室", 用于研究多细胞 tubulogenesis 中的单细胞和亚 apicobasal 膜和腔生物。本协议描述了如何结合标准标记, 功能的遗传/rna 干扰和微观的方法来解剖这些过程的分子水平。

C. 线虫小肠作为细胞间腔形态发生和体内极化膜生物单细胞水平的模型: 抗体染色、rna 干扰功能分析和成像标记

Multicellular tubes, fundamental units of all internal organs, are composed of polarized epithelial or endothelial cells, with apical membranes lining the ...

the-c-elegans-intestine-as-model-for-intercellular-lumen

Research

JoVE Journal

Developmental Biology

The C. elegans Intestine As a Model for Intercellular Lumen Morphogenesis and In Vivo Polarized Membrane Biogenesis at the Single-cell Level: Labeling by Antibody Staining, RNAi Loss-of-function Analysis and Imaging

13.3K 次观看.  Harvard Medical School. 透明的C. 线虫小肠可作为 "体内组织室", 用于研究多细胞 tubulogenesis 中的单细胞和亚 apicobasal 膜和腔生物。本协议描述了如何结合标准标记, 功能的遗传/rna 干扰和微观的方法来解剖这些过程的分子水平。

视频: The C. elegans Intestine As a Model for Intercellular Lumen Morphogenesis and In Vivo Polarized Membrane Biogenesis at the Single-cell Level: Labeling by Antibody Staining, RNAi Loss-of-function Analy

Imaging Cleared Embryonic and Postnatal Hearts at Single-cell Resolution

Single clonal tracing and analysis at the whole-heart level can determine cardiac progenitor cell behavior and differentiation during cardiac development, and allow for the study of the cellular and molecular basis of normal and abnormal cardiac morphogenesis. Recent emerging technologies of retrospective single clonal analyses make the study of cardiac morphogenesis at single cell resolution feasible. However, tissue opacity and light scattering of the heart as imaging depth is increased hinder whole-heart imaging at single cell resolution. To overcome these obstacles, a whole-embryo clearing system that can render the heart highly transparent for both illumination and detection must be developed. Fortunately, in the last several years, many methodologies for whole-organism clearing systems such as CLARITY, Scale, SeeDB, ClearT, 3DISCO, CUBIC, DBE, BABB and PACT have been reported. This lab is interested in the cellular and molecular mechanisms of cardiac morphogenesis. Recently, we established single cell lineage tracing via the ROSA26-CreERT2; ROSA26-Confetti system to sparsely label cells during cardiac development. We adapted several whole embryo-clearing methodologies including Scale and CUBIC (clear, unobstructed brain imaging cocktails and computational analysis) to clear the embryo in combination with whole mount staining to image single clones inside the heart. The heart was successfully imaged at single cell resolution. We found that Scale can clear the embryonic heart, but cannot effectively clear the postnatal heart, while CUBIC can clear the postnatal heart, but damages the embryonic heart by dissolving the tissue. The methods described here will permit the study of gene function at a single clone resolution during cardiac morphogenesis, which, in turn, can reveal the cellular and molecular basis of congenital heart defects.

We describe a protocol to volumetrically image fluorescent protein labeled cells deep inside intact embryonic and postnatal hearts. Utilizing tissue-clearing methods in combination with whole mount staining, single fluorescent protein-labeled cells inside an embryonic or postnatal heart can be imaged clearly and accurately.

成像清除胚胎和出生后心脏在单细胞分辨率

Single clonal tracing and analysis at the whole-heart level can determine cardiac progenitor cell behavior and differentiation during cardiac development, ...

科研

发育生物学

Methods to Examine the Lymph Gland and Hemocytes in Drosophila Larvae

Many parallels exist between the Drosophila and mammalian hematopoietic systems, even though Drosophila lack the lymphoid lineage that characterize mammalian adaptive immunity. Drosophila and mammalian hematopoiesis occur in spatially and temporally distinct phases to produce several blood cell lineages. Both systems maintain reservoirs of blood cell progenitors with which to expand or replace mature lineages. The hematopoietic system allows Drosophila and mammals to respond to and to adapt to immune challenges. Importantly, the transcriptional regulators and signaling pathways that control the generation, maintenance, and function of the hematopoietic system are conserved from flies to mammals. These similarities allow Drosophila to be used to genetically model hematopoietic development and disease.
Here we detail assays to examine the hematopoietic system of Drosophila larvae. In particular, we outline methods to measure blood cell numbers and concentration, visualize a specific mature lineage in vivo, and perform immunohistochemistry on blood cells in circulation and in the hematopoietic organ. These assays can reveal changes in gene expression and cellular processes including signaling, survival, proliferation, and differentiation and can be used to investigate a variety of questions concerning hematopoiesis. Combined with the genetic tools available in Drosophila, these assays can be used to evaluate the hematopoietic system upon defined genetic alterations. While not specifically outlined here, these assays can also be used to examine the effect of environmental alterations, such as infection or diet, on the hematopoietic system.

Methods to Examine the Lymph Gland and Hemocytes in Drosophila Larvae

Drosophila and mammalian hematopoietic systems share many common features, making Drosophila an attractive genetic model to study hematopoiesis. Here we demonstrate dissection and mounting of the major larval hematopoietic organ for immunohistochemistry. We also describe methods to assay various larval hematopoietic compartments including circulating hemocytes and sessile crystal cells.

方法来检查淋巴腺和血细胞中<em&gt;果蝇</em&gt;幼虫

Many parallels exist between the Drosophila and mammalian hematopoietic systems, even though Drosophila lack the lymphoid lineage that characterize ...

Spatial and Temporal Analysis of Active ERK in the C. elegans Germline

The evolutionarily conserved&#160;extracellular signal transducing RTK-RAS-ERK pathway is an important kinase-signaling cascade that controls multiple cellular and developmental processes principally via activation of ERK, the terminal kinase of the pathway. Tight regulation of ERK activity is essential for normal development and homeostasis; overly active ERK results in excessive cellular proliferation, while underactive ERK causes cell death. C. elegans is a powerful model system that has helped characterize the function and regulation of RTK-RAS-ERK signaling pathway during development. In particular, the RTK-RAS-ERK pathway is essential for C. elegans germline development, which is the focus of this method. Using antibodies specific to the active, diphosphorylated form of ERK (dpERK), the stereotypical localization pattern can be visualized within the germline. Because this pattern is both spatially and temporally controlled, the ability to reproducibly assay dpERK is useful to identify regulators of the pathway that affect dpERK signal duration and amplitude and thus germline development. Here we demonstrate how to successfully dissect, stain, and image dpERK within the C. elegans gonad. This method can be adapted for spatial localization of any signaling or structural protein in the C. elegans gonad, provided an antibody compatible with immunofluorescence is available.

Spatial and Temporal Analysis of Active ERK in the C. elegans Germline

We present an immunofluorescence&#160;imaging-based method for spatial and temporal localization of active ERK in the dissected C. elegans gonad. The protocol described here can be adapted for visualization of any signaling or structural protein in the C. elegans gonad, provided a suitable antibody reagent is available.

在Active ERK的时空分析<em&gt;℃。线虫</em&gt;生殖系

The evolutionarily conserved&#160;extracellular signal transducing RTK-RAS-ERK pathway is an important kinase-signaling cascade that controls multiple ...

Using In Vivo and Tissue and Cell Explant Approaches to Study the Morphogenesis and Pathogenesis of the Embryonic and Perinatal Aorta

The aorta is the largest artery in the body. The aortic wall is composed of an inner layer of endothelial cells, a middle layer of alternating elastic lamellae and smooth muscle cells (SMCs), and an outer layer of fibroblasts and extracellular matrix. In contrast to the widespread study of pathological models (e.g., atherosclerosis) in the adult aorta, much less is known about the embryonic and perinatal aorta. Here, we focus on SMCs and provide protocols for the analysis of the morphogenesis and pathogenesis of embryonic and perinatal aortic SMCs in normal development and disease. Specifically, the four protocols included are: i) in vivo embryonic fate mapping and clonal analysis; ii) explant embryonic aorta culture; iii) SMC isolation from the perinatal aorta; and iv) subcutaneous osmotic mini-pump placement in pregnant (or non-pregnant) mice. Thus, these approaches facilitate the investigation of the origin(s), fate, and clonal architecture of SMCs in the aorta in vivo. They allow for modulating embryonic aorta morphogenesis in utero by continuous exposure to pharmacological agents. In addition, isolated aortic tissue explants or aortic SMCs can be used to gain insights into the role of specific gene targets during fundamental processes such as muscularization, proliferation, and migration. These hypothesis-generating experiments on isolated SMCs and the explanted aorta can then be assessed in the in vivo context through pharmacological and genetic approaches.

Using In Vivo and Tissue and Cell Explant Approaches to Study the Morphogenesis and Pathogenesis of the Embryonic and Perinatal Aorta

Protocols for studying the embryonic and perinatal murine aorta using in vivo clonal analysis and fate mapping, aortic explants, and isolated smooth muscle cells are detailed here. These diverse approaches facilitate the investigation of the morphogenesis of the embryonic and perinatal aorta in normal development and the pathogenesis in disease.

使用体内和组织和细胞外植体方法研究胚胎和围产期主动脉的形态和发病机制

The aorta is the largest artery in the body. The aortic wall is composed of an inner layer of endothelial cells, a middle layer of alternating elastic ...

The C. elegans Excretory Canal as a Model for Intracellular Lumen Morphogenesis and In Vivo Polarized Membrane Biogenesis in a Single Cell: labeling by GFP-fusions, RNAi Interaction Screen and Imaging

The four C. elegans excretory canals are narrow tubes extended through the length of the animal from a single cell, with almost equally far extended intracellular endotubes that build and stabilize the lumen with a membrane and submembraneous cytoskeleton of apical character. The excretory cell expands its length approximately 2,000 times to generate these canals, making this model unique for the in vivo assessment of de novo polarized membrane biogenesis, intracellular lumen morphogenesis and unicellular tubulogenesis. The protocol presented here shows how to combine standard labeling, gain- and loss-of-function genetic or RNA interference (RNAi)-, and microscopic approaches to use this model to visually dissect and functionally analyze these processes on a molecular level. As an example of a labeling approach, the protocol outlines the generation of transgenic animals with fluorescent fusion proteins for live analysis of tubulogenesis. As an example of a genetic approach, it highlights key points of a visual RNAi-based interaction screen designed to modify a gain-of-function cystic canal phenotype. The specific methods described are how to: label and visualize the canals by expressing fluorescent proteins; construct a targeted RNAi library and strategize RNAi screening for the molecular analysis of canal morphogenesis; visually assess modifications of canal phenotypes; score them by dissecting fluorescence microscopy; characterize subcellular canal components at higher resolution by confocal microscopy; and quantify visual parameters. The approach is useful for the investigator who is interested in taking advantage of the C. elegans excretory canal for identifying and characterizing genes involved in the phylogenetically conserved processes of intracellular lumen and unicellular tube morphogenesis.

The C. elegans Excretory Canal as a Model for Intracellular Lumen Morphogenesis and In Vivo Polarized Membrane Biogenesis in a Single Cell: labeling by GFP-fusions, RNAi Interaction Screen and Imaging

The C. elegans excretory canal is a unique single-cell model for the visual in vivo analysis of de novo polarized membrane biogenesis. This protocol describes a combination of standard genetic/RNAi and imaging approaches, adaptable for the identification and characterization of molecules directing unicellular tubulogenesis, and apical membrane and lumen biogenesis.

C. 线虫排泄管作为细胞内腔形态发生和体内极化膜生物在单个细胞中的模型: GFP-融合、rna 干扰界面和成像的标记

The four C. elegans excretory canals are narrow tubes extended through the length of the animal from a single cell, with almost equally far extended ...

Dissection and Immunofluorescent Staining of Mushroom Body and Photoreceptor Neurons in Adult Drosophila melanogaster Brains

Nervous system development involves a sequential series of events that are coordinated by several signaling pathways and regulatory networks. Many of the proteins involved in these pathways are evolutionarily conserved between mammals and other eukaryotes, such as the fruit fly Drosophila melanogaster, suggesting that similar organizing principles exist during the development of these organisms. Importantly, Drosophila has been used extensively to identify cellular and molecular mechanisms regulating processes that are required in mammals including neurogenesis, differentiation, axonal guidance, and synaptogenesis. Flies have also been used successfully to model a variety of human neurodevelopmental diseases. Here we describe a protocol for the step-by-step microdissection, fixation, and immunofluorescent localization of proteins within the adult Drosophila brain. This protocol focuses on two example neuronal populations, mushroom body neurons and retinal photoreceptors, and includes optional steps to trace individual mushroom body neurons using Mosaic Analysis with a Repressible Cell Marker (MARCM) technique. Example data from both wild-type and mutant brains are shown along with a brief description of a scoring criteria for axonal guidance defects. While this protocol highlights two well-established antibodies for investigating the morphology of mushroom body and photoreceptor neurons, other Drosophila brain regions and the localization of proteins within other brain regions can also be investigated using this protocol.

Dissection and Immunofluorescent Staining of Mushroom Body and Photoreceptor Neurons in Adult Drosophila melanogaster Brains

This protocol describes the dissection and immunostaining of adult Drosophila melanogaster brain tissues. Specifically, this protocol highlights the use of Drosophila mushroom body and photoreceptor neurons as example neuronal subsets that can be accurately used to uncover general principles underlying many aspects of neuronal development.

成人果蝇脑内蘑菇体和感光神经元的解剖和荧光染色

Nervous system development involves a sequential series of events that are coordinated by several signaling pathways and regulatory networks. Many of the ...

Application of RNAi and Heat-shock-induced Transcription Factor Expression to Reprogram Germ Cells to Neurons in C. elegans

Studying the cell biological processes during converting the identities of specific cell types provides important insights into mechanism that maintain and protect cellular identities. The conversion of germ cells into specific neurons in the nematode Caenorhabditis elegans (C. elegans) is a powerful tool for performing genetic screens in order to dissect regulatory pathways that safeguard established cell identities. Reprogramming of germ cells to a specific type of neurons termed ASE requires transgenic animals that allow broad over-expression of the Zn-finger transcription factor (TF) CHE-1. Endogenous CHE-1 is expressed exclusively in two head neurons and is required to specify the glutamatergic ASE neurons fate, which can easily be visualized by the gcy-5prom::gfp reporter. A trans gene containing the heat-shock promoter-driven che-1 gene expression construct allows broad mis-expression of CHE-1 in the entire animal upon heat-shock treatment. The combination of RNAi against the chromatin-regulating factor LIN-53 and heat-shock-induced che-1 over-expression leads to reprogramming of germ cell into ASE neuron-like cells. We describe here the specific RNAi procedure and appropriate conditions for heat-shock treatment of transgenic animals in order to successfully induce germ cell to neuron conversion.

Application of RNAi and Heat-shock-induced Transcription Factor Expression to Reprogram Germ Cells to Neurons in C. elegans

This protocol describes how to study cellular processes during cell fate conversion in Caenorhabditis elegans in vivo. Using transgenic animals, allowing heat-shock promoter-driven overexpression of the neuron fate-inducing transcription factor CHE-1 and RNAi-mediated depletion of the chromatin-regulating factor LIN-53 germ cell to neuron reprogramming can be observed in vivo.

rna 干扰和热休克诱导转录因子表达在重编程生殖细胞到神经元中的应用C. 线虫

Studying the cell biological processes during converting the identities of specific cell types provides important insights into mechanism that maintain ...

CRISPR-mediated Loss of Function Analysis in Cerebellar Granule Cells Using In Utero Electroporation-based Gene Transfer

Brain malformation is often caused by genetic mutations. Deciphering the mutations in patient-derived tissues has identified potential causative factors of the diseases. To validate the contribution of a dysfunction of the mutated genes to disease development, the generation of animal models carrying the mutations is one obvious approach. While germline genetically engineered mouse models (GEMMs) are popular biological tools and exhibit reproducible results, it is restricted by time and costs. Meanwhile, non-germline GEMMs often enable exploring gene function in a more feasible manner. Since some brain diseases (e.g., brain tumors) appear to result from somatic but not germline mutations, non-germline chimeric mouse models, in which normal and abnormal cells coexist, could be helpful for disease-relevant analysis. In this study, we report a method for the induction of CRISPR-mediated somatic mutations in the cerebellum. Specifically, we utilized conditional knock-in mice, in which Cas9 and GFP are chronically activated by the CAG (CMV enhancer/chicken &#223;-actin) promoter after Cre-mediated recombination of the genome. The self-designed single-guide RNAs (sgRNAs) and the Cre recombinase sequence, both encoded in a single plasmid construct, were delivered into cerebellar stem/progenitor cells at an embryonic stage using in utero electroporation. Consequently, transfected cells and their daughter cells were labeled with green fluorescent protein (GFP), thus facilitating further phenotypic analyses. Hence, this method is not only showing electroporation-based gene delivery into embryonic cerebellar cells but also proposing a novel quantitative approach to assess CRISPR-mediated loss-of-function phenotypes.

CRISPR-mediated Loss of Function Analysis in Cerebellar Granule Cells Using In Utero Electroporation-based Gene Transfer

Conventional loss-of-function studies of genes using knockout animals have often been costly and time-consuming. Electroporation-based CRISPR-mediated somatic mutagenesis is a powerful tool to understand gene functions in vivo. Here, we report a method to analyze knockout phenotypes in proliferating cells of the cerebellum.

CRISPR 介导的小脑颗粒细胞在宫内电穿孔基因转移中的功能分析损失

Brain malformation is often caused by genetic mutations. Deciphering the mutations in patient-derived tissues has identified potential causative factors ...

Assessing Lysosomal Alkalinization in the Intestine of Live Caenorhabditis elegans

The nematode Caenorhabditis elegans (C. elegans) is a model system that is widely used to study longevity and developmental pathways. Such studies are facilitated by the transparency of the animal, the ability to do forward and reverse genetic assays, the relative ease of generating fluorescently labeled proteins, and the use of fluorescent dyes that can either be microinjected into the early embryo or incorporated into its food (E. coli strain OP50) to label cellular organelles (e.g. 9-diethylamino-5H-benzo(a)phenoxazine-5-one and (3-{2-[(1H,1&#39;H-2,2&#39;-bipyrrol-5-yl-kappaN(1))methylidene]-2H-pyrrol-5-yl-kappaN}-N-[2-(dimethylamino)ethyl]propanamidato)(difluoro)boron). Here, we present the use of a fluorescent pH-sensitive dye that stains intestinal lysosomes, providing a visual readout of dynamic, physiological changes in lysosomal acidity in live worms. This protocol does not measure lysosomal pH, but rather aims to establish a reliable method of assessing physiological relevant variations in lysosomal acidity. cDCFDA is a cell-permeant compound that is converted to the fluorescent fluorophore 5-(and-6)-carboxy-2&#39;,7&#39;-dichlorofluorescein (cDCF) upon hydrolysis by intracellular esterases. Protonation inside lysosomes traps cDCF in these organelles, where it accumulates. Due to its low pKa of 4.8, this dye has been used as a pH sensor in yeast. Here we describe the use of cDCFDA as a food supplement to assess the acidity of intestinal lysosomes in C. elegans. This technique allows for the detection of alkalinizing lysosomes in live animals, and has a broad range of experimental applications including studies on aging, autophagy, and lysosomal biogenesis.

Assessing Lysosomal Alkalinization in the Intestine of Live Caenorhabditis elegans

A step-by-step guide to probe loss of lysosomal acidity in the intestine of C. elegans using the pH-sensitive vital dye 5(6)-carboxy-2&#39;,7&#39;-dichlorofluorescein diacetate (cDCFDA)

评估溶酶体碱化在活的秀丽线虫中的肠道

The nematode Caenorhabditis elegans (C. elegans) is a model system that is widely used to study longevity and developmental pathways. Such studies are ...

Cell Cycle Analysis in the C. elegans Germline with the Thymidine Analog EdU

Cell cycle analysis in eukaryotes frequently utilizes chromosome morphology, expression and/or localization of gene products required for various phases of the cell cycle, or the incorporation of nucleoside analogs. During S-phase, DNA polymerases incorporate thymidine analogs such as EdU or BrdU into chromosomal DNA, marking the cells for analysis. For C. elegans, the nucleoside analog EdU is fed to the worms during regular culture and is compatible with immunofluorescent techniques. The germline of C. elegans is a powerful model system for the studies of signaling pathways, stem cells, meiosis, and cell cycle because it is transparent, genetically facile, and meiotic prophase and cellular differentiation/gametogenesis occur in a linear assembly-like fashion. These features make EdU a great tool to study dynamic aspects of mitotically cycling cells and germline development. This protocol describes how to successfully prepare EdU bacteria, feed them to wild-type C. elegans hermaphrodites, dissect the hermaphrodite gonad, stain for EdU incorporation into DNA, stain with antibodies to detect various cell cycle and developmental markers, image the gonad and analyze the results. The protocol describes the variations in the method and analysis for the measurement of S-phase index, M-phase index, G2 duration, cell cycle duration, rate of meiotic entry, and rate of meiotic prophase progression. This method can be adapted to study the cell cycle or cell history in other tissues, stages, genetic backgrounds, and physiological conditions.

Cell Cycle Analysis in the C. elegans Germline with the Thymidine Analog EdU

An imaging-based method is described that can be used to identify S-phase and analyze cell cycle dynamics in the C. elegans hermaphrodite germline using the thymidine analog EdU. This method requires no transgenes and is compatible with immunofluorescent staining.

种系线虫与胸苷模拟教育的细胞周期分析

Cell cycle analysis in eukaryotes frequently utilizes chromosome morphology, expression and/or localization of gene products required for various phases ...