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><tbody><tr><td>&lt;strong&gt;Antibody staining&lt;/strong&gt;</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>&lt;strong&gt;Primary antibodies&lt;/strong&gt;</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 Mario &lt;em&gt;de&lt;/em&gt; Bono (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>&lt;strong&gt;Secondary antibodies&lt;/strong&gt;</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>&lt;strong&gt;&lt;em&gt;C. elegans&lt;/em&gt; related&lt;/strong&gt;</td><td></td><td></td><td>see reference&lt;sup&gt;29 &lt;/sup&gt;for standard &lt;em&gt;C. elegans&lt;/em&gt; culture and maintenance procedures.</td></tr><tr><td>&lt;strong&gt;LB Medium and plates&lt;/strong&gt;</td><td></td><td></td><td>see reference&lt;sup&gt;29&lt;/sup&gt; 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>&lt;strong&gt;M9 Medium&lt;/strong&gt;</td><td></td><td></td><td>see reference&lt;sup&gt;29&lt;/sup&gt; for protocols.</td></tr><tr><td>NaCl</td><td>Sigma</td><td>S7653</td><td></td></tr><tr><td>KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;</td><td>Sigma</td><td>P0662</td><td></td></tr><tr><td>Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;</td><td>Sigma</td><td>S7907</td><td></td></tr><tr><td>MgSO&lt;sub&gt;4&lt;/sub&gt;</td><td>Sigma</td><td>M2773</td><td></td></tr><tr><td>&lt;strong&gt;NGM plates&lt;/strong&gt;</td><td></td><td></td><td>see reference&lt;sup&gt;29&lt;/sup&gt; 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>MgSO&lt;sub&gt;4&lt;/sub&gt;</td><td>Sigma</td><td>M2773</td><td></td></tr><tr><td>CaCl&lt;sub&gt;2&lt;/sub&gt;</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>K&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;</td><td>Sigma</td><td>P3786</td><td></td></tr><tr><td>KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;</td><td>Sigma</td><td>P0662</td><td></td></tr><tr><td>&lt;strong&gt;RNAi plates&lt;/strong&gt;</td><td></td><td></td><td>see reference&lt;sup&gt;30&lt;/sup&gt; 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>MgSO&lt;sub&gt;4&lt;/sub&gt;</td><td>Sigma</td><td>M2773</td><td></td></tr><tr><td>CaCl&lt;sub&gt;2&lt;/sub&gt;</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>K&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;</td><td>Sigma</td><td>P3786</td><td></td></tr><tr><td>KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;</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>&lt;strong&gt;Bacteria&lt;/strong&gt;</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&lt;sup&gt; 30,49,50&lt;/sup&gt;)</td><td>Source BioScience</td><td></td><td></td></tr><tr><td>&lt;em&gt;C. elegans&lt;/em&gt; ORF-RNAi feeding library (ref&lt;sup&gt;51&lt;/sup&gt;)</td><td>Source BioScience</td><td></td><td></td></tr><tr><td>&lt;strong&gt;Imaging related&lt;/strong&gt;</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>&lt;strong&gt;Equipment&lt;/strong&gt;</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

Universidad San Sebastian

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

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 ...

科研

发育生物学

Immuno-fluorescent Labeling of Microtubules and Centrosomal Proteins in Ex Vivo Intestinal Tissue and 3D In Vitro Intestinal Organoids

The advent of 3D in vitro organoids that mimic the in vivo tissue architecture and morphogenesis has greatly advanced the ability to study key biological questions in cell and developmental biology. In addition, organoids together with recent technical advances in gene editing and viral gene delivery promises to advance medical research and development of new drugs for treatment of diseases. Organoids grown in vitro in basement matrix provide powerful model systems for studying the behavior and function of various proteins and are well suited for live-imaging of fluorescent-tagged proteins. However, establishing the expression and localization of the endogenous proteins in ex vivo tissue and in in vitro organoids is important to verify the behavior of the tagged proteins. To this end we have developed and modified tissue isolation, fixation, and immuno-labeling protocols for localization of microtubules, centrosomal, and associated proteins in ex vivo intestinal tissue and in in vitro intestinal organoids. The aim was for the fixative to preserve the 3D architecture of the organoids/tissue while also preserving antibody antigenicity and enabling good penetration and clearance of fixative and antibodies. Exposure to cold depolymerizes all but stable microtubules and this was a key factor when modifying the various protocols. We found that increasing the ethylenediaminetetraacetic acid (EDTA) concentration from 3 mM to 30 mM gave efficient detachment of villi and crypts in the small intestine while 3 mM EDTA was sufficient for colonic crypts. The developed formaldehyde/methanol fixation protocol gave very good structural preservation while also preserving antigenicity for effective labeling of microtubules, actin, and the end-binding (EB) proteins. It also worked for the centrosomal protein ninein although the methanol protocol worked more consistently. We further established that fixation and immuno-labeling of microtubules and associated proteins could be achieved with organoids isolated from or remaining within the basement matrix.

Immuno-fluorescent Labeling of Microtubules and Centrosomal Proteins in Ex Vivo Intestinal Tissue and 3D In Vitro Intestinal Organoids

We present protocols for isolation of intestinal 3D structures from in vivo tissue and in vitro basement matrix embedded organoids, and detail different fixation and staining protocols optimized for immuno-labeling of microtubule, centrosomal, and junctional proteins as well as cell markers including the stem cell protein Lgr5.

免疫荧光标记的微管和中心蛋白在体内肠道组织和 3D体外肠道 Organoids

The advent of 3D in vitro organoids that mimic the in vivo tissue architecture and morphogenesis has greatly advanced the ability to study key biological ...

Isotropic Light-Sheet Microscopy and Automated Cell Lineage Analyses to Catalogue Caenorhabditis elegans Embryogenesis with Subcellular Resolution

Caenorhabditis elegans (C. elegans) stands out as the only organism in which the challenge of understanding the cellular origins of an entire nervous system can be observed, with single cell resolution, in vivo. Here, we present an integrated protocol for the examination of neurodevelopment in C. elegans embryos. Our protocol combines imaging, lineaging and neuroanatomical tracing of single cells in developing embryos. We achieve long-term, four-dimensional (4D) imaging of living C. elegans&#160;embryos with nearly isotropic spatial resolution through the use of Dual-view Inverted Selective Plane Illumination Microscopy (diSPIM). Nuclei and neuronal structures in the nematode embryos are imaged and isotropically fused to yield images with resolution of ~330 nm in all three dimensions. These minute-by-minute high-resolution 4D data sets are then analyzed to correlate definitive cell-lineage identities with gene expression and morphological dynamics at single-cell and subcellular levels of detail. Our protocol is structured to enable modular implementation of each of the described steps and enhance studies on embryogenesis, gene expression, or neurodevelopment.

Here, we present a combinatorial approach using high-resolution microscopy, computational tools, and single-cell labeling in living C. elegans embryos to understand single cell dynamics during neurodevelopment.

亚细胞分辨率的各向同性光片显微镜及细胞自动动力学分析

Caenorhabditis elegans (C. elegans) stands out as the only organism in which the challenge of understanding the cellular origins of an entire nervous ...

Visualizing Neuroblast Cytokinesis During C. elegans Embryogenesis

This protocol describes the use of fluorescence microscopy to image dividing cells within developing Caenorhabditis elegans embryos. In particular, this protocol focuses on how to image dividing neuroblasts, which are found underneath the epidermal cells and may be important for epidermal morphogenesis. Tissue formation is crucial for metazoan development&#160;and relies on external cues from neighboring tissues. C. elegans is an excellent model organism to study tissue morphogenesis in vivo due to its transparency and simple organization, making its tissues easy to study via microscopy. Ventral enclosure is the process where the ventral surface of the embryo is covered by a single layer of epithelial cells. This event is thought to be facilitated by the underlying neuroblasts, which provide chemical guidance cues to mediate migration of the overlying epithelial cells. However, the neuroblasts are highly proliferative and also may act as a mechanical substrate for the ventral epidermal cells. Studies using this experimental protocol could uncover the importance of intercellular communication during tissue formation, and could be used to reveal the roles of genes involved in cell division within developing tissues.

Visualizing Neuroblast Cytokinesis During C. elegans Embryogenesis

This protocol describes how to image dividing cells within a tissue in Caenorhabditis elegans embryos. While several protocols describe how to image cell division in the early embryo, this protocol describes how to image cell division within a developing tissue during mid late embryogenesis.

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 ...

神经科学

A Method for Culturing Embryonic C. elegans Cells

C. elegans is a powerful model system, in which genetic and molecular techniques are easily applicable. Until recently though, techniques that require direct access to cells and isolation of specific cell types, could not be applied in C. elegans. This limitation was due to the fact that tissues are confined within a pressurized cuticle which is not easily digested by treatment with enzymes and/or detergents. Based on early pioneer work by Laird Bloom, Christensen and colleagues 1 developed a robust method for culturing C. elegans embryonic cells in large scale. Eggs are isolated from gravid adults by treatment with bleach/NaOH and subsequently treated with chitinase to remove the eggshells. Embryonic cells are then dissociated by manual pipetting and plated onto substrate-covered glass in serum-enriched media. Within 24 hr of isolation cells begin to differentiate by changing morphology and by expressing cell specific markers. C. elegans cells cultured using this method survive for up 2 weeks in vitro and have been used for electrophysiological, immunochemical, and imaging analyses as well as they have been sorted and used for microarray profiling.

A Method for Culturing Embryonic C. elegans Cells

We describe here a revised protocol for large-scale culture of embryonic C. elegans cells. Embryonic C. elegans cells cultured in vitro using this method, appear to differentiate and recapitulate the expression of genes in a cell specific manner. Techniques that require direct access to the cells or isolation of specific cell types from the other tissues can be applied on C. elegans cultured cells.

一种用于培养胚胎<em&gt; C。线虫</em&gt;细胞

C. elegans is a powerful model system, in which genetic and molecular techniques are easily applicable. Until recently though, techniques that require ...

生物学

RNA Fluorescence in situ Hybridization (FISH) to Visualize Microbial Colonization and Infection in Caenorhabditis elegans Intestines

The intestines of wild Caenorhabditis nematodes are inhabited by a variety of microorganisms, including gut microbiome bacteria and pathogens, such as microsporidia and viruses. Because of the similarities between Caenorhabditis elegans and mammalian intestinal cells, as well as the power of the C. elegans system, this host has emerged as a model system to study host intestine-microbe interactions in vivo. While it is possible to observe some aspects of these interactions with bright-field microscopy, it is difficult to accurately classify microbes and characterize the extent of colonization or infection without more precise tools. RNA fluorescence in situ hybridization (FISH) can be used as a tool to identify and visualize microbes in nematodes from the wild or to experimentally characterize and quantify infection in nematodes infected with microbes in the lab. FISH probes, labeling the highly abundant small subunit ribosomal RNA, produce a bright signal for bacteria and microsporidian cells. Probes designed to target conserved regions of ribosomal RNA common to many species can detect a broad range of microbes, whereas targeting divergent regions of the ribosomal RNA is useful for narrower detection. Similarly, probes can be designed to label viral RNA. A protocol for RNA FISH staining with either paraformaldehyde (PFA) or acetone fixation is presented. PFA fixation is ideal for nematodes associated with bacteria, microsporidia, and viruses, whereas acetone fixation is necessary for the visualization of microsporida spores. Animals were first washed and fixed in paraformaldehyde or acetone. After fixation, FISH probes were incubated with samples to allow for the hybridization of probes to the desired target. The animals were again washed and then examined on microscope slides or using automated approaches. Overall, this FISH protocol enables detection, identification, and quantification of the microbes that inhabit the C. elegans intestine, including microbes for which there are no genetic tools available.

RNA Fluorescence in situ Hybridization FISH to Visualize Microbial Colonization and Infection in Caenorhabditis elegans Intestines

Intestinal microbes, including extracellular bacteria and intracellular pathogens like the Orsay virus and microsporidia (fungi), are often associated with wild Caenorhabditis nematodes. This article presents a protocol for detecting and quantifying microbes that colonize and/or infect C. elegans nematodes, and for measuring pathogen load after controlled infections in the lab.

RNA荧光 原位 杂交（FISH）可视化 秀丽隐杆线虫 肠道中的微生物定植和感染

The intestines of wild Caenorhabditis nematodes are inhabited by a variety of microorganisms, including gut microbiome bacteria and pathogens, such as ...

免疫与感染

Measuring the Effects of Bacteria and Chemicals on the Intestinal Permeability of Caenorhabditis elegans

In living organisms, intestinal hyperpermeability is a serious symptom that leads to many inflammatory bowel diseases (IBDs). Caenorhabditis elegans is a nonmammalian animal model that is widely used as an assay system due to its short lifespan, transparency, cost-effectiveness, and lack of animal ethics issues. In this study, a method was developed to investigate the effects of different bacteria and 3,3'-diindolylmethane (DIM) on the intestinal permeability of C. elegans with a high-throughput image analysis system. The worms were infected with different gut bacteria or cotreated with DIM for 48 h and fed with fluorescein isothiocyanate (FITC)-dextran overnight. Then, the intestinal permeability was examined by comparing the fluorescence images and the fluorescence intensity inside the worm bodies. This method may also have the potential to identify probiotic and pathogenic intestinal bacteria that affect intestinal permeability in the animal model and is effective for examining the effects of harmful or health-promoting chemicals on intestinal permeability and intestinal health. However, this protocol also has some considerable limitations at the genetic level, especially for determining which genes are altered to control illness, because this method is mostly used for phenotypic determination. In addition, this method is limited to determining exactly which pathogenic substrates cause inflammation or increase the permeability of the worms' intestines during infection. Therefore, further in-depth studies, including investigation of the molecular genetic mechanism using mutant bacteria and nematodes as well as chemical component analysis of bacteria, are required to fully evaluate the function of bacteria and chemicals in determining intestinal permeability.

Measuring the Effects of Bacteria and Chemicals on the Intestinal Permeability of Caenorhabditis elegans

This protocol describes how to measure intestinal permeability of Caenorhabditis elegans. This method is helpful for basic biological research on intestinal health related to the interaction between intestinal bacteria and their host and for screening to identify probiotic and chemical agents to cure leaky gut syndrome and inflammatory bowel diseases.

测定细菌和化学物质对开牛结不二病的肠道渗透性的影响

In living organisms, intestinal hyperpermeability is a serious symptom that leads to many inflammatory bowel diseases (IBDs). Caenorhabditis elegans is a ...

医学

Tracking and Quantifying Developmental Processes in C. elegans Using Open-source Tools

Quantitatively capturing developmental processes is crucial to derive mechanistic models and key to identify and describe mutant phenotypes. Here protocols are presented for preparing embryos and adult C. elegans animals for short- and long-term time-lapse microscopy and methods for tracking and quantification of developmental processes. The methods presented are all based on C. elegans strains available from the Caenorhabditis Genetics Center and on open-source software that can be easily implemented in any laboratory independently of the microscopy system used. A reconstruction of a 3D cell-shape model using the modelling software IMOD, manual tracking of fluorescently-labeled subcellular structures using the multi-purpose image analysis program Endrov, and an analysis of cortical contractile flow using PIVlab (Time-Resolved Digital Particle Image Velocimetry Tool for MATLAB) are shown. It is discussed how these methods can also be deployed to quantitatively capture other developmental processes in different models, e.g., cell tracking and lineage tracing, tracking of vesicle flow.

Tracking and Quantifying Developmental Processes in C. elegans Using Open-source Tools

Here it is shown how to track and quantify developmental processes in C. elegans. The methods presented are based on open-source tools that can be easily implemented. It is demonstrated how to reconstruct 3D cell-shape models, how to manually track subcellular structures, and how to analyze cortical contractile flow.

跟踪和量化的发育过程<em&gt;℃。线虫</em&gt;使用开源工具

Quantitatively capturing developmental processes is crucial to derive mechanistic models and key to identify and describe mutant phenotypes. Here ...

Generation of Murine Primary Colon Epithelial Monolayers from Intestinal Crypts

The intestinal epithelium is comprised of a single layer of cells that act as a barrier between the gut lumen and the interior of the body. Disruption in the continuity of this barrier can result in inflammatory disorders such as inflammatory bowel disease. One of the limitations in the study of intestinal epithelial biology has been the lack of primary cell culture models, which has obliged researchers to use model cell lines derived from carcinomas. The advent of three dimensional (3D) enteroids has given epithelial biologists a powerful tool to generate primary cell cultures, nevertheless, these structures are embedded in extracellular matrix and lack the maturity characteristic of differentiated intestinal epithelial cells. Several techniques to generate intestinal epithelial monolayers have been published, but most are derived from established 3D enteroids making the process laborious and expensive. Here we describe a protocol to generate primary epithelial colon monolayers directly from murine intestinal crypts. We also detail experimental approaches that can be used with this model such as the generation of confluent cultures on permeable filters, confluent monolayer for scratch wound healing studies and sparse and confluent monolayers for immunofluorescence analysis.

In this protocol, we describe how to generate murine primary epithelial colon monolayers directly from intestinal crypts. We provide experimental approaches to generate confluent monolayers on permeable filters, confluent monolayers for scratch wound healing and biochemical studies, and sparse and confluent monolayers for immunofluorescence analysis.

来自肠道密码的穆林原结肠上皮单层的生成

The intestinal epithelium is comprised of a single layer of cells that act as a barrier between the gut lumen and the interior of the body. Disruption in ...

Culture Methods to Study Apical-Specific Interactions using Intestinal Organoid Models

The lining of the gut epithelium is made up of a simple layer of specialized epithelial cells that expose their apical side to the lumen and respond to external cues. Recent optimization of in vitro culture conditions allows for the re-creation of the intestinal stem cell niche and the development of advanced 3-dimensional (3D) culture systems that recapitulate the cell composition and the organization of the epithelium. Intestinal organoids embedded in an extracellular matrix (ECM) can be maintained for long-term and self-organize to generate a well-defined, polarized epithelium that encompasses an internal lumen and an external exposed basal side. This restrictive nature of the intestinal organoids presents challenges in accessing the apical surface of the epithelium in vitro and limits the investigation of biological mechanisms such as nutrient uptake and host-microbiota/host-pathogen interactions. Here, we describe two methods that facilitate access to the apical side of the organoid epithelium and support the differentiation of specific intestinal cell types. First, we show how ECM removal induces an inversion of the epithelial cell polarity and allows for the generation of apical-out 3D organoids. Second, we describe how to generate 2-dimensional (2D) monolayers from single cell suspensions derived from intestinal organoids, comprised of&#160;mature and differentiated cell types. These techniques provide novel tools to study apical-specific interactions of the epithelium with external cues in vitro and promote the use of organoids as a platform to facilitate precision medicine.

Here we present two protocols that allow the modeling of intestinal apical-specific interactions. Organoid-derived intestinal monolayers and Air-Liquid Interface (ALI) cultures facilitate the generation of well-differentiated epithelia accessible from both luminal and basolateral sides, whereas polarity-inverted intestinal organoids expose their apical side and are amenable to high throughput assays.

使用肠道器官模型研究猿特异性相互作用的文化方法

The lining of the gut epithelium is made up of a simple layer of specialized epithelial cells that expose their apical side to the lumen and respond to ...

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

摘要

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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

免疫荧光标记的微管和中心蛋白在体内肠道组织和 3D体外肠道 Organoids

亚细胞分辨率的各向同性光片显微镜及细胞自动动力学分析

可视化的神经细胞胞质分裂过程中

一种用于培养胚胎

RNA荧光原位杂交（FISH）可视化秀丽隐杆线虫肠道中的微生物定植和感染

测定细菌和化学物质对开牛结不二病的肠道渗透性的影响

跟踪和量化的发育过程

来自肠道密码的穆林原结肠上皮单层的生成

使用肠道器官模型研究猿特异性相互作用的文化方法

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

摘要

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此视频中的章节

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

免疫荧光标记的微管和中心蛋白在体内肠道组织和 3D体外肠道 Organoids

亚细胞分辨率的各向同性光片显微镜及细胞自动动力学分析

可视化的神经细胞胞质分裂过程中

一种用于培养胚胎

RNA荧光 原位 杂交（FISH）可视化 秀丽隐杆线虫 肠道中的微生物定植和感染

测定细菌和化学物质对开牛结不二病的肠道渗透性的影响

跟踪和量化的发育过程

来自肠道密码的穆林原结肠上皮单层的生成

使用肠道器官模型研究猿特异性相互作用的文化方法

RNA荧光原位杂交（FISH）可视化秀丽隐杆线虫肠道中的微生物定植和感染