This work was supported by grants from the Natural Sciences and Engineering Research Council (NSERC) of Canada and The Canada Foundation for Innovation. Thanks to Dr Paul Mains (University of Calgary, Calgary, Canada) for let-502(sb118ts) strain. Some of the strains were provided by the Caenorhabditis Genetics Center, which is funded by NIH Office of Research Infrastructure Programs (P40 OD010440).

1.在WT和突变体动物早期伸长缺陷的表征<ol><li> 安装胚胎Normarski DIC显微镜 <ol><li>准备下列培养基和材料: <ol><li> M9缓冲液，溶解12.8克/升的 Na 2 HPO 4•7H 2 O，3g / L的KH 2 PO 4，5g / L的氯化钠，0.25克/升MgSO 4上•7H 2在蒸馏水和高压釜消毒O操作。 </li><li> NGM板，溶解3g / L的氯化钠16克/升琼脂，2.5g / L的细菌用蛋白胨在DDH 2 O高压灭菌，并添加1mM的MgSO 4上，1mM的氯化钙 ，1 mM磷酸盐缓冲液和5微克/毫升胆固醇。倒为60 mm培养皿6毫升NGM的。允许介质固化，在室温下24小时。添加大肠杆菌的饱和培养几滴大肠杆菌 OP50细菌生长在Luria培养基（LB）。让细菌生长48小时，将板储存在4℃。 </li><li>要生成蠕虫PI放，安装在一个短巴斯德吸管一个"直径0.01铂丝。通过加热该末端与苯环燃烧器紧固铂线对玻璃吸管的末端。拼合线的末端，以产生一种铲。 </li></ol></li><li>长60毫米，OP50 NGM板蠕虫应变在20℃。 注意:热敏等位基因可能需要在高达摄氏25.5度正以非许可温度动物。板应包含为了追求协议很多的年轻人，仍然充足的食物。 </li><li>将通过遮蔽胶带（ 图1A）的两层涉及两个间隔幻灯片之间载玻片。 </li><li>由30秒期间的微波加热0.6克琼脂糖粉末溶解在20毫升的M9缓冲准备在M9缓冲液中的3％的琼脂糖溶液（重量/体积）。使该溶液冷却5分钟。 注:琼脂糖溶液可以用微波炉融化后用于几天。它也可以等分并储存在4℃几周。 </li><li>放置在位于两个间隔物幻灯片之间的载玻片热3％的琼脂糖溶液一滴。避免形成气泡。 </li><li>迅速覆盖另一个滑动琼脂糖如图1B和轻轻下压。顶部滑动将变平的琼脂糖滴与生成用胶带的两层的厚度的垫。 </li><li>允许琼脂糖固化在室温下至少1分钟，或直至胚胎是准备要传送到垫。 </li><li>使用蠕虫挑，转20〜30以及美联储从年轻人身上NGM板到含有400微升M9的缓冲区的微型离心管中。 </li><li>允许蠕虫通过重力为约5分钟沉降并使用微量去除上清。这一步的目的是去除最高挑的线虫细菌。 </li><li>加入200μl的M9缓冲液中向每个管中。 </li><li>使用PIP巴斯德ETTE，管（缓冲和线虫）的内容传送到一个​​表玻璃。 </li><li>使用一个或两个25G 5/8"针头切动物在中间主体部（精囊和外阴之间）。 注:一种手术刀刀片也可以使用以替代针（多个）。做到这一点的游泳线虫和可能需要一些练习。该想法是使用针作为剪刀或取决于许多针是如何使用一把刀。一旦动物被剖开，雌雄同体释放他们的胚胎到缓冲区。 </li><li>在表玻璃的通过产生一个圆形涡流的液体内的中心集中胚胎。 </li><li>从使用微量的表玻璃除去大部分的M9。留出大约30微升的M9与胚胎。 </li><li>卸下滑动它关闭垫覆盖琼脂糖垫（ 图1B）的幻灯片。 </li><li>切割垫，以便刀片能够用盖玻片完全覆盖它（图1C）。 </li><li>使用巴斯德吸管，所有的胚胎（和蠕虫碎片）转移到琼脂糖垫。 </li><li>组的胚胎在利用在用于200微升微量的尖端的末端胶合睫毛垫的中心。 </li><li>慢慢地放在垫，避免泡沫形成盖玻片并密封。 注意:几个封闭剂可以使用。我们运用拉丝在艺术和工艺品商店发现胶（常用作掩蔽水彩画画胶）。此胶被使用，因为它是亲水性的并固化相当快并且没有毒性的蠕虫。简报也可以用指甲油或VALAP（由凡士林，羊毛脂和石蜡蜡的混合物）密封。 </li><li>允许约15分钟的拉伸胶干燥。 </li></ol></li><li> 录音采用四维Normarski DIC显微镜早期伸长率 注意:该步骤的目的是从逗号早伸长记录到晚伸长的开始- 当体壁肌肉开始收缩定义为时刻。我们还致力于记录当时不止一个胚胎。八个小时记录的通常需要记录早期伸长的一组非同步的胚胎。 <ol><li>放置在显微镜阶段安装的滑动。确保显微镜配备了10X 60X和目标以及DIC透镜，棱镜，照相机和捕捉软件，使时间推移显微镜和代Z-栈（自动Z-平台）。 注意:确保温度在显微镜室20和23℃之间不变。加热 - 冷却室可以用热敏突变体尤其是当需要在显微镜舞台上。 </li><li>识别一组胚胎在使用10倍的目标形态前阶段。 </li><li>一旦找到，滑出10倍的目标，并添加浸油一滴在幻灯片上。 </li><li>使用60X的目标，确定顶部和e的底部mbryos设置了Z-叠加成像参数。 注意:不要犹豫，设置Z堆栈比胚胎的厚度。设置两个相邻平面之间的距离为0.8微米。这将使覆盖胚胎的总深度在35至50的Z-计划。 注意:如果在显微镜包含一个自动化的xy平台，位于所述垫的不同位置的胚胎可以同时记录。这样做，记录的xy坐标上希望在实验的过程中，分析垫的每个位置的。确保设置记录参数时，选择XY，并按照协议的其余部分所示。记录期间的胚胎薄的Z-切片不一定需要对该协议详述的测量。然而，记录重量和突变的动物胚胎发育的最大分辨率是为了建立录音能够支持额外分析库一个很好的做法。 </li><li>组了时间推移与两次收购持续8小时之间2分钟的间隔。 注:暴露时间将取决于在显微镜设置的光强度。早在延长细胞的运动速度很慢;曝光时间可以是每秒或更小大约一帧。如果胚胎早期阶段的形态（背夹层，腹外壳），早期伸长率会1小时之内被记录。确保光快门每次采集之间关闭。 </li><li>运行采集和保存。 </li></ol></li><li> 使用图像分析早期伸长缺陷的测量 <ol><li>打开斐济的ImageJ软件（ <a href="http://fiji.sc/Fiji">v1.48o - http://fiji.sc/Fiji</a> ）。 </li><li>在菜单中选择分析/集测量，选择外围。对于每个胚，调整的Z比例尺专注于胚胎的咽， 如图2A所示 。这将确保你专注于胚胎的中心。 </li&gt; <li>为了测量胚的长度，调整时间比例尺有感兴趣的胚胎在早期伸长的开始（逗号阶段; 图2A）。注意时间（"时间的开始"）。 </li><li>选择的分割行工具。绘制从胚胎的嘴的前端的分割线至其尾巴尖的胚胎（ 图2A）的中线以下。使用菜单选项卡分析/测量，获取画线的长度（"长度的开始"）。 </li><li>在早期的延伸的终端重复这种测量对相同的胚胎（时刻时肌肉开始收缩）。注意时间（"时间的终结"），测量胚胎以上（ 图2B）详见（"长度端"）的长度。 </li><li>在这个阶段计算初延伸率和长度的增加（L）的持续时间（D）的，如下所示: D ="时间尽头" - "时间开始。" ＆＃160; L ="长度高端" - "长 - 开始" </li><li>为了测量头的宽度，调整时间比例尺具有在感兴趣（1.2倍阶段或早期伸长的端）的阶段的胚胎。选择直线工具。绘制的头部的横向（背腹轴）中线。这一部分是胚胎（ 图2C）的最厚的部分。使用菜单分析/测量，获得对应于所述头部宽度的线的长度。 </li><li>在相同的时间点，通过绘制尾部的横向中线重复此步骤（肠阀和尾巴尖之间中部线; 图2C），并测量它来获取尾部的宽度。 注:使用该测量在不同的表型同台比较胚胎。以保证该测量的再现性，确保找到周围设动物的尾部的中间线，可以很容易地从一个动物识别到一个位置诺特尔。 </li><li>要计算头尾宽度的比例，除以尾宽头的宽度。 </li></ol></li></ol> 2.晚伸长缺陷表征图像分析<ol><li> 使用碱性次氯酸钠处理L1幼虫同步 <ol><li>从含有许多妊娠成人非饥饿60毫米板，通过使用巴氏吸管1毫升M9缓冲液与洗涤线虫断板收集在微型离心管中的所有蠕虫。 </li><li>沉积物线虫在3500 xg离心在室温3分钟，除去使用微量上清液而不扰乱蠕虫粒料。 </li><li> 1毫升次氯酸钠溶液添加到每个管（0.4M的次氯酸钠，0.5M氢氧化钠），并摇动3分钟。 注:碱性次氯酸盐溶液应新鲜配制和胚胎在这个解决方案应该轻轻搅动，但不断优化成年人溶解和氧合电磁制动的YOS。经过3分钟搅拌，大多数成年人应该释放他们的鸡蛋到溶液中。 </li><li>转速为3500 XG 3分钟，在室温和快速清除使用微量上清而不干扰沉淀。 </li><li>用1ml的M9缓冲液中，随后通过离心3500 xg离心3分钟洗四次。 </li><li>第四洗涤后，除去在700微升的M9缓冲液中的上清，重悬鸡蛋不吹吸卵（如鸡蛋会粘到尖端的塑料）。 </li><li>磁带上在适当的生长温度置于培养箱3毫米轨道摇床的管中，在600rpm下摇动过夜。 </li></ol></li><li> 同步幼虫长度测量 <ol><li>离心机被捕L1幼虫在3500 xg离心在室温3分钟，去除上清。悬浮幼虫在100μl的M9缓冲液中，并将它们传送到使用巴斯德吸管琼脂糖垫（琼脂糖焊盘应准备-d作为在1.1.2节描述1.1.8）。广场上的垫盖玻片。 注:垫并不需要与胶密封该实验涉及短的采集。 </li><li>使用10X物镜和相衬照明来测量幼虫的长度，如果使用一个高分辨率照相机。增加光的强度，以便能够在几毫秒内捕获图像，从而获得幼虫（ 图2D）的清晰图象。 注意:当通过琼脂糖垫减少幼虫的游泳象垃圾一样清除，他们仍然感动。因此，快速记录是必不可少的，以获得一个清晰的图像。 </li><li>打开斐济的ImageJ并重复步骤1.3.1。为了测量幼虫的长度，选择分割行工具。绘制从头部向上的尖 ​​端到尾尖幼虫（ 图2D，右图）的中线以下一个分段线。 </li><li>使用菜单分析/测量，获得的长度对应于微米幼虫的长度划线。 </li></ol></li></ol> 3.后伸长缺陷表征采用流式细胞仪<ol><li> 幼虫同步 <ol><li>净化用碱性次氯酸钠处理的胚胎作为从衣食无忧的年轻人整整100 MM平板2.1节所述并根据应变胚胎孵化和L1逮捕M9的缓冲区16至24小时，在20或25.5℃，让进行分析。 注:每应变以及馈成年人的一个满盘足以用于下面描述的分析。 </li></ol></li><li> 蠕虫分拣机标定 注:细胞仪这里所用的流是大颗粒流式细胞仪（以下简称为蜗杆分拣机）。该蠕虫分拣机包括一个670纳米的红色二极管激光器，它位于消光探测器前。蜗杆分拣机还包含用于荧光激发的多线的氩激光器。在STANDARD文书有三个光电倍增管来检测荧光发射在光谱的绿色，黄色和红色区域（光电倍增管）的荧光检测器。在这里所描述的协议中，飞行时间将被用于测量幼虫的大小，红色通道将被用于确定将与碘化丙锭（PI）染色死虫。 GP（通用）高的荧光控制颗粒，用于校准仪器，并在我们的实验中的内部控制，绿色，黄色和红色显示高荧光。它们将与在绿色通道检测高发射分析的样品中的唯一目的和基于该特征也将随之被识别。 注:活的动物是基于在绿色和红色信道，以及对与TOF没有荧光的标识。从幼虫的肠自体荧光发射不在L1阶段检测到。 <ol><li>切换激光块，计算机，压缩机的上d是蠕虫分拣机的仪器。按下开蠕虫分拣机软件的START按钮，在仪器的使用说明书指示。 </li><li>观察氩激光控制弹出窗口。选择运行模式，等待的激光功率达到10±1毫瓦。选择完成激光控制窗口。 </li><li>设置护套和样品压力分别5.10±0.02和5.51±0.01。允许压力平衡至少15分钟，然后按一下压力确定复选框。 注:流速取决于护套和样品的压力。 </li><li>确保消除存在于所述样品杯与所述分析室之间的流路管的所有气泡。要做到这一点，请点击ACQUIRE，直到无气泡被收购（如采集窗口看到的）轻轻一抖小管。 </li><li>荧光和TOF测量和检测如下所有参数的设置: <ol><li>设置TOF，分机，绿色，黄色和红色的鳞表1。设置PMT控制在700为绿色和黄色，并以900为红色描述256集增益。 注意:两个激发滤光片是可用的（488纳米和514纳米），可以被用于激励或绿色荧光蛋白（GFP）或黄色荧光蛋白（YFP），或在这些波长与多行氩激光激发的任何荧光染料。适当的过滤器需要在该设备的过滤室被插入。我们使用了488nm的过滤器用于以下实验。 </li></ol></li><li>要校准蠕虫分类器，选择工具菜单中的"运行控制粒子"。把20毫升1X GP（通用）高的荧光颗粒控制在杯（这些颗粒尺寸精确的荧光颗粒，由厂家仪出售给校准他们的文书）。 </li><li>点击ACQUIRE按钮开始鞘和样品流。 注:期望与21±6和交流的控制颗粒分布的平均围绕平均值（CV）≤11变异oefficient。如果CV值&gt; 11，平均为&gt; 27尝试通过点击清洁按钮几次或通过轻弹的流路来清洁管。 </li></ol></li><li> 动物TOF收购 注意:当一个对象在所述激光源和消光检测器之间的流动单元通过光散射。花费的光的时间，以通过用于测量物体的轴向长度的飞行物体（飞行时间，TOF）被散射。由对象（消光，EXT）掩蔽光的程度被用作其不透明度/光密度的量度。 <ol><li>放置10微升同步的野生型（WT）中的L1表玻璃，并估计使用解剖显微镜死卵的百分比。 注:同步的L1 重量显示高EMB（高于20％）不应该被用于进一步的分析。 </li><li>转让从离心同步L1（一pproximately 700微升含有L1的M9）到15毫升锥形管中。以10微克/毫升（稀释从1毫克/毫升储液1/100）的终浓度添加碘化丙啶（PI）和孵育在室温下30分钟。 注:死卵和幼虫会使用PI进行染色并检测为使用红色通道高荧光对象。 </li><li>加入10 mL M9缓冲液稀释染色的人群。取5毫升稀释种群并通过加入15ml的M9稀释它们四次。把这个稀释样品杯中。 </li><li>通过运行ACQUIRE点击样品流并观察流速。 注:为了有精确的测量，流速应保持对象15至25 /秒。 </li><li>如果需要的话，调整动物的量在杯以达到所需的流速。 注:该流率将取决于在3.2.3和幼虫我的浓度表明是恒定的压力（鞘和样品）n个杯子。如果流速高于25对象/秒在杯中加一些缓冲。如果它低于15的对象/秒加浓L1（从步骤3.3.3）在杯中。 </li><li>一旦达到目标的适当的浓度，评估在杯的体积，并添加该卷在高荧光的GP控制颗粒4倍溶液的1/4 次 。 </li><li>调整门和排序参数。要做到这一点，点击"门控"菜单，选择"TOF与&#39;和"绿色"。点击菜单中的"排序"，然后选择"TOF与"和"红色"。然后选通和排序的图形将出现如图3A中 。 注意:这将允许控制粒子（在绿色和红色的排放）的可视化，通过PI和泡沫染色死动物（发射红色，而不是绿色）和活体动物（既不环保，也不红色通道零排放）（ 图3A ）。 </li><li>通过点击ACQ运行实验UIRE。 注:要确定幼虫之间的小尺寸上的差异，分析大约10,000个对象，所以约8000动物。停止试验，并通过点击STORE将数据导出为.txt格式。 </li></ol></li><li> 生活的动物的种群同步的相对大小的测量 <ol><li>使用能够管理大型数据表软件中打开.txt文件。 </li><li>从分析的对象提取物，在绿色通道的特点是发射超过100任意单位较高的控制颗粒相关的TOF值（此值取决于第3.2.5节设置的参数）。创建一个新的列，并把它称作&#39;控制粒子的。创建一个包含名为除外"样本"控制颗粒的所有其他对象的列。注:新粒子一批需要荧光发射在启动收购前进行验证。这将设置门槛荧光使鉴定在L1S控制颗粒。 </li><li>对于每个分析应变能辨认出流量已被改变（由于鸡蛋例如插头的存在下）的实验的一部分。要做到这一点: <ol><li>积"控制颗粒"的飞行时间为每个样品作为时间的因素（ 图3B）。 </li><li>通过绘制趋势线工具线性趋势线，并在图表上显示的公式。 注意:控制颗粒的飞行时间应随时间常数（水平线， 图3B）。考虑到对数据y = AX + B绘出的线性趋势线方程，确保了&#39;A&#39;值较低的10 -4。显示在控制粒子的取向断裂时间段内进行的所有测量应该从分析中除去。 </li></ol></li><li>去除死的胚胎和气泡（发射15高于在红色），以及小的碎片和大蛋塞（TOF低于10和高呃比70分别）从"样本"的测量。 </li><li>积分析每一株"控制粒子的分布。 如图3C看到这些分布应该覆盖几乎完美。这保证了对不同菌株得到TOF测量可比拟。注:样品元件的正常化TOF过控制颗粒可以，如果控制颗粒的样品中的分布不与对照样品的重叠使用。作为后面标准化TOF计算: <ol><li> TOF正常化基因型G =（对于G样品TOF）/（平均"控制粒子"对于G TOF）×（平均"控制粒子"TOF的重量 ），其中重量是对照样品。 </li></ol></li><li>绘制每一株，包括对照样品幼虫TOF分布，在我们的例子重量的动物（ 图3D）。测量的平均值和平均值（SEM）的标准误差为每个群体（ <stro纳克&gt;图5A和B）。 </li></ol></li></ol>

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The authors declare that they have no competing financial interests.

本协议描述的新的指标来表征早期胚胎和伸长率的后期阶段。 在部分1中，关键的步骤是细菌在垫中可能存在。将胚气密封闭垫和图像采集期间盖玻片之间。密封滑动时需要避免采集，持续两个多小时期间动物的干燥。据我们所知，没有用于安装滑动和盖玻片之间琼脂糖垫密封剂是透气的。因此，当大量的细菌（或胚胎）的存在于垫，它们可以在几个小时导致其过早死亡后缺?...

近50年来的线虫确立了自己作为一个强大的模型来研究发展，神经生物学，进化，宿主-病原体相互作用等重要问题1这种模式在发展研究的优势在于:它的生命周期短的3天;与这些动物可以遗传改造的容易性;其透明度，使细胞的位移和形态的观察活体动物，其​​发展的主要是多余的子宫。线虫的发育阶段涉及胚胎和四个幼虫期（L1到L4），其次是成年。在胚胎发育期间，表皮形态吸引了相当多的关注其能更好地理解如何上皮细胞迁移作为一个群体，他们是如何重新组织他们的路口和修改其个人的形态和功能性上皮细胞内的相对定位能力。表皮形态被分为四个阶段:背侧嵌入由背表皮细胞的重组，称为皮下组织;腹侧外壳，包括在朝向腹侧中线从而包围在上皮细胞单层胚胎腹侧皮下细胞的迁移;早期和晚期延伸改造豆形的胚胎成蠕虫状幼虫。继形态，胚胎孵化和幼虫L1在开始他们的直接环境中使用有益菌喂养。 因此胚胎伸长率是胚胎发育的后期阶段。它由沿其纵向轴线的胚胎的延伸和减少其横向直径的。这涉及皮下细胞形状的急剧改变。伸长分为早期和晚期。早期阶段开始于逗号阶段结束时，体壁肌肉开始在以W 1.75倍承包期ILD型（wt）的胚胎-对应于1.75倍的长度相比，非细长的胚胎的胚胎。在该阶段发生的形态发生的过程，主要由设在皮下细胞的顶磁极丝状肌动蛋白束（FB）的驱动其伸长沿胚胎2的前-后轴线的收缩驱动。 FB的收缩是通过控制肌球蛋白轻链的磷酸三激酶LET-502 / ROCK，MRCK-1和PAK-1 5。伸长的后期，开始时身体壁肌肉变得功能，并开始承包。它涉及从体内壁肌肉的背部和腹部皮下细胞机械传导信令和结束时动物孵化3。 伸长缺陷通常的特征在于由动物死于胚胎的百分率（胚胎致死性; EMB）和那些阻止其为L1幼虫（幼虫被捕表型的发展;吕a）和比重量显著缩短。发育停滞阶段的鉴定需要死胚显微镜观察并逮捕幼虫3-6。 据最近表明，一些基因，如Cdc42的/ Rac的调节和效应PIX-1和PAK-1，早期和晚期伸长3,7-期间控制形态发生的过程。我们最近还表明，形态发生过程早期伸长3 7期间沿胚的前-后轴线不同。这些发现促使新指标专门针对早期或晚期阶段的延伸和其他指标，可早在伸长沿前 - 后轴上胚胎的形态表征的发展。 这些新颖的方法包括在测量胚的长度在开始和早期伸长的末端以及它们的宽度，他广告和尾巴。7两个协议还制定了衡量初孵幼虫的长度，在L1阶段7同步。 胚胎的蛋壳保护它们免受碱性次氯酸盐处理而幼虫，成虫和存在于培养基的细菌通过处理溶解。然后，该处理用于从含有多数精心喂养成人8的非同步人口纯化胚胎。食物相克是用来初孵幼虫同步。测量这些幼虫的长度，然后用于检测伸长缺陷。这种测量优于在培养皿被捕幼虫的测量，因为幼虫从孵化非充分拉长胚胎可以恢复喂奶时"正常的长度"，但在缺乏食物时被捕将保持它们的尺寸减小。 在这里，我们提出详细的协议，使的乐测量延长使用时间推移DIC显微镜和图像分析（方案1）的胚胎，以及他们的头部的宽度和尾部NGTH。我们还提供了详细的协议，来测量使用图像分析（协议2）和流式细胞仪（协议3）同步幼虫的长度。

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novel metrics to characterize embryonic elongation of the nematode caenorhabditis elegans

Dissecting the signaling pathways that control the alteration of morphogenic processes during embryonic development requires robust and sensitive metrics. Embryonic elongation of the nematode Caenorhabditis elegans is a late developmental stage consisting of the elongation of the embryo along its longitudinal axis. This developmental stage is controlled by intercellular communication between hypodermal cells and underlying body-wall muscles. These signaling mechanisms control the morphology of hypodermal cells by remodeling the cytoskeleton and the cell-cell junctions. Measurement of embryonic lethality and developmental arrest at larval stages as well as alteration of cytoskeleton and cell-cell adhesion structures in hypodermal and muscle cells are classical phenotypes that have been used for more than 25 years to dissect these signaling pathways. Recent studies required the development of novel metrics specifically targeting either early or late elongation and characterizing morphogenic defects along the antero-posterior axis of the embryo. Here, we provide detailed protocols enabling the accurate measurement of the length and the width of the elongating embryos as well as the length of synchronized larvae. These methods constitute useful tools to identify genes controlling elongation, to assess whether these genes control both early and late phases of this stage and are required evenly along the antero-posterior axis of the embryo.

平视，Tail-和头部/尾部宽比是稳健的指标。 这里所描述的协议已被成功地用于表征调节和Rho GTP酶PIX-1的效应的功能，PAK-1和早期伸长7期间让-502。PIX-1和分别PAK-1的代码为鸟嘌呤交?...

Watch this Scientific Journal Video about Novel Metrics to Characterize Embryonic Elongation of the Nematode Caenorhabditis elegans at JoVE.com

Novel Metrics to Characterize Embryonic Elongation of the Nematode Caenorhabditis elegans

新颖的指标来表征线虫胚胎伸长率<em&gt;秀丽隐杆线虫</em

Here we detail protocols specifically designed to monitor morphogenic defects that occur during early and late phases of embryonic elongation of the nematode Caenorhabditis elegans. Ultimately, these protocols are designed to identify genes that regulate these phases and to characterize their differential requirements along the antero-posterior axis of the embryo.

Dissecting the signaling pathways that control the alteration of morphogenic processes during embryonic development requires robust and sensitive metrics. ...

novel-metrics-to-characterize-embryonic-elongation-nematode

Research

JoVE Journal

Neuroscience

Novel Metrics to Characterize Embryonic Elongation of the Nematode Caenorhabditis elegans

6.0K 次观看.  Université du Québec à Montréal. Here we detail protocols specifically designed to monitor morphogenic defects that occur during early and late phases of embryonic elongation of the nematode Caenorhabditis elegans. Ultimately, these protocols are designed to identify genes that regulate these phases and to characterize their differential requirements along the antero-posterior axis of the embryo. 

视频: Novel Metrics to Characterize Embryonic Elongation of the Nematode Caenorhabditis elegans

Multi-unit Recording Methods to Characterize Neural Activity in the Locust (Schistocerca Americana) Olfactory Circuits

Detection and interpretation of olfactory cues are critical for the survival of many organisms. Remarkably, species across phyla have strikingly similar olfactory systems suggesting that the biological approach to chemical sensing has been optimized over evolutionary time1. In the insect olfactory system, odorants are transduced by olfactory receptor neurons (ORN) in the antenna, which convert chemical stimuli into trains of action potentials. Sensory input from the ORNs is then relayed to the antennal lobe (AL; a structure analogous to the vertebrate olfactory bulb). In the AL, neural representations for odors take the form of spatiotemporal firing patterns distributed across ensembles of principal neurons (PNs; also referred to as projection neurons)2,3. The AL output is subsequently processed by Kenyon cells (KCs) in the downstream mushroom body (MB), a structure associated with olfactory memory and learning4,5. Here, we present electrophysiological recording techniques to monitor odor-evoked neural responses in these olfactory circuits.
First, we present a single sensillum recording method to study odor-evoked responses at the level of populations of ORNs6,7. We discuss the use of saline filled sharpened glass pipettes as electrodes to extracellularly monitor ORN responses. Next, we present a method to extracellularly monitor PN responses using a commercial 16-channel electrode3. A similar approach using a custom-made 8-channel twisted wire tetrode is demonstrated for Kenyon cell recordings8. We provide details of our experimental setup and present representative recording traces for each of these techniques.

Multi-unit Recording Methods to Characterize Neural Activity in the Locust Schistocerca Americana Olfactory Circuits

We demonstrate variations of the extracellular multi-unit recording technique to characterize odor-evoked responses in the first three stages of the invertebrate olfactory pathway. These techniques can easily be adapted to examine ensemble activity in other neural systems as well.

多单位记录方法来描述蝗虫的神经活动（<em&gt; Schistocerca美洲</em&gt;）嗅觉电路

Detection and interpretation of olfactory cues are critical for the survival  of many organisms. Remarkably, species across phyla have strikingly similar ...

科研

神经科学

A Caenorhabditis elegans Model System for Amylopathy Study

Amylopathy is a term that describes abnormal synthesis and accumulation of amyloid beta (A&beta;) in tissues with time. A&beta; is a hallmark of Alzheimer's disease (AD) and is found in Lewy body dementia, inclusion body myositis and cerebral amyloid angiopathy 1-4. Amylopathies progressively develop with time. For this reason simple organisms with short lifespans may help to elucidate molecular aspects of these conditions. Here, we describe experimental protocols to study A&beta;-mediated neurodegeneration using the worm Caenorhabditis elegans. Thus, we construct transgenic worms by injecting DNA encoding human A&beta;42 into the syncytial gonads of adult hermaphrodites. Transformant lines are stabilized by a mutagenesis-induced integration. Nematodes are age synchronized by collecting and seeding their eggs. The function of neurons expressing A&beta;42 is tested in opportune behavioral assays (chemotaxis assays). Primary neuronal cultures obtained from embryos are used to complement behavioral data and to test the neuroprotective effects of anti-apoptotic compounds.

A Caenorhabditis elegans Model System for Amylopathy Study

We describe methods to study aspects of amylopathies in the worm C. elegans. We show how to construct worms expressing human A&beta;42 in neurons and how to test their function in behavioral assays. We further show how to obtain primary neuronal cultures that can be used for pharmacological testing.

一<em&gt;线虫</em&gt;研究Amylopathy模型系统

Amylopathy is a term that describes abnormal synthesis and accumulation of amyloid beta (A&beta;) in tissues with time. A&beta; is a hallmark of ...

Methods for Studying the Mechanisms of Action of Antipsychotic Drugs in Caenorhabditis elegans

Caenorhabditis elegans is a simple genetic organism amenable to large-scale forward and reverse genetic screens and chemical genetic screens. The C. elegans genome includes potential antipsychotic drug (APD) targets conserved in humans, including genes encoding proteins required for neurotransmitter synthesis and for synaptic structure and function. APD exposure produces developmental delay and/or lethality in nematodes in a concentration-dependent manner. These phenotypes are caused, in part, by APD-induced inhibition of pharyngeal pumping1,2. Thus, the developmental phenotype has a neuromuscular basis, making it useful for pharmacogenetic studies of neuroleptics. Here we demonstrate detailed procedures for testing APD effects on nematode development and pharyngeal pumping. For the developmental assay, synchronized embryos are placed on nematode growth medium (NGM) plates containing APDs, and the stages of developing animals are then scored daily. For the pharyngeal pumping rate assay, staged young adult animals are tested on NGM plates containing APDs. The number of pharyngeal pumps per unit time is recorded, and the pumping rate is calculated. These assays can be used for studying many other types of small molecules or even large molecules.

Methods for Studying the Mechanisms of Action of Antipsychotic Drugs in Caenorhabditis elegans

Approaches for testing the effects of antipsychotic drugs (APDs) in Caenorhabditis elegans are demonstrated. Assays are described for testing drug effects on development and viability and on pharyngeal pumping rate. These methods are also applicable for pharmacogenetic experiments with drug classes other than APDs.

方法研究抗精神病药物的作用机制在<em&gt;秀丽隐杆线虫</em

Caenorhabditis elegans is a simple genetic organism amenable to  large-scale forward and reverse genetic screens and chemical genetic screens. The C.  ...

An Assay for Measuring the Effects of Ethanol on the Locomotion Speed of Caenorhabditis elegans

Alcohol use disorders are a significant public health concern, for which there are few effective treatment strategies. One difficulty that has delayed the development of more effective treatments is the relative lack of understanding of the molecular underpinnings of the effects of ethanol on behavior. The nematode, Caenorhabditis elegans (C.&#160;elegans), provides a useful model in which to generate and test hypotheses about the molecular effects of ethanol. Here, we describe an assay that has been developed and used to examine the roles of particular genes and environmental factors in behavioral responses to ethanol, in which locomotion is the behavioral output. Ethanol dose-dependently causes an acute depression of crawling on an agar surface. The effects are dynamic; animals exposed to a high concentration demonstrate an initial strong depression of crawling, referred to here as initial sensitivity, and then partially recover locomotion speed despite the continued presence of the drug. This ethanol-induced behavioral plasticity is referred to here as the development of acute functional tolerance. This assay has been used to demonstrate that these two phenotypes are distinct and genetically separable. The straightforward locomotion assay described here is suitable for examining the effects of both genetic and environmental manipulations on these acute behavioral responses to ethanol in C.&#160;elegans.

An Assay for Measuring the Effects of Ethanol on the Locomotion Speed of Caenorhabditis elegans

C. elegans is a useful model for studying the effects of ethanol on behavior. We present a behavioral assay that quantifies the effects of ethanol on the locomotion speed of crawling worms; both initial sensitivity and the development of acute functional tolerance to ethanol can be measured with this assay.

测量乙醇的影响上的步态速度的测定<em&gt;秀丽隐杆线虫</em

Alcohol use disorders are a significant public health concern, for which there are few effective treatment strategies. One difficulty that has delayed the ...

Agarose Microchambers for Long-term Calcium Imaging of Caenorhabditis elegans

Behavior is controlled by the nervous system. Calcium imaging is a straightforward method in the transparent nematode Caenorhabditis elegans to measure the activity of neurons during various behaviors. To correlate neural activity with behavior, the animal should not be immobilized but should be able to move. Many behavioral changes occur during long time scales and require recording over many hours of behavior. This also makes it necessary to culture the worms in the presence of food. How can worms be cultured and their neural activity imaged over long time scales? Agarose Microchamber Imaging (AMI) was previously developed to culture and observe small larvae and has now been adapted to study all life stages from early L1 until the adult stage of C. elegans. AMI can be performed on various life stages of C. elegans. Long-term calcium imaging is achieved without immobilizing the animals by using short externally triggered exposures combined with an electron multiplying charge-coupled device (EMCCD) camera recording. Zooming out or scanning can scale up this method to image up to 40 worms in parallel. Thus, a method is described to image behavior and neural activity over long time scales in all life stages of C. elegans.

Agarose Microchambers for Long-term Calcium Imaging of Caenorhabditis elegans

Imaging behavior and neural activity over long time scales without immobilization of the animal is a prerequisite to understand behavior. Agarose microfluidic chambers imaging (AMI) can be used to image neural activity and behavior for all life stages of Caenorhabditis elegans.

琼脂糖微室的长期钙成像<em&gt;秀丽隐杆线虫</em

Behavior is controlled by the nervous system. Calcium imaging is a straightforward method in the transparent nematode Caenorhabditis elegans to measure ...

Dynamic Quantitative Sensory Testing to Characterize Central Pain Processing

Central facilitation and modulation of incoming nociceptive signals play an important role in the perception of pain. Disruption in central pain processing is present in many chronic pain conditions and can influence responses to specific therapies. Thus, the ability to precisely describe the state of central pain processing has profound clinical significance in both prognosis and prediction. Because it is not practical to record neuronal firings directly in the human spinal cord, surrogate behavior tests become an important tool to assess the state of central pain processing. Dynamic QST is one such test, and can probe both the ascending facilitation and descending modulation of incoming nociceptive signals via TS and CPM, respectively. Due to the large between-individual variability in the sensitivity to noxious signals, standardized TS and CPM tests may not yield any meaningful data in up to 50% of the population due to floor or ceiling effects. We present methodologies to individualize TS and CPM so we can capture these measures in a broader range of individuals than previously possible. We have used these methods successfully in several studies at the lab, and data from one ongoing study will be presented to demonstrate feasibility and potential applications of the methods.

By assessing pain in response to repetitive or different types of standardized stimuli, dynamic quantitative sensory testing (QST) can reveal changes in the central processing of pain. We present methods to optimize and individualize two dynamic QST measures: temporal summation (TS) and conditioned pain modulation (CPM).

动态定量感觉测试表征中央疼痛处理

Central facilitation and modulation of incoming nociceptive signals play an important role in the perception of pain. Disruption in central pain ...

A Caenorhabditis elegans Nutritional-status Based Copper Aversion Assay

To ensure survival, organisms must be capable of avoiding unfavorable habitats while ensuring a consistent food source. Caenorhabditis elegans alter their locomotory patterns upon detection of diverse environmental stimuli and can modulate their suite of behavioral responses in response to starvation conditions. Nematodes typically exhibit a decreased aversive response when removed from a food source for over 30 min. Observation of behavioral changes in response to a changing nutritional status can provide insight into the mechanisms that regulate the transition from a well-fed to starved state.
We have developed an assay that measures a nematode&#39;s ability to cross an aversive barrier (i.e. copper) then reach a food source over a prolonged period of time. This protocol builds upon previous work by integrating multiple variables in a manner that allows for continued data collection as the organisms shift towards an increasingly starved condition. Moreover, this assay permits an increased sample size so that larger populations of nematodes can be simultaneously evaluated.
Organisms defective for the ability to detect or respond to copper immediately cross the chemical barrier, while wild type nematodes are initially repelled. As wild type worms are increasingly starved, they begin to cross the barrier and reach the food source. We designed this assay to evaluate a mutant that is incapable of responding to diverse environmental cues, including food sensation or detection of aversive chemicals. When evaluated via this protocol, the defective organisms immediately crossed the barrier, but were also incapable of detecting a food source. Hence, these mutants repeatedly cross the chemical barrier despite temporarily reaching a food source. This assay can straightforwardly test populations of worms to evaluate potential pathway defects related to aversion and starvation.

A Caenorhabditis elegans Nutritional-status Based Copper Aversion Assay

Here, we present a Caenorhabditis elegans-specific assay designed to evaluate changes in copper aversion behavior and the ability to locate a common food source, as the organism progresses from a well-fed to starved nutritional state.

一个<em秀丽隐杆线虫</em基于营养状态的铜反转测定

To ensure survival, organisms must be capable of avoiding unfavorable habitats while ensuring a consistent food source. Caenorhabditis elegans alter their ...

Ratiometric Calcium Imaging of Individual Neurons in Behaving Caenorhabditis Elegans

It has become increasingly clear that neural circuit activity in behaving animals differs substantially from that seen in anesthetized or immobilized animals. Highly sensitive, genetically encoded fluorescent reporters of Ca2+ have revolutionized the recording of cell and synaptic activity using non-invasive optical approaches in behaving animals. When combined with genetic and optogenetic techniques, the molecular mechanisms that modulate cell and circuit activity during different behavior states can be identified.
Here we describe methods for ratiometric Ca2+ imaging of single neurons in freely behaving Caenorhabditis elegans worms. We demonstrate a simple mounting technique that gently overlays worms growing on a standard Nematode Growth Media (NGM) agar block with a glass coverslip, permitting animals to be recorded at high-resolution during unrestricted movement and behavior. With this technique, we use the sensitive Ca2+ reporter GCaMP5 to record changes in intracellular Ca2+ in the serotonergic Hermaphrodite Specific Neurons (HSNs) as they drive egg-laying behavior. By co-expressing mCherry, a Ca2+-insensitive fluorescent protein, we can track the position of the HSN within ~ 1 &#181;m and correct for fluctuations in fluorescence caused by changes in focus or movement. Simultaneous, infrared brightfield imaging allows for behavior recording and animal tracking using a motorized stage. By integrating these microscopic techniques and data streams, we can record Ca2+ activity in the C. elegans egg-laying circuit as it progresses between inactive and active behavior states over tens of minutes.

Ratiometric Calcium Imaging of Individual Neurons in Behaving Caenorhabditis Elegans

This protocol describes the use of genetically encoded Ca2+ reporters to record changes in neural activity in behaving Caenorhabditis elegans worms. &#8195;

比率钙显像对单个神经元的行为线虫线虫

It has become increasingly clear that neural circuit activity in behaving animals differs substantially from that seen in anesthetized or immobilized ...

Imaging Dendritic Spines in Caenorhabditis elegans

Dendritic spines are specialized sites of synaptic innervation modulated by activity and serve as substrates for learning and memory. Recently, dendritic spines have been described for DD GABAergic neurons as the input sites from presynaptic cholinergic neurons in the motor circuit of Caenorhabditis elegans. This synaptic circuit can now serve as a powerful new in vivo model of spine morphogenesis and function that exploits the facile genetics and ready accessibility of C. elegans to live-cell imaging. 
This protocol describes experimental strategies for assessing DD spine structure and function. In this approach, a super-resolution imaging strategy is used to visualize the intricate shapes of actin-rich dendritic spines. To evaluate the DD spine function, the light-activated opsin, Chrimson, stimulates the presynaptic cholinergic neurons, and the calcium indicator, GCaMP, reports the evoked calcium transients in postsynaptic DD spines. Together, these methods comprise powerful approaches for identifying genetic determinants of dendritic spines in C. elegans that could also direct spine morphogenesis and function in the brain.

Imaging Dendritic Spines in Caenorhabditis elegans

Dendritic spines are important cellular features of the nervous system. Here live imaging methods are described for assessing the structure and function of dendritic spines in C. elegans.&#160;These approaches support the development of mutant screens for genes that define dendritic spine shape or function.

秀丽隐杆线虫树突棘成像

Dendritic spines are specialized sites of synaptic innervation modulated by activity and serve as substrates for learning and memory. Recently, dendritic ...

In vivo Assessment of Microtubule Dynamics and Orientation in Caenorhabditis elegans Neurons

In neurons, microtubule orientation has been a key assessor to identify axons that have plus-end out microtubules and dendrites that generally have mixed orientation. Here we describe methods to label, image, and analyze the microtubule dynamics and growth during the development and regeneration of touch neurons in C. elegans. Using genetically encoded fluorescent reporters of microtubule tips, we imaged the axonal microtubules. The local changes in microtubule behavior that initiates axon regeneration following axotomy can be quantified using this protocol. This assay is adaptable to other neurons and genetic backgrounds to investigate the regulation of microtubule dynamics in various cellular processes.

In vivo Assessment of Microtubule Dynamics and Orientation in Caenorhabditis elegans Neurons

A protocol for imaging the dynamic microtubules in vivo using fluorescently labeled End binding protein has been presented. We described the methods to label, image, and analyze the dynamic microtubules in the Posterior lateral microtubule (PLM) neuron of C. elegans.

在体内卡诺哈布德炎电子神经元微管动力学和方向评估

In neurons, microtubule orientation has been a key assessor to identify axons that have plus-end out microtubules and dendrites that generally have mixed ...

新颖的指标来表征线虫胚胎伸长率

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在体内卡诺哈布德炎电子神经元微管动力学和方向评估