Work in the Arur Lab is supported by grants from the National Institutes of Health, NIHGM98200; Cancer Prevention Research Institute of Texas, CPRIT RP160023; the American Cancer Society Research Scholar Award (ACS RSG014-044-DDC); and by the Anna Fuller Funds.

这里描述的协议主要是对无脊椎动物模型系统C.线虫 ，并遵循所有由该机构规定的道德准则。 1.动物保养<ol><li> C.维护线虫线虫生长培养基20,21（NGM，见材料表 ）工作储备培养琼脂接种E.大肠杆菌 OP50。 </li><li>在16℃至25℃之间的温度下保持蠕虫股票。 注:在较快的增长速度，经常，生殖异常发展并降低育雏规模较高的温度导致的培养蠕虫。此外，温度敏感的基因型显示在不同温度下的不同的表型。 </li><li>转移蠕虫新NGM板，每2 - 1天根据它们的生长速率，以维持良好的供给蠕虫的恒定供给。 注意:对于涉及dpERK水平的任何给定的实验，蠕虫病毒不应该从一个饿死或人群获得ED板。拥挤或饥饿影响胰岛素的蠕虫22信令和胰岛素信号调节ERK 12活性。因此，从饥饿或拥挤的板蠕虫会导致可变和难以再生染色模式。 </li></ol> 2.解剖成虫的性腺获得<ol><li>挑100 - 150的WT（N 2）或在1.5毫升微量离心管中100微升的M9缓冲的期望和特定发育阶段（L1，L2，L3，L4，成人等 ）蠕虫的期望的基因型。 </li><li>装满M9的缓冲区的900微升的离心管（见材料表 ）。离心在1000 xg离心管在微量在环境温度下1分钟。 </li><li>轻轻取下M9的缓冲区900μL并丢弃。在解剖显微镜下去掉缓冲，以确保蠕虫不会无意中除去。 </li><li>重复步骤2.2 - 每次2.3两次用新鲜的M9的缓冲区。这确保了与蠕虫结转，任何细菌都有效地冲走。 </li><li>最后一次洗涤后从管中，并弃删除M9缓冲区的900微升。 </li><li>包含100剩下的100 M9的微升缓冲传输 - 150蠕虫用200微升，枪头，以平底玻璃表镜菜。确保枪头在使用前在10微升切痕。不切割尖端将导致蠕虫剪切。 </li><li>加入1 - 0.1M的左旋咪唑3微升包含在M9的缓冲区蠕虫玻璃手表菜。轻轻地摇晃左旋咪唑和M9，使搅拌均匀，直至蠕虫停止移动。 注:左旋咪唑股可以储存在-20℃下长期贮存。这里，可使用的1高浓度- 3mM的比已经在0.2的文献23描述了-左旋咪唑0.25毫为了获得在动物移动性迅速丧失。如果动物剥周围太升翁在液体悬浮液，dpERK在性腺所得图案可以是可变的（由于压力或营养缺乏或两者）。更重现染色模式已经实现了与1 - 3毫米左旋咪唑解剖。 </li><li>连接两个地下25针两个1毫升注射器（注射器的大小并不重要，所述针的型号是非常关键的）。在各手位置的一个注射器（ 图2）。 </li><li>在解剖显微镜下，每一个定位下针，并在每一个蠕虫病毒， 如图2，将一细切附近的剪刀状运动第二咽灯泡的蠕虫病毒。在蜗杆一个切口后移动到下一个动物直到在盘中的所有动物都被切割至少一次。 注意:要留意的是，步骤2.8 - 2.9对时间敏感。解剖的菜虫都在五分钟的重现性dpERK模式。较长解剖倍将导致关闭dpERK信号的，特别是沿Z1 1.调整每轮夹层（ 即 50蠕虫与 150），如果有必要的蠕虫的数量保持在规定的时间框架。 <ol><li>如果挤压性腺是不可见的解剖过程中，不要试图在同一动物的另一个晋级。通常，这将仅剪切动物和不会导致性腺的挤压。相反，移动到下一个动物。蠕虫那里的性腺不挤压会出现这样的将在第6条提出，并可以成像过程中忽略的幻灯片 注:通过所有的解剖和处理步骤，但记住，性腺将保持附着在身体/胴体来承担是非常重要的。不要强迫性腺和外体用针。很多时候，在性腺组织的异常撕裂，企图从主体切断性腺可导致杂散抗体信号。主体/胎体可以在成像工序（第6节）被忽略，并且只有生殖腺成像。此外，索姆etimes肠细胞也可以作为内部控制/躯体控制该抗体被测定。 </li></ol></li></ol> 3.固定性腺<ol><li>解剖完成后，加入2毫升的3％多聚甲醛（PFA）直接到含有100微升的M9的玻璃表皿和解剖动物和用Parafilm覆盖。放置在一个化学通风柜被覆手表玻璃盘。 注意:PFA是一种危险化学品。从而，定影应该在化学通风橱中进行。 </li><li>孵育在室温10分钟。 </li><li>使用一次性9"巴斯德玻璃吸管通过绘制PFA溶液与1X PBS-T与解剖动物进入巴斯德添加1X PBS-T（见材料表 ）3毫升含解剖动物手表玻璃盘，混合吸管和释放轻轻放回手表玻璃盘，洗涤液时不要涡旋管。 </li><li>使用FREsh的玻璃巴斯德吸管吸取5mL的液体（含有解剖蠕虫，PFA和1x PBS-T）从手表玻璃皿放入巴斯德吸管和内容传送到一个新的5毫升的玻璃锥形管中。 </li><li>离心30秒在临床离心机在1000×g下的锥形管中。使用玻璃巴斯德吸液管和锥形管作为解剖性腺坚持塑料。 </li><li>取出并弃去上清液小心，以免打扰解剖动物。从锥形管在解剖显微镜下每次洗涤后除去液体，以便不影响解剖性腺，并最大限度地减少其损失。 </li><li>用一个新的巴斯德吸管，加入5 1X毫升PBS-T的锥形管。离心机在1000×g的临床离心机30秒的锥形管中。取出并弃去上清液小心，以免打扰解剖动物。 </li><li>重复步骤3.7，共三次。 </li><li>用一个新的巴斯德吸管，加入2毫升的100％甲醇管，轻轻地通过绘制成巴斯德吸管混合与甲醇的性腺。孵育在-20℃的管最少1小时。 注:解剖性腺可以存储在甲醇长达2天的dpERK染色。存储超过2天，最多2周是与其他抗体染色的应用，如抗拉明抗体兼容，但不与dpERK抗体。 </li><li>所需的温育时间之后，重复步骤3.7，共三次以洗涤性腺。在洗涤过程中不要涡管。在最后一次洗涤后离开1X的PBS-T 500微升的5毫升锥形管中。 </li><li>用一个新的巴斯德吸管锥形玻璃管的内容物转移到新鲜1毫升的玻璃管（6毫米×50毫米）。允许解剖动物通过重力5安顿 - 10分钟。 </li><li>用一个新的巴斯德吸管和在解剖显微镜下，除去尽可能多的洗涤尽可能从1毫升玻璃管机智豪特扰乱解剖性腺。 </li></ol> 4.阻塞和主要抗体治疗<ol><li>加入100微升的30％在1×PBS-T稀释正常山羊血清（NGS）（见材料表 ），以在1毫升的玻璃管中的解剖动物。 30％NGS用作封闭缓冲液。覆盖用封口膜的管，以避免该液体的蒸发。孵育在室温下1小时或在4℃下过夜。 </li><li>后培养的所需时间，删除使用新鲜的巴氏吸管的封闭缓冲液，除去尽可能多的液体尽可能。使用解剖显微镜，以确保性腺未在巴斯德吸管制定。 </li><li>稀释MAPKYT抗体（见材料表 ，在该方法称为dpERK）以1:400在30％NGS。准备足够的抗体稀释到每管使用100微升。未使用的，稀释的抗体可被储存在4℃下一周。 </li><li>加入100微升的稀释的抗dpERK抗体的这样lution到包含解剖动物1mL的玻璃试管中。密封用封口膜管。孵育在4℃下过夜管。 </li><li>过夜孵育后，添加1×PBST 800μL到在RT管。 </li><li>画出样品上在一个新的玻璃巴斯德吸管和释放轻轻确保性腺均匀悬浮在1×PBS-T。让性腺解决与重力底部。去除上清。这确保了解剖性腺洗涤，但在此过程中不被损坏。 </li><li>重复步骤4.5 - 4.6总共洗涤三次。在洗涤过程中不要涡管。第三次洗涤后，请务必拆除和丢弃尽可能多上清尽可能不干扰动物解剖。确保新鲜巴氏吸管每次使用。 </li></ol> 5.二级抗体治疗<ol><li>在第一抗体洗涤准备稀释的二级抗体。稀释的抗小鼠本身在1 condary抗体:500在30％NGS。作为与第一抗体，使用每管100微升。未使用的抗体可以储存在4℃下一周。 </li><li>添加100μL的二级抗体溶液含有解剖动物1mL的玻璃试管中。覆盖各管用Parafilm，然后包裹在铝箔的管，以保持所述管的内容物在黑暗中。孵育在RT管2小时，或者在4℃过夜。 </li><li>后培养的所需时间，加入1倍的PBS-T 800μL到管，并重复步骤4.5 - 4.6总共三次。最后一次洗涤后，除去，并用新鲜的巴氏吸管丢弃一个解剖显微镜下尽可能多的上清液尽可能的。 </li><li>准备洗涤为二次抗体中的DAPI溶液中。稀释的DAPI 1微升（从1000倍库存1毫克/毫升的储存在-20℃）合1毫升1×PBST的。 </li><li>添加稀释的DAPI溶液的800微升到包含所述管剖分动物。覆盖用封口膜的管口，并且包裹在铝箔每个管。孵育在室温20分钟。 </li><li>用DAPI培养后，除去尽可能多的DAPI溶液尽可能用新鲜的巴氏吸管的，在解剖显微镜下，以便不打扰解剖动物。 </li><li>加安装溶液在管子1滴。包装在铝箔每管。 注:对于可视化dpERK染色，染色性腺应立即安装显微镜。对于其它抗体的应用，如磷酸化丝氨酸10组蛋白H3，性腺可以保持在黑暗中在4℃下固定媒体长达2周。 </li></ol> 6.组装幻灯片和成像<ol><li>放置实验室带的一层纵向，在上面，作为如图3所示两个玻璃显微镜载片（25毫米×75毫米×1毫米）。将新鲜干净的玻璃显微镜载片在两个录音幻灯片之间。 注:厚度磁带的有助于产生在中间滑动琼脂糖垫。琼脂糖垫的厚度几乎等于该带的，作为磁带用作用于创建琼脂糖垫的隔板。 </li><li>在中间的显微镜载玻片一滴熔融的2％琼脂糖的〜200微升（在蒸馏水中制成）。快速地将新鲜干净的玻片，垂直和顶部，琼脂糖。 </li><li>让琼脂糖固化（1 - 2分钟）。 </li><li>非常温和地去除顶端显微镜载玻片，使得琼脂糖垫不被破坏。琼脂糖垫可保持连接或者到顶部或底部滑动。不要紧其中滑动琼脂糖垫保持附连到的，只要它是琼脂糖的光滑表面。 </li><li>使用巴斯德吸管解剖动物转移到琼脂糖垫。删除用解剖显微镜下巴斯德吸管滑动任何多余的液体。 </li><li>使用睫毛头发或精拉毛细管，推性腺和肠子从以传播性腺出的第一张幻灯片的另一种方式。 </li><li>覆盖24毫米×50毫米大小的盖玻片显微镜载玻片。小心确保盖玻片轻轻降低到与盖玻片与载玻片之间形成极小的气泡的幻灯片。 </li><li>储存于4℃过夜在滑动箱（与载玻片平躺，盖玻片了一个不透明滑动箱），以允许多余的液体蒸发和性腺变得有些扁平（由于盖玻片上性腺的重量）。性腺的略有缩小使得原本一轮性腺管的更好的成像。 <ol><li>一旦盖玻片被安装时，不施加压力，以用手指盖玻片的顶部。通常这导致性腺和dpERK信号丢失的失真。 </li><li>拼合性腺隔夜只为更有效的成像。幻灯片准备尽快它们组装查看。要立即采取图片，轻轻吸盖玻片并从使用干净的实验室组织的拐角滑动之间的多余的液体。 </li></ol></li><li>在过夜后，密封该滑动并与盖玻片和滑动边界的四个角透明指甲油面漆盖玻片。指甲油/盖玻片密封，确保盖玻片不同时成像移动和防止样品的破坏。 </li><li>图像与63X复式显微镜性腺。然而物镜和显微镜可以基于显微镜和用户应用程序的可用性而变化。因为整个性腺从增殖区域的大小的卵母细胞比可以在一个图像被捕获时，所述图像被取为一蒙太奇重叠边界， 如图4。11-14，18。 <ol><li>在许多情况下，编辑胎体最终成像和组装之后，只要没有大的变化都与种系图像制成。店里的T沿着组装的形象，他原始图像，使&#39;前&#39;装配/编辑的对比和图像的组装"后"/编辑。 注:显微图像不能蒙太奇的装配过程中被改变的信号强度或饱和度。 </li></ol></li></ol>

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The authors declared that no competing interests exist.

主动ERK（dpERK）遵循了C的刻板空间和动力定位模式线虫生殖细胞。这种定型dpERK空间图案（ 图5），和振幅中的成人C.线虫性腺，可以与ERK的调节许多生物过程被有效地相关。例如，无法激活ERK在2区导致逮捕在卵母细胞减数分裂，在不指定精子，因而不激活成熟卵母细胞ERK（ 图5）女性生殖细胞系观察到的表型的前期。与男性"在2区在女性生殖细胞系11,13 dpERK的有效积累，加上卵母细胞成熟和排卵的发生结果交配。因此，空间格局，并在一定的遗传扰动（如RNA干扰介导的基因失活）或化学处理dpERK的时间激活分析，便可以对REGU因素识别晚ERK信号，从而ERK依赖的生物学过程。这样的分析也使信号通路之间新的遗传相互作用的发现。 用于任何成像基于分析的关键瓶颈是官能试剂的可用性如特定于应用程序的抗体。如果那么这样的试剂的存在，如上述的一种方法，可以很容易地适于本地化模式的分析为任何感兴趣的蛋白质。应用程序因此通过一个功能抗体的可用性的限制。此外，由于该方法描述了在给定的发育时间解剖和染色，它不仅能使的信息在一时间帧的捕捉，以及实时或蛋白质周转速率的动态或蛋白质调控不线索。 上述方法适于从Francis 等。23，其是从Seydoux和唐恩m个不同ethod 24性腺挤压和抗体染色。根据弗朗西斯等人的方法。将被称为"悬浮法"并称为"冻结破解方法"进行比较的Seydoux和邓恩方法。该悬浮液的方法是非常有效的在我们手中用于获得信号传导分子如dpERK，这是固有苛刻的操作条件更敏感的可重复的定位模式。在dpERK定位的情况下，根据我们的经验，在1区的信号与冻结破解方法几乎检测不到。此外，样品烘干，而这往往与冻结破解方法出现，结果中的虚假抗体信号。该悬浮液的方法最大程度地减少样品烘干，由于性腺悬浮于液体的整个过程。我们还发现，悬浮法是上一张幻灯片成像多个不同基因型有用。例如，如果两个基因型，如雌雄同体VS女性必须直接用于dpERK定位相比，两种基因型可以混合在一起，并进行处理。因为表型是不同的，并且可以通过 DAPI形态区分开来，这是可能的。染色在同一管，随后通过成像在同一滑动允许从不同基因型性腺之间直接比较。或者，如果DAPI形态没有区别，则一个两步抗体染色可以采用。首先每个个体基因型与两个不同的抗体治疗，抗体，一种WT和抗体B突变体（两种抗体需要从dpERK抗体不同的宿主，以升高）。一旦两个基因型已染色与第一抗体，并洗涤，可以将它们一起在一个试管混合并立即与dpERK抗体染色，接着通过二次抗体处理可视化在管的所有抗体。能力为D时，不同基因型上一张幻灯片比较，特别是的激活模式eductions正在作出保证不会受到明显的染色条件准确的解释。 虽然有利，也有整个悬浮法多个步骤，需要谨慎操作。至关重要的是，发生在时间效率的方式解剖;重要的是，夹层不应该接管5分钟。较长解剖时间导致可变dpERK信号，其可以经常被误认为在ERK活化调节的变化，而不是作为技术故障。因为在各个洗涤步骤性腺可以容易地从由于移液误差管失去150动物为每个解剖 - 它是先从100是至关重要的。 （如每50动物三批）可以组合，或通过解剖一个大批量的150可以通过在多个小批量解剖减小性腺损耗的另一个挑战是越来越性腺趴在良好隔开编队在滑动，以便整个远侧性腺可以成像。为了实现这一目标，使用睫毛上的火柴或拉吸管太空，性腺。然而，应小心以免在此过程中损坏性腺。常与这些技术需要多次尝试掌握解剖以及性腺的安排上滑动。 一旦此技术已经被掌握，它用作用于多样生物分析的有效方法，并可以用于研究不仅仅是dpERK水平以上。例如，这方法可用于可视化活性p38的水平，或活性型CDK的水平，或为其中抗体试剂存在的任何蛋白质。另外，该方法可以配上在整体动物的化学抑制屏幕测定为新型抑制剂或途径的活化， 通过化验为dpERK水平。这将允许两个反应和敏感性可能诠释任何抑制剂的体内的读出与RTK-RAS-ERK信号通路eract。此外，这种方法可以在具有多个信号输出抗体组合了都可以使用，并且在同一时间可视化中使用。使用多种抗体允许多种途径，其激活状态之间的相关性，和结果都是一样的组织内，从而更好地理解这些相互作用的。这些是这种方法的另一个应用的几个例子，但该系统可以修改为研究多种研究兴趣。

的受体酪氨酸激酶（RTK） -大鼠肉瘤（RAS）-Extracellular信号调节激酶（ERK）途径通过一个保守的激酶级联，其导致ERK 1-3的磷酸化和激活中继外信号。 ERK蛋白的保守脯氨酸定向的丝氨酸/苏氨酸的MAP（促分裂原活化蛋白）激酶家族成员，和由MEK 经由在保守TEY基序的苏氨酸（T）和酪氨酸（Y）的双重磷酸化被直接激活。活性ERK（简称diphosphorylated ERK，或dpERK），然后通过其下游底物1-3的电池的磷酸化调节许多细胞和发育过程。因此异常ERK活性导致许多细胞和发育缺陷4-7。 ERK活性严厉的调控是正常发育的关键:在哺乳动物系统太多ERK活性导致过度的细胞增殖领先到致癌增长;活动太少导致细胞死亡4,6。另外，在ERK活性的持续时间的变化也可导致不同的结果:在PC12细胞中，ERK活化为60分钟或更多诱导神经元分化8,9 30分钟或更小诱导细胞增殖，但ERK活化。 ERK活性的紧缩调控因而是正常发育和体内平衡显然是必不可少的。 线虫是一个强大的，和基因可塑性模型系统解剖RTK-RAS-ERK信号通路3,10-15的功能和调节。相对于哺乳动物系统，其中包含的RAS和ERK，C多个基因线虫包含一个RAS基因（ 让-60）和一个ERK基因（MPK - 1），使得它在其中剖析该途径3,10-14的功能的基因更容易的系统。 C.在线虫种系本质上是一个管，它由有丝分裂的茎在其远端并在其近端（ 图1）16细胞成熟的卵母细胞。生殖细胞发起减数分裂只是通过扩展减数分裂前期（粗线），之后他们开始形成在环区域的卵母细胞，最后进行卵母细胞成熟中的近端区域16近端向远端有丝分裂区，和进步。 从多个实验室，包括我们自己的遗传学研究，证明了RTK-RAS-ERK信号通路是在生殖C.发展至关重要线虫 12,13,15,17-19。具体来说，我们发现，ERK控制和种系发育过程坐标至少九个不同的生物过程，从发育开关，如生殖细胞凋亡与细胞生物过程，如卵母细胞的生长11,18。很像在哺乳动物系统中，C.太多ERK活性线虫生殖细胞导致生产多个小卵母细胞中，而李太ttle活动结果于一体的大型卵母细胞11。因此dpERK的严格监管是正常的生殖细胞发展至关重要。在成熟再次dpERK中期粗线期间是高（ 区1，图1），低的环区和高:ERK的活性形式，如可视化通过特定dpERK的抗体，显示一个定型的，动态的，双峰的定位图案卵母细胞（ 区域2，图1）。最近，我们发现，营养作用通过胰岛素样生长因子受体1（DAF-2）在1区12来激活ERK;以前的工作表明，精子的信号（ 通过肝配蛋白受体酪氨酸激酶）在2区13激活ERK。 鉴于活性ERK的作为在种系变阻器来调节卵母细胞的生长，空间和时间定位，以及dpERK的幅度，是关键是理解它的正常信令结果。使用此处描述的方法中，在改变dpERK的刻板定位可以很容易地监控并在环境或遗传扰动对ERK活性，因此功能的影响，得出的预测。因此，测定为dpERK使得其作用的种系发育期间的全面理解。

<table><tbody><tr><td>Agarose</td><td>Sigma Inc.</td><td>A9539</td><td>Dissolve 2 g in 100 mL to make the 2% solution</td></tr><tr><td>Flat bottom glass watch dish</td><td>Agar Scientific</td><td>AGL4161</td><td>We use glass because dissected gonads often stick to plastic</td></tr><tr><td>25 G needles</td><td>BD PrecisionGlide</td><td>305122</td><td></td></tr><tr><td>Syringes (could be 1 or 5 mL)</td><td>BD Syringes</td><td>1 mL: BD 309659</td><td></td></tr><tr><td>Microscope slides (25 x 75 x 1.0 mm)</td><td>Fisherbrand</td><td>12-550-343</td><td></td></tr><tr><td>Coverslips (24 x 50 mm)</td><td>Corning</td><td>2935-245</td><td></td></tr><tr><td>5 mL glass conical tube</td><td>Corning</td><td>8060-5</td><td></td></tr><tr><td>9&quot; disposable Pasteur pipette</td><td>Fisherbrand</td><td>13-678-20D</td><td></td></tr><tr><td>Clinical bench top centrifuge</td><td></td><td></td><td></td></tr><tr><td>Glass&amp;nbsp; tubes (6 x 50 mm)</td><td>Fisherbrand</td><td>14-958-A</td><td></td></tr><tr><td>*M9 solution</td><td></td><td></td><td></td></tr><tr><td>Levamisole</td><td>Sigma</td><td>L9756</td><td></td></tr><tr><td>3% Paraformaldehyde (PFA)</td><td>Electron microscopy services</td><td>17500</td><td>Obtained as 16% solution in ampoules, and diluted to 3% in Potassium Phosphate Buffer</td></tr><tr><td>1x PBS-T</td><td>1x PBS with 0.1% Tween 20</td><td></td><td></td></tr><tr><td>Methanol</td><td>Electron microscopy services</td><td>18510</td><td></td></tr><tr><td>Normal goat serum (NGS)</td><td>Cell Signaling</td><td>5425</td><td>Diluted to 30% in 1x PBS-T</td></tr><tr><td>MAPKYT (dpERK) antibody&amp;nbsp;</td><td>Sigma Inc.</td><td>M9692</td><td>Dilution 1:400 in 30% NGS</td></tr><tr><td>Secondary antibody</td><td>Invitrogen</td><td>A-11005</td><td>Dilution 1:500 in 30% NGS</td></tr><tr><td>DAPI</td><td>Sigma</td><td>D9542</td><td></td></tr><tr><td>Vectashield</td><td>Vector Labs</td><td>H-1000</td><td></td></tr><tr><td>*M9 Buffer Recipe</td><td></td><td></td><td>3 g KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;, 6 g Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;, 5 g NaCl, 1 mL 1 M MgSO&lt;sub&gt;4&lt;/sub&gt;, H&lt;sub&gt;2&lt;/sub&gt;O to 1 L.&amp;nbsp;</td></tr><tr><td>**PBS (1x)</td><td></td><td></td><td>8 g NaCl, 0.2 g KCl, 1.44 g Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;, 0.24 g KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;, 0.133 g CaCl&lt;sub&gt;2&lt;/sub&gt;, 0.10 g MgCl&lt;sub&gt;2&lt;/sub&gt;, H&lt;sub&gt;2&lt;/sub&gt;O to 1 L</td></tr><tr><td>Nematode Growth Medium (NGM)</td><td></td><td></td><td>3 g NaCl, 17 g Agar, 2.5 g Peptone, 975 mL of water in 2 L Erlenmayer Flask. Autoclave for 50 min. Cool, and add 1 mL of 1 M CaCl&lt;sub&gt;2&lt;/sub&gt;, 1 mL of 5 mg/mL cholesterol, 1 mL of 1 M MgSO&lt;sub&gt;4&lt;/sub&gt; and 25 mL of 1 M KPO&lt;sub&gt;4&lt;/sub&gt;.&amp;nbsp;</td></tr></tbody></table>

spatial and temporal analysis of active erk in the c. elegans germline

在进化上保守的细胞外信号转导的RTK-RAS-ERK途径是重要的激酶信号级联经由 ERK，所述通路的末端激酶的激活主要控制多个蜂窝和发育过程。 ERK活性的紧缩调控是正常发育和体内平衡至关重要;过度的细胞增殖过度活跃ERK的结果，但不够活跃的ERK导致细胞死亡。C.线虫是一种强大的模型系统，帮助开发过程中表征RTK-RAS-ERK信号通路的功能和调节。特别是，RTK-RAS-ERK途径为C.必不可少线虫种系的发展，这是该方法的焦点，利用特定于ERK（dpERK）的活性，diphosphorylated形式的抗体，所述定型本地化模式可以与种系内被可视化。因为该模式是在空间和时间控制研究版，可重复地测定dpERK的能力是有用的，以确定影响dpERK信号持续时间和幅度，因而种系发育途径的调节剂。在这里，我们将演示如何成功地解剖，染色和图像dpERK的内C.线虫性腺。这种方法可以适用于任何信号的空间定位或结构中的蛋白质C.线虫性腺，提供兼容的免疫抗体是可用的。

在成年野生动物雌雄同体，dpERK通常是通过-4或-5（2区）可视中旬粗线区，1区，并在从-1最成熟的卵母细胞。在该激活模式扰动反映改变到信号通路。母种系不指定精子，因此不会在第2区显示的ERK的活化，仅在1区弱活化这些在图5表示。 <img alt="图1" src="/files/ftp_upload/54901/54901fig1.jpg" /> 图1:C。线虫生殖形态，用白线概述一个U形形的性腺手臂成人雌雄同体的微分干涉对比（DIC）的形象。成年两性性腺包含在远侧尖端有丝分裂的细胞。有丝分裂的细胞通过减数分裂（粗线期）发展成为matur进入减数分裂和进步Ë卵母细胞在近端性腺。在成人两性性腺dpERK的1区和2马克刻板的本地化。规模:20微米<a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54901/54901fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/54901/54901fig2.jpg" /> 图2:解剖过程中针位的示范左 :过程解剖的照片。右:参照蠕虫针位置<a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54901/54901fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图3" src="/files/ftp_upload/54901/54901fig3.jpg" /> 图3:琼脂糖准备幻灯片演示。 （a）将磁带lengthwisË在2个干净的显微镜载玻片。放置两个幻灯片与磁带之间的新鲜干净的显微镜载玻片，如图所示。三个幻灯片彼此相邻纵向。（B）添加琼脂糖熔化，在中心的幻灯片。（C）将新鲜的显微镜载玻片垂直，以及，中间滑板顶部，承载琼脂糖的下降。（ D）允许琼脂糖凝固。（E）拆下顶部滑动留下凝固的琼脂糖凝胶垫后面。使用有安装解剖和种系染色琼脂糖垫的底部幻灯片。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54901/54901fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图4" src="/files/ftp_upload/54901/54901fig4.jpg" /> 图4:成像幻灯片作为野生型种系用DAPI 一个蒙太奇蒙太奇（ 吨 。运算）和dpERK（ 底部 ）信道。在63X重叠边界拍摄的图像，是在两个不同的通道同时取出白色框面板A和B以及绿盒面板B和C的DAPI和dpERK图像为每个面板。组装的图像示于图5A。比例尺:20微米<a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54901/54901fig4large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图5" src="/files/ftp_upload/54901/54901fig5.jpg" /> 图5:动态时空的ERK /空间激活。 （AC）WT（N2）成人（发展24小时过去年年L4期）染色的DNA（B，DAPI，白）和dpERK雌雄同体种系（C，红色）。在1区，粗线区和2区，成熟的卵母细胞的dpERK信号被高亮显示。（DF）雾-2（oz40）女性生殖细胞系（在8小时的发展过去年年L4期）染色的DNA（E，DAPI）和dpERK（F）。年轻女性生殖细胞系显示在1区弱dpERK，并在精子独立的方式单一的卵母细胞。区2是精子依赖性的，并因此在阴种系不存在。比例尺:20微米<a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54901/54901fig5large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about Spatial and Temporal Analysis of Active ERK in the C. elegans Germline at JoVE.com

线虫种系中活性 ERK 的时空分析发育生物学|视频

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

我们目前在解剖C.积极ERK的时间和空间定位的免疫基于成像的方法线虫性腺。这里所描述的协议可以适于任何信令的可视化或结构蛋白中的C.线虫性腺，提供了合适的抗体试剂是可用的。

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 ...

spatial-and-temporal-analysis-of-active-erk-in-the-c-elegans-germline

Nanyang Technological University

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

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10.1K 次观看.  Baylor College of Medicine. 我们目前在解剖C.积极ERK的时间和空间定位的免疫基于成像的方法线虫性腺。这里所描述的协议可以适于任何信令的可视化或结构蛋白中的C.线虫性腺，提供了合适的抗体试剂是可用的。 

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

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

Identification of EGFR and RAS Inhibitors using Caenorhabditis elegans

The changes in the plasma membrane localization of the epidermal growth factor receptor (EGFR) and its downstream effector RAS have been implicated in several diseases including cancer. The free-living nematode C. elegans possesses an evolutionary and functionally conserved EGFR-RAS-ERK MAP signal cascade which is central for the development of the vulva. Gain of function mutations in RAS homolog LET-60 and EGFR homolog LET-23 induce the generation of visible nonfunctional ectopic pseudovulva along the ventral body wall of these worms. Previously, the multivulval (Muv) phenotype in these worms has been shown to be inhibited by small chemical molecules. Here we describe a protocol for using the worm in a liquid-based assay to identify inhibitors that abolish the activities of EGFR and RAS proteins. Using this assay, we show R-fendiline, an indirect inhibitor of K-RAS, suppresses the Muv phenotype expressed in the let-60(n1046) and let-23(sa62) mutant worms. The assay is simple, inexpensive, is not time consuming to setup, and can be used as an initial platform for the discovery of anticancer therapeutics.

Identification of EGFR and RAS Inhibitors using Caenorhabditis elegans

The genetically tractable nematode Caenorhabditis elegans can be used as a simple and inexpensive model for drug discovery. Described here is a protocol to identify anticancer therapeutics that inhibit the downstream signaling of RAS and EGFR proteins.

使用卡诺哈布迪炎埃莱甘斯识别 EGFR 和 RAS 抑制剂

The changes in the plasma membrane localization of the epidermal growth factor receptor (EGFR) and its downstream effector RAS have been implicated in ...

癌症研究

Computational Analysis of the Caenorhabditis elegans Germline to Study the Distribution of Nuclei, Proteins, and the Cytoskeleton

The Caenorhabditis elegans (C. elegans) germline is used to study several biologically important processes including stem cell development, apoptosis, and chromosome dynamics. While the germline is an excellent model, the analysis is often two dimensional due to the time and labor required for three-dimensional analysis. Major readouts in such studies are the number/position of nuclei and protein distribution within the germline. Here, we present a method to perform automated analysis of the germline using confocal microscopy and computational approaches to determine the number and position of nuclei in each region of the germline. Our method also analyzes germline protein distribution that enables the three-dimensional examination of protein expression in different genetic backgrounds. Further, our study shows variations in cytoskeletal architecture in distinct regions of the germline that may accommodate specific spatial developmental requirements. Finally, our method enables automated counting of the sperm in the spermatheca of each germline. Taken together, our method enables rapid and reproducible phenotypic analysis of the C. elegans germline.

Computational Analysis of the Caenorhabditis elegans Germline to Study the Distribution of Nuclei, Proteins, and the Cytoskeleton

We present an automated method for three-dimensional reconstruction of the Caenorhabditis elegans germline. Our method determines the number and position of each nucleus within the germline and analyses germline protein distribution and cytoskeletal structure.

秀丽线虫生殖的计算分析研究细胞核、蛋白质和细胞骨架的分布

The Caenorhabditis elegans (C. elegans) germline is used to study several biologically important processes including stem cell development, apoptosis, and ...

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 ...

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 ...

A Semi-high-throughput Imaging Method and Data Visualization Toolkit to Analyze C. elegans Embryonic Development

C. elegans is the premier system for the systematic analysis of cell fate specification and morphogenetic events during embryonic development. One challenge is that embryogenesis dynamically unfolds over a period of about 13 h; this half day-long timescale has constrained the scope of experiments by limiting the number of embryos that can be imaged. Here, we describe a semi-high-throughput protocol that allows for the simultaneous 3D time-lapse imaging of development in 80&#8211;100 embryos at moderate time resolution, from up to 14 different conditions, in a single overnight run. The protocol is straightforward and can be implemented by any laboratory with access to a microscope with point visiting capacity. The utility of this protocol is demonstrated by using it to image two custom-built strains expressing fluorescent markers optimized to visualize key aspects of germ-layer specification and morphogenesis. To analyze the data, a custom program that crops individual embryos out of a broader field of view in all channels, z-steps, and timepoints and saves the sequences for each embryo into a separate tiff stack was built. The program, which includes a user-friendly graphical user interface (GUI), streamlines data processing by isolating, pre-processing, and uniformly orienting individual embryos in preparation for visualization or automated analysis. Also supplied is an ImageJ macro that compiles individual embryo data into a multi-panel file that displays maximum intensity fluorescence projection and brightfield images for each embryo at each time point. The protocols and tools described herein were validated by using them to characterize embryonic development following knock-down of 40 previously described developmental genes; this analysis visualized previously annotated developmental phenotypes and revealed new ones. In summary, this work details a semi-high-throughput imaging method coupled with a cropping program and ImageJ visualization tool that, when combined with strains expressing informative fluorescent markers, greatly accelerates experiments to analyze embryonic development.

A Semi-high-throughput Imaging Method and Data Visualization Toolkit to Analyze C. elegans Embryonic Development

This work describes a semi-high-throughput protocol that allows simultaneous 3D time-lapse imaging of embryogenesis in 80&#8211;100 C. elegans embryos in a single overnight run. Additionally, image processing and visualization tools are included to streamline data analysis. The combination of these methods with custom reporter strains enables detailed monitoring of embryogenesis.

半高通量成像方法和数据可视化工具包，用于分析C. elegans胚胎发育

C. elegans is the premier system for the systematic analysis of cell fate specification and morphogenetic events during embryonic development. One ...

In Situ Detection of Ribonucleoprotein Complex Assembly in the C. elegans Germline using Proximity Ligation Assay

Understanding when and where protein-protein interactions (PPIs) occur is critical to understanding protein function in the cell and how broader processes such as development are affected. The Caenorhabditis elegans germline is a great model system for studying PPIs that are related to the regulation of stem cells, meiosis, and development. There are a variety of well-developed techniques that allow proteins of interest to be tagged for recognition by standard antibodies, making this system advantageous for proximity ligation assay (PLA) reactions. As a result, the PLA is able to show where PPIs occur in a spatial and temporal manner in germlines more effectively than alternative approaches. Described here is a protocol for the application and quantification of this technology to probe PPIs in the C. elegans germline.

In Situ Detection of Ribonucleoprotein Complex Assembly in the C. elegans Germline using Proximity Ligation Assay

This protocol demonstrates use of the proximity ligation assay to probe for protein-protein interactions in situ in the C. elegans germline.

使用接近连接测定在C.elegans Germline中对核糖蛋白复合体装配的实地检测

Understanding when and where protein-protein interactions (PPIs) occur is critical to understanding protein function in the cell and how broader processes ...

An Efficient Protocol to Assess ERK Activity Modulation in Early Zebrafish Noonan Syndrome Models via Live FRET Microscopy and Immunofluorescence

RASopathies are genetic syndromes caused by ERK hyperactivation and resulting in multisystemic diseases that can also lead to cancer predisposition. Despite a broad genetic heterogeneity, germline gain-of-function mutations in key regulators of the RAS-MAPK pathway underlie the majority of the cases, and, thanks to advanced sequencing techniques, potentially pathogenic variants affecting the RAS-MAPK pathway continue to be identified. Functional validation of the pathogenicity of these variants, essential for accurate diagnosis, requires fast and reliable protocols, preferably in vivo. Given the scarcity of effective treatments in early childhood, such protocols, especially if scalable in cost-effective animal models, can be instrumental in offering a preclinical ground for drug repositioning/repurposing. 
Here we describe step-by-step the protocol for rapid generation of transient RASopathy models in zebrafish embryos and direct inspection of live disease-associated ERK activity changes occurring already during gastrulation through real-time multispectral F&#246;rster resonance energy transfer (FRET) imaging. The protocol uses a transgenic ERK reporter recently established and integrated with the hardware of commercial microscopes. We provide an example application for Noonan syndrome (NS) zebrafish models obtained by expression of the Shp2D61G. We describe a straightforward method that enables registration of ERK signal change in the NS fish model before and after pharmacological signal modulation by available low-dose MEK inhibitors. We detail how to generate, retrieve, and assess ratiometric FRET signals from multispectral acquisitions before and after treatment and how to cross-validate the results via classical immunofluorescence on whole embryos at early stages. We then describe how, via examining standard morphometric parameters, to query late changes in embryo shape, indicative of a resulting impairment of gastrulation, in the same embryos whose ERK activity is assessed by live FRET at 6 h post fertilization.

RASopathies are multisystem genetic syndromes caused by RAS-MAPK pathway hyperactivation. Potentially pathogenic variants awaiting validation emerge continuously while poor preclinical evidence limits therapy. Here, we describe our in vivo protocol to test and cross-validate RASopathy-associated ERK activation levels and its pharmacological modulation during embryogenesis by live FRET imaging in Teen-reporter zebrafish.

通过实时 FRET 显微镜和免疫荧光评估早期斑马鱼 Noonan 综合征模型中 ERK 活性调节的有效方案

RASopathies are genetic syndromes caused by ERK hyperactivation and resulting in multisystemic diseases that can also lead to cancer predisposition. ...

生物化学

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 ...

生物学

通过共聚焦成像对 ERL1 动力学进行时空研究

Image-Based Methods to Study Membrane Trafficking Events in Stomatal Lineage Cells

In eukaryotic cells, membrane components, including proteins and lipids, are spatiotemporally transported to their destination within the endomembrane system. This includes the secretory transport of newly synthesized proteins to the cell surface or the outside of the cell, the endocytic transport of extracellular cargoes or plasma membrane components into the cell, and the recycling or shuttling transport of cargoes between the subcellular organelles, etc. Membrane trafficking events are crucial to the development, growth, and environmental adaptation of all eukaryotic cells and, thus, are under stringent regulation. Cell-surface receptor kinases, which perceive ligand signals from the extracellular space, undergo both secretory and endocytic transport. Commonly used approaches to study the membrane trafficking events using a plasma membrane-localized leucine-rich-repeat receptor kinase, ERL1, are described here. The approaches include plant material preparation, pharmacological treatment, and confocal imaging setup. To monitor the spatiotemporal regulation of ERL1, this study describes the co-localization analysis between ERL1 and a multi-vesicular body marker protein, RFP-Ara7, the time series analysis of these two proteins, and the z-stack analysis of ERL1-YFP treated with the membrane trafficking inhibitors brefeldin A and wortmannin.

作者聚焦：研究气孔谱系细胞中膜运输事件的基于图像的方法  

Several commonly used methods are introduced here to study the membrane trafficking events of a plasma membrane receptor kinase. This manuscript describes detailed protocols including the plant material preparation, pharmacological treatment, and confocal imaging setup.

基于图像的方法研究气孔谱系细胞中的膜运输事件

In eukaryotic cells, membrane components, including proteins and lipids, are spatiotemporally transported to their destination within the endomembrane ...

在Active ERK的时空分析

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