这项工作是由纽约干细胞科学（NYSTEM C028107）格兰特GHT的支持。

1.温度，营养和光/暗周期调节动物<ol><li>来自世界各地的众多Nematostella实验室之一，还是一个非营利性的供应商获取Nematostella vectensis成人（ 表1） </li><li>在黑暗中在恒定温度（通常介于18和21℃）保持Nematostella，在"1/3×"人造海水（ASW）中的12份每千（ppt）的盐度。保持培养物在简单的钠钙玻璃培养皿，通常为250毫升或1.5升容量11。 注意:这些简单的培养条件通常是学习Nematostella实验室中使用，但文化的护理也可以自动的19。 </li><li>饲料Nematostella刚孵出的卤虫无节幼体2 - 4倍每星期。孵化丰年虫卵完全强度（36个百分点），或者1 / 3X ASW在30℃，在一个矩形浅玻璃盘20或任何一种规模小，商业或自制盐水虾孵化场的。如果一个孵化器不可用，虾孵化在室温下，但这样做的更慢。 注意:这通常需要完成超过24小时。 </li><li>每周更换海葵培养水至少一次。为了获得最佳成人健康，彻底清洁（不含肥皂）文化周累积粘液分泌物，这大衣碗，可以捕获吃剩的食物和垃圾，并套住动物碗一次。 </li></ol> 2.选择和营养学放松空调动物<ol><li>大致相同的长度的选择大小匹配息肉（3 - 5厘米，当自然松弛），并放置在从菌落中分离为截肢前三天碗。 注意:选择用于切割的动物的数量将通过实验正在进行当然，确定，但一般而言，我们推荐每个采样点的至少五只动物具有六个重复。从而，在典型的实验中的最低30的动物将被预选。一般情况下，这是明智的选择，因为相比那些不规则的（见下文）可以在以后影响得分截肢的最低数量（30）更多。 </li><li>从培养箱截肢之前去除所选动物的盘进入房间的灯的至少一小时。 注意:暴露于室内光线和振动的处理可能会导致动物收缩，所以他们需要适应或在实验台上孵化"轻松"。动物将变得难治触摸并曝光，并在该点可通过温和移液移动。 </li><li>可选:通过添加7.5％的MgCl 2（1/3×ASW）麻醉动物。轻轻地用标准的塑料5毫升吸管加入氯化镁溶液到碗里。 注意:虽然动物最终将成为习惯于光且物理操纵，它可能是有利的麻醉动物，以维持或"修理"吨他放松的状态后，他们已成为拉长16,21,22。 </li><li>使用宽孔（&gt; 0.5，厘米）的塑料吸管转移（在1/3×ASW）的五只动物从池中被截肢，到含有12个百分点ASW的直径100毫米的无菌玻璃切割盘的底部。放置培养皿上以10之间可变放大倍率立体显微镜的阶段 - 40X。 注意:如果动物没有被麻醉和轻松的切割，它们仍可能响应触摸和体视镜照明，因此可能需要几分钟的时间来重新变得轻松。 </li></ol> 3.截肢<ol><li>用无菌解剖刀，从每个息肉锯掉的反口physa，其目标是获得是只要大约是它的宽度和不含肠系膜physa的截面。 注:理想的切割位点就是反口的肠系膜终止。在切割面有来自肠系膜适当薄L的过渡对应于每个mesenterial插入（ 见图1，箭头）分级表。肠系膜缺席是至关重要的，因为它产生可便于&#39;堵&#39;的孔17,30粘液。 <ol><li>放置手术刀刀片在截肢的期望部位与动物接触。做到这一点无论求告无门（写意），或用5号钳（杜蒙风格或类似）轻轻地抓动物的身体。 </li><li>穿过组织通过利用手术刀的弯曲叶片横跨主体上的"摇摆"运动切割。 注:组织应断绝干净的手术刀震撼，解放physa从捐赠者的所需部分。然而，如果一个小块组织仍然连接在主体和physa，用手术刀切割。不要试图通过拉至所连接件分开，因为这可能会损坏physa。 </li></ol></li><li>从盘中取出每个截肢&#39;供体&#39;息肉 ，并返回到国家环保总局标有"汇集截肢者一碗率;迄今为止，碗，并返回到股市文化。 注意:离断息肉会愈合一天之内伤口反口，然后就可以正常喂食。他们将在两周在这一点上，如果需要，physa可以再次截肢内再生一个正常的期待physa。 </li><li>冲洗切除physa保留在12个百分点ASW切割盘，那么每个physa转移到单独的无菌孔中已经有10毫升中各12个百分点ASW的良好的多孔细胞培养板。 注:此示例使用一个六孔板，每孔持股10毫升海水和五个切除physa。一般来说海水中应涵盖physa足以避免暴露在空气中因处理和潜在蒸发的运动。该板或孔应具有一个盖子。 </li><li>重复步骤3.1 - 3.3，以收集至少5 physa在每口井保留每个实验治疗。 </li><li>孵育physa在将提供图片的温度下IDE再生的实验计划审问最好的速率。放置含physa成温度调节培养箱的板，在由再生的所希望的速率来确定一个固定的温度。 注:physa将重新生成缺少的组织，当温度15至27℃之间培养形成一个完整的息肉。再生的速率依赖于温度，除了前两个阶段。为达到第4阶段为所有的温度的平均天是第7天在切割之后，这也与在21℃再生重合。在27℃，第4阶段达到较早约3天，并在15℃，第4阶段是由大约3天在21℃（也参见参考文献10）延迟相比再生。 </li></ol> 4.评估与Nematostella再生分期系统再生（NRSS） <ol><li>采用可变庄重一个立体复式显微镜得分physa通货膨胀（10 - 80X）。比分新鲜切割Nematostella physa为0级，并继续得分在同一时间使用NRSS 10每一天后截肢（DPA）。 注意:对于关键的分期标准和详情，请参阅参考文献10。 <ol><li>如果一个新鲜切割physa显示为杯形肿块形似气球弛缓分数physa为0级（伤口），用一个开放的伤口部位可能是可见的。 注意:伤口边缘也可能会从一开始就粘在一起，但组织仍然会折叠和缺乏刚性。开放性伤口的边缘可以观察到发生径向收缩的伤口愈合。 </li><li>分数physa为第1阶段（伤口闭合），如果截肢伤口出现关闭。 注意:伤口位置将对应于未来的口服极。围绕未来口服磁极的外表面可开始显示对应于底层径向对称内胚mesenterial插入不同拱门。 </li><li>分数pH值YSA作为第2阶段（径向弓）如果口服极表面出现膨胀，露出排列在径向对称模式和沟分隔8上调拱门。在拱门的顶点观察小，半球形泡。他们将约高大宽，可能是瞬态的，一开始由单一的外胚层细胞层组成。 注:在某些情况下双层泡可能稳定下来。注:在这个或后期阶段粘膜层可能会出现封装physa（ 图2），在膜"鞘"。此封装材料应当被除去以促进得分。 </li><li>如果在至少一些径向弓的口腔端形成稳定的内胚层含有外胚层和组织层的触角芽分数physa为第3阶段（触须）。 注:触角更长比他们宽，是最低限度能动。该physa将显示上升，但通货膨胀变量使肠系膜雏形可能会变得可见IBLE从mesenterial插入延伸进入体腔（coelenteron）。 </li><li>得分physa如阶段4（线性肠系膜）如physa包含在体壁延伸进入coelenteron从插入，与口服-反口长度是比从那里出现连接两次测量它们的径向宽度更八个不同的，可见肠系膜在其反口端（enterostome）咽。 注:四个或更少的肠系膜有"褶"内部自由边。咽是可见的。超过八只触角是可见的，能动的，有时他们收缩到体内。 </li><li>得分physa如阶段5（主要百褶肠系膜）如physa有四个以上的肠系膜与打褶，并且打褶比在阶段4的动物用具有几乎"正常的"成人外观更充分和蜿蜒，但是没有可见的性腺细胞。 </li></ol></li></ol>

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Q., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+imaging+of+Nematostella+vectensis+embryogenesis+and+late+development+using+fluorescent+probes.">In vivo imaging of Nematostella vectensis embryogenesis and late development using fluorescent probes.</a> BMC Cell Biol. 15, (2014).</li><li>Kaur, J., Debnath, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autophagy+at+the+crossroads+of+catabolism+and+anabolism.">Autophagy at the crossroads of catabolism and anabolism.</a> Nat Rev Mol Cell Biol. 16 (8), 461-472 (2015).</li><li>Carroll, B., Korolchuk, V. I., Sarkar, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amino+acids+and+autophagy:+cross-talk+and+co-operation+to+control+cellular+homeostasis.">Amino acids and autophagy: cross-talk and co-operation to control cellular homeostasis.</a> Amino Acids. 47 (10), 2065-2088 (2015).</li><li>Glick, D., Barth, S., Macleod, K. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autophagy:+cellular+and+molecular+mechanisms.">Autophagy: cellular and molecular mechanisms.</a> J Pathol. 221 (1), 3-12 (2010).</li><li>Rodolfo, C., Di Bartolomeo, S., Cecconi, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autophagy+in+stem+and+progenitor+cells.">Autophagy in stem and progenitor cells.</a> Cell Mol Life Sci. 73 (3), 475-496 (2016).</li><li>Guan, J. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autophagy+in+stem+cells.">Autophagy in stem cells.</a> Autophagy. 9 (6), 830-849 (2013).</li><li>Phadwal, K., Watson, A. S., Simon, A. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tightrope+act:+autophagy+in+stem+cell+renewal,+differentiation,+proliferation,+and+aging.">Tightrope act: autophagy in stem cell renewal, differentiation, proliferation, and aging.</a> Cell Mol Life Sci. 70 (1), 89-103 (2013).</li><li>Varga, M., Fodor, E., Vellai, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autophagy+in+zebrafish.">Autophagy in zebrafish.</a> Methods. 75, 172-180 (2015).</li><li>Varga, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autophagy+is+required+for+zebrafish+caudal+fin+regeneration.">Autophagy is required for zebrafish caudal fin regeneration.</a> Cell Death Differ. 21 (4), 547-556 (2014).</li><li>Gonzalez-Estevez, C., Salo, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autophagy+and+apoptosis+in+planarians.">Autophagy and apoptosis in planarians.</a> Apoptosis. 15 (3), 279-292 (2010).</li><li>Buzgariu, W., Chera, S., Galliot, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methods+to+investigate+autophagy+during+starvation+and+regeneration+in+hydra.">Methods to investigate autophagy during starvation and regeneration in hydra.</a> Methods Enzymol. 451, 409-437 (2008).</li><li>Tettamanti, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autophagy+in+invertebrates:+insights+into+development,+regeneration+and+body+remodeling.">Autophagy in invertebrates: insights into development, regeneration and body remodeling.</a> Curr Pharm Des. 14 (2), 116-125 (2008).</li></ol>

The authors have nothing to disclose.

使用Nematostella的伤口愈合和再生的一个模型，变得越来越流行。因而，为了能够之前有效的细胞和分子分析，以可视特定协议的形态学模式是很重要的可以分配和比较。 Nematostella具有高度的再生"灵活性"，能够改革几乎在任何地点任何截肢缺少结构，在人生的浮浪幼虫后阶段。因此，各种调查人员检查从再生导致截肢或息肉的不同区域伤人，在不同的年龄和大小7-18。 <p cl...

再生在一个单一的水螅的观察是在生物学的出现为一门实验科学1,2开创性的事件。再生仍然非常广泛的吸引力生物学家的现象，都奠定人。对发育生物学家，医生，生物医学家和组织工程师了解并克服对人体再生的限制的潜力，使再生生物学超过内在有趣。 现在，利用新兴技术，如基因组测序和增益和功能的工具丢失，该领域有望梳理出的再生机制，并最终理解物种如何各种可以再生，有些则不能。在分子，细胞和形态响应的通用化程度有待阐明，但到目前为止，似乎动物中可再生基本的反应比本来意马更相似斯内德就在10年前3。 尤其是腔肠动物处于际形态多样性的广谱几乎所有的身体部位的再生简便。从孤淡水水螅，水螅与建造巨大的珊瑚礁，到复杂的殖民管水母，如葡萄牙文-O-战争的微小海洋息肉以来，再生往往是复制的模式，除了建立机制修复或改革免受伤害和掠夺造成损坏或丢失的身体部位。无论是刺胞动物门的物种多样使用类似或不同的机制再生是一个从根本上有趣的问题4-6。 我们和其他人已经开发了珊瑚虫，Nematostella vectensis作为再生7-17的典范。我们最近开发出一种分期系统描述从abora平分的形态均匀一块组织的整个身体的再生息肉10 l的结束。而当在任何级别等分Nematostella息肉可再生，我们选择在最形态简单地区，physa一个反口位置切割成人，部分是因为这是接近自然无性裂变18的正常平面，并且还因为它许可证观察和如何将整个体从最简单的形态部件重新组装分子分析。 所述Nematostella再生分期系统（NRSS）提供了一个相对简单的设置，可以被用于通过一个截去physa得分再生的任何方面的进展形态基准，正常培养条件下或在实验微扰的情况下，例如小分子治疗，基因操作或环境的改变。如预期的，在NRSS正在成为采用为在其上再生的细胞和分子事件可以被引用一个形态的支架10。 最后，我们的切割方法产生要比在最近的研究中使用的17针点刺更高的大洞几个数量级，但都是伤口愈合约有6小时。记录伤口闭合的视觉拦阻和不同的相应建议的实验方法来解释伤口的尺寸和花费，关闭时间的表观独立性。因此，反口截肢过程有更深入直观的了解，通过这种协议提供，将有助于进一步调查这种模式再生系统，拓宽使用Nematostella vectensis这种分期系统的应用。

<table><tbody><tr><td>&lt;em&gt;Nematostella vectensis,&lt;/em&gt; adults</td><td>Marine Biological Lab (MBL)</td><td></td><td>non-profit supplier</td></tr><tr><td>Glass Culture Dish, 250 mL</td><td>Carolina Biological Supply</td><td>741004</td><td>250 mL</td></tr><tr><td>Glass Culture Dish, 1,500 mL</td><td>Carolina Biological Supply</td><td>741006</td><td>1,500 mL</td></tr><tr><td>Polyethylene transfer pipette, 5&amp;nbsp;mL</td><td>USA Scientific&amp;nbsp;</td><td>1022-2500</td><td>narrow bore, graduated</td></tr><tr><td>Polyethylene transfer pipet, tapered</td><td>Samco</td><td>202-205</td><td>cut off 1 inch of tip to make wide bore</td></tr><tr><td>Disposable Scalpel</td><td>Feather Safety Razor Co. Ltd</td><td>no. 10</td><td>blade should be curved</td></tr><tr><td>#5 Dumont Fine point tweezers</td><td>Roboz</td><td>RS5045</td><td>alternative suppliers available</td></tr><tr><td>Pyrex Petri dish, 100 mm diameter</td><td>Corning</td><td>3160</td><td>can substitute other glass Petri plates</td></tr><tr><td>Sterile 6-well plate</td><td>Corning Falcon&amp;nbsp;</td><td>353046</td><td>or similar from other manufacturer</td></tr><tr><td>Sterile 12-well plate</td><td>Nunc&amp;nbsp;</td><td>150628</td><td>or similar from other manufacturer</td></tr><tr><td>Sterile 24-well plate</td><td>Cellstar, Greiner bio-one</td><td>662-160</td><td>or similar from other manufacturer</td></tr><tr><td>Brine shrimp hathery kit</td><td>San Francisco Bay; drsfostersmith.com</td><td>CD-154005</td><td>option for growing brine shrimp</td></tr><tr><td>pyrex baking dish</td><td>common in grocery stores</td><td></td><td>option for growing brine shrimp</td></tr><tr><td>artificial seawater mix 50 gal or more&amp;nbsp;</td><td>Instant Ocean; drsfoster-smith.com</td><td>CD-116528</td><td>others brands may suffice</td></tr><tr><td>Plastic tub for stock ASW preparation</td><td>various</td><td></td><td>common 25 gallon plastic trash can OK</td></tr><tr><td>Polypropylene Carboy</td><td>Carolina Biological Supply</td><td>716391</td><td>For working stock of ASW @ 12 ppt</td></tr><tr><td>Beaker, Graduated, 4,000 mL</td><td>PhytoTechnology Laboratories</td><td>B199</td><td>For dilution of 36 ppt ASW to 12 ppt</td></tr><tr><td>Stereomicroscope and light source</td><td>various&amp;nbsp;</td><td></td><td>with continuous 1 - 40X magnification</td></tr></tbody></table>

inducing complete polyp regeneration from the aboral physa of the starlet sea anemone nematostella vectensis

腔肠动物，特别水润 ，却显示出了再生受损的或损坏的结构，实际上这样的研究可以说是通过推出的托尼川伯利工作的现代生物调查超过250多年前，第一批动物。目前再生的研究已经看到了同时使用"经典"再生生物，如水螅 ，涡虫和有尾类的复苏，以及物种跨越后生动物的范围的扩大范围，从海绵通过哺乳动物。除了其内在的兴趣作为一种生物现象，了解再生多样的品种是如何工作的通知我们友情再生过程是否具有共同特点和/或种或上下文特定的细胞和分子机制。在明星海葵，Nematostella vectensis，是再生一个新兴的模式生物。如同水润 ，Nematostella是古门，刺胞动物门中的一员，但在T内他类珊瑚虫，一个妹妹分支到是更进化的基础水螅。在Nematostella再生因而方面将是有趣的比较和与九头蛇和其他腔肠动物的对比。在这篇文章中，我们提出要平分，观察和分类Nematostella成人，这就是所谓的physa的反口结束再生的方法。该physa自然发生裂变为无性繁殖的手段，无论是与自然裂变或physa手动触发截肢复杂形态的再生长和改造。在这里，我们编纂了Nematostella再生分期系统（在NRSS）这些简单的形态变化。我们用NRSS测试氯喹的影响，溶酶体功能阻断自噬的抑制剂。的结果表明，当自噬被抑制息肉的结构，特别是肠系膜，再生是不正常的。

在切断physa再生过程中的形态的事件的进程示于图1A中 ，其中包括在每个NRSS阶段physa的代表性的观点。典型physa切削部位被表示在成年（箭头）。 图1a中的照片显示，从新鲜切割physa通过完全形成息肉口头和身体结构的渐进式再生。 图1B，C示出内部隔垫，肠系膜的布置中，在阶段4和阶段5，分别。请注意，某些肠系膜阶段4将缺乏"打褶"?...

Watch this Scientific Journal Video about Inducing Complete Polyp Regeneration from the Aboral Physa of the Starlet Sea Anemone Nematostella vectensis at JoVE.com

Inducing Complete Polyp Regeneration from the Aboral Physa of the Starlet Sea Anemone Nematostella vectensis

Here we demonstrate how to induce and monitor regeneration in the Starlet Sea Anemone Nematostella vectensis, a model cnidarian anthozoan. We demonstrate how to amputate and categorize regeneration using a morphological staging system, and we use this system to reveal a requirement for autophagy in regenerating polyp structures.

从斯塔利特海葵的反口Physa完全诱导再生息肉<em&gt; Nematostella vectensis</em

Cnidarians, and specifically Hydra, were the first animals shown to regenerate damaged or severed structures, and indeed such studies arguably launched ...

inducing-complete-polyp-regeneration-from-aboral-physa-starlet-sea

Universidad de Cartagena UDEC

Research

JoVE Journal

Developmental Biology

Inducing Complete Polyp Regeneration from the Aboral Physa of the Starlet Sea Anemone Nematostella vectensis

8.5K 次观看.  Stony Brook University. Here we demonstrate how to induce and monitor regeneration in the Starlet Sea Anemone Nematostella vectensis, a model cnidarian anthozoan. We demonstrate how to amputate and categorize regeneration using a morphological staging system, and we use this system to reveal a requirement for autophagy in regenerating polyp structures.

视频: Inducing Complete Polyp Regeneration from the Aboral Physa of the Starlet Sea Anemone Nematostella vectensis

Chemical Amputation and Regeneration of the Pharynx in the Planarian Schmidtea mediterranea

Planarians are flatworms that are extremely efficient at regeneration. They owe this ability to a large number of stem cells that can rapidly respond to any type of injury. Common injury models in these animals remove large amounts of tissue, which damages multiple organs. To overcome this broad tissue damage, we describe here a method to selectively remove a single organ, the pharynx, in the planarian Schmidtea mediterranea. We achieve this by soaking animals in a solution containing the cytochrome oxidase inhibitor sodium azide. Brief exposure to sodium azide causes extrusion of the pharynx from the animal, which we call &#34;chemical amputation.&#34;&#160;Chemical amputation removes the entire pharynx, and generates a small wound where the pharynx attaches to the intestine. After extensive rinsing, all amputated animals regenerate a fully functional pharynx in approximately one week. Stem cells in the rest of the body drive regeneration of the new pharynx. Here, we provide a detailed protocol for chemical amputation, and describe both histological and behavioral methods to assess successful amputation and regeneration.

Chemical Amputation and Regeneration of the Pharynx in the Planarian Schmidtea mediterranea

The planarian Schmidtea mediterranea is an excellent model for studying stem cells and tissue regeneration. This publication describes a method to selectively remove one organ, the pharynx, by exposing animals to the chemical sodium azide. This protocol also outlines methods for monitoring pharynx regeneration.

真涡虫的化学截肢和咽部再生Schmidtea mediterranea

Planarians are flatworms that are extremely efficient at regeneration. They owe this ability to a large number of stem cells that can rapidly respond to ...

科研

发育生物学

Methods for the Study of Regeneration in Stentor

Cells need to be able to regenerate their parts to recover from external perturbations. The unicellular ciliate Stentor coeruleus is an excellent model organism to study wound healing and subsequent cell regeneration. The Stentor genome became available recently, along with modern molecular biology methods, such as RNAi. These tools make it possible to study single-cell regeneration at the molecular level. The first section of the protocol covers establishing Stentor cell cultures from single cells or cell fragments, along with general guidelines for maintaining Stentor cultures. Culturing Stentor in large quantities allows for the use of valuable tools like biochemistry, sequencing, and mass spectrometry. Subsequent sections of the protocol cover different approaches to inducing regeneration in Stentor. Manually cutting cells with a glass needle allows studying the regeneration of large cell parts, while treating cells with either sucrose or urea allows studying the regeneration of specific structures located at the anterior end of the cell. A method for imaging individual regenerating cells is provided, along with a rubric for staging and analyzing the dynamics of regeneration. The entire process of regeneration is divided in three stages. By visualizing the dynamics of the progression of a population of cells through the stages, the heterogeneity in regeneration timing is demonstrated.

Methods for the Study of Regeneration in Stentor

The giant ciliate, Stentor coeruleus, is an excellent system to study regeneration and wound healing. We present procedures for establishing Stentor cell cultures from single cells or cell fragments, inducing regeneration by cutting cells, chemically inducing the regeneration of membranellar band and oral apparatus, imaging, and analysis of cell regeneration.

Stentor再生的研究方法

Cells need to be able to regenerate their parts to recover from external perturbations. The unicellular ciliate Stentor coeruleus is an excellent model ...

Genotyping of Sea Anemone during Early Development

Described here is a PCR-based protocol to genotype the gastrula stage embryo of the anthozoan cnidarian Nematostella vectensis without sacrificing the life of the animal. Following in vitro fertilization and de-jellying, zygotes are allowed to develop for 24 h at room temperature to reach the early- to mid-gastrula stage. The gastrula embryos are then placed on an agarose gel bed in a Petri dish containing seawater. Under the dissecting microscope, a tungsten needle is used to surgically separate an aboral tissue fragment from each embryo. Post-surgery embryos are then allowed to heal and continue development. Genomic DNA is extracted from the isolated tissue fragment and used as a template for locus-specific PCR. The genotype can be determined based on the size of PCR products or presence/absence of allele-specific PCR products. Post-surgery embryos are then sorted according to the genotype. The duration of the entire genotyping process depends on the number of embryos to be screened, but it minimally requires 4&#8211;5 h. This method can be used to identify knockout mutants from a genetically heterogeneous population of embryos and enables analyses of phenotypes during development.

The goal of this protocol is to genotype the sea anemone Nematostella vectensis during gastrulation without sacrificing the embryo.

早期开发期间海葵基因分型

Described here is a PCR-based protocol to genotype the gastrula stage embryo of the anthozoan cnidarian Nematostella vectensis without sacrificing the ...

Identification of Hemolytic and Phospholipase Activity in Crude Extracts from Sea Anemones by Straightforward Bioassays

Sea anemone venom composition includes polypeptide and non-proteins molecules. Cytolytic components have a high biotechnological and biomedical potential for designing new molecular tools. Sea anemone venom locates in glandular cells from ectoderm and sub-cellular structures called nematocysts, both of which are distributed throughout the sea anemone body. This characteristic implies challenges because the cells and nematocyst must be lysed to release the venom components with other non-toxic molecules. Therefore, first, the venom is derived from a crude extract (mixture of different and diverse molecules and tissue debris). The next step is to detect polypeptides with specific bioactivities. Here, we describe an efficient strategy to obtain the sea anemone crude extract and bioassay to identify the presence of cytolysins. The first step involves inexpensive and straightforward techniques (stirred and freeze-thaw cycle) to release cytolysins. We obtained the highest cytolytic activity and protein (~500 mg of protein from 20 g of dry weight). Next, the polypeptide complexity of the extract was analyzed by SDS-PAGE gel detecting proteins with molecular weights between 10 kDa and 250 kDa. In the hemolytic assay, we used sheep red blood cells and determined HU50 (11.1 &plusmn; 0.3 &#181;g/mL). In contrast, the presence of phospholipases in the crude extract was determined using egg yolk as a substrate in a solid medium with agarose. Overall, this study uses an efficient and inexpensive protocol to prepare the crude extract and applies replicable bioassays to identify cytolysins, molecules with biotechnological and biomedical interests.

Here, we describe a protocol to obtain crude venom extract from sea anemone and detect its hemolytic and phospholipase activity.

通过直接生物测定法鉴定海葵粗提取物中的溶血和磷脂酶活性

Sea anemone venom composition includes polypeptide and non-proteins molecules. Cytolytic components have a high biotechnological and biomedical potential ...

生物化学

Pharmacological and Functional Genetic Assays to Manipulate Regeneration of the Planarian Dugesia japonica

Free-living planarian flatworms have a long history of experimental usage owing to their remarkable regenerative abilities1. Small fragments excised from these animals reform the original body plan following regeneration of missing body structures. For example if a 'trunk' fragment is cut from an intact worm, a new 'head' will regenerate anteriorly and a 'tail' will regenerate posteriorly restoring the original 'head-to-tail' polarity of body structures prior to amputation (Figure 1A).
Regeneration is driven by planarian stem cells, known as 'neoblasts' which differentiate into ~30 different cell types during normal body homeostasis and enforced tissue regeneration. This regenerative process is robust and easy to demonstrate. Owing to the dedication of several pioneering labs, many tools and functional genetic methods have now been optimized for this model system. Consequently, considerable recent progress has been made in understanding and manipulating the molecular events underpinning planarian developmental plasticity2-9.
The planarian model system will be of interest to a broad range of scientists. For neuroscientists, the model affords the opportunity to study the regeneration of an entire nervous system, rather than simply the regrowth/repair of single nerve cell process that typically are the focus of study in many established models. Planarians express a plethora of neurotransmitters10, represent an important system for studying evolution of the central nervous system11, 12 and have behavioral screening potential13, 14. 
Regenerative outcomes are amenable to manipulation by pharmacological and genetic apparoaches. For example, drugs can be screened for effects on regeneration simply by placing body fragments in drug-containing solutions at different time points after amputation. The role of individual genes can be studied using knockdown methods (in vivo RNAi), which can be achieved either through cycles of microinjection or by feeding bacterially-expressed dsRNA constructs8, 9, 15. Both approaches can produce visually striking phenotypes at high penetrance- for example, regeneration of bipolar animals16-21. To facilitate adoption of this model and implementation of such methods, we showcase in this video article protocols for pharmacological and genetic assays (in vivo RNAi by feeding) using the planarian Dugesia japonica.

Pharmacological and Functional Genetic Assays to Manipulate Regeneration of the Planarian Dugesia japonica

An attractive model for studying stem cell differentiation within a live animal is the planarian flatworm. Regeneration is studied by simple amputation experiments that are easily performed in a basic laboratory and are amenable to pharmacological and genetic (in vivo RNAi) manipulation as detailed by protocols in this article.

药理和功能的遗传分析处理涡虫的再生日本三角涡虫</em

Free-living planarian flatworms have a long history of experimental usage owing to their remarkable regenerative abilities1. Small fragments excised from ...

生物学

Generation and Long-term Maintenance of Nerve-free Hydra

The interstitial cell lineage of Hydra includes multipotent stem cells, and their derivatives: gland cells, nematocytes, germ cells, and nerve cells. The interstitial cells can be eliminated through two consecutive treatments with colchicine, a plant-derived toxin that kills dividing cells, thus erasing the potential for renewal of the differentiated cells that are derived from the interstitial stem cells. This allows for the generation of Hydra that lack nerve cells. A nerve-free polyp cannot open its mouth to feed, egest, or regulate osmotic pressure. Such animals, however, can survive and be cultured indefinitely in the laboratory if regularly force-fed and burped. The lack of nerve cells allows for studies of the role of the nervous system in regulating animal behavior and regeneration. Previously published protocols for nerve-free Hydra maintenance involve outdated techniques such as mouth-pipetting with hand-pulled micropipette tips to feed and clean the Hydra. Here, an improved protocol for maintenance of nerve-free Hydra is introduced. Fine-tipped forceps are used to force open the mouth and insert freshly killed Artemia. Following force-feeding, the body cavity of the animal is flushed with fresh medium using a syringe and hypodermic needle to remove undigested material, referred to here as "burping". This new method of force-feeding and burping nerve-free Hydra through the use of forceps and syringes eliminates the need for mouth-pipetting using hand-pulled micropipette tips. It thus makes the process safer and significantly more time efficient. To ensure that the nerve cells in the hypostome have been eliminated, immunohistochemistry using anti-tyrosine-tubulin is conducted.

Generation and Long-term Maintenance of Nerve-free Hydra

Through a double treatment with colchicine, a plant-derived toxin that kills dividing cells, nerve-free Hydra vulgaris can be generated. These Hydra cannot feed or egest on their own. This paper describes an improved method for long-term maintenance of nerve free Hydra vulgaris in the laboratory.

无神经的一代和长期维持<em&gt;水润</em

The interstitial cell lineage of Hydra includes multipotent stem cells, and their derivatives: gland cells, nematocytes, germ cells, and nerve cells. The ...

神经科学

Planarian Immobilization, Partial Irradiation, and Tissue Transplantation

The planarian, a freshwater flatworm, has proven to be a powerful system for dissecting metazoan regeneration and stem cell biology1,2. Planarian regeneration of any missing or damaged tissues is made possible by adult stem cells termed neoblasts3. Although these stem cells have been definitively shown to be pluripotent and singularly capable of reconstituting an entire animal4, the heterogeneity within the stem cell population and the dynamics of their cellular behaviors remain largely unresolved. Due to the large number and wide distribution of stem cells throughout the planarian body plan, advanced methods for manipulating subpopulations of stem cells for molecular and functional study in vivo are needed.
Tissue transplantation and partial irradiation are two methods by which a subpopulation of planarian stem cells can be isolated for further study. Each technique has distinct advantages. Tissue transplantation allows for the introduction of stem cells, into a na&iuml;ve host, that are either inherently genetically distinct or have been previously treated pharmacologically. Alternatively, partial irradiation allows for the isolation of stem cells within a host, juxtaposed to tissue devoid of stem cells, without the introduction of a wound or any breech in tissue integrity. Using these two methods, one can investigate the cell autonomous and non-autonomous factors that control stem cell functions, such as proliferation, differentiation, and migration.
Both tissue transplantation5,6 and partial irradiation7 have been used historically in defining many of the questions about planarian regeneration that remain under study today. However, these techniques have remained underused due to the laborious and inconsistent nature of previous methods. The protocols presented here represent a large step forward in decreasing the time and effort necessary to reproducibly generate large numbers of grafted or partially irradiated animals with efficacies approaching 100 percent. We cover the culture of large animals, immobilization, preparation for partial irradiation, tissue transplantation, and the optimization of animal recovery. Furthermore, the work described here demonstrates the first application of the partial irradiation method for use with the most widely studied planarian, Schmidtea mediterranea. Additionally, efficient tissue grafting in planaria opens the door for the functional testing of subpopulations of na&iuml;ve or treated stem cells in repopulation assays, which has long been the gold-standard method of assaying adult stem cell potential in mammals8. Broad adoption of these techniques will no doubt lead to a better understanding of the cellular behaviors of adult stem cells during tissue homeostasis and regeneration.

An effective method for grafting tissue of defined and consistent size between planaria is described. Also included is a description of how the immobilization technique used for transplantation can be adapted, in conjunction with lead shields, for the partial irradiation of live animals.

涡固定，局部照射，组织移植

The planarian, a freshwater flatworm, has proven to be a powerful system for dissecting metazoan regeneration and stem cell biology1,2. Planarian ...

A Method for Microinjection of Patiria minata Zygotes

Echinoderms have long been a favorite model system for studies of reproduction and development, and more recently for the study of gene regulation and evolution of developmental processes. The sea star, Patiria miniata, is gaining prevalence as a model system for these types of studies which were previously performed almost exclusively in the sea urchins, Strongylocentrotus purpuratus and Lytechinus variegatus. An advantage of these model systems is the ease of producing modified embryos in which a particular gene is up or downregulated, labeling a group of cells, or introducing a reporter gene. A single microinjection method is capable of creating a wide variety of such modified embryos. Here, we present a method for obtaining gametes from P. miniata, producing zygotes, and introducing perturbing reagents via microinjection. Healthy morphant embryos are subsequently isolated for quantitative and qualitative studies of gene function. The availability of genome and transcriptome data for this organism has increased the types of studies that are performed and the ease of executing them.

A Method for Microinjection of Patiria minata Zygotes

Methods that produce morphant embryos are essential to study developmental mechanisms and gene regulatory networks. The sea star Patiria miniata is an emerging model system for these studies. Here we present a protocol for obtaining gametes, producing cultures of embryos, and rapid microinjection of zygotes from this species.

用于显微注射的方法<em&gt; Patiria minata</em&gt;合子

Echinoderms have long been a favorite model system for studies of reproduction and development, and more recently for the study of gene regulation and ...

Inducing Polyp Bail-out in Coral Colonies to Obtain Individualized Micropropagates for Laboratory Experimental Use

Corals are colonial animals formed by modular units called polyps. Coral polyps are physiologically linked and connected by tissue. The phenomenon of polyp bail-out is a process induced by acute stress, in which coral polyps digest the tissue connecting them to the rest of the colony and ultimately detach from the skeleton to continue living as separate individuals. Coral biologists have acknowledged the process of polyp bail-out for years, but only recently the micropropagates generated by this process have been recognized as a model system for coral biology studies. The use of polyp bail-out can create a high number of clonal units from a single coral fragment. Another benefit is that single polyps or patches of polyps can be easily visualized under a microscope and maintained in highly standardized low-cost environments such as Petri dishes, flasks, and microfluidic chips. The present protocol demonstrates reproducible methods capable of inducing coral micropropagation and different approaches for maintaining the single polyps alive in the long term. This methodology was capable of successfully cultivating polyps of the coral species Pocillopora verrucosa for up to 8 weeks after bail-out, exhibiting the practicality of using individual coral polyps for coral research.

Polyp bail-out is a process induced by acute stress, in which coral polyps digest the tissue connecting them to their colony and detach from it to live as individuals. The present protocol describes how to induce coral micropropagation by bail-out using hypersaline or calcium-free seawater treatments.

诱导珊瑚群落息肉获救，获得个体化微繁殖物，用于实验室实验用途

Corals are colonial animals formed by modular units called polyps. Coral polyps are physiologically linked and connected by tissue. The phenomenon of ...

Characterizing Epithelial Wound Healing In Vivo Using the Cnidarian Model Organism Clytia hemisphaerica

All animal organs, from the skin to eyes to intestines, are covered with sheets of epithelial cells that allow them to maintain homeostasis while protecting them from infection. Therefore, it is not surprising that the ability to repair epithelial wounds is critical to all metazoans. Epithelial wound healing in vertebrates involves overlapping processes, including inflammatory responses, vascularization, and re-epithelialization. Regulation of these processes involves complex interactions between epithelial cells, neighboring cells, and the extracellular matrix (ECM); the ECM contains structural proteins, regulatory proteins, and active small molecules. This complexity, together with the fact that most animals have opaque tissues and inaccessible ECMs, makes wound healing difficult to study in live animals. Much work on epithelial wound healing is therefore performed in tissue culture systems, with a single epithelial cell-type plated as a monolayer on an artificial matrix. Clytia hemisphaerica (Clytia) provides a unique and exciting complement to these studies, allowing epithelial wound healing to be studied in an intact animal with an authentic ECM. The ectodermal epithelium of Clytia is a single layer of large squamous epithelial cells, allowing high-resolution imaging using differential interfering contrast (DIC) microscopy in living animals. The absence of migratory fibroblasts, vasculature, or inflammatory responses makes it possible to dissect the critical events in re-epithelialization in vivo. The healing of various types of wounds can be analyzed, including single-cell microwounds, small and large epithelial wounds, and wounds that damage the basement membrane. Lamellipodia formation, purse string contraction, cell stretching, and collective cell migration can all be observed in this system. Furthermore, pharmacological agents can be introduced via the ECM to modify cell:ECM interactions and cellular processes in vivo. This work shows methods for creating wounds in live Clytia, capturing movies of healing, and probing healing mechanisms by microinjecting reagents into the ECM.

Characterizing Epithelial Wound Healing In Vivo Using the Cnidarian Model Organism Clytia hemisphaerica

This paper describes a method to create wounds in the epithelium of a live Clytia hemisphaerica medusa and image wound healing at a high resolution in vivo. Additionally, a technique to introduce dyes and drugs to perturb signaling processes in the epithelial cells and extracellular matrix during wound healing is presented.

从斯塔利特海葵的反口Physa完全诱导再生息肉

摘要

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此视频中的章节

真涡虫的化学截肢和咽部再生Schmidtea mediterranea

Stentor再生的研究方法

早期开发期间海葵基因分型

通过直接生物测定法鉴定海葵粗提取物中的溶血和磷脂酶活性

药理和功能的遗传分析处理涡虫的再生日本三角涡虫

无神经的一代和长期维持

涡固定，局部照射，组织移植

用于显微注射的方法

诱导珊瑚群落息肉获救，获得个体化微繁殖物，用于实验室实验用途

使用刺胞动物模式生物Clytia hemisphaerica表征体内上皮伤口愈合