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>

<ol>
	<li>Lenhoff, S. G., Lenhoff, H. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Hydra and the Birth of Experimental Biology: Abraham Trembley's Memoirs Concerning the Natural History of a Type of Freshwater Polyp with Arms Shaped like Horns. , (1986).</li><li>Trembley, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> M&#233;moires pour servir &#224; l'histoire d'un genre de polypes d'eau douce, &#224; bras en forme de cornes. , (1744).</li><li>Poss, K. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advances+in+understanding+tissue+regenerative+capacity+and+mechanisms+in+animals.">Advances in understanding tissue regenerative capacity and mechanisms in animals.</a> Nat Rev Genet. 11 (10), 710-722 (2010).</li><li>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=Hydra,+a+fruitful+model+system+for+270+years.">Hydra, a fruitful model system for 270 years.</a> Int J Dev Biol. 56 (6-8), 411-423 (2012).</li><li>Gold, D. A., Jacobs, D. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stem+cell+dynamics+in+Cnidaria:+are+there+unifying+principles?.">Stem cell dynamics in Cnidaria: are there unifying principles?.</a> Dev Genes Evol. 233 (1-2), 53-66 (2013).</li><li>Holstein, T. W., Hobmayer, E., Technau, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cnidarians:+an+evolutionarily+conserved+model+system+for+regeneration?.">Cnidarians: an evolutionarily conserved model system for regeneration?.</a> Dev Dyn. 226 (2), 257-267 (2003).</li><li>Amiel, A. R., 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=Characterization+of+Morphological+and+Cellular+Events+Underlying+Oral+Regeneration+in+the+Sea+Anemone,+Nematostella+vectensis.">Characterization of Morphological and Cellular Events Underlying Oral Regeneration in the Sea Anemone, Nematostella vectensis.</a> Int J Mol Sci. 16 (12), 28449-28471 (2015).</li><li>Warren, C. R., 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=Evolution+of+the+perlecan/HSPG2+gene+and+its+activation+in+regenerating+Nematostella+vectensis.">Evolution of the perlecan/HSPG2 gene and its activation in regenerating Nematostella vectensis.</a> PLoS One. 10 (4), e0124578 (2015).</li><li>Gong, 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=Integrins+of+the+starlet+sea+anemone+Nematostella+vectensis.">Integrins of the starlet sea anemone Nematostella vectensis.</a> Biol Bull. 227 (3), 211-220 (2014).</li><li>Bossert, P. E., Dunn, M. P., Thomsen, G. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+staging+system+for+the+regeneration+of+a+polyp+from+the+aboral+physa+of+the+anthozoan+Cnidarian+Nematostella+vectensis.">A staging system for the regeneration of a polyp from the aboral physa of the anthozoan Cnidarian Nematostella vectensis.</a> Dev Dyn. 242 (11), 1320-1331 (2013).</li><li>Stefanik, D. J., Friedman, L. E., Finnerty, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Collecting,+rearing,+spawning+and+inducing+regeneration+of+the+starlet+sea+anemone,+Nematostella+vectensis.">Collecting, rearing, spawning and inducing regeneration of the starlet sea anemone, Nematostella vectensis.</a> Nat Protoc. 8 (5), 916-923 (2013).</li><li>Tucker, R. P., 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=A+thrombospondin+in+the+anthozoan+Nematostella+vectensis+is+associated+with+the+nervous+system+and+upregulated+during+regeneration.">A thrombospondin in the anthozoan Nematostella vectensis is associated with the nervous system and upregulated during regeneration.</a> Biol Open. 2 (2), 217-226 (2013).</li><li>Passamaneck, Y. J., Martindale, M. Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+proliferation+is+necessary+for+the+regeneration+of+oral+structures+in+the+anthozoan+cnidarian+Nematostella+vectensis.">Cell proliferation is necessary for the regeneration of oral structures in the anthozoan cnidarian Nematostella vectensis.</a> BMC Dev Biol. 12, (2012).</li><li>Trevino, M., Stefanik, D. J., Rodriguez, R., Harmon, S., Burton, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+of+canonical+Wnt+signaling+by+alsterpaullone+is+sufficient+for+oral+tissue+fate+during+regeneration+and+embryogenesis+in+Nematostella+vectensis.">Induction of canonical Wnt signaling by alsterpaullone is sufficient for oral tissue fate during regeneration and embryogenesis in Nematostella vectensis.</a> Dev Dyn. 240 (12), 2673-2679 (2011).</li><li>Renfer, E., Amon-Hassenzahl, A., Steinmetz, P. R., Technau, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+muscle-specific+transgenic+reporter+line+of+the+sea+anemone,+Nematostella+vectensis.">A muscle-specific transgenic reporter line of the sea anemone, Nematostella vectensis.</a> Proc Natl Acad Sci U S A. 107 (1), 104-108 (2010).</li><li>Burton, P. M., Finnerty, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conserved+and+novel+gene+expression+between+regeneration+and+asexual+fission+in+Nematostella+vectensis.">Conserved and novel gene expression between regeneration and asexual fission in Nematostella vectensis.</a> Dev Genes Evol. 219 (2), 79-87 (2009).</li><li>DuBuc, T. Q., Traylor-Knowles, N., Martindale, M. Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Initiating+a+regenerative+response;+cellular+and+molecular+features+of+wound+healing+in+the+cnidarian+Nematostella+vectensis.">Initiating a regenerative response; cellular and molecular features of wound healing in the cnidarian Nematostella vectensis.</a> BMC Biol. 12, (2014).</li><li>Hand, C., Uhlinger, K. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Asexual+reproduction+by+transverse+fission+and+some+anomalies+in+the+sea+anemone+Nematostella+vectensis.">Asexual reproduction by transverse fission and some anomalies in the sea anemone Nematostella vectensis.</a> Invert Biol. 114, 9-18 (1995).</li><li>Fritzenwanker, J. H., Technau, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+of+gametogenesis+in+the+basal+cnidarian+Nematostella+vectensis(Anthozoa).">Induction of gametogenesis in the basal cnidarian Nematostella vectensis(Anthozoa).</a> Dev Genes Evol. 212 (2), 99-103 (2002).</li><li>Magie, C., Bossert, P., Aramli, L., Thomsen, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Science's+super+star:+The+starlet+sea+anemone+is+an+ideal+tool+for+student+inquiry.">Science's super star: The starlet sea anemone is an ideal tool for student inquiry.</a> The Science Teacher. 83 (3), 33-40 (2016).</li><li>Genikhovich, G., Technau, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+situ+hybridization+of+starlet+sea+anemone+(Nematostella+vectensis)+embryos,+larvae,+and+polyps.">In situ hybridization of starlet sea anemone (Nematostella vectensis) embryos, larvae, and polyps.</a> Cold Spring Harb Protoc. (9), (2009).</li><li>Magie, C. R., Pang, K., Martindale, M. Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genomic+inventory+and+expression+of+Sox+and+Fox+genes+in+the+cnidarian+Nematostella+vectensis.">Genomic inventory and expression of Sox and Fox genes in the cnidarian Nematostella vectensis.</a> Dev Genes Evol. 215 (12), 618-630 (2005).</li><li>Chera, S., Kaloulis, K., 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=The+cAMP+response+element+binding+protein+(CREB)+as+an+integrative+HUB+selector+in+metazoans:+clues+from+the+hydra+model+system.">The cAMP response element binding protein (CREB) as an integrative HUB selector in metazoans: clues from the hydra model system.</a> Biosystems. 87 (2-3), 191-203 (2007).</li><li>Reitzel, A. M., Burton, P. M., Krone, C., Finnerty, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+developmental+trajectories+in+the+starlet+sea+anemone+Nematostella+vectensis:+embryogenesis,+regeneration,+and+two+forms+of+asexual+fission.">Comparison of developmental trajectories in the starlet sea anemone Nematostella vectensis: embryogenesis, regeneration, and two forms of asexual fission.</a> Invertebr Biol. 126, 99-112 (2007).</li><li>Ikmi, A., McKinney, S. A., Delventhal, K. M., Gibson, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TALEN+and+CRISPR/Cas9-mediated+genome+editing+in+the+early-branching+metazoan+Nematostella+vectensis.">TALEN and CRISPR/Cas9-mediated genome editing in the early-branching metazoan Nematostella vectensis.</a> Nat Commun. 5, 5486 (2014).</li><li>Jahnel, S. M., Walzl, M., Technau, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+and+epithelial+organisation+of+muscle+cells+in+the+sea+anemone+Nematostella+vectensis.">Development and epithelial organisation of muscle cells in the sea anemone Nematostella vectensis.</a> Front Zool. 11, 44 (2014).</li><li>Kelava, I., Rentzsch, F., Technau, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evolution+of+eumetazoan+nervous+systems:+insights+from+cnidarians.">Evolution of eumetazoan nervous systems: insights from cnidarians.</a> Philos Trans R Soc Lond B Biol Sci. 370 (1684), (2015).</li><li>Nakanishi, N., Renfer, E., Technau, U., Rentzsch, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nervous+systems+of+the+sea+anemone+Nematostella+vectensis+are+generated+by+ectoderm+and+endoderm+and+shaped+by+distinct+mechanisms.">Nervous systems of the sea anemone Nematostella vectensis are generated by ectoderm and endoderm and shaped by distinct mechanisms.</a> Development. 139 (2), 347-357 (2012).</li><li>Richards, G. S., Rentzsch, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transgenic+analysis+of+a+SoxB+gene+reveals+neural+progenitor+cells+in+the+cnidarian+Nematostella+vectensis.">Transgenic analysis of a SoxB gene reveals neural progenitor cells in the cnidarian Nematostella vectensis.</a> Development. 141 (24), 4681-4689 (2014).</li><li>DuBuc, T. 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。 这里所描述的分期系统遵循physa的形态均匀节仅缺少生殖组织的转变，从一个成年人的反口结束截肢，一个完整的解剖息肉的少年（我们还没有确定，当这些青少年再生性行为米ATURE）。这种方法与组织的简单雏形至接近最大复杂性的一种检验的再生。包括五physa作为建议的最低组大小正常化潜在的个体差异和生存。当然，这个数字可以调整，以适应特定的实验的目标，但是在NRSS协议允许2毫升每physa介质的否定像已报道影响再生水螅23一个潜在的"体积效应"。 研究Nematostella再生其他方法已经用一分为二的成年人身体中部或在口头结束，或少年4触手阶段息肉平分身体中部7,11-18。两项研究审查physa自然裂变16,18解放再生的方法，并且采用截肢physa最近与NRSS 8,9出现了。每个这些不同的方法都有自己的优点，并且可以解决有关regenerati唯一的问题已经在不同的截肢或伤人的政权发生和中老年人不同动物的过程。在NRSS协议physa截肢和得分，在目前的研究中所示，产生用于系统的研究一相对均匀的组physa的并避免了在physa尺寸变化，组织成分，并与自然横向裂变18,24观察到再生的后续进展。虽然physa截肢有所对应于无性繁殖的Nematostella的固有模式，不同分子已注意到在中体或在physa 16,18,24自然裂变和截肢导致再生之间。无论是这里描述截肢或自然裂变出了这种差异的方法产生physa仍有待确定。 这里有描述的是与饲养截肢成功的关键的几个问题，和得分技巧。选择截肢寿息肉LD被保持在相同的温度，为physa大小，营养历史匹配，如果可能，年龄（尽管后者还没有被系统进行测试，以确定是否有在步骤级数上变异的年龄效应）。获得一个开放的伤口与具有弧形刀片锋利无菌解剖刀为0段和Stage 1之间伤口愈合的形态学观察重要当physa膨胀和手术刀的弯曲叶片被摇动在成年组织中，它被截肢在一个运动并能很好地从切割盘被迅速转移到治疗。截肢包括肠系膜或是广泛的物理操纵的结果应该被丢弃。 实践分期未处理physa得到在群体的各个变化的感被测试。个人physa之间的差异是至少在最初阶段。例如，所有physa在舞台0 0 DPA。以相同的方式，所有physa到达阶段1在1 DPA同步。第2阶段的外观可能更难观察者分辨，因为"通货膨胀"然而就是实现由2 DPA几乎没有变化相对条件。真正的触角痕进展的外观舞台3.再生触手可通过膜"破茧"即下阻碍触手芽可视化的外观所掩盖。如果膜状覆盖先前没有除去，应当所以现在完成。用细尖镊子膜去除将解放再生physa。阶段4和5之间的区别是打褶肠系膜的数目。阶段4具有四个或更少的打褶肠系膜和阶段5具有五到八打褶肠系膜。而可以在侧向视图中观察到打褶，打褶肠系膜的确切数目是与反口视图更好地观察。 在从PHY学习成人Nematostella再生一个挑战SA雏形，并确实与其他截肢部位切断的组织，是活体组织的不同清晰度。该physa的灯泡是完整的成人比较清晰，但它变得相当不透明由于截肢后的组织收缩。清晰度逐渐返回（第2阶段）一旦伤口闭合（阶段1）和动物开始膨胀，但即使再绕伤口部位，其中组织和结构都在积极再生遗体由致密组织（特别是第3阶段）有所遮蔽的区域。增加通货膨胀通常伴随阶段4和5固定，随后通过光学澄清几乎肯定会解决什么是在口服端发生，但更多的信息可以是能够对荧光进行监测和更容易地可视化15带电，组织特异性基因的记者， 25-30。 截肢physa显然不能养活因为它缺乏触角，嘴和肠系膜（其中怀有消化腺），从而requi丢失的车身结构环再生，从非食品来源调集营养储备来完成。该physa可以潜在做到这是由自噬，其中细胞质，细胞器和其它细胞组分在细胞内吞噬并通过溶酶体依赖机制处理，以产生能量和化合物对合成代谢过程31-33。我们发现，与溶酶体酶抑制剂，氯喹，治疗physa引起肠系膜和触角异常再生，一般的身体形态，表明自噬是需要口服和身体结构的正常再生。自噬调控干细胞的功能34-36，并发挥再生重要作用长蛇 ，涡虫，和斑马鱼37-41。进一步分析，以了解如何自噬在细胞和分子水平影响Nematostella再生，但我们的第一遍实验示出了使用NR的效用SS作为可能影响再生的小分子快速筛选方法。 在Nematostella调节再生遗传，分子和细胞过程是仅在谅解的初步阶段，但是对于再生该紧急模式具有的基因组和基因表达分析工具越来越剧目。凭借其注释的基因组，地区和组织特异性遗传标记过多，而对于转基因，诱变，组织学和显微镜稳健的方法，Nematostella有望揭示执政珊瑚虫刺胞动物的再生机制，揭开它的再生过程是否刺胞动物和后生动物是相似的或独特的一般来说。

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"，但有资格作为必须让多数人已经开发出褶一阶段5。 图1D示出了在其中可以用钳子（ 图1E）被移除粘膜的膜包围的physa。虽然通常不会伤害到再生的（除非它不合理捕捉动物），膜会阻碍得分physa做ING实验操作，如显微镜，样品固定或收获分子/生化分析。这是第一阶段之后最好的去除，或更高版本，如果它的改革。 图2示出分期系统如何可以被用来评分的实验的结果，以评估抑制自噬的效果。 Physa被切断，并在10，50和100微米与氯喹处理过的，或physa是未处理的（对照）。氯喹抑制溶酶体的功能，这是必需的自噬。该NRSS标准来得分超过physa再生和结果的过程中， 如图2E绘制。作了当控制到达阶段5.氯喹处理过的动物以外阶段4没有进展，并且它们通常表现出不完全的肠系膜regenera -代表性的控制（ 图2A）和氯喹处理过的physa（D图2 B）的照片化（最缺乏打褶），短触手大小，并且在某些情况下，短体长度。 <img alt="图1" src="/files/ftp_upload/54626/54626fig1.jpg" /> 图1. 再生的特点。 （A）physa再生口腔和身体结构的例子根据NRSS上演。标记成人面板显示触须（T）咽（PH），肠系膜（m）和肠系膜插入（MI）成熟的动物的特征。白色箭头表示肠系膜过渡到肠系膜插入，延伸到反口末端内胚层的脊，其中打褶区。这一地区构成了physa。黄色箭头表示理想physa等分站点。 面板 0示出了对分经过五physa分钟， 面板 0&#39;是那些physa的一个的放大图，在中心开放性伤口，定义的阶段0NRSS。 面板 1显示了伤口physa已经结束，第二阶段定义面板 1 2显示与周围的口腔极提出径向弓，下面虚增组织physa，对应阶段2（板0 - 2是享有口腔年底）。 面板3显示触手芽出现在口头极（向右）伸长和膨胀physa的，现在阶段3.注意基本的肠系膜元素可见咽部是在口头制止的黑暗区域形成。 面板 4显示了真正的触须在口头极的出现，以及短暂的&#39;泡&#39;，定义阶段4.线性系膜处于膨胀physa可见。大圆形肿块息肉内可见来源不明，并会通过口腔排出。 面板5显示了几乎完全再生特点是四个多褶肠系膜，一个完全形成的pHarynx和八个或更多的触手，将其定义为第5阶段。 （B，C）个人physa的反口视图显示肠系膜的biradial布局和形态。这种观点认为，肠系膜褶似乎没有中间组织的隆起（绿色箭头）。具有阶段4 physa具有四个或更少打褶肠系膜和阶段5 physa具有多于四个（C）。带或不带褶裥肠系膜用绿色或黄色的箭头指示的分别。黑箭头（C）中指向一个打褶肠系膜已经径向伸展。从physa粘液鞘（D，E）拆卸所示。白色箭头指示得分再生之前，周围应该作为（E）要删除的physa（D）粘液护套。有时残留组织（黄色箭头）可能被困在护套内，这也应该被删除。星号表示口服极，其中标志编辑。红色的大小酒吧0.5mm的所有面板除了A5（1.0毫米）。 图 B和该图的Ç已被修改，经许可转载自参考10. <a href="http://ecsource.jove.com/files/ftp_upload/54626/54626fig1large.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/54626/54626fig2.jpg" /> 图 2. 再生氯喹的影响。为了证明NRSS的应用，我们测试了氯喹，自噬的抑制剂的效果。 Physa被截肢，并立即置于含有0.1％DMSO（对照）或氯在10，50，100μM1/3×ASW。 Physa是在使用NRSS 24小时的间隔得分。 （A），当他们到达第5阶段采取控制端点代表的图像。 <strong&gt;（B - D）即达4阶段氯喹"再生高原"氯喹治疗physa的代表形象造成了再生类似的缺陷在所有测试的剂量。最显着的问题是缺少肠系膜和触角完全再生的。异常体形态（如发育迟缓）也偶尔提到（C）。存在（白色箭头）和氯喹治疗physa褶的缺乏（黑色箭头）以D所示（触角被部分撤销）。 （E）的所有physa暂存数据绘制为时间的函数（在23℃）。 <a href="http://ecsource.jove.com/files/ftp_upload/54626/54626fig2large.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/54626/54626fig3.jpg" /> 图3的概要在NRSS阶段的主要特点。该图显示，在NRSS定义每个阶段的关键形态变化。运动方向由绿色箭头红色箭头和功能说明。舞台0描述只是切割（A）和T）后开放性伤口。在伤口边缘经受闭合径向收缩朝向中心（B）。阶段1的特征在于，所述伤口（C）和面向口服-脊（D）之间的拱门高程的全封闭;口腔表面（箭头）的中心是郁闷。第2阶段有口腔表面（箭头，E）的一个明显的拱起;的physa始于触手芽和泡在拱（箭头）的顶点可见伸长和变窄（F）。阶段3具有稳定的触手芽（箭头，G）和再生咽可被视为在口服极的密度（橙色质量，箭头H中</strong&gt;）。阶段4表示直链和打褶肠系膜（Ⅰ），该应尽可能长的至少两倍，因为它们是在咽（I&#39;）的结高大的存在。在mesenterial长丝（J，J&#39;） -四或更少的肠系膜可以在他们的内边缘打褶。阶段5的特征在于，多于四个褶肠系膜（K），其可以通过从反口端（K&#39;）观看被最佳评估。重新打印与许可参考10. <a href="http://ecsource.jove.com/files/ftp_upload/54626/54626fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

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

Nanyang Technological University

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

An Assay for Lateral Line Regeneration in Adult Zebrafish

Due to the clinical importance of hearing and balance disorders in man, model organisms such as the zebrafish have been used to study lateral line development and regeneration. The zebrafish is particularly attractive for such studies because of its rapid development time and its high regenerative capacity. To date, zebrafish studies of lateral line regeneration have mainly utilized fish of the embryonic and larval stages because of the lower number of neuromasts at these stages. This has made quantitative analysis of lateral line regeneration/and or development easier in the earlier developmental stages. Because many zebrafish models of neurological and non-neurological diseases are studied in the adult fish and not in the embryo/larvae, we focused on developing a quantitative lateral line regenerative assay in adult zebrafish so that an assay was available that could be applied to current adult zebrafish disease models. Building on previous studies by Van Trump et al.17&#160;that described procedures for ablation of hair cells in adult Mexican blind cave fish and zebrafish (Danio rerio), our assay was designed to allow quantitative comparison between control and experimental groups. This was accomplished by developing a regenerative neuromast standard curve based on the percent of neuromast reappearance over a 24 hr time period following gentamicin-induced necrosis of hair cells in a defined region of the lateral line. The assay was also designed to allow extension of the analysis to the individual hair cell level when a higher level of resolution is required.

Because many zebrafish models of neurological and non-neurological diseases are studied in the adult fish rather than the embryo/larvae, we developed a quantitative lateral line regenerative assay that can be applied to adult zebrafish disease models. The assay involved resolution at the 1) neuromast and 2) individual hair cell levels.

对于侧线再生成年斑马鱼的含量

Due to the clinical importance of hearing and balance disorders in man, model organisms such as the zebrafish have been used to study lateral line ...

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

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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

对于侧线再生成年斑马鱼的含量

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