从斯塔利特海葵的反口Physa完全诱导再生息肉<em> Nematostella vectensis</em
概要
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.
Abstract
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。
Introduction
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。
Protocol
1.温度，营养和光/暗周期调节动物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） 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?…
Representative Results
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…
Discussion
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。
<…開示
The authors have nothing to disclose.
Acknowledgements
这项工作是由纽约干细胞科学（NYSTEM C028107）格兰特GHT的支持。
Materials
| Nematostella vectensis, adults | Marine Biological Lab (MBL) | non-profit supplier | |
| Glass Culture Dish, 250 ml | Carolina Biological Supply | 741004 | 250 ml |
| Glass Culture Dish, 1,500 ml | Carolina Biological Supply | 741006 | 1,500 ml |
| Polyethylene transfer pipette, 5ml | USA Scientific | 1022-2500 | narrow bore, graduated |
| Polyethylene transfer pipet, tapered | Samco | 202-205 | cut off 1 inch of tip to make wide bore |
| Disposable Scalpel | Feather Safety Razor Co. Ltd | no. 10 | blade should be curved |
| #5 Dumont Fine point tweezers | Roboz | RS5045 | alternative suppliers available |
| Pyrex petri dish, 100 mm diameter | Corning | 3160 | can substitute other glass petri plates |
| Sterile 6 well plate | Corning Falcon | 353046 | or similar from other manufacturer |
| Sterile 12 well plate | Nunc | 150628 | or similar from other manufacturer |
| Sterile 24 well plate | Cellstar, Greiner bio-one | 662-160 | or similar from other manufacturer |
| Brine shrimp hathery kit | San Francisco Bay; drsfostersmith.com | CD-154005 | option for growing brine shrimp |
| pyrex baking dish | common in grocery stores | option for growing brine shrimp | |
| artificial seawater mix 50 gal or more | Instant Ocean; drsfoster-smith.com | CD-116528 | others brands may suffice |
| Plastic tub for stock ASW preparation | various | common 25 gallon plastic trash can OK | |
| Polypropylene Carboy | Carolina Biological Supply | 716391 | For working stock of ASW @ 12 ppt |
| Beaker, Graduated, 4,000ml | PhytoTechnology Laboratories | B199 | For dilution of 36 ppt ASW to 12 ppt |
| Stereomicroscope and light source | various | with continuous 1 – 40x magnification |
参考文献
- Lenhoff, S. G., Lenhoff, H. M. . 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).
- Trembley, A. . Mémoires pour servir à l’histoire d’un genre de polypes d’eau douce, à bras en forme de cornes. , (1744).
- Poss, K. D. Advances in understanding tissue regenerative capacity and mechanisms in animals. Nat Rev Genet. 11 (10), 710-722 (2010).
- Galliot, B. Hydra, a fruitful model system for 270 years. Int J Dev Biol. 56 (6-8), 411-423 (2012).
- Gold, D. A., Jacobs, D. K. Stem cell dynamics in Cnidaria: are there unifying principles?. Dev Genes Evol. 233 (1-2), 53-66 (2013).
- Holstein, T. W., Hobmayer, E., Technau, U. Cnidarians: an evolutionarily conserved model system for regeneration?. Dev Dyn. 226 (2), 257-267 (2003).
- Amiel, A. R., et al. Characterization of Morphological and Cellular Events Underlying Oral Regeneration in the Sea Anemone, Nematostella vectensis. Int J Mol Sci. 16 (12), 28449-28471 (2015).
- Warren, C. R., et al. Evolution of the perlecan/HSPG2 gene and its activation in regenerating Nematostella vectensis. PLoS One. 10 (4), e0124578 (2015).
- Gong, Q., et al. Integrins of the starlet sea anemone Nematostella vectensis. Biol Bull. 227 (3), 211-220 (2014).
- Bossert, P. E., Dunn, M. P., Thomsen, G. H. A staging system for the regeneration of a polyp from the aboral physa of the anthozoan Cnidarian Nematostella vectensis. Dev Dyn. 242 (11), 1320-1331 (2013).
- Stefanik, D. J., Friedman, L. E., Finnerty, J. R. Collecting, rearing, spawning and inducing regeneration of the starlet sea anemone, Nematostella vectensis. Nat Protoc. 8 (5), 916-923 (2013).
- Tucker, R. P., et al. A thrombospondin in the anthozoan Nematostella vectensis is associated with the nervous system and upregulated during regeneration. Biol Open. 2 (2), 217-226 (2013).
- Passamaneck, Y. J., Martindale, M. Q. Cell proliferation is necessary for the regeneration of oral structures in the anthozoan cnidarian Nematostella vectensis. BMC Dev Biol. 12, (2012).
- Trevino, M., Stefanik, D. J., Rodriguez, R., Harmon, S., Burton, P. M. Induction of canonical Wnt signaling by alsterpaullone is sufficient for oral tissue fate during regeneration and embryogenesis in Nematostella vectensis. Dev Dyn. 240 (12), 2673-2679 (2011).
- Renfer, E., Amon-Hassenzahl, A., Steinmetz, P. R., Technau, U. A muscle-specific transgenic reporter line of the sea anemone, Nematostella vectensis. Proc Natl Acad Sci U S A. 107 (1), 104-108 (2010).
- Burton, P. M., Finnerty, J. R. Conserved and novel gene expression between regeneration and asexual fission in Nematostella vectensis. Dev Genes Evol. 219 (2), 79-87 (2009).
- DuBuc, T. Q., Traylor-Knowles, N., Martindale, M. Q. Initiating a regenerative response; cellular and molecular features of wound healing in the cnidarian Nematostella vectensis. BMC Biol. 12, (2014).
- Hand, C., Uhlinger, K. R. Asexual reproduction by transverse fission and some anomalies in the sea anemone Nematostella vectensis. Invert Biol. 114, 9-18 (1995).
- Fritzenwanker, J. H., Technau, U. Induction of gametogenesis in the basal cnidarian Nematostella vectensis(Anthozoa). Dev Genes Evol. 212 (2), 99-103 (2002).
- Magie, C., Bossert, P., Aramli, L., Thomsen, G. Science’s super star: The starlet sea anemone is an ideal tool for student inquiry. The Science Teacher. 83 (3), 33-40 (2016).
- Genikhovich, G., Technau, U. In situ hybridization of starlet sea anemone (Nematostella vectensis) embryos, larvae, and polyps. Cold Spring Harb Protoc. (9), (2009).
- Magie, C. R., Pang, K., Martindale, M. Q. Genomic inventory and expression of Sox and Fox genes in the cnidarian Nematostella vectensis. Dev Genes Evol. 215 (12), 618-630 (2005).
- Chera, S., Kaloulis, K., Galliot, B. The cAMP response element binding protein (CREB) as an integrative HUB selector in metazoans: clues from the hydra model system. Biosystems. 87 (2-3), 191-203 (2007).
- Reitzel, A. M., Burton, P. M., Krone, C., Finnerty, J. R. Comparison of developmental trajectories in the starlet sea anemone Nematostella vectensis: embryogenesis, regeneration, and two forms of asexual fission. Invertebr Biol. 126, 99-112 (2007).
- Ikmi, A., McKinney, S. A., Delventhal, K. M., Gibson, M. C. TALEN and CRISPR/Cas9-mediated genome editing in the early-branching metazoan Nematostella vectensis. Nat Commun. 5, 5486 (2014).
- Jahnel, S. M., Walzl, M., Technau, U. Development and epithelial organisation of muscle cells in the sea anemone Nematostella vectensis. Front Zool. 11, 44 (2014).
- Kelava, I., Rentzsch, F., Technau, U. Evolution of eumetazoan nervous systems: insights from cnidarians. Philos Trans R Soc Lond B Biol Sci. 370 (1684), (2015).
- Nakanishi, N., Renfer, E., Technau, U., Rentzsch, F. Nervous systems of the sea anemone Nematostella vectensis are generated by ectoderm and endoderm and shaped by distinct mechanisms. Development. 139 (2), 347-357 (2012).
- Richards, G. S., Rentzsch, F. Transgenic analysis of a SoxB gene reveals neural progenitor cells in the cnidarian Nematostella vectensis. Development. 141 (24), 4681-4689 (2014).
- DuBuc, T. Q., et al. In vivo imaging of Nematostella vectensis embryogenesis and late development using fluorescent probes. BMC Cell Biol. 15, (2014).
- Kaur, J., Debnath, J. Autophagy at the crossroads of catabolism and anabolism. Nat Rev Mol Cell Biol. 16 (8), 461-472 (2015).
- Carroll, B., Korolchuk, V. I., Sarkar, S. Amino acids and autophagy: cross-talk and co-operation to control cellular homeostasis. Amino Acids. 47 (10), 2065-2088 (2015).
- Glick, D., Barth, S., Macleod, K. F. Autophagy: cellular and molecular mechanisms. J Pathol. 221 (1), 3-12 (2010).
- Rodolfo, C., Di Bartolomeo, S., Cecconi, F. Autophagy in stem and progenitor cells. Cell Mol Life Sci. 73 (3), 475-496 (2016).
- Guan, J. L., et al. Autophagy in stem cells. Autophagy. 9 (6), 830-849 (2013).
- Phadwal, K., Watson, A. S., Simon, A. K. Tightrope act: autophagy in stem cell renewal, differentiation, proliferation, and aging. Cell Mol Life Sci. 70 (1), 89-103 (2013).
- Varga, M., Fodor, E., Vellai, T. Autophagy in zebrafish. Methods. 75, 172-180 (2015).
- Varga, M., et al. Autophagy is required for zebrafish caudal fin regeneration. Cell Death Differ. 21 (4), 547-556 (2014).
- Gonzalez-Estevez, C., Salo, E. Autophagy and apoptosis in planarians. Apoptosis. 15 (3), 279-292 (2010).
- Buzgariu, W., Chera, S., Galliot, B. Methods to investigate autophagy during starvation and regeneration in hydra. Methods Enzymol. 451, 409-437 (2008).
- Tettamanti, G., et al. Autophagy in invertebrates: insights into development, regeneration and body remodeling. Curr Pharm Des. 14 (2), 116-125 (2008).
