Chan John D, Marchant Jonathan S
Department of Pharmacology and The Stem Cell Institute, University of Minnesota Medical School, USA.
J Vis Exp. 2011 Aug 31(54):3058. doi: 10.3791/3058.
Free-living planarian flatworms have a long history of experimental usage owing to their remarkable regenerative abilities. 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. 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 plasticity. 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 neurotransmitters, represent an important system for studying evolution of the central nervous system and have behavioral screening potential. 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 constructs. Both approaches can produce visually striking phenotypes at high penetrance--for example, regeneration of bipolar animals. 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.
由于其卓越的再生能力，自由生活的涡虫纲扁形虫有着悠久的实验应用历史。从这些动物身上切下的小片段在缺失身体结构再生后会重新形成原来的身体结构。例如，如果从一条完整的涡虫身上切下一个“躯干”片段，新的“头部”会在前端再生，“尾部”会在后端再生，恢复截肢前身体结构原来的“从头到尾”极性。再生由涡虫干细胞驱动，这些干细胞被称为“新生细胞”，在正常身体稳态和强制组织再生过程中分化为约30种不同的细胞类型。这个再生过程强大且易于展示。由于几个开创性实验室的努力，现在许多工具和功能基因方法已针对这个模型系统进行了优化。因此，最近在理解和操纵涡虫发育可塑性背后的分子事件方面取得了相当大的进展。涡虫模型系统将引起广泛科学家的兴趣。对于神经科学家来说，这个模型提供了研究整个神经系统再生的机会，而不仅仅是研究许多现有模型中通常关注的单个神经细胞突起的再生/修复。涡虫表达大量神经递质，是研究中枢神经系统进化的重要系统，并且具有行为筛选潜力。再生结果适合通过药理学和遗传学方法进行操纵。例如，只需在截肢后的不同时间点将身体片段置于含药溶液中，就可以筛选药物对再生的影响。可以使用敲低方法（体内RNA干扰）研究单个基因的作用，这可以通过显微注射循环或喂食细菌表达的dsRNA构建体来实现。这两种方法都可以在高穿透率下产生视觉上显著的表型，例如双极动物的再生。为了促进这个模型的采用和此类方法的实施，我们在这篇视频文章中展示了使用日本三角涡虫进行药理学和遗传学分析（通过喂食进行体内RNA干扰）的方案。
