MOLECULAR BASIS OF PLANARIAN REGENERATION

这个提议的目的是使用真涡虫作为后生动物的模型 解剖和操纵动物的分子基础的系统 再生 Planarians被选为模型系统，因为 它们在后生动物生殖中的关键地位， 能力，以及这些动物再生能力的全能性， 干细胞群（新生细胞）。 首先，我们建议确定和 描述参与指定和维持前额叶的基因， 和后部再生的命运。 我们会达到这个目标 使用基于聚合酶链反应的基因筛选。 的方法 涉及来自再生的cDNA的消减杂交， 非再生组织，然后扩增富集的cDNA， 每个组织（见初步结果）。 基因筛选分离 cDNA将通过测序、发育Northerns和 原位杂交。 其次，我们将利用无性 和有性生殖策略，以及它们的 全能再生干细胞开发方法 生产转基因动物所必需的。转基因将 允许对那些基因进行功能测试，这些基因的序列，发育， 表达谱和组织特异性表明， 在再生过程中的作用。 一旦确定， 真涡虫再生特异基因的功能研究 将垂直整合到更高的再生研究中， 有机体 最终，在真涡虫中发现的基因， 再生过程中的相互作用将定义一系列有用的 分子模板将有助于解开更复杂的表观遗传 脊椎动物再生的过程，并揭示了使 再生允许在一些，但不是所有的后生动物。

The objective of this proposal is to use planarians as a metazoan model system in which to dissect and manipulate the molecular basis of animal regeneration. Planarians have been chosen as a model system because of their key position in metazoan phylogeny, their remarkable regeneration abilities, and the totipotential nature of these animals' regenerative stem cell population (neoblasts). First, we propose to identify and characterize the genes involved in specifying and maintaining anterior and posterior regeneration fates. We will accomplish this objective using a polymerase chain reaction-based gene-screen. The methodology involves the subtractive hybridization of cDNAs from regenerating and non-regenerating tissues, and then amplifying the enriched cDNAs unique to each tissue (see preliminary results). The gene-screen-isolated cDNAs will be characterized by sequencing, developmental Northerns and in situ hybridizations. Secondly, we will take advantage of the asexual and sexual reproductive strategies of planarians, and of their totipotential regenerative stem cells to develop the methodologies necessary for the production of transgenic animals. Transgenesis will allow functional testing of those genes whose sequence, developmental expression profile, and tissue specificity indicate potential regulatory roles during the process of regeneration. Once identified, the functional characterization of regeneration-specific genes in planarians will be vertically integrated to the study of regeneration in higher organisms. Ultimately, the genes identified in planarians, and their interactions during regeneration will define a series of useful molecular templates which will help unravel the more complex epigenetic processes of vertebrate regeneration, and uncover the factors which make regeneration permissive in some, but not all metazoans.