The molecular basis of planarian regeneration

描述（由申请人提供）：所提出的实验旨在使用Schmidtea medialacea作为模型系统来鉴定和功能表征调节后生动物再生的基因和遗传途径。之所以选择Planarians，是因为它们是最简单的生物体的代表，展示了双边对称性，头部化和复杂的器官系统，并被赋予了一个显着的全能干细胞（neoblasts），他们从中获得非凡的再生能力。考虑到许多仍然是了解再生的分子基础，研究这个问题的最佳策略是进行大规模的时空分析的基因表达沿着功能丧失的遗传筛选。使用微阵列，我们将确定从来源于S克隆系4（CIW 4）的头部、头部胚基和新生细胞（干细胞）cDNA文库获得的~ 4，500个非冗余cDNA的表达谱。地中海。将通过杂交来自在再生的不同时间点获得的组织的Cy-labeled cDNA，生成打印在微阵列上的基因的时间序列表达谱。将对数据进行聚类分析，以鉴定具有相似表达动力学的基因组。微阵列结果将通过阵列中代表的基因的空间表征来补充。通过整体原位杂交获得的空间表达数据将根据组织和/或细胞类型特异性进行聚类。将空间基因表达簇与微阵列衍生的时间表达簇进行比较，以鉴定细胞特异性和表达动力学的重叠。这将导致根据基因表达的时间和地点来识别可能属于相同遗传途径的基因子集。为了测试基因功能，并确定在再生事件中的作用，我们将使用RNAi进行反向遗传筛选。基于RNAi的筛选将用于：1）测试时空关系; 2）鉴定其功能废除能够干扰再生的基因;和3）鉴定微阵列分析所遗漏的基因，其产物活性的变化而不是表达水平的变化负责调节再生事件。最后，我们将尝试建立上位相互作用的基因确定为发挥关键作用的再生过程中，通过定义表达谱的RNAi治疗的动物在稳态微阵列研究。

DESCRIPTION (provided by applicant): The experiments proposed aim to identify and functionally characterize genes and genetic pathways regulating metazoan regeneration using Schmidtea mediterranea as a model system. Planarians were chosen because they are representatives of the simplest organisms displaying bilateral symmetry, cephalization, and complex organ systems and are endowed with a remarkable population of totipotential stem cells (neoblasts) from which they derive extraordinary regenerative abilities. Considering that much remains to be learned about the molecular basis of regeneration, the best strategy to study this problem is to perform large-scale temporo-spatial analyses of gene expression along with loss-of-function genetic screens. Using microarrays, we will define the expression profile of -4,500 non-redundant cDNAs obtained from head, head blastema and neoblast (stem cells) cDNA libraries derived from clonal line 4 (CIW4) of S. mediterranea. A time-series expression profile of the genes printed on the microarrays will be generated by hybridizing Cy-labeled cDNAs from tissues obtained at various time points of regeneration. The data will be subjected to cluster analyses to identify groups of genes sharing similar expression kinetics. The microarray results will be complemented by the spatial characterization of the genes represented in the arrays. Spatial expression data obtained by whole-mount in situ hybridizations will be clustered according to tissue and/or cell type specificity. The spatial gene expression clusters will be compared to microarray-derived temporal expression clusters to identify overlaps in cell specificity and expression kinetics. This should result in the identification of subsets of genes that may belong to the same genetic pathways based on when and where they are expressed. To test for gene function, and to determine roles in regenerative events, we will carry out reverse genetic screens using RNAi. The RNAi-based screen will serve to: 1) test the temporo-spatial relationships; 2) identify genes whose functional abrogation are capable of perturbing regeneration; and 3) identify genes missed by microarray analyses to which changes in the activity of their products, rather than changes in expression levels are responsible for modulating regenerative events. Finally, we will attempt to establish epistatic interactions for those genes identified as playing key roles in the progress of regeneration by defining expression profiles of RNAi-treated animals in steady-state microarray studies.