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.
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