P83. grimp, a novel gene required for stem cell proliferation at an initial stage of regeneration in Enchytraeus japonensis (Enchytraeidae, Oligochaete)

Abstract

Enchytraeus japonensis is a small oligochaete species that proliferates asexually via fragmentation and regeneration. As sexual reproduction can also be induced, it is a good model system for the study of both regenerative and germline stem cells. It has been shown by histological study that putative mesodermal stem cells called neoblasts, and dedifferentiated epidermal and endodermal cells are involved in blastema formation. To clarify the molecular biological mechanism operating at an initial stage of regeneration in E. japonensis , we isolated five genes whose expression levels changed in the regeneration process occurring between growing and early regenerating worms by cDNA subtraction method. One of the isolated genes (a novel gene named grimp ) was expressed transiently from 3 to 12 h post-amputation only in neoblasts and a population of mesodermal cells (the non-neoblast grimp-expressing cells) incorporating BrdU simultaneously showed mitotic activity. We carried out functional analysis by using RNA interference (RNAi). In knock-down worms, the number of BrdU-positive neoblasts and the non-neoblast grimp-expressing cells in the coelom drastically decreased. Also the elongation and segmentation of blastema were inhibited, while no statistically significant inhibitory effect was observed in epidermal and intestinal cells. These results suggest that grimp is required for initial proliferation of neoblasts and some mesodermal cells for regeneration. We also investigate the function of grimp at the protein level. cDNA sequence of grimp contains a putative open reading frame and poly (A). The deduced polypeptide contains a triplicate repeat of a very similar sequence containing integrin-binding RGDS sequences and protein kinase C phosphorylation sites. However, it is hard to predict its function from the sequence. Thus we took several approaches to clarify the localization and the function of grimp protein.