Functional analysis of grimp, a novel gene required for mesodermal cell proliferation at an initial stage of regeneration in Enchytraeus japonensis (Enchytraeidae, Oligochaete).
Enchytraeus japonensis is a small oligochaete species, which has a remarkable regeneration capacity. It has been proposed as a new model animal for the study of regeneration, and some histological studies of this species have been carried out. On the other hand, the molecular biological mechanism of regeneration is almost unknown in this species. To clarify the molecular biological mechanism operating at an initial stage of regeneration in E. japonensis, we isolated by the cDNA subtraction method five genes whose expression levels changed in the regeneration process occurring between growing and early regenerating worms. 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 succeeded in inhibiting grimp expression by RNA interference (RNAi), thus applying this technique for the first time in Oligochaeta. In knock-down worms, the number of BrdU-positive neoblasts and the non-neoblast grimp-expressing cells in the coelom drastically decreased. Moreover, the elongation and the segmentation of blastemas 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.
Takeo
Makoto
M
Department of Natural History Sciences, Faculty of Science, Hokkaido University, Sapporo, Japan. tko@bio.sci.hokudai.ac.jp
Yoshida-Noro
Chikako
C
Tochinai
Shin
S
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern; D001973:Bromodeoxyuridine; D002454:Cell Differentiation; D049109:Cell Proliferation; D003001:Cloning, Molecular; D018507:Gene Expression Regulation, Developmental; D005796:Genes / Q000502:physiology*; D007124:Immunoenzyme Techniques; D017403:In Situ Hybridization; D008648:Mesoderm / Q000166:cytology* / Q000502:physiology; D008969:Molecular Sequence Data; D009835:Oligochaeta / Q000235:genetics* / Q000254:growth & development*; D034622:RNA Interference; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D012038:Regeneration / Q000502:physiology*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013382:Subtraction Technique
D012333:RNA, Messenger; D001973:Bromodeoxyuridine