Stem cells in asexual reproduction of Enchytraeus japonensis (Oligochaeta, Annelid): proliferation and migration of neoblasts.

Enchytraeus japonensis is a small oligochaete that reproduces mainly asexually by fragmentation (autotomy) and regeneration. As sexual reproduction can also be induced, it is a good animal model for the study of both somatic and germline stem cells. To clarify the features of stem cells in regeneration, we investigated the proliferation and lineage of stem cells in E. japonensis. Neoblasts, which have the morphological characteristics of undifferentiated cells, were found to firmly adhere to the posterior surface of septa in each trunk segment. Also, smaller neoblast-like cells, which are designated as N-cells in this study, were located dorsal to the neoblasts on the septa. By conducting 5-bromo-2'-deoxyuridine (BrdU)-labeling-experiments, we have shown that neoblasts are slow-cycling (or quiescent) in intact growing worms, but proliferate rapidly in response to fragmentation. N-cells proliferate more actively than do neoblasts in intact worms. The results of pulse-chase experiments indicated that neoblast and N-cell lineage mesodermal cells that incorporated BrdU early in regeneration migrated toward the autotomized site to form the mesodermal region of the blastema, while the epidermal and intestinal cells also contributed to the blastema locally near the autotomized site. We have also shown that neoblasts have stem cell characteristics by expressing Ej-vlg2 and by the activity of telomerase during regeneration. Telomerase activity was high in the early stage of regeneration and correlated with the proliferation activity in the neoblast lineage of mesodermal stem cells. Taken together, our results indicate that neoblasts are mesodermal stem cells involved in the regeneration of E. japonensis.

Exploration of embryonic origins of germline stem cells and neoblasts in Enchytraeus japonensis (Oligochaeta, Annelida).

An oligochaete annelid species, Enchytraeus japonensis, reproduces not only asexually but also sexually. It has been reported that putative mesodermal stem cells called neoblasts contribute to blastema formation and that Ej-piwi(+) germline stem cells participate in gonadal regeneration. To delineate the origin and formation of both of these stem cells, we isolated two vasa-related genes (Ej-vlg1 and Ej-vlg2) and analyzed the expression of each along with that of germline marker gene Ej-piwi. In adults, Ej-vlg1 and Ej-vlg2 were expressed in Ej-piwi(+) germline stem cells and germ cells in gonads, while only Ej-vlg2 mRNAs were detected in neoblasts. Expression analysis during embryogenesis indicated that clusters of Ej-vlg1(+)/Ej-vlg2(+) cells, located at the posterior ventral region in late embryos, became Ej-vlg1(+)/Ej-vlg2(+)/Ej-piwi(+) germline stem cells just after embryogenesis. On the other hand, Ej-vlg2 single positive cells with morphological characteristics of neoblasts became detectable much later after embryogenesis at the ventral position on each septum where adult neoblasts exist, although these early detected cells were much smaller in size than adult neoblasts. The present results suggest that (1) germline stem cells specified just after embryogenesis are derived from Ej-vlg1(+)/Ej-vlg2(+) cells which appear at the posterior ventral region in late embryos, and that (2) neoblasts appear much later in development.

Early segregation of germ and somatic lineages during gonadal regeneration in the annelid Enchytraeus japonensis.

Although regeneration studies are useful for understanding how organs renew, little information is available about regeneration of reproductive organs and germ cells. We here describe the behavior of germ-cell precursors during regeneration of the oligochaete annelid worm Enchytraeus japonensis, which has the remarkable feature of undergoing asexual (by fission) and sexual reproduction . We first found that the gonad can regenerate from any body fragment yielded by fission during asexual reproduction. We then examined behavior of germ-cell lineage during this regenerative process, by using a homolog of the Piwi gene (Ej-piwi) as a marker. We found that in asexually growing animals, specialized cells expressing Ej-piwi are distributed widely in the body as single cells. These cells seem to serve as a reservoir of germ-cell precursors because during asexual propagation these cells migrate into the regenerating tissue, where they ultimately settle in the prospective gonads, and give rise to germ cells upon sexualization. These cells are distinct from the neoblasts, thought to be stem cells in other animals. This is the first report to directly show that the germ and somatic lineages are segregated in asexually growing animals and behave differently during regeneration.