Isaeva V.V., A.V. Akhmadieva A, Ya.N. Alexandrova, A.I. Shukalyuk
(Institute of Marine Biology, FEB RAS, Vladivostok, Russia; e-mail: vv_isaeva@mail.ru
Laboratory of Stem Cell Biology and Functional Genomics, University of Toronto, Canada)
Studies on human embryonic stem cells have become one of the hottest areas in biotechnology because of the potential for medicine. However the molecular, sub-cellular, and cellular bases for totipotency and immortality, “stemness” in stem and germ cells remains a mystery.
In sexually reproducing animals the line of germ cells is segregated early in embryogenesis, but in invertebrates, capable for asexual reproduction, the line of self-renewing totipotent (at least, pluripotent) stem cells maintains continuously throughout the life. Totipotent stem cells retain the ability to differentiate into all somatic cell types as well as germ cells.
Our study points towards elucidation of the evolutionary conservative sub-cellular and molecular bases of «stemness», focusing on the comparative investigation of stem and germ cells in asexually reproducing invertebrates: colonial parasitic rhizocephalan crustaceans (Arthropoda), colonial hydroids (Cnidaria), colonial ascidians (Chordata), planarians (Turbellaria), and also mammalian embryonic stem cells as standard reference, using in vitro culture, electron microscopic, histological, histochemical, immunochemical, and molecular methods. In studied colonial rhizocephalans we found stem cells, which give rise to somatic as well as germ cells so providing the cellular basis for realizing of the reproductive strategy with alternating asexual and asexual reproduction and colonial organization - unique among arthropods. We found stem cells in stolons, buds and ovary rudiments of the colonial endoparasitic interna of the rhizocephalan Peltogasterella gracilis, Polyascus polygenea and Thylacoplethus isaevae, in the colonial hydroid Obelia longissima and in early buds of the colonial ascidian Botryllus tuberatus. Stem and primary germ cells in representatives of such different taxa have common features typical for embryonic stem and germ cells in other studied Metazoa: a high nucleocytoplasmic ratio, large, light nucleus with diffuse chromatin and large nucleolus; thin rim of basophilic cytoplasm with numerous free ribosomes, mitochondria and also specific electron-dense structures referred to as germinal bodies or nuage (germ plasm determinants) in other studied animals. We have found typical germinal bodies surrounded by mitochondria in stem and germ cells of the studied rhizocephalans, the planarian Dugesia tigrina, the cnidarian Obelia longissima as well as a “nuage” complex in stem cells of B. tuberatus. In mouse embryonic stem cells in vitro germ granules and nuage were revealed. All or most embryonic cells in cleaving embryos (8-16 blastomeres) of Polyascus polygenea have germinal bodies near cell nuclei. So in the rizocephalan P. polygenea an early segregation of germ plasm determinants is absent in contrast to other crustaceans and all arthropods; egg cleavage in this colonial rhizocephalan ceased to be strictly determined in connection with secondary emergence of asexual reproduction. We found the evolutionary conservative gene vasa and other members of the DEAD-box family in the DNA of studied Rhizocephala. The vasa gene product (RNA-helicase) is known to be marker components of the germ plasm granules in a wide range of multi-cellular animals from nematodes to vertebrates and is supposedly involved in determination and maintenance of cell totipotency.
We have shown intensive selective expression of the vasa-like gene in stem and germ cells as well as in germinal granules of early blastomeres of P. polygenea; vasa-like gene expression was found also in germinal granules and nuage in mouse ESC. We have carried out immunocytochemical test using proliferating cell nuclear antigen (PCNA) to detect self-renewing cells that have not left the mitotic cycle in interna of P. gracilis and budding colonial hydroid O. longissima; stem cells were PCNA-positive cells, i.e. reproducing cells. We have shown that the stem cells in the rhizocephalan P. polygenea, P. gracilis and Th. isaevae, stem (interstitial) and germ cells in the colonial cnidarian O. longissima, hemoblasts and stem cells in early buds in the ascidian protochordates B. tuberatus selectively express alkaline phosphatase activity, a well-known histochemical marker for mammalian embryonic stem and primary germ cells in vivo and in vitro. During early cleavage in P. polygenea alkaline phosphatase activity is localized in germinal bodies (germ determinants) of blastomeres.
The intensity of the cytochemical reaction in stem cells of studied invertebrates is comparable to that revealed under similar conditions in mouse embryonic stem cells in vitro. The cytochemical manifestation of alkaline phosphatase activity in the stem cells is the first evidence of this activity in stem cells of invertebrates and it is being a feature common to stem cells of animals belonging to such diverse taxa as Cnidaria, Arthropoda and Chordata.
Thus, our results along with literature data allow suggest the existence of evolutionary conservative, common for all studied representatives of metazoans cellular, sub-cellular and molecular bases of totipotency and “immortality” of stem and germ cells. Totipotent stem cells in colonial invertebrates share morphological and functional features presumably involving in maintenance of totipotency and potential immortality of metazoan embryonic stem and germ-line cells. In colonial invertebrates stem cells provides the cell source for asexual and sexual reproduction and the cellular basis for realization of the life strategy including both alternating reproductive modes.
Some publications:
Isaeva V., Alexandrova Ya., Reunov A. Interaction between chromatoid bodies and mitochondria in neoblasts and gonial cells of the asexual and spontaneously sexualized planarian Girardia (Dugesia) tigrina. Invertebrate Reproduction and Development. 2005. Vol. 48(1-3). P. 119-128.
Shukalyuk A., Isaeva V., Kizilova E., Baiborodin S. Stem cells in reproductive strategy of colonial rhizocephalan crustaceans (Crustacea: Cirripedia: Rhizocephala). Invertebrate Reproduction and Development. 2005. Vol. 48. P. 41-53.
Shukalyuk A.I., Golovnina K.A., Baiborodin S.I., Gunbin K., Blinov A.G., Isaeva V.V. vasa-related genes and their expression in stem cells of colonial parasitic rhizocephalan barnacle Polyascus polygenea (Arthropoda: Crustacea: Cirripedia: Rhizocephala). Cell Biology International 2007 (in print).
Shukalyuk A.I., Isaeva V.V. Stem cells are found in colonial rhizocephalans (Crustacea: Rhizocephala). Molecular Biology of the Cell. 2003. Vol. 14 (Suppl.): Abstracts. 43th Annual Meeting of the American Society for Cell Biology, San Francisco, 2003). P. 523a.
Shukalyuk A., Isaeva V., Golovnina K., Blinov A., Baiborodin S. Stem cells in reproductive strategy of rhizocephalans (Crustacea: Cirripedia: Rhizocephala). (Abstracts. 44th Annual Meeting of the American Society for Cell Biology. Washington, DC, 2004). Molecular Biology of the Cell. 2004. Vol. 15. N 11 (Suppl.) P. 341a.
Shukalyuk A.I., Isaeva V.V. Stem cells in invertebrate animals: evolutionary conservative mechanism of totipotency. International Society for Stem Cell Research: 3rd Annual Meeting (San Francisco, June, 2005). Abstracts. P.214-215.
Shukalyuk A.I., Isaeva V.V., Golovnina K.A., Baiborodin S.I., Blinov A.G. vasa-related genes and their expression in stem cells of colonial Polyascus polygenea (Crustacea: Cirripedia: Rhizocephala). International Society for Stem Cell Research: 3rd Annual Meeting (San Francisco, June, 2005). Abstracts. P. 215.
Shukalyuk A.I., Isaeva V.V. Stem and germ cells in asexually reproducing animals: a comparative study. The American Society for Cell Biology. 45th Annual Meeting (San Francisco, December, 2005). Molecular Biology of the Cell. 2005. Vol. 16. N 11 (Suppl.). P. 752a.
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