Germinal granules in interstitial cells of the colonial hydroids <Emphasis Type="Italic">Obelia longissima</Emphasis> pallas, 1766 and <Emphasis Type="Italic">Ectopleura crocea</Emphasis> Agassiz, 1862

Electron-dense germinal granules, which are usually regarded as markers and key organelles of germline cells, were revealed in the interstitial (stem) cells of the colonial hydroids Obelia longissima and Ectopleura crocea. The interstitial cells of O. longissima displayed intense alkaline phosphatase activity, a histochemical marker for vertebrate embryonic stem and primary germ cells, as well as positive reaction to proliferating cell nuclear antigen (PCNA), which is an immunochemical marker for cell reproduction. Our findings and the literature data suggest the evolutionary conservation and similarity of the morphological and functional organization of potentially gametogenic stem cells in asexually reproducing invertebrates and germ cells in all studied Metazoa. The self-renewing pool of such stem cells provides the cellular source for blastogenesis and gametogenesis and the cellular basis for life functions, including both asexual and sexual reproduction.     

Stem cells in reproductive strategy of asexually reproducing invertebrates

Original and literature data supporting the evolutionary conservation of the morphofunctional organization of totipotent cells of germ and stem lineages in metazoan animals are reviewed. We studied stem cells of the colonial rhizocephalans, Peltogasterella gracilis, Polyascus polygenea and Thylacoplethus isaevae, the turbellarian Dugesia tigrina, the colonial hydroid Obelia longissima, and cultured embryonic stem cells of mouse. The typical germinal granules of germ plasm, selective expression of the activity of alkaline phosphatase and of proliferating cell nuclear antigen (PCNA), which are known as markers of stem and primary germ cells of vertebrates, and the specific expression of the protein product of the vasa gene in cells of rhizocephalans, which is a marker of cells of germ and stem lineages of various metazoans, specified the stem cells of invertebrates of such different taxa. The self-renewing pool of totipotent stem cells is the cellular basis of the reproductive strategy, including sexual and asexual reproduction; such cells share morphofunctional features of embryonic stem and germline cells of Metazoa.     

Forward and reverse genetics through derivation of haploid mouse embryonic stem cells

All somatic mammalian cells carry two copies of chromosomes (diploidy), whereas organisms with a single copy of their genome, such as yeast, provide a basis for recessive genetics. Here we report the generation of haploid mouse ESC lines from parthenogenetic embryos. These cells carry 20 chromosomes, express stem cell markers, and develop into all germ layers in vitro and in vivo. We also developed a reversible mutagenesis protocol that allows saturated genetic recessive screens and results in homozygous alleles. This system allowed us to generate a knockout cell line for the microRNA processing enzyme Drosha. In a forward genetic screen, we identified Gpr107 as a molecule essential for killing by ricin, a toxin being used as a bioweapon. Our results open the possibility of combining the power of a haploid genome with pluripotency of embryonic stem cells to uncover fundamental biological processes in defined cell types at a genomic scale.     

Regeneration of the Digestive Tube in the Holothurian Apostichopus japonicusafter Evisceration

We performed a TEM study of regeneration of the intestine in the Far Eastern trepang, the holothurian Apostichopus japonicus, after evisceration. The following stages were distinguished in the restoration process of the digestive tube: the growth of connective tissue along the margin of the mesenterium, in the place of rupture; dedifferentiation of cells and their migration and proliferation; the rooting of the esophagus lining into the connective tissue anlage; and the transformation of esophagus cells into cells of the middle part of the intestine. The migration of epithelium into the area of regeneration takes place through a solid cellular layer, without breaking of the cell contacts. The mitotic activity was registered in all stages of restoration; the dividing cells were located chaotically, without the development of a blastema.     

vasa-related genes and their expression in stem cells of colonial parasitic rhizocephalan barnacle Polyascus polygenea (Arthropoda: Crustacea: Cirripedia: Rhizocephala)

vasa (vas)-related genes are members of the DEAD-box protein family and are expressed in the germ cells of many Metazoa. We cloned vasa-related genes (PpVLG, CpVLG) and other DEAD-box family related genes (PpDRH1, PpDRH2, CpDRH, AtDRHr) from the colonial parasitic rhizocephalan barnacle Polyascus polygenea, the non-colonial Clistosaccus paguri (Crustacea: Cirripedia: Rhizocephala), and the parasitic isopodan Athelgis takanoshimensis (Crustacea: Isopoda). The colonial Polyascus polygenea, a parasite of the coastal crabs Hemigrapsus sanguineus and Hemigrapsus longitarsis was used as a model object for further detailed investigations. Phylogenetic analysis suggested that PpVLG and CpVLG are closely related to vasa-like genes of other Arthropoda. The rest of the studied genes form their own separate branch on the phylogenetic tree and have a common ancestry with the p68 and PL10 subfamilies. We suppose this group may be a new subfamily of the DEAD-box RNA helicases that is specific for parasitic Crustacea. We found PpVLG and PpDRH1 expression products in stem cells from stolons and buds of internae, during asexual reproduction of colonial P. polygenea, and in germ cells from sexually reproducing externae, including male spermatogenic cells and female oogenic cells.     

Infestation Level of the Crab Hemigrapsus sanguineus by a Parasitic Crustacean Polyascus polygenea (Crustacea: Cirripedia) in Vostok Bay, Sea of Japan

We studied the infestation level of the coastal crab Hemigrapsus sanguineus by a cirripede barnacle Polyascus polygenea in Peter the Great Bay (Vostok Bay, Sea of Japan) in 1998–1999. It is shown that the externae of the parasite usually appear on the surface of the host’s body at the time when the crab attains sexual maturity (at a carapace width of 14–15 mm). More often we encountered infested crabs of a medium size, with a carapace width of 25–29 mm. The level of crab invasion did not depend on the sex of the host. In the summer period the proportion of infested crabs in different habitats ranged from 7 to 84%. The greatest number of crabs bearing the externae of the parasite was registered in an area with a boulder-pebble surface and small surf waves.Original Russian Text Copyright © 2005 by Biologiya Morya, Korn, Akhmadieva, Rybakov, Shukalyuk.     

Effects of the Briarosaccus callosus infestation on the commercial golden king crab Lithodes aequispina

Commercial crab populations off the Kamchatka coasts are infested to a considerable degree by the rhizocephalan parasite Briarosaccus callosus: of 769 Lithodes aequispina males examined, 43 (5.7%) were parasitized. Infestations result in the feminization of the crabs, a significant decrease in the cheliped length, and a significant decrease in the carapace length and width. We suggest that commercial selection of healthy males, and the returning of unsuitable crabs, including infested ones, back into the sea, results in an increase of the proportion of infested crabs in the population, their elimination from reproduction, and, eventually, the gradual degradation of a whole population. To minimize as far as possible the negative effects of commercial crab harvesting, all infested crab specimens caught must be destroyed, either aboard or elsewhere, instead of throwing them back into the sea.     

Organization of the Interna of the Rhizocephalan Barnacle Peltogasterella gracilis

We carried out an in vitro histological and TEM investigation of the organization of the interna of Peltogasterella gracilis(Crustacea: Rhizocephala), a parasite of the hermit crab. The colonial interna of P. gracilisincludes a reproductive system with multiple nuclei (externa rudiments) and a trophic system of absorbing lampbrush and transportation canals. The nucleus forms a cluster of embryonic stem cells. In the distal parts of the trophic system, there are cells that function in the absorption, processing, and storage of trophic substances. Cells filled with trophic resources disintegrate (evidently by apoptosis), releasing trophic substances and cell remnants into the canal cavity formed through this process. The parasitic phase of the P. gracilislife cycle is characterized by the loss of the basic morphological features and complete pattern of organization of the arthropods and by chaos and fractalization in the interna.     

CD133 protein N-glycosylation processing contributes to cell surface recognition of the primitive cell marker AC133 epitope

The AC133 epitope expressed on the CD133 glycoprotein has been widely used as a cell surface marker of numerous stem cell and cancer stem cell types. It has been recently proposed that posttranslational modification and regulation of CD133 may govern cell surface AC133 recognition. Therefore, we performed a large scale pooled RNA interference (RNAi) screen to identify genes involved in cell surface AC133 expression. Gene hits could be validated at a rate of 70.5% in a secondary assay using an orthogonal RNAi system, demonstrating that our primary RNAi screen served as a powerful genetic screening approach. Within the list of hits from the primary screen, genes involved in N-glycan biosynthesis were significantly enriched as determined by Ingenuity Canonical Pathway analyses. Indeed, inhibiting biosynthesis of the N-glycan precursor using the small molecule tunicamycin or inhibiting its transfer to CD133 by generating N-glycan-deficient CD133 mutants resulted in undetectable cell surface AC133. Among the screen hits involved in N-glycosylation were genes involved in complex N-glycan processing, including the poorly characterized MGAT4C, which we demonstrate to be a positive regulator of cell surface AC133 expression. Our study identifies a set of genes involved in CD133 N-glycosylation as a direct contributing factor to cell surface AC133 recognition and provides biochemical evidence for the function and structure of CD133 N-glycans.     

Stem cells in asexual reproduction of the colonial ascidian <Emphasis Type="Italic">Botryllus tubaratus</Emphasis> (Tunicata: Ascidiacea)

Histological, cytochemical and ultrastructure research on the budding of the colonial ascidian Botryllus tuberatus aimed at searching for stem cells was performed. A dense mass of undifferentiated cells and the connection of the outer epidermal and inner atrial epithelia were revealed for the first time in the early buds of B. tuberatus. Undifferentiated cells revealed in the early buds and vascular system of the colony had morphological features of the stem and primary germ cells of metazoans. Intensive expression of alkaline phosphatase, the cytochemical marker of embryonal stem and primary germ cells of vertebrate animals, was revealed in developing buds and in some cells of the hematocyte population. Based on the literature and the author’s data it is hypothesized that the self-renewing pool of stem cells of the colonial ascidian B. tuberatus is the cellular basis of its reproductive strategy, including both sexual and asexual reproduction.