Gene expression in pluripotent stem cells of invertebrates
In this review, morphological and some functional properties of stem cells in different representatives of animals with asexual reproduction (sponges, hydroids, planaria, colonial rhizocephalan crustaceans and colonial ascidia) are considered in comparison with metazoan germline cells and
Stem cells are an essential and defining feature developed during evolution of all multicellular organisms (Lohman, 2008; Batygina, 2010; Funayama et al., 2010). The study of mammalian embryonic stem cells has become a hot, intensely developing field in biology, biotechnology, and biomedicine. However, comparative studies of stem cells in various multicellular organisms are required to understand molecular mechanisms of maintaining pluri/totipotency, “stemness”, which still remain far from clear, as well as mechanisms that regulate gametogenesis, reproduction, development and regeneration.
According to the generally accepted view, stem cells are cells of embryos or adult organisms capable of self-renewing by mitotic reproduction and differentiation into specialized cell types (Weissman et al., 2001; Cogle et al 2003; Müller, 2006; Lohman 2008; Rinkevich et al., 2009; Sköld et al., 2009; Funayama et al., 2010).
Two main types of stem cells should be considered: the cells of the germline and the somatic stem cell lineages (Hogan, 2001; Rinkevich, 2009; Srouji & Extavour, 2011). Germ cells are the only cell type capable of generating a new whole organism in animals; everlasting germline cycle continues from one generation to the next, thus the germline escapes the mortality that all somatic cells of an organism ultimately confront (the common view from Weismann, 1892, to Cinalli et al., 2008). The true innovation in the evolution was the generation of gametogenic germ line, the loss of gametogenic potential from the majority of cells of the organism and protection of the germ line throughout development (Extavour, 2008; Strouji & Extavour, 2011). During development, germ cells are set aside from all somatic cells of the embryo (Cinalli et al., 2008).
E. Davidson and colleagues have developed the “set-aside” hypothesis (Davidson et al., 1995; Cameron et al., 1998; Jenner, 2000; Collins & Valentine, 2001). Cameron et al. (1998) called “set-aside cells” the patches of larval cells that give rise to the adult body plan in animals with indirect development while most of the larval cells have a dead-end fate. These cells are developmentally set aside from the embryo-larva differentiation process, have an essentially unlimited division capacity, and they produce new populations of cells that are organized into the parts of the adult body plan (Cameron et al., 1998). So many adult organs are not derived from cells within larval organs, but from pluripotent cells sequestered, set aside during larval life as primordia from which adult structures form, such as imaginal discs of insects (Collins & Valentine, 2001).
Generally, we can regard all stem cells as “set-aside cells”, reserve cells.
The reproductive strategy of multicellular organisms can include sexual and asexual reproduction. In organisms with exclusively sexual reproduction, primary germ cells become segregated during embryogenesis. Two main types of germline cell specification were defined as preformation (early specification of primary germ cells by means of asymmetric distribution of maternal cytoplasmic determinants) and epigenesis, i.e. later specification of germ lineage cells by inductive signals (Extavour & Akam, 2003; Travis, 2007; Frank et al., 2009). Recently, Strouji and Extavour (2011) refer to former “preformation” as “inheritance mode”, and former “epigenesis” as “inductive mode”. Besides, a third variant of germ cell specification, along with preformation and epigenesis, is recognized as somatic embryogenesis, in asexually reproducing animals, which have stem cells able to differentiate into germ and somatic cells throughout the life of an individual or a colony (Buss, 1987, 1999; Blackstone & Jasker, 2003; Frank et al., 2009; Rinckevich et al., 2009). Pluri/totipotent stem cells of these animals provide a cell source for gametogenesis, asexual reproduction and regeneration (Isaeva et al., 2008b, 2009; Frank et al., 2009; Sköld et al., 2009). Stem cells that have the potential to become both somatic and primary germ cells and are morphologically indistinguishable from the latter were defined as “primary stem cells” (Sköld et al., 2009).
In animals with asexual reproduction by somatic embryogenesis, the germ lineage remains non-segregated even in the adult organism, the gametes of which differentiate from stem cells (Blackstone & Jasker, 2003; Rinkevich, 2009; Sköld et al., 2009). Examples of such reserve stem cells, capable of differentiating both into germ and somatic cells, include sponge archaeocytes, cnidarian interstitial cells, planarian neoblasts, and stem cells of colonial ascidians (reviews: Isaeva et al., 2008b, 2009; Frank et al., 2009; Rinkevich et al., 2009; Sköld et al., 2009; Isaeva, 2010; Srouji, Extavour, 2011). These self-renewing stem cells maintains continuously throughout the life of an individual or a colony, being predecessors of germ cells and all the types (or a wide spectrum) of somatic cells, so ensuring both sexual and asexual reproduction. Thus, in invertebrates with asexual reproduction, no early segregation of a germ cell lineage takes place, and a self-renewing reserve of stem cells with broad or unlimited morphogenetic potential is maintained over the entire lifespan.
Similarly in plants, floral stem cells arise from stem cells of shoot apical meristema (Verdeil et al., 2007; Lohman, 2008; Batygina, 2010). Somatic embryogenesis is viewed as a condition characteristic to the lower metazoans; in the course of evolution, metazoans switch from somatic embryogenesis to preformation and epigenesis, and a subsequent return to somatic embryogenesis is a rare event (Blackstone & Jasker, 2003). If epigenesis was used by Urbilateria to specify the germ line, then preformation must have evolved convergently several times during the bilaterian radiation (Extavour, 2008).
In organisms with asexual, agamous development, the organism that has developed from the zygote is capable of natural cloning and forming numerous genetically identical individuals or modular units of a colony. Clonal morphogenesis, called somatic embryogenesis, as applied to animals, was usually termed blastogenesis (Berrill, 1961; Ivanova-Kazas, 1996) while in plants it was termed somatic embryogenesis or embryoidogenesis (see Verdeil et al., 2007; Batygina, 2010). In the life cycle of colonial animals, one generation of oozooid (individual that has developed from an egg) alternates with numerous generations of blastozooids, with respectively alternating morphogenetic processes: embryogenesis and blastogenesis (Ivanova-Kazas, 1996). In animal kingdom, natural cloning is a widespread phenomenon that includes polyembryony, budding, fission (architomy, paratomy, autotomy) etc.
Both striking similarities and considerable differences in stem cell systems have been observed between plants and animals (Lohman, 2008; Batygina, 2010; Sablowski, 2010). Lohman (2008) considers as the most important differences the capacity of plants to maintain totipotent stem cells throughout their entire lives and the dramatic developmental plasticity of plant cells; besides, plant cells are unable to move within the organism by active migration. Similarities in the stem cell pools of plants and animals suggest that there might have been strong evolutionary constraints that shaped the path for the development of stem cell systems (Lohman, 2008; Sablowski, 2010).
Our study points towards elucidation of the evolutionary conservative cellular, sub-cellular and molecular bases of “stemness”, focusing on the comparative investigation of pluripotent stem cells in reproducing asexually representatives of five metazoan types: archаeocytes in the sponge
Sponge archaeocytes, cnidarian interstitial cells, and planarian neoblasts are classical, long-explored stem cells. Rhizocephalan crustaceans (Arthropoda: Crustacea: Cirripedia: Rhizocephala) parasitizing free-living crustaceans, mainly decapods, until now are often considered as incapable of asexual reproduction and coloniality (for example, Blackstone & Jasker, 2003; Sköld et al., 2009). However, at the parasitic stage of the life cycle, many species of rhizocephalan have asexual reproduction without separation of blastozooids, resulting in the emergence of colonial organization (Høeg, 1992; Høeg & Lützen, 1995; Isaeva et al., 2003, 2004; Shukalyuk et al., 2005, 2007, 2011) that is unique among crustaceans, all arthropods, and all Ecdysozoa. Direct evidence of colonial organization at the parasitic stage of life cycle has been obtained only for a few rhizocephalan species. We visualized asexual reproduction, revealed and studied stem cells in the stolons and buds of the colonial rhizocephalans
The data of our team showed that studied pluripotent reserve stem cells serve as the predecessors of germ and somatic cells and display some evolutionarily conserved features of the morphological and functional organization typical also for cells of the germ line and embryonic stem cells (Isaeva et al., 2003, 2004, 2005, 2008b, 2009, 2011; Akhmadieva et al., 2007; Shukalyuk, Isaeva, 2005; Shukalyuk et al., 2005, 2007, 2011; Isaeva, 2010; Isaeva, Akhmadieva, 2011).
2. Stem cells in asexually reproducing animals share common features with germ cells and embryonic stem cells
In animals with asexual reproduction, the differentiation of “primary” stem cells into germ and somatic cells is delayed, the germ lineage in these animals is not segregated (Tuzet, 1964; Buss, 1987, 1999; Sköld et al., 2009); these stem cells serve as the cellular source of asexual and sexual reproduction as well as of regeneration (Isaeva et al., 2008b, 2009; Frank et al., 2009). Pluripotent stem cells can differentiate into a very wide spectrum of somatic cells in adult organisms.
Depending on the width of the potential range of cell differentiation, totipotent, pluripotent, multipotent, oligopotent, and unipotent stem cells are distinguished, but the usage of this terminology is not unified (Müller, 2006; Newton, 2006; Isaeva et al., 2008b, 2009; Rinkevich et al., 2009; Sköld et al., 2009). Totipotent cells can give rise to all cell types of a developing organism; the zygote and the cells of the early mammalian embryo are totipotent. Stem cells of invertebrates reproducing asexually are traditionally often but not always referred to as totipotent, if their ability to differentiate into gametes and all somatic cells of the organism is shown (reviews: Isaeva et al., 2008b, 2009; Rinkevich et al., 2009; Sköld et al., 2009). For instance, archaeocytes of sponges are regarded as totipotent (Simpson, 1984; Müller, 2006) or pluripotent (Funayama, 2008; Funayama et al., 2010). Funayama believes that both archaeocytes and choanocytes of sponges are pluripotent. Neoblasts of planarians are considered totipotent (Shibata et al., 1999; Gschwentner et al., 2001; Peter et al., 2001; Sköld et al., 2009) or pluripotent (Shibata et al., 2010). Similarly, stem cells of colonial rhizocephalan crustaceans can also be considered totipotent (Isaeva et al., 2004, 2008b, 2009; Shukalyuk et al., 2005, 2007) or pluripotent. The stem cells in colonial ascidians are named both totipotent and pluripotent cells (Stoner et al., 1999; Weissman, 2000; Laird & Weissman, 2004; Laird et al., 2005; Sunanaga et al., 2006). Stem cells giving rise to germline and many but not all somatic cell types are referred to as multipotent. Cnidarian interstitial cells are usually believed to be multipotent, especially in the genus
Estimations of the potentiality of female germ line cells are also contradictory: such cells can be qualified as unipotent, since they produce only one type of differentiated cells, and totipotent, taking into account their potential of developing into a whole organism (Hogan, 2001; Seydoux & Braun, 2006; Strome & Lehman, 2007). Nussbaum (1880) recognized the germ line cells as totipotent and principally different from somatic cells with their limited potency. Accepting the concept of the maintenance of totipotency by cells of the female germ lineage, the author believes that the ability of the stem cells in asexually reproducing invertebrates to differentiate into female gametes and potentially to develop into a whole organism gives grounds for considering them as totipotent independently of the width of their somatic derivates (Isaeva et al., 2008b, 2009).
If we understand cell totipotency as the ability of a single cell to produce a whole organism, only the zygote and the blastomeres of the early embryo of mammals and other animals with regulative type of development are totipotent, having the potential to form an entire living organism. Mammalian embryonic stem cells derived from inner cell mass of the blastocyst have pluripotency: they are able to differentiate into tissues of all three germ layers but cannot produce a whole embryo (Cogle et al 2003). On the other hand, mammalian embryonic stem cells are able to give oogenic cells and oocytes entering meiosis and parthenogenetically producing blastocyst-like cell masses (Hübner et al., 2003; Daley, 2007; Kerkis et al., 2007).
In plants, a new individual can develop from one totipotent somatic cell (Verdeil et al., 2007; Lohman, 2008; Batygina, 2010; Sablowski, 2010). In the asexual reproduction of plants, for instance in polyembryony, a new individual develops from one stem cell, and the pattern of the cell divisions is similar to the cleavage of the zygote (Batygina, 2010). Thus, a single totipotent stem cell of plants may be similar to zygote. The ability of plants to maintain totipotent stem cells over the entire life span of the organism is considered to be their fundamental difference from animal stem cells (Lohman, 2008; Verdeil et al., 2007).
As for the stem cells of invertebrates with asexual reproduction, traditionally named totipotent, it is usually not one stem cell, but some kind of a complex, an aggregate of the stem cells gives rise to the new organism or zooid in asexual reproduction (Blackstone & Jasker, 2003; Rinkevich et al., 2009). It was experimentally shown that one stem cell of a trypsinized cysticercus of the parasitic cestode
The problem of cell line having unlimited morphogenetic potential stems from A. Weismann’ “germ plasm” theory. August Weismann (1834–1914) was the first to discover and describe metazoan stem cells (
2.2. Self-renewal of stem cells
The term “self-renewal” denotes the ability of stem cells to reproduce mitotically during a long period, and in the case of the stem cells of adult organisms, during entire life span of the organism (Weissman et al., 2001; Lohman 2008; Rinkevich et al., 2009; Sköld et al., 2009). Particularly, pluripotent stem, primary germ, and gonial cells have a common property – self-renewal through mitotic reproduction over long periods or throughout life span of the organism (Houston & King, 2000; Hogan, 2000; Sköld et al., 2009).
For instance, sponge archaeocytes are self-renewing, mitotically active, telomerase-positive and bromodeoxyuridine incorporating cells (Müller, 2006; Funayama et al., 2010). Interstitial cells in hydra and other cnidarians can produce both germline cells and some but not all somatic cell types, since epidermal and gastrodermal cells are also capable of mitotic reproduction; so the stem cell system in hydroids includes interstitial, epidermal and gastrodermal stem cells continuously undergoing the mitotic cycle (Campbell, 1974; Thomas & Edwards, 1991; Bode, 1996). For the identification of stem cells capable of mitotic reproduction, bromodeoxyuridine, a thymidine analogue, was successfully used to reveal DNA synthesis in interstitial cells in hydra (Teragawa & Bode, 1990), and neoblasts in flatworms (Gschwentner et al., 2001; Peter et al., 2001). Ethynyl deoxyurudine, another thymidine analogue, was employed for the same purpose in the ctenophore
PCNA (proliferating cell nuclear antigen) assay is used to reveal cells that do not cease to divide mitotically (Hall & Woods, 1990). The PCNA assay was also used to identify neoblasts in planarian; it has been shown that such a test is a reliable tool for neoblast identification in
Mammalian embryonic stem cells express telomerase, the protein associated with a pluripotent and immortal phenotype (Cogle et al 2003).
2.3. Gametogenic potentiality
The pluripotent stem cells ensure both sexual and asexual reproduction, being predecessors of the germ and all the somatic cells. The ability to differentiate into gametogenic and somatic cells was shown for archaeocytes and choanocytes in sponges (Simpson, 1984; Müller, 2006; Funayama, 2008; Funayama et al., 2010), interstitial cells in hydra and other cnidarians (Thomas & Edwards, 1991; Bode, 1996; Isaeva et al., 2011), turbellarian neoblasts (Shibata et al., 1999; Peter et al., 2001; Isaeva et al., 2005), stem cells of colonial rhizocephalans (Isaeva et al., 2004; Shukalyuk et al., 2005), ascidian stem cells (Pancer et al., 1995; Stoner & Weissman, 1996; Stoner et al., 1999; Weissman, 2000). So, pluripotent stem cells in invertebrates with asexual reproduction are potentially gametogenic cells.
Particularly, sponges have no permanent germline; archaeocytes and choanocytes are gametogenic cells (Tuzet, 1964; Blackstone & Jasker, 2003; Sköld et al., 2009). Sponge archaeocytes are considered to be the main cell source in sexual and asexual reproduction (Simpson, 1984; Müller, 2006; Funayama, 2008). Probably, the stem system of sponges includes two types of pluripotent stem cells: archaeocytes and choanocytes; both cell types are able to differentiate into germ and somatic cells; choanocytes can transform to archaeocytes, which later produce other cell types (Funayama, 2008; Funayama et al., 2010).
Cnidarian interstitial cells can produce germline cells (Campbell, 1974; Thomas & Edwards, 1991; Bode, 1996). The gonial cells and early oocytes developing from interstitial cells are distinct from them only by a greater size (Thomas & Edwards, 1991).
Gametogenic potentiality was most convincingly displayed using planarians (Baguña et al., 1989; Peter et al., 2001; Shibata et al., 1999). Neoblasts of asexually reproducing planarians and other Turbellaria are able to differentiate into germ and somatic cells of all types (Agata & Watanabe, 1999; Shibata et al., 1999; Peter et al., 2001; Orii et al., 2005). According to our data, neoblasts can become gonial cells: among individuals in asexual race of
Pluripotent stem cells of colonial rhizocephalans are also the predecessors of somatic and germ cells, so ensuring the reproductive strategy with alternation of asexual and sexual reproduction. In the colonial rhizocephalans
In colonial ascidians, germline as well as somatic cells differentiate from circulating hemoblasts (Pancer et al., 1995; Stoner & Weissman, 1996; Stoner et al., 1999). The differentiation of
Pluripotent embryonic stem cells of mammals have capability to differentiate
2.4. Morphological features of pluripotent stem cells
Pluripotent stem cells of asexually reproducing invertebrates and germline cells of all studied metazoan animals share common morphological and functional features: a high nuclear/cytoplasmic ratio, a large rounded nucleus with diffuse chromatin and a prominent nucleolus, thin rim of undifferentiated basophilic cytoplasm, including specific electron-dense cytoplasmic granules or nuage, and a set of specific regulatory molecules (Shukalyuk & Isaeva, 2005; Isaeva et al., 2008b, 2009, 2011; Extavour, 2008; Rinkevich et al., 2009; Isaeva, 2010; Srouji & Extavour, 2011; Shukalyuk et al., 2011). Germ cells can almost always be unambiguously distinguished from somatic cells by the same characteristic morphology (Extavour, 2008).
The morphological organization of pluripotent stem and gonial cells in the studied representatives of Porifera, Cnidaria, Platyhelminthes, Arthropoda, and Chordata shares common features typical for germline cells in other studied Metazoa (Isaeva et al., 2003, 2004, 2005, 2008b, 2009; Akhmadieva et al., 2007; Shukalyuk & Isaeva, 2005; Shukalyuk et al., 2005, 2007, 2011; Isaeva et al., 2011). For example, archaeocytes of the sponge
2.4.1. Germinal granules (nuage)
The cells of the germ line can be identified and retraced during development of an organism owing to the availability of the “germ plasm” as cytoplasmic markers presented by granular or fibrillar material not surrounded by a membrane. “Germ plasm”, Weismann’s famous term (Weismann, 1892, 1893) now is understood metaphorically. According to this modern understanding, the “germ plasm” contains electron-dense, RNA-enriched material structured as compact germinal granules or a more dispersed “nuage”, a specific ultrastructural marker of metazoan germline cells (see Matova & Cooley, 2001; Seydoux & Braun, 2006; Strome & Lehman, 2007). The germinal granules or nuage are considered key organelles of germline cells (Ikenishi, 1998; Amikura et al., 2001; Matova & Cooley, 2001; Chuma et al., 2006; Seydoux & Braun, 2006; Lim & Kai, 2007; Strome & Lehman, 2007).
The specific electron-dense material of germinal granules was denoted in the early XX century by the German terms
The presence of germinal (perinuclear) granules is an evolutionary conserved feature of germline cells in multicellular animals. These specific organelles have been found in more than 80 species of seven animal types (Eddy, 1975). The structure of these organelles is similar, but they can be represented in cells of different organisms and at different life cycle stages as either a few large granules (bodies) or as a cloud (nuage) of fine-dispersed material. In oogenesis, the germinal bodies transform morphologically but do not disappear in female germ cells throughout the life cycle: for instance, the polar granules are gradually replaced with nuage during polar cell migration in
In the plant kingdom, oogonial cells of the brown alga
Germlinal granules in pluripotent stem cells
In some asexually reproducing invertebrates, stem cells capable to differentiate into germ and somatic cells can be also identified by the presence of specific electron-dense cytoplasmic structures, morphologically similar or identical to germinal granules or nuage in germline cells. The “germ plasm,” containing germinal granules or dispersed “nuage” material, becomes acceptable as a specific ultrastructural marker and a key organelle in pluripotent, potentially gametogenic stem cells of asexually reproducing invertebrates (Shibata et al., 1999; Mochizuki et al., 2001; Isaeva et al., 2008b, 2009, 2011; Frank et al., 2009; Srouji & Extavour, 2011), although the data concerning the structural and molecular organization of germ determinants in the cells of various metazoan taxa are rather fragmentary.
The germinal granules in stem cells of invertebrates, whose life cycle includes asexual reproduction, were earlier have been revealed in the interstitial cells of
Typical electron-dense germinal granules have not been previously described in the archaeocytes or any other cells of sponges. Dense fibrillar bodies were found in the oogonia and oocytes of different sponges (see Tuzet, 1964; Isaeva, Akhmadieva, 2011). In the cytoplasm of archaeocytes in the sponge
The cytoplasm of embryonic and stem cells in the studied rhizocephalans
In the colonial ascidian
So, pluripotent gametogenic stem cells in studied asexually reproducing sponges, cnidarians, turbellarians, arthropods, and chordates feature the presence of the germinal granules. So germinal granules (or more dispersed nuage material) can be used as a specific ultrastructural marker and a key organelle of pluripotent stem cells of asexually reproducing invertebrates (Shibata et al., 1999; Mochizuki et al., 2001; Isaeva et al., 2008b, 2009, 2011; Frank et al., 2009; Isaeva, 2010; Isaeva & Akhmadieva, 2011).
Electron-dense granular structures were observed also in embryonic stem cells of mouse (Shukalyuk et al., 2011). Thus, germinal granules were found not only in cells of the germ line but also in pluripotent stem cells of asexually reproducing invertebrates (sponges, hydroids, turbellarians, colonial rhizocephalan crustaceans and ascidians) and pluripotent mESC
In some somatic metazoan cells, processing bodies (P-bodies) have been found; their function is translation and they are considered as a structural and functional analog of the germinal granules (see Seydoux & Braun, 2006; Kotaja et al., 2006).
2.5. Molecular markers of stem cells
The ultrastructural and molecular organization of germinal granules of germline cells is evolutionarily conserved in all studied representatives of the animal kingdom from sponges to mammals (Ding & Lipshitz, 1993; Ikenishi, 1998; Houston & King, 2000; Matova & Cooley, 2001; Mochizuki et al., 2001; Extavour & Akam, 2003; Juliano et al., 2006; Seydoux & Braun, 2006; Strome & Lehman, 2007; Extavour, 2008; Ewen-Campen et al. 2010; Srouji & Extavour, 2011). It has been shown that some molecules localized in germinal granules are involved in specification of germline cells, and some genes encoding them are highly conserved evolutionary in all studied metazoans (Mahowald, 2001; Matova & Cooley, 2001; Mochizuki et al., 2001; Sato et al., 2001; Seydoux & Braun, 2006; Strome & Lehman, 2007). Germinal granules components include proteins, mRNAs, and noncoding RNAs; as far as is known, RNA-binding proteins are involved in mRNA localization, protection, and translation control (Extavour & Akam, 2003; Leatherman & Jongens, 2003; Chuma et al., 2006; Seydoux & Braun, 2006; Hayashi et al., 2007; Lim & Kai, 2007; Strome & Lehman, 2007; Ewen-Camden et al., 2010). The germinal granules are thought to function as a specific cytoplasmic regulatory center preventing the expression of somatic differentiation genes, maintaining the totipotency in germline cells, necessary for the conception of a new organism, preventing somatic gene expression and protecting the cells from somatic differentiation, that confirmed by data on the transcription “silence” of germline cells (Leatherman & Jongens, 2003; Chuma et al., 2006; Seydoux & Braun, 2006; Strome & Lehman, 2007; Cinalli et al., 2008; Extavour, 2008).
Germline cells can be distinguished from somatic cells by localization of mRNA or protein products of germ-cell-specific genes, notably the
Several conserved molecules are expressed in both germ and pluripotent stem cells; these include Piwi family proteins, Tudor family proteins, and
The piRNA-binding proteins of Argonaute subfamily, coding by
So stem cells of invertebrates with asexual reproduction, as well as cells of the germ lineage, also display the expression of proteins related to Piwi, Nanos, and some others (reviews: Rinkevich et al., 2009; Sköld et al., 2009; Srouji & Extavour, 2011). In stem cells of the sponge
Mammalian embryonic stem cells express gene
Vasaand other members of DEAD-box family
The first identified component of the granules of germ plasm was the protein product of the
In the polyembryonic wasp
The presence of a Vasa-like protein was demonstrated not only in germline cells but also in large interstitial cells of hydra
We have revealed the evolutionarily conserved sites of genes of the DEAD family, particularly
Recently, Alié et al. (2011) found the expression of
The data on
2.5.2. Mitochondrial components of germinal granules
The material of germinal granules (nuage) includes products of the nuclear genome; besides, there is evidence for the mitochondrial origin of some molecular components of germinal granules. The contact with mitochondria is a typical property of structured germinal granules in diverse multicellular animals (Isaeva & Reunov, 2001; Matova & Cooley, 2001; Carré et al., 2002). Ribosomal RNAs of mitochondrial origin and several other products of
|Porifera||Funayama et al., 2010|
|Cnidaria||Mochizuki et al., 2001; Rebscher et al., 2008||Mochizuki et al., 2001||Seipel et al., 2004|
|Ctenophora||Alié et al., 2011||Alié et al., 2011||Alié et al., 2011|
|Plathelminthes||Agata et al., 2006; Pfister et al., 2008||Shibata et al., 1999||Shibata et al. 2010|
|Annelida||Rebscher et al., 2007||Rebscher et al., 2007||Rebscher et al., 2007|
|Shukalyuk et al., 2007, 2011||Shukalyuk et al., 2007, 2011|
|Echinodermata||Juliano & Wessel, 2009||Juliano et al., 2006|
|Chordata (Tunicata)||Rosner et al., 2009||Rosner et al., 2005||Brown et al., 2009|
the mitochondrial genome were revealed in the germinal granules of
The presence of mitochondrial rRNAs outside of the mitochondria in association with germinal granules has been generally accepted; it becomes apparent that mitochondrial rRNAs and other products of the mitochondrial genome are involved in the formation of germline cells in diverse multicellular animals (Ikenishi, 1998; Kloc et al., 2000; Mahowald, 2001; Amikura et al., 2001; Matova & Cooley, 2001; Leatherman & Jongens, 2003; Seydoux & Braun, 2006). It has been suggested that products of both the nuclear and mitochondrial genomes are essential for the structural organization and functioning of the germinal granules of germ plasm (Kobayashi et al., 1998, 2005; Ding & Lipshitz, 1993; Isaeva & Reunov, 2001; Isaeva et al., 2005, 2011).
The export of mitochondrial rRNA from mitochondria to the polar granules in
The export of the ribosomal RNAs from mitochondria to the germinal granules is no longer questioned, but the mechanism underlying a transport is considered unprecedented and enigmatic (Kashikawa et al., 1999; Ding & Lipshitz, 1993; Amikura et al., 2001). Our ultrastructural data indicating the disruption of the outer mitochondrial membrane and the transformation of the mitochondrial matrix with inner membrane cristae into material of germinal granules in representatives of various animal taxa may clarify the mechanism of the export of mitochondrial components into germinal granules (Reunov et al., 2000; Reunov et al., 2004; Isaeva et al., 2005, 2011; Isaeva & Akhmadieva, 2011). This phenomenon enables us to suppose the participation of mitochondria in the biogenesis of the germinal granules (Isaeva & Reunov, 2001; Isaeva et al., 2005, 2011).
Destruction of the outer mitochondrial membrane and transformation of the mitochondrial matrix to the material of germinal granules or nuage have been revealed in the gonial cells of echinoderms and vertebrates (Reunov et al., 2000, 2004), planarian (Isaeva et al., 2005), sponge (Isaeva & Akhmadieva, 2011) and hydroids (Isaeva et al., 2011). The ultrastructural evidence of mitochondrial origin of the germinal granules (chromatoid bodies) in gonial cells and neoblasts of planarian
2.5.3. Alkaline phosphatase activity in stem cells
The histochemically detectable high level of alkaline phosphatase activity has become an empirical marker of mammalian primary germ and embryonic stem cells
No similar research has been carried out on invertebrates until quite recently. We applied cytochemical methods to show alkaline phosphatase activity for stem cell identification in the rhizocephalans
High alkaline phosphatase activity has also been recorded in interstitial and gonial cells of the colonial hydroid
Thus, the stem and gonial cells in rhizocephalans, hydroids and ascidians selectively express alkaline phosphatase activity. Specific brick red staining of stem cells in the studied representatives of colonial cnidarians, arthropods, and chordates was similar in color and intensity to that of cultured mouse embryonic stem cells used as “standard reference” (Isaeva et al., 2003; Shukalyuk et al., 2005). Our data is the evidence of the common functional characteristic of stem cells in such distant taxa as chordates, arthropods and cnidarians. We applied this classical histochemical method developed on the mammalian embryonic germ and stem cells to identify invertebrate stem cells, that reveals an opportunity for the application of this cytochemical reaction to the specific marking of stem cells of invertebrates in other taxonomic groups.
So classical reaction revealing the activity of alkaline phosphatase, earlier used for the identification of primary germ cells and embryonic stem cells in vertebrates, became applicable as a cytochemical marker of both gametogenic and pluri/totipotent stem cells of invertebrates (Isaeva et al., 2003; Laird et al., 2005; Shukalyuk et al., 2005; Akhmadieva et al., 2007; Rinkevich et al., 2009; Sköld et al., 2009). Among plants, a high alkaline phosphatase activity was found in the early gametangia of the brown alga
2.6. Amoeboid cell motility of stem cells
Archaeocytes of sponges are characterized by amoeboid motility and active migration (Simpson, 1984; Müller, 2006; Funayama, 2008; Funayama et al., 2010). Archaeocytes are defined as large amoeboid cells actively migrating within the mesohyl (Funayama, 2008; Funayama et al., 2010). According to our data, migrating archaeocytes morphologically similar to those described previously in other sponge species participate in
In hydra and other cnidarians, interstitial cells are capable of active migration (Campbell, 1974; Thomas & Edwards, 1991; Bode, 1996). Migration of numerous interstitial stem and oogonial cells inside the stolon and their participation in the formation of medusoid generation was also observed in
Turbellarian neoblasts can migrate to the injured surface and sites of gonad formation (Rieger et al., 1991; Auladell et al., 1993; Agata & Watanabe, 1999; Shibata et al., 1999); amoeboid neoblasts and gonial cells in planarian
Undifferentiated rhizocephalan stem cells have been found inside each early stolon bud; similar cells migrate within the stolons in
The primary germline cells are known to emerge outside of the future gonad and later traverse through several developing somatic tissues on their journey to the emerging gonad using both amoeboid motility and passive morphogenetic movements (Matova & Cooley, 2001; Kunwar & Lehmann, 2003; Travis, 2007; Cinalli et al., 2008).
Thus, pluripotent stem cells of asexually reproducing invertebrates are similar to primary germ cells in their ability to amoeboid movement and extensive migrations within the organism, directed to asexual reproduction sites, to the wound surface resulting from fission or damage, or to the gonads, respectively (Isaeva et al., 2009; Isaeva, 2010). In contrast, plant stem cells, with the rigid cellulose wall, are unable to migrate within the organism, and only passively moving together with the tissue, due to cell proliferation and expansion (Lohman, 2008).
2.7. Plasticity of stem cells in morphogenesis
Comparison of normal morphogenesis with its experimental changes helps us understand the plasticity of embryogenesis and blastogenesis. The plasticity of early animal embryogenesis is clearly shown in experiments with dissociated cells
So changes in the initial conditions of morphogenesis
The embryonic stem cells of mammals
Sexual reproduction and early stages of embryogenesis are relatively conservative in all the animal kingdom due to the monophyly of metazoans (Sköld et al., 2009). Since asexual reproduction emerged in the course of the evolution of different metazoan lineages repeatedly and independently, asexual reproduction is more variable and less conservative than embryogenesis. The stage of cleavage is missing in blastogenesis, and stem cells can be likened to the embryonic cells of the morula stage. The integration of blastogenesis in the process of early embryogenesis in animals with polyembryony disrupts the conservatism of embryonic development (Isaeva, 2010). Polyembryony and the breaking of the conservatism of embryogenesis are known also in plants (Batygina, 2010).
The data on the asexual reproduction in some arthropods and chordates contradicts the dogma that asexual reproduction is common exclusively among the lower animals. In particular, the statement that vertebrates are incapable of natural cloning (Blackstone & Jasker, 2003) is disproved by known facts about facultative polyembryony in mammals, which has become obligate in some armadillo species, e.g. in
The self-renewing pool of totipotent stem cells in colonial invertebrates provides the cellular basis for realization of the reproductive strategy including both asexual and sexual reproduction. The principal difference between the reproductive strategy that includes asexual reproduction and the strategy with exclusively sexual reproduction concerns the maintenance of the pluri/totipotent stem cell lineage with gametogenic potential during the entire life span of an asexually reproducing organism; a self-renewing reserve of pluripotent stem cells is the cellular source ensuring the reproductive strategy that includes sexual and asexual reproduction.
The problem of cells dedifferentiation in asexual reproduction and regeneration is less obvious, and the solution of this problem requires special markers. The notion of the high plasticity of the development and fate of cells in colonial animals (Frank et al., 2009; Rinkevich et al., 2009; Sköld et al., 2009), similar to that found in plants (Skold et al., 2009), appears sufficiently justified. In plants, however, differentiated cells retain the ability to dedifferentiate and become totipotent stem cells (Lohman, 2008; Batygina, 2010); animal cells at the stage of terminal differentiation usually have no such ability.
2.8. Evolutionary transition from preformation to epigenesis in colonial Rhizacephala
Extavour (2008) considered the transition from epigenesis to preformation as the repeated evolutionary event, but she thinks that examples of epigenesis in phyla where preformation is plesiomorphic never observed. However, the blastogenesis in colonial species of rhizocephalan crustaceans (Arthropoda: Crustacea: Cirripedia: Rhizocephala) involves a deep reorganization of development; we observe evolutionary secondary transition from preformation to epigenesis. We found germinal bodies in all or most blastomeres of cleaving embryos of
In recent reviews (Blackstone & Jasker, 2003; Sköld et al., 2009) and in modern textbooks, crustaceans as well as all other arthropods and the entire Ecdysozoa clade, are considered as acolonial and aclonal, though in some rhizocephalan crustaceans the colonial organization as result of asexual cloning has already been described (Høeg & Lützen, 1995; Isaeva et al., 2003, 2004, 2008; Glenner et al., 2003; Shukalyuk et al., 2005). We have shown budding of a stolon filled with stem cells in colonial rhizocephalans
On the morphological and gene expression levels, germ cells and stem cells are very similar (Shukalyuk & Isaeva, 2005; Extavour, 2008; Isaeva et al., 2008b, 2009, 2011; Rinkevich et al., 2009; Sköld et al., 2009; Isaeva, 2010; Srouji & Extavour, 2011). Pluri/totipotent gametogenic stem cells are similar to germ and embryonic stem cells; evidence of the evolutionary conserved morphological and functional characteristics of pluripotent stem cells typical also to cells of the germ line have been obtained in representatives of such various metazoan phyla as Porifera, Cnidaria, Plathelminthes, Arthropoda and Chordata (Isaeva et al., 2003, 2004, 2005, 2008b, 2009, 2011; Shukalyuk et al., 2005, 2007, 2011; Akhmadieva et al., 2007; Isaeva & Akhmadieva, 2011).
The data supported our hypothesis that pluripotent, potentially gametogenic stem cells display evolutionarily conserved features of the morphological and functional organization typical for cells of the germ line and embryonic stem cells. In asexually reproducing invertebrates, from sponges and hydroids to some arthropods and chordates, stem cells share with cells of early embryos evolutionary conserved features presumably involved in maintenance of pluri/totipotency, including the gametogenic program. Such invertebrate cells capable of both gametogenesis and asexual reproduction (blastogenesis) are similar in their potential to mammalian embryonic stem cells. We propose that evolutionary and ontogenetically related cells of early embryos and pluripotent stem cells belong to populations of cells that retain a wide or unlimited morphogenetic potential.
Our results along with literature data allow suggest the existence of evolutionary conservative, common for all studied metazoan representatives, from sponges to chordates, cellular, sub-cellular and molecular bases of pluripotency and “stemness” of stem and germ cells.
Many authors called pluripotent stem cells of animals with asexual reproduction
Stem cells of animals with asexual reproduction, as well as cells of the germ lineage, probably originate in the early embryogenesis either from the early totipotent blastomeres or from their derivates that retain pluri/totipotency. The author believes that the evolutionarily and ontogenetically related cells of early embryos, primary stem and primary germ cells belong to cell populations capable of realizing the developmental program, including gametogenesis (and, potentially, subsequent embryogenesis) and blastogenesis (Isaeva et al., 2008b, 2009; Isaeva, 2010).
Thus, published and original data indicate evolutionary conservation and similarity of the studied morphofunctional properties of stem cells in metazoans with asexual reproduction (from sponges and cnidarians to chordates), germline and embryonic stem cells. In invertebrates with asexual reproduction, stem cells can differentiate into both germline and somatic cells; these pluri/totipotent stem cells represent a source of cells for the life strategy realization including sexual and asexual reproduction. Further research on the stem cells of various metazoan animals may reveal the evolutionary conserved basis of cellular totipotency and potential immortality.
This study was supported by a grant from the Russian Federation for Basic Research (no. 09-04-00019).I am most grateful always to my collegues Andrey Shukalyuk, Anna Akhmadieva, Yana Alexandrova, Alexey Chernyshev and Arkady Reunov for our collective work.