Evidence for Involvement of Wnt Signalling in Body Polarities,Cell Proliferation,and the Neuro-Sensory System in an Adult Ctenophore

Signalling through the Wnt family of secreted proteins originated in a common metazoan ancestor and greatly influenced the evolution of animal body plans. In bilaterians, Wnt signalling plays multiple fundamental roles during embryonic development and in adult tissues, notably in axial patterning, neural development and stem cell regulation. Studies in various cnidarian species have particularly highlighted the evolutionarily conserved role of the Wnt/β-catenin pathway in specification and patterning of the primary embryonic axis. However in another key non-bilaterian phylum, Ctenophora, Wnts are not involved in early establishment of the body axis during embryogenesis. We analysed the expression in the adult of the ctenophore Pleurobrachia pileus of 11 orthologues of Wnt signalling genes including all ctenophore Wnt ligands and Fz receptors and several members of the intracellular β-catenin pathway machinery. All genes are strongly expressed around the mouth margin at the oral pole, evoking the Wnt oral centre of cnidarians. This observation is consistent with primary axis polarisation by the Wnts being a universal metazoan feature, secondarily lost in ctenophores during early development but retained in the adult. In addition, local expression of Wnt signalling genes was seen in various anatomical structures of the body including in the locomotory comb rows, where their complex deployment suggests control by the Wnts of local comb polarity. Other important contexts of Wnt involvement which probably evolved before the ctenophore/cnidarian/bilaterian split include proliferating stem cells and progenitors irrespective of cell types, and developing as well as differentiated neuro-sensory structures.     

Independent specialisation of myosin II paralogues in muscle vs. non-muscle functions during early animal evolution: a ctenophore perspective

ABSTRACT: BACKGROUND: Myosin II (or Myosin Heavy Chain II, MHCII) is a family of molecular motors involved in the contractile activity of animal muscle cells but also in various other cellular processes in non-muscle cells. Previous phylogenetic analyses of bilaterian MHCII genes identified two main clades associated respectively with smooth/non-muscle cells (MHCIIa) and striated muscle cells (MHCIIb). Muscle cells are generally thought to have originated only once in ancient animal history, and decisive insights about their early evolution are expected to come from expression studies of Myosin II genes in the two non-bilaterian phyla that possess muscles, the Cnidaria and Ctenophora. RESULTS: We have uncovered three MHCII paralogues in the ctenophore species Pleurobrachia pileus. Phylogenetic analyses indicate that the MHCIIa / MHCIIb duplication is more ancient than the divergence between extant metazoan lineages. The ctenophore MHCIIa gene (PpiMHCIIa) has an expression pattern akin to that of "stem cell markers" (Piwi, Vasa...) and is expressed in proliferating cells. We identified two MHCIIb genes that originated from a ctenophore-specific duplication. PpiMHCIIb1 represents the exclusively muscular form of myosin II in ctenophore, while PpiMHCIIb2 is expressed in non-muscle cells of various types. In parallel, our phalloidin staining and TEM observations highlight the structural complexity of ctenophore musculature and emphasize the experimental interest of the ctenophore tentacle root, in which myogenesis is spatially ordered and strikingly similar to striated muscle formation in vertebrates. CONCLUSION: MHCIIa expression in putative stem cells/proliferating cells probably represents an ancestral trait, while specific involvement of some MHCIIa genes in smooth muscle fibres is a uniquely derived feature of the vertebrates. That one ctenophore MHCIIb paralogue (PpiMHCIIb2) has retained MHCIIa-like expression features furthermore suggests that muscular expression of the other paralogue, PpiMHCIIb1, was the result of neofunctionalisation within the ctenophore lineage, making independent origin of ctenophore muscle cells a likely option.     

Expansion of the SOX gene family predated the emergence of the Bilateria

Members of the SOX gene family are involved in regulating many developmental processes including neuronal determination and differentiation, and in carcinogenesis. So far they have only been identified in species from the Bilateria (deuterostomes and protostomes). To understand the origins of the SOX family, we used a PCR-based strategy to obtain 28 new sequences of SOX gene HMG domains from four non-bilaterian Metazoa: two sponge species, one ctenophore and one cnidarian. One additional SOX sequence was retrieved from EST sequences of the cnidarian species Clytia hemisphaerica. Unexpected SOX gene diversity was found in these species, especially in the cnidarian and the ctenophore. The topology of gene relationships deduced by Maximum Likelihood analysis, although not supported by bootstrap values, suggested that the SOX family started to diversify in the metazoan stem branch prior to the divergence of demosponges, and that further diversification occurred in the eumetazoan branch, as well as later in calcisponges, ctenophores, cnidarians and vertebrates. In contrast, gene loss appears to have occurred in the nematode and probably in other protostome lineages, explaining their lower number of SOX genes.     

The early human germ cell lineage does not express SOX2 during in vivo development or upon in vitro culture

NANOG, POU5F1, and SOX2 are required by the inner cell mass of the blastocyst and act cooperatively to maintain pluripotency in both mouse and human embryonic stem cells. Inadequacy of any one of them causes loss of the undifferentiated state. Mouse primordial germ cells (PGCs), from which pluripotent embryonic germ cells (EGCs) are derived, also express POU5F1, NANOG, and SOX2. Thus, a similar expression profile has been predicted for human PGCs. Here we show by RT-PCR, immunoblotting, and immunohistochemistry that human PGCs express POU5F1 and NANOG but not SOX2, with no evidence of redundancy within the group B family of human SOX genes. Although lacking SOX2, proliferative human germ cells can still be identified in situ during early development and are capable of culture in vitro. Surprisingly, with the exception of FGF4, many stem cell-restricted SOX2 target genes remained detected within the human SOX2-negative germ cell lineage. These studies demonstrate an unexpected difference in gene expression between human and mouse. The human PGC is the first primary cell type described to express POU5F1 and NANOG but not SOX2. The data also provide a new reference point for studies attempting to turn human stem cells into gametes by normal developmental pathways for the treatment of infertility.     

A Combination of Culture Conditions and Gene Expression Analysis Can Be Used to Investigate and Predict hES Cell Differentiation Potential towards Male Gonadal Cells

Human embryonic stem cell differentiation towards various cell types belonging to ecto-, endo- and mesodermal cell lineages has been demonstrated, with high efficiency rates using standardized differentiation protocols. However, germ cell differentiation from human embryonic stem cells has been very inefficient so far. Even though the influence of various growth factors has been evaluated, the gene expression of different cell lines in relation to their differentiation potential has not yet been extensively examined. In this study, the potential of three male human embryonic stem cell lines to differentiate towards male gonadal cells was explored by analysing their gene expression profiles. The human embryonic stem cell lines were cultured for 14 days as monolayers on supporting human foreskin fibroblasts or as spheres in suspension, and were differentiated using BMP7, or spontaneous differentiation by omitting exogenous FGF2. TLDA analysis revealed that in the undifferentiated state, these cell lines have diverse mRNA profiles and exhibit significantly different potentials for differentiation towards the cell types present in the male gonads. This potential was associated with important factors directing the fate of the male primordial germ cells in vivo to form gonocytes, such as SOX17 or genes involved in the NODAL/ACTIVIN pathway, for example. Stimulation with BMP7 in suspension culture resulted in up-regulation of cytoplasmic SOX9 protein expression in all three lines. The observation that human embryonic stem cells differentiate towards germ and somatic cells after spontaneous and BMP7-induced stimulation in suspension emphasizes the important role of somatic cells in germ cell differentiation in vitro.     

A dominant-negative form of mouse SOX2 induces trophectoderm differentiation and progressive polyploidy in mouse embryonic stem cells

SOX2 plays an important role in early embryogenesis by cooperating with OCT4 in regulating gene expression in fertilized eggs, yet the precise mechanism through which SOX2 accomplishes this important function remains poorly understood. Here, we describe the identification of two nuclear localization signals (NLS) in SOX2 and the generation of a dominant-negative mutant (Dmu-mSox2) by mutating these two NLS in its high mobility group domain. Characterization of this mutant demonstrated that SOX2 shuttles between the cytoplasm and nucleus using these two NLS. The mutant has lost its ability to interact with OCT4, but remains competent to interact with wild-type SOX2. Functionally, Dmu-mSox2 is inactive and unable to cooperate with OCT4 in transactivating target promoters bearing its binding sites. However, Dmu-mSox2 is able to inhibit the activity of wild-type SOX2 and subsequently suppress the activity of downstream genes such as Oct4 and Nanog. When stably expressed in embryonic stem (ES) cells, Dmu-mSox2 triggered progressive doublings of cell ploidy (>8N), leading to differentiation into the trophectoderm lineage. Knockdown of Sox2 by small interfering RNA also induced trophectoderm differentiation and polyploid formation in mouse ES cells. These results suggest that SOX2 maintains stem cell pluripotency by shuttling between the nucleus and cytoplasm in cooperation with OCT4 to prevent trophectoderm differentiation and polyploid formation in ES cells.     

Somatic stem cells express Piwi and Vasa genes in an adult ctenophore: ancient association of "germline genes" with stemness

Stem cells are essential for animal development and adult tissue homeostasis, and the quest for an ancestral gene fingerprint of stemness is a major challenge for evolutionary developmental biology. Recent studies have indicated that a series of genes, including the transposon silencer Piwi and the translational activator Vasa, specifically involved in germline determination and maintenance in classical bilaterian models (e.g., vertebrates, fly, nematode), are more generally expressed in adult multipotent stem cells in other animals like flatworms and hydras. Since the progeny of these multipotent stem cells includes both somatic and germinal derivatives, it remains unclear whether Vasa, Piwi, and associated genes like Bruno and PL10 were ancestrally linked to stemness, or to germinal potential. We have investigated the expression of Vasa, two Piwi paralogues, Bruno and PL10 in Pleurobrachia pileus, a member of the early-diverging phylum Ctenophora, the probable sister group of cnidarians. These genes were all expressed in the male and female germlines, and with the exception of one of the Piwi paralogues, they showed similar expression patterns within somatic territories (tentacle root, comb rows, aboral sensory complex). Cytological observations and EdU DNA-labelling and long-term retention experiments revealed concentrations of stem cells closely matching these gene expression areas. These stem cell pools are spatially restricted, and each specialised in the production of particular types of somatic cells. These data unveil important aspects of cell renewal within the ctenophore body and suggest that Piwi, Vasa, Bruno, and PL10 belong to a gene network ancestrally acting in two distinct contexts: (i) the germline and (ii) stem cells, whatever the nature of their progeny.     

Androgen receptor expression reduces stemness characteristics of prostate cancer cells (PC3) by repression of CD44 and SOX2

Studies have shown that a subgroup of tumor cells possess stemness characteristics having self-renewal capacity and the ability to form new tumors. We sought to identify the plausible stemness factor that determines the “molecular signature” of prostate cancer (PCa) cells derived from different metastases (PC3, PCa2b, LNCaP, and DU145) and whether androgen receptor (AR) influences the maintenance of stemness features. Here we show sex-determining region Y (SRY)-box 2 (SOX2) as a putative stem cell marker in PC3 PCa cells and not in DU145, PCa2b, or LNCaP cells. PCa2b and PC3 cells were derived from bone metastases. PCa2b cells which are positive for the AR failed to demonstrate the expression of either cluster of differentiation 44 (CD44) or SOX2. Knockdown (KD) of AR in these cells did not affect the expression of either CD44 or SOX2. Conversely, PC3 cells, which are negative for AR, expressed both CD44 and SOX2. However, the expression of AR downregulated the expression of both CD44 and SOX2 in PC3 cells. CD44 regulates SOX2 expression as KD of CD44 and reduces SOX2 levels considerably. SOX2 KD attenuated not only the expression of SNAIL and SLUG but also the migration and tumorsphere formation in PC3 cells. Collectively, our findings underscore a novel role of CD44 signaling in the maintenance of stemness and progression of cancer through SOX2 in AR-independent PC3 cells. SOX2 has a role in the regulation of expression of SNAIL and SLUG. SOX2 could be a potential therapeutic target to thwart the progression of SOX2-positive cancer cells or recurrence of androgen-independent PCa.     

Stem cells of marine invertebrates: Regulation of proliferation and induction of differentiation in vitro

The review surveys own and the literature data on the plasticity of marine invertebrate stem cells. Stem and embryonic cell cultures of marine invertebrates are a novel model system characterized by a high level of physiological and synthetic processes. The production of biologically active substances in vitro may be an alternative to chemical synthesis and aquaculture. The factors involved in determination and maintenance of the pluripotency of marine invertebrate stem cells have been analyzed. The technology of the directed differentiation of marine invertebrate stem cells into certain functionally active cells in vitro embraces the use of different growth factors, various natural and artificial substrates, and unique bioactive compounds from marine invertebrate tissues. To increase the expression levels of regulatory genes, we applied genetically engineered constructions with foreign genes. The regulation of growth and differentiation of marine invertebrate stem cells opens new prospective uses for their application in marine biotechnology and is helpful for research in developmental biology.